research article open access incongruence …...another widespread species according to veron [3] is...
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RESEARCH ARTICLE Open Access
Incongruence between morphotypes andgenetically delimited species in the coral genusStylophora phenotypic plasticity morphologicalconvergence morphological stasis or interspecifichybridizationJean-Franccedilois Flot1234 Jean Blanchot5 Loiumlc Charpy6 Corinne Cruaud2 Wilfredo Y Licuanan7 Yoshikatsu Nakano8Claude Payri9 and Simon Tillier3
Abstract
Background Morphological data suggest that unlike most other groups of marine organisms scleractinian coralsof the genus Stylophora are more diverse in the western Indian Ocean and in the Red Sea than in the central Indo-Pacific However the morphology of corals is often a poor predictor of their actual biodiversity hence weconducted a genetic survey of Stylophora corals collected in Madagascar Okinawa the Philippines and NewCaledonia in an attempt to find out the true number of species in these various locations
Results A molecular phylogenetic analysis of the mitochondrial ORF and putative control region concurs with ahaploweb analysis of nuclear ITS2 sequences in delimiting three species among our dataset species A and B arefound in Madagascar whereas species C occurs in Okinawa the Philippines and New Caledonia Comparison ofITS1 sequences from these three species with data available online suggests that species C is also found on theGreat Barrier Reef in Malaysia in the South China Sea and in Taiwan and that a distinct species D occurs in theRed Sea Shallow-water morphs of species A correspond to the morphological description of Stylophoramadagascarensis species B presents the morphology of Stylophora mordax whereas species C comprises variousmorphotypes including Stylophora pistillata and Stylophora mordax
Conclusions Genetic analysis of the coral genus Stylophora reveals species boundaries that are not congruentwith morphological traits Of the four hypotheses that may explain such discrepancy (phenotypic plasticitymorphological stasis morphological convergence and interspecific hybridization) the first two appear likely to playa role but the fourth one is rejected since mitochondrial and nuclear markers yield congruent species delimitationsThe position of the root in our molecular phylogenies suggests that the center of origin of Stylophora is located inthe western Indian Ocean which probably explains why this genus presents a higher biodiversity in thewesternmost part of its area of distribution than in the ldquoCoral Trianglerdquo
Correspondence jflotuni-goettingende1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen GermanyFull list of author information is available at the end of the article
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
copy 2011 Flot et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution and reproduction inany medium provided the original work is properly cited
BackgroundThe reason why most marine life forms including cor-als display their peak of biodiversity in the so calledlsquoCoral Trianglersquo in Southeast Asia remains mysteriousand much debated [12] The rare examples of sea crea-tures that do not conform to this general pattern mayoffer information crucial for our understanding of itsroot causes provided that a solid taxonomic frameworkis available to interpret their present and past distribu-tion (which is unfortunately rarely the case) Severalsuch exceptions to the lsquoCoral Trianglersquo rule can befound in the scleractinian coral family Pocilloporidaethat comprises the three genera Pocillopora Seriatoporaand Stylophora although morphospecies of Seriatoporaare most diverse in the Coral Triangle and thereforeseem to follow the rule Pocillopora ldquohas what appearsto be many regional endemics especially in the centraland far eastern Pacificrdquo and Stylophora ldquohas a higherdiversity in the western Indian Ocean and Red Sea thanin the central Indo-Pacificrdquo [3] However ongoinggenetic studies of species boundaries in Pocillopora andSeriatopora suggest that even though morphologicaldescriptions of pocilloporid corals appear well foundedin some locations [4-6] in others places current taxon-omy is a poor predictor of the actual number of species[7-11]Molecular studies of Stylophora have focused so far on
a single species the ldquolab ratrdquo Stylophora pistillata (Esper1797) without delving into the delineation of interspeci-fic boundaries [1213] S pistillata was originallydescribed ldquoaus den ostindischen Meerenrdquo [14] (from theEast Indian seas in the ldquoCoral Trianglerdquo) as character-ized by short twisted dichotomous branches
(Madrepora aggregata ramulis conglomeratis brevibusdichotomis stellis crenatis profundis pistillo in medioelongato erectordquo [14] Figure 1 left side) and is consid-ered to occur over the entire Indo-Pacific (from the RedSea to Polynesia) The distinction between S pistillataand Stylophora mordax (Dana 1846) has long beendebated in the literature S mordax is considered bysome authors as a junior synonym of S pistillata[15163] and by others as a distinct species [17-24] Smordax was originally described from Fiji as havingldquobranches nearly simple much compressed not thinnerat apex scarcely flabellate 12 to 1 inch broad and 13of an inch thick polyps with a pale yellowish disk andshort tentacles of a bright green colour deep brown atbase Corallum with the cells strongly vaulted and thesurface therefore decidedly scabrousrdquo (Figure 1 rightside) [25] however intermediary forms between S mor-dax and S pistillata are commonly found in the fieldand the areas of distribution of these two morphotypesare nearly identical further adding to the confusionAnother widespread species according to Veron [3] is
Stylophora subseriata (Ehrenberg 1834) also foundacross the Indo-Pacific region whereas the five othermorphospecies of this genus are restricted to the RedSea and the Gulf of Aden (Stylophora kuehlmanniScheer and Pillai 1983 Stylophora danae MilneEdwards and Haime 1850 Stylophora mamillata Scheerand Pillai 1983) to Madagascar (Stylophora madagas-carensis Veron 2000) or to both of these regions (Stylo-phora wellsi Scheer 1964) Hence the pattern ofoccurrence of the various species of Stylophora seems tocontradict strongly the common ldquoCoral Trianglerdquo centerof biodiversity model However recent reports of S
Figure 1 Drawings of the type specimens of S pistillata and S mordax The drawing of the type of S pistillata (left) is from [25] thedrawing of the type of S mordax (right) is from [14]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 2 of 14
danae and S kuehlmanni from the Philippines [26] havestarted to question this pattern raising further concernthat morphological species may not correspond to actualgenetic entities (and indeed Sheppard and Sheppard[27] considered S danae S kuehlmanni and S subser-iata as ecomorphs of S pistillata)Experimental work has shown that S pistillata is phe-
notypically plastic [2829] under the influence of envir-onmental parameters such as gravity [30] and waterflow [31] Moreover evidence of intergeneric hybridiza-tion between Stylophora and Pocillopora has beenreported in the literature [32] suggesting that hybridiza-tion may a fortiori occur among various Stylophora spe-cies Finally morphological stasis and phenotypicconvergence albeit not reported yet in the genus Stylo-phora may lead to further underestimation of the actualnumber of species in this genus by causing severalgenetically distinct species to be lumped under a singlename To find out whether phenotypic plasticity inter-specific hybridization morphological stasis and phenoty-pic convergence obscure the taxonomy of Stylophorawe conducted a genetic analysis of 70 corals of this
genus collected in Madagascar Okinawa the Philippinesand New Caledonia published sequences of Stylophoraindividuals from Australia Malaysia the South ChinaSea Taiwan and Okinawa were also scrutinized (Figure2) As a first step towards a future taxonomic revisionof this genus we report here the result of our attemptto determine the true number of Stylophora speciesoccurring at these various locations and to test whetherthe unusual biogeographic pattern reported for thisgenus holds true
ResultsPhylogenetic analysis of mitochondrial sequences revealsthe presence of two Stylophora clades in Madagascar vsa single one in the Pacific OceanWe successfully sequenced from all individuals sampleda portion of the mitochondrial ORF and the putativecontrol region (CR) two markers that had previouslybeen characterised as highly variable in the closelyrelated genus Pocillopora [33] (but see [345] for a dif-ferent interpretation of these regions) Since these mar-kers gave results that were completely congruent only
Figure 2 Map of the area of distribution of the genus Stylophora showing our sampling locations Full circles show the locations wherewe collected samples whereas empty circles mark locations where sequences were obtained from the literature The area of distribution of thegenus Stylophora (modified from [3] and [68]) is shown in blue (basemap from httpd-mapscom)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 3 of 14
the result of their combined analysis is presented here(Figure 3) The reciprocal monophyly of each pocillo-porid genus received very strong bootstrap support(100 using maximum likelihood neighbor-joining andparsimony) whereas three clades of Stylophora emergedfrom the phylogenetic analysis clade A comprised 12individuals all from Madagascar clade B comprised 7individuals also all from Madagascar whereas clade Ccomprised the remaining 51 individuals from New Cale-donia Okinawa and the Philippines plus one previouslypublished sequence (from the complete mitochondrialgenome of one Stylophora pistillata individual from Tai-wan [34]) All three clades were very strongly supported(100 bootstrap support using all three methods)
Haploweb analysis of nuclear ITS2 sequences shows thatthese three clades represent distinct speciesWe obtained nuclear internal transcribed spacer 2 (ITS2)sequences from all individuals sampled and analyzedthem together with the single published Stylophora ITS2sequence available from GenBank (also from a Stylophorapistillata individual from Taiwan [35]) Despite its multi-ple-copy nature and its concerted mode of evolution[36] the ITS2 behaved in the present study just like aldquoregularrdquo single-copy nuclear marker with each indivi-dual harboring either one or two sequence types More-over most ITS2 sequences types found co-occurring insome individuals were also observed occurring alone inother coral colonies suggesting that these sequence typeswere allelic and segregated in a Mendelian fashion forthis reason we decided to call ldquoheterozygotesrdquo all indivi-duals found to possess two different ITS2 types eventhough we could not be totally sure that all ITS2sequences obtained were really allelic (in the closelyrelated genus Pocillopora for instance three ITS2sequences types were observed in one individual [6])Molecular phylogenetic analyses revealed many clades
of sequences several of which were very strongly sup-ported (Figure 4) In order to determine which of theseclades were conspecific and which ones belonged to dif-ferent species curves were added connecting sequencesfound co-occurring in heterozygous individuals therebyconverting the ITS2 tree into a haploweb [9] Thisapproach revealed three distinct pools of ITS2sequences corresponding to the three clades obtainedfrom mitochondrial DNA The monophyly of clades Aand B in our ITS2 dataset received very strong bootstrapsupport using all three methods whereas the monophylyof clade C was only weakly supported using maximumlikelihood (lt50 bootstrap support) and not at all sup-ported using neighbor-joining and parsimony None ofthe 43 heterozygous individuals in our dataset containedITS2 sequences from two different clades thereforeclades A B and C correspond to three distinct species
according to the mutual allelic exclusivity criterion[379]
Phylogenetic analysis of nuclear ITS1 sequences revealsthe existence of a fourth species of Stylophora in the RedSeaEven though the ITS2 of scleractinian corals is generallyeasier to align and more informative than ITS1 [35] and istherefore more commonly used the vast majority of ITSsequences available to date for the genus Stylophora areITS1 sequences In order to compare the results of ourstudy with those previously published data the ITS1regions of 14 representative individuals from our molecu-larly defined species A B and C were sequenced andaligned with sequences available in GenBank Alignmentwas indeed markedly more uncertain for ITS1 than forITS2 and the resulting alignment was rather short (345positions including many gaps vs 700 positions for ITS2and 2645 positions for the combined mitochondrial mar-kers) nevertheless the resulting phylogeny revealed inter-esting patterns and is therefore presented here (Figure 5)The three species delimited from the mitochondrial
and ITS2 datasets were recovered as distinct clades ofITS1 sequences the monophyly of species B was verystrongly supported (gt98 bootstrap support using allthree methods) whereas the monophyly of species Creceived weaker bootstrap support and the monophyly ofspecies A was very weakly supported All previously pub-lished ITS1 sequences from Australia Malaysia theSouth China Sea Taiwan and Japan turned out to belongto species C whereas all published Stylophora sequencesfrom the Red Sea fell in a well supported fourth clade D(gt90 bootstrap support using maximum likelihood andneighbor-joining) that can be considered a distinct spe-cies following the criterion of reciprocal monophyly
DiscussionHaplowebs are useful tools to delineate speciesThe present study confirms the usefulness of ourrecently proposed haploweb approach to deal withsequences of nuclear markers [9] Whereas the corre-sponding phylogenetic tree supported the delineation ofclades A and B but revealed a large number of cladesamong our sequences from the Pacific Ocean haplowebanalysis (Figure 4) showed that these various PacificOcean clades are actually conspecific (since many het-erozygous individuals harbor sequences from two differ-ent clades) The monophyly of species C was only veryweakly supported in the maximum-likelihood phylogeny(with a bootstrap value of only 43) and not at all sup-ported using neighbor-joining and parsimony thereforethis species would probably not have been detected inour ITS2 data if we had not taken into account theinformation provided by the co-occurrence of
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 4 of 14
001
04NC130
Pocillopora meandrina (02Oahu18)
04NC436
04NC071
EU400214 (Taiwan)
07Mad157
Seriatopora Cluster 3 (04NC360)
04NC145
04Oki195
04NC365
04NC452
Pocillopora damicornis (02Oahu08)
07Mad160
04NC440
04NC282
04NC232
04NC253
04NC141
Seriatopora Cluster 2 (04Oki112)
07Mad159
07Mad189
04NC439
07Mad172
04NC199
04NC336
04NC139
04NC379
04NC152
04NC237
04NC131
04NC087
04Oki140
07Mad088
07Mad071
04NC189
07Mad151
04NC170
Pocillopora eydouxi (02Oahu32)
Seriatopora Cluster 1 (04NC432)
07Mad086
04NC132
07Mad082
04NC024
04NC233
07Mad161
04NC289
04NC101
04NC105
04NC251
07Mad150
07Mad079
04NC43404NC323
04NC182
04NC009
04Oki136
05Phil53
07Mad188
04NC064
04NC012
04NC324
04NC404
Seriatopora Cluster 4 (05Phil77)
07Mad073
04NC455
07Mad074
04NC230
07Mad070
07Mad087
04NC373
04NC187
07Mad170
04NC266
04NC441
04Oki115
04NC149
04NC140
05Phil19
93100100
100100100
100100100
100100100
835298
100100100
100100100
100100100
100100100
A
B
C
Figure 3 Phylogenetic tree of mitochondrial DNA sequences (ORF and CR combined) Outgroup sequences are from [6] and [7] whereasone ingroup sequence comes from the complete mitochondrial genome of one Stylophora pistillata individual from the Penghu Islands nearTaiwan [34] This haplotree was generated with PhyML using the TPM3uf+G model suggested by jModelTest Bootstrap values obtained usingmaximum likelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The three main clades aredelineated with brackets and labeled A B and C
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 5 of 14
001
07Mad188b
04NC139b
04NC009a
04NC064a
04NC182a
07Mad070
07Mad082
04NC009b
04NC230
04NC266
04NC071b
07Mad172a
04NC199
04NC282b
07Mad161b
04NC439
04NC441a
04NC189a
07Mad074b
04NC452
07Mad087a
04NC434a
05Phil19
04NC141
04NC251b
04NC145b
04NC289a
04NC071a
04NC012b
07Mad189b
07Mad071
04NC434b
04NC189b04NC187
04NC441b
07Mad150
04NC105
07Mad157b
04NC012a
04NC436a
04NC024b
04NC336b
04NC379b
04NC130a
04NC101
04NC087a
04NC149a
07Mad159
04NC152a
04NC289b
04NC404
07Mad073a
04NC130b
04NC140
04NC233b
04NC182b
07Mad087b
07Mad188a
AY722795 (Taiwan)
04NC064b
04Oki115b
04NC251a
04NC323
07Mad088a
04NC087b
04Oki195a
04NC440b
07Mad088b
04NC336a
04Oki140
07Mad189a
04NC149b
07Mad151a
04Oki115a
07Mad086b
04NC132b
07Mad157a
04NC440a
04NC282a
04NC237a
04Oki136b
07Mad160b
07Mad172b
04NC365a
04Oki195b
07Mad074a
07Mad073b
04NC253a
07Mad086a
04NC170
07Mad170
04NC436b
04NC455
07Mad161a
04NC324a04NC365b
05Phil53
04NC253b
04NC024a
04NC233a
04NC152b
07Mad160a
04NC131
04Oki136a
07Mad151b
04NC139a
04NC132a
04NC237b
04NC324b
04NC373
04NC145a
04NC232
04NC379a
07Mad079
10010099
10010099
43--
993100100
99510098
828360
828983
99610097
7958-
808372
7583-
A
B
C
Figure 4 Haploweb of ITS2 sequences This graph was derived from a phylogenetic tree (obtained with PhyML using the TIM2+G modelsuggested by jModelTest) by drawing curves connecting haplotypes found co-occurring in heterozygous individuals [9] The sequence of oneStylophora pistillata individual from the Penghu Islands near Taiwan [35] is included in the figure and bootstrap values obtained using maximumlikelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The position of the root was inferred fromthe mitochondrial DNA phylogeny (Figure 3) This approach delineated three reproductively isolated pools of Stylophora sequences labeled A Band C as each of these groups corresponded to one of the mitochondrial clades of Figure 3
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 6 of 14
phylogenetically distant alleles in some individuals Incontrast haploweb analysis delineated three groups ofalleles among our ITS2 sequences a result perfectlycongruent with the mitochondrial phylogeny obtainedfrom the same set of individualsUnlike in our previous article [9] the haploweb pre-
sented here was built on a tree rather than a networkIndeed tree-based haplowebs are more straightforwardto draw than their network-based counterparts and arealso more informative since they display the genotype ofeach sequenced individual However precisely due to
their larger information content tree-based haplowebstend to become messy when dealing with large datasetsandor non-monophyletic species in such cases net-work-based haplowebs often turn out to be faster todraw and easier to interpret than tree-based ones
Are molecularly delimited species of Stylophoracongruent with morphologyMost of the 12 individuals from species A displayed thecharacteristic morphology of S madagascarensis (someexamples are shown on Figure 6) ldquocolonies have thin
D
005
AF416161 Stylophora pistillata brown Heron Island
AJ619830 Stylophora pistillata Red Sea
07Mad157a Madagascar
04Oki140 Okinawa
AF416224 Stylophora pistillata pink Sesoko
AJ619838 Stylophora pistillata Red Sea
AF416170 Stylophora pistillata brown One Tree Island
07Mad150 Madagascar
AJ865618 Stylophora pistillata Red Sea
AF416150 Stylophora pistillata pink Akajima
AF416229 Stylophora pistillata brown Terengganu
AF416151 Stylophora pistillata pink Akajima
AF416147 Stylophora pistillata brown Akajima
AF416194 Stylophora pistillata brown Moulter Cay
AF416222 Stylophora pistillata pink Sesoko
AJ619837 Stylophora pistillata Red Sea
AF416219 Stylophora pistillata brown Sesoko
AF416162 Stylophora pistillata pink Heron Island
AJ619844 Stylophora pistillata Red Sea
AJ865621 Stylophora pistillata Red Sea
AJ865631 Stylophora pistillata Red Sea
AJ619831 Stylophora pistillata Red Sea
AF416202 Stylophora pistillata pink Moulter Cay
AF416227 Stylophora pistillata brown Terengganu
AJ619841 Stylophora pistillata Red Sea
07Mad160a Madagascar
AF416182 Stylophora pistillata brown Raine Island
AJ619817 Stylophora pistillata Red Sea
AF416232 Stylophora pistillata brown Terengganu
AJ619811 Stylophora pistillata Red Sea
AF416193 Stylophora pistillata brown Raine Island
AF416231 Stylophora pistillata brown Terengganu
AF416201 Stylophora pistillata brown Moulter Cay
