Research ArticleResolving the Taxonomic Status of Chamelea gallinaand C striatula (Veneridae Bivalvia) A Combined MolecularCytogenetic and Phylogenetic Approach

Daniel Garciacutea-Souto Vesa Qarkaxhija and Juan J Pasantes

Departamento Bioquımica Xenetica e Inmunoloxıa Universidade de Vigo Vigo Spain

Correspondence should be addressed to Juan J Pasantes pasantesuvigoes

Received 31 January 2017 Revised 27 March 2017 Accepted 3 April 2017 Published 7 May 2017

Academic Editor Daniele Corsaro

Copyright copy 2017 Daniel Garcıa-Souto et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The striped venus clams Chamelea gallina and C striatula are commercially important bivalves inhabiting European and NorthAfrican coastal waters The taxonomic status of these taxa has been the subject of debate for decades In order to elucidate thisissue we generated 5S and 28S ribosomal RNA and H3 histone gene probes and mapped them by fluorescent in situ hybridizationto the chromosomes of morphologically identified striped venus clams collected from four geographically distant Atlantic andMediterranean populations The nucleotide variation at the three DNA markers that is the nuclear internal transcribed spacer2 (ITS2) the mitochondrial cytochrome c oxidase subunit I (COI) and the large ribosomal subunit rRNA (16S) fragments wasalso studied and the resultant phylogenetic trees were evaluated Striking differences in both the chromosome distribution of thesegenes and the clustering of the samples on the phylogenetic trees observed provide clear evidence that C gallina and C striatulaare separated species

1 Introduction

The striped venus clams Chamelea gallina (Linnaeus 1758)and C striatula (da Costa 1778) (Bivalvia Veneridae) theonly extant species in the genus Chamelea Morch 1853 arefilter feeding sand burrowing bivalves inhabiting Europeanand North African coastal waters Even though these taxaare commercially exploited and economically valuable fordecades their taxonomic status has been amatter of debate [1]that is still not completely settledThe confused status of thesetaxa is partially due to the use of variable shell and siphoncharacteristics to identify them C striatula is distinguishedfromC gallina by possessing a more pointed and ridged shelland longer siphons [2] and by residing in the Atlantic [3] Onthe basis of shell and siphonmorphologiesC striatula andCgallina have been considered members of a single polymor-phic species two geographically isolated subspecies or twodifferent species The lack of modern genetic studies appliedto differentiate C gallina and C striatula also contributes totheir uncertain status The genetic distances estimated after

studying seven polymorphic enzymes in samples obtainedfrom striped venus clam mixed beds in the south of Portugal[2] supporting their status as separated species are as yetthe only genetic study applied to these taxa

Although shell shape analyses [4] and geometricmorpho-metric methods [3] have helped in accurately predicting theorigin of someC gallina samples anddifferentiatingC gallinafrom C striatula establishing taxonomic boundaries withinbivalves has always been hindered by a lack of diagnosticmorphological features [5] This is due to an extensiveparallelism of interspecific variability as a result of convergentevolution in response to the same environment alongsidea degree of phenotypic divergence among populations of asingle taxon developed on different substrates Therefore itis inaccurate to rely on morphology alone to delimit speciesboundaries and further criteria have to be analyzed [5]

In comparison with many other groups of organismscytogenetic analyses in clams of the family Veneridae arescarce All species studied to date show adiploid chromosomenumber of 2119899 = 38 [6 7] and their karyotypes are mostly

HindawiBioMed Research InternationalVolume 2017 Article ID 7638790 8 pageshttpsdoiorg10115520177638790

2 BioMed Research International

Chamelea striatula

Chamelea gallina

Chamelea gallina

Chamelea gallina

(Pontevedra)

(

(Valencia)

(Italy)aacutedi

Figure 1 Collection localities and representative shells of the striped venus analyzed

composed of chromosome pairs showing small differencesin size and morphology therefore making their accurateidentification almost impossible [7] As a resolution to thisproblem along the last two decades highly conserved repet-itive DNA sequences have been used as probes in fluorescentin situ hybridization (FISH) experiments to identify chromo-some pairs in bivalve species [8 9] some of which were venusclams [7 10ndash14] Most commonly used DNA sequences forthese types of studies are ribosomal RNA (rRNA) and histonegenes as they are usually organized in tandems and clusteredat one or more chromosomal positions The employment ofthis approach inmussels of theMytilus edulis species complexhas allowed for differentiating M edulis and M galloprovin-cialis from M trossulus by the chromosomal location of oneof the H3 histone gene clusters and the number and locationof the major rRNA gene (rDNA) clusters [15]

Chromosomal studies on striped venus clams are rarerthan in other venerid clams To the best of our knowl-edge there is no published karyological information for Cstriatula For C gallina information is mostly limited totwo reports describing chromosome number and karyotypecomposition and the location of major rDNA 5S rDNA andH3 histone gene clusters [7 16]

In regard to molecular phylogenetic analysis many stud-ies have utilized the mitochondrial cytochrome c oxidasesubunit I (COI) gene as the standard most accurate markerfor delimitating and clarifying the taxonomic status of animalspecies including venerid clams [17] The mitochondrial16S rRNA gen has also been successfully employed forspecies identification in clams [17 18] These mitochondrialsequences are known to evolve faster than nuclear genesmaking them ideal tools for detecting differences amongclosely related species and among populations within aspecies In addition the sequences of the internal transcribedspacer 2 (ITS2) of the 18S-58S-28S nuclear rDNA and theircorresponding secondary structures of the ITS2 rRNAs have

also proved suitable for assessing phylogenetic and phylogeo-graphic relationships amongmany animal taxa some of themvenerid clams [19]

In order to contribute to a better understanding of theevolutionary history and diversification within the genusChamelea we used an integrated approach based on theone hand on the comparison of the karyotypes constructedafter mapping by FISH three tandemly repeated gene families(5S rDNA 28S rDNA and H3 histone genes) and on theother hand on the molecular phylogenies obtained from onenuclear (ITS2) and two mitochondrial (COI and 16S rRNAgenes) sequences in striped venus clams collected from twoAtlantic populations (Rıa de Pontevedra NW Spain Gulfof Cadiz S Spain) and two Mediterranean ones (Gulf ofValencia E Spain Adriatic Sea E Italy)The genetic evidenceobtained in this work confirms the consideration ofC gallinaand C striatula as separated species

2 Materials and Methods

21 Sampling and Identification Specimens of striped venusclams were collected (Figure 1) from different localities in Rıade Pontevedra (Atlantic coast NW Spain) and identified asC striatula through their morphological attributes Otherstriped venus clam samples identified as C gallina werecollected from natural beds in Valencia (Gulf of Valen-cia Mediterranean coast E Spain) and from local marketplaces in coastal towns of the Gulf of Cadiz (Atlanticcoast S Spain) and the Adriatic Sea (Mediterranean coastE Italy) The nomenclature utilized for these taxa followsthe World Register of Marine Species (WoRMS) database(httpwwwmarinespeciesorg) In all cases striped venusclams were transported alive to the lab maintained in tanksof 5 L filtered seawater at 18 plusmn 1∘C and fed on microalgae topromote somatic growth

BioMed Research International 3

22 Chromosome Preparation Chromosome spreads wereobtained as previously published [20 21] Following an invivo colchicine (0005 10 h) treatment striped venus clamswere dissected and gills and gonads were immersed in dilutedsea water (50 20min 25 20min) After fixation inethanolacetic acid (3 1 3 times 20min) pieces of tissue weredisaggregated in 60 acetic acid and the cell suspensionsspread onto warm microscope slides [22 23]

23 DNA Extraction and PCR Amplification DNA wasextracted from adductormuscles using a phenol-chloroform-isoamyl alcohol method [24] A fragment of the mitochon-drial COI gene was amplified by PCR employing the standardbarcoding primers LCO1490 and HCO2198 [25] A fragmentof the mitochondrial 16S rRNA gene was amplified by meansof primers 16L29 [26] and 16SBr [27] The complete ITS2 ofthe major rDNA was amplified using primers ITS3 and ITS4[28] On FISH mapping purposes universal primers LR10Rand LR12 retrieved from Vilgalys lab website [29] were usedto amplify a fragment of the 28S rDNA Amplifications ofthe entire 5S rDNA repeat and the H3 histone genes usedprimers described by Perez-Garcıa et al [22 23] and Giribetand Distel [30] respectively

DNA sequences were amplified in a GeneAmp PCR sys-tem 9700 (Applied Biosystems) in 50 120583L solutions containing125 ng of genomic DNA 50120583M each dNTP 50 120583M eachprimer 1xPCR buffer 15 120583M MgCl

2 and 5U of JumpStart

Taq DNA Polymerase (Sigma) Amplifications included aninitial denaturation step at 95∘C (2min) 35 amplificationcycles (Supplementary Table 1 in Supplementary Materialavailable online at httpsdoiorg1011552017 7638790) anda final extension at 72∘C (5min) PCR products were exam-ined by electrophoresis on 2 agarose gels

