research article open access incongruence …...another widespread species according to veron [3] is...

RESEARCH ARTICLE Open Access

Incongruence between morphotypes andgenetically delimited species in the coral genusStylophora phenotypic plasticity morphologicalconvergence morphological stasis or interspecifichybridizationJean-Franccedilois Flot1234 Jean Blanchot5 Loiumlc Charpy6 Corinne Cruaud2 Wilfredo Y Licuanan7 Yoshikatsu Nakano8Claude Payri9 and Simon Tillier3

Abstract

Background Morphological data suggest that unlike most other groups of marine organisms scleractinian coralsof the genus Stylophora are more diverse in the western Indian Ocean and in the Red Sea than in the central Indo-Pacific However the morphology of corals is often a poor predictor of their actual biodiversity hence weconducted a genetic survey of Stylophora corals collected in Madagascar Okinawa the Philippines and NewCaledonia in an attempt to find out the true number of species in these various locations

Results A molecular phylogenetic analysis of the mitochondrial ORF and putative control region concurs with ahaploweb analysis of nuclear ITS2 sequences in delimiting three species among our dataset species A and B arefound in Madagascar whereas species C occurs in Okinawa the Philippines and New Caledonia Comparison ofITS1 sequences from these three species with data available online suggests that species C is also found on theGreat Barrier Reef in Malaysia in the South China Sea and in Taiwan and that a distinct species D occurs in theRed Sea Shallow-water morphs of species A correspond to the morphological description of Stylophoramadagascarensis species B presents the morphology of Stylophora mordax whereas species C comprises variousmorphotypes including Stylophora pistillata and Stylophora mordax

Conclusions Genetic analysis of the coral genus Stylophora reveals species boundaries that are not congruentwith morphological traits Of the four hypotheses that may explain such discrepancy (phenotypic plasticitymorphological stasis morphological convergence and interspecific hybridization) the first two appear likely to playa role but the fourth one is rejected since mitochondrial and nuclear markers yield congruent species delimitationsThe position of the root in our molecular phylogenies suggests that the center of origin of Stylophora is located inthe western Indian Ocean which probably explains why this genus presents a higher biodiversity in thewesternmost part of its area of distribution than in the ldquoCoral Trianglerdquo

Correspondence jflotuni-goettingende1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen GermanyFull list of author information is available at the end of the article

Flot et al BMC Ecology 2011 1122httpwwwbiomedcentralcom1472-67851122

copy 2011 Flot et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution and reproduction inany medium provided the original work is properly cited

BackgroundThe reason why most marine life forms including cor-als display their peak of biodiversity in the so calledlsquoCoral Trianglersquo in Southeast Asia remains mysteriousand much debated [12] The rare examples of sea crea-tures that do not conform to this general pattern mayoffer information crucial for our understanding of itsroot causes provided that a solid taxonomic frameworkis available to interpret their present and past distribu-tion (which is unfortunately rarely the case) Severalsuch exceptions to the lsquoCoral Trianglersquo rule can befound in the scleractinian coral family Pocilloporidaethat comprises the three genera Pocillopora Seriatoporaand Stylophora although morphospecies of Seriatoporaare most diverse in the Coral Triangle and thereforeseem to follow the rule Pocillopora ldquohas what appearsto be many regional endemics especially in the centraland far eastern Pacificrdquo and Stylophora ldquohas a higherdiversity in the western Indian Ocean and Red Sea thanin the central Indo-Pacificrdquo [3] However ongoinggenetic studies of species boundaries in Pocillopora andSeriatopora suggest that even though morphologicaldescriptions of pocilloporid corals appear well foundedin some locations [4-6] in others places current taxon-omy is a poor predictor of the actual number of species[7-11]Molecular studies of Stylophora have focused so far on

a single species the ldquolab ratrdquo Stylophora pistillata (Esper1797) without delving into the delineation of interspeci-fic boundaries [1213] S pistillata was originallydescribed ldquoaus den ostindischen Meerenrdquo [14] (from theEast Indian seas in the ldquoCoral Trianglerdquo) as character-ized by short twisted dichotomous branches

(Madrepora aggregata ramulis conglomeratis brevibusdichotomis stellis crenatis profundis pistillo in medioelongato erectordquo [14] Figure 1 left side) and is consid-ered to occur over the entire Indo-Pacific (from the RedSea to Polynesia) The distinction between S pistillataand Stylophora mordax (Dana 1846) has long beendebated in the literature S mordax is considered bysome authors as a junior synonym of S pistillata[15163] and by others as a distinct species [17-24] Smordax was originally described from Fiji as havingldquobranches nearly simple much compressed not thinnerat apex scarcely flabellate 12 to 1 inch broad and 13of an inch thick polyps with a pale yellowish disk andshort tentacles of a bright green colour deep brown atbase Corallum with the cells strongly vaulted and thesurface therefore decidedly scabrousrdquo (Figure 1 rightside) [25] however intermediary forms between S mor-dax and S pistillata are commonly found in the fieldand the areas of distribution of these two morphotypesare nearly identical further adding to the confusionAnother widespread species according to Veron [3] is

Stylophora subseriata (Ehrenberg 1834) also foundacross the Indo-Pacific region whereas the five othermorphospecies of this genus are restricted to the RedSea and the Gulf of Aden (Stylophora kuehlmanniScheer and Pillai 1983 Stylophora danae MilneEdwards and Haime 1850 Stylophora mamillata Scheerand Pillai 1983) to Madagascar (Stylophora madagas-carensis Veron 2000) or to both of these regions (Stylo-phora wellsi Scheer 1964) Hence the pattern ofoccurrence of the various species of Stylophora seems tocontradict strongly the common ldquoCoral Trianglerdquo centerof biodiversity model However recent reports of S

Figure 1 Drawings of the type specimens of S pistillata and S mordax The drawing of the type of S pistillata (left) is from [25] thedrawing of the type of S mordax (right) is from [14]


Page 2 of 14

danae and S kuehlmanni from the Philippines [26] havestarted to question this pattern raising further concernthat morphological species may not correspond to actualgenetic entities (and indeed Sheppard and Sheppard[27] considered S danae S kuehlmanni and S subser-iata as ecomorphs of S pistillata)Experimental work has shown that S pistillata is phe-

notypically plastic [2829] under the influence of envir-onmental parameters such as gravity [30] and waterflow [31] Moreover evidence of intergeneric hybridiza-tion between Stylophora and Pocillopora has beenreported in the literature [32] suggesting that hybridiza-tion may a fortiori occur among various Stylophora spe-cies Finally morphological stasis and phenotypicconvergence albeit not reported yet in the genus Stylo-phora may lead to further underestimation of the actualnumber of species in this genus by causing severalgenetically distinct species to be lumped under a singlename To find out whether phenotypic plasticity inter-specific hybridization morphological stasis and phenoty-pic convergence obscure the taxonomy of Stylophorawe conducted a genetic analysis of 70 corals of this

genus collected in Madagascar Okinawa the Philippinesand New Caledonia published sequences of Stylophoraindividuals from Australia Malaysia the South ChinaSea Taiwan and Okinawa were also scrutinized (Figure2) As a first step towards a future taxonomic revisionof this genus we report here the result of our attemptto determine the true number of Stylophora speciesoccurring at these various locations and to test whetherthe unusual biogeographic pattern reported for thisgenus holds true

ResultsPhylogenetic analysis of mitochondrial sequences revealsthe presence of two Stylophora clades in Madagascar vsa single one in the Pacific OceanWe successfully sequenced from all individuals sampleda portion of the mitochondrial ORF and the putativecontrol region (CR) two markers that had previouslybeen characterised as highly variable in the closelyrelated genus Pocillopora [33] (but see [345] for a dif-ferent interpretation of these regions) Since these mar-kers gave results that were completely congruent only

Figure 2 Map of the area of distribution of the genus Stylophora showing our sampling locations Full circles show the locations wherewe collected samples whereas empty circles mark locations where sequences were obtained from the literature The area of distribution of thegenus Stylophora (modified from [3] and [68]) is shown in blue (basemap from httpd-mapscom)


Page 3 of 14

the result of their combined analysis is presented here(Figure 3) The reciprocal monophyly of each pocillo-porid genus received very strong bootstrap support(100 using maximum likelihood neighbor-joining andparsimony) whereas three clades of Stylophora emergedfrom the phylogenetic analysis clade A comprised 12individuals all from Madagascar clade B comprised 7individuals also all from Madagascar whereas clade Ccomprised the remaining 51 individuals from New Cale-donia Okinawa and the Philippines plus one previouslypublished sequence (from the complete mitochondrialgenome of one Stylophora pistillata individual from Tai-wan [34]) All three clades were very strongly supported(100 bootstrap support using all three methods)

