systematics of hypnea asiatica sp. nov. (hypneaceae, rhodophyta) based on morphology and nrdna ssu,...

606

TAXON 58 (2) bull May 2009 606ndash616Geraldino amp al bull Hypnea asiatica sp nov

INTRODUCTIONHypnea JV Lamouroux a genus comprising about 53

species of annual red algae is abundant in intertidal and subtidal zones of the tropical and warm temperate regions The genus is economically important as food and for the production of kappa carrageenan (Mshigeni amp Chapman 1994) Hypnea is characterized by terete to compressed much branched thalli with short lateral branchlets globu-lar cystocarps and zonate tetrasporangia on short laterals (Lamouroux 1813 Mshigeni 1978 Womersley 1994) However since the genus has a wide pantropical distribu-tion and considerable morphological variation there is a need for critical reassessment of species delimitation using extensive preserved collections including those collected from the type localities (Masuda amp al 1997)

Hypnea charoides JV Lamouroux is a widespread and taxonomically confusing species (Womersley 1994 Yamagishi amp Masuda 1997 2000) It is one of the five species of the genus described by Lamouroux (1813) from samples collected off the southwestern or southeastern coasts of Australia (Womersley 1994) Hypnea charoi-des is characterized by branched axes branchlets curving abruptly toward the main axis and by the lack of lenticular thickening in medullary cell walls (Womersley 1994) The species is generally believed to be ubiquitous outside Australia ie in Korea (Shin amp Boo 1994) Japan (Yam-agishi amp Masuda 1997) China (Xia amp Wang 1997) and Malaysia (Tani amp al 2003) However Womersley (1994)

doubted the speciesrsquo occurrence outside Australia noting that H charoides appears to be essentially a western spe-cies occurring along southern Australia

Progress in molecular systematics has led to increas-ing use of multiple and newly developed genes for iden-tifying species with widespread distributions or without clear-cut morphological diagnostic criteria For example red algal nuclear ribosomal DNA and plastid large sub-unit of ribulose-15-bisphosphate carboxylaseoxygenase (rbcL) have been used for phylogenetic analysis and for species identification (Freshwater amp al 1994 Ragan amp al 1994) The cox1 mitochondrial gene is used as DNA barcode to catalog the diversity of red algal species (Saunders 2005) The approach is especially useful for dealing with widespread species with distribution ranges spanning geographical barriers Molecular techniques allow us to answer questions on the origins of populations on different sides of the barriers eg to determine whether the populations are the product of speciesrsquo long distance dispersal or are in fact different species or are in different phyletic clades within a species

In this study we investigated a species identified as H charoides from Korea by Shin amp Boo (1994) We used detailed morphological information and analysis of genes from three different genomes the nuclear small subunit ribosomal DNA (SSU rDNA) the plastid rbcL and the mitochondrial cox1 The published SSU sequence of the species from Australia (Saunders amp al 2004) was avail-able for our study The many rbcL sequences available

Systematics of Hypnea asiatica sp nov (Hypneaceae Rhodophyta) based on morphology and nrDNA SSU plastid rbcL and mitochondrial cox1

Paul John L Geraldino1 Eun Chan Yang1 Myung Sook Kim2 amp Sung Min Boo1

1 Department of Biology Chungnam National University Daejeon 305-764 Korea smboocnuackr (author for correspondence)

2 Department of Biology Cheju National University Jeju 690-756 Korea

Hypnea is an economically and ecologically important red alga including about 53 species worldwide Here we describe H asiatica sp nov from Korea Japan and Taiwan Specimens of this species from the northwestern Pacific have often been assigned to H charoides Hypnea asiatica is distinguished by percurrent main axes branches with abruptly curved adaxial branchlets and the presence of lenticular thickening in the walls of medullary cells It is abundant in wave-exposed sites from summer through fall and produces tetrasporangial sori in fall We analyzed three genes from three different genomes nuclear SSU rDNA from 27 specimens plastid rbcL from 51 specimens and mitochondrial cox1 from 41 specimens including putative relatives and H charoides from Australia All analyzed DNA datasets produced congruent trees in which H asiatica was always separated from H charoides Network analysis of cox1 haplotypes indicated geographic structuring across the species distribution range Although Hypnea was found to be monophyletic the distant relationship between H asiatica and the other species raises questions about divergence pathways within the genus

KEYWORDS cox1 Hypnea asiatica rbcL Rhodophyta SSU rDNA systematics

607

Geraldino amp al bull Hypnea asiatica sp novTAXON 58 (2) bull May 2009 606ndash616

for Hypnea (Freshwater amp al 1994 Yamagish amp Ma-suda 2000Hommersand amp Fredericq 2003 Tani amp al 2003 Yamagishi amp al 2003 Geraldino amp al 2006) al-lowed us to compare our results directly with those from other species in the genus and with putative relatives We tested the suitability of the cox1 gene for identification of H flexicaulis Yamagishi amp Masuda from other species of the genus (Geraldino amp al 2006) The three genes we studied are in different cellular compartments thus providing three separate analytic approaches Here we describe H asiatica sp nov from Korea Japan and Tai-wan and conclude that H charoides should be removed from the northwestern Pacific marine flora

MATERIALS AND METHODSField observations and collections of H asiatica were

made at six locations around Korea and at sites in Japan and Taiwan Representative specimens of H asiatica were collected in the intertidal zone from the following locali-ties Korea Gampo Gyeongju (2 October 2005) Gijang Busan (17 October 2005) Tongyoung (15 July 2004) Guryongpo Gyeongju (2 October 2005) Wolpo Pohang (3 October 2005) Jeju (13 November 2004) Taiwan Dali Keelung (30 March 2006) Japan Choshi Chiba Prefecture (30 July 2004)

The phenology of H asiatica was monitored in situ in Gampo Korea every two months from June 2006 to June 2007 Tissues were sectioned using a freezing microtome FX-802A (Coper Electronics Co Ltd Kanagawa Japan) and the sectioned preparations were stained with 1 aque-ous aniline blue acidified with a drop of 1 HCl and mounted in 30 glycerinndashseawater Photographs were taken with a FX-35DX camera (Nikon Tokyo Japan) at-tached to a microscope (Vanox AHBT3 Olympus Tokyo Japan) Voucher specimens are housed at the herbarium of Chungnam National University (CNUK) Daejeon Korea