24 DNA Sequencing and Phylogenetic Analysis Ampli-fied mitochondrial 16S rRNA and COI genes and nuclearITS2 rDNA sequences were purified (FavorPrep GELPCRPurification Kit Favorgen) and sequenced (CACTI Univer-sity ofVigo) in both directions in anABI PRISM3730GeneticAnalyzer (Applied Biosystems) using a BigDye Termina-tor v 31 Cycle Sequencing Kit (Applied Biosystems) Thesequences were edited with BioEdit v 7111 [31] and alignedwith Muscle set to default parameters using MEGA7 [32]Sequence similarity searches were performed using the BasicLocal Alignment Search Tool (BLAST) algorithm available atthe National Center for Biotechnology Information (NCBI)(httpwwwncbinlmnihgovblast) The MegaBLAST algo-rithm set to default parameters was employed against bothNCBI nucleotide collection and NCBI nucleotide collectionand Barcode of Life Data System (BOLD) databases Afterremoving primers maximum-likelihood (ML) phylogeneticanalyses were performedThe best-fit nucleotide substitutionmodels were selected (COI gene JC + G 16S rRNA geneHKY+G ITS2 T92) by the AIC criterion employing JMod-elTest 2 [33 34] ML reliability was assessed with 500 boot-strap replicates All phylogenetic analyses were performedon MEGA7 [32] Nucleotide diversity (pi) and uncorrectedpairwise p-distanceswere estimated usingDnaSP v 5 [35] andMEGA7 [32] respectively

25 Fluorescence In Situ Hybridization (FISH) Single dou-ble and sequential FISH experiments using 5S and 28SrDNA and H3 histone gene probes were performed onmetaphase chromosome spreads obtained from striped venusclams collected in all four regions Biotin and digoxigeninlabeled probes were generated either directly by PCR or bynick translation [36] Chromosome slides were digested withRNase and pepsin before denaturation (70∘C 2min) andhybridized overnight at 37∘C Biotin was detected with fluo-rescein isothiocyanate (FITC) conjugated avidin and biotiny-lated anti-avidin (Vector) whereas digoxigenin was detectedwith anti-digoxigenin antibodies conjugated with tetram-ethylrhodamine isothiocyanate (TRITC) (Sigma) Chromo-some slides were counterstained with 41015840-6-diamidino-2-phenylindole (DAPI 014 120583gmL in 2xSSC) and mountedwith antifade (Vectashield Vector)

Chromosome preparations were examined with a NikonEclipse-800 microscope equipped with an epifluorescencesystem [36] Separated images for each fluorochrome wererecorded and pseudocolored using a DS-Qi1Mc CCD camera(Nikon) controlled by the NIS-Elements software (Nikon)Merging of the images was performed with Adobe Photo-shop

3 Results

31 Karyotypes and Chromosomal Mapping of rDNA andH3 Histone Gene Clusters All striped venus clams analyzedshowed mitotic metaphase plates presenting 38 chromo-somes (Figure 2) The karyotypes constructed for the fourstriped venus clam populations (Figure 2) were roughlysimilar for 18 of the chromosome pairs (7 metacentric 6metasubmetacentric 2 submetacentric and 3 subtelocen-tric) but the remaining one (number 19 for comparative pur-poses) showed morphological differences This chromosomepair was subtelocentric in both C striatula and the ItalianC gallina and metacentric in the two Spanish C gallinapopulations

FISH mapping of 5S rDNA probes showed intercalarysignals located in two metacentric chromosome pairs (5pand 9q) in all striped venus clams regardless of origin andwhether they were morphologically identified as C gallinaor C striatula (Figure 2) In contrast the number andthe distribution of the signals corresponding to both 28SrDNA and H3 histone gene probes evinced some differencesAll striped venus clam specimens presented at minimum asingle 28S rDNA signal situated in the neighborhood of thecentromere on chromosome pair 19 whereas this is the onlysignal in C gallina an additional signal subterminal to 8qappeared in C striatula (Figure 2) Likewise although H3histone gene probes were subterminal to 15q and 17q in allstriped venus clams further signals were present in both theItalian C gallina population (6q) and in C striatula (6q and5q)

Double-color FISH mapping using 5S rDNA and H3histone gene probes also confirmed the presence of signalsfor both probes on C striatula chromosome pair 5 Thischromosome pair only bears 5S rDNA clusters in C gallina

4 BioMed Research International

5S H3 28S

5S H3 28S

5S H3 28S

5S H3 28S

5S H3 28S

5S H3 28S

5S H3 28S

5S H3 28S

5

5S

5S

5S

5S

5S

5S

5S

SH3

H3

H3

H3

H3 H3

H3H3

H3

H3

H3

28S

28S

28S

28S

28S

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18 19

5 6 8 9 15 17 19

5 9 15 17 19

5 9 15 17 19

5 6 9 15 17 19

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18 19

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18 19

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18 19

CST Pon

CGA Cad

CGA Val

CGA Ita

CST Pon

CGA Cad

CGA Val

CGA Ita

Figure 2 FISH mapping of 5S rRNA (5S red) 28S rRNA (28S magenta) and H3 histone gene (H3 green) probes to mitotic metaphasechromosomes of striped venus clams Chamelea striatula from Pontevedra (CST Pon) and Chamelea gallina from Cadiz (CGA Cad) Valencia(CGAVal) and Italy (CGA Ita) counterstained with DAPIThe corresponding karyotypes and schematic representations of the signal bearingchromosomes are also included Note that all chromosome pairs present a single signal with the exception of CST Pon chromosome pair 5that bears both 5S rDNA and H3 histone gene clusters Scale bars 5 120583m

In all four populations variation in signal patterning waslow and aberrance was reduced to a pericentric inversion oran additional 28S rDNA signal in four of the 55 C striatulaspecimens and one additional 28S rDNA signal in two of the28 specimens of the Italian C gallina

32 DNA Sequence Variation and Genetic Divergence A frag-ment of 629 bp excluding primers of the mitochondrial COIgenewas sequenced for 20 striped venus clams five per popu-lation (GenBank acc numbers KY547747 to KY547766) Thesequences showed 89 polymorphic sites and 78 differentiallyfixedmutationsThe nucleotide diversity was 00556 (Table 1)and the genetic distance between taxa was 1320 (Table 2)

The comparison of these sequences with those stored inGenBank showed that ourC striatula sequences coincided ina 99 with those fromNorth Sea C gallina samples [37]TheC gallina COI sequences displayed a high level of homology(98) with samples from the Adriatic Sea (GenBank accnumbers DQ458474 and KR078004) and a market placein the Canary Islands (GenBank acc number DQ184835)Pairwise genetic distances between populations inferred fromthe mitochondrial COI gene sequences (Table 2) showedthat C striatula is clearly distinct from the three C gallinapopulations Likewise the ML tree recovered by MEGA onthese sequences (using Dosinia exoleta as outgroup) revealedtwo well supported clades one formed by the specimens

BioMed Research International 5

COI

005

CST Pon 05

CST Pon 08CST Pon 02CST Pon 01

CST Pon 135660

ITS2

CGA Cad 04CGA Cad 01CGA Cad 02CGA Cad 05CGA Cad 03

CGA Val 05CGA Val 04

86 82 CGA Val 03CGA Val 02

58 CGA Val 01CGA lta 0236 CGA lta 03 92

CST Pon 03CST Pon 02CST Pon 01

CST Pon 06

CST Pon 04CST Pon 14

100CST Pon 07

CST Pon 15CST Pon 05CST Pon 03CST Pon 09

CGA Cad 05CGA Cad 01CGA Cad 02 100

CGA Cad 03CGA Cad 04

CGA Val 05

84CGA Val 03CGA Val 02CGA Val 01CGA lta 05 98CGA lta 04

CGA lta 0150 CGA lta 04

CGA lta 05

CGA lta 03CGA lta 02CGA lta 01

Dosinia exoleta DQ458478 Dosinia exoleta HE965776

002

CST Pon

CGA Cad

CGA Val

CGA Ita

CST Pon 10

CGA Val 04

CST Pon 12

100

56 CST Pon 04

CST Pon 1128

Figure 3 Maximum-likelihood trees based on mitochondrial COI gene and nuclear ITS2 sequences of striped venus clams using Dosiniaexoleta as outgroup together with a schematic representation of the main chromosomal differences Numbers in internal nodes indicatemaximum-likelihood bootstrap support values (500 replicates)

Table 1 Nucleotide diversity (pi) of COI gene 16S rDNA and ITS2sequences in Chamelea gallina and Chamelea striatula

COI gene 16S rDNA ITS2119899 pi 119899 pi 119899 pi

Chamelea striatulaCST Pon 5 00022 5 00000 15 00021

Chamelea gallinaCST Cad 5 00000 5 00000 5 00000CST Val 5 00000 5 00000 5 00000CST Ita 5 00057 5 00035 5 00000All CST 15 00060 15 00030 15 00063