Haploweb analysis of nuclear ITS2 sequences shows thatthese three clades represent distinct speciesWe obtained nuclear internal transcribed spacer 2 (ITS2)sequences from all individuals sampled and analyzedthem together with the single published Stylophora ITS2sequence available from GenBank (also from a Stylophorapistillata individual from Taiwan [35]) Despite its multi-ple-copy nature and its concerted mode of evolution[36] the ITS2 behaved in the present study just like aldquoregularrdquo single-copy nuclear marker with each indivi-dual harboring either one or two sequence types More-over most ITS2 sequences types found co-occurring insome individuals were also observed occurring alone inother coral colonies suggesting that these sequence typeswere allelic and segregated in a Mendelian fashion forthis reason we decided to call ldquoheterozygotesrdquo all indivi-duals found to possess two different ITS2 types eventhough we could not be totally sure that all ITS2sequences obtained were really allelic (in the closelyrelated genus Pocillopora for instance three ITS2sequences types were observed in one individual [6])Molecular phylogenetic analyses revealed many clades

of sequences several of which were very strongly sup-ported (Figure 4) In order to determine which of theseclades were conspecific and which ones belonged to dif-ferent species curves were added connecting sequencesfound co-occurring in heterozygous individuals therebyconverting the ITS2 tree into a haploweb [9] Thisapproach revealed three distinct pools of ITS2sequences corresponding to the three clades obtainedfrom mitochondrial DNA The monophyly of clades Aand B in our ITS2 dataset received very strong bootstrapsupport using all three methods whereas the monophylyof clade C was only weakly supported using maximumlikelihood (lt50 bootstrap support) and not at all sup-ported using neighbor-joining and parsimony None ofthe 43 heterozygous individuals in our dataset containedITS2 sequences from two different clades thereforeclades A B and C correspond to three distinct species

according to the mutual allelic exclusivity criterion[379]

Phylogenetic analysis of nuclear ITS1 sequences revealsthe existence of a fourth species of Stylophora in the RedSeaEven though the ITS2 of scleractinian corals is generallyeasier to align and more informative than ITS1 [35] and istherefore more commonly used the vast majority of ITSsequences available to date for the genus Stylophora areITS1 sequences In order to compare the results of ourstudy with those previously published data the ITS1regions of 14 representative individuals from our molecu-larly defined species A B and C were sequenced andaligned with sequences available in GenBank Alignmentwas indeed markedly more uncertain for ITS1 than forITS2 and the resulting alignment was rather short (345positions including many gaps vs 700 positions for ITS2and 2645 positions for the combined mitochondrial mar-kers) nevertheless the resulting phylogeny revealed inter-esting patterns and is therefore presented here (Figure 5)The three species delimited from the mitochondrial

and ITS2 datasets were recovered as distinct clades ofITS1 sequences the monophyly of species B was verystrongly supported (gt98 bootstrap support using allthree methods) whereas the monophyly of species Creceived weaker bootstrap support and the monophyly ofspecies A was very weakly supported All previously pub-lished ITS1 sequences from Australia Malaysia theSouth China Sea Taiwan and Japan turned out to belongto species C whereas all published Stylophora sequencesfrom the Red Sea fell in a well supported fourth clade D(gt90 bootstrap support using maximum likelihood andneighbor-joining) that can be considered a distinct spe-cies following the criterion of reciprocal monophyly

DiscussionHaplowebs are useful tools to delineate speciesThe present study confirms the usefulness of ourrecently proposed haploweb approach to deal withsequences of nuclear markers [9] Whereas the corre-sponding phylogenetic tree supported the delineation ofclades A and B but revealed a large number of cladesamong our sequences from the Pacific Ocean haplowebanalysis (Figure 4) showed that these various PacificOcean clades are actually conspecific (since many het-erozygous individuals harbor sequences from two differ-ent clades) The monophyly of species C was only veryweakly supported in the maximum-likelihood phylogeny(with a bootstrap value of only 43) and not at all sup-ported using neighbor-joining and parsimony thereforethis species would probably not have been detected inour ITS2 data if we had not taken into account theinformation provided by the co-occurrence of


Page 4 of 14

001

04NC130

Pocillopora meandrina (02Oahu18)

04NC436

04NC071

EU400214 (Taiwan)

07Mad157

Seriatopora Cluster 3 (04NC360)

04NC145

04Oki195

04NC365

04NC452

Pocillopora damicornis (02Oahu08)

07Mad160

04NC440

04NC282

04NC232

04NC253

04NC141

Seriatopora Cluster 2 (04Oki112)

07Mad159

07Mad189

04NC439

07Mad172

04NC199

04NC336

04NC139

04NC379

04NC152

04NC237

04NC131

04NC087

04Oki140

07Mad088

07Mad071

04NC189

07Mad151

04NC170

Pocillopora eydouxi (02Oahu32)


07Mad086

04NC132

07Mad082

04NC024

04NC233

07Mad161

04NC289

04NC101

04NC105

04NC251

07Mad150

07Mad079

04NC43404NC323

04NC182

04NC009

04Oki136

05Phil53

07Mad188

04NC064

04NC012

04NC324

04NC404

Seriatopora Cluster 4 (05Phil77)

07Mad073

04NC455

07Mad074

04NC230

07Mad070

07Mad087

04NC373

04NC187

07Mad170

04NC266

04NC441

04Oki115

04NC149

04NC140

05Phil19

93100100

100100100

100100100

100100100

835298

100100100

100100100

100100100

100100100

A

B

C

Figure 3 Phylogenetic tree of mitochondrial DNA sequences (ORF and CR combined) Outgroup sequences are from [6] and [7] whereasone ingroup sequence comes from the complete mitochondrial genome of one Stylophora pistillata individual from the Penghu Islands nearTaiwan [34] This haplotree was generated with PhyML using the TPM3uf+G model suggested by jModelTest Bootstrap values obtained usingmaximum likelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The three main clades aredelineated with brackets and labeled A B and C


Page 5 of 14

001

07Mad188b

04NC139b

04NC009a

04NC064a

04NC182a

07Mad070

07Mad082

04NC009b

04NC230

04NC266

04NC071b

07Mad172a

04NC199

04NC282b

07Mad161b

04NC439

04NC441a

04NC189a

07Mad074b

04NC452

07Mad087a

04NC434a

05Phil19

04NC141

04NC251b

04NC145b

04NC289a

04NC071a

04NC012b

07Mad189b

07Mad071

04NC434b

04NC189b04NC187

04NC441b

07Mad150

04NC105

07Mad157b

04NC012a

04NC436a

04NC024b

04NC336b

04NC379b

04NC130a

04NC101

04NC087a

04NC149a

07Mad159

04NC152a

04NC289b

04NC404

07Mad073a

04NC130b

04NC140

04NC233b

04NC182b

07Mad087b

07Mad188a

AY722795 (Taiwan)

04NC064b

04Oki115b

04NC251a

04NC323

07Mad088a

04NC087b

04Oki195a

04NC440b

07Mad088b

04NC336a

04Oki140

07Mad189a

04NC149b

07Mad151a

04Oki115a

07Mad086b

04NC132b

07Mad157a

04NC440a

04NC282a

04NC237a

04Oki136b

07Mad160b

07Mad172b

04NC365a

04Oki195b

07Mad074a

07Mad073b

04NC253a

07Mad086a

04NC170

07Mad170

04NC436b

04NC455

07Mad161a

04NC324a04NC365b

05Phil53

04NC253b

04NC024a

04NC233a

04NC152b

07Mad160a

04NC131

04Oki136a

07Mad151b

04NC139a

04NC132a

04NC237b

04NC324b

04NC373

04NC145a

04NC232

04NC379a

07Mad079

10010099

10010099

43--

993100100

99510098

828360

828983

99610097

7958-

808372

7583-

A

B

C

Figure 4 Haploweb of ITS2 sequences This graph was derived from a phylogenetic tree (obtained with PhyML using the TIM2+G modelsuggested by jModelTest) by drawing curves connecting haplotypes found co-occurring in heterozygous individuals [9] The sequence of oneStylophora pistillata individual from the Penghu Islands near Taiwan [35] is included in the figure and bootstrap values obtained using maximumlikelihood neighbor-joining and parsimony (1000 replicates each) are displayed next to each node The position of the root was inferred fromthe mitochondrial DNA phylogeny (Figure 3) This approach delineated three reproductively isolated pools of Stylophora sequences labeled A Band C as each of these groups corresponded to one of the mitochondrial clades of Figure 3


Page 6 of 14

phylogenetically distant alleles in some individuals Incontrast haploweb analysis delineated three groups ofalleles among our ITS2 sequences a result perfectlycongruent with the mitochondrial phylogeny obtainedfrom the same set of individualsUnlike in our previous article [9] the haploweb pre-

sented here was built on a tree rather than a networkIndeed tree-based haplowebs are more straightforwardto draw than their network-based counterparts and arealso more informative since they display the genotype ofeach sequenced individual However precisely due to

their larger information content tree-based haplowebstend to become messy when dealing with large datasetsandor non-monophyletic species in such cases net-work-based haplowebs often turn out to be faster todraw and easier to interpret than tree-based ones

Are molecularly delimited species of Stylophoracongruent with morphologyMost of the 12 individuals from species A displayed thecharacteristic morphology of S madagascarensis (someexamples are shown on Figure 6) ldquocolonies have thin