DNA extraction mdash 51 specimens were available for our molecular study (Appendix) Total DNA was extracted from approximately 5 mg of dried thalli ground in liquid nitrogen using a DNeasy Plant Mini Kit (Qiagen GmbH Hilden Germany) following manufacturerrsquos instructions Extracts were dissolved in 150 mL of distilled water The extracted DNA was stored at ndash20degC and used to amplify SSU rbcL and cox1 genes Voucher specimens used for molecular systematics were deposited in the herbarium of Chungnam National University (CNUK) Daejeon Korea

Analysis of SSU rbcL and cox1 regions mdash For am-plification and sequencing reaction of each gene specific primer pairs were used as follows for SSU G01-G10 G02-G14 G04-G13 and G06-G07 (Saunders amp Kraft 1994) for rbcL F7-R753 and F645-RrbcS start (Freshwater amp Rueness 1994 Lin amp al 2001 Gavio amp Fredericq 2002)

and for cox1 cox143F-cox11549R (Geraldino amp al 2006) and C622F-C880R (Yang amp al 2008) All PCR amplifi-cations were carried out with a TechGene thermal cycler (Techne Ltd Duxford Cambridge UK) using a TaKaRa Ex Taq reaction kit (Takara Shuzo Shiga Japan) a total volume of 25 μL consisted of 25 μL 10times Ex Taq Buffer 20 μL 25 mM MgCl2 10 μM dNTP mixture 015 μM of each primer 0625 units of TaKaRa Ex Taq and 30 μM DNA solution (containing 05ndash10 μg DNA) PCR was per-formed with an initial denaturation step at 94degC for 10 min followed by 35 cycles of 30 s at 90degC 30 s at 50degC and 2 min at 72degC with a final 10-min extension cycle at 72degC

The PCR products were purified using a High Pure PCR Product Purification Kit (Roche Diagnostics GmbH Mannheim Germany) according to the manufacturerrsquos instructions Sequences of the forward and reverse strands were determined for all PCR products using an ABI PRISMtrade 377 DNA Sequencer (Applied Biosystems Foster City California USA) at the Research Center CNUK Both electropherogram outputs from each sample were edited using the program Sequence Navigator ver 101 (Applied Biosystems)

Twenty-nine SSU sequences (27 new and 2 published) 75 rbcL sequences (51 new and 24 published) and 44 cox1 sequences (41 new and 3 published) of Hypnea were col-lated using the multiple-sequence editing program SeqPup (Gilbert 1995) and aligned visually For rbcL data outgroups were Gigartina pinnata J Agardh (AF385663) Chondrus ocellatus Holmes (AF294815) Grateloupia imbricata Holmes (AF299252) and Gracilaria caudata J Agardh (AY049331) All sequences used in this study are presented in the Appendix None of the alignments posed problems as no gaps were observed

Phylogenetic analyses mdash All datasets from SSU rbcL and cox1 genes were utilized Maximum parsimony (MP) trees were constructed for each dataset with PAUP 40b10 (Swofford 2002) using a heuristic search algo-rithm with the following settings 1000 random sequence additions tree bisection-reconnection (TBR) branch swapping MulTrees all characters unordered and un-weighted and branches with a maximum length of zero collapsed to yield polytomies Bootstrap values for the resulting nodes were assessed using 1000 bootstrapping replicates with ten random sequence additions

For maximum likelihood (ML) and Bayesian analyses the best models for individual datasets were determined under Modeltest 308b (Posada amp Crandall 1998) with the following settings for all datasets Kimura three-parameter model unequal frequencies (K3Puf) proportion of invari-able sites (I) and the γ shape parameter (G) For Modeltest model scores were estimated on the basis of the model block for PAUP (in the Modeltest package) The best model was selected using the Akaike Information Criterion (AIC) as recommended by Posada amp Buckley (2004) ML analyses

608


were performed by heuristic searches with 100 random sequence additions TBR branch swapping and MulTrees options Bootstrap analysis was conducted by performing replicate maximum likelihood searches (with two random sequence-addition replicates) using the search conditions described above A 50 majority rule consensus bootstrap tree was estimated by aggregating and weighting trees ac-cording to the number of trees found in each bootstrap replicate so that the bootstrap replicates had equal weight

Bayesian analyses were conducted with MrBayes v31 software (Ronquist amp Huelsenbeck 2003) using the Metropolis-coupled Markov chain Monte Carlo method (MC3 ) with the GTR + Γ + I model for individual datasets For the SSU matrix 1 million generations in two indepen-dent runs were performed with four chains and trees were sampled every 100th generation 3 million and 4 million generations were run for the rbcL and cox1 matrix re-spectively The burn-in period was identified graphically by tracking likelihoods at each generation to determine whether the likelihood values had reached a plateau the burn-in periods were 5880 for SSU 1172 for cox1 and 9010 for rbcL The 15151 trees for SSU 41980 trees for rbcL and 77857 for cox1 sampled at stationarity were used to infer the Bayesian posterior probability Majority-rule consensus trees were calculated using PAUP

A statistical parsimony network was drawn for cox1 haplotypes of H asiatica using the program TCS Version 121 (Clement amp al 2000) This TCS program calculates the minimal number of mutational steps by which the sequences can be joined with gt 95 confidence Nucleo-tide diversity (Pi) and haplotype diversity (Hd) of each gene were calculated using the DnaSP program (Rozas amp Rozas 2000)

RESULTS Hypnea asiatica PJL Geraldino EC Yang amp Boo sp

novPlantae erectae subcartilagineae rubrae in coloure

usque ad 18 cm altae axes principales percurrentes teres ramos laterals numorosos ordinum usque ad quin-que primorum in modo altero-spirali ad angulos circa 90deg ramuli flexum adaxialem conspicuum praebentes incrassationes lenticulares in parietibus cellularum peri-axialium et corticalium presentes Tetrasporangia in soris inflatis proximalibus ramulorum ultimorum formata tetrasporangia matura sporis zonatim dispositis Cysto-carpia et spermatangia ignota Data de sequentiis = Gen-Bank accession number nrDNA SSU = EU240858 rbcL = EU240824 cox1 = EU240798