AF416173 Stylophora pistillata brown One Tree Island
AF416196 Stylophora pistillata brown Moulter Cay
AJ619829 Stylophora pistillata Red Sea
AJ619850 Stylophora pistillata Red Sea
AF416156 Stylophora pistillata brown Heron Island
AJ619820 Stylophora pistillata Red Sea
AJ865608 Stylophora pistillata Red Sea
AJ865625 Stylophora pistillata Red Sea
AF416209 Stylophora pistillata pink Sabah
AF416154 Stylophora pistillata pink Akajima
AF416181 Stylophora pistillata brown Raine Island
AF416175 Stylophora pistillata pink One Tree Island
AF416210 Stylophora pistillata pink Sabah
AJ619840 Stylophora pistillata Red Sea
AJ619832 Stylophora pistillata Red Sea
AF416164 Stylophora pistillata pink Heron Island
AJ865633 Stylophora pistillata Red Sea
AF416187 Stylophora pistillata brown Raine Island
AJ619808 Stylophora pistillata Red Sea
AF416199 Stylophora pistillata brown Moulter Cay
AF416172 Stylophora pistillata brown One Tree Island
AJ619816 Stylophora pistillata Red Sea
AJ619814 Stylophora pistillata Red Sea
AF416171 Stylophora pistillata brown One Tree Island
AF416179 Stylophora pistillata pink One Tree IslandAF416176 Stylophora pistillata pink One Tree Island
AJ865630 Stylophora pistillata Red Sea
AF416183 Stylophora pistillata brown Raine Island
AF416212 Stylophora pistillata pink Sabah
AF416228 Stylophora pistillata brown Terengganu
AJ865614 Stylophora pistillata Red Sea
AF416146 Stylophora pistillata brown Akajima
04NC101 New Caledonia
AF416216 Stylophora pistillata brown Sesoko
AF416206 Stylophora pistillata brown Sabah
07Mad160b Madagascar
AJ619845 Stylophora pistillata Red Sea
AJ865605 Stylophora pistillata Red Sea
AJ619833 Stylophora pistillata Red Sea
07Mad082 Madagascar
AF416178 Stylophora pistillata pink One Tree Island
AF416223 Stylophora pistillata pink Sesoko
AJ619825 Stylophora pistillata Red Sea
AF416163 Stylophora pistillata pink Heron Island
AF416157 Stylophora pistillata brown Heron Island
AF416155 Stylophora pistillata pink Akajima
AJ619839 Stylophora pistillata Red Sea
AF416217 Stylophora pistillata brown Sesoko
AF038909 Stylophora pistillata One Tree Island
AJ619848 Stylophora pistillata Red Sea
AJ865616 Stylophora pistillata Red Sea
07Mad073a Madagascar
AF416159 Stylophora pistillata brown Heron Island
04NC140a New Caledonia
HM013855 Seriatopora hystrix Xisha Islands
AJ619852 Stylophora pistillata Red Sea
AF416218 Stylophora pistillata brown Sesoko
AF416204 Stylophora pistillata brown Sabah
AJ619846 Stylophora pistillata Red Sea
AJ619819 Stylophora pistillata Red Sea
AJ619828 Stylophora pistillata Red Sea
AJ619810 Stylophora pistillata Red Sea
AJ619818 Stylophora pistillata Red SeaAJ619826 Stylophora pistillata Red Sea
AJ619835 Stylophora pistillata Red Sea
AJ619806 Stylophora pistillata Red Sea
AF038908 Stylophora pistillata One Tree Island
AJ865620 Stylophora pistillata Red Sea
05Phil53 Philippines
AF416160 Stylophora pistillata brown Heron IslandAF038907 Stylophora pistillata One Tree Island
AF416177 Stylophora pistillata pink One Tree Island
AF416168 Stylophora pistillata brown One Tree Island
AJ865617 Stylophora pistillata Red Sea
AF416174 Stylophora pistillata pink One Tree Island
AJ865626 Stylophora pistillata Red Sea
AF416208 Stylophora pistillata brown Sabah
04NC140b New Caledonia
AJ865623 Stylophora pistillata Red Sea
AF416189 Stylophora pistillata pink Raine Island
AF416220 Stylophora pistillata brown Sesoko
AJ865624 Stylophora pistillata Red Sea
AF416184 Stylophora pistillata brown Raine Island
AJ865627 Stylophora pistillata Red Sea
AJ619822 Stylophora pistillata Red Sea
AF416167 Stylophora pistillata pink Heron Island
AJ619842 Stylophora pistillata Red Sea
AJ865622 Stylophora pistillata Red Sea
HM013847 Pocillopora damicornis Xisha Islands
AY722795 Stylophora pistillata Penghu Island
AJ619824 Stylophora pistillata Red Sea
AF416226 Stylophora pistillata pink Sesoko
AJ865607 Stylophora pistillata Red Sea
AJ619805 Stylophora pistillata Red Sea
AJ619807 Stylophora pistillata Red Sea
AF416213 Stylophora pistillata pink Sabah
AJ865606 Stylophora pistillata Red Sea
AF416207 Stylophora pistillata brown Sabah
AJ619827 Stylophora pistillata Red Sea
AF416203 Stylophora pistillata brown Sabah
07Mad157b Madagascar
AJ619836 Stylophora pistillata Red Sea
07Mad073b Madagascar
AF416192 Stylophora pistillata brown Raine Island
AJ619812 Stylophora pistillata Red Sea
07Mad070 Madagascar
04NC105 New Caledonia
AF416195 Stylophora pistillata brown Moulter Cay
AJ865604 Stylophora pistillata Red Sea
HM013856 Stylophora pistillata Xisha Islands
AJ619843 Stylophora pistillata Red Sea
AJ619834 Stylophora pistillata Red Sea
07Mad188a Madagascar
AF416152 Stylophora pistillata pink Akajima
AF416186 Stylophora pistillata brown Raine Island
AJ865628 Stylophora pistillata Red Sea
AF416205 Stylophora pistillata brown Sabah
AF416149 Stylophora pistillata brown Akajima
AJ619821 Stylophora pistillata Red Sea
07Mad188b Madagascar
AJ619813 Stylophora pistillata Red Sea
AJ619847 Stylophora pistillata Red Sea
AJ619809 Stylophora pistillata Red Sea
AJ865615 Stylophora pistillata Red Sea
AF416225 Stylophora pistillata pink Sesoko
AF416190 Stylophora pistillata brown Raine Island
AJ865610 Stylophora pistillata Red Sea
AF416198 Stylophora pistillata brown Moulter Cay
AJ865629 Stylophora pistillata Red Sea
AF416221 Stylophora pistillata pink Sesoko
AF416145 Stylophora pistillata brown Akajima
AJ865611 Stylophora pistillata Red Sea
AF416169 Stylophora pistillata brown One Tree Island
AF416148 Stylophora pistillata brown Akajima
AF416214 Stylophora pistillata pink Sabah
AJ865613 Stylophora pistillata Red Sea
AJ865609 Stylophora pistillata Red Sea
AF416158 Stylophora pistillata brown Heron Island
AJ865619 Stylophora pistillata Red Sea
AF416165 Stylophora pistillata pink Heron Island
AF416191 Stylophora pistillata brown Raine Island
AJ619815 Stylophora pistillata Red Sea
AF416215 Stylophora pistillata brown Sesoko
AF416153 Stylophora pistillata pink Akajima
07Mad071 Madagascar
AF416185 Stylophora pistillata pink Raine Island
05Phil19 Philippines
AF416200 Stylophora pistillata brown Moulter Cay
AJ619851 Stylophora pistillata Red Sea
AJ619849 Stylophora pistillata Red Sea
AJ865632 Stylophora pistillata Red Sea
AF416211 Stylophora pistillata pink Sabah
AF416188 Stylophora pistillata brown Raine Island
AF416197 Stylophora pistillata brown Moulter Cay
AJ865612 Stylophora pistillata Red Sea
AF416230 Stylophora pistillata brown Terengganu
AJ619823 Stylophora pistillata Red Sea
AF416166 Stylophora pistillata pink Heron Island
AF416144 Stylophora pistillata brown Akajima
9129979
6937118
94490-
297--
9809966
345-76
9879998
A
B
C
Figure 5 ITS1 molecular phylogeny of the genus Stylophora In addition to ITS1 sequences from selected individuals of species A B and Cdata from published articles [69123513] and from an unpublished study by Feng You and Hui Huang [GenBank HM013847 HM013855HM013856] were included in the haplotree (generated with PhyML using the TPM2+G model suggested by jModelTest)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 7 of 14
(up to 5 mm diameter) straight compact branches Cor-allites are crowded and uniformly spaced on branchsides and ends They have a slight development ofhoods towards branch ends They have small style-likecolumellae and six primary septa which are fused withthe columellae The coenosteum is covered with finespicules Tentacles are not extended during the dayColour Uniform tan sometimes with pinkish branchbasesrdquo [38] Since Veronrsquos holotype was collected fromthe very same location in Madagascar where we con-ducted our sampling (approximately 4 m depth Tuleacutearsouth-west Madagascarrdquo [38]) there is little doubt thatspecies A and S madagascarensis are conspecificAccording to Veronrsquos description this species wasldquorecorded only from shallow reef environments exposedto some wave action and in south-west Madagascar inshallow sheltered lagoonsrdquo [38] In contrast we foundthis species down to a depth of 30 meters where it dis-played less compact and thicker branches comparedwith the typical S madagacarensis morphology (Figure7) hence S madagascarensis appears to be more ecolo-gically widespread and morphologically variable thanpreviously thought
All 7 individuals of species B from Madagascar hadflattened stout branches (Figure 8) This correspondsunambiguously to the morphological description of Smordax but since S mordax was originally describedfrom Fiji in the Pacific Ocean it is dubious whether thisname may be assigned to species BSpecies C gathered all individuals collected from loca-
tions in the Pacific Ocean It is characterized by exten-sive morphological variation comprising individualsattributable to S pistillata and others attributable to S
Figure 6 Morphology of species A From top to bottom colonies07Mad074 sampled at 5 meters depth colony 07Mad086 sampledat 6 meters depth and colony 07Mad170 sampled at 11 metersdepth display the typical morphology of S madagascarensis (thinstraight compact branches with small hoods) The green wire onthe pictures of the skeletons has a diameter of 09 mm
Figure 7 Deep-water morphs of species A Colony 07Mad088(left) and colony 07Mad097 (right) were both sampled at 30 metersdepth and have thicker less compact branches compared withtypical S madagascarensis morphology
Figure 8 Morphology of species B From top to bottom colony07Mad150 collected at 8 meters depth colony 07Mad071 collectedat 7 meters depth and colony 07Mad160 collected at 3 metersdepth all display the typical morphology of S mordax (shortcompressed branches with well-developed hoods) The green wireon the pictures of the skeletons has a diameter of 09 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 8 of 14
mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described
from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])
What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both
species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to
pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation
Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia
Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm
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Page 9 of 14
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
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Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
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Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
BackgroundThe reason why most marine life forms including cor-als display their peak of biodiversity in the so calledlsquoCoral Trianglersquo in Southeast Asia remains mysteriousand much debated [12] The rare examples of sea crea-tures that do not conform to this general pattern mayoffer information crucial for our understanding of itsroot causes provided that a solid taxonomic frameworkis available to interpret their present and past distribu-tion (which is unfortunately rarely the case) Severalsuch exceptions to the lsquoCoral Trianglersquo rule can befound in the scleractinian coral family Pocilloporidaethat comprises the three genera Pocillopora Seriatoporaand Stylophora although morphospecies of Seriatoporaare most diverse in the Coral Triangle and thereforeseem to follow the rule Pocillopora ldquohas what appearsto be many regional endemics especially in the centraland far eastern Pacificrdquo and Stylophora ldquohas a higherdiversity in the western Indian Ocean and Red Sea thanin the central Indo-Pacificrdquo [3] However ongoinggenetic studies of species boundaries in Pocillopora andSeriatopora suggest that even though morphologicaldescriptions of pocilloporid corals appear well foundedin some locations [4-6] in others places current taxon-omy is a poor predictor of the actual number of species[7-11]Molecular studies of Stylophora have focused so far on
a single species the ldquolab ratrdquo Stylophora pistillata (Esper1797) without delving into the delineation of interspeci-fic boundaries [1213] S pistillata was originallydescribed ldquoaus den ostindischen Meerenrdquo [14] (from theEast Indian seas in the ldquoCoral Trianglerdquo) as character-ized by short twisted dichotomous branches
(Madrepora aggregata ramulis conglomeratis brevibusdichotomis stellis crenatis profundis pistillo in medioelongato erectordquo [14] Figure 1 left side) and is consid-ered to occur over the entire Indo-Pacific (from the RedSea to Polynesia) The distinction between S pistillataand Stylophora mordax (Dana 1846) has long beendebated in the literature S mordax is considered bysome authors as a junior synonym of S pistillata[15163] and by others as a distinct species [17-24] Smordax was originally described from Fiji as havingldquobranches nearly simple much compressed not thinnerat apex scarcely flabellate 12 to 1 inch broad and 13of an inch thick polyps with a pale yellowish disk andshort tentacles of a bright green colour deep brown atbase Corallum with the cells strongly vaulted and thesurface therefore decidedly scabrousrdquo (Figure 1 rightside) [25] however intermediary forms between S mor-dax and S pistillata are commonly found in the fieldand the areas of distribution of these two morphotypesare nearly identical further adding to the confusionAnother widespread species according to Veron [3] is
Stylophora subseriata (Ehrenberg 1834) also foundacross the Indo-Pacific region whereas the five othermorphospecies of this genus are restricted to the RedSea and the Gulf of Aden (Stylophora kuehlmanniScheer and Pillai 1983 Stylophora danae MilneEdwards and Haime 1850 Stylophora mamillata Scheerand Pillai 1983) to Madagascar (Stylophora madagas-carensis Veron 2000) or to both of these regions (Stylo-phora wellsi Scheer 1964) Hence the pattern ofoccurrence of the various species of Stylophora seems tocontradict strongly the common ldquoCoral Trianglerdquo centerof biodiversity model However recent reports of S
Figure 1 Drawings of the type specimens of S pistillata and S mordax The drawing of the type of S pistillata (left) is from [25] thedrawing of the type of S mordax (right) is from [14]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 2 of 14
danae and S kuehlmanni from the Philippines [26] havestarted to question this pattern raising further concernthat morphological species may not correspond to actualgenetic entities (and indeed Sheppard and Sheppard[27] considered S danae S kuehlmanni and S subser-iata as ecomorphs of S pistillata)Experimental work has shown that S pistillata is phe-
notypically plastic [2829] under the influence of envir-onmental parameters such as gravity [30] and waterflow [31] Moreover evidence of intergeneric hybridiza-tion between Stylophora and Pocillopora has beenreported in the literature [32] suggesting that hybridiza-tion may a fortiori occur among various Stylophora spe-cies Finally morphological stasis and phenotypicconvergence albeit not reported yet in the genus Stylo-phora may lead to further underestimation of the actualnumber of species in this genus by causing severalgenetically distinct species to be lumped under a singlename To find out whether phenotypic plasticity inter-specific hybridization morphological stasis and phenoty-pic convergence obscure the taxonomy of Stylophorawe conducted a genetic analysis of 70 corals of this
genus collected in Madagascar Okinawa the Philippinesand New Caledonia published sequences of Stylophoraindividuals from Australia Malaysia the South ChinaSea Taiwan and Okinawa were also scrutinized (Figure2) As a first step towards a future taxonomic revisionof this genus we report here the result of our attemptto determine the true number of Stylophora speciesoccurring at these various locations and to test whetherthe unusual biogeographic pattern reported for thisgenus holds true
ResultsPhylogenetic analysis of mitochondrial sequences revealsthe presence of two Stylophora clades in Madagascar vsa single one in the Pacific OceanWe successfully sequenced from all individuals sampleda portion of the mitochondrial ORF and the putativecontrol region (CR) two markers that had previouslybeen characterised as highly variable in the closelyrelated genus Pocillopora [33] (but see [345] for a dif-ferent interpretation of these regions) Since these mar-kers gave results that were completely congruent only
Figure 2 Map of the area of distribution of the genus Stylophora showing our sampling locations Full circles show the locations wherewe collected samples whereas empty circles mark locations where sequences were obtained from the literature The area of distribution of thegenus Stylophora (modified from [3] and [68]) is shown in blue (basemap from httpd-mapscom)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 3 of 14
the result of their combined analysis is presented here(Figure 3) The reciprocal monophyly of each pocillo-porid genus received very strong bootstrap support(100 using maximum likelihood neighbor-joining andparsimony) whereas three clades of Stylophora emergedfrom the phylogenetic analysis clade A comprised 12individuals all from Madagascar clade B comprised 7individuals also all from Madagascar whereas clade Ccomprised the remaining 51 individuals from New Cale-donia Okinawa and the Philippines plus one previouslypublished sequence (from the complete mitochondrialgenome of one Stylophora pistillata individual from Tai-wan [34]) All three clades were very strongly supported(100 bootstrap support using all three methods)
Haploweb analysis of nuclear ITS2 sequences shows thatthese three clades represent distinct speciesWe obtained nuclear internal transcribed spacer 2 (ITS2)sequences from all individuals sampled and analyzedthem together with the single published Stylophora ITS2sequence available from GenBank (also from a Stylophorapistillata individual from Taiwan [35]) Despite its multi-ple-copy nature and its concerted mode of evolution[36] the ITS2 behaved in the present study just like aldquoregularrdquo single-copy nuclear marker with each indivi-dual harboring either one or two sequence types More-over most ITS2 sequences types found co-occurring insome individuals were also observed occurring alone inother coral colonies suggesting that these sequence typeswere allelic and segregated in a Mendelian fashion forthis reason we decided to call ldquoheterozygotesrdquo all indivi-duals found to possess two different ITS2 types eventhough we could not be totally sure that all ITS2sequences obtained were really allelic (in the closelyrelated genus Pocillopora for instance three ITS2sequences types were observed in one individual [6])Molecular phylogenetic analyses revealed many clades
of sequences several of which were very strongly sup-ported (Figure 4) In order to determine which of theseclades were conspecific and which ones belonged to dif-ferent species curves were added connecting sequencesfound co-occurring in heterozygous individuals therebyconverting the ITS2 tree into a haploweb [9] Thisapproach revealed three distinct pools of ITS2sequences corresponding to the three clades obtainedfrom mitochondrial DNA The monophyly of clades Aand B in our ITS2 dataset received very strong bootstrapsupport using all three methods whereas the monophylyof clade C was only weakly supported using maximumlikelihood (lt50 bootstrap support) and not at all sup-ported using neighbor-joining and parsimony None ofthe 43 heterozygous individuals in our dataset containedITS2 sequences from two different clades thereforeclades A B and C correspond to three distinct species
according to the mutual allelic exclusivity criterion[379]
Phylogenetic analysis of nuclear ITS1 sequences revealsthe existence of a fourth species of Stylophora in the RedSeaEven though the ITS2 of scleractinian corals is generallyeasier to align and more informative than ITS1 [35] and istherefore more commonly used the vast majority of ITSsequences available to date for the genus Stylophora areITS1 sequences In order to compare the results of ourstudy with those previously published data the ITS1regions of 14 representative individuals from our molecu-larly defined species A B and C were sequenced andaligned with sequences available in GenBank Alignmentwas indeed markedly more uncertain for ITS1 than forITS2 and the resulting alignment was rather short (345positions including many gaps vs 700 positions for ITS2and 2645 positions for the combined mitochondrial mar-kers) nevertheless the resulting phylogeny revealed inter-esting patterns and is therefore presented here (Figure 5)The three species delimited from the mitochondrial