D

005

AF416161 Stylophora pistillata brown Heron Island

AJ619830 Stylophora pistillata Red Sea

07Mad157a Madagascar

04Oki140 Okinawa

AF416224 Stylophora pistillata pink Sesoko


AF416170 Stylophora pistillata brown One Tree Island

07Mad150 Madagascar


AF416150 Stylophora pistillata pink Akajima

AF416229 Stylophora pistillata brown Terengganu


AF416147 Stylophora pistillata brown Akajima

AF416194 Stylophora pistillata brown Moulter Cay



AF416219 Stylophora pistillata brown Sesoko

AF416162 Stylophora pistillata pink Heron Island





AF416202 Stylophora pistillata pink Moulter Cay




AF416182 Stylophora pistillata brown Raine Island















AF416209 Stylophora pistillata pink Sabah



AF416175 Stylophora pistillata pink One Tree Island













AF416179 Stylophora pistillata pink One Tree IslandAF416176 Stylophora pistillata pink One Tree Island







04NC101 New Caledonia


AF416206 Stylophora pistillata brown Sabah

07Mad160b Madagascar




07Mad082 Madagascar









AF038909 Stylophora pistillata One Tree Island





04NC140a New Caledonia

HM013855 Seriatopora hystrix Xisha Islands








AJ619818 Stylophora pistillata Red SeaAJ619826 Stylophora pistillata Red Sea





05Phil53 Philippines

AF416160 Stylophora pistillata brown Heron IslandAF038907 Stylophora pistillata One Tree Island







04NC140b New Caledonia


AF416189 Stylophora pistillata pink Raine Island









HM013847 Pocillopora damicornis Xisha Islands

AY722795 Stylophora pistillata Penghu Island
















07Mad070 Madagascar




HM013856 Stylophora pistillata Xisha Islands



































07Mad071 Madagascar















9129979

6937118

94490-

297--

9809966

345-76

9879998

A

B

C

Figure 5 ITS1 molecular phylogeny of the genus Stylophora In addition to ITS1 sequences from selected individuals of species A B and Cdata from published articles [69123513] and from an unpublished study by Feng You and Hui Huang [GenBank HM013847 HM013855HM013856] were included in the haplotree (generated with PhyML using the TPM2+G model suggested by jModelTest)


Page 7 of 14

(up to 5 mm diameter) straight compact branches Cor-allites are crowded and uniformly spaced on branchsides and ends They have a slight development ofhoods towards branch ends They have small style-likecolumellae and six primary septa which are fused withthe columellae The coenosteum is covered with finespicules Tentacles are not extended during the dayColour Uniform tan sometimes with pinkish branchbasesrdquo [38] Since Veronrsquos holotype was collected fromthe very same location in Madagascar where we con-ducted our sampling (approximately 4 m depth Tuleacutearsouth-west Madagascarrdquo [38]) there is little doubt thatspecies A and S madagascarensis are conspecificAccording to Veronrsquos description this species wasldquorecorded only from shallow reef environments exposedto some wave action and in south-west Madagascar inshallow sheltered lagoonsrdquo [38] In contrast we foundthis species down to a depth of 30 meters where it dis-played less compact and thicker branches comparedwith the typical S madagacarensis morphology (Figure7) hence S madagascarensis appears to be more ecolo-gically widespread and morphologically variable thanpreviously thought

All 7 individuals of species B from Madagascar hadflattened stout branches (Figure 8) This correspondsunambiguously to the morphological description of Smordax but since S mordax was originally describedfrom Fiji in the Pacific Ocean it is dubious whether thisname may be assigned to species BSpecies C gathered all individuals collected from loca-

tions in the Pacific Ocean It is characterized by exten-sive morphological variation comprising individualsattributable to S pistillata and others attributable to S

Figure 6 Morphology of species A From top to bottom colonies07Mad074 sampled at 5 meters depth colony 07Mad086 sampledat 6 meters depth and colony 07Mad170 sampled at 11 metersdepth display the typical morphology of S madagascarensis (thinstraight compact branches with small hoods) The green wire onthe pictures of the skeletons has a diameter of 09 mm

Figure 7 Deep-water morphs of species A Colony 07Mad088(left) and colony 07Mad097 (right) were both sampled at 30 metersdepth and have thicker less compact branches compared withtypical S madagascarensis morphology

Figure 8 Morphology of species B From top to bottom colony07Mad150 collected at 8 meters depth colony 07Mad071 collectedat 7 meters depth and colony 07Mad160 collected at 3 metersdepth all display the typical morphology of S mordax (shortcompressed branches with well-developed hoods) The green wireon the pictures of the skeletons has a diameter of 09 mm


Page 8 of 14

mordax as well as morphotypes somehow intermediaryin shape (Figure 9) Several colonies of this species col-lected in a very muddy location (Banc des Japonais inNew Caledonia) displayed a peculiar phenotype charac-terized by extremely elongated thin branches (Figure10) Published sequences of S pistillata from Taiwan(ITS2 ORF) and from Okinawa Taiwan the SouthChina Sea Malaysia and Australia (ITS1) grouped withour sequences from species C further supporting itsputative identification as S pistillataFinally all published ITS1 sequences from the Red Sea

fell in a distinct well-supported species D The coralcolonies sequenced were collected at depths of 2-4meters in the Gulf of Aqaba and reported under thename S pistillata [13] but according to another studythe main morphospecies of Stylophora found in shallowwaters in Aqaba is actually S mordax [21] thereforespecies D probably comprises a mixture of S pistillataand S mordax morphotypes Since the type localities ofS pistillata and S mordax are both in the PacificOcean another name will be required for species D(possibly S subseriata since this species was described

from the Red Sea and was considered by some authorsas a synonym of S pistillata [27])

What causes the discrepancy between morphological andmolecular species delimitations in StylophoraOur study revealed extensive morphological variationwithin species A and C a more detailed genetic inves-tigation using a larger number of variable markerssuch as microsatellites will be required to find outwhether these variations are due to phenotypic plasti-city or to underlying intraspecific genetic differencesHowever phenotypic plasticity is well documented inStylophora [30312829] and is therefore likely to beresponsible for at least part of the observed morpholo-gical variationsThe occurrence of the S mordax morphotype in both

species B and C can hardly be explained by intra-specificvariation alone but may rather result from phenotypicconvergence (whereby two non-sister species indepen-dently evolve similar morphologies) andor morphologicalstasis (whereby the appearance of the common ancestor oftwo sister species is passed on to both of them) Since thesister-species relationship between B and C was verystrongly supported by all molecular markers analyzedmorphological stasis appears more likely than phenotypicconvergence to explain the similar appearance of species Band of some morphs of species CEven though the data available are not yet sufficient to

pin down completely the causes of the interspecificmorphological similarities and intraspecific phenotypicvariations observed in Stylophora the observed congru-ence between nuclear and mitochondrial phylogeniesallows us to reject the hypothesis that hybridizationcould be responsible for the discrepancy between mor-phological and genetic species boundaries in this genusThis contrasts with previous reports that hybridizationmay be rampant in corals (eg [39-50]) instead ourresults concur with two recent articles on the closelyrelated genus Pocillopora [911] in suggesting that manysuch reports actually result from improper delineation

Figure 10 Extremely elongated morph of species C from a verymuddy site in New Caledonia Colonies 04NC149 (left) and04NC170 (right) were sampled at 16 and 15 meters depth at theBanc des Japonais in New Caledonia

Figure 9 Morphology of species C From top to bottom colony04NC253 was collected at 19 meters depth and displays the typicalS mordax morphology colony 04NC323 was collected at 6 metersdepth and displays the typical S pistillata morphology colony04NC282 was collected at 6 meters depth and is intermediate inshape between S mordax and S pistillata The distance betweentwo white lines in the background is 30 mm


Page 9 of 14

of species boundaries and not from actual introgressionbetween distinct genetic entities

A new light on the biogeography and biodiversity ofStylophora in the Indo-Pacific OceanSurprisingly the incongruence between morphologicalspecies delimitations and genetic species boundariesrevealed in our study does not seem to affect the generalpicture of the biogeographic distribution of Stylophoraspecies with a least three species in the westernmostpart of its area of occurrence versus a single one so farin the ldquoCoral Trianglerdquo Stylophora stands confirmed asa blatant exception to the usual biodiversity patternobserved in tropical marine invertebrates The mito-chondrial and ITS2 phylogenies of Stylophora compriseonly species A B and C and do not contradict thetopology of the more complete (but less resolved) ITS1phylogeny the sister-group relationship of species B andC is strongly supported by all markers whereas the rootof the mitochondrial and ITS1 phylogenies fall betweenspecies A (from Madagascar) and species B and C(respectively from Madagascar and from the PacificOcean) Even though the sister-group of species D couldnot be determined unambiguously due to the currentlack of mitochondrial and ITS2 sequences for this spe-cies the position of the root in our molecular phyloge-nies suggests that the center of origin of Stylophora islocated in the western Indian Ocean This hypothesiswill need to be confirmed by analyzing more samplesfrom key locations in the Red Sea the Gulf of Aden andthe Indian Ocean but would explain well the unusualconcentration of the biodiversity of this genus in thewesternmost part of his area of distributionWhile waiting for a global taxonomic revision of the

genus Stylophora for the sake of taxonomic stability werecommend that the preliminary results presented herenot be translated yet into nomenclature but that eachgenetically delimited species be provisionally designatedby a letter (ie ldquoStylophora sp Ardquo ldquoStylophora sp BrdquoldquoStylophora sp Crdquo and ldquoStylophora sp Drdquo) It is onlywhen a complete picture of the species boundaries ofStylophora over its whole area of distribution becomesavailable that names will be reliably assigned to eachspecies for instance even though the name S madagas-carensis appears suitable for species A given its morpho-logical traits and the location where it was collected thisspecies may very well have been described first underanother name in a different location in which case Smadagascarensis would become a junior synonym of theactual name of this species