Plants erect subcartilaginous red in color up to 18 cm high main axes percurrent terete bearing numerous lateral branches lateral branches of up to five

orders in an alternate-spiral manner at the angle of about 90deg branchlets showing pronounced adaxial bending Lenticular thickenings present in the walls of periaxial and cortical cells Tetrasporangia formed in proximal swollen sori of ultimate branchlets mature tetrasporangia with zonately arranged spores Cystocarps and spermatangia unknown GenBank accession number nrDNA SSU = EU240858 rbcL = EU240824 cox1 = EU240798

Type mdash C50012 intertidal zone off Gampo Gyeongju Korea 2 October 2005 (Fig 1) Herbarium of Chungnam National University (CNUK) Daejeon Korea

Paratypes mdash C50035 Wolpo Pohang Korea 3 Oc-tober 2005 C50067 Gijang Busan Korea 17 October 2005 C50046 Guryongpo Gyeongju Korea 2 October 2005 C50052 Tongyoung Gyeongnam Korea 15 July 2004 C50512 Dali Keelung Taiwan 31 March 2006 C50509 Lonedome Keelung Taiwan 31 March 2006 C50078 Kurohae Beach Choshi Japan 30 July 2004 C50080 Ashikajima Beach Chosi Japan 31 July 2004

Etymology mdash The specific epithet refers to Asia which encompasses the geographical range of the speci-mens we examined

Morphology mdash Plants are up to 18 cm high subcarti-laginous and red in color (Fig 2A) Main axis arises from a primary discoid holdfast terete throughout strongly per-current and usually longer than any lateral branch 13ndash15 mm in diameter in the proximal half and slightly less dis-tally branching alternately-spiral at angles of 90deg from the

Fig 1 Type specimen of Hypnea asiatica collected in Gam-po Korea deposited in the Herbarium of Chungnam Na-tional University (CNUK) Daejeon Korea

609


parent branch (Fig 2B) First-order branches are up to 6 cm in length bearing abruptly shorter and progressively more slender branches of up to five orders at angles similar to those coming off the main axis Branches of any order are often abruptly curved adaxially (Fig 2C) Axes and branches of lower orders produce short simple or long divided adventitious branchlets usually directed upward Adventitious branchlets are formed from surface cells and often abruptly curved in an adaxial direction (Fig 2D) Thalli are uniaxial with an obvious protruding apical cell (Fig 3A) at the tips of main axes ordinary branches and adventitious branchlets Axial cells are elongate and slen-der in longitudinal sections without small cells 73ndash109 μm wide up to 25 mm long with lengths 15ndash30 times the width of the lower axes (Fig 3B) Periaxial cells are

circular elliptical or broadly obovate and up to 287ndash465 μm wide in transverse sections Periaxial cells produce shorter and more slender cells outward forming a layer of 3ndash6 cells thick in the middle to lower axes and branches (Fig 3B) Lateral and longitudinal secondary pit connec-tions are frequently present between adjacent periaxial and cortical cells (Fig 3C) Lenticular thickenings are present in the walls of cortical and periaxial cells (Fig 3D) Super-ficial cortical cells are circular semicircular or irregular in transverse view 16ndash18 μm in diameter Tetrasporangia cut off from the outermost cortical cells and are found in proximal swollen sori 730ndash1435 μm long by 331ndash636 μm wide (Fig 2D) sori develop on ultimate branchlets Mature tetrasporangia consist of zonately arranged spores and are 29ndash60 μm long by 21ndash36 μm wide (Fig 3E)

Fig 2 Morphology of Hypnea asiatica A thalli collected from a wave-exposed site B vegetative thallus with adaxially curved branches (arrows) C tetrasporangial thallus D branches with tetrasporangial sori (arrows)

610


Habit and phenology mdash Thalli grow abundantly on a diversity of other algae boulders and bedrocks in the lower intertidal and upper subtidal zones in wave-exposed sites Thalli are clumped but are easily separated into single tuft Thalli were collected in June August Octo-ber and December 2004 to 2006 off the coast at Gampo Korea and also in January and June 2005 to 2007 Young thalli begin to appear in June and bear tetrasporangial sori from August through October when plants achieve their largest size In January thalli were small and scarce No specimens were collected in February and April

Phylogeny of SSU rDNA rbcL and cox1 mdash A 1487-nucleotide portion of the SSU gene was aligned for 29 sequences representing five species of Hypnea The se-quences from 19 isolates of H asiatica from Korea Japan and Taiwan were almost identical However H asiatica dif-fered by 4minus6 bp (percent divergence 027minus04) from H charoides and H spinella (C Agardh) Kuumltzing All isolates of H asiatica from Korea Japan and Taiwan were strongly monophyletic and were clearly separated from H charoides in the phylogenetic tree (not shown)

Seventy-five sequences representing 16 species of Hypnea were aligned using a 1364-nucleotide portion of the rbcL gene Variable sites occurred at 457 positions (335) and among them 340 positions (249) were parsimoniously informative Twenty-eight H asiatica sequences from Korea Japan and Taiwan were almost identical with minor differences of up to 5 bp (037) The five specimens of H charoides from Australia were identical However H asiatica differed by 62ndash66 bp (455ndash484) from H charoides and by 58ndash96 bp (425ndash701) from the rest of the species tested in the present study In the phylogenetic tree (Fig 4) H asiatica from Korea Japan and Taiwan formed a monophyletic clade with strong support Hypnea yamadae Tanaka and H volubilis Searles formed a sister group with strong support Hypnea cornuta (Kuumltzing) J Agardh and H stellulifera J Agardh were clustered within the clade of H musciformis (Wulfen) Lamouroux H flagelliformis J Agardh and H chordacea Kuumltzing The sister rela-tionship between H pannosa J Agardh and H japonica Tanaka was also strongly supported

Fig 3 Transverse sections of Hypnea asiatica thalli A ultimate branchlet showing apical cell (arrow) B axis with small axial (ac) and large periaxial cells (pa) C axis with pit connections (arrows) D medullary cells with lenticular thicken-ings (arrow) E tetrasporangial sorus with zonately divided tetrasporangia (arrow)

611


Fig 4 ML tree inferred from the rbcL sequence calculated using the GTR + Γ + I model of evolution [ndashlnL = 5540484096 substitu-tion rate matrix RAC = 13092 RAG = 68566 RAT = 37471 RCG = 17516 RCT = 202451 RGT = 1 base frequen-cies πA = 03025 πC = 01365 πG = 02138 πT = 03472 shape parameter (α) = 12588 proportion of invariable site (I) = 04977] Values above each clade refer to ML and MP bootstrap values and Bayesian posterior prob-abilities