All Chamelea 20 00556 20 00443 30 00202

morphologically identified as C striatula and the other bythe specimens identified as C gallina with bootstrap valuesof 100 and 86 respectively (Figure 3)

The sequenced 16S rRNA gene fragments were 468 bplong in the specimens collected from Pontevedra (C striat-ula) and 466 bp long in all C gallina specimens (GenBankacc numbers KY547767 to KY547786) The difference inlength was due to six between-taxa gaps two single nucle-otide and one dinucleotide insertions in C striatula and twosingle nucleotide insertions in C gallina There were 51 poly-morphic sites 48 of them taxa specific providing a geneticdistance between taxa of 1144The nucleotide diversity was00443 (Table 1) These 16S rRNA gene sequences were also

Table 2 Pairwise p-distances between striped venus clam popu-lations using mitochondrial COI and 16S rRNA genes and nuclearITS2 sequences Interspecific distances in bold

Populations COI gene 16S rDNA ITS2CST Pon CGA Cad 01304 01121 00396CST Pon CGA Val 01320 01164 00330CST Pon CGA Ita 01335 01147 00330CGA Cad CGA Val 00095 00043 00132CGA Cad CGA Ita 00086 00047 00132CGA Val CGA Ita 00048 00022 00000CST CGA 01320 01144 00352

compared with the six sequences stored in GenBank The Cstriatula sequences were identical to sequences from England(GenBank acc numbers DQ280041 and KX713203) whereasthose from C gallina matched the sequences of specimensfrom Turkey (GenBank acc number AM085110) AdriaticSea (GenBank acc number AJ548762) NW Spain (GenBankacc number JF808193) and a market in the Canary Islands(GenBank acc number DQ184735) Again both the pairwisedistances (Table 2) and the ML tree recovered (not shown)differentiated specimens belonging to the two taxa (bootstrapvalues of 100 and 72)

The amplified ITS2 fragments (GenBank acc numbersKY508254 to KY508283) were the most inconsistent in

6 BioMed Research International

length While all specimens of the Mediterranean popu-lations of C gallina displayed 498 bp long fragments inthe Atlantic specimens the ITS2 was 496 bp long thesedifferences in length were a result of a dinucleotide insertion(or deletion) and six nucleotide substitutions In contrastfive specimens of theC striatula population displayed 498 bplong ITS2 fragments whereas the remaining 10 exhibited495 bp long fragments due to a trinucleotide insertion andtwo point mutations Sequence analysis of the ITS2 forall striped venus clams revealed 34 polymorphic sites andexcluding gaps 13 differentially fixed mutations betweenthe two taxa The nucleotide diversity was 00202 (Table 1)and the genetic distance between taxa was 352 (Table 2)While the Atlantic C gallina sequences obtained in thiswork were identical to those from Tyrrhenian Sea specimens(GenBank acc numbers HE965773 and HE965774) the twoMediterranean populations ITS2 sequences coincided withthose from Adriatic Sea samples (GenBank acc numbersHE965771 and HE965772) No C striatula ITS2 sequenceswere found in GenBank The pairwise genetic distancesbetween populations inferred from nuclear ITS2 sequences(Table 2) also depicted that C striatula is clearly separatedfrom the three C gallina populations and the ML tree alsorecovered two clades with bootstrap values of 100 and 84(Figure 3)

In addition the predicted folding shapes of the C gallinaITS2 rRNAs (Supplementary Figure 1) were identical to thosepreviously published for the same species [19] those for Cstriatula were concordant with the structure proposed forVeneridae [19] Most point mutations detected (17 of a totalof 20) including those differentiating taxa and populationswere clustered on theDIV-DVI stems the less conserved areain terms of primary sequence [19]

4 Discussion and Conclusion

The taxonomic status of the striped venus clams C gallinaand C striatula has been a matter of debate for decades[1] Although the World Register of Marine Species haverecognized them as separated species since 2004 [38 39] thedistribution ranges of these taxa displayed in WoRMs fromthe North Atlantic to the EasternMediterranean still overlapin almost their entirety Moreover even as recently as lastyear COI gene sequences obtained from North Sea samplestherefore C striatula were stored in GenBank under thespecific name C gallina [37] In order to resolve this issue weapplied a molecular cytogenetic approach from the perspec-tive of chromosomal distribution of three gene families [7 12ndash14] alongside comparing the sequences ofmitochondrial andnuclear DNA markers that are increasingly being utilized inphylogenetic studies [5 17ndash19 40 41] to further insight andstrengthen the evidence

The diploid chromosome number of 2119899 = 38 obtainedin this work is in accordance with those previously describedfor C gallina [7 16] and all other Veneridae studied to date[6ndash8 12ndash14] The karyotype compositions obtained were alsofundamentally coincidental with that proposed for C gallina[16]

Discordant with their conserved chromosome numbersthe species of Veneridae present clear differences in thenumber and distribution of rDNA and histone gene clusterson their chromosomes [7 12ndash14] These differences arealso present in closely related congeneric species and areusually accompanied with an almost complete absence ofintraspecific variability [7 12ndash14]

Our results demonstrated that this is also the case for thetwo striped venus clam taxa studied The consistency in the5S rDNA signal pattern and the mapping differences for both28S rDNA and H3 histone gene signals found between Cgallina and C striatula are of a similar magnitude to thosereported forVenus casinaV verrucosa andDosinia exoletaDlupinus [7] Conversely although we found some mappingdifferences among C gallina populations and among Cstriatula specimens these differences were comparativelynarrower than those between taxa and similar to those foundin other bivalve species [23 36] thusly constituting thestandard intraspecific variation

The sequence data obtained also indicated that these twotaxa are separated species All individual ML trees recoveredby MEGA on the COI gene 16S rRNA gene and ITS2sequences (using Dosinia exoleta as outgroup) revealed twoclearly separated well supported clades one formed by thespecimens morphologically identified as C striatula and theother by the specimens identified as C gallina Furthermorethe genetic distances between the C striatula population andany of the C gallina populations were indubitably higherthan those between any two C gallina populations for bothmitochondrial and nuclear sequences The magnitudes ofthese genetic distances were fully concordant with thosepreviously reported for congeneric species of Veneridae bothfor the mitochondrial COI gene [5] and the nuclear ITS2 [19]sequences analyzed

In conclusion the results obtained in this study afteremploying twomitochondrial and one nuclear DNAmarkerstogether with three chromosomal markers in four geograph-ically distant populations of striped venus clams clearlydemonstrate that C gallina and C striatula are well differen-tiated species

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors wish to thank M Lastra and M Rodrıguezfor kindly providing some of the striped venus clams Thiswork was partly funded by grants from Xunta de Gali-cia and Fondos FEDER ldquoUnha maneira de facer Europardquo(08MMA023310PR Axudas do programa de consolidacione estruturacion de unidades de investigacions competitivasdo SUG ED431C 2016-037) D Garcıa-Souto was partiallysupported by a FPU fellowship fromMinisterio de Educacion(Spain)

BioMed Research International 7

References

[1] P Bouchet ldquoThe magnitude of marine diversityrdquo in TheExploration of Marine Diversity Scientific and TechnologicalChallenges C Duarte Ed pp 31ndash64 Fundacion BBVA BilbaoSpain 2006

[2] T Backeljau and S Gofas ldquoGenetic variation systematics anddistribution of the venerid clam Chamelea gaeeinardquo Journal oftheMarine Biological Association of the United Kingdom vol 74no 1 pp 211ndash223 1994

[3] M M Rufino M B Gaspar A M Pereira and P VasconcelosldquoUse of shape to distinguish Chamelea gallina and Chameleastriatula (Bivalvia Veneridae) linear and geometric morpho-metric methodsrdquo Journal of Morphology vol 267 no 12 pp1433ndash1440 2006

[4] M Palmer G X Pons and M Linde ldquoDiscriminating betweengeographical groups of a Mediterranean commercial clam(Chamelea gallina (L) Veneridae) by shape analysisrdquo FisheriesResearch vol 67 no 1 pp 93ndash98 2004

[5] J Chen Q Li L Kong and H Yu ldquoHow DNA barcodescomplement taxonomy and explore species diversity the casestudy of a poorly understood marine faunardquo PLoS ONE vol 6no 6 Article ID e21326 2011

[6] C Thiriot-Quievreux ldquoAdvances in cytogenetics of aquaticorganismsrdquo in Genetics and Evolution of Aquatic Organisms AR Beaumont Ed pp 369ndash388 Chapman and Hall LondonUK 1994

[7] D Garcıa-Souto C Perez-Garcıa P Moran and J J PasantesldquoDivergent evolutionary behavior ofH3 histone gene and rDNAclusters in venerid clamsrdquo Molecular Cytogenetics vol 8 no 1article 40 2015