ConclusionsGenetic analysis of the coral genus Stylophora revealsspecies boundaries that are not congruent with

morphology Of the four hypotheses capable of explain-ing such discrepancy (phenotypic plasticity morphologi-cal stasis morphological convergence and interspecifichybridization) the first two seem likely to play a rolebut the fourth one is rejected since mitochondrial andnuclear markers yield congruent species delimitationsThe center of origin of Stylophora appears to be locatedin the Indian Ocean which probably explains why thisgenus presents a higher biodiversity in the westernmostpart of its area of distribution than in the ldquoCoralTrianglerdquo

MethodsSample collection and processingFragments from 70 Stylophora coral colonies were col-lected while scuba diving or snorkeling on reefs of NewCaledonia Okinawa (Japan) Bolinao (Philippines) andToliara (Madagascar) between 2004 and 2007 Each col-ony sampled was photographed underwater and itsdepth recorded (Table 1) Coral tissues were preservedin a buffered guanidium thiocyanate solution [5152]and their DNA purified on an ABI Prism 6100 NucleicAcid PrepStation

PCR amplification and sequencingThree DNA markers previously developed in the closelyrelated genus Pocillopora [33] were amplified andsequenced for each individual the nuclear ribosomalintergenic transcribed spacer 2 (ITS2) the mitochondrialORF and the putative control region (CR) In additionnew primers were used to amplify the ITS1 region of afew selected individuals (Table 2) Amplifications wereperformed in 25 μl reaction mixes containing 1x Red Taqbuffer 264 μM dNTP 5 DMSO 03 μM PCR primers03 units Red Taq (Sigma) and 10-50 ng DNA PCR con-ditions comprised an initial denaturation step of 60 s at94degC followed by 40-50 cycles (30 s denaturation at 94degC 30 s annealing at 53degC 75 s elongation at 72degC) and afinal 5-min elongation step at 72degC PCR products weresequenced in both directions with the same primers asfor amplification and chromatograms were assembledand cleaned using Sequencher 4 (Gene Codes)

Determination of nuclear haplotypesThe ITS2 chromatogram pairs obtained from 43 indivi-duals contained double peaks indicating that each ofthese individuals harbored two sequence types Findingout the sequence types was trivial for 5 individualswhose chromatograms contained only one double peakFurthermore 21 other chromatogram pairs had numer-ous double peaks a situation typical of length-variantheterozygotes [5354] that allowed direct deconvolutionof their superposed sequences using the programCHAMPURU [55] (available online at httpwwwmnhn


0 of 14

frjfflotchampuru) The remaining 17 chromatogramspairs had several double peaks (at most 9) as expectedfrom heterozygotes with no intra-individual length varia-tion we first attempted to resolve their haplotypes sta-tistically by reference to the rest of the dataset usingSeqPHASE [56] (available online at httpwwwmnhnfrjfflotseqphase) and PHASE [57] but only 7 individualswere phased unambiguously ie with posterior prob-abilities equal or nearly equal to 1 (04NC064 04NC18204NC251 04NC282 04NC324 04NC436 07Mad087)Among the 10 remaining heterozygotes the haplotypes

of 2 individuals (07Mad151 07Mad189) were deduceddirectly from their chromatograms thanks to clear-cutdifferences in peak sizes (reflecting either differences incopy number in ribosomal DNA arrays or differentialamplification during PCR) and the sequences of the 8others (04NC024 04NC132 04NC365 04NC37907Mad073 07Mad074 07Mad088 07Mad157) wereinferred using Clarkrsquos method [58] Length-variant het-erozygosity was also observed in the ITS1 chromato-grams of five individuals all of which were resolvedusing CHAMPURU

Table 1 Localization and depth of each Stylophora sample analyzed

Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)

04Oki115 (26deg12rsquo10N 127deg1912E) nr 04NC323 (20deg42rsquo39S 165deg09rsquo14E) 6



04Oki195 (26deg14rsquo25N 127deg27rsquo50E) nr 04NC365 (20deg40rsquo12S 164deg11rsquo19E) 22

04NC009 (22deg26rsquo54S 166deg22rsquo23E) 2 04NC373 (20deg40rsquo12S 164deg11rsquo19E) 15










04NC132 (22deg22rsquo59S 167deg05rsquo50E) 1 05Phil19 (16deg23rsquo46N 119deg54rsquo03E) 24


04NC140 (22deg22rsquo59S 167deg05rsquo50E) 1 07Mad070 (23deg25rsquo01S 43deg38rsquo36E) 8





04NC170 (22deg15rsquo27S 166deg24rsquo33E) 15 07Mad082 ( 23deg23rsquo07S 43deg38rsquo18E) 8














nr = not recorded


Page 11 of 14

Phylogenetic analyses and haploweb constructionAll haplotype sequences were deposited in public data-bases [GenBank JN558840-JN559111] ORF sequenceswere aligned in MEGA5 [59] by taking advantage ofthe high degree of conservation of their aminoacidtranslations all sequences from Stylophora were firstaligned by hand as there were only few indels beforealigning them with outgroup sequences from Pocillo-pora and Seriatopora using the MEGA5 implementa-tion of MUSCLE [60] CR ITS1 and ITS2 sequenceswere aligned using MAFFTrsquos Q-INS-I option [6162]Since the two mitochondrial markers ORF and CRyielded congruent phylogenies they were concatenatedand only the result of the combined analysis is pre-sented here The best suited nucleotide model among88 possible ones was determined for each dataset fol-lowing the Bayesian Information Criterion [63] asimplemented in jModelTest [64] and used to performmaximum-likelihood phylogenetic analyses in PhyML[65] with 1000 bootstrap replicates [66] Additionalbootstrap analyses (1000 replicates) using neighbor-joining (K2P model pairwise deletion) and parsimony(dataset collapsed using FaBox [67] complete deletion)were conducted in MEGA5 The Newick format haplo-type trees (haplotreesrdquo) produced by PhyML wereconverted into enhanced metafiles (emf) using the pro-gram FigTree 131 (available online at httptreebioedacuksoftwarefigtree) then imported in MicrosoftPowerPoint The ITS2 haploweb was obtained fromthe corresponding haplotree by drawing connectionsbetween haplotypes found co-occurring in heterozy-gous individuals [9]

AcknowledgementsThanks to Annie Tillier Josie Lambourdiegravere and Ceacuteline Bonillo (Service deSysteacutematique Moleacuteculaire CNRS UMS 2700 MNHN) for assistance with labwork and to Eric Folcher Catherine Geoffray Jean-Louis Menou and MarkVergara for helping with sample collection Fieldwork in New Caledonia andMadagascar was financed by grants from the MNHN programs ldquoStructure eteacutevolution des eacutecosystegravemesrdquo et ldquoEacutetat et structure phylogeacuteneacutetique de labiodiversiteacute actuelle et fossile thanks to Man Wai Rabenevanana director ofthe Institut Halieutique et des Sciences Marines for his logistic support in Tuleacutear(Toliara) Thanks also to two anonymous reviewers for their useful comments

This project was part of agreement ndeg200567 between Genoscope andMNHN on the project lsquoMacrophylogeny of lifersquo directed by GuillaumeLecointre support from the Consortium National de Recherche en Geacutenomiqueis gratefully acknowledged This is contribution ndeg78 from the CourantResearch Center ldquoGeobiologyrdquo funded by the German Initiative of Excellence

Author details1Courant Research Center ldquoGeobiologyrdquo University of GoumlttingenGoldschmidtstraszlige 3 37077 Goumlttingen Germany 2CEA-Institut deGeacutenomique GENOSCOPE Centre National de Seacutequenccedilage 2 rue GastonCreacutemieux CP5706 91057 Evry Cedex France 3UMR UPMC-CNRS-MNHN-IRD7138 Deacutepartement Systeacutematique et Eacutevolution Museacuteum National drsquoHistoireNaturelle Case Postale 26 57 rue Cuvier 75231 Paris Cedex 05 France4URBO Department of Biology University of Namur Rue de Bruxelle 615000 Namur Belgium 5UMR LOBP Centre drsquoOceacuteanologie de MarseilleCampus de Luminy Case 901 13288 Marseille Cedex 09 France 6UMR LOBPCentre IRD de Tahiti BP 529 98713 Papeete French Polynesia 7Br AlfredShields FSC Marine Station and Biology Department De La Salle UniversityManila 1004 Philippines 8Sesoko Station Tropical Biosphere Research CenterUniversity of the Ryukyus Okinawa 3422 Japan 9UR COREUS IRD BP A598848 Noumeacutea New Caledonia