612


A 1362-nucleotide portion of the cox1 gene was aligned for 44 specimens representing four species of Hypnea Variable sites occurred at 387 positions (284) and among them 366 positions (269) were parsimoni-ously informative Sequence divergence of H asiatica isolates ranged up to 28 bp (206) However H asiatica differed significantly by 157minus165 bp (1153ndash1212) from H charoides The phylogenetic tree of cox1 (Fig 5) revealed that all 27 sequences of H asiatica from Korea

Japan and Taiwan were monophyletic with high bootstrap support Hypnea asiatica was more related to H spinella than to H charoides although this was poorly supported A strong relationship was observed between H boerge-senii and H flexicaulis the clade being positioned close to H japonica

Haplotype analyses of rbcL and cox1 mdash Twenty-eight samples of H asiatica were used for haplotype analy-ses of rbcL The nucleotide and haplotype diversities were

Fig 5 ML tree inferred from the cox1 sequence calculated using the GTR + Γ + I model of evolution [ndashlnL = 3889079871 substitution rate matrix RAC = 14224 RAG = 70097 RAT = 21341 RCG = 00486 RCT = 99098 RGT = 1 base fre-quencies πA = 02877 πC = 01267 πG = 01763 πT = 04093 shape parameter (α) = 01874 propor-tion of invariable site (I) = 00013] Values above each clade refer to ML and MP bootstrap values and Bayesian posterior probabilities

613


0001 and 0775 respectively The statistical parsimony net-work revealed seven haplotypes four from Korea one from Japan and three from Taiwan) Of these two isolates from Korea share the same haplotype from Taiwan (not shown)

Twenty-seven sequences of H asiatica were used for haplotype analyses of cox1 The nucleotide and haplotype diversities were 0008 and 0912 respectively The statis-tical parsimony network revealed 15 haplotypes (seven from Korea two from Japan and six from Taiwan) shown in Fig 6 All haplotypes from each of the three countries belonged to its own geographic group However one haplotype from Korea was found in the group of Taiwan

Paired t-tests using JMP 402 (SAS Institute Inc Cary North Carolina USA) revealed significant dif-ferences between p distances of cox1 and rbcL (t = 1517 p lt 00001)

DISCUSSIONThrough broad molecular analyses and detailed mor-

phological observations we answered the question posed by Womersley (1994) on the occurrence of H charoides in the northwest Pacific Ocean region We demonstrated marked differences between H charoides and H asiatica Morphologically H asiatica is distinguished by percur-rent main axes branches having abruptly curved adaxial branchlets and the presence of lenticular thickening in the walls of medullary cells The species is abundant from summer to fall in the lower intertidal and subtidal zone often inhabiting exposed sites All analyses of SSU rbcL

and cox1 sequences consistently separated H asiatica (from Korea Japan and Taiwan) from other species of the genus including H charoides Specimens from Japan un-der the name H charoides should be reassigned to H asi-atica because their habit and other morphological traits (see Figs 10minus15 in Yamagishi amp Masuda 2000) agree well with our description of H asiatica Furthermore the rbcL sequence (AB033159) of putative material of H charoides from Japan is identical to that of H asiatica in the present study The description of H asiatica in Korea Japan and Taiwan leads us to question previous reports of H charoides in southeast Asia (eg Silva amp al 1996 Chiang 1997 Lewmanomont 1997)

A comparison of H asiatica with similar species is given in Table 1 Hypnea charoides is characterized by branched axes branches and branchlets curving abruptly toward the main axis and a lack of lenticular thicken-ing in the medullary cell walls (Womersley 1994) Our SSU sequences of H charoides from Perth Australia were identical to those of Saunders amp al (2004) In ad-dition in all phylogenetic trees for SSU rbcL and cox1 H charoides was more related to H spinella than to H asiatica Hypnea valentiae (Turner) Montagne has relatively few spinous branchlets which are directed up-ward rather than at right angles (Yamagishi amp Masuda 1997) and the type locality of the species is the Red Sea which is biogeographically separated from the northeast Asia Hypnea spinella is different from H asiatica in hav-ing slender branches and branchlets with sharply pointed spines (Yamagishi amp Masuda 1997) The Mediterranean H furnariana Cormaci Alongi amp Dinaro (1993) has much

Fig 6 Statistical parsimony networks of cox1 haplotypes Small black circles represent missing haplotypes and each line represents a single mutation Circle size is pro-portional to haplotype frequency

614


thinner branches and branchlets which are tightly com-pact and matted close to the substratum and its tetraspo-rangial sori are located in the middle to subapical parts of the swollen branch or branchlet Hypnea flexicaulis grows in a habitat closely similar to that of H asiatica in Korea and the two are sometimes confused However H flexi-caulis is characterized by flexuous branched axes with wide angles branchlets with pronounced abaxial bending and antler-like branches and the species usually occurs in sheltered areas and tide pools (Geraldino amp al 2006)

The distributional pattern detected among H asiatica specimens was congruent for both rbcL and cox1 hap-lotypes However the cox1 gene with 15 haplotypes is more geographically structured than rbcL (7 haplotypes) The presence of two Korean isolates in the Taiwan group of cox1 data reflects gene flow between two countries Although a general theory explaining the distribution pat-terns of red algae along the northwestern Pacific region is as yet fragmentary (due to the lack of intensive sam-pling) we suggest that ocean currents are likely major influences on the distribution of H asiatica Despite the close proximity of Korea and Taiwan Korea is much cooler Judging from herbarium specimens deposited in CNUK and extensive collections during the last three years the distribution of H asiatica in Korea is limited to the southern coast which is influenced by the Kuroshio Current The current begins in the western Pacific Ocean off the east coast of Taiwan and flows past the southern coast of Korea toward Honshu Japan carrying thalli or propagules of marine algae (Kim amp al 2006) Additional sampling of the species from Taiwan and Japan will elucidate the phylogeographical structure of the species