[8] A Leitao and R Chaves ldquoBanding for chromosomal identi-fication in bivalves a 20-year historyrdquo Dynamic BiochemistryProcess Biotechnology and Molecular Biology vol 2 no 1 pp44ndash49 2008

[9] D Garcıa-Souto C Perez-Garcıa J Kendall and J J PasantesldquoMolecular cytogenetics in trough shells (Mactridae Bivalvia)divergent GC-rich heterochromatin contentrdquo Genes vol 7 no8 article 47 2016

[10] N S Hurtado and J J Pasantes ldquoSurface spreading of synap-tonemal complexes in the clam Dosinia exoleta (MolluscaBivalvia)rdquo Chromosome Research vol 13 no 6 pp 575ndash5802005

[11] Y Wang and X Guo ldquoChromosomal mapping of major ribo-somal rRNA genes in the hard clam (Mercenaria mercenaria)using fluorescence in situ hybridizationrdquo Marine Biology vol150 no 6 pp 1183ndash1189 2007

[12] J Carrilho C Perez-Garcıa A Leitao I Malheiro and J J Pas-antes ldquoCytogenetic characterization and mapping of rDNAscore histone genes and telomeric sequences in Venerupis aureaand Tapes rhomboides (Bivalvia Veneridae)rdquo Genetica vol 139no 6 pp 823ndash831 2011

[13] N S Hurtado C Perez-Garcıa P Moran and J J PasantesldquoGenetic and cytological evidence of hybridization betweennative Ruditapes decussatus and introduced Ruditapes philip-pinarum (Mollusca Bivalvia Veneridae) in NW Spainrdquo Aqua-culture vol 311 no 1ndash4 pp 123ndash128 2011

[14] C Perez-Garcıa N S Hurtado P Moran and J J PasantesldquoEvolutionary dynamics of rDNA clusters in chromosomes offive clam species belonging to the family Veneridae (MolluscaBivalvia)rdquo BioMed Research International vol 2014 Article ID754012 9 pages 2014

[15] C Perez-Garcıa P Moran and J J Pasantes ldquoKaryotypic diver-sification inMytilus mussels (Bivalvia Mytilidae) inferred fromchromosomal mapping of rRNA and histone gene clustersrdquoBMC Genetics vol 15 article 84 2014

[16] M G Corni andM Trentini ldquoA chromosomic study of Chame-lea gallina (L) (Bivalvia Venerihdae)rdquo Bolletino di zoologia vol53 no 1 pp 23ndash24 1986

[17] J Chen Q Li L Kong and X Zheng ldquoMolecular phylogenyof venus clams (Mollusca Bivalvia Veneridae) with emphasison the systematic position of taxa along the coast of mainlandChinardquo Zoologica Scripta vol 40 no 3 pp 260ndash271 2011

[18] A Canapa S Schiaparelli I Marota andM Barucca ldquoMolecu-lar data from the 16S rRNA gene for the phylogeny of Veneridae(Mollusca Bivalvia)rdquo Marine Biology vol 142 no 6 pp 1125ndash1130 2003

[19] D Salvi and P Mariottini ldquoMolecular phylogenetics in 2DITS2 rRNA evolution and sequence-structure barcode fromVeneridae to Bivalviardquo Molecular Phylogenetics and Evolutionvol 65 no 2 pp 792ndash798 2012

[20] J Pasantes M J Martinez-exposito A Martinez-lage andJ Mendez ldquoChromosomes of galician musselsrdquo Journal ofMolluscan Studies vol 56 no 1 pp 123ndash126 1990

[21] M J Martınez-Exposito J J Pasantes and J Mendez ldquoProlif-eration kinetics of mussel (Mytilus galloprovincialis) gill cellsrdquoMarine Biology vol 120 no 1 pp 41ndash45 1994

[22] C Perez-Garcıa J M Cambeiro P Moran and J J PasantesldquoChromosomal mapping of rDNAs core histone genes andtelomeric sequences in Perumytilus purpuratus (BivalviaMytil-idae)rdquo Journal of Experimental Marine Biology and Ecology vol395 no 1-2 pp 199ndash205 2010

[23] C Perez-Garcıa J Guerra-Varela P Moran and J J PasantesldquoChromosomalmapping of rRNAgenes core histone genes andtelomeric sequences in Brachidontes puniceus and Brachidontesrodriguezi (Bivalvia Mytilidae)rdquo BMC Genetics vol 11 article109 2010

[24] B Winnepenninckx T Backeljau and R de Wachter ldquoExtrac-tion of high molecular weight DNA from molluscsrdquo Trends inGenetics vol 9 no 12 p 407 1993

[25] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[26] C D Schubart J E Conde C Carmona-Suarez R Roblesand D L Felder ldquoLack of divergence between 16S mtDNAsequences of the swimming crabs Callinectes bocourti andC maracaiboensis (Brachyura Portunidae) from VenezuelardquoFishery Bulletin vol 99 no 3 pp 475ndash481 2001

[27] S R Palumbi ldquoNucleic acids IIThe polymerase chain reactionrdquoin Molecular Systematics D M Hillis C Moritz and B KMable Eds pp 205ndash247 Sinauer Associates SunderlandMassUSA 2nd edition 1996

[28] T J White T Burms S Lee and J W Taylor ldquoAmplificationand direct sequences of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Inmus D H Guelfand J J Sminsky andT J White Eds pp 315ndash322 Academic Press New York NYUSA 1990

[29] R Vilgalys Duke University Durham NC USA httpwwwbiologydukeedufungimycolabprimershtm

[30] G Giribet and D Distel ldquoBivalve phylogeny and molec-ular datardquo in Systematics and Phylogeography of Molluscks

BioMed Research International 5

COI

005

CST Pon 05

CST Pon 08CST Pon 02CST Pon 01

CST Pon 135660

ITS2

CGA Cad 04CGA Cad 01CGA Cad 02CGA Cad 05CGA Cad 03

CGA Val 05CGA Val 04

86 82 CGA Val 03CGA Val 02

58 CGA Val 01CGA lta 0236 CGA lta 03 92

CST Pon 03CST Pon 02CST Pon 01

CST Pon 06

CST Pon 04CST Pon 14

100CST Pon 07

CST Pon 15CST Pon 05CST Pon 03CST Pon 09

CGA Cad 05CGA Cad 01CGA Cad 02 100

CGA Cad 03CGA Cad 04

CGA Val 05

84CGA Val 03CGA Val 02CGA Val 01CGA lta 05 98CGA lta 04

CGA lta 0150 CGA lta 04

CGA lta 05

CGA lta 03CGA lta 02CGA lta 01

Dosinia exoleta DQ458478 Dosinia exoleta HE965776

002

CST Pon

CGA Cad

CGA Val

CGA Ita

CST Pon 10

CGA Val 04

CST Pon 12

100

56 CST Pon 04

CST Pon 1128

Figure 3 Maximum-likelihood trees based on mitochondrial COI gene and nuclear ITS2 sequences of striped venus clams using Dosiniaexoleta as outgroup together with a schematic representation of the main chromosomal differences Numbers in internal nodes indicatemaximum-likelihood bootstrap support values (500 replicates)

Table 1 Nucleotide diversity (pi) of COI gene 16S rDNA and ITS2sequences in Chamelea gallina and Chamelea striatula

COI gene 16S rDNA ITS2119899 pi 119899 pi 119899 pi

Chamelea striatulaCST Pon 5 00022 5 00000 15 00021

Chamelea gallinaCST Cad 5 00000 5 00000 5 00000CST Val 5 00000 5 00000 5 00000CST Ita 5 00057 5 00035 5 00000All CST 15 00060 15 00030 15 00063

All Chamelea 20 00556 20 00443 30 00202

morphologically identified as C striatula and the other bythe specimens identified as C gallina with bootstrap valuesof 100 and 86 respectively (Figure 3)

The sequenced 16S rRNA gene fragments were 468 bplong in the specimens collected from Pontevedra (C striat-ula) and 466 bp long in all C gallina specimens (GenBankacc numbers KY547767 to KY547786) The difference inlength was due to six between-taxa gaps two single nucle-otide and one dinucleotide insertions in C striatula and twosingle nucleotide insertions in C gallina There were 51 poly-morphic sites 48 of them taxa specific providing a geneticdistance between taxa of 1144The nucleotide diversity was00443 (Table 1) These 16S rRNA gene sequences were also

Table 2 Pairwise p-distances between striped venus clam popu-lations using mitochondrial COI and 16S rRNA genes and nuclearITS2 sequences Interspecific distances in bold