Authorsrsquo contributionsJFF carried out fieldwork DNA extractions and PCR amplifications analyzedthe results and drafted the manuscript CC sequenced all PCR products JBLC WL YN and CP provided logistic support participated in fieldwork andrevised the manuscript ST supervised the study and revised the manuscriptthe final version of which was read and approved by all authors

Received 25 March 2011 Accepted 4 October 2011Published 4 October 2011

References1 Veron JEN Corals in Space and Time Biogeography amp Evolution of the

Scleractinia Sydney Australia University of New South Wales Press 19952 Hoeksema B Delineation of the Indo-Malayan centre of maximum

marine biodiversity the Coral Triangle In Biogeography Time and PlaceDistributions Barriers and Islands Edited by Renema W Dordrecht SpringerNetherlands 2007117-178

3 Veron JEN Stafford-Smith M Corals of the World Australian Institute ofMarine Science 2000

4 Flot JF Tillier S Molecular phylogeny and systematics of the scleractiniancoral genus Pocillopora in Hawaii Proceedings of the 10th InternationalCoral Reef Symposium 2006 124-29

5 Chen C Dai CF Plathong S Chiou CY Chen CA The completemitochondrial genomes of needle corals Seriatopora spp (ScleractiniaPocilloporidae) an idiosyncratic atp8 duplicated trnW gene andhypervariable regions used to determine species phylogenies andrecently diverged populations Molecular Phylogenetics and Evolution 20084619-33

6 Flot JF Magalon H Cruaud C Couloux A Tillier S Patterns of geneticstructure among Hawaiian corals of the genus Pocillopora yield clustersof individuals that are compatible with morphology Comptes RendusBiologies 2008 331239-247

Table 2 Primers used for DNA amplification and sequencing

Marker Primer name Primer sequence Reference

ITS1 (nuclear) F18S1 5rsquo-CGATYGAAYGGTTTAGTGAGGC-3rsquo this study

ITS1 (nuclear) ITSc1-3 5rsquo- CATTTGCGTTCAAAGATTCG-3rsquo this study

ITS2 (nuclear) ITSc2-5 5rsquo-AGCCAGCTGCGATAAGTAGTG-3rsquo [4]

ITS2 (nuclear) R28S1 5rsquo-GCTGCAATCCCAAACAACCC-3rsquo [4]

ORF (mitochondrial) FATP61 5rsquo-TTTGGGSATTCGTTTAGCAG-3rsquo [6]

ORF (mitochondrial) RORF 5rsquo-SCCAATATGTTAAACASCATGTCA-3rsquo [6]

CR (mitochondrial) FNAD52deg 5rsquo-GCCYAGRGGTGTTGTTCAAT-3rsquo [6]

CR (mitochondrial) RCOI3deg 5rsquo-CGCAGAAAGCTCBARTCGTA-3rsquo [7]


Page 12 of 14

7 Flot JF Licuanan W Nakano Y Payri C Cruaud C Tillier S Mitochondrialsequences of Seriatopora corals show little agreement with morphologyand reveal the duplication of a tRNA gene near the control region CoralReefs 2008 27789-794

8 Bongaerts P Riginos C Ridgway T Sampayo EM van Oppen MJHEnglebert N Vermeulen F Hoegh-Guldberg O Genetic divergence acrosshabitats in the widespread coral Seriatopora hystrix and its associatedSymbiodinium PLoS ONE 2010 5e10871

9 Flot JF Couloux A Tillier S Haplowebs as a graphical tool for delimitingspecies a revival of Doylersquos ldquofield for recombinationrdquo approach and itsapplication to the coral genus Pocillopora in Clipperton BMC EvolutionaryBiology 2010 10372

10 Souter P Hidden genetic diversity in a key model species of coralMarine Biology 2010 157875-885

11 Pinzoacuten JH LaJeunesse TC Species delimitation of common reef corals inthe genus Pocillopora using nucleotide sequence phylogeniespopulation genetics and symbiosis ecology Molecular Ecology 201120311-325

12 Takabayashi M Carter DA Lopez JV Hoegh-Guldberg O Genetic variationof the scleractinian coral Stylophora pistillata from western Pacific reefsCoral Reefs 2003 2217-22

13 Zvuloni A Mokady O Al-Zibdah M Bernardi G Gaines SD Abelson A Localscale genetic structure in coral populations a signature of selectionMarine Pollution Bulletin 2008 56430-438

14 Esper EJC Fortsetzungen der Pflanzenthiere in Abbildungen nach der Naturmit Farben erleuchtet nebst Beschreibungen Erster Theil Nuumlrnberg RaspischeBuchhandlung 1797

15 Veron JEN Pichon M Scleractinia of eastern Australia I FamiliesThamnasteriidae Astrocoeniidae Pocilloporidae Australian Institute ofMarine Science Monograph Series 1976 11-86

16 Scheer G Pillai CSG Report on the stony corals from the Red SeaZoologica 1983 451-198

17 Scheer G Pillai CSG Report on the Scleractinia from the Nicobar IslandsResults of the Xarifa Expedition 195758 of the International Institute forSubmarine Research Vaduz Liechtenstein (Director Dr Hans Hass)Zoologica 1974 421-75

18 Pillai CSG Scheer G Report on the stony corals from the MaldiveArchipelago Results of the Xarifa Expedition 195758 of theInternational Institute for Submarine Research Vaduz Liechtenstein(Director Dr Hans Hass) Zoologica 1976 431-83

19 Faure G Recherche sur les peuplements de Scleacuteractiniaires des reacutecifscoralliens de lrsquoarchipel des Mascareignes (Oceacutean Indien occidental)Volume 2 - Systeacutematique PhD thesis Universiteacute drsquoAix-Marseille II Faculteacutedes Sciences de Luminy 1982

20 Hamilton HGH Brakel WH Structure and coral fauna of East African reefsBulletin of Marine Science 1984 34248-266

21 Gattuso JP Pichon M Jaubert J Physiology and taxonomy of scleractiniancorals a case study in the genus Stylophora Coral Reefs 1991 9173-182

22 Hidaka M Use of nematocyst morphology for taxonomy of some relatedspecies of scleractinian corals Galaxea 1992 1121-28

23 Cairns SD Hoeksema BW van der Land J Appendix list of extant stonycorals Atoll Research Bulletin 1999 45913-46

24 Pichon M Scleractinia of New Caledonia check list of reef dwellingspecies In Compendium of marine species of New Caledonia DocumentsScientifiques et Techniques II7 2 edition Edited by Payri CE Richer deForges B Noumeacutea IRD 2007149-157

25 Dana JD United States Exploring Expedition Vol VII Zoophytes PhiladelphiaC Sherman 1846

26 Licuanan WY Capili EB New records of stony corals from the Philippinespreviously known from peripheral areas of the Indo-Pacific The RafflesBulletin of Zoology 2004 52285-288

27 Sheppard CRC Sheppard ALS Corals and coral communities of ArabiaFauna of Saudi Arabia 1991 123-170

28 Shaish L Abelson A Rinkevich B Branch to colony trajectory in a modularorganism pattern formation in the Indo-Pacific coral Stylophorapistillata Developmental Dynamics 2006 2352111-2121

29 Shaish L Abelson A Rinkevich B How plastic can phenotypic plasticitybe The branching coral Stylophora pistillata as a model system PLoSONE 2007 2e644

30 Meroz E Brickner I Loya Y Peretzman-Shemer A Ilan M The effect ofgravity on coral morphology Proceedings of the Royal Society of LondonSeries B Biological Sciences 2002 269717-720

31 Nakamura T Yamasaki H Morphological changes of pocilloporid coralsexposed to water flow Proceedings of the 10th International Coral ReefSymposium 2006 1872-875

32 Miller KJ Ayre DJ The role of sexual and asexual reproduction instructuring high latitude populations of the reef coral Pocilloporadamicornis Heredity 2004 92557-568

33 Flot JF Tillier S The mitochondrial genome of Pocillopora (CnidariaScleractinia) contains two variable regions The putative D-loop and anovel ORF of unknown function Gene 2007 40180-87

34 Chen C Chiou CY Dai CF Chen CA Unique mitogenomic features in thescleractinian family Pocilloporidae (Scleractinia Astrocoeniina) MarineBiotechnology 2008 10538-553

35 Chen CA Chang CC Wei NV Chen CH Lein YT Lin HE Dai CF Wallace CCSecondary structure and phylogenetic utility of the ribosomal internaltranscribed spacer 2 (ITS2) in scleractinian corals Zoological Studies 200443759-771