The monophyly of Hypnea is strongly supported in the rbcL tree Although H asiatica formed a branch within the clade of H yamadae Tanaka and H volubi-lis the resolution was not supported Hypnea asiatica

has erect thalli while H yamadae and H volubilis have complanate mat-forming habits (Schneider amp Searles 1976) Hypnea charoides is also distantly related to other Hypnea species The distant relationships of H asiatica and H charoides from the putative relatives raises questions about divergence pathways within the genus which will be reinvestigated through additional taxon sampling within the genus However H flagelliformis H chordacea H musciformis H cornuta and H stellulifera formed a well resolved clade containing H flexicaulis and H borgesenii Hypnea flexicaulis and H borgesenii have lenticular thickenings in their medullary cells that are absent from members of the closely related clade comprising H flagelliformis H chordacea H musciformis H corn-uta and H stellulifera (Tanaka 1941 Yamagishi amp Masuda 2000 Geraldino amp al 2006) However all seven species in the clade have a percurrent axis (Tanaka 1941 Mshigeni 1978 Womersley 1994 Yamagishi amp Masuda 2000) which is regarded as a synapomorphic character Although H pannosa has relatively complanate thalli with matted growth it is morphologically distinct from H japonica by the possession of cylindrical thalli with hooked branches During our collections we noted that both species have similar brilliant blue iridescence when submerged (Tanaka 1941) The presence of hooked branches occurring in H japonica and H musciformis may be regarded as character reversal because of distant relationship between both species but is beyond this study

In conclusion the analysis of SSU rbcL and cox1 regions in the present study reinforces the taxonomic dis-crimination of H asiatica from other species in the genus As expected SSU is a slowly evolving gene and cox1 is the most variable gene among the three that we analyzed Analysis of the cox1 gene in rhodophytes is in its infancy but it has proven valuable for DNA barcoding within the phylum (Saunders 2005 Geraldino amp al 2006 Robba amp al

Table 1 Distinguishing characters of Hypnea asiatica and similar species

Morphological characteristics H asiatica H charoides H flexicaulis H spinellaSize (height) 11ndash18 cm 5ndash20 cm 5ndash35 cm 15ndash3 cm Color Red-browndeep red Medium redred-brown Yellowish-greenred-brown Red to scarletTexture Fleshysubcartilaginous Fleshysubcartilaginous Fleshsubcartilaginous FleshysubcartilaginousWidth of axes 12ndash14 mm in diameter 01ndash03 mm in diameter 07ndash25 mm in diameter 03ndash05 mm in diameterShape of main axes Alternate-spiral Branched irregularly Alternate-spiral Alternate-spiral Main axes Strongly percurrent Less percurrent Less percurrent (flexuous) Non-percurrentShape of lateral branches Adaxially curved (spinous) Slender (spinous) Adaxially curved (spinous) Sharply pointed spinesDirection of branching Approx 90deg angle Approx 90deg angle 45degndash150deg angle Forming loose webSpecial branches Secondary holdfasts Secondary holdfasts Antler-like upper branches Secondary holdfastsLenticular thickenings Present Absent Absent PresentPosition of tetrasporangial sori Base of the branchlets Base of the branchlets Throughout branchlets Throughout branchlets

615


Chiang YM 1997 Species of Hypnea Lamouroux (Gigartina-les Rhodophyta) from Taiwan Pp 163ndash177 in Abbott IA (ed) Taxonomy of Economic Seaweeds vol 6 California Sea Grant College System La Jolla

Clement M Posada D amp Crandall KA 2000 TCS a computer program to estimate gene genealogies Molec Ecol 9 1657ndash1669

Cormaci M Alongi G amp Dinaro R 1993 Hypnea furnari-ana sp nov (Hypneaceae Rhodophyta) from eastern Sicily (Mediterranean Sea) Nord J Bot 13 227ndash231

Freshwater DW Fredericq S Butler BS Hommersand MH amp Chase MW 1994 A gene phylogeny of the red algae (Rhodophyta) based on plastid rbcL Proc Natl Acad Sci USA 91 7281ndash7285

Freshwater DW amp Rueness J 1994 Phylogenetic relation-ships of some European Gelidium (Gelidiales Rhodophyta) species based on rbcL nucleotide sequence analysis Phy-cologia 33 187ndash194

Gavio B amp Fredericq S 2002 Grateloupia turuturu (Haly-meniaceae Rhodophyta) is the correct name of the non-native species in the Atlantic known as Grateloupia dory-phora Eur J Phycol 37 349ndash360

Geraldino PJL Yang EC amp Boo SM 2006 Morphology and molecular phylogeny of Hypnea flexicaulis (Gigartina-les Rhodophyta) from Korea Algae 21 417ndash423

Gilbert DG 1995 SeqPup A Biological Sequence Editor and Analysis Program for Macintosh Computers Biology Department Indiana University Bloomington

Hommersand MH amp Fredericq S 2003 Biogeography of the marine red algae of the South African west coast a molecular approach Pp 325ndash336 In Anthony RO Chapman ARO Anderson RJ Vreeland VJ amp Davison IR (eds) Seventeenth International Seaweed Symposium Proceedings of the XVIIth International

Seaweed Symposium Cape Town South Africa 28 Januaryndash2 February 2001 Oxford University Press Oxford

Kim HS Yang EC amp Boo SM 2006 The occurrence of Griffithsia okiensis (Ceramiaceae Rhodophyta) from Korea on the basis of morphology and molecular data Algae 21 91ndash101

Lamouroux JVF 1813 Essai sur les genres de la famille des thalassiophytes non articuleacutees Ann Mus Hist Nat Paris 20 21ndash47 115ndash139 267ndash293

Lewmanomont K 1997 Species of Hypnea from Thailand Pp 179ndash191 in Abbott IA (ed) Taxonomy of Economic Sea-weeds vol 6 California Sea Grant College System La Jolla

Lin SM Fredericq S amp Hommersand MH 2001 System-atics of the Delesseriaceae (Ceramiales Rhodophyta) based on large subunit rDNA and rbcL sequences including the Phycodryoideae subfam nov J Phycol 37 881ndash899

Masuda M Yamagishi Y Chiang YM Lewmanomont K amp Xia B 1997 Overview of Hypnea (Rhodophyta Hypneaceae) Pp 127ndash133 in Abbott IA (ed) Taxonomy of Economic Seaweeds vol 6 California Sea Grant Col-lege System La Jolla

Mshigeni KE 1978 The biology and ecology of benthic ma-rine algae with special reference to Hypnea (Rhodophyta Gigartinales) a review of the literature Biblioth Phycol 37 1ndash168