and ITS2 datasets were recovered as distinct clades ofITS1 sequences the monophyly of species B was verystrongly supported (gt98 bootstrap support using allthree methods) whereas the monophyly of species Creceived weaker bootstrap support and the monophyly ofspecies A was very weakly supported All previously pub-lished ITS1 sequences from Australia Malaysia theSouth China Sea Taiwan and Japan turned out to belongto species C whereas all published Stylophora sequencesfrom the Red Sea fell in a well supported fourth clade D(gt90 bootstrap support using maximum likelihood andneighbor-joining) that can be considered a distinct spe-cies following the criterion of reciprocal monophyly
DiscussionHaplowebs are useful tools to delineate speciesThe present study confirms the usefulness of ourrecently proposed haploweb approach to deal withsequences of nuclear markers [9] Whereas the corre-sponding phylogenetic tree supported the delineation ofclades A and B but revealed a large number of cladesamong our sequences from the Pacific Ocean haplowebanalysis (Figure 4) showed that these various PacificOcean clades are actually conspecific (since many het-erozygous individuals harbor sequences from two differ-ent clades) The monophyly of species C was only veryweakly supported in the maximum-likelihood phylogeny(with a bootstrap value of only 43) and not at all sup-ported using neighbor-joining and parsimony thereforethis species would probably not have been detected inour ITS2 data if we had not taken into account theinformation provided by the co-occurrence of
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 4 of 14
001
04NC130
Pocillopora meandrina (02Oahu18)
04NC436
04NC071
EU400214 (Taiwan)
07Mad157
Seriatopora Cluster 3 (04NC360)
04NC145
04Oki195
04NC365
04NC452
Pocillopora damicornis (02Oahu08)
07Mad160
04NC440
04NC282
04NC232
04NC253
04NC141
Seriatopora Cluster 2 (04Oki112)
07Mad159
07Mad189
04NC439
07Mad172
04NC199
04NC336
04NC139
04NC379
04NC152
04NC237
04NC131
04NC087
04Oki140
07Mad088
07Mad071
04NC189
07Mad151
04NC170
Pocillopora eydouxi (02Oahu32)
Seriatopora Cluster 1 (04NC432)
07Mad086
04NC132
07Mad082
04NC024
04NC233
07Mad161
04NC289
04NC101
04NC105
04NC251
07Mad150
07Mad079
04NC43404NC323
04NC182
04NC009
04Oki136
05Phil53
07Mad188
04NC064
04NC012
04NC324
04NC404
Seriatopora Cluster 4 (05Phil77)
07Mad073
04NC455
07Mad074
04NC230
07Mad070
07Mad087
04NC373
04NC187
07Mad170
04NC266
04NC441
04Oki115
04NC149
04NC140
05Phil19
93100100
100100100
100100100
100100100
835298
100100100
100100100
100100100
100100100
A
B
C
Figure 3 Phylogenetic tree of mitochondrial DNA sequences (ORF and CR combined) Outgroup sequences are from [6] and [7] whereasone ingroup sequence comes from the complete mitochondrial genome of one Stylophora pistillata individual from the Penghu Islands nearTaiwan [34] This haplotree was generated with PhyML using the TPM3uf+G model suggested by jModelTest Bootstrap values obtained usingmaximum likelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The three main clades aredelineated with brackets and labeled A B and C
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 5 of 14
001
07Mad188b
04NC139b
04NC009a
04NC064a
04NC182a
07Mad070
07Mad082
04NC009b
04NC230
04NC266
04NC071b
07Mad172a
04NC199
04NC282b
07Mad161b
04NC439
04NC441a
04NC189a
07Mad074b
04NC452
07Mad087a
04NC434a
05Phil19
04NC141
04NC251b
04NC145b
04NC289a
04NC071a
04NC012b
07Mad189b
07Mad071
04NC434b
04NC189b04NC187
04NC441b
07Mad150
04NC105
07Mad157b
04NC012a
04NC436a
04NC024b
04NC336b
04NC379b
04NC130a
04NC101
04NC087a
04NC149a
07Mad159
04NC152a
04NC289b
04NC404
07Mad073a
04NC130b
04NC140
04NC233b
04NC182b
07Mad087b
07Mad188a
AY722795 (Taiwan)
04NC064b
04Oki115b
04NC251a
04NC323
07Mad088a
04NC087b
04Oki195a
04NC440b
07Mad088b
04NC336a
04Oki140
07Mad189a
04NC149b
07Mad151a
04Oki115a
07Mad086b
04NC132b
07Mad157a
04NC440a
04NC282a
04NC237a
04Oki136b
07Mad160b
07Mad172b
04NC365a
04Oki195b
07Mad074a
07Mad073b
04NC253a
07Mad086a
04NC170
07Mad170
04NC436b
04NC455
07Mad161a
04NC324a04NC365b
05Phil53
04NC253b
04NC024a
04NC233a
04NC152b
07Mad160a
04NC131
04Oki136a
07Mad151b
04NC139a
04NC132a
04NC237b
04NC324b
04NC373
04NC145a
04NC232
04NC379a
07Mad079
10010099
10010099
43--
993100100
99510098
828360
828983
99610097
7958-
808372
7583-
A
B
C
Figure 4 Haploweb of ITS2 sequences This graph was derived from a phylogenetic tree (obtained with PhyML using the TIM2+G modelsuggested by jModelTest) by drawing curves connecting haplotypes found co-occurring in heterozygous individuals [9] The sequence of oneStylophora pistillata individual from the Penghu Islands near Taiwan [35] is included in the figure and bootstrap values obtained using maximumlikelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The position of the root was inferred fromthe mitochondrial DNA phylogeny (Figure 3) This approach delineated three reproductively isolated pools of Stylophora sequences labeled A Band C as each of these groups corresponded to one of the mitochondrial clades of Figure 3
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 6 of 14
phylogenetically distant alleles in some individuals Incontrast haploweb analysis delineated three groups ofalleles among our ITS2 sequences a result perfectlycongruent with the mitochondrial phylogeny obtainedfrom the same set of individualsUnlike in our previous article [9] the haploweb pre-
sented here was built on a tree rather than a networkIndeed tree-based haplowebs are more straightforwardto draw than their network-based counterparts and arealso more informative since they display the genotype ofeach sequenced individual However precisely due to
their larger information content tree-based haplowebstend to become messy when dealing with large datasetsandor non-monophyletic species in such cases net-work-based haplowebs often turn out to be faster todraw and easier to interpret than tree-based ones
Are molecularly delimited species of Stylophoracongruent with morphologyMost of the 12 individuals from species A displayed thecharacteristic morphology of S madagascarensis (someexamples are shown on Figure 6) ldquocolonies have thin
D
005
AF416161 Stylophora pistillata brown Heron Island
AJ619830 Stylophora pistillata Red Sea
07Mad157a Madagascar
04Oki140 Okinawa
AF416224 Stylophora pistillata pink Sesoko
AJ619838 Stylophora pistillata Red Sea
AF416170 Stylophora pistillata brown One Tree Island
07Mad150 Madagascar
AJ865618 Stylophora pistillata Red Sea
AF416150 Stylophora pistillata pink Akajima
AF416229 Stylophora pistillata brown Terengganu
AF416151 Stylophora pistillata pink Akajima
AF416147 Stylophora pistillata brown Akajima
AF416194 Stylophora pistillata brown Moulter Cay
AF416222 Stylophora pistillata pink Sesoko
AJ619837 Stylophora pistillata Red Sea
AF416219 Stylophora pistillata brown Sesoko
AF416162 Stylophora pistillata pink Heron Island
AJ619844 Stylophora pistillata Red Sea
AJ865621 Stylophora pistillata Red Sea
AJ865631 Stylophora pistillata Red Sea
AJ619831 Stylophora pistillata Red Sea
AF416202 Stylophora pistillata pink Moulter Cay
AF416227 Stylophora pistillata brown Terengganu
AJ619841 Stylophora pistillata Red Sea
07Mad160a Madagascar
AF416182 Stylophora pistillata brown Raine Island
AJ619817 Stylophora pistillata Red Sea
AF416232 Stylophora pistillata brown Terengganu
AJ619811 Stylophora pistillata Red Sea
AF416193 Stylophora pistillata brown Raine Island
AF416231 Stylophora pistillata brown Terengganu
AF416201 Stylophora pistillata brown Moulter Cay
AF416173 Stylophora pistillata brown One Tree Island
AF416196 Stylophora pistillata brown Moulter Cay
AJ619829 Stylophora pistillata Red Sea
AJ619850 Stylophora pistillata Red Sea
AF416156 Stylophora pistillata brown Heron Island
AJ619820 Stylophora pistillata Red Sea
AJ865608 Stylophora pistillata Red Sea
AJ865625 Stylophora pistillata Red Sea
AF416209 Stylophora pistillata pink Sabah
AF416154 Stylophora pistillata pink Akajima
AF416181 Stylophora pistillata brown Raine Island
AF416175 Stylophora pistillata pink One Tree Island
AF416210 Stylophora pistillata pink Sabah
AJ619840 Stylophora pistillata Red Sea
AJ619832 Stylophora pistillata Red Sea
AF416164 Stylophora pistillata pink Heron Island
AJ865633 Stylophora pistillata Red Sea
AF416187 Stylophora pistillata brown Raine Island
AJ619808 Stylophora pistillata Red Sea
AF416199 Stylophora pistillata brown Moulter Cay
AF416172 Stylophora pistillata brown One Tree Island
AJ619816 Stylophora pistillata Red Sea
AJ619814 Stylophora pistillata Red Sea
AF416171 Stylophora pistillata brown One Tree Island
AF416179 Stylophora pistillata pink One Tree IslandAF416176 Stylophora pistillata pink One Tree Island
AJ865630 Stylophora pistillata Red Sea
AF416183 Stylophora pistillata brown Raine Island
AF416212 Stylophora pistillata pink Sabah
AF416228 Stylophora pistillata brown Terengganu
AJ865614 Stylophora pistillata Red Sea
AF416146 Stylophora pistillata brown Akajima
04NC101 New Caledonia
AF416216 Stylophora pistillata brown Sesoko
AF416206 Stylophora pistillata brown Sabah
07Mad160b Madagascar
AJ619845 Stylophora pistillata Red Sea
AJ865605 Stylophora pistillata Red Sea
AJ619833 Stylophora pistillata Red Sea
07Mad082 Madagascar
AF416178 Stylophora pistillata pink One Tree Island
AF416223 Stylophora pistillata pink Sesoko
AJ619825 Stylophora pistillata Red Sea
AF416163 Stylophora pistillata pink Heron Island
AF416157 Stylophora pistillata brown Heron Island
AF416155 Stylophora pistillata pink Akajima
AJ619839 Stylophora pistillata Red Sea
AF416217 Stylophora pistillata brown Sesoko
AF038909 Stylophora pistillata One Tree Island
AJ619848 Stylophora pistillata Red Sea
AJ865616 Stylophora pistillata Red Sea
07Mad073a Madagascar
AF416159 Stylophora pistillata brown Heron Island
04NC140a New Caledonia
HM013855 Seriatopora hystrix Xisha Islands
AJ619852 Stylophora pistillata Red Sea
AF416218 Stylophora pistillata brown Sesoko
AF416204 Stylophora pistillata brown Sabah
AJ619846 Stylophora pistillata Red Sea
AJ619819 Stylophora pistillata Red Sea
AJ619828 Stylophora pistillata Red Sea
AJ619810 Stylophora pistillata Red Sea
AJ619818 Stylophora pistillata Red SeaAJ619826 Stylophora pistillata Red Sea
AJ619835 Stylophora pistillata Red Sea
AJ619806 Stylophora pistillata Red Sea
AF038908 Stylophora pistillata One Tree Island
AJ865620 Stylophora pistillata Red Sea
05Phil53 Philippines
AF416160 Stylophora pistillata brown Heron IslandAF038907 Stylophora pistillata One Tree Island
AF416177 Stylophora pistillata pink One Tree Island
AF416168 Stylophora pistillata brown One Tree Island
AJ865617 Stylophora pistillata Red Sea
AF416174 Stylophora pistillata pink One Tree Island
AJ865626 Stylophora pistillata Red Sea
AF416208 Stylophora pistillata brown Sabah
04NC140b New Caledonia
AJ865623 Stylophora pistillata Red Sea
AF416189 Stylophora pistillata pink Raine Island
AF416220 Stylophora pistillata brown Sesoko
AJ865624 Stylophora pistillata Red Sea
AF416184 Stylophora pistillata brown Raine Island
AJ865627 Stylophora pistillata Red Sea
AJ619822 Stylophora pistillata Red Sea
AF416167 Stylophora pistillata pink Heron Island
AJ619842 Stylophora pistillata Red Sea
AJ865622 Stylophora pistillata Red Sea
HM013847 Pocillopora damicornis Xisha Islands
AY722795 Stylophora pistillata Penghu Island
AJ619824 Stylophora pistillata Red Sea
AF416226 Stylophora pistillata pink Sesoko
AJ865607 Stylophora pistillata Red Sea
AJ619805 Stylophora pistillata Red Sea
AJ619807 Stylophora pistillata Red Sea
AF416213 Stylophora pistillata pink Sabah
AJ865606 Stylophora pistillata Red Sea
AF416207 Stylophora pistillata brown Sabah
AJ619827 Stylophora pistillata Red Sea
AF416203 Stylophora pistillata brown Sabah
07Mad157b Madagascar
AJ619836 Stylophora pistillata Red Sea
07Mad073b Madagascar
AF416192 Stylophora pistillata brown Raine Island
AJ619812 Stylophora pistillata Red Sea
07Mad070 Madagascar
04NC105 New Caledonia
AF416195 Stylophora pistillata brown Moulter Cay
AJ865604 Stylophora pistillata Red Sea
HM013856 Stylophora pistillata Xisha Islands
AJ619843 Stylophora pistillata Red Sea
AJ619834 Stylophora pistillata Red Sea
07Mad188a Madagascar
AF416152 Stylophora pistillata pink Akajima
AF416186 Stylophora pistillata brown Raine Island
AJ865628 Stylophora pistillata Red Sea
AF416205 Stylophora pistillata brown Sabah
AF416149 Stylophora pistillata brown Akajima
AJ619821 Stylophora pistillata Red Sea
07Mad188b Madagascar
AJ619813 Stylophora pistillata Red Sea
AJ619847 Stylophora pistillata Red Sea
AJ619809 Stylophora pistillata Red Sea
AJ865615 Stylophora pistillata Red Sea
AF416225 Stylophora pistillata pink Sesoko
AF416190 Stylophora pistillata brown Raine Island
AJ865610 Stylophora pistillata Red Sea
AF416198 Stylophora pistillata brown Moulter Cay
AJ865629 Stylophora pistillata Red Sea
AF416221 Stylophora pistillata pink Sesoko
AF416145 Stylophora pistillata brown Akajima
AJ865611 Stylophora pistillata Red Sea
AF416169 Stylophora pistillata brown One Tree Island
AF416148 Stylophora pistillata brown Akajima
AF416214 Stylophora pistillata pink Sabah
AJ865613 Stylophora pistillata Red Sea
AJ865609 Stylophora pistillata Red Sea
AF416158 Stylophora pistillata brown Heron Island
AJ865619 Stylophora pistillata Red Sea
AF416165 Stylophora pistillata pink Heron Island
AF416191 Stylophora pistillata brown Raine Island
AJ619815 Stylophora pistillata Red Sea
AF416215 Stylophora pistillata brown Sesoko
AF416153 Stylophora pistillata pink Akajima
07Mad071 Madagascar
AF416185 Stylophora pistillata pink Raine Island
05Phil19 Philippines
AF416200 Stylophora pistillata brown Moulter Cay
AJ619851 Stylophora pistillata Red Sea
AJ619849 Stylophora pistillata Red Sea
AJ865632 Stylophora pistillata Red Sea
AF416211 Stylophora pistillata pink Sabah
AF416188 Stylophora pistillata brown Raine Island
AF416197 Stylophora pistillata brown Moulter Cay
AJ865612 Stylophora pistillata Red Sea
AF416230 Stylophora pistillata brown Terengganu
AJ619823 Stylophora pistillata Red Sea
AF416166 Stylophora pistillata pink Heron Island
AF416144 Stylophora pistillata brown Akajima
9129979
6937118
94490-
297--
9809966
345-76
9879998
A
B
C
Figure 5 ITS1 molecular phylogeny of the genus Stylophora In addition to ITS1 sequences from selected individuals of species A B and Cdata from published articles [69123513] and from an unpublished study by Feng You and Hui Huang [GenBank HM013847 HM013855HM013856] were included in the haplotree (generated with PhyML using the TPM2+G model suggested by jModelTest)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 7 of 14
(up to 5 mm diameter) straight compact branches Cor-allites are crowded and uniformly spaced on branchsides and ends They have a slight development ofhoods towards branch ends They have small style-likecolumellae and six primary septa which are fused withthe columellae The coenosteum is covered with finespicules Tentacles are not extended during the dayColour Uniform tan sometimes with pinkish branchbasesrdquo [38] Since Veronrsquos holotype was collected fromthe very same location in Madagascar where we con-ducted our sampling (approximately 4 m depth Tuleacutearsouth-west Madagascarrdquo [38]) there is little doubt thatspecies A and S madagascarensis are conspecificAccording to Veronrsquos description this species wasldquorecorded only from shallow reef environments exposedto some wave action and in south-west Madagascar inshallow sheltered lagoonsrdquo [38] In contrast we foundthis species down to a depth of 30 meters where it dis-played less compact and thicker branches comparedwith the typical S madagacarensis morphology (Figure7) hence S madagascarensis appears to be more ecolo-gically widespread and morphologically variable thanpreviously thought
All 7 individuals of species B from Madagascar hadflattened stout branches (Figure 8) This correspondsunambiguously to the morphological description of Smordax but since S mordax was originally describedfrom Fiji in the Pacific Ocean it is dubious whether thisname may be assigned to species BSpecies C gathered all individuals collected from loca-
tions in the Pacific Ocean It is characterized by exten-sive morphological variation comprising individualsattributable to S pistillata and others attributable to S
Figure 6 Morphology of species A From top to bottom colonies07Mad074 sampled at 5 meters depth colony 07Mad086 sampledat 6 meters depth and colony 07Mad170 sampled at 11 metersdepth display the typical morphology of S madagascarensis (thinstraight compact branches with small hoods) The green wire onthe pictures of the skeletons has a diameter of 09 mm
Figure 7 Deep-water morphs of species A Colony 07Mad088(left) and colony 07Mad097 (right) were both sampled at 30 metersdepth and have thicker less compact branches compared withtypical S madagascarensis morphology
Figure 8 Morphology of species B From top to bottom colony07Mad150 collected at 8 meters depth colony 07Mad071 collectedat 7 meters depth and colony 07Mad160 collected at 3 metersdepth all display the typical morphology of S mordax (shortcompressed branches with well-developed hoods) The green wireon the pictures of the skeletons has a diameter of 09 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 8 of 14
mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described
from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])
What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both
species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to
pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation
Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia
Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 9 of 14
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
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Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
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Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
danae and S kuehlmanni from the Philippines [26] havestarted to question this pattern raising further concernthat morphological species may not correspond to actualgenetic entities (and indeed Sheppard and Sheppard[27] considered S danae S kuehlmanni and S subser-iata as ecomorphs of S pistillata)Experimental work has shown that S pistillata is phe-
notypically plastic [2829] under the influence of envir-onmental parameters such as gravity [30] and waterflow [31] Moreover evidence of intergeneric hybridiza-tion between Stylophora and Pocillopora has beenreported in the literature [32] suggesting that hybridiza-tion may a fortiori occur among various Stylophora spe-cies Finally morphological stasis and phenotypicconvergence albeit not reported yet in the genus Stylo-phora may lead to further underestimation of the actualnumber of species in this genus by causing severalgenetically distinct species to be lumped under a singlename To find out whether phenotypic plasticity inter-specific hybridization morphological stasis and phenoty-pic convergence obscure the taxonomy of Stylophorawe conducted a genetic analysis of 70 corals of this
genus collected in Madagascar Okinawa the Philippinesand New Caledonia published sequences of Stylophoraindividuals from Australia Malaysia the South ChinaSea Taiwan and Okinawa were also scrutinized (Figure2) As a first step towards a future taxonomic revisionof this genus we report here the result of our attemptto determine the true number of Stylophora speciesoccurring at these various locations and to test whetherthe unusual biogeographic pattern reported for thisgenus holds true
ResultsPhylogenetic analysis of mitochondrial sequences revealsthe presence of two Stylophora clades in Madagascar vsa single one in the Pacific OceanWe successfully sequenced from all individuals sampleda portion of the mitochondrial ORF and the putativecontrol region (CR) two markers that had previouslybeen characterised as highly variable in the closelyrelated genus Pocillopora [33] (but see [345] for a dif-ferent interpretation of these regions) Since these mar-kers gave results that were completely congruent only
Figure 2 Map of the area of distribution of the genus Stylophora showing our sampling locations Full circles show the locations wherewe collected samples whereas empty circles mark locations where sequences were obtained from the literature The area of distribution of thegenus Stylophora (modified from [3] and [68]) is shown in blue (basemap from httpd-mapscom)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 3 of 14