36 Hillis DM Dixon MT Ribosomal DNA molecular evolution andphylogenetic inference Quarterly Review of Biology 1991 66411-453

37 Doyle JJ The irrelevance of allele tree topologies for speciesdelimitation and a non-topological alternative Systematic Botany 199520574-588

38 Veron JEN New species described in Corals of the World AustralianInstitute of Marine Science Monograph Series 2002 111-206

39 Kenyon JC Models of reticulate evolution in the coral genus Acroporabased on chromosome numbers parallels with plants Evolution 199751756-767

40 Szmant AM Weil E Miller MW Coloacuten DE Hybridization within the speciescomplex of the scleractinan coral Montastraea annularis Marine Biology1997 129561-572

41 Hatta M Fukami H Wang WQ Omori M Shimoike K Hayashibara T Ina YSugiyama T Reproductive and genetic evidence for a reticulateevolutionary history of mass-spawning corals Molecular Biology andEvolution 1999 161607-1613

42 Diekmann OE Bak RPM Stam WT Olsen JL Molecular genetic evidencefor probable reticulate speciation in the coral genus Madracis from aCaribbean fringing reef slope Marine Biology 2001 139221-233

43 van Oppen MJH McDonald BJ Willis B Miller DJ The evolutionary historyof the coral genus Acropora (Scleractinia Cnidaria) based on amitochondrial and a nuclear marker reticulation incomplete lineagesorting or morphological convergence Molecular Biology and Evolution2001 181315-1329

44 Maacuterquez LM Van Oppen MJH Willis BL Reyes A Miller DJ The highlycross-fertile coral species Acropora hyacinthus and Acropora cythereaconstitute statistically distinguishable lineages Molecular Ecology 2002111339-1349

45 van Oppen MJH Willis BL Van Rheede T Miller DJ Spawning timesreproductive compatibilities and genetic structuring in the Acroporaaspera group evidence for natural hybridization and semi-permeablespecies boundaries in corals Molecular Ecology 2002 111363-1376

46 Vollmer SV Palumbi SR Hybridization and the evolution of reef coraldiversity Science 2002 2962023-2025

47 Miller DJ van Oppen MJH A lsquofair gorsquo for coral hybridization MolecularEcology 2003 12805-807

48 Willis BL van Oppen MJH Miller DJ Vollmer SV Ayre DJ The role ofhybridization in the evolution of reef corals Annual Review of EcologyEvolution and Systematics 2006 37489-517

49 Combosch DJ Guzman HM Schuhmacher H Vollmer SV Interspecifichybridization and restricted trans-Pacific gene flow in the TropicalEastern Pacific Pocillopora Molecular Ecology 2008 171304-1312

50 Frade PR Reyes-Nivia MC Faria J Kaandorp JA Luttikhuizen PC Bak RPMSemi-permeable species boundaries in the coral genus MadracisIntrogression in a brooding coral system Molecular Phylogenetics andEvolution 2010 571072-1090

51 Sargent TD Jamrich M Dawid IB Cell interactions and the control ofgene activity during early development of Xenopus laevis DevelopmentalBiology 1986 114238-246


Page 13 of 14

52 Fukami H Budd AF Levitan DR Jara J Kersanach R Knowlton NGeographic differences in species boundaries among members of theMontastraea annularis complex based on molecular and morphologicalmarkers Evolution 2004 38324-337

53 Creer S Malhotra A Thorpe RS Pook CE Targeting optimal introns forphylogenetic analyses in non-model taxa experimental results in Asianpitvipers Cladistics 2005 21390-395

54 Flot JF Tillier A Samadi S Tillier S Phase determination from directsequencing of length-variable DNA regions Molecular Ecology Notes 20066627-630

55 Flot JF Champuru 10 a computer software for unraveling mixtures oftwo DNA sequences of unequal lengths Molecular Ecology Notes 20077974-977

56 Flot JF SeqPHASE a web tool for interconverting PHASE inputoutputfiles and FASTA sequence alignments Molecular Ecology Resources 201010162-166

57 Stephens M Smith NJ Donnelly P A new statistical method for haplotypereconstruction from population data The American Journal of HumanGenetics 2001 68978-989

58 Clark A Inference of haplotypes from PCR-amplified samples of diploidpopulations Molecular Biology and Evolution 1990 7111-122

59 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5Molecular Evolutionary Genetics Analysis using maximum likelihoodevolutionary distance and maximum parsimony methods MolecularBiology and Evolution

60 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Research 2004 321792-1797

61 Katoh K Misawa K Kuma Ki Miyata T MAFFT a novel method for rapidmultiple sequence alignment based on fast Fourier transform NucleicAcids Research 2002 303059-3066

62 Katoh K Toh H Improved accuracy of multiple ncRNA alignment byincorporating structural information into a MAFFT-based frameworkBMC Bioinformatics 2008 9212

63 Schwarz G Estimating the dimension of a model The Annals of Statistics1978 6461-464

64 Posada D jModelTest Phylogenetic model averaging Molecular Biologyand Evolution 2008 251253-1256

65 Guindon S Gascuel O A simple fast and accurate algorithm to estimatelarge phylogenies by maximum likelihood Systematic Biology 200352696-704

66 Felsenstein J Confidence limits on phylogenies an approach using thebootstrap Evolution 1985 39783-791

67 Villesen P FaBox an online toolbox for fasta sequences Molecular EcologyNotes 2007 7965-968

68 Veron JEN Stafford-Smith M Coral ID Release 1 Australian Institute ofMarine Science and CRR Qld Pty Ltd 2002

69 Takabayashi M Carter DA Loh WKW Hoegh-Guldberg O A coral-specificprimer for PCR amplification of the internal transcribed spacer region inribosomal DNA Molecular Ecology 1998 7925-931

doi1011861472-6785-11-22Cite this article as Flot et al Incongruence between morphotypes andgenetically delimited species in the coral genus Stylophora phenotypicplasticity morphological convergence morphological stasis orinterspecific hybridization BMC Ecology 2011 1122

Submit your next manuscript to BioMed Centraland take full advantage of

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Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results

Phylogenetic analysis of mitochondrial sequences reveals the presence of two Stylophora clades in Madagascar vs a single one in the Pacific Ocean

Haploweb analysis of nuclear ITS2 sequences shows that these three clades represent distinct species

Phylogenetic analysis of nuclear ITS1 sequences reveals the existence of a fourth species of Stylophora in the Red Sea

Discussion

Haplowebs are useful tools to delineate species

Are molecularly delimited species of Stylophora congruent with morphology

What causes the discrepancy between morphological and molecular species delimitations in Stylophora

A new light on the biogeography and biodiversity of Stylophora in the Indo-Pacific Ocean

Conclusions

Methods

Sample collection and processing

PCR amplification and sequencing

Determination of nuclear haplotypes

Phylogenetic analyses and haploweb construction

Acknowledgements

Author details

Authors contributions

References

BackgroundThe reason why most marine life forms including cor-als display their peak of biodiversity in the so calledlsquoCoral Trianglersquo in Southeast Asia remains mysteriousand much debated [12] The rare examples of sea crea-tures that do not conform to this general pattern mayoffer information crucial for our understanding of itsroot causes provided that a solid taxonomic frameworkis available to interpret their present and past distribu-tion (which is unfortunately rarely the case) Severalsuch exceptions to the lsquoCoral Trianglersquo rule can befound in the scleractinian coral family Pocilloporidaethat comprises the three genera Pocillopora Seriatoporaand Stylophora although morphospecies of Seriatoporaare most diverse in the Coral Triangle and thereforeseem to follow the rule Pocillopora ldquohas what appearsto be many regional endemics especially in the centraland far eastern Pacificrdquo and Stylophora ldquohas a higherdiversity in the western Indian Ocean and Red Sea thanin the central Indo-Pacificrdquo [3] However ongoinggenetic studies of species boundaries in Pocillopora andSeriatopora suggest that even though morphologicaldescriptions of pocilloporid corals appear well foundedin some locations [4-6] in others places current taxon-omy is a poor predictor of the actual number of species[7-11]Molecular studies of Stylophora have focused so far on

a single species the ldquolab ratrdquo Stylophora pistillata (Esper1797) without delving into the delineation of interspeci-fic boundaries [1213] S pistillata was originallydescribed ldquoaus den ostindischen Meerenrdquo [14] (from theEast Indian seas in the ldquoCoral Trianglerdquo) as character-ized by short twisted dichotomous branches