Mshigeni KE amp Chapman DJ 1994 Hypnea (Gigartinales Rhodophyta) Pp 245ndash281 in Akatsuka I (ed) Biology of Economic Algae SPB Academic Publishing The Hague

Posada D amp Buckley TR 2004 Model selection and model averaging in phylogenetics analysis of Akaike information criterion and Bayesian approaches over likelihood ratio tests Syst Biol 53 793ndash808

Posada D amp Crandall KA 1998 ModelTest testing the model of DNA substitution Bioinformatics 14 817ndash818

Ragan MA Bird CJ Rice EL Gutell RR Murphy CA amp Singh RK 1994 A molecular phylogeny of the marine red algae (Rhodophyta) based on the nuclear small subunit rRNA gene Proc Natl Acad Sci USA 91 7276ndash7280

Robba L Russell SJ Barker GL amp Brodie J 2006 Assessing the use of the mitochondrial cox1 marker for use in DNA barcoding of red algae (Rhodophyta) Amer J Bot 93 1101ndash1108

Ronquist F amp Huelsenbeck JP 2003 MrBayes 3 Bayesian phylogenetic inference under mixed models Bioinformat-ics 19 1572ndash1574

Rozas J amp Rozas R 2000 DnaSP version 3 an integrated program for molecular population genetics and molecular evolution analysis Bioinformatics 15 174ndash175

Saunders GW 2005 Applying DNA barcoding to red mac-roalgae a preliminary appraisal holds promise for future applications Philos Trans Ser B 360 1879ndash1888

Saunders GW Chiovitti A amp Kraft GT 2004 Small-subunit rRNA gene sequences from representatives of selected families of the Gigartinales and Rhodymenia-les (Rhodophyta) 3 Recognizing the Gigartinales sensu stricto Canad J Bot 82 43ndash74

Saunders GW amp Kraft GT 1994 Small-subunit rRNA gene sequences from representatives of selected families of the Gigartinales and Rhodymeniales (Rhodophyta) 1 Evidence for the Plocamiales ord nov Canad J Bot 72 1250ndash1263

2006 Yang amp al 2008) There remain species of uncertain affinities in the genus eg H spinella from Vietnam whose rbcL sequences were identical to a sequence from Japan but quite different from that of North American samples It will be very rewarding to investigate other cosmopolitan species of red algae that have phenotypic variation or a lack of morphologically clear-cut characters by comparing type material and molecular analysis Analyzing more than one gene is essential for an accurate assessment of species within large widespread genera of red algae

ACKNOWLEDGEMENTSWe thank John Huisman for help with the collection in

Australia Il Ki Hwang for collecting specimens in Vietnam Giovanni Furnari for critical comments and Lawrence Liao for reading the first draft This research was supported by MarineBio21 program grants from the Ministry of Maritime Affairs amp Fisheries Korea

LITERATURE CITED

616


Appendix List of taxa investigated supplied with voucher information or reference (in parentheses) and GenBank accession num-bers for each DNA region Dash indicates the absence of the sequence analyzed

Species (voucher specimen code or reference) locality GenBank accession SSU rbcL cox1