Populations COI gene 16S rDNA ITS2CST Pon CGA Cad 01304 01121 00396CST Pon CGA Val 01320 01164 00330CST Pon CGA Ita 01335 01147 00330CGA Cad CGA Val 00095 00043 00132CGA Cad CGA Ita 00086 00047 00132CGA Val CGA Ita 00048 00022 00000CST CGA 01320 01144 00352

compared with the six sequences stored in GenBank The Cstriatula sequences were identical to sequences from England(GenBank acc numbers DQ280041 and KX713203) whereasthose from C gallina matched the sequences of specimensfrom Turkey (GenBank acc number AM085110) AdriaticSea (GenBank acc number AJ548762) NW Spain (GenBankacc number JF808193) and a market in the Canary Islands(GenBank acc number DQ184735) Again both the pairwisedistances (Table 2) and the ML tree recovered (not shown)differentiated specimens belonging to the two taxa (bootstrapvalues of 100 and 72)

The amplified ITS2 fragments (GenBank acc numbersKY508254 to KY508283) were the most inconsistent in

6 BioMed Research International

length While all specimens of the Mediterranean popu-lations of C gallina displayed 498 bp long fragments inthe Atlantic specimens the ITS2 was 496 bp long thesedifferences in length were a result of a dinucleotide insertion(or deletion) and six nucleotide substitutions In contrastfive specimens of theC striatula population displayed 498 bplong ITS2 fragments whereas the remaining 10 exhibited495 bp long fragments due to a trinucleotide insertion andtwo point mutations Sequence analysis of the ITS2 forall striped venus clams revealed 34 polymorphic sites andexcluding gaps 13 differentially fixed mutations betweenthe two taxa The nucleotide diversity was 00202 (Table 1)and the genetic distance between taxa was 352 (Table 2)While the Atlantic C gallina sequences obtained in thiswork were identical to those from Tyrrhenian Sea specimens(GenBank acc numbers HE965773 and HE965774) the twoMediterranean populations ITS2 sequences coincided withthose from Adriatic Sea samples (GenBank acc numbersHE965771 and HE965772) No C striatula ITS2 sequenceswere found in GenBank The pairwise genetic distancesbetween populations inferred from nuclear ITS2 sequences(Table 2) also depicted that C striatula is clearly separatedfrom the three C gallina populations and the ML tree alsorecovered two clades with bootstrap values of 100 and 84(Figure 3)

In addition the predicted folding shapes of the C gallinaITS2 rRNAs (Supplementary Figure 1) were identical to thosepreviously published for the same species [19] those for Cstriatula were concordant with the structure proposed forVeneridae [19] Most point mutations detected (17 of a totalof 20) including those differentiating taxa and populationswere clustered on theDIV-DVI stems the less conserved areain terms of primary sequence [19]

4 Discussion and Conclusion

The taxonomic status of the striped venus clams C gallinaand C striatula has been a matter of debate for decades[1] Although the World Register of Marine Species haverecognized them as separated species since 2004 [38 39] thedistribution ranges of these taxa displayed in WoRMs fromthe North Atlantic to the EasternMediterranean still overlapin almost their entirety Moreover even as recently as lastyear COI gene sequences obtained from North Sea samplestherefore C striatula were stored in GenBank under thespecific name C gallina [37] In order to resolve this issue weapplied a molecular cytogenetic approach from the perspec-tive of chromosomal distribution of three gene families [7 12ndash14] alongside comparing the sequences ofmitochondrial andnuclear DNA markers that are increasingly being utilized inphylogenetic studies [5 17ndash19 40 41] to further insight andstrengthen the evidence

The diploid chromosome number of 2119899 = 38 obtainedin this work is in accordance with those previously describedfor C gallina [7 16] and all other Veneridae studied to date[6ndash8 12ndash14] The karyotype compositions obtained were alsofundamentally coincidental with that proposed for C gallina[16]

Discordant with their conserved chromosome numbersthe species of Veneridae present clear differences in thenumber and distribution of rDNA and histone gene clusterson their chromosomes [7 12ndash14] These differences arealso present in closely related congeneric species and areusually accompanied with an almost complete absence ofintraspecific variability [7 12ndash14]

Our results demonstrated that this is also the case for thetwo striped venus clam taxa studied The consistency in the5S rDNA signal pattern and the mapping differences for both28S rDNA and H3 histone gene signals found between Cgallina and C striatula are of a similar magnitude to thosereported forVenus casinaV verrucosa andDosinia exoletaDlupinus [7] Conversely although we found some mappingdifferences among C gallina populations and among Cstriatula specimens these differences were comparativelynarrower than those between taxa and similar to those foundin other bivalve species [23 36] thusly constituting thestandard intraspecific variation

The sequence data obtained also indicated that these twotaxa are separated species All individual ML trees recoveredby MEGA on the COI gene 16S rRNA gene and ITS2sequences (using Dosinia exoleta as outgroup) revealed twoclearly separated well supported clades one formed by thespecimens morphologically identified as C striatula and theother by the specimens identified as C gallina Furthermorethe genetic distances between the C striatula population andany of the C gallina populations were indubitably higherthan those between any two C gallina populations for bothmitochondrial and nuclear sequences The magnitudes ofthese genetic distances were fully concordant with thosepreviously reported for congeneric species of Veneridae bothfor the mitochondrial COI gene [5] and the nuclear ITS2 [19]sequences analyzed

In conclusion the results obtained in this study afteremploying twomitochondrial and one nuclear DNAmarkerstogether with three chromosomal markers in four geograph-ically distant populations of striped venus clams clearlydemonstrate that C gallina and C striatula are well differen-tiated species

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors wish to thank M Lastra and M Rodrıguezfor kindly providing some of the striped venus clams Thiswork was partly funded by grants from Xunta de Gali-cia and Fondos FEDER ldquoUnha maneira de facer Europardquo(08MMA023310PR Axudas do programa de consolidacione estruturacion de unidades de investigacions competitivasdo SUG ED431C 2016-037) D Garcıa-Souto was partiallysupported by a FPU fellowship fromMinisterio de Educacion(Spain)

BioMed Research International 7

References

[1] P Bouchet ldquoThe magnitude of marine diversityrdquo in TheExploration of Marine Diversity Scientific and TechnologicalChallenges C Duarte Ed pp 31ndash64 Fundacion BBVA BilbaoSpain 2006

[2] T Backeljau and S Gofas ldquoGenetic variation systematics anddistribution of the venerid clam Chamelea gaeeinardquo Journal oftheMarine Biological Association of the United Kingdom vol 74no 1 pp 211ndash223 1994

[3] M M Rufino M B Gaspar A M Pereira and P VasconcelosldquoUse of shape to distinguish Chamelea gallina and Chameleastriatula (Bivalvia Veneridae) linear and geometric morpho-metric methodsrdquo Journal of Morphology vol 267 no 12 pp1433ndash1440 2006

[4] M Palmer G X Pons and M Linde ldquoDiscriminating betweengeographical groups of a Mediterranean commercial clam(Chamelea gallina (L) Veneridae) by shape analysisrdquo FisheriesResearch vol 67 no 1 pp 93ndash98 2004

[5] J Chen Q Li L Kong and H Yu ldquoHow DNA barcodescomplement taxonomy and explore species diversity the casestudy of a poorly understood marine faunardquo PLoS ONE vol 6no 6 Article ID e21326 2011

[6] C Thiriot-Quievreux ldquoAdvances in cytogenetics of aquaticorganismsrdquo in Genetics and Evolution of Aquatic Organisms AR Beaumont Ed pp 369ndash388 Chapman and Hall LondonUK 1994

[7] D Garcıa-Souto C Perez-Garcıa P Moran and J J PasantesldquoDivergent evolutionary behavior ofH3 histone gene and rDNAclusters in venerid clamsrdquo Molecular Cytogenetics vol 8 no 1article 40 2015

[8] A Leitao and R Chaves ldquoBanding for chromosomal identi-fication in bivalves a 20-year historyrdquo Dynamic BiochemistryProcess Biotechnology and Molecular Biology vol 2 no 1 pp44ndash49 2008

[9] D Garcıa-Souto C Perez-Garcıa J Kendall and J J PasantesldquoMolecular cytogenetics in trough shells (Mactridae Bivalvia)divergent GC-rich heterochromatin contentrdquo Genes vol 7 no8 article 47 2016

[10] N S Hurtado and J J Pasantes ldquoSurface spreading of synap-tonemal complexes in the clam Dosinia exoleta (MolluscaBivalvia)rdquo Chromosome Research vol 13 no 6 pp 575ndash5802005

[11] Y Wang and X Guo ldquoChromosomal mapping of major ribo-somal rRNA genes in the hard clam (Mercenaria mercenaria)using fluorescence in situ hybridizationrdquo Marine Biology vol150 no 6 pp 1183ndash1189 2007

[12] J Carrilho C Perez-Garcıa A Leitao I Malheiro and J J Pas-antes ldquoCytogenetic characterization and mapping of rDNAscore histone genes and telomeric sequences in Venerupis aureaand Tapes rhomboides (Bivalvia Veneridae)rdquo Genetica vol 139no 6 pp 823ndash831 2011