the result of their combined analysis is presented here(Figure 3) The reciprocal monophyly of each pocillo-porid genus received very strong bootstrap support(100 using maximum likelihood neighbor-joining andparsimony) whereas three clades of Stylophora emergedfrom the phylogenetic analysis clade A comprised 12individuals all from Madagascar clade B comprised 7individuals also all from Madagascar whereas clade Ccomprised the remaining 51 individuals from New Cale-donia Okinawa and the Philippines plus one previouslypublished sequence (from the complete mitochondrialgenome of one Stylophora pistillata individual from Tai-wan [34]) All three clades were very strongly supported(100 bootstrap support using all three methods)
Haploweb analysis of nuclear ITS2 sequences shows thatthese three clades represent distinct speciesWe obtained nuclear internal transcribed spacer 2 (ITS2)sequences from all individuals sampled and analyzedthem together with the single published Stylophora ITS2sequence available from GenBank (also from a Stylophorapistillata individual from Taiwan [35]) Despite its multi-ple-copy nature and its concerted mode of evolution[36] the ITS2 behaved in the present study just like aldquoregularrdquo single-copy nuclear marker with each indivi-dual harboring either one or two sequence types More-over most ITS2 sequences types found co-occurring insome individuals were also observed occurring alone inother coral colonies suggesting that these sequence typeswere allelic and segregated in a Mendelian fashion forthis reason we decided to call ldquoheterozygotesrdquo all indivi-duals found to possess two different ITS2 types eventhough we could not be totally sure that all ITS2sequences obtained were really allelic (in the closelyrelated genus Pocillopora for instance three ITS2sequences types were observed in one individual [6])Molecular phylogenetic analyses revealed many clades
of sequences several of which were very strongly sup-ported (Figure 4) In order to determine which of theseclades were conspecific and which ones belonged to dif-ferent species curves were added connecting sequencesfound co-occurring in heterozygous individuals therebyconverting the ITS2 tree into a haploweb [9] Thisapproach revealed three distinct pools of ITS2sequences corresponding to the three clades obtainedfrom mitochondrial DNA The monophyly of clades Aand B in our ITS2 dataset received very strong bootstrapsupport using all three methods whereas the monophylyof clade C was only weakly supported using maximumlikelihood (lt50 bootstrap support) and not at all sup-ported using neighbor-joining and parsimony None ofthe 43 heterozygous individuals in our dataset containedITS2 sequences from two different clades thereforeclades A B and C correspond to three distinct species
according to the mutual allelic exclusivity criterion[379]
Phylogenetic analysis of nuclear ITS1 sequences revealsthe existence of a fourth species of Stylophora in the RedSeaEven though the ITS2 of scleractinian corals is generallyeasier to align and more informative than ITS1 [35] and istherefore more commonly used the vast majority of ITSsequences available to date for the genus Stylophora areITS1 sequences In order to compare the results of ourstudy with those previously published data the ITS1regions of 14 representative individuals from our molecu-larly defined species A B and C were sequenced andaligned with sequences available in GenBank Alignmentwas indeed markedly more uncertain for ITS1 than forITS2 and the resulting alignment was rather short (345positions including many gaps vs 700 positions for ITS2and 2645 positions for the combined mitochondrial mar-kers) nevertheless the resulting phylogeny revealed inter-esting patterns and is therefore presented here (Figure 5)The three species delimited from the mitochondrial
and ITS2 datasets were recovered as distinct clades ofITS1 sequences the monophyly of species B was verystrongly supported (gt98 bootstrap support using allthree methods) whereas the monophyly of species Creceived weaker bootstrap support and the monophyly ofspecies A was very weakly supported All previously pub-lished ITS1 sequences from Australia Malaysia theSouth China Sea Taiwan and Japan turned out to belongto species C whereas all published Stylophora sequencesfrom the Red Sea fell in a well supported fourth clade D(gt90 bootstrap support using maximum likelihood andneighbor-joining) that can be considered a distinct spe-cies following the criterion of reciprocal monophyly
DiscussionHaplowebs are useful tools to delineate speciesThe present study confirms the usefulness of ourrecently proposed haploweb approach to deal withsequences of nuclear markers [9] Whereas the corre-sponding phylogenetic tree supported the delineation ofclades A and B but revealed a large number of cladesamong our sequences from the Pacific Ocean haplowebanalysis (Figure 4) showed that these various PacificOcean clades are actually conspecific (since many het-erozygous individuals harbor sequences from two differ-ent clades) The monophyly of species C was only veryweakly supported in the maximum-likelihood phylogeny(with a bootstrap value of only 43) and not at all sup-ported using neighbor-joining and parsimony thereforethis species would probably not have been detected inour ITS2 data if we had not taken into account theinformation provided by the co-occurrence of
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 4 of 14
001
04NC130
Pocillopora meandrina (02Oahu18)
04NC436
04NC071
EU400214 (Taiwan)
07Mad157
Seriatopora Cluster 3 (04NC360)
04NC145
04Oki195
04NC365
04NC452
Pocillopora damicornis (02Oahu08)
07Mad160
04NC440
04NC282
04NC232
04NC253
04NC141
Seriatopora Cluster 2 (04Oki112)
07Mad159
07Mad189
04NC439
07Mad172
04NC199
04NC336
04NC139
04NC379
04NC152
04NC237
04NC131
04NC087
04Oki140
07Mad088
07Mad071
04NC189
07Mad151
04NC170
Pocillopora eydouxi (02Oahu32)
Seriatopora Cluster 1 (04NC432)
07Mad086
04NC132
07Mad082
04NC024
04NC233
07Mad161
04NC289
04NC101
04NC105
04NC251
07Mad150
07Mad079
04NC43404NC323
04NC182
04NC009
04Oki136
05Phil53
07Mad188
04NC064
04NC012
04NC324
04NC404
Seriatopora Cluster 4 (05Phil77)
07Mad073
04NC455
07Mad074
04NC230
07Mad070
07Mad087
04NC373
04NC187
07Mad170
04NC266
04NC441
04Oki115
04NC149
04NC140
05Phil19
93100100
100100100
100100100
100100100
835298
100100100
100100100
100100100
100100100
A
B
C
Figure 3 Phylogenetic tree of mitochondrial DNA sequences (ORF and CR combined) Outgroup sequences are from [6] and [7] whereasone ingroup sequence comes from the complete mitochondrial genome of one Stylophora pistillata individual from the Penghu Islands nearTaiwan [34] This haplotree was generated with PhyML using the TPM3uf+G model suggested by jModelTest Bootstrap values obtained usingmaximum likelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The three main clades aredelineated with brackets and labeled A B and C
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 5 of 14
001
07Mad188b
04NC139b
04NC009a
04NC064a
04NC182a
07Mad070
07Mad082
04NC009b
04NC230
04NC266
04NC071b
07Mad172a
04NC199
04NC282b
07Mad161b
04NC439
04NC441a
04NC189a
07Mad074b
04NC452
07Mad087a
04NC434a
05Phil19
04NC141
04NC251b
04NC145b
04NC289a
04NC071a
04NC012b
07Mad189b
07Mad071
04NC434b
04NC189b04NC187
04NC441b
07Mad150
04NC105
07Mad157b
04NC012a
04NC436a
04NC024b
04NC336b
04NC379b
04NC130a
04NC101
04NC087a
04NC149a
07Mad159
04NC152a
04NC289b
04NC404
07Mad073a
04NC130b
04NC140
04NC233b
04NC182b
07Mad087b
07Mad188a
AY722795 (Taiwan)
04NC064b
04Oki115b
04NC251a
04NC323
07Mad088a
04NC087b
04Oki195a
04NC440b
07Mad088b
04NC336a
04Oki140
07Mad189a
04NC149b
07Mad151a
04Oki115a
07Mad086b
04NC132b
07Mad157a
04NC440a
04NC282a
04NC237a
04Oki136b
07Mad160b
07Mad172b
04NC365a
04Oki195b
07Mad074a
07Mad073b
04NC253a
07Mad086a
04NC170
07Mad170
04NC436b
04NC455
07Mad161a
04NC324a04NC365b
05Phil53
04NC253b
04NC024a
04NC233a
04NC152b
07Mad160a
04NC131
04Oki136a
07Mad151b
04NC139a
04NC132a
04NC237b
04NC324b
04NC373
04NC145a
04NC232
04NC379a
07Mad079
10010099
10010099
43--
993100100
99510098
828360
828983
99610097
7958-
808372
7583-
A
B
C
Figure 4 Haploweb of ITS2 sequences This graph was derived from a phylogenetic tree (obtained with PhyML using the TIM2+G modelsuggested by jModelTest) by drawing curves connecting haplotypes found co-occurring in heterozygous individuals [9] The sequence of oneStylophora pistillata individual from the Penghu Islands near Taiwan [35] is included in the figure and bootstrap values obtained using maximumlikelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The position of the root was inferred fromthe mitochondrial DNA phylogeny (Figure 3) This approach delineated three reproductively isolated pools of Stylophora sequences labeled A Band C as each of these groups corresponded to one of the mitochondrial clades of Figure 3
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 6 of 14
phylogenetically distant alleles in some individuals Incontrast haploweb analysis delineated three groups ofalleles among our ITS2 sequences a result perfectlycongruent with the mitochondrial phylogeny obtainedfrom the same set of individualsUnlike in our previous article [9] the haploweb pre-
sented here was built on a tree rather than a networkIndeed tree-based haplowebs are more straightforwardto draw than their network-based counterparts and arealso more informative since they display the genotype ofeach sequenced individual However precisely due to
their larger information content tree-based haplowebstend to become messy when dealing with large datasetsandor non-monophyletic species in such cases net-work-based haplowebs often turn out to be faster todraw and easier to interpret than tree-based ones
Are molecularly delimited species of Stylophoracongruent with morphologyMost of the 12 individuals from species A displayed thecharacteristic morphology of S madagascarensis (someexamples are shown on Figure 6) ldquocolonies have thin
D
005
AF416161 Stylophora pistillata brown Heron Island
AJ619830 Stylophora pistillata Red Sea
07Mad157a Madagascar
04Oki140 Okinawa
AF416224 Stylophora pistillata pink Sesoko
AJ619838 Stylophora pistillata Red Sea
AF416170 Stylophora pistillata brown One Tree Island
07Mad150 Madagascar
AJ865618 Stylophora pistillata Red Sea
AF416150 Stylophora pistillata pink Akajima
AF416229 Stylophora pistillata brown Terengganu
AF416151 Stylophora pistillata pink Akajima
AF416147 Stylophora pistillata brown Akajima
AF416194 Stylophora pistillata brown Moulter Cay
AF416222 Stylophora pistillata pink Sesoko
AJ619837 Stylophora pistillata Red Sea
AF416219 Stylophora pistillata brown Sesoko
AF416162 Stylophora pistillata pink Heron Island
AJ619844 Stylophora pistillata Red Sea
AJ865621 Stylophora pistillata Red Sea
AJ865631 Stylophora pistillata Red Sea
AJ619831 Stylophora pistillata Red Sea
AF416202 Stylophora pistillata pink Moulter Cay
AF416227 Stylophora pistillata brown Terengganu
AJ619841 Stylophora pistillata Red Sea
07Mad160a Madagascar
AF416182 Stylophora pistillata brown Raine Island
AJ619817 Stylophora pistillata Red Sea
AF416232 Stylophora pistillata brown Terengganu
AJ619811 Stylophora pistillata Red Sea
AF416193 Stylophora pistillata brown Raine Island
AF416231 Stylophora pistillata brown Terengganu
AF416201 Stylophora pistillata brown Moulter Cay
AF416173 Stylophora pistillata brown One Tree Island
AF416196 Stylophora pistillata brown Moulter Cay
AJ619829 Stylophora pistillata Red Sea
AJ619850 Stylophora pistillata Red Sea
AF416156 Stylophora pistillata brown Heron Island
AJ619820 Stylophora pistillata Red Sea
AJ865608 Stylophora pistillata Red Sea
AJ865625 Stylophora pistillata Red Sea
AF416209 Stylophora pistillata pink Sabah
AF416154 Stylophora pistillata pink Akajima
AF416181 Stylophora pistillata brown Raine Island
AF416175 Stylophora pistillata pink One Tree Island
AF416210 Stylophora pistillata pink Sabah
AJ619840 Stylophora pistillata Red Sea
AJ619832 Stylophora pistillata Red Sea
AF416164 Stylophora pistillata pink Heron Island
AJ865633 Stylophora pistillata Red Sea
AF416187 Stylophora pistillata brown Raine Island
AJ619808 Stylophora pistillata Red Sea
AF416199 Stylophora pistillata brown Moulter Cay
AF416172 Stylophora pistillata brown One Tree Island
AJ619816 Stylophora pistillata Red Sea
AJ619814 Stylophora pistillata Red Sea
AF416171 Stylophora pistillata brown One Tree Island
AF416179 Stylophora pistillata pink One Tree IslandAF416176 Stylophora pistillata pink One Tree Island
AJ865630 Stylophora pistillata Red Sea
AF416183 Stylophora pistillata brown Raine Island
AF416212 Stylophora pistillata pink Sabah
AF416228 Stylophora pistillata brown Terengganu
AJ865614 Stylophora pistillata Red Sea
AF416146 Stylophora pistillata brown Akajima
04NC101 New Caledonia
AF416216 Stylophora pistillata brown Sesoko
AF416206 Stylophora pistillata brown Sabah
07Mad160b Madagascar
AJ619845 Stylophora pistillata Red Sea
AJ865605 Stylophora pistillata Red Sea
AJ619833 Stylophora pistillata Red Sea
07Mad082 Madagascar
AF416178 Stylophora pistillata pink One Tree Island
AF416223 Stylophora pistillata pink Sesoko
AJ619825 Stylophora pistillata Red Sea
AF416163 Stylophora pistillata pink Heron Island
AF416157 Stylophora pistillata brown Heron Island
AF416155 Stylophora pistillata pink Akajima
AJ619839 Stylophora pistillata Red Sea
AF416217 Stylophora pistillata brown Sesoko
AF038909 Stylophora pistillata One Tree Island
AJ619848 Stylophora pistillata Red Sea
AJ865616 Stylophora pistillata Red Sea
07Mad073a Madagascar
AF416159 Stylophora pistillata brown Heron Island
04NC140a New Caledonia
HM013855 Seriatopora hystrix Xisha Islands
AJ619852 Stylophora pistillata Red Sea
AF416218 Stylophora pistillata brown Sesoko
AF416204 Stylophora pistillata brown Sabah
AJ619846 Stylophora pistillata Red Sea
AJ619819 Stylophora pistillata Red Sea
AJ619828 Stylophora pistillata Red Sea
AJ619810 Stylophora pistillata Red Sea
AJ619818 Stylophora pistillata Red SeaAJ619826 Stylophora pistillata Red Sea
AJ619835 Stylophora pistillata Red Sea
AJ619806 Stylophora pistillata Red Sea
AF038908 Stylophora pistillata One Tree Island
AJ865620 Stylophora pistillata Red Sea
05Phil53 Philippines
AF416160 Stylophora pistillata brown Heron IslandAF038907 Stylophora pistillata One Tree Island
AF416177 Stylophora pistillata pink One Tree Island
AF416168 Stylophora pistillata brown One Tree Island
AJ865617 Stylophora pistillata Red Sea
AF416174 Stylophora pistillata pink One Tree Island
AJ865626 Stylophora pistillata Red Sea
AF416208 Stylophora pistillata brown Sabah
04NC140b New Caledonia
AJ865623 Stylophora pistillata Red Sea
AF416189 Stylophora pistillata pink Raine Island
AF416220 Stylophora pistillata brown Sesoko
AJ865624 Stylophora pistillata Red Sea
AF416184 Stylophora pistillata brown Raine Island
AJ865627 Stylophora pistillata Red Sea
AJ619822 Stylophora pistillata Red Sea
AF416167 Stylophora pistillata pink Heron Island
AJ619842 Stylophora pistillata Red Sea
AJ865622 Stylophora pistillata Red Sea
HM013847 Pocillopora damicornis Xisha Islands
AY722795 Stylophora pistillata Penghu Island
AJ619824 Stylophora pistillata Red Sea
AF416226 Stylophora pistillata pink Sesoko
AJ865607 Stylophora pistillata Red Sea
AJ619805 Stylophora pistillata Red Sea
AJ619807 Stylophora pistillata Red Sea
AF416213 Stylophora pistillata pink Sabah
AJ865606 Stylophora pistillata Red Sea
AF416207 Stylophora pistillata brown Sabah
AJ619827 Stylophora pistillata Red Sea
AF416203 Stylophora pistillata brown Sabah
07Mad157b Madagascar
AJ619836 Stylophora pistillata Red Sea
07Mad073b Madagascar
AF416192 Stylophora pistillata brown Raine Island
AJ619812 Stylophora pistillata Red Sea
07Mad070 Madagascar
04NC105 New Caledonia
AF416195 Stylophora pistillata brown Moulter Cay
AJ865604 Stylophora pistillata Red Sea
HM013856 Stylophora pistillata Xisha Islands
AJ619843 Stylophora pistillata Red Sea
AJ619834 Stylophora pistillata Red Sea
07Mad188a Madagascar
AF416152 Stylophora pistillata pink Akajima
AF416186 Stylophora pistillata brown Raine Island
AJ865628 Stylophora pistillata Red Sea
AF416205 Stylophora pistillata brown Sabah
AF416149 Stylophora pistillata brown Akajima
AJ619821 Stylophora pistillata Red Sea
07Mad188b Madagascar
AJ619813 Stylophora pistillata Red Sea
AJ619847 Stylophora pistillata Red Sea
AJ619809 Stylophora pistillata Red Sea
AJ865615 Stylophora pistillata Red Sea
AF416225 Stylophora pistillata pink Sesoko
AF416190 Stylophora pistillata brown Raine Island
AJ865610 Stylophora pistillata Red Sea
AF416198 Stylophora pistillata brown Moulter Cay
AJ865629 Stylophora pistillata Red Sea
AF416221 Stylophora pistillata pink Sesoko
AF416145 Stylophora pistillata brown Akajima
AJ865611 Stylophora pistillata Red Sea
AF416169 Stylophora pistillata brown One Tree Island
AF416148 Stylophora pistillata brown Akajima
AF416214 Stylophora pistillata pink Sabah
AJ865613 Stylophora pistillata Red Sea
AJ865609 Stylophora pistillata Red Sea
AF416158 Stylophora pistillata brown Heron Island
AJ865619 Stylophora pistillata Red Sea
AF416165 Stylophora pistillata pink Heron Island
AF416191 Stylophora pistillata brown Raine Island
AJ619815 Stylophora pistillata Red Sea
AF416215 Stylophora pistillata brown Sesoko
AF416153 Stylophora pistillata pink Akajima
07Mad071 Madagascar
AF416185 Stylophora pistillata pink Raine Island
05Phil19 Philippines
AF416200 Stylophora pistillata brown Moulter Cay
AJ619851 Stylophora pistillata Red Sea
AJ619849 Stylophora pistillata Red Sea
AJ865632 Stylophora pistillata Red Sea
AF416211 Stylophora pistillata pink Sabah
AF416188 Stylophora pistillata brown Raine Island
AF416197 Stylophora pistillata brown Moulter Cay
AJ865612 Stylophora pistillata Red Sea
AF416230 Stylophora pistillata brown Terengganu
AJ619823 Stylophora pistillata Red Sea
AF416166 Stylophora pistillata pink Heron Island
AF416144 Stylophora pistillata brown Akajima
9129979
6937118
94490-
297--
9809966
345-76
9879998
A
B
C
Figure 5 ITS1 molecular phylogeny of the genus Stylophora In addition to ITS1 sequences from selected individuals of species A B and Cdata from published articles [69123513] and from an unpublished study by Feng You and Hui Huang [GenBank HM013847 HM013855HM013856] were included in the haplotree (generated with PhyML using the TPM2+G model suggested by jModelTest)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 7 of 14
(up to 5 mm diameter) straight compact branches Cor-allites are crowded and uniformly spaced on branchsides and ends They have a slight development ofhoods towards branch ends They have small style-likecolumellae and six primary septa which are fused withthe columellae The coenosteum is covered with finespicules Tentacles are not extended during the dayColour Uniform tan sometimes with pinkish branchbasesrdquo [38] Since Veronrsquos holotype was collected fromthe very same location in Madagascar where we con-ducted our sampling (approximately 4 m depth Tuleacutearsouth-west Madagascarrdquo [38]) there is little doubt thatspecies A and S madagascarensis are conspecificAccording to Veronrsquos description this species wasldquorecorded only from shallow reef environments exposedto some wave action and in south-west Madagascar inshallow sheltered lagoonsrdquo [38] In contrast we foundthis species down to a depth of 30 meters where it dis-played less compact and thicker branches comparedwith the typical S madagacarensis morphology (Figure7) hence S madagascarensis appears to be more ecolo-gically widespread and morphologically variable thanpreviously thought
All 7 individuals of species B from Madagascar hadflattened stout branches (Figure 8) This correspondsunambiguously to the morphological description of Smordax but since S mordax was originally describedfrom Fiji in the Pacific Ocean it is dubious whether thisname may be assigned to species BSpecies C gathered all individuals collected from loca-
tions in the Pacific Ocean It is characterized by exten-sive morphological variation comprising individualsattributable to S pistillata and others attributable to S
Figure 6 Morphology of species A From top to bottom colonies07Mad074 sampled at 5 meters depth colony 07Mad086 sampledat 6 meters depth and colony 07Mad170 sampled at 11 metersdepth display the typical morphology of S madagascarensis (thinstraight compact branches with small hoods) The green wire onthe pictures of the skeletons has a diameter of 09 mm
Figure 7 Deep-water morphs of species A Colony 07Mad088(left) and colony 07Mad097 (right) were both sampled at 30 metersdepth and have thicker less compact branches compared withtypical S madagascarensis morphology