Hypnea asiatica sp nov PJL Geraldino EC Yang amp Boo (PH35) Wolpo Pohang Korea EU240861 EU240827 EU240804 (PH65) Gijang Busan Korea ndash EU346001 EU345978 (PH66) Gijang Busan Korea ndash EU346000 EU345979 (PH67) Gijang Busan Korea EU240864 EU240828 EU240802 (PH46) Guryongpo Gyeongju Korea EU240862 EU240829 EU240803 (PH10) Gampo Gyeongju Korea EU240858 EU240824 EU240798 (PH1) Gampo Gyeongju Korea ndash EU346007 EU345975 (PH9) Gampo Gyeongju Korea ndash EU346005 EU345976 (PH11) Gampo Gyeongju Korea ndash EU346006 EU345977 (PH12) Gampo Gyeongju Korea EU240859 EU240825 EU240799 (PH36) Wolpo Pohang Korea EU240860 EU240826 EU240800 (PH52) Tongyoung Gyeongnam Korea EU240863 EU240830 EU240801 (PH91) Gijang Busan Korea ndash EU345998 EU345982 (PH130-2) Hengchun Pintung Taiwan ndash EU346008 EU345981 (PH0509) Lonedome Keelung Taiwan EU240869 EU240831 EU240809 (PH0511) Lonedome Keelung Taiwan EU240870 EU240832 EU240810 (PH0512) Dali Keelung Taiwan EU240871 EU240833 EU240811 (PH0513) Dali Keelung Taiwan EU240872 EU240834 EU240812 (PH0514) Dali Keelung Taiwan EU240873 EU240835 EU240813 (PH0517) Pinglang bridge Keelung Taiwan EU240874 EU240836 EU240814 (PH0518) Pinglang bridge Keelung Taiwan EU240875 EU240837 EU240815 (PH0519) Pinglang bridge Keelung Taiwan ndash EU345997 EU345980 (PH0523) Daisanglan Keelung Taiwan EU240876 EU240838 EU240816 (PHH078) Kurohae Beach Choshi Chiba Japan EU240867 EU240839 EU240805 (PHH079) Kurohae Beach Choshi Chiba Japan EU240868 EU240840 EU240806 (PHH080) Ashikajima Beach Choshi Chiba Japan EU240866 EU240841 EU240808 (PHH081) Nagasaki Choshi Chiba Japan EU240865 EU240842 EU240807 (Yamagishi amp Masuda 200) Nabeta Shimoda Shizuoka Japan ndash AB033159 ndash Hypnea boergesenii Tanaka (PH030) Seongsan Jeju Korea ndash EU346010 ndash (PH95) Jeju Korea ndash EU346009 ndash (PH0826) Gimyeong Jeju Korea ndash EU345994 EU345983 (Hommersand amp Fredericq 2001) Taiwan ndash AF385634 ndash (Geraldino amp al 2006) Daisanglan Keelung Taiwan ndash EF136612 EF136608 Hypnea charoides JV Lamouroux (PH0922) Point Peron Perth Australia EU240852 EU240844 EU240819 (PH0923) Point Peron Perth Australia EU240851 EU240845 EU240820 (PH0926) Point Peron Perth Australia EU240854 EU240846 EU240823 (PH0927) Point Peron Perth Australia EU240853 EU240847 EU240821 (PH0930) Point Peron Perth Australia EU240855 EU240843 EU240822 (Saunders amp al 2004) Pinnaroo Pt WA Australia AY437682 ndash ndash Hypnea chordacea Kuumltzing (Yamagishi amp Ma-suda 2000) Shirahama Shimoda Shizuoka Japan ndash AB033160 ndash Hypnea cornuta (Kuumltzing) J Agardh (PH0901) Bali Indonesia ndash EU345991 ndash (PH0902) Bali Indonesia ndash EU345993 ndash (PH0920) Point Peron Perth Australia ndash EU345990 ndash (PH0931) Point Peron Perth Australia ndash EU345992 ndash (Yamigishi amp al 2003) Sukuji Ishigaki Island Okinawa Japan ndash AB095911 ndash (Yamagishi amp Masuda 2000) Teguma Nagasaki Nagasaki Prefecture Japan ndash AB033161 ndash (Yamagishi amp al 2003) Taranto Italy ndash AB095912 ndash Hypnea flagelliformis J Agardh (Yamagishi amp Masuda 2000) Fukaura Aomori Prefecture Japan ndash AB033162 ndash Hypnea flexicaulis Yamagishi amp Masuda (Yamagishi amp Masuda 2000) Shirahama Shimoda Shizuoka Japan ndash AB033163 ndash (Geraldino amp al 2006) Wolpo Pohang Korea ndash EF136623 ndash (Geraldino amp al 2006) Dancalan Bulusan Philippines ndash EF136632 EF136591 (Geraldino amp al 2006) EU240850 EF136628 EF136594 (Geraldino amp al 2006) Gampo Gyeongju Korea ndash EF136627 ndash Hypnea japonica Tanaka (PH7) Gampo Gyeongju Korea ndash EU346003 EU345986 (PH8) Gampo Gyeongju Korea ndash EU346002 EU345987 (PH0506) Lonedome Keelung Taiwan ndash EU345996 EU345988 (PH0507) Dali Keelung Taiwan ndash EU345995 EU345989 (Yamagishi amp Masuda 2000) Banshobana Ei Kagoshima Japan ndash AB033164 ndash Hypnea musciformis (Wulfen) JV Lamouroux (Hmus1) Cap Ferrat Villefranche France ndash EU346011 ndash (Hmus2) Theoule Cannes France ndash EU346012 (Hmus3) Antibes France ndash EU346013 ndash (Hmus4) Antibes France ndash EU346014 ndash (Hommersand amp Fredericq 2001) New Hanover North Carolina USA ndash U04179 ndash Hypnea pannosa J Agardh (Yamagishi amp Masuda 2000) Hedo-misaki Okinawa Prefecture Japan ndash AB033165 ndash Hypnea ramentacea (C Agardh) J Agardh (Saunders amp al 2004) Port Macdonnell SA Australia AY437683 ndash ndash Hypnea stellulifera J Agardh (PH0101) Panglao Bohol Philippines ndash EU346004 EU345984 (PH0122) Panglao Bohol Philippines ndash EU345999 EU345985 (Yamigishi amp al 2003) Pulau Sipadan Sabah Malaysia ndash AB095915 ndash (Yamagishi amp al 2003) Pulau Sipadan Sabah Malaysia ndash AB095914 ndash (Yamagishi amp al 2003) Pulau Besar Melaka Malaysia ndash AB095913 ndash Hypnea spinella (C Agardh) Kuumltzing (PH0504) Nha Trang Panang Bay Vietnam EU240856 EU240848 EU240818 (PH0527) Nha Trang Panang Bay Vietnam EU240857 EU240849 EU240817 (Yamagishi amp Masuda 2000) Sesoko Island Okinawa Prefecture Japan ndash AB033166 ndash (Hommersand amp Fredericq 2001) Florida United States of America ndash AF385635 ndash Hypnea sp (Yamagishi amp Masuda 2000) Izumozaki Kushimoto Wakayama Japan ndash AB033167 ndash Hypnea volubilis Searles (Hommersand amp Fredericq 2001) Los Angeles USA ndash AF385636 ndash Hypnea yamadae Tanaka (Yamigishi amp al 2003) Nomozaki Nagasaki Prefecture Japan ndash AB095916 ndash

Schneider CW amp Searles RB 1976 North Carolina marine algae VII New species of Hypnea and Petroglossum (Rhodophyta Gigartinales) and additional records of other Rhodophyta Phycologia 15 51ndash60

Shin WG amp Boo SM 1994 A systematic study on the genus Hypnea (Gigartinales Rhodophyta) in Korea Algae 9 7ndash20

Silva PC Basson PW amp Moe RL 1996 Catalogue of the benthic marine algae of the Indian Ocean Univ Calif Publ Bot 79 1ndash1259

Swofford DL 2002 PAUP Phylogenetic Analysis Using Parsimony (and Other Methods) vers 40b10 Sinauer Sunderland

Tanaka T 1941 The genus Hypnea from Japan Sci Pap Inst Algol Res Fac Sci Hokkaido Univ 2 227ndash250

Tani M Yamagishi Y Masuda M Kogame K Kawagu-chi S amp Phang SM 2003 Taxonomic notes on marine algae from Malaysia IX Four species of Rhodophyceae with the description of Chondria decidua sp nov Bot Mar 46 24ndash35

Womersley HBS 1994 The Marine Benthic Flora of Southern Australia part IIIA Bangiophyceae and Florideophyceae (Acrochaetiales Nemaliales Gelidiales Hildenbrandiales

and Gigartinales sensu lato) Australian Biological Re-sources Study Canberra

Xia BM amp Wang YQ 1997 Some species of the genus Hyp-nea (Gigartinales Rhodophyta) from China Pp 193ndash206 in Abbott IA (ed) Taxonomy of Economic Seaweeds vol 6 California Sea Grant College System La Jolla

Yamagishi Y amp Masuda M 1997 Species of Hypnea from Japan Pp 135ndash162 in Abbott IA (ed) Taxonomy of Economic Seaweeds vol 6 California Sea Grant College System La Jolla

Yamagishi Y amp Masuda M 2000 A taxonomic revision of a Hypnea charoides-valentiae complex (Rhodophyta Gigar-tinales) in Japan with a description of Hypnea flexicaulis sp nov Phycol Res 48 27ndash35