(Madrepora aggregata ramulis conglomeratis brevibusdichotomis stellis crenatis profundis pistillo in medioelongato erectordquo [14] Figure 1 left side) and is consid-ered to occur over the entire Indo-Pacific (from the RedSea to Polynesia) The distinction between S pistillataand Stylophora mordax (Dana 1846) has long beendebated in the literature S mordax is considered bysome authors as a junior synonym of S pistillata[15163] and by others as a distinct species [17-24] Smordax was originally described from Fiji as havingldquobranches nearly simple much compressed not thinnerat apex scarcely flabellate 12 to 1 inch broad and 13of an inch thick polyps with a pale yellowish disk andshort tentacles of a bright green colour deep brown atbase Corallum with the cells strongly vaulted and thesurface therefore decidedly scabrousrdquo (Figure 1 rightside) [25] however intermediary forms between S mor-dax and S pistillata are commonly found in the fieldand the areas of distribution of these two morphotypesare nearly identical further adding to the confusionAnother widespread species according to Veron [3] is

Stylophora subseriata (Ehrenberg 1834) also foundacross the Indo-Pacific region whereas the five othermorphospecies of this genus are restricted to the RedSea and the Gulf of Aden (Stylophora kuehlmanniScheer and Pillai 1983 Stylophora danae MilneEdwards and Haime 1850 Stylophora mamillata Scheerand Pillai 1983) to Madagascar (Stylophora madagas-carensis Veron 2000) or to both of these regions (Stylo-phora wellsi Scheer 1964) Hence the pattern ofoccurrence of the various species of Stylophora seems tocontradict strongly the common ldquoCoral Trianglerdquo centerof biodiversity model However recent reports of S

Figure 1 Drawings of the type specimens of S pistillata and S mordax The drawing of the type of S pistillata (left) is from [25] thedrawing of the type of S mordax (right) is from [14]


of 14







Page 3 of 14









Page 4 of 14

001

04NC130


04NC436

04NC071

EU400214 (Taiwan)

07Mad157


04NC145

04Oki195

04NC365

04NC452


07Mad160

04NC440

04NC282

04NC232

04NC253

04NC141


07Mad159

07Mad189

04NC439

07Mad172

04NC199

04NC336

04NC139

04NC379

04NC152

04NC237

04NC131

04NC087

04Oki140

07Mad088

07Mad071

04NC189

07Mad151

04NC170



07Mad086

04NC132

07Mad082

04NC024

04NC233

07Mad161

04NC289

04NC101

04NC105

04NC251

07Mad150

07Mad079

04NC43404NC323

04NC182

04NC009

04Oki136

05Phil53

07Mad188

04NC064

04NC012

04NC324

04NC404


07Mad073

04NC455

07Mad074

04NC230

07Mad070

07Mad087

04NC373

04NC187

07Mad170

04NC266

04NC441

04Oki115

04NC149

04NC140

05Phil19

93100100

100100100

100100100

100100100

835298

100100100

100100100

100100100

100100100

A

B

C



Page 5 of 14

001

07Mad188b

04NC139b

04NC009a

04NC064a

04NC182a

07Mad070

07Mad082

04NC009b

04NC230

04NC266

04NC071b

07Mad172a

04NC199

04NC282b

07Mad161b

04NC439

04NC441a

04NC189a

07Mad074b

04NC452

07Mad087a

04NC434a

05Phil19

04NC141

04NC251b

04NC145b

04NC289a

04NC071a

04NC012b

07Mad189b

07Mad071

04NC434b

04NC189b04NC187

04NC441b

07Mad150

04NC105

07Mad157b

04NC012a

04NC436a

04NC024b

04NC336b

04NC379b

04NC130a

04NC101

04NC087a

04NC149a

07Mad159

04NC152a

04NC289b

04NC404

07Mad073a

04NC130b

04NC140

04NC233b

04NC182b

07Mad087b

07Mad188a

AY722795 (Taiwan)

04NC064b

04Oki115b

04NC251a

04NC323

07Mad088a

04NC087b

04Oki195a

04NC440b

07Mad088b

04NC336a

04Oki140

07Mad189a

04NC149b

07Mad151a

04Oki115a

07Mad086b

04NC132b

07Mad157a

04NC440a

04NC282a

04NC237a

04Oki136b

07Mad160b

07Mad172b

04NC365a

04Oki195b

07Mad074a

07Mad073b

04NC253a

07Mad086a

04NC170

07Mad170

04NC436b

04NC455

07Mad161a

04NC324a04NC365b

05Phil53

04NC253b

04NC024a

04NC233a

04NC152b

07Mad160a

04NC131

04Oki136a

07Mad151b

04NC139a

04NC132a

04NC237b

04NC324b

04NC373

04NC145a

04NC232

04NC379a

07Mad079

10010099

10010099

43--

993100100

99510098

828360

828983

99610097

7958-

808372

7583-

A

B

C



Page 6 of 14





D

005




04Oki140 Okinawa




07Mad150 Madagascar
































































07Mad082 Madagascar
































































07Mad070 Madagascar







































07Mad071 Madagascar















9129979

6937118

94490-

297--

9809966

345-76

9879998

A

B

C



Page 7 of 14








Page 8 of 14










Page 9 of 14










Page 10 of 14








































nr = not recorded


Page 11 of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References







of 14









Page 4 of 14

001

04NC130


04NC436

04NC071

EU400214 (Taiwan)

07Mad157


04NC145

04Oki195

04NC365

04NC452


07Mad160

04NC440

04NC282

04NC232

04NC253

04NC141


07Mad159

07Mad189

04NC439

07Mad172

04NC199

04NC336

04NC139

04NC379

04NC152

04NC237

04NC131

04NC087

04Oki140

07Mad088

07Mad071

04NC189

07Mad151

04NC170



07Mad086

04NC132

07Mad082

04NC024

04NC233

07Mad161

04NC289

04NC101

04NC105

04NC251

07Mad150

07Mad079

04NC43404NC323

04NC182

04NC009

04Oki136

05Phil53

07Mad188

04NC064

04NC012

04NC324

04NC404


07Mad073

04NC455

07Mad074

04NC230

07Mad070

07Mad087

04NC373

04NC187

07Mad170

04NC266

04NC441

04Oki115

04NC149

04NC140

05Phil19

93100100

100100100

100100100

100100100

835298

100100100

100100100

100100100

100100100

A

B

C



Page 5 of 14

001

07Mad188b

04NC139b

04NC009a

04NC064a

04NC182a

07Mad070

07Mad082

04NC009b

04NC230

04NC266

04NC071b

07Mad172a

04NC199

04NC282b

07Mad161b

04NC439

04NC441a

04NC189a

07Mad074b

04NC452

07Mad087a

04NC434a

05Phil19

04NC141

04NC251b

04NC145b

04NC289a

04NC071a

04NC012b

07Mad189b

07Mad071

04NC434b

04NC189b04NC187

04NC441b

07Mad150

04NC105

07Mad157b

04NC012a

04NC436a

04NC024b

04NC336b

04NC379b

04NC130a

04NC101

04NC087a

04NC149a

07Mad159

04NC152a

04NC289b

04NC404

07Mad073a

04NC130b

04NC140

04NC233b

04NC182b

07Mad087b

07Mad188a

AY722795 (Taiwan)

04NC064b

04Oki115b

04NC251a

04NC323

07Mad088a

04NC087b

04Oki195a

04NC440b

07Mad088b

04NC336a

04Oki140

07Mad189a

04NC149b

07Mad151a

04Oki115a

07Mad086b

04NC132b

07Mad157a

04NC440a

04NC282a

04NC237a

04Oki136b

07Mad160b

07Mad172b

04NC365a

04Oki195b

07Mad074a

07Mad073b

04NC253a

07Mad086a

04NC170

07Mad170

04NC436b

04NC455

07Mad161a

04NC324a04NC365b

05Phil53

04NC253b

04NC024a

04NC233a

04NC152b

07Mad160a

04NC131

04Oki136a

07Mad151b

04NC139a

04NC132a

04NC237b

04NC324b

04NC373

04NC145a

04NC232

04NC379a

07Mad079

10010099

10010099

43--

993100100

99510098

828360

828983

99610097

7958-

808372

7583-

A

B

C



Page 6 of 14





D

005




04Oki140 Okinawa




07Mad150 Madagascar
































































07Mad082 Madagascar
































































07Mad070 Madagascar







































07Mad071 Madagascar















9129979

6937118

94490-

297--

9809966

345-76

9879998

A

B

C



Page 7 of 14








Page 8 of 14










Page 9 of 14










Page 10 of 14








































nr = not recorded


Page 11 of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References









of 14

001

04NC130


04NC436

04NC071

EU400214 (Taiwan)