[13] N S Hurtado C Perez-Garcıa P Moran and J J PasantesldquoGenetic and cytological evidence of hybridization betweennative Ruditapes decussatus and introduced Ruditapes philip-pinarum (Mollusca Bivalvia Veneridae) in NW Spainrdquo Aqua-culture vol 311 no 1ndash4 pp 123ndash128 2011

[14] C Perez-Garcıa N S Hurtado P Moran and J J PasantesldquoEvolutionary dynamics of rDNA clusters in chromosomes offive clam species belonging to the family Veneridae (MolluscaBivalvia)rdquo BioMed Research International vol 2014 Article ID754012 9 pages 2014

[15] C Perez-Garcıa P Moran and J J Pasantes ldquoKaryotypic diver-sification inMytilus mussels (Bivalvia Mytilidae) inferred fromchromosomal mapping of rRNA and histone gene clustersrdquoBMC Genetics vol 15 article 84 2014

[16] M G Corni andM Trentini ldquoA chromosomic study of Chame-lea gallina (L) (Bivalvia Venerihdae)rdquo Bolletino di zoologia vol53 no 1 pp 23ndash24 1986

[17] J Chen Q Li L Kong and X Zheng ldquoMolecular phylogenyof venus clams (Mollusca Bivalvia Veneridae) with emphasison the systematic position of taxa along the coast of mainlandChinardquo Zoologica Scripta vol 40 no 3 pp 260ndash271 2011

[18] A Canapa S Schiaparelli I Marota andM Barucca ldquoMolecu-lar data from the 16S rRNA gene for the phylogeny of Veneridae(Mollusca Bivalvia)rdquo Marine Biology vol 142 no 6 pp 1125ndash1130 2003

[19] D Salvi and P Mariottini ldquoMolecular phylogenetics in 2DITS2 rRNA evolution and sequence-structure barcode fromVeneridae to Bivalviardquo Molecular Phylogenetics and Evolutionvol 65 no 2 pp 792ndash798 2012

[20] J Pasantes M J Martinez-exposito A Martinez-lage andJ Mendez ldquoChromosomes of galician musselsrdquo Journal ofMolluscan Studies vol 56 no 1 pp 123ndash126 1990

[21] M J Martınez-Exposito J J Pasantes and J Mendez ldquoProlif-eration kinetics of mussel (Mytilus galloprovincialis) gill cellsrdquoMarine Biology vol 120 no 1 pp 41ndash45 1994

[22] C Perez-Garcıa J M Cambeiro P Moran and J J PasantesldquoChromosomal mapping of rDNAs core histone genes andtelomeric sequences in Perumytilus purpuratus (BivalviaMytil-idae)rdquo Journal of Experimental Marine Biology and Ecology vol395 no 1-2 pp 199ndash205 2010

[23] C Perez-Garcıa J Guerra-Varela P Moran and J J PasantesldquoChromosomalmapping of rRNAgenes core histone genes andtelomeric sequences in Brachidontes puniceus and Brachidontesrodriguezi (Bivalvia Mytilidae)rdquo BMC Genetics vol 11 article109 2010

[24] B Winnepenninckx T Backeljau and R de Wachter ldquoExtrac-tion of high molecular weight DNA from molluscsrdquo Trends inGenetics vol 9 no 12 p 407 1993

[25] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[26] C D Schubart J E Conde C Carmona-Suarez R Roblesand D L Felder ldquoLack of divergence between 16S mtDNAsequences of the swimming crabs Callinectes bocourti andC maracaiboensis (Brachyura Portunidae) from VenezuelardquoFishery Bulletin vol 99 no 3 pp 475ndash481 2001

[27] S R Palumbi ldquoNucleic acids IIThe polymerase chain reactionrdquoin Molecular Systematics D M Hillis C Moritz and B KMable Eds pp 205ndash247 Sinauer Associates SunderlandMassUSA 2nd edition 1996

[28] T J White T Burms S Lee and J W Taylor ldquoAmplificationand direct sequences of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Inmus D H Guelfand J J Sminsky andT J White Eds pp 315ndash322 Academic Press New York NYUSA 1990

[29] R Vilgalys Duke University Durham NC USA httpwwwbiologydukeedufungimycolabprimershtm

[30] G Giribet and D Distel ldquoBivalve phylogeny and molec-ular datardquo in Systematics and Phylogeography of Molluscks

6 BioMed Research International

length While all specimens of the Mediterranean popu-lations of C gallina displayed 498 bp long fragments inthe Atlantic specimens the ITS2 was 496 bp long thesedifferences in length were a result of a dinucleotide insertion(or deletion) and six nucleotide substitutions In contrastfive specimens of theC striatula population displayed 498 bplong ITS2 fragments whereas the remaining 10 exhibited495 bp long fragments due to a trinucleotide insertion andtwo point mutations Sequence analysis of the ITS2 forall striped venus clams revealed 34 polymorphic sites andexcluding gaps 13 differentially fixed mutations betweenthe two taxa The nucleotide diversity was 00202 (Table 1)and the genetic distance between taxa was 352 (Table 2)While the Atlantic C gallina sequences obtained in thiswork were identical to those from Tyrrhenian Sea specimens(GenBank acc numbers HE965773 and HE965774) the twoMediterranean populations ITS2 sequences coincided withthose from Adriatic Sea samples (GenBank acc numbersHE965771 and HE965772) No C striatula ITS2 sequenceswere found in GenBank The pairwise genetic distancesbetween populations inferred from nuclear ITS2 sequences(Table 2) also depicted that C striatula is clearly separatedfrom the three C gallina populations and the ML tree alsorecovered two clades with bootstrap values of 100 and 84(Figure 3)

In addition the predicted folding shapes of the C gallinaITS2 rRNAs (Supplementary Figure 1) were identical to thosepreviously published for the same species [19] those for Cstriatula were concordant with the structure proposed forVeneridae [19] Most point mutations detected (17 of a totalof 20) including those differentiating taxa and populationswere clustered on theDIV-DVI stems the less conserved areain terms of primary sequence [19]

4 Discussion and Conclusion

The taxonomic status of the striped venus clams C gallinaand C striatula has been a matter of debate for decades[1] Although the World Register of Marine Species haverecognized them as separated species since 2004 [38 39] thedistribution ranges of these taxa displayed in WoRMs fromthe North Atlantic to the EasternMediterranean still overlapin almost their entirety Moreover even as recently as lastyear COI gene sequences obtained from North Sea samplestherefore C striatula were stored in GenBank under thespecific name C gallina [37] In order to resolve this issue weapplied a molecular cytogenetic approach from the perspec-tive of chromosomal distribution of three gene families [7 12ndash14] alongside comparing the sequences ofmitochondrial andnuclear DNA markers that are increasingly being utilized inphylogenetic studies [5 17ndash19 40 41] to further insight andstrengthen the evidence

The diploid chromosome number of 2119899 = 38 obtainedin this work is in accordance with those previously describedfor C gallina [7 16] and all other Veneridae studied to date[6ndash8 12ndash14] The karyotype compositions obtained were alsofundamentally coincidental with that proposed for C gallina[16]

Discordant with their conserved chromosome numbersthe species of Veneridae present clear differences in thenumber and distribution of rDNA and histone gene clusterson their chromosomes [7 12ndash14] These differences arealso present in closely related congeneric species and areusually accompanied with an almost complete absence ofintraspecific variability [7 12ndash14]

Our results demonstrated that this is also the case for thetwo striped venus clam taxa studied The consistency in the5S rDNA signal pattern and the mapping differences for both28S rDNA and H3 histone gene signals found between Cgallina and C striatula are of a similar magnitude to thosereported forVenus casinaV verrucosa andDosinia exoletaDlupinus [7] Conversely although we found some mappingdifferences among C gallina populations and among Cstriatula specimens these differences were comparativelynarrower than those between taxa and similar to those foundin other bivalve species [23 36] thusly constituting thestandard intraspecific variation

The sequence data obtained also indicated that these twotaxa are separated species All individual ML trees recoveredby MEGA on the COI gene 16S rRNA gene and ITS2sequences (using Dosinia exoleta as outgroup) revealed twoclearly separated well supported clades one formed by thespecimens morphologically identified as C striatula and theother by the specimens identified as C gallina Furthermorethe genetic distances between the C striatula population andany of the C gallina populations were indubitably higherthan those between any two C gallina populations for bothmitochondrial and nuclear sequences The magnitudes ofthese genetic distances were fully concordant with thosepreviously reported for congeneric species of Veneridae bothfor the mitochondrial COI gene [5] and the nuclear ITS2 [19]sequences analyzed

In conclusion the results obtained in this study afteremploying twomitochondrial and one nuclear DNAmarkerstogether with three chromosomal markers in four geograph-ically distant populations of striped venus clams clearlydemonstrate that C gallina and C striatula are well differen-tiated species