Figure 8 Morphology of species B From top to bottom colony07Mad150 collected at 8 meters depth colony 07Mad071 collectedat 7 meters depth and colony 07Mad160 collected at 3 metersdepth all display the typical morphology of S mordax (shortcompressed branches with well-developed hoods) The green wireon the pictures of the skeletons has a diameter of 09 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 8 of 14
mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described
from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])
What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both
species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to
pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation
Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia
Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm
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Page 9 of 14
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
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Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
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Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
the result of their combined analysis is presented here(Figure 3) The reciprocal monophyly of each pocillo-porid genus received very strong bootstrap support(100 using maximum likelihood neighbor-joining andparsimony) whereas three clades of Stylophora emergedfrom the phylogenetic analysis clade A comprised 12individuals all from Madagascar clade B comprised 7individuals also all from Madagascar whereas clade Ccomprised the remaining 51 individuals from New Cale-donia Okinawa and the Philippines plus one previouslypublished sequence (from the complete mitochondrialgenome of one Stylophora pistillata individual from Tai-wan [34]) All three clades were very strongly supported(100 bootstrap support using all three methods)
Haploweb analysis of nuclear ITS2 sequences shows thatthese three clades represent distinct speciesWe obtained nuclear internal transcribed spacer 2 (ITS2)sequences from all individuals sampled and analyzedthem together with the single published Stylophora ITS2sequence available from GenBank (also from a Stylophorapistillata individual from Taiwan [35]) Despite its multi-ple-copy nature and its concerted mode of evolution[36] the ITS2 behaved in the present study just like aldquoregularrdquo single-copy nuclear marker with each indivi-dual harboring either one or two sequence types More-over most ITS2 sequences types found co-occurring insome individuals were also observed occurring alone inother coral colonies suggesting that these sequence typeswere allelic and segregated in a Mendelian fashion forthis reason we decided to call ldquoheterozygotesrdquo all indivi-duals found to possess two different ITS2 types eventhough we could not be totally sure that all ITS2sequences obtained were really allelic (in the closelyrelated genus Pocillopora for instance three ITS2sequences types were observed in one individual [6])Molecular phylogenetic analyses revealed many clades
of sequences several of which were very strongly sup-ported (Figure 4) In order to determine which of theseclades were conspecific and which ones belonged to dif-ferent species curves were added connecting sequencesfound co-occurring in heterozygous individuals therebyconverting the ITS2 tree into a haploweb [9] Thisapproach revealed three distinct pools of ITS2sequences corresponding to the three clades obtainedfrom mitochondrial DNA The monophyly of clades Aand B in our ITS2 dataset received very strong bootstrapsupport using all three methods whereas the monophylyof clade C was only weakly supported using maximumlikelihood (lt50 bootstrap support) and not at all sup-ported using neighbor-joining and parsimony None ofthe 43 heterozygous individuals in our dataset containedITS2 sequences from two different clades thereforeclades A B and C correspond to three distinct species
according to the mutual allelic exclusivity criterion[379]
Phylogenetic analysis of nuclear ITS1 sequences revealsthe existence of a fourth species of Stylophora in the RedSeaEven though the ITS2 of scleractinian corals is generallyeasier to align and more informative than ITS1 [35] and istherefore more commonly used the vast majority of ITSsequences available to date for the genus Stylophora areITS1 sequences In order to compare the results of ourstudy with those previously published data the ITS1regions of 14 representative individuals from our molecu-larly defined species A B and C were sequenced andaligned with sequences available in GenBank Alignmentwas indeed markedly more uncertain for ITS1 than forITS2 and the resulting alignment was rather short (345positions including many gaps vs 700 positions for ITS2and 2645 positions for the combined mitochondrial mar-kers) nevertheless the resulting phylogeny revealed inter-esting patterns and is therefore presented here (Figure 5)The three species delimited from the mitochondrial
and ITS2 datasets were recovered as distinct clades ofITS1 sequences the monophyly of species B was verystrongly supported (gt98 bootstrap support using allthree methods) whereas the monophyly of species Creceived weaker bootstrap support and the monophyly ofspecies A was very weakly supported All previously pub-lished ITS1 sequences from Australia Malaysia theSouth China Sea Taiwan and Japan turned out to belongto species C whereas all published Stylophora sequencesfrom the Red Sea fell in a well supported fourth clade D(gt90 bootstrap support using maximum likelihood andneighbor-joining) that can be considered a distinct spe-cies following the criterion of reciprocal monophyly
DiscussionHaplowebs are useful tools to delineate speciesThe present study confirms the usefulness of ourrecently proposed haploweb approach to deal withsequences of nuclear markers [9] Whereas the corre-sponding phylogenetic tree supported the delineation ofclades A and B but revealed a large number of cladesamong our sequences from the Pacific Ocean haplowebanalysis (Figure 4) showed that these various PacificOcean clades are actually conspecific (since many het-erozygous individuals harbor sequences from two differ-ent clades) The monophyly of species C was only veryweakly supported in the maximum-likelihood phylogeny(with a bootstrap value of only 43) and not at all sup-ported using neighbor-joining and parsimony thereforethis species would probably not have been detected inour ITS2 data if we had not taken into account theinformation provided by the co-occurrence of
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 4 of 14
001
04NC130
Pocillopora meandrina (02Oahu18)
04NC436
04NC071
EU400214 (Taiwan)
07Mad157
Seriatopora Cluster 3 (04NC360)
04NC145
04Oki195
04NC365
04NC452
Pocillopora damicornis (02Oahu08)
07Mad160
04NC440
04NC282
04NC232
04NC253
04NC141
Seriatopora Cluster 2 (04Oki112)
07Mad159
07Mad189
04NC439
07Mad172
04NC199
04NC336
04NC139
04NC379
04NC152
04NC237
04NC131
04NC087
04Oki140
07Mad088
07Mad071
04NC189
07Mad151
04NC170
Pocillopora eydouxi (02Oahu32)
Seriatopora Cluster 1 (04NC432)
07Mad086
04NC132
07Mad082
04NC024
04NC233
07Mad161
04NC289
04NC101
04NC105
04NC251
07Mad150
07Mad079
04NC43404NC323
04NC182
04NC009
04Oki136
05Phil53
07Mad188
04NC064
04NC012
04NC324
04NC404
Seriatopora Cluster 4 (05Phil77)
07Mad073
04NC455
07Mad074
04NC230
07Mad070
07Mad087
04NC373
04NC187
07Mad170
04NC266
04NC441
04Oki115
04NC149
04NC140
05Phil19
93100100
100100100
100100100
100100100
835298
100100100
100100100
100100100
100100100
A
B
C
Figure 3 Phylogenetic tree of mitochondrial DNA sequences (ORF and CR combined) Outgroup sequences are from [6] and [7] whereasone ingroup sequence comes from the complete mitochondrial genome of one Stylophora pistillata individual from the Penghu Islands nearTaiwan [34] This haplotree was generated with PhyML using the TPM3uf+G model suggested by jModelTest Bootstrap values obtained usingmaximum likelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The three main clades aredelineated with brackets and labeled A B and C
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 5 of 14
001
07Mad188b
04NC139b
04NC009a
04NC064a
04NC182a
07Mad070
07Mad082
04NC009b
04NC230
04NC266
04NC071b
07Mad172a
04NC199
04NC282b
07Mad161b
04NC439
04NC441a
04NC189a
07Mad074b
04NC452
07Mad087a
04NC434a
05Phil19
04NC141
04NC251b
04NC145b
04NC289a
04NC071a
04NC012b
07Mad189b
07Mad071
04NC434b
04NC189b04NC187
04NC441b
07Mad150
04NC105
07Mad157b
04NC012a
04NC436a
04NC024b
04NC336b
04NC379b
04NC130a
04NC101
04NC087a
04NC149a
07Mad159
04NC152a
04NC289b
04NC404
07Mad073a
04NC130b
04NC140
04NC233b
04NC182b
07Mad087b
07Mad188a
AY722795 (Taiwan)
04NC064b
04Oki115b
04NC251a
04NC323
07Mad088a
04NC087b
04Oki195a
04NC440b
07Mad088b
04NC336a
04Oki140
07Mad189a
04NC149b
07Mad151a
04Oki115a
07Mad086b
04NC132b
07Mad157a
04NC440a
04NC282a
04NC237a
04Oki136b
07Mad160b
07Mad172b
04NC365a
04Oki195b
07Mad074a
07Mad073b
04NC253a
07Mad086a
04NC170
07Mad170
04NC436b
04NC455
07Mad161a
04NC324a04NC365b
05Phil53
04NC253b
04NC024a
04NC233a
04NC152b
07Mad160a
04NC131
04Oki136a
07Mad151b
04NC139a
04NC132a
04NC237b
04NC324b
04NC373
04NC145a
04NC232
04NC379a
07Mad079
10010099
10010099
43--
993100100
99510098
828360
828983
99610097
7958-
808372
7583-
A
B
C
Figure 4 Haploweb of ITS2 sequences This graph was derived from a phylogenetic tree (obtained with PhyML using the TIM2+G modelsuggested by jModelTest) by drawing curves connecting haplotypes found co-occurring in heterozygous individuals [9] The sequence of oneStylophora pistillata individual from the Penghu Islands near Taiwan [35] is included in the figure and bootstrap values obtained using maximumlikelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The position of the root was inferred fromthe mitochondrial DNA phylogeny (Figure 3) This approach delineated three reproductively isolated pools of Stylophora sequences labeled A Band C as each of these groups corresponded to one of the mitochondrial clades of Figure 3
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 6 of 14
phylogenetically distant alleles in some individuals Incontrast haploweb analysis delineated three groups ofalleles among our ITS2 sequences a result perfectlycongruent with the mitochondrial phylogeny obtainedfrom the same set of individualsUnlike in our previous article [9] the haploweb pre-
sented here was built on a tree rather than a networkIndeed tree-based haplowebs are more straightforwardto draw than their network-based counterparts and arealso more informative since they display the genotype ofeach sequenced individual However precisely due to
their larger information content tree-based haplowebstend to become messy when dealing with large datasetsandor non-monophyletic species in such cases net-work-based haplowebs often turn out to be faster todraw and easier to interpret than tree-based ones
Are molecularly delimited species of Stylophoracongruent with morphologyMost of the 12 individuals from species A displayed thecharacteristic morphology of S madagascarensis (someexamples are shown on Figure 6) ldquocolonies have thin
D
005
AF416161 Stylophora pistillata brown Heron Island
AJ619830 Stylophora pistillata Red Sea
07Mad157a Madagascar
04Oki140 Okinawa
AF416224 Stylophora pistillata pink Sesoko
AJ619838 Stylophora pistillata Red Sea
AF416170 Stylophora pistillata brown One Tree Island
07Mad150 Madagascar
AJ865618 Stylophora pistillata Red Sea
AF416150 Stylophora pistillata pink Akajima
AF416229 Stylophora pistillata brown Terengganu
AF416151 Stylophora pistillata pink Akajima
AF416147 Stylophora pistillata brown Akajima
AF416194 Stylophora pistillata brown Moulter Cay
AF416222 Stylophora pistillata pink Sesoko
AJ619837 Stylophora pistillata Red Sea
AF416219 Stylophora pistillata brown Sesoko
AF416162 Stylophora pistillata pink Heron Island
AJ619844 Stylophora pistillata Red Sea
AJ865621 Stylophora pistillata Red Sea
AJ865631 Stylophora pistillata Red Sea
AJ619831 Stylophora pistillata Red Sea
AF416202 Stylophora pistillata pink Moulter Cay
AF416227 Stylophora pistillata brown Terengganu
AJ619841 Stylophora pistillata Red Sea
07Mad160a Madagascar
AF416182 Stylophora pistillata brown Raine Island
AJ619817 Stylophora pistillata Red Sea
AF416232 Stylophora pistillata brown Terengganu
AJ619811 Stylophora pistillata Red Sea
AF416193 Stylophora pistillata brown Raine Island
AF416231 Stylophora pistillata brown Terengganu
AF416201 Stylophora pistillata brown Moulter Cay
AF416173 Stylophora pistillata brown One Tree Island
AF416196 Stylophora pistillata brown Moulter Cay
AJ619829 Stylophora pistillata Red Sea
AJ619850 Stylophora pistillata Red Sea
AF416156 Stylophora pistillata brown Heron Island
AJ619820 Stylophora pistillata Red Sea
AJ865608 Stylophora pistillata Red Sea
AJ865625 Stylophora pistillata Red Sea
AF416209 Stylophora pistillata pink Sabah
AF416154 Stylophora pistillata pink Akajima
AF416181 Stylophora pistillata brown Raine Island
AF416175 Stylophora pistillata pink One Tree Island
AF416210 Stylophora pistillata pink Sabah
AJ619840 Stylophora pistillata Red Sea
AJ619832 Stylophora pistillata Red Sea
AF416164 Stylophora pistillata pink Heron Island
AJ865633 Stylophora pistillata Red Sea
AF416187 Stylophora pistillata brown Raine Island
AJ619808 Stylophora pistillata Red Sea
AF416199 Stylophora pistillata brown Moulter Cay
AF416172 Stylophora pistillata brown One Tree Island
AJ619816 Stylophora pistillata Red Sea
AJ619814 Stylophora pistillata Red Sea
AF416171 Stylophora pistillata brown One Tree Island
AF416179 Stylophora pistillata pink One Tree IslandAF416176 Stylophora pistillata pink One Tree Island
AJ865630 Stylophora pistillata Red Sea
AF416183 Stylophora pistillata brown Raine Island
AF416212 Stylophora pistillata pink Sabah
AF416228 Stylophora pistillata brown Terengganu
AJ865614 Stylophora pistillata Red Sea
AF416146 Stylophora pistillata brown Akajima
04NC101 New Caledonia
AF416216 Stylophora pistillata brown Sesoko
AF416206 Stylophora pistillata brown Sabah
07Mad160b Madagascar
AJ619845 Stylophora pistillata Red Sea
AJ865605 Stylophora pistillata Red Sea
AJ619833 Stylophora pistillata Red Sea
07Mad082 Madagascar
AF416178 Stylophora pistillata pink One Tree Island
AF416223 Stylophora pistillata pink Sesoko
AJ619825 Stylophora pistillata Red Sea
AF416163 Stylophora pistillata pink Heron Island
AF416157 Stylophora pistillata brown Heron Island
AF416155 Stylophora pistillata pink Akajima
AJ619839 Stylophora pistillata Red Sea
AF416217 Stylophora pistillata brown Sesoko
AF038909 Stylophora pistillata One Tree Island
AJ619848 Stylophora pistillata Red Sea
AJ865616 Stylophora pistillata Red Sea
07Mad073a Madagascar
AF416159 Stylophora pistillata brown Heron Island
04NC140a New Caledonia
HM013855 Seriatopora hystrix Xisha Islands
AJ619852 Stylophora pistillata Red Sea
AF416218 Stylophora pistillata brown Sesoko
AF416204 Stylophora pistillata brown Sabah
AJ619846 Stylophora pistillata Red Sea
AJ619819 Stylophora pistillata Red Sea
AJ619828 Stylophora pistillata Red Sea
AJ619810 Stylophora pistillata Red Sea
AJ619818 Stylophora pistillata Red SeaAJ619826 Stylophora pistillata Red Sea
AJ619835 Stylophora pistillata Red Sea
AJ619806 Stylophora pistillata Red Sea
AF038908 Stylophora pistillata One Tree Island
AJ865620 Stylophora pistillata Red Sea
05Phil53 Philippines
AF416160 Stylophora pistillata brown Heron IslandAF038907 Stylophora pistillata One Tree Island
AF416177 Stylophora pistillata pink One Tree Island
AF416168 Stylophora pistillata brown One Tree Island
AJ865617 Stylophora pistillata Red Sea
AF416174 Stylophora pistillata pink One Tree Island
AJ865626 Stylophora pistillata Red Sea
AF416208 Stylophora pistillata brown Sabah
04NC140b New Caledonia
AJ865623 Stylophora pistillata Red Sea
AF416189 Stylophora pistillata pink Raine Island
AF416220 Stylophora pistillata brown Sesoko
AJ865624 Stylophora pistillata Red Sea
AF416184 Stylophora pistillata brown Raine Island
AJ865627 Stylophora pistillata Red Sea
AJ619822 Stylophora pistillata Red Sea
AF416167 Stylophora pistillata pink Heron Island
AJ619842 Stylophora pistillata Red Sea
AJ865622 Stylophora pistillata Red Sea
HM013847 Pocillopora damicornis Xisha Islands
AY722795 Stylophora pistillata Penghu Island
AJ619824 Stylophora pistillata Red Sea
AF416226 Stylophora pistillata pink Sesoko
AJ865607 Stylophora pistillata Red Sea
AJ619805 Stylophora pistillata Red Sea
AJ619807 Stylophora pistillata Red Sea
AF416213 Stylophora pistillata pink Sabah
AJ865606 Stylophora pistillata Red Sea
AF416207 Stylophora pistillata brown Sabah
AJ619827 Stylophora pistillata Red Sea
AF416203 Stylophora pistillata brown Sabah
07Mad157b Madagascar
AJ619836 Stylophora pistillata Red Sea
07Mad073b Madagascar
AF416192 Stylophora pistillata brown Raine Island
AJ619812 Stylophora pistillata Red Sea
07Mad070 Madagascar
04NC105 New Caledonia
AF416195 Stylophora pistillata brown Moulter Cay
AJ865604 Stylophora pistillata Red Sea
HM013856 Stylophora pistillata Xisha Islands
AJ619843 Stylophora pistillata Red Sea
AJ619834 Stylophora pistillata Red Sea
07Mad188a Madagascar
AF416152 Stylophora pistillata pink Akajima
AF416186 Stylophora pistillata brown Raine Island
AJ865628 Stylophora pistillata Red Sea
AF416205 Stylophora pistillata brown Sabah
AF416149 Stylophora pistillata brown Akajima
AJ619821 Stylophora pistillata Red Sea
07Mad188b Madagascar
AJ619813 Stylophora pistillata Red Sea
AJ619847 Stylophora pistillata Red Sea
AJ619809 Stylophora pistillata Red Sea
AJ865615 Stylophora pistillata Red Sea
AF416225 Stylophora pistillata pink Sesoko
AF416190 Stylophora pistillata brown Raine Island
AJ865610 Stylophora pistillata Red Sea
AF416198 Stylophora pistillata brown Moulter Cay
AJ865629 Stylophora pistillata Red Sea
AF416221 Stylophora pistillata pink Sesoko
AF416145 Stylophora pistillata brown Akajima
AJ865611 Stylophora pistillata Red Sea
AF416169 Stylophora pistillata brown One Tree Island
AF416148 Stylophora pistillata brown Akajima
AF416214 Stylophora pistillata pink Sabah
AJ865613 Stylophora pistillata Red Sea
AJ865609 Stylophora pistillata Red Sea
AF416158 Stylophora pistillata brown Heron Island
AJ865619 Stylophora pistillata Red Sea
AF416165 Stylophora pistillata pink Heron Island
AF416191 Stylophora pistillata brown Raine Island
AJ619815 Stylophora pistillata Red Sea
AF416215 Stylophora pistillata brown Sesoko
AF416153 Stylophora pistillata pink Akajima
07Mad071 Madagascar
AF416185 Stylophora pistillata pink Raine Island
05Phil19 Philippines
AF416200 Stylophora pistillata brown Moulter Cay
AJ619851 Stylophora pistillata Red Sea
AJ619849 Stylophora pistillata Red Sea
AJ865632 Stylophora pistillata Red Sea
AF416211 Stylophora pistillata pink Sabah
AF416188 Stylophora pistillata brown Raine Island
AF416197 Stylophora pistillata brown Moulter Cay
AJ865612 Stylophora pistillata Red Sea
AF416230 Stylophora pistillata brown Terengganu
AJ619823 Stylophora pistillata Red Sea
AF416166 Stylophora pistillata pink Heron Island
AF416144 Stylophora pistillata brown Akajima
9129979
6937118
94490-
297--
9809966
345-76
9879998
A
B
C
Figure 5 ITS1 molecular phylogeny of the genus Stylophora In addition to ITS1 sequences from selected individuals of species A B and Cdata from published articles [69123513] and from an unpublished study by Feng You and Hui Huang [GenBank HM013847 HM013855HM013856] were included in the haplotree (generated with PhyML using the TPM2+G model suggested by jModelTest)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 7 of 14
(up to 5 mm diameter) straight compact branches Cor-allites are crowded and uniformly spaced on branchsides and ends They have a slight development ofhoods towards branch ends They have small style-likecolumellae and six primary septa which are fused withthe columellae The coenosteum is covered with finespicules Tentacles are not extended during the dayColour Uniform tan sometimes with pinkish branchbasesrdquo [38] Since Veronrsquos holotype was collected fromthe very same location in Madagascar where we con-ducted our sampling (approximately 4 m depth Tuleacutearsouth-west Madagascarrdquo [38]) there is little doubt thatspecies A and S madagascarensis are conspecificAccording to Veronrsquos description this species wasldquorecorded only from shallow reef environments exposedto some wave action and in south-west Madagascar inshallow sheltered lagoonsrdquo [38] In contrast we foundthis species down to a depth of 30 meters where it dis-played less compact and thicker branches comparedwith the typical S madagacarensis morphology (Figure7) hence S madagascarensis appears to be more ecolo-gically widespread and morphologically variable thanpreviously thought
All 7 individuals of species B from Madagascar hadflattened stout branches (Figure 8) This correspondsunambiguously to the morphological description of Smordax but since S mordax was originally describedfrom Fiji in the Pacific Ocean it is dubious whether thisname may be assigned to species BSpecies C gathered all individuals collected from loca-
tions in the Pacific Ocean It is characterized by exten-sive morphological variation comprising individualsattributable to S pistillata and others attributable to S
Figure 6 Morphology of species A From top to bottom colonies07Mad074 sampled at 5 meters depth colony 07Mad086 sampledat 6 meters depth and colony 07Mad170 sampled at 11 metersdepth display the typical morphology of S madagascarensis (thinstraight compact branches with small hoods) The green wire onthe pictures of the skeletons has a diameter of 09 mm
Figure 7 Deep-water morphs of species A Colony 07Mad088(left) and colony 07Mad097 (right) were both sampled at 30 metersdepth and have thicker less compact branches compared withtypical S madagascarensis morphology
Figure 8 Morphology of species B From top to bottom colony07Mad150 collected at 8 meters depth colony 07Mad071 collectedat 7 meters depth and colony 07Mad160 collected at 3 metersdepth all display the typical morphology of S mordax (shortcompressed branches with well-developed hoods) The green wireon the pictures of the skeletons has a diameter of 09 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 8 of 14
mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described
from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])
What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both
species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to
pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation
Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia
Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 9 of 14
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
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52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
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doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
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Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
001
04NC130
Pocillopora meandrina (02Oahu18)
04NC436
04NC071
EU400214 (Taiwan)
07Mad157
Seriatopora Cluster 3 (04NC360)
04NC145
04Oki195
04NC365
04NC452
Pocillopora damicornis (02Oahu08)
07Mad160
04NC440
04NC282
04NC232
04NC253
04NC141
Seriatopora Cluster 2 (04Oki112)
07Mad159
07Mad189
04NC439
07Mad172
04NC199
04NC336
04NC139
04NC379
04NC152
04NC237
04NC131
04NC087
04Oki140
07Mad088
07Mad071
04NC189
07Mad151
04NC170
Pocillopora eydouxi (02Oahu32)
Seriatopora Cluster 1 (04NC432)
07Mad086
04NC132
07Mad082
04NC024
04NC233
07Mad161
04NC289
04NC101
04NC105
04NC251
07Mad150
07Mad079
04NC43404NC323
04NC182
04NC009
04Oki136
05Phil53
07Mad188
04NC064
04NC012
04NC324
04NC404
Seriatopora Cluster 4 (05Phil77)
07Mad073
04NC455
07Mad074
04NC230
07Mad070
07Mad087
04NC373
04NC187
07Mad170
04NC266
04NC441
04Oki115
04NC149
04NC140
05Phil19
93100100
100100100
100100100
100100100
835298
100100100
100100100
100100100
100100100
A
B
C
Figure 3 Phylogenetic tree of mitochondrial DNA sequences (ORF and CR combined) Outgroup sequences are from [6] and [7] whereasone ingroup sequence comes from the complete mitochondrial genome of one Stylophora pistillata individual from the Penghu Islands nearTaiwan [34] This haplotree was generated with PhyML using the TPM3uf+G model suggested by jModelTest Bootstrap values obtained usingmaximum likelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The three main clades aredelineated with brackets and labeled A B and C
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 5 of 14
001
07Mad188b
04NC139b
04NC009a
04NC064a
04NC182a
07Mad070
07Mad082
04NC009b
04NC230
04NC266
04NC071b
07Mad172a
04NC199
04NC282b
07Mad161b
04NC439
04NC441a
04NC189a
07Mad074b
04NC452
07Mad087a
04NC434a
05Phil19
04NC141
04NC251b
04NC145b
04NC289a
04NC071a
04NC012b
07Mad189b
07Mad071
04NC434b
04NC189b04NC187
04NC441b
07Mad150
04NC105
07Mad157b
04NC012a
04NC436a
04NC024b
04NC336b
04NC379b
04NC130a
04NC101
04NC087a
04NC149a
07Mad159
04NC152a
04NC289b
04NC404
07Mad073a
04NC130b
04NC140
04NC233b
04NC182b
07Mad087b
07Mad188a
AY722795 (Taiwan)
04NC064b
04Oki115b
04NC251a
04NC323
07Mad088a
04NC087b
04Oki195a
04NC440b
07Mad088b
04NC336a
04Oki140
07Mad189a
04NC149b
07Mad151a
04Oki115a
07Mad086b
04NC132b
07Mad157a
04NC440a
04NC282a
04NC237a
04Oki136b
07Mad160b
07Mad172b
04NC365a
04Oki195b
07Mad074a
07Mad073b
04NC253a
07Mad086a
04NC170
07Mad170
04NC436b
04NC455
07Mad161a
04NC324a04NC365b
05Phil53
04NC253b
04NC024a
04NC233a
04NC152b
07Mad160a
04NC131
04Oki136a
07Mad151b
04NC139a
04NC132a
04NC237b
04NC324b
04NC373
04NC145a
04NC232
04NC379a
07Mad079
10010099
10010099
43--
993100100
99510098
828360
828983
99610097
7958-
808372
7583-
A
B
C
Figure 4 Haploweb of ITS2 sequences This graph was derived from a phylogenetic tree (obtained with PhyML using the TIM2+G modelsuggested by jModelTest) by drawing curves connecting haplotypes found co-occurring in heterozygous individuals [9] The sequence of oneStylophora pistillata individual from the Penghu Islands near Taiwan [35] is included in the figure and bootstrap values obtained using maximumlikelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The position of the root was inferred fromthe mitochondrial DNA phylogeny (Figure 3) This approach delineated three reproductively isolated pools of Stylophora sequences labeled A Band C as each of these groups corresponded to one of the mitochondrial clades of Figure 3
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 6 of 14
phylogenetically distant alleles in some individuals Incontrast haploweb analysis delineated three groups ofalleles among our ITS2 sequences a result perfectlycongruent with the mitochondrial phylogeny obtainedfrom the same set of individualsUnlike in our previous article [9] the haploweb pre-
sented here was built on a tree rather than a networkIndeed tree-based haplowebs are more straightforwardto draw than their network-based counterparts and arealso more informative since they display the genotype ofeach sequenced individual However precisely due to
their larger information content tree-based haplowebstend to become messy when dealing with large datasetsandor non-monophyletic species in such cases net-work-based haplowebs often turn out to be faster todraw and easier to interpret than tree-based ones
Are molecularly delimited species of Stylophoracongruent with morphologyMost of the 12 individuals from species A displayed thecharacteristic morphology of S madagascarensis (someexamples are shown on Figure 6) ldquocolonies have thin
D
005
AF416161 Stylophora pistillata brown Heron Island
AJ619830 Stylophora pistillata Red Sea
07Mad157a Madagascar
04Oki140 Okinawa
AF416224 Stylophora pistillata pink Sesoko
AJ619838 Stylophora pistillata Red Sea
AF416170 Stylophora pistillata brown One Tree Island
07Mad150 Madagascar
AJ865618 Stylophora pistillata Red Sea
AF416150 Stylophora pistillata pink Akajima
AF416229 Stylophora pistillata brown Terengganu
AF416151 Stylophora pistillata pink Akajima
AF416147 Stylophora pistillata brown Akajima
AF416194 Stylophora pistillata brown Moulter Cay
AF416222 Stylophora pistillata pink Sesoko
AJ619837 Stylophora pistillata Red Sea
AF416219 Stylophora pistillata brown Sesoko
AF416162 Stylophora pistillata pink Heron Island
AJ619844 Stylophora pistillata Red Sea
AJ865621 Stylophora pistillata Red Sea
AJ865631 Stylophora pistillata Red Sea
AJ619831 Stylophora pistillata Red Sea
AF416202 Stylophora pistillata pink Moulter Cay
AF416227 Stylophora pistillata brown Terengganu
AJ619841 Stylophora pistillata Red Sea
07Mad160a Madagascar
AF416182 Stylophora pistillata brown Raine Island
AJ619817 Stylophora pistillata Red Sea
AF416232 Stylophora pistillata brown Terengganu
AJ619811 Stylophora pistillata Red Sea
AF416193 Stylophora pistillata brown Raine Island
AF416231 Stylophora pistillata brown Terengganu
AF416201 Stylophora pistillata brown Moulter Cay
AF416173 Stylophora pistillata brown One Tree Island
AF416196 Stylophora pistillata brown Moulter Cay
AJ619829 Stylophora pistillata Red Sea
AJ619850 Stylophora pistillata Red Sea
AF416156 Stylophora pistillata brown Heron Island
AJ619820 Stylophora pistillata Red Sea
AJ865608 Stylophora pistillata Red Sea
AJ865625 Stylophora pistillata Red Sea
AF416209 Stylophora pistillata pink Sabah
AF416154 Stylophora pistillata pink Akajima
AF416181 Stylophora pistillata brown Raine Island
AF416175 Stylophora pistillata pink One Tree Island
AF416210 Stylophora pistillata pink Sabah
AJ619840 Stylophora pistillata Red Sea
AJ619832 Stylophora pistillata Red Sea
AF416164 Stylophora pistillata pink Heron Island
AJ865633 Stylophora pistillata Red Sea
AF416187 Stylophora pistillata brown Raine Island
AJ619808 Stylophora pistillata Red Sea
AF416199 Stylophora pistillata brown Moulter Cay
AF416172 Stylophora pistillata brown One Tree Island
AJ619816 Stylophora pistillata Red Sea
AJ619814 Stylophora pistillata Red Sea
AF416171 Stylophora pistillata brown One Tree Island
AF416179 Stylophora pistillata pink One Tree IslandAF416176 Stylophora pistillata pink One Tree Island
AJ865630 Stylophora pistillata Red Sea
AF416183 Stylophora pistillata brown Raine Island
AF416212 Stylophora pistillata pink Sabah
AF416228 Stylophora pistillata brown Terengganu
AJ865614 Stylophora pistillata Red Sea
AF416146 Stylophora pistillata brown Akajima
04NC101 New Caledonia
AF416216 Stylophora pistillata brown Sesoko
AF416206 Stylophora pistillata brown Sabah
07Mad160b Madagascar
AJ619845 Stylophora pistillata Red Sea
AJ865605 Stylophora pistillata Red Sea
AJ619833 Stylophora pistillata Red Sea
07Mad082 Madagascar
AF416178 Stylophora pistillata pink One Tree Island
AF416223 Stylophora pistillata pink Sesoko
AJ619825 Stylophora pistillata Red Sea
AF416163 Stylophora pistillata pink Heron Island
AF416157 Stylophora pistillata brown Heron Island
AF416155 Stylophora pistillata pink Akajima
AJ619839 Stylophora pistillata Red Sea
AF416217 Stylophora pistillata brown Sesoko
AF038909 Stylophora pistillata One Tree Island
AJ619848 Stylophora pistillata Red Sea
AJ865616 Stylophora pistillata Red Sea
07Mad073a Madagascar
AF416159 Stylophora pistillata brown Heron Island
04NC140a New Caledonia
HM013855 Seriatopora hystrix Xisha Islands
AJ619852 Stylophora pistillata Red Sea
AF416218 Stylophora pistillata brown Sesoko
AF416204 Stylophora pistillata brown Sabah
AJ619846 Stylophora pistillata Red Sea
AJ619819 Stylophora pistillata Red Sea
AJ619828 Stylophora pistillata Red Sea
AJ619810 Stylophora pistillata Red Sea
AJ619818 Stylophora pistillata Red SeaAJ619826 Stylophora pistillata Red Sea
AJ619835 Stylophora pistillata Red Sea
AJ619806 Stylophora pistillata Red Sea
AF038908 Stylophora pistillata One Tree Island
AJ865620 Stylophora pistillata Red Sea
05Phil53 Philippines
AF416160 Stylophora pistillata brown Heron IslandAF038907 Stylophora pistillata One Tree Island
AF416177 Stylophora pistillata pink One Tree Island
AF416168 Stylophora pistillata brown One Tree Island
AJ865617 Stylophora pistillata Red Sea
AF416174 Stylophora pistillata pink One Tree Island
AJ865626 Stylophora pistillata Red Sea
AF416208 Stylophora pistillata brown Sabah
04NC140b New Caledonia
AJ865623 Stylophora pistillata Red Sea
AF416189 Stylophora pistillata pink Raine Island
AF416220 Stylophora pistillata brown Sesoko
AJ865624 Stylophora pistillata Red Sea
AF416184 Stylophora pistillata brown Raine Island
AJ865627 Stylophora pistillata Red Sea
AJ619822 Stylophora pistillata Red Sea
AF416167 Stylophora pistillata pink Heron Island
AJ619842 Stylophora pistillata Red Sea
AJ865622 Stylophora pistillata Red Sea
HM013847 Pocillopora damicornis Xisha Islands
AY722795 Stylophora pistillata Penghu Island
AJ619824 Stylophora pistillata Red Sea
AF416226 Stylophora pistillata pink Sesoko
AJ865607 Stylophora pistillata Red Sea
AJ619805 Stylophora pistillata Red Sea
AJ619807 Stylophora pistillata Red Sea
AF416213 Stylophora pistillata pink Sabah
AJ865606 Stylophora pistillata Red Sea
AF416207 Stylophora pistillata brown Sabah
AJ619827 Stylophora pistillata Red Sea
AF416203 Stylophora pistillata brown Sabah
07Mad157b Madagascar
AJ619836 Stylophora pistillata Red Sea
07Mad073b Madagascar
AF416192 Stylophora pistillata brown Raine Island
AJ619812 Stylophora pistillata Red Sea
07Mad070 Madagascar
04NC105 New Caledonia
AF416195 Stylophora pistillata brown Moulter Cay
AJ865604 Stylophora pistillata Red Sea
HM013856 Stylophora pistillata Xisha Islands
AJ619843 Stylophora pistillata Red Sea
AJ619834 Stylophora pistillata Red Sea
07Mad188a Madagascar
AF416152 Stylophora pistillata pink Akajima
AF416186 Stylophora pistillata brown Raine Island
AJ865628 Stylophora pistillata Red Sea
AF416205 Stylophora pistillata brown Sabah
AF416149 Stylophora pistillata brown Akajima
AJ619821 Stylophora pistillata Red Sea
07Mad188b Madagascar
AJ619813 Stylophora pistillata Red Sea
AJ619847 Stylophora pistillata Red Sea
AJ619809 Stylophora pistillata Red Sea
AJ865615 Stylophora pistillata Red Sea
AF416225 Stylophora pistillata pink Sesoko
AF416190 Stylophora pistillata brown Raine Island
AJ865610 Stylophora pistillata Red Sea
AF416198 Stylophora pistillata brown Moulter Cay
AJ865629 Stylophora pistillata Red Sea
AF416221 Stylophora pistillata pink Sesoko
AF416145 Stylophora pistillata brown Akajima
AJ865611 Stylophora pistillata Red Sea
AF416169 Stylophora pistillata brown One Tree Island
AF416148 Stylophora pistillata brown Akajima
AF416214 Stylophora pistillata pink Sabah
AJ865613 Stylophora pistillata Red Sea
AJ865609 Stylophora pistillata Red Sea
AF416158 Stylophora pistillata brown Heron Island
AJ865619 Stylophora pistillata Red Sea
AF416165 Stylophora pistillata pink Heron Island
AF416191 Stylophora pistillata brown Raine Island
AJ619815 Stylophora pistillata Red Sea
AF416215 Stylophora pistillata brown Sesoko
AF416153 Stylophora pistillata pink Akajima
07Mad071 Madagascar
AF416185 Stylophora pistillata pink Raine Island
05Phil19 Philippines
AF416200 Stylophora pistillata brown Moulter Cay
AJ619851 Stylophora pistillata Red Sea
AJ619849 Stylophora pistillata Red Sea
AJ865632 Stylophora pistillata Red Sea
AF416211 Stylophora pistillata pink Sabah
AF416188 Stylophora pistillata brown Raine Island
AF416197 Stylophora pistillata brown Moulter Cay
AJ865612 Stylophora pistillata Red Sea
AF416230 Stylophora pistillata brown Terengganu
AJ619823 Stylophora pistillata Red Sea
AF416166 Stylophora pistillata pink Heron Island
AF416144 Stylophora pistillata brown Akajima
9129979
6937118
94490-
297--
9809966
345-76
9879998
A
B
C
Figure 5 ITS1 molecular phylogeny of the genus Stylophora In addition to ITS1 sequences from selected individuals of species A B and Cdata from published articles [69123513] and from an unpublished study by Feng You and Hui Huang [GenBank HM013847 HM013855HM013856] were included in the haplotree (generated with PhyML using the TPM2+G model suggested by jModelTest)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 7 of 14
(up to 5 mm diameter) straight compact branches Cor-allites are crowded and uniformly spaced on branchsides and ends They have a slight development ofhoods towards branch ends They have small style-likecolumellae and six primary septa which are fused withthe columellae The coenosteum is covered with finespicules Tentacles are not extended during the dayColour Uniform tan sometimes with pinkish branchbasesrdquo [38] Since Veronrsquos holotype was collected fromthe very same location in Madagascar where we con-ducted our sampling (approximately 4 m depth Tuleacutearsouth-west Madagascarrdquo [38]) there is little doubt thatspecies A and S madagascarensis are conspecificAccording to Veronrsquos description this species wasldquorecorded only from shallow reef environments exposedto some wave action and in south-west Madagascar inshallow sheltered lagoonsrdquo [38] In contrast we foundthis species down to a depth of 30 meters where it dis-played less compact and thicker branches comparedwith the typical S madagacarensis morphology (Figure7) hence S madagascarensis appears to be more ecolo-gically widespread and morphologically variable thanpreviously thought
All 7 individuals of species B from Madagascar hadflattened stout branches (Figure 8) This correspondsunambiguously to the morphological description of Smordax but since S mordax was originally describedfrom Fiji in the Pacific Ocean it is dubious whether thisname may be assigned to species BSpecies C gathered all individuals collected from loca-
tions in the Pacific Ocean It is characterized by exten-sive morphological variation comprising individualsattributable to S pistillata and others attributable to S
Figure 6 Morphology of species A From top to bottom colonies07Mad074 sampled at 5 meters depth colony 07Mad086 sampledat 6 meters depth and colony 07Mad170 sampled at 11 metersdepth display the typical morphology of S madagascarensis (thinstraight compact branches with small hoods) The green wire onthe pictures of the skeletons has a diameter of 09 mm
Figure 7 Deep-water morphs of species A Colony 07Mad088(left) and colony 07Mad097 (right) were both sampled at 30 metersdepth and have thicker less compact branches compared withtypical S madagascarensis morphology
Figure 8 Morphology of species B From top to bottom colony07Mad150 collected at 8 meters depth colony 07Mad071 collectedat 7 meters depth and colony 07Mad160 collected at 3 metersdepth all display the typical morphology of S mordax (shortcompressed branches with well-developed hoods) The green wireon the pictures of the skeletons has a diameter of 09 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 8 of 14
mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described
from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])
What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both
species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to
pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation
Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia
Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 9 of 14
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
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Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
001
07Mad188b
04NC139b
04NC009a
04NC064a
04NC182a
07Mad070
07Mad082
04NC009b
04NC230
04NC266
04NC071b
07Mad172a
04NC199
04NC282b
07Mad161b
04NC439
04NC441a
04NC189a
07Mad074b
04NC452
07Mad087a
04NC434a
05Phil19
04NC141
04NC251b
04NC145b
04NC289a
04NC071a
04NC012b
07Mad189b
07Mad071
04NC434b
04NC189b04NC187
04NC441b
07Mad150
04NC105
07Mad157b
04NC012a
04NC436a
04NC024b
04NC336b
04NC379b
04NC130a
04NC101
04NC087a
04NC149a
07Mad159
04NC152a
04NC289b
04NC404
07Mad073a
04NC130b
04NC140
04NC233b
04NC182b
07Mad087b
07Mad188a
AY722795 (Taiwan)
04NC064b
04Oki115b
04NC251a
04NC323
07Mad088a
04NC087b
04Oki195a
04NC440b
07Mad088b
04NC336a
04Oki140
07Mad189a
04NC149b
07Mad151a
04Oki115a
07Mad086b
04NC132b
07Mad157a
04NC440a
04NC282a
04NC237a
04Oki136b
07Mad160b
07Mad172b
04NC365a
04Oki195b
07Mad074a
07Mad073b
04NC253a
07Mad086a
04NC170
07Mad170
04NC436b
04NC455
07Mad161a
04NC324a04NC365b
05Phil53
04NC253b
04NC024a
04NC233a
04NC152b
07Mad160a
04NC131
04Oki136a
07Mad151b
04NC139a
04NC132a
04NC237b
04NC324b
04NC373
04NC145a
04NC232
04NC379a
07Mad079
10010099
10010099
43--
993100100
99510098
828360
828983
99610097
7958-
808372
7583-
A
B
C
Figure 4 Haploweb of ITS2 sequences This graph was derived from a phylogenetic tree (obtained with PhyML using the TIM2+G modelsuggested by jModelTest) by drawing curves connecting haplotypes found co-occurring in heterozygous individuals [9] The sequence of oneStylophora pistillata individual from the Penghu Islands near Taiwan [35] is included in the figure and bootstrap values obtained using maximumlikelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The position of the root was inferred fromthe mitochondrial DNA phylogeny (Figure 3) This approach delineated three reproductively isolated pools of Stylophora sequences labeled A Band C as each of these groups corresponded to one of the mitochondrial clades of Figure 3
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 6 of 14