Yamagishi Y Masuda M Abe T Uwai S Kogame K Kawaguchi S amp Phang SM 2003 Taxonomic notes on marine algae from Malaysia XI Four species of Rhodo-phyceae Bot Mar 46 534ndash547

Yang EC Kim MS Geraldino PJL Sahoo D Shin JA amp Boo SM 2008 Mitochondrial cox1 and plastid rbcL genes of Gracilaria vermiculophylla (Gracilariaceae Rhodophyta) J Appl Phycol 20 161ndash168

607


for Hypnea (Freshwater amp al 1994 Yamagish amp Ma-suda 2000Hommersand amp Fredericq 2003 Tani amp al 2003 Yamagishi amp al 2003 Geraldino amp al 2006) al-lowed us to compare our results directly with those from other species in the genus and with putative relatives We tested the suitability of the cox1 gene for identification of H flexicaulis Yamagishi amp Masuda from other species of the genus (Geraldino amp al 2006) The three genes we studied are in different cellular compartments thus providing three separate analytic approaches Here we describe H asiatica sp nov from Korea Japan and Tai-wan and conclude that H charoides should be removed from the northwestern Pacific marine flora

MATERIALS AND METHODSField observations and collections of H asiatica were

made at six locations around Korea and at sites in Japan and Taiwan Representative specimens of H asiatica were collected in the intertidal zone from the following locali-ties Korea Gampo Gyeongju (2 October 2005) Gijang Busan (17 October 2005) Tongyoung (15 July 2004) Guryongpo Gyeongju (2 October 2005) Wolpo Pohang (3 October 2005) Jeju (13 November 2004) Taiwan Dali Keelung (30 March 2006) Japan Choshi Chiba Prefecture (30 July 2004)

The phenology of H asiatica was monitored in situ in Gampo Korea every two months from June 2006 to June 2007 Tissues were sectioned using a freezing microtome FX-802A (Coper Electronics Co Ltd Kanagawa Japan) and the sectioned preparations were stained with 1 aque-ous aniline blue acidified with a drop of 1 HCl and mounted in 30 glycerinndashseawater Photographs were taken with a FX-35DX camera (Nikon Tokyo Japan) at-tached to a microscope (Vanox AHBT3 Olympus Tokyo Japan) Voucher specimens are housed at the herbarium of Chungnam National University (CNUK) Daejeon Korea

DNA extraction mdash 51 specimens were available for our molecular study (Appendix) Total DNA was extracted from approximately 5 mg of dried thalli ground in liquid nitrogen using a DNeasy Plant Mini Kit (Qiagen GmbH Hilden Germany) following manufacturerrsquos instructions Extracts were dissolved in 150 mL of distilled water The extracted DNA was stored at ndash20degC and used to amplify SSU rbcL and cox1 genes Voucher specimens used for molecular systematics were deposited in the herbarium of Chungnam National University (CNUK) Daejeon Korea

Analysis of SSU rbcL and cox1 regions mdash For am-plification and sequencing reaction of each gene specific primer pairs were used as follows for SSU G01-G10 G02-G14 G04-G13 and G06-G07 (Saunders amp Kraft 1994) for rbcL F7-R753 and F645-RrbcS start (Freshwater amp Rueness 1994 Lin amp al 2001 Gavio amp Fredericq 2002)

and for cox1 cox143F-cox11549R (Geraldino amp al 2006) and C622F-C880R (Yang amp al 2008) All PCR amplifi-cations were carried out with a TechGene thermal cycler (Techne Ltd Duxford Cambridge UK) using a TaKaRa Ex Taq reaction kit (Takara Shuzo Shiga Japan) a total volume of 25 μL consisted of 25 μL 10times Ex Taq Buffer 20 μL 25 mM MgCl2 10 μM dNTP mixture 015 μM of each primer 0625 units of TaKaRa Ex Taq and 30 μM DNA solution (containing 05ndash10 μg DNA) PCR was per-formed with an initial denaturation step at 94degC for 10 min followed by 35 cycles of 30 s at 90degC 30 s at 50degC and 2 min at 72degC with a final 10-min extension cycle at 72degC

The PCR products were purified using a High Pure PCR Product Purification Kit (Roche Diagnostics GmbH Mannheim Germany) according to the manufacturerrsquos instructions Sequences of the forward and reverse strands were determined for all PCR products using an ABI PRISMtrade 377 DNA Sequencer (Applied Biosystems Foster City California USA) at the Research Center CNUK Both electropherogram outputs from each sample were edited using the program Sequence Navigator ver 101 (Applied Biosystems)

Twenty-nine SSU sequences (27 new and 2 published) 75 rbcL sequences (51 new and 24 published) and 44 cox1 sequences (41 new and 3 published) of Hypnea were col-lated using the multiple-sequence editing program SeqPup (Gilbert 1995) and aligned visually For rbcL data outgroups were Gigartina pinnata J Agardh (AF385663) Chondrus ocellatus Holmes (AF294815) Grateloupia imbricata Holmes (AF299252) and Gracilaria caudata J Agardh (AY049331) All sequences used in this study are presented in the Appendix None of the alignments posed problems as no gaps were observed

Phylogenetic analyses mdash All datasets from SSU rbcL and cox1 genes were utilized Maximum parsimony (MP) trees were constructed for each dataset with PAUP 40b10 (Swofford 2002) using a heuristic search algo-rithm with the following settings 1000 random sequence additions tree bisection-reconnection (TBR) branch swapping MulTrees all characters unordered and un-weighted and branches with a maximum length of zero collapsed to yield polytomies Bootstrap values for the resulting nodes were assessed using 1000 bootstrapping replicates with ten random sequence additions

For maximum likelihood (ML) and Bayesian analyses the best models for individual datasets were determined under Modeltest 308b (Posada amp Crandall 1998) with the following settings for all datasets Kimura three-parameter model unequal frequencies (K3Puf) proportion of invari-able sites (I) and the γ shape parameter (G) For Modeltest model scores were estimated on the basis of the model block for PAUP (in the Modeltest package) The best model was selected using the Akaike Information Criterion (AIC) as recommended by Posada amp Buckley (2004) ML analyses

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systematics of hypnea asiatica sp. nov. (hypneaceae, rhodophyta) based on morphology and nrdna ssu,...

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