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

The authors wish to thank M Lastra and M Rodrıguezfor kindly providing some of the striped venus clams Thiswork was partly funded by grants from Xunta de Gali-cia and Fondos FEDER ldquoUnha maneira de facer Europardquo(08MMA023310PR Axudas do programa de consolidacione estruturacion de unidades de investigacions competitivasdo SUG ED431C 2016-037) D Garcıa-Souto was partiallysupported by a FPU fellowship fromMinisterio de Educacion(Spain)

BioMed Research International 7

References

[1] P Bouchet ldquoThe magnitude of marine diversityrdquo in TheExploration of Marine Diversity Scientific and TechnologicalChallenges C Duarte Ed pp 31ndash64 Fundacion BBVA BilbaoSpain 2006

[2] T Backeljau and S Gofas ldquoGenetic variation systematics anddistribution of the venerid clam Chamelea gaeeinardquo Journal oftheMarine Biological Association of the United Kingdom vol 74no 1 pp 211ndash223 1994

[3] M M Rufino M B Gaspar A M Pereira and P VasconcelosldquoUse of shape to distinguish Chamelea gallina and Chameleastriatula (Bivalvia Veneridae) linear and geometric morpho-metric methodsrdquo Journal of Morphology vol 267 no 12 pp1433ndash1440 2006

[4] M Palmer G X Pons and M Linde ldquoDiscriminating betweengeographical groups of a Mediterranean commercial clam(Chamelea gallina (L) Veneridae) by shape analysisrdquo FisheriesResearch vol 67 no 1 pp 93ndash98 2004

[5] J Chen Q Li L Kong and H Yu ldquoHow DNA barcodescomplement taxonomy and explore species diversity the casestudy of a poorly understood marine faunardquo PLoS ONE vol 6no 6 Article ID e21326 2011

[6] C Thiriot-Quievreux ldquoAdvances in cytogenetics of aquaticorganismsrdquo in Genetics and Evolution of Aquatic Organisms AR Beaumont Ed pp 369ndash388 Chapman and Hall LondonUK 1994

[7] D Garcıa-Souto C Perez-Garcıa P Moran and J J PasantesldquoDivergent evolutionary behavior ofH3 histone gene and rDNAclusters in venerid clamsrdquo Molecular Cytogenetics vol 8 no 1article 40 2015

[8] A Leitao and R Chaves ldquoBanding for chromosomal identi-fication in bivalves a 20-year historyrdquo Dynamic BiochemistryProcess Biotechnology and Molecular Biology vol 2 no 1 pp44ndash49 2008

[9] D Garcıa-Souto C Perez-Garcıa J Kendall and J J PasantesldquoMolecular cytogenetics in trough shells (Mactridae Bivalvia)divergent GC-rich heterochromatin contentrdquo Genes vol 7 no8 article 47 2016

[10] N S Hurtado and J J Pasantes ldquoSurface spreading of synap-tonemal complexes in the clam Dosinia exoleta (MolluscaBivalvia)rdquo Chromosome Research vol 13 no 6 pp 575ndash5802005

[11] Y Wang and X Guo ldquoChromosomal mapping of major ribo-somal rRNA genes in the hard clam (Mercenaria mercenaria)using fluorescence in situ hybridizationrdquo Marine Biology vol150 no 6 pp 1183ndash1189 2007

[12] J Carrilho C Perez-Garcıa A Leitao I Malheiro and J J Pas-antes ldquoCytogenetic characterization and mapping of rDNAscore histone genes and telomeric sequences in Venerupis aureaand Tapes rhomboides (Bivalvia Veneridae)rdquo Genetica vol 139no 6 pp 823ndash831 2011

[13] N S Hurtado C Perez-Garcıa P Moran and J J PasantesldquoGenetic and cytological evidence of hybridization betweennative Ruditapes decussatus and introduced Ruditapes philip-pinarum (Mollusca Bivalvia Veneridae) in NW Spainrdquo Aqua-culture vol 311 no 1ndash4 pp 123ndash128 2011

[14] C Perez-Garcıa N S Hurtado P Moran and J J PasantesldquoEvolutionary dynamics of rDNA clusters in chromosomes offive clam species belonging to the family Veneridae (MolluscaBivalvia)rdquo BioMed Research International vol 2014 Article ID754012 9 pages 2014

[15] C Perez-Garcıa P Moran and J J Pasantes ldquoKaryotypic diver-sification inMytilus mussels (Bivalvia Mytilidae) inferred fromchromosomal mapping of rRNA and histone gene clustersrdquoBMC Genetics vol 15 article 84 2014

[16] M G Corni andM Trentini ldquoA chromosomic study of Chame-lea gallina (L) (Bivalvia Venerihdae)rdquo Bolletino di zoologia vol53 no 1 pp 23ndash24 1986

[17] J Chen Q Li L Kong and X Zheng ldquoMolecular phylogenyof venus clams (Mollusca Bivalvia Veneridae) with emphasison the systematic position of taxa along the coast of mainlandChinardquo Zoologica Scripta vol 40 no 3 pp 260ndash271 2011

[18] A Canapa S Schiaparelli I Marota andM Barucca ldquoMolecu-lar data from the 16S rRNA gene for the phylogeny of Veneridae(Mollusca Bivalvia)rdquo Marine Biology vol 142 no 6 pp 1125ndash1130 2003

[19] D Salvi and P Mariottini ldquoMolecular phylogenetics in 2DITS2 rRNA evolution and sequence-structure barcode fromVeneridae to Bivalviardquo Molecular Phylogenetics and Evolutionvol 65 no 2 pp 792ndash798 2012

[20] J Pasantes M J Martinez-exposito A Martinez-lage andJ Mendez ldquoChromosomes of galician musselsrdquo Journal ofMolluscan Studies vol 56 no 1 pp 123ndash126 1990

[21] M J Martınez-Exposito J J Pasantes and J Mendez ldquoProlif-eration kinetics of mussel (Mytilus galloprovincialis) gill cellsrdquoMarine Biology vol 120 no 1 pp 41ndash45 1994

[22] C Perez-Garcıa J M Cambeiro P Moran and J J PasantesldquoChromosomal mapping of rDNAs core histone genes andtelomeric sequences in Perumytilus purpuratus (BivalviaMytil-idae)rdquo Journal of Experimental Marine Biology and Ecology vol395 no 1-2 pp 199ndash205 2010

[23] C Perez-Garcıa J Guerra-Varela P Moran and J J PasantesldquoChromosomalmapping of rRNAgenes core histone genes andtelomeric sequences in Brachidontes puniceus and Brachidontesrodriguezi (Bivalvia Mytilidae)rdquo BMC Genetics vol 11 article109 2010

[24] B Winnepenninckx T Backeljau and R de Wachter ldquoExtrac-tion of high molecular weight DNA from molluscsrdquo Trends inGenetics vol 9 no 12 p 407 1993

[25] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[26] C D Schubart J E Conde C Carmona-Suarez R Roblesand D L Felder ldquoLack of divergence between 16S mtDNAsequences of the swimming crabs Callinectes bocourti andC maracaiboensis (Brachyura Portunidae) from VenezuelardquoFishery Bulletin vol 99 no 3 pp 475ndash481 2001

[27] S R Palumbi ldquoNucleic acids IIThe polymerase chain reactionrdquoin Molecular Systematics D M Hillis C Moritz and B KMable Eds pp 205ndash247 Sinauer Associates SunderlandMassUSA 2nd edition 1996

[28] T J White T Burms S Lee and J W Taylor ldquoAmplificationand direct sequences of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Inmus D H Guelfand J J Sminsky andT J White Eds pp 315ndash322 Academic Press New York NYUSA 1990

[29] R Vilgalys Duke University Durham NC USA httpwwwbiologydukeedufungimycolabprimershtm

[30] G Giribet and D Distel ldquoBivalve phylogeny and molec-ular datardquo in Systematics and Phylogeography of Molluscks

BioMed Research International 7

References

[1] P Bouchet ldquoThe magnitude of marine diversityrdquo in TheExploration of Marine Diversity Scientific and TechnologicalChallenges C Duarte Ed pp 31ndash64 Fundacion BBVA BilbaoSpain 2006

[2] T Backeljau and S Gofas ldquoGenetic variation systematics anddistribution of the venerid clam Chamelea gaeeinardquo Journal oftheMarine Biological Association of the United Kingdom vol 74no 1 pp 211ndash223 1994

[3] M M Rufino M B Gaspar A M Pereira and P VasconcelosldquoUse of shape to distinguish Chamelea gallina and Chameleastriatula (Bivalvia Veneridae) linear and geometric morpho-metric methodsrdquo Journal of Morphology vol 267 no 12 pp1433ndash1440 2006

[4] M Palmer G X Pons and M Linde ldquoDiscriminating betweengeographical groups of a Mediterranean commercial clam(Chamelea gallina (L) Veneridae) by shape analysisrdquo FisheriesResearch vol 67 no 1 pp 93ndash98 2004