07Mad157


04NC145

04Oki195

04NC365

04NC452


07Mad160

04NC440

04NC282

04NC232

04NC253

04NC141


07Mad159

07Mad189

04NC439

07Mad172

04NC199

04NC336

04NC139

04NC379

04NC152

04NC237

04NC131

04NC087

04Oki140

07Mad088

07Mad071

04NC189

07Mad151

04NC170



07Mad086

04NC132

07Mad082

04NC024

04NC233

07Mad161

04NC289

04NC101

04NC105

04NC251

07Mad150

07Mad079

04NC43404NC323

04NC182

04NC009

04Oki136

05Phil53

07Mad188

04NC064

04NC012

04NC324

04NC404


07Mad073

04NC455

07Mad074

04NC230

07Mad070

07Mad087

04NC373

04NC187

07Mad170

04NC266

04NC441

04Oki115

04NC149

04NC140

05Phil19

93100100

100100100

100100100

100100100

835298

100100100

100100100

100100100

100100100

A

B

C



Page 5 of 14

001

07Mad188b

04NC139b

04NC009a

04NC064a

04NC182a

07Mad070

07Mad082

04NC009b

04NC230

04NC266

04NC071b

07Mad172a

04NC199

04NC282b

07Mad161b

04NC439

04NC441a

04NC189a

07Mad074b

04NC452

07Mad087a

04NC434a

05Phil19

04NC141

04NC251b

04NC145b

04NC289a

04NC071a

04NC012b

07Mad189b

07Mad071

04NC434b

04NC189b04NC187

04NC441b

07Mad150

04NC105

07Mad157b

04NC012a

04NC436a

04NC024b

04NC336b

04NC379b

04NC130a

04NC101

04NC087a

04NC149a

07Mad159

04NC152a

04NC289b

04NC404

07Mad073a

04NC130b

04NC140

04NC233b

04NC182b

07Mad087b

07Mad188a

AY722795 (Taiwan)

04NC064b

04Oki115b

04NC251a

04NC323

07Mad088a

04NC087b

04Oki195a

04NC440b

07Mad088b

04NC336a

04Oki140

07Mad189a

04NC149b

07Mad151a

04Oki115a

07Mad086b

04NC132b

07Mad157a

04NC440a

04NC282a

04NC237a

04Oki136b

07Mad160b

07Mad172b

04NC365a

04Oki195b

07Mad074a

07Mad073b

04NC253a

07Mad086a

04NC170

07Mad170

04NC436b

04NC455

07Mad161a

04NC324a04NC365b

05Phil53

04NC253b

04NC024a

04NC233a

04NC152b

07Mad160a

04NC131

04Oki136a

07Mad151b

04NC139a

04NC132a

04NC237b

04NC324b

04NC373

04NC145a

04NC232

04NC379a

07Mad079

10010099

10010099

43--

993100100

99510098

828360

828983

99610097

7958-

808372

7583-

A

B

C



Page 6 of 14





D

005




04Oki140 Okinawa




07Mad150 Madagascar
































































07Mad082 Madagascar
































































07Mad070 Madagascar







































07Mad071 Madagascar















9129979

6937118

94490-

297--

9809966

345-76

9879998

A

B

C



Page 7 of 14








Page 8 of 14










Page 9 of 14










Page 10 of 14








































nr = not recorded


Page 11 of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References

001

04NC130


04NC436

04NC071

EU400214 (Taiwan)

07Mad157


04NC145

04Oki195

04NC365

04NC452


07Mad160

04NC440

04NC282

04NC232

04NC253

04NC141


07Mad159

07Mad189

04NC439

07Mad172

04NC199

04NC336

04NC139

04NC379

04NC152

04NC237

04NC131

04NC087

04Oki140

07Mad088

07Mad071

04NC189

07Mad151

04NC170



07Mad086

04NC132

07Mad082

04NC024

04NC233

07Mad161

04NC289

04NC101

04NC105

04NC251

07Mad150

07Mad079

04NC43404NC323

04NC182

04NC009

04Oki136

05Phil53

07Mad188

04NC064

04NC012

04NC324

04NC404


07Mad073

04NC455

07Mad074

04NC230

07Mad070

07Mad087

04NC373

04NC187

07Mad170

04NC266

04NC441

04Oki115

04NC149

04NC140

05Phil19

93100100

100100100

100100100

100100100

835298

100100100

100100100

100100100

100100100

A

B

C



of 14

001

07Mad188b

04NC139b

04NC009a

04NC064a

04NC182a

07Mad070

07Mad082

04NC009b

04NC230

04NC266

04NC071b

07Mad172a

04NC199

04NC282b

07Mad161b

04NC439

04NC441a

04NC189a

07Mad074b

04NC452

07Mad087a

04NC434a

05Phil19

04NC141

04NC251b

04NC145b

04NC289a

04NC071a

04NC012b

07Mad189b

07Mad071

04NC434b

04NC189b04NC187

04NC441b

07Mad150

04NC105

07Mad157b

04NC012a

04NC436a

04NC024b

04NC336b

04NC379b

04NC130a

04NC101

04NC087a

04NC149a

07Mad159

04NC152a

04NC289b

04NC404

07Mad073a

04NC130b

04NC140

04NC233b

04NC182b

07Mad087b

07Mad188a

AY722795 (Taiwan)

04NC064b

04Oki115b

04NC251a

04NC323

07Mad088a

04NC087b

04Oki195a

04NC440b

07Mad088b

04NC336a

04Oki140

07Mad189a

04NC149b

07Mad151a

04Oki115a

07Mad086b

04NC132b

07Mad157a

04NC440a

04NC282a

04NC237a

04Oki136b

07Mad160b

07Mad172b

04NC365a

04Oki195b

07Mad074a

07Mad073b

04NC253a

07Mad086a

04NC170

07Mad170

04NC436b

04NC455

07Mad161a

04NC324a04NC365b

05Phil53

04NC253b

04NC024a

04NC233a

04NC152b

07Mad160a

04NC131

04Oki136a

07Mad151b

04NC139a

04NC132a

04NC237b

04NC324b

04NC373

04NC145a

04NC232

04NC379a

07Mad079

10010099

10010099

43--

993100100

99510098

828360

828983

99610097

7958-

808372

7583-

A

B

C



Page 6 of 14





D

005




04Oki140 Okinawa




07Mad150 Madagascar
































































07Mad082 Madagascar
































































07Mad070 Madagascar







































07Mad071 Madagascar















9129979

6937118

94490-

297--

9809966

345-76

9879998

A

B

C



Page 7 of 14








Page 8 of 14










Page 9 of 14










Page 10 of 14








































nr = not recorded


Page 11 of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References

001

07Mad188b

04NC139b

04NC009a

04NC064a

04NC182a

07Mad070

07Mad082

04NC009b

04NC230

04NC266

04NC071b

07Mad172a

04NC199

04NC282b

07Mad161b

04NC439

04NC441a

04NC189a

07Mad074b

04NC452

07Mad087a

04NC434a

05Phil19

04NC141

04NC251b

04NC145b

04NC289a

04NC071a

04NC012b

07Mad189b

07Mad071

04NC434b

04NC189b04NC187

04NC441b

07Mad150

04NC105

07Mad157b

04NC012a

04NC436a

04NC024b

04NC336b

04NC379b

04NC130a

04NC101

04NC087a

04NC149a

07Mad159

04NC152a

04NC289b

04NC404

07Mad073a

04NC130b

04NC140

04NC233b

04NC182b

07Mad087b

07Mad188a

AY722795 (Taiwan)

04NC064b

04Oki115b

04NC251a

04NC323

07Mad088a

04NC087b

04Oki195a

04NC440b

07Mad088b

04NC336a

04Oki140

07Mad189a

04NC149b

07Mad151a

04Oki115a

07Mad086b

04NC132b

07Mad157a

04NC440a

04NC282a

04NC237a

04Oki136b

07Mad160b

07Mad172b

04NC365a

04Oki195b

07Mad074a

07Mad073b

04NC253a

07Mad086a

04NC170

07Mad170

04NC436b

04NC455

07Mad161a

04NC324a04NC365b

05Phil53

04NC253b

04NC024a

04NC233a

04NC152b

07Mad160a

04NC131

04Oki136a

07Mad151b

04NC139a

04NC132a

04NC237b

04NC324b

04NC373

04NC145a

04NC232

04NC379a

07Mad079

10010099

10010099

43--

993100100

99510098

828360

828983

99610097

7958-

808372

7583-

A

B

C



of 14





D

005




04Oki140 Okinawa




07Mad150 Madagascar
































































07Mad082 Madagascar
































































07Mad070 Madagascar







































07Mad071 Madagascar















9129979

6937118

94490-

297--

9809966

345-76

9879998

A

B

C



Page 7 of 14








Page 8 of 14










Page 9 of 14










Page 10 of 14








































nr = not recorded


Page 11 of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References





D

005




04Oki140 Okinawa




07Mad150 Madagascar
































































07Mad082 Madagascar
































































07Mad070 Madagascar







































07Mad071 Madagascar















9129979

6937118

94490-

297--

9809966

345-76

9879998

A

B

C



of 14








Page 8 of 14










Page 9 of 14










Page 10 of 14








































nr = not recorded


Page 11 of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References








of 14










Page 9 of 14










Page 10 of 14








































nr = not recorded


Page 11 of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References










of 14










Page 10 of 14








































nr = not recorded


Page 11 of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References










of 14








































nr = not recorded


Page 11 of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References








































nr = not recorded


of 14

























Page 12 of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References

























of 14















































Page 13 of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References















































of 14





























Page 14 of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References





























of 14

Abstract

Background

Results

Conclusions

Background

Results




Discussion





Conclusions

Methods





Acknowledgements

Author details


References