phylogenetically distant alleles in some individuals Incontrast haploweb analysis delineated three groups ofalleles among our ITS2 sequences a result perfectlycongruent with the mitochondrial phylogeny obtainedfrom the same set of individualsUnlike in our previous article [9] the haploweb pre-
sented here was built on a tree rather than a networkIndeed tree-based haplowebs are more straightforwardto draw than their network-based counterparts and arealso more informative since they display the genotype ofeach sequenced individual However precisely due to
their larger information content tree-based haplowebstend to become messy when dealing with large datasetsandor non-monophyletic species in such cases net-work-based haplowebs often turn out to be faster todraw and easier to interpret than tree-based ones
Are molecularly delimited species of Stylophoracongruent with morphologyMost of the 12 individuals from species A displayed thecharacteristic morphology of S madagascarensis (someexamples are shown on Figure 6) ldquocolonies have thin
D
005
AF416161 Stylophora pistillata brown Heron Island
AJ619830 Stylophora pistillata Red Sea
07Mad157a Madagascar
04Oki140 Okinawa
AF416224 Stylophora pistillata pink Sesoko
AJ619838 Stylophora pistillata Red Sea
AF416170 Stylophora pistillata brown One Tree Island
07Mad150 Madagascar
AJ865618 Stylophora pistillata Red Sea
AF416150 Stylophora pistillata pink Akajima
AF416229 Stylophora pistillata brown Terengganu
AF416151 Stylophora pistillata pink Akajima
AF416147 Stylophora pistillata brown Akajima
AF416194 Stylophora pistillata brown Moulter Cay
AF416222 Stylophora pistillata pink Sesoko
AJ619837 Stylophora pistillata Red Sea
AF416219 Stylophora pistillata brown Sesoko
AF416162 Stylophora pistillata pink Heron Island
AJ619844 Stylophora pistillata Red Sea
AJ865621 Stylophora pistillata Red Sea
AJ865631 Stylophora pistillata Red Sea
AJ619831 Stylophora pistillata Red Sea
AF416202 Stylophora pistillata pink Moulter Cay
AF416227 Stylophora pistillata brown Terengganu
AJ619841 Stylophora pistillata Red Sea
07Mad160a Madagascar
AF416182 Stylophora pistillata brown Raine Island
AJ619817 Stylophora pistillata Red Sea
AF416232 Stylophora pistillata brown Terengganu
AJ619811 Stylophora pistillata Red Sea
AF416193 Stylophora pistillata brown Raine Island
AF416231 Stylophora pistillata brown Terengganu
AF416201 Stylophora pistillata brown Moulter Cay
AF416173 Stylophora pistillata brown One Tree Island
AF416196 Stylophora pistillata brown Moulter Cay
AJ619829 Stylophora pistillata Red Sea
AJ619850 Stylophora pistillata Red Sea
AF416156 Stylophora pistillata brown Heron Island
AJ619820 Stylophora pistillata Red Sea
AJ865608 Stylophora pistillata Red Sea
AJ865625 Stylophora pistillata Red Sea
AF416209 Stylophora pistillata pink Sabah
AF416154 Stylophora pistillata pink Akajima
AF416181 Stylophora pistillata brown Raine Island
AF416175 Stylophora pistillata pink One Tree Island
AF416210 Stylophora pistillata pink Sabah
AJ619840 Stylophora pistillata Red Sea
AJ619832 Stylophora pistillata Red Sea
AF416164 Stylophora pistillata pink Heron Island
AJ865633 Stylophora pistillata Red Sea
AF416187 Stylophora pistillata brown Raine Island
AJ619808 Stylophora pistillata Red Sea
AF416199 Stylophora pistillata brown Moulter Cay
AF416172 Stylophora pistillata brown One Tree Island
AJ619816 Stylophora pistillata Red Sea
AJ619814 Stylophora pistillata Red Sea
AF416171 Stylophora pistillata brown One Tree Island
AF416179 Stylophora pistillata pink One Tree IslandAF416176 Stylophora pistillata pink One Tree Island
AJ865630 Stylophora pistillata Red Sea
AF416183 Stylophora pistillata brown Raine Island
AF416212 Stylophora pistillata pink Sabah
AF416228 Stylophora pistillata brown Terengganu
AJ865614 Stylophora pistillata Red Sea
AF416146 Stylophora pistillata brown Akajima
04NC101 New Caledonia
AF416216 Stylophora pistillata brown Sesoko
AF416206 Stylophora pistillata brown Sabah
07Mad160b Madagascar
AJ619845 Stylophora pistillata Red Sea
AJ865605 Stylophora pistillata Red Sea
AJ619833 Stylophora pistillata Red Sea
07Mad082 Madagascar
AF416178 Stylophora pistillata pink One Tree Island
AF416223 Stylophora pistillata pink Sesoko
AJ619825 Stylophora pistillata Red Sea
AF416163 Stylophora pistillata pink Heron Island
AF416157 Stylophora pistillata brown Heron Island
AF416155 Stylophora pistillata pink Akajima
AJ619839 Stylophora pistillata Red Sea
AF416217 Stylophora pistillata brown Sesoko
AF038909 Stylophora pistillata One Tree Island
AJ619848 Stylophora pistillata Red Sea
AJ865616 Stylophora pistillata Red Sea
07Mad073a Madagascar
AF416159 Stylophora pistillata brown Heron Island
04NC140a New Caledonia
HM013855 Seriatopora hystrix Xisha Islands
AJ619852 Stylophora pistillata Red Sea
AF416218 Stylophora pistillata brown Sesoko
AF416204 Stylophora pistillata brown Sabah
AJ619846 Stylophora pistillata Red Sea
AJ619819 Stylophora pistillata Red Sea
AJ619828 Stylophora pistillata Red Sea
AJ619810 Stylophora pistillata Red Sea
AJ619818 Stylophora pistillata Red SeaAJ619826 Stylophora pistillata Red Sea
AJ619835 Stylophora pistillata Red Sea
AJ619806 Stylophora pistillata Red Sea
AF038908 Stylophora pistillata One Tree Island
AJ865620 Stylophora pistillata Red Sea
05Phil53 Philippines
AF416160 Stylophora pistillata brown Heron IslandAF038907 Stylophora pistillata One Tree Island
AF416177 Stylophora pistillata pink One Tree Island
AF416168 Stylophora pistillata brown One Tree Island
AJ865617 Stylophora pistillata Red Sea
AF416174 Stylophora pistillata pink One Tree Island
AJ865626 Stylophora pistillata Red Sea
AF416208 Stylophora pistillata brown Sabah
04NC140b New Caledonia
AJ865623 Stylophora pistillata Red Sea
AF416189 Stylophora pistillata pink Raine Island
AF416220 Stylophora pistillata brown Sesoko
AJ865624 Stylophora pistillata Red Sea
AF416184 Stylophora pistillata brown Raine Island
AJ865627 Stylophora pistillata Red Sea
AJ619822 Stylophora pistillata Red Sea
AF416167 Stylophora pistillata pink Heron Island
AJ619842 Stylophora pistillata Red Sea
AJ865622 Stylophora pistillata Red Sea
HM013847 Pocillopora damicornis Xisha Islands
AY722795 Stylophora pistillata Penghu Island
AJ619824 Stylophora pistillata Red Sea
AF416226 Stylophora pistillata pink Sesoko
AJ865607 Stylophora pistillata Red Sea
AJ619805 Stylophora pistillata Red Sea
AJ619807 Stylophora pistillata Red Sea
AF416213 Stylophora pistillata pink Sabah
AJ865606 Stylophora pistillata Red Sea
AF416207 Stylophora pistillata brown Sabah
AJ619827 Stylophora pistillata Red Sea
AF416203 Stylophora pistillata brown Sabah
07Mad157b Madagascar
AJ619836 Stylophora pistillata Red Sea
07Mad073b Madagascar
AF416192 Stylophora pistillata brown Raine Island
AJ619812 Stylophora pistillata Red Sea
07Mad070 Madagascar
04NC105 New Caledonia
AF416195 Stylophora pistillata brown Moulter Cay
AJ865604 Stylophora pistillata Red Sea
HM013856 Stylophora pistillata Xisha Islands
AJ619843 Stylophora pistillata Red Sea
AJ619834 Stylophora pistillata Red Sea
07Mad188a Madagascar
AF416152 Stylophora pistillata pink Akajima
AF416186 Stylophora pistillata brown Raine Island
AJ865628 Stylophora pistillata Red Sea
AF416205 Stylophora pistillata brown Sabah
AF416149 Stylophora pistillata brown Akajima
AJ619821 Stylophora pistillata Red Sea
07Mad188b Madagascar
AJ619813 Stylophora pistillata Red Sea
AJ619847 Stylophora pistillata Red Sea
AJ619809 Stylophora pistillata Red Sea
AJ865615 Stylophora pistillata Red Sea
AF416225 Stylophora pistillata pink Sesoko
AF416190 Stylophora pistillata brown Raine Island
AJ865610 Stylophora pistillata Red Sea
AF416198 Stylophora pistillata brown Moulter Cay
AJ865629 Stylophora pistillata Red Sea
AF416221 Stylophora pistillata pink Sesoko
AF416145 Stylophora pistillata brown Akajima
AJ865611 Stylophora pistillata Red Sea
AF416169 Stylophora pistillata brown One Tree Island
AF416148 Stylophora pistillata brown Akajima
AF416214 Stylophora pistillata pink Sabah
AJ865613 Stylophora pistillata Red Sea
AJ865609 Stylophora pistillata Red Sea
AF416158 Stylophora pistillata brown Heron Island
AJ865619 Stylophora pistillata Red Sea
AF416165 Stylophora pistillata pink Heron Island
AF416191 Stylophora pistillata brown Raine Island
AJ619815 Stylophora pistillata Red Sea
AF416215 Stylophora pistillata brown Sesoko
AF416153 Stylophora pistillata pink Akajima
07Mad071 Madagascar
AF416185 Stylophora pistillata pink Raine Island
05Phil19 Philippines
AF416200 Stylophora pistillata brown Moulter Cay
AJ619851 Stylophora pistillata Red Sea
AJ619849 Stylophora pistillata Red Sea
AJ865632 Stylophora pistillata Red Sea
AF416211 Stylophora pistillata pink Sabah
AF416188 Stylophora pistillata brown Raine Island
AF416197 Stylophora pistillata brown Moulter Cay
AJ865612 Stylophora pistillata Red Sea
AF416230 Stylophora pistillata brown Terengganu
AJ619823 Stylophora pistillata Red Sea
AF416166 Stylophora pistillata pink Heron Island
AF416144 Stylophora pistillata brown Akajima
9129979
6937118
94490-
297--
9809966
345-76
9879998
A
B
C
Figure 5 ITS1 molecular phylogeny of the genus Stylophora In addition to ITS1 sequences from selected individuals of species A B and Cdata from published articles [69123513] and from an unpublished study by Feng You and Hui Huang [GenBank HM013847 HM013855HM013856] were included in the haplotree (generated with PhyML using the TPM2+G model suggested by jModelTest)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 7 of 14
(up to 5 mm diameter) straight compact branches Cor-allites are crowded and uniformly spaced on branchsides and ends They have a slight development ofhoods towards branch ends They have small style-likecolumellae and six primary septa which are fused withthe columellae The coenosteum is covered with finespicules Tentacles are not extended during the dayColour Uniform tan sometimes with pinkish branchbasesrdquo [38] Since Veronrsquos holotype was collected fromthe very same location in Madagascar where we con-ducted our sampling (approximately 4 m depth Tuleacutearsouth-west Madagascarrdquo [38]) there is little doubt thatspecies A and S madagascarensis are conspecificAccording to Veronrsquos description this species wasldquorecorded only from shallow reef environments exposedto some wave action and in south-west Madagascar inshallow sheltered lagoonsrdquo [38] In contrast we foundthis species down to a depth of 30 meters where it dis-played less compact and thicker branches comparedwith the typical S madagacarensis morphology (Figure7) hence S madagascarensis appears to be more ecolo-gically widespread and morphologically variable thanpreviously thought
All 7 individuals of species B from Madagascar hadflattened stout branches (Figure 8) This correspondsunambiguously to the morphological description of Smordax but since S mordax was originally describedfrom Fiji in the Pacific Ocean it is dubious whether thisname may be assigned to species BSpecies C gathered all individuals collected from loca-
tions in the Pacific Ocean It is characterized by exten-sive morphological variation comprising individualsattributable to S pistillata and others attributable to S
Figure 6 Morphology of species A From top to bottom colonies07Mad074 sampled at 5 meters depth colony 07Mad086 sampledat 6 meters depth and colony 07Mad170 sampled at 11 metersdepth display the typical morphology of S madagascarensis (thinstraight compact branches with small hoods) The green wire onthe pictures of the skeletons has a diameter of 09 mm
Figure 7 Deep-water morphs of species A Colony 07Mad088(left) and colony 07Mad097 (right) were both sampled at 30 metersdepth and have thicker less compact branches compared withtypical S madagascarensis morphology
Figure 8 Morphology of species B From top to bottom colony07Mad150 collected at 8 meters depth colony 07Mad071 collectedat 7 meters depth and colony 07Mad160 collected at 3 metersdepth all display the typical morphology of S mordax (shortcompressed branches with well-developed hoods) The green wireon the pictures of the skeletons has a diameter of 09 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 8 of 14
mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described
from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])
What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both
species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to
pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation
Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia
Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 9 of 14
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
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Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
phylogenetically distant alleles in some individuals Incontrast haploweb analysis delineated three groups ofalleles among our ITS2 sequences a result perfectlycongruent with the mitochondrial phylogeny obtainedfrom the same set of individualsUnlike in our previous article [9] the haploweb pre-
sented here was built on a tree rather than a networkIndeed tree-based haplowebs are more straightforwardto draw than their network-based counterparts and arealso more informative since they display the genotype ofeach sequenced individual However precisely due to
their larger information content tree-based haplowebstend to become messy when dealing with large datasetsandor non-monophyletic species in such cases net-work-based haplowebs often turn out to be faster todraw and easier to interpret than tree-based ones
Are molecularly delimited species of Stylophoracongruent with morphologyMost of the 12 individuals from species A displayed thecharacteristic morphology of S madagascarensis (someexamples are shown on Figure 6) ldquocolonies have thin
D
005
AF416161 Stylophora pistillata brown Heron Island
AJ619830 Stylophora pistillata Red Sea
07Mad157a Madagascar
04Oki140 Okinawa
AF416224 Stylophora pistillata pink Sesoko
AJ619838 Stylophora pistillata Red Sea
AF416170 Stylophora pistillata brown One Tree Island
07Mad150 Madagascar
AJ865618 Stylophora pistillata Red Sea
AF416150 Stylophora pistillata pink Akajima
AF416229 Stylophora pistillata brown Terengganu
AF416151 Stylophora pistillata pink Akajima
AF416147 Stylophora pistillata brown Akajima
AF416194 Stylophora pistillata brown Moulter Cay
AF416222 Stylophora pistillata pink Sesoko
AJ619837 Stylophora pistillata Red Sea
AF416219 Stylophora pistillata brown Sesoko
AF416162 Stylophora pistillata pink Heron Island
AJ619844 Stylophora pistillata Red Sea
AJ865621 Stylophora pistillata Red Sea
AJ865631 Stylophora pistillata Red Sea
AJ619831 Stylophora pistillata Red Sea
AF416202 Stylophora pistillata pink Moulter Cay
AF416227 Stylophora pistillata brown Terengganu
AJ619841 Stylophora pistillata Red Sea
07Mad160a Madagascar
AF416182 Stylophora pistillata brown Raine Island
AJ619817 Stylophora pistillata Red Sea
AF416232 Stylophora pistillata brown Terengganu
AJ619811 Stylophora pistillata Red Sea
AF416193 Stylophora pistillata brown Raine Island
AF416231 Stylophora pistillata brown Terengganu
AF416201 Stylophora pistillata brown Moulter Cay
AF416173 Stylophora pistillata brown One Tree Island
AF416196 Stylophora pistillata brown Moulter Cay
AJ619829 Stylophora pistillata Red Sea
AJ619850 Stylophora pistillata Red Sea
AF416156 Stylophora pistillata brown Heron Island
AJ619820 Stylophora pistillata Red Sea
AJ865608 Stylophora pistillata Red Sea
AJ865625 Stylophora pistillata Red Sea
AF416209 Stylophora pistillata pink Sabah
AF416154 Stylophora pistillata pink Akajima
AF416181 Stylophora pistillata brown Raine Island
AF416175 Stylophora pistillata pink One Tree Island
AF416210 Stylophora pistillata pink Sabah
AJ619840 Stylophora pistillata Red Sea
AJ619832 Stylophora pistillata Red Sea
AF416164 Stylophora pistillata pink Heron Island
AJ865633 Stylophora pistillata Red Sea
AF416187 Stylophora pistillata brown Raine Island
AJ619808 Stylophora pistillata Red Sea
AF416199 Stylophora pistillata brown Moulter Cay
AF416172 Stylophora pistillata brown One Tree Island
AJ619816 Stylophora pistillata Red Sea
AJ619814 Stylophora pistillata Red Sea
AF416171 Stylophora pistillata brown One Tree Island
AF416179 Stylophora pistillata pink One Tree IslandAF416176 Stylophora pistillata pink One Tree Island
AJ865630 Stylophora pistillata Red Sea
AF416183 Stylophora pistillata brown Raine Island
AF416212 Stylophora pistillata pink Sabah
AF416228 Stylophora pistillata brown Terengganu
AJ865614 Stylophora pistillata Red Sea
AF416146 Stylophora pistillata brown Akajima
04NC101 New Caledonia
AF416216 Stylophora pistillata brown Sesoko
AF416206 Stylophora pistillata brown Sabah
07Mad160b Madagascar
AJ619845 Stylophora pistillata Red Sea
AJ865605 Stylophora pistillata Red Sea
AJ619833 Stylophora pistillata Red Sea
07Mad082 Madagascar
AF416178 Stylophora pistillata pink One Tree Island
AF416223 Stylophora pistillata pink Sesoko
AJ619825 Stylophora pistillata Red Sea
AF416163 Stylophora pistillata pink Heron Island
AF416157 Stylophora pistillata brown Heron Island
AF416155 Stylophora pistillata pink Akajima
AJ619839 Stylophora pistillata Red Sea
AF416217 Stylophora pistillata brown Sesoko
AF038909 Stylophora pistillata One Tree Island
AJ619848 Stylophora pistillata Red Sea
AJ865616 Stylophora pistillata Red Sea
07Mad073a Madagascar
AF416159 Stylophora pistillata brown Heron Island
04NC140a New Caledonia
HM013855 Seriatopora hystrix Xisha Islands
AJ619852 Stylophora pistillata Red Sea
AF416218 Stylophora pistillata brown Sesoko
AF416204 Stylophora pistillata brown Sabah
AJ619846 Stylophora pistillata Red Sea
AJ619819 Stylophora pistillata Red Sea
AJ619828 Stylophora pistillata Red Sea
AJ619810 Stylophora pistillata Red Sea
AJ619818 Stylophora pistillata Red SeaAJ619826 Stylophora pistillata Red Sea
AJ619835 Stylophora pistillata Red Sea
AJ619806 Stylophora pistillata Red Sea
AF038908 Stylophora pistillata One Tree Island
AJ865620 Stylophora pistillata Red Sea
05Phil53 Philippines
AF416160 Stylophora pistillata brown Heron IslandAF038907 Stylophora pistillata One Tree Island
AF416177 Stylophora pistillata pink One Tree Island
AF416168 Stylophora pistillata brown One Tree Island
AJ865617 Stylophora pistillata Red Sea
AF416174 Stylophora pistillata pink One Tree Island
AJ865626 Stylophora pistillata Red Sea
AF416208 Stylophora pistillata brown Sabah
04NC140b New Caledonia
AJ865623 Stylophora pistillata Red Sea
AF416189 Stylophora pistillata pink Raine Island
AF416220 Stylophora pistillata brown Sesoko
AJ865624 Stylophora pistillata Red Sea
AF416184 Stylophora pistillata brown Raine Island
AJ865627 Stylophora pistillata Red Sea
AJ619822 Stylophora pistillata Red Sea
AF416167 Stylophora pistillata pink Heron Island
AJ619842 Stylophora pistillata Red Sea
AJ865622 Stylophora pistillata Red Sea
HM013847 Pocillopora damicornis Xisha Islands
AY722795 Stylophora pistillata Penghu Island
AJ619824 Stylophora pistillata Red Sea
AF416226 Stylophora pistillata pink Sesoko
AJ865607 Stylophora pistillata Red Sea
AJ619805 Stylophora pistillata Red Sea
AJ619807 Stylophora pistillata Red Sea
AF416213 Stylophora pistillata pink Sabah
AJ865606 Stylophora pistillata Red Sea
AF416207 Stylophora pistillata brown Sabah
AJ619827 Stylophora pistillata Red Sea
AF416203 Stylophora pistillata brown Sabah
07Mad157b Madagascar
AJ619836 Stylophora pistillata Red Sea
07Mad073b Madagascar
AF416192 Stylophora pistillata brown Raine Island
AJ619812 Stylophora pistillata Red Sea
07Mad070 Madagascar
04NC105 New Caledonia
AF416195 Stylophora pistillata brown Moulter Cay
AJ865604 Stylophora pistillata Red Sea
HM013856 Stylophora pistillata Xisha Islands
AJ619843 Stylophora pistillata Red Sea
AJ619834 Stylophora pistillata Red Sea
07Mad188a Madagascar
AF416152 Stylophora pistillata pink Akajima
AF416186 Stylophora pistillata brown Raine Island
AJ865628 Stylophora pistillata Red Sea
AF416205 Stylophora pistillata brown Sabah
AF416149 Stylophora pistillata brown Akajima
AJ619821 Stylophora pistillata Red Sea
07Mad188b Madagascar
AJ619813 Stylophora pistillata Red Sea
AJ619847 Stylophora pistillata Red Sea
AJ619809 Stylophora pistillata Red Sea
AJ865615 Stylophora pistillata Red Sea
AF416225 Stylophora pistillata pink Sesoko
AF416190 Stylophora pistillata brown Raine Island
AJ865610 Stylophora pistillata Red Sea
AF416198 Stylophora pistillata brown Moulter Cay
AJ865629 Stylophora pistillata Red Sea
AF416221 Stylophora pistillata pink Sesoko
AF416145 Stylophora pistillata brown Akajima
AJ865611 Stylophora pistillata Red Sea
AF416169 Stylophora pistillata brown One Tree Island
AF416148 Stylophora pistillata brown Akajima
AF416214 Stylophora pistillata pink Sabah
AJ865613 Stylophora pistillata Red Sea
AJ865609 Stylophora pistillata Red Sea
AF416158 Stylophora pistillata brown Heron Island
AJ865619 Stylophora pistillata Red Sea
AF416165 Stylophora pistillata pink Heron Island
AF416191 Stylophora pistillata brown Raine Island
AJ619815 Stylophora pistillata Red Sea
AF416215 Stylophora pistillata brown Sesoko
AF416153 Stylophora pistillata pink Akajima
07Mad071 Madagascar
AF416185 Stylophora pistillata pink Raine Island
05Phil19 Philippines
AF416200 Stylophora pistillata brown Moulter Cay
AJ619851 Stylophora pistillata Red Sea
AJ619849 Stylophora pistillata Red Sea
AJ865632 Stylophora pistillata Red Sea
AF416211 Stylophora pistillata pink Sabah
AF416188 Stylophora pistillata brown Raine Island
AF416197 Stylophora pistillata brown Moulter Cay
AJ865612 Stylophora pistillata Red Sea
AF416230 Stylophora pistillata brown Terengganu
AJ619823 Stylophora pistillata Red Sea
AF416166 Stylophora pistillata pink Heron Island
AF416144 Stylophora pistillata brown Akajima
9129979
6937118
94490-
297--
9809966
345-76
9879998
A
B
C
Figure 5 ITS1 molecular phylogeny of the genus Stylophora In addition to ITS1 sequences from selected individuals of species A B and Cdata from published articles [69123513] and from an unpublished study by Feng You and Hui Huang [GenBank HM013847 HM013855HM013856] were included in the haplotree (generated with PhyML using the TPM2+G model suggested by jModelTest)
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 7 of 14
(up to 5 mm diameter) straight compact branches Cor-allites are crowded and uniformly spaced on branchsides and ends They have a slight development ofhoods towards branch ends They have small style-likecolumellae and six primary septa which are fused withthe columellae The coenosteum is covered with finespicules Tentacles are not extended during the dayColour Uniform tan sometimes with pinkish branchbasesrdquo [38] Since Veronrsquos holotype was collected fromthe very same location in Madagascar where we con-ducted our sampling (approximately 4 m depth Tuleacutearsouth-west Madagascarrdquo [38]) there is little doubt thatspecies A and S madagascarensis are conspecificAccording to Veronrsquos description this species wasldquorecorded only from shallow reef environments exposedto some wave action and in south-west Madagascar inshallow sheltered lagoonsrdquo [38] In contrast we foundthis species down to a depth of 30 meters where it dis-played less compact and thicker branches comparedwith the typical S madagacarensis morphology (Figure7) hence S madagascarensis appears to be more ecolo-gically widespread and morphologically variable thanpreviously thought
All 7 individuals of species B from Madagascar hadflattened stout branches (Figure 8) This correspondsunambiguously to the morphological description of Smordax but since S mordax was originally describedfrom Fiji in the Pacific Ocean it is dubious whether thisname may be assigned to species BSpecies C gathered all individuals collected from loca-
tions in the Pacific Ocean It is characterized by exten-sive morphological variation comprising individualsattributable to S pistillata and others attributable to S
Figure 6 Morphology of species A From top to bottom colonies07Mad074 sampled at 5 meters depth colony 07Mad086 sampledat 6 meters depth and colony 07Mad170 sampled at 11 metersdepth display the typical morphology of S madagascarensis (thinstraight compact branches with small hoods) The green wire onthe pictures of the skeletons has a diameter of 09 mm
Figure 7 Deep-water morphs of species A Colony 07Mad088(left) and colony 07Mad097 (right) were both sampled at 30 metersdepth and have thicker less compact branches compared withtypical S madagascarensis morphology
Figure 8 Morphology of species B From top to bottom colony07Mad150 collected at 8 meters depth colony 07Mad071 collectedat 7 meters depth and colony 07Mad160 collected at 3 metersdepth all display the typical morphology of S mordax (shortcompressed branches with well-developed hoods) The green wireon the pictures of the skeletons has a diameter of 09 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 8 of 14
mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described
from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])
What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both
species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to
pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation
Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia
Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 9 of 14
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
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Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
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Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
(up to 5 mm diameter) straight compact branches Cor-allites are crowded and uniformly spaced on branchsides and ends They have a slight development ofhoods towards branch ends They have small style-likecolumellae and six primary septa which are fused withthe columellae The coenosteum is covered with finespicules Tentacles are not extended during the dayColour Uniform tan sometimes with pinkish branchbasesrdquo [38] Since Veronrsquos holotype was collected fromthe very same location in Madagascar where we con-ducted our sampling (approximately 4 m depth Tuleacutearsouth-west Madagascarrdquo [38]) there is little doubt thatspecies A and S madagascarensis are conspecificAccording to Veronrsquos description this species wasldquorecorded only from shallow reef environments exposedto some wave action and in south-west Madagascar inshallow sheltered lagoonsrdquo [38] In contrast we foundthis species down to a depth of 30 meters where it dis-played less compact and thicker branches comparedwith the typical S madagacarensis morphology (Figure7) hence S madagascarensis appears to be more ecolo-gically widespread and morphologically variable thanpreviously thought
All 7 individuals of species B from Madagascar hadflattened stout branches (Figure 8) This correspondsunambiguously to the morphological description of Smordax but since S mordax was originally describedfrom Fiji in the Pacific Ocean it is dubious whether thisname may be assigned to species BSpecies C gathered all individuals collected from loca-
tions in the Pacific Ocean It is characterized by exten-sive morphological variation comprising individualsattributable to S pistillata and others attributable to S
Figure 6 Morphology of species A From top to bottom colonies07Mad074 sampled at 5 meters depth colony 07Mad086 sampledat 6 meters depth and colony 07Mad170 sampled at 11 metersdepth display the typical morphology of S madagascarensis (thinstraight compact branches with small hoods) The green wire onthe pictures of the skeletons has a diameter of 09 mm
Figure 7 Deep-water morphs of species A Colony 07Mad088(left) and colony 07Mad097 (right) were both sampled at 30 metersdepth and have thicker less compact branches compared withtypical S madagascarensis morphology
Figure 8 Morphology of species B From top to bottom colony07Mad150 collected at 8 meters depth colony 07Mad071 collectedat 7 meters depth and colony 07Mad160 collected at 3 metersdepth all display the typical morphology of S mordax (shortcompressed branches with well-developed hoods) The green wireon the pictures of the skeletons has a diameter of 09 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 8 of 14
mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described
from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])
What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both
species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to
pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation
Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia
Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 9 of 14
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
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Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
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52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
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Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described
from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])
What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both
species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to
pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation
Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia
Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 9 of 14
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
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Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
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bull Research which is freely available for redistribution
Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
of species boundaries and not from actual introgressionbetween distinct genetic entities
A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the
genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species
ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with
morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo
MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation
PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)
Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 10 of 14
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
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Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
bull Convenient online submission
bull Thorough peer review
bull No space constraints or color figure charges
bull Immediate publication on acceptance
bull Inclusion in PubMed CAS Scopus and Google Scholar
bull Research which is freely available for redistribution
Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes
of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki136 (26deg12rsquo10N 127deg1912E) nr 04NC324 (20deg42rsquo39S 165deg09rsquo14E) 6
04Oki140 (26deg12rsquo10N 127deg1912E) nr 04NC336 (20deg35rsquo42S 165deg10rsquo40E) 26
04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22
04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15
04NC012 (22deg26rsquo54S 166deg22rsquo23E) 6 04NC379 (20deg40rsquo12S 164deg11rsquo19E) 14
04NC024 (22deg26rsquo54S 166deg22rsquo23E) 1 04NC404 (20deg40rsquo20S 164deg14rsquo53E) 4
04NC064 (22deg38rsquo30S 166deg34rsquo40E) 16 04NC434 (20deg39rsquo58S 164deg15rsquo26E) 25
04NC071 (22deg40rsquo54S 168deg36rsquo24E) 1 04NC436 (20deg41rsquo39S 164deg14rsquo50E) 24
04NC087 (22deg37rsquo20S 166deg36rsquo53E) 5 04NC439 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC101 (22deg37rsquo20S 166deg36rsquo53E) 9 04NC440 (20deg41rsquo39S 164deg14rsquo50E) 23
04NC105 (22deg01rsquo01S 165deg55rsquo10E) 26 04NC441 (20deg41rsquo39S 164deg14rsquo50E) 21
04NC130 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC452 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC131 (22deg22rsquo59S 167deg05rsquo50E) 1 04NC455 (20deg41rsquo39S 164deg14rsquo50E) 1
04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24
04NC139 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil53 (16deg26rsquo22N 119deg56rsquo33E) 13
04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC141 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad071 (23deg25rsquo01S 43deg38rsquo36E) 7
04NC145 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad073 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC149 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad074 (23deg25rsquo01S 43deg38rsquo36E) 5
04NC152 (22deg15rsquo27S 166deg24rsquo33E) 16 07Mad079 (23deg25rsquo01S 43deg38rsquo36E) 8
04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8
04NC182 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad086 ( 23deg23rsquo07S 43deg38rsquo18E) 6
04NC187 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad087 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC189 (22deg12rsquo31S 166deg24rsquo55E) 4 07Mad088 (23deg30rsquo20S 43deg41rsquo10E) 30
04NC199 (22deg18rsquo40S 166deg27rsquo26E) 1 07Mad150 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC230 (20deg46rsquo18S 165deg16rsquo30E) 14 07Mad151 (23deg23rsquo29S 43deg37rsquo38E) 8
04NC232 (20deg46rsquo18S 165deg16rsquo30E) 13 07Mad157 (23deg23rsquo29S 43deg37rsquo38E) 7
04NC233 (20deg46rsquo18S 165deg16rsquo30E) 9 07Mad159 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC237 (20deg46rsquo18S 165deg16rsquo30E) 7 07Mad160 (23deg23rsquo29S 43deg37rsquo38E) 3
04NC251 (20deg34rsquo59S 165deg08rsquo11E) 21 07Mad161 (23deg23rsquo29S 43deg37rsquo38E) 2
04NC253 (20deg34rsquo59S 165deg08rsquo11E) 19 07Mad170 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC266 (20deg34rsquo59S 165deg08rsquo11E) 7 07Mad172 (23deg22rsquo58S 43deg38rsquo11E) 11
04NC282 (20deg34rsquo59S 165deg08rsquo11E) 6 07Mad188 (23deg22rsquo58S 43deg38rsquo11E) 5
04NC289 (20deg34rsquo59S 165deg08rsquo11E) 2 07Mad189 (23deg22rsquo58S 43deg38rsquo11E) 3
nr = not recorded
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 11 of 14
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
bull Convenient online submission
bull Thorough peer review
bull No space constraints or color figure charges
bull Immediate publication on acceptance
bull Inclusion in PubMed CAS Scopus and Google Scholar
bull Research which is freely available for redistribution
Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]
AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments
This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence
Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia
Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors
Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011
References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the
Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum
marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178
3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000
4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29
5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33
6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247
Table 2 Primers used for DNA amplification and sequencing
Marker Primer name Primer sequence Reference
ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study
ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study
ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]
ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]
ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]
ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]
CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]
CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 12 of 14
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
bull Convenient online submission
bull Thorough peer review
bull No space constraints or color figure charges
bull Immediate publication on acceptance
bull Inclusion in PubMed CAS Scopus and Google Scholar
bull Research which is freely available for redistribution
Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794
8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871
9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372
10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885
11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325
12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22
13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438
14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797
15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86
16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198
17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75
18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83
19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982
20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266
21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182
22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28
23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46
24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157
25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846
26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288
27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170
28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121
29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644
30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720
31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875
32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568
33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87
34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553
35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771
36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453
37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588
38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206
39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767
40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572
41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613
42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233
43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329
44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349
45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376
46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025
47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807
48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517
49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312
50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090
51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 13 of 14
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
bull Convenient online submission
bull Thorough peer review
bull No space constraints or color figure charges
bull Immediate publication on acceptance
bull Inclusion in PubMed CAS Scopus and Google Scholar
bull Research which is freely available for redistribution
Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-
52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337
53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395
54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630
55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977
56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166
57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989
58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122
59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution
60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797
61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066
62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212
63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464
64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256
65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704
66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791
67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968
68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002
69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931
doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122
Submit your next manuscript to BioMed Centraland take full advantage of
bull Convenient online submission
bull Thorough peer review
bull No space constraints or color figure charges
bull Immediate publication on acceptance
bull Inclusion in PubMed CAS Scopus and Google Scholar
bull Research which is freely available for redistribution
Submit your manuscript at wwwbiomedcentralcomsubmit
Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122
Page 14 of 14
- Abstract
-
- Background
- Results
- Conclusions
-
- Background
- Results
-
- Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean
- Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species
- Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea
-
- Discussion
-
- Haplowebs are useful tools to delineate species
- Are molecularly delimited species of Stylophora congruent with morphology
- What causes the discrepancy between morphological and molecular species delimitations in Stylophora
- A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean
-
- Conclusions
- Methods
-
- Sample collection and processing
- PCR amplification and sequencing
- Determination of nuclear haplotypes
- Phylogenetic analyses and haploweb construction
-
- Acknowledgements
- Author details
- Authors contributions
- References
-