[5] J Chen Q Li L Kong and H Yu ldquoHow DNA barcodescomplement taxonomy and explore species diversity the casestudy of a poorly understood marine faunardquo PLoS ONE vol 6no 6 Article ID e21326 2011

[6] C Thiriot-Quievreux ldquoAdvances in cytogenetics of aquaticorganismsrdquo in Genetics and Evolution of Aquatic Organisms AR Beaumont Ed pp 369ndash388 Chapman and Hall LondonUK 1994

[7] D Garcıa-Souto C Perez-Garcıa P Moran and J J PasantesldquoDivergent evolutionary behavior ofH3 histone gene and rDNAclusters in venerid clamsrdquo Molecular Cytogenetics vol 8 no 1article 40 2015

[8] A Leitao and R Chaves ldquoBanding for chromosomal identi-fication in bivalves a 20-year historyrdquo Dynamic BiochemistryProcess Biotechnology and Molecular Biology vol 2 no 1 pp44ndash49 2008

[9] D Garcıa-Souto C Perez-Garcıa J Kendall and J J PasantesldquoMolecular cytogenetics in trough shells (Mactridae Bivalvia)divergent GC-rich heterochromatin contentrdquo Genes vol 7 no8 article 47 2016

[10] N S Hurtado and J J Pasantes ldquoSurface spreading of synap-tonemal complexes in the clam Dosinia exoleta (MolluscaBivalvia)rdquo Chromosome Research vol 13 no 6 pp 575ndash5802005

[11] Y Wang and X Guo ldquoChromosomal mapping of major ribo-somal rRNA genes in the hard clam (Mercenaria mercenaria)using fluorescence in situ hybridizationrdquo Marine Biology vol150 no 6 pp 1183ndash1189 2007

[12] J Carrilho C Perez-Garcıa A Leitao I Malheiro and J J Pas-antes ldquoCytogenetic characterization and mapping of rDNAscore histone genes and telomeric sequences in Venerupis aureaand Tapes rhomboides (Bivalvia Veneridae)rdquo Genetica vol 139no 6 pp 823ndash831 2011

[13] N S Hurtado C Perez-Garcıa P Moran and J J PasantesldquoGenetic and cytological evidence of hybridization betweennative Ruditapes decussatus and introduced Ruditapes philip-pinarum (Mollusca Bivalvia Veneridae) in NW Spainrdquo Aqua-culture vol 311 no 1ndash4 pp 123ndash128 2011

[14] C Perez-Garcıa N S Hurtado P Moran and J J PasantesldquoEvolutionary dynamics of rDNA clusters in chromosomes offive clam species belonging to the family Veneridae (MolluscaBivalvia)rdquo BioMed Research International vol 2014 Article ID754012 9 pages 2014

[15] C Perez-Garcıa P Moran and J J Pasantes ldquoKaryotypic diver-sification inMytilus mussels (Bivalvia Mytilidae) inferred fromchromosomal mapping of rRNA and histone gene clustersrdquoBMC Genetics vol 15 article 84 2014

[16] M G Corni andM Trentini ldquoA chromosomic study of Chame-lea gallina (L) (Bivalvia Venerihdae)rdquo Bolletino di zoologia vol53 no 1 pp 23ndash24 1986

[17] J Chen Q Li L Kong and X Zheng ldquoMolecular phylogenyof venus clams (Mollusca Bivalvia Veneridae) with emphasison the systematic position of taxa along the coast of mainlandChinardquo Zoologica Scripta vol 40 no 3 pp 260ndash271 2011

[18] A Canapa S Schiaparelli I Marota andM Barucca ldquoMolecu-lar data from the 16S rRNA gene for the phylogeny of Veneridae(Mollusca Bivalvia)rdquo Marine Biology vol 142 no 6 pp 1125ndash1130 2003

[19] D Salvi and P Mariottini ldquoMolecular phylogenetics in 2DITS2 rRNA evolution and sequence-structure barcode fromVeneridae to Bivalviardquo Molecular Phylogenetics and Evolutionvol 65 no 2 pp 792ndash798 2012

[20] J Pasantes M J Martinez-exposito A Martinez-lage andJ Mendez ldquoChromosomes of galician musselsrdquo Journal ofMolluscan Studies vol 56 no 1 pp 123ndash126 1990

[21] M J Martınez-Exposito J J Pasantes and J Mendez ldquoProlif-eration kinetics of mussel (Mytilus galloprovincialis) gill cellsrdquoMarine Biology vol 120 no 1 pp 41ndash45 1994

[22] C Perez-Garcıa J M Cambeiro P Moran and J J PasantesldquoChromosomal mapping of rDNAs core histone genes andtelomeric sequences in Perumytilus purpuratus (BivalviaMytil-idae)rdquo Journal of Experimental Marine Biology and Ecology vol395 no 1-2 pp 199ndash205 2010

[23] C Perez-Garcıa J Guerra-Varela P Moran and J J PasantesldquoChromosomalmapping of rRNAgenes core histone genes andtelomeric sequences in Brachidontes puniceus and Brachidontesrodriguezi (Bivalvia Mytilidae)rdquo BMC Genetics vol 11 article109 2010

[24] B Winnepenninckx T Backeljau and R de Wachter ldquoExtrac-tion of high molecular weight DNA from molluscsrdquo Trends inGenetics vol 9 no 12 p 407 1993

[25] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[26] C D Schubart J E Conde C Carmona-Suarez R Roblesand D L Felder ldquoLack of divergence between 16S mtDNAsequences of the swimming crabs Callinectes bocourti andC maracaiboensis (Brachyura Portunidae) from VenezuelardquoFishery Bulletin vol 99 no 3 pp 475ndash481 2001

[27] S R Palumbi ldquoNucleic acids IIThe polymerase chain reactionrdquoin Molecular Systematics D M Hillis C Moritz and B KMable Eds pp 205ndash247 Sinauer Associates SunderlandMassUSA 2nd edition 1996

[28] T J White T Burms S Lee and J W Taylor ldquoAmplificationand direct sequences of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Inmus D H Guelfand J J Sminsky andT J White Eds pp 315ndash322 Academic Press New York NYUSA 1990

[29] R Vilgalys Duke University Durham NC USA httpwwwbiologydukeedufungimycolabprimershtm

[30] G Giribet and D Distel ldquoBivalve phylogeny and molec-ular datardquo in Systematics and Phylogeography of Molluscks

8 BioMed Research International

C Lydeard and D R Lindberg Eds pp 45ndash90 SmithsonianBooks Washington Wash USA 2003

[31] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[32] S Kumar G Stecher and K Tamura ldquoMEGA7 molecularevolutionary genetics analysis version 70 for bigger datasetsrdquoMolecular Biology and Evolution vol 33 no 7 pp 1870ndash18742016

[33] D Darriba G L Taboada R Doallo and D Posada ldquoJMod-elTest 2 more models new heuristics and parallel computingrdquoNature Methods vol 9 no 8 article 772 p 772 2012

[34] S Guindon and O Gascuel ldquoA simple fast and accurate algo-rithm to estimate large phylogenies by maximum likelihoodrdquoSystematic Biology vol 52 no 5 pp 696ndash704 2003

[35] P Librado and J Rozas ldquoDnaSP v5 a software for comprehen-sive analysis of DNA polymorphism datardquo Bioinformatics vol25 pp 1451ndash1452 2009

[36] C Perez-Garcıa P Moran and J J Pasantes ldquoCytogeneticcharacterization of the invasive mussel species Xenostrobussecuris Lmk (Bivalvia Mytilidae)rdquo Genome vol 54 no 9 pp771ndash778 2011

[37] A Barco M J Raupach S Laakmann H Neumann and TKnebelsberger ldquoIdentification of North Seamolluscs with DNAbarcodingrdquoMolecular Ecology Resources vol 16 no 1 pp 288ndash297 2016

[38] S Gofas ldquoChamelea gallina (Linnaeus 1758)rdquo in MolluscaBase(2016) 2004 World Register of Marine Species httpwwwmarinespeciesorgaphiaphpp=taxdetailsampampid=141907

[39] S Gofas ldquoChamelea striatula (da Costa 1778)rdquo inMolluscaBase(2016) 2004 World Register of Marine Species httpwwwmarinespeciesorgaphiaphpp=taxdetailsampampid=141908

[40] I Kappner and R Bieler ldquoPhylogeny of venus clams (BivalviaVenerinae) as inferred from nuclear and mitochondrial genesequencesrdquo Molecular Phylogenetics and Evolution vol 40 no2 pp 317ndash331 2006

[41] P M Mikkelsen R Bieler I Kappner and T A RawlingsldquoPhylogeny of Veneroidea (Mollusca Bivalvia) based on mor-phology and moleculesrdquo Zoological Journal of the LinneanSociety vol 148 no 3 pp 439ndash521 2006

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

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International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology