RESEARCH ARTICLE Open Access
Incongruence between morphotypes and
genetically delimited species in the coral genus
Stylophora: phenotypic plasticity, morphological
convergence, morphological stasis or interspecific
hybridization?
Jean-François Flot1,2,3,4*, Jean Blanchot5, Loïc Charpy6, Corinne Cruaud2, Wilfredo Y Licuanan7, Yoshikatsu Nakano8,
Claude Payri9 and Simon Tillier3
Abstract
Background: Morphological data suggest that, unlike most other groups of marine organisms, scleractinian corals
of 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, we
conducted a genetic survey of Stylophora corals collected in Madagascar, Okinawa, the Philippines and New
Caledonia 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 a
haploweb analysis of nuclear ITS2 sequences in delimiting three species among our dataset: species A and B are
found in Madagascar whereas species C occurs in Okinawa, the Philippines and New Caledonia. Comparison of
ITS1 sequences from these three species with data available online suggests that species C is also found on the
Great Barrier Reef, in Malaysia, in the South China Sea and in Taiwan, and that a distinct species D occurs in the
Red Sea. Shallow-water morphs of species A correspond to the morphological description of Stylophora
madagascarensis, species B presents the morphology of Stylophora mordax, whereas species C comprises various
morphotypes including Stylophora pistillata and Stylophora mordax.
Conclusions: Genetic analysis of the coral genus Stylophora reveals species boundaries that are not congruent
with morphological traits. Of the four hypotheses that may explain such discrepancy (phenotypic plasticity,
morphological stasis, morphological convergence, and interspecific hybridization), the first two appear likely to play
a role but the fourth one is rejected since mitochondrial and nuclear markers yield congruent species delimitations.
The position of the root in our molecular phylogenies suggests that the center of origin of Stylophora is located in
the western Indian Ocean, which probably explains why this genus presents a higher biodiversity in the
westernmost part of its area of distribution than in the “Coral Triangle”.
* Correspondence: jflot@uni-goettingen.de
1Courant Research Center “Geobiology”, University of Göttingen,
Goldschmidtstraße 3, 37077 Göttingen, Germany
Full list of author information is available at the end of the article
Flot et al. BMC Ecology 2011, 11:22
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© 2011 Flot et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Background
The reason why most marine life forms, including cor-
als, display their peak of biodiversity in the so called
‘Coral Triangle’ in Southeast Asia remains mysterious
and much debated [1,2]. The rare examples of sea crea-
tures that do not conform to this general pattern may
offer information crucial for our understanding of its
root causes, provided that a solid taxonomic framework
is available to interpret their present and past distribu-
tion (which is unfortunately rarely the case). Several
such exceptions to the ‘Coral Triangle’ rule can be
found in the scleractinian coral family Pocilloporidae
that comprises the three genera Pocillopora, Seriatopora
and Stylophora: although morphospecies of Seriatopora
are most diverse in the Coral Triangle and therefore
seem to follow the rule, Pocillopora “has what appears
to be many regional endemics, especially in the central
and far eastern Pacific” and Stylophora “has a higher
diversity in the western Indian Ocean and Red Sea than
in the central Indo-Pacific” [3]. However, ongoing
genetic studies of species boundaries in Pocillopora and
Seriatopora suggest that, even though morphological
descriptions of pocilloporid corals appear well founded
in 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 “lab rat” Stylophora pistillata (Esper,
1797), without delving into the delineation of interspeci-
fic boundaries [12,13]. S. pistillata was originally
described “aus den ostindischen Meeren” [14] (from the
East Indian seas, in the “Coral Triangle”) as character-
ized by short twisted dichotomous branches
("Madrepora aggregata, ramulis conglomeratis brevibus,
dichotomis; stellis crenatis profundis, pistillo in medio
elongato erecto” [14]; Figure 1, left side), and is consid-
ered to occur over the entire Indo-Pacific (from the Red
Sea to Polynesia). The distinction between S. pistillata
and Stylophora mordax (Dana, 1846) has long been
debated in the literature: S. mordax is considered by
some authors as a junior synonym of S. pistillata
[15,16,3] and by others as a distinct species [17-24]. S.
mordax was originally described from Fiji as having
“branches nearly simple, much compressed, not thinner
at apex, scarcely flabellate, 1/2 to 1 inch broad, and 1/3
of an inch thick; polyps with a pale yellowish disk, and
short tentacles of a bright green colour, deep brown at
base. Corallum with the cells strongly vaulted, and the
surface, therefore, decidedly scabrous” (Figure 1, right
side) [25]; however, intermediary forms between S. mor-
dax and S. pistillata are commonly found in the field,
and the areas of distribution of these two morphotypes
are nearly identical, further adding to the confusion.
Another widespread species according to Veron [3] is
Stylophora subseriata (Ehrenberg, 1834), also found
across the Indo-Pacific region, whereas the five other
morphospecies of this genus are restricted to the Red
Sea and the Gulf of Aden (Stylophora kuehlmanni
Scheer and Pillai, 1983; Stylophora danae Milne
Edwards and Haime, 1850; Stylophora mamillata Scheer
and Pillai, 1983), to Madagascar (Stylophora madagas-
carensis Veron, 2000) or to both of these regions (Stylo-
phora wellsi Scheer, 1964). Hence, the pattern of
occurrence of the various species of Stylophora seems to
contradict strongly the common “Coral Triangle” center
of 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], the
drawing of the type of S. mordax (right) is from [14].
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danae and S. kuehlmanni from the Philippines [26] have
started to question this pattern, raising further concern
that morphological species may not correspond to actual
genetic 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 [28,29] under the influence of envir-
onmental parameters such as gravity [30] and water
flow [31]. Moreover, evidence of intergeneric hybridiza-
tion between Stylophora and Pocillopora has been
reported in the literature [32], suggesting that hybridiza-
tion may a fortiori occur among various Stylophora spe-
cies. Finally, morphological stasis and phenotypic
convergence, albeit not reported yet in the genus Stylo-
phora, may lead to further underestimation of the actual
number of species in this genus by causing several
genetically distinct species to be lumped under a single
name. To find out whether phenotypic plasticity, inter-
specific hybridization, morphological stasis and phenoty-
pic convergence obscure the taxonomy of Stylophora,
we conducted a genetic analysis of 70 corals of this
genus collected in Madagascar, Okinawa, the Philippines
and New Caledonia; published sequences of Stylophora
individuals from Australia, Malaysia, the South China
Sea, Taiwan and Okinawa were also scrutinized (Figure
2). As a first step towards a future taxonomic revision
of this genus, we report here the result of our attempt
to determine the true number of Stylophora species
occurring at these various locations and to test whether
the unusual biogeographic pattern reported for this
genus holds true.
Results
Phylogenetic analysis of mitochondrial sequences reveals
the presence of two Stylophora clades in Madagascar vs.
a single one in the Pacific Ocean
We successfully sequenced from all individuals sampled
a portion of the mitochondrial ORF and the putative
control region (CR), two markers that had previously
been characterised as highly variable in the closely
related genus Pocillopora [33] (but see [34,5] 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 where
we collected samples, whereas empty circles mark locations where sequences were obtained from the literature. The area of distribution of the
genus Stylophora (modified from [3] and [68]) is shown in blue (basemap from http://d-maps.com/).
Flot et al. BMC Ecology 2011, 11:22
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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 and
parsimony), whereas three clades of Stylophora emerged
from the phylogenetic analysis: clade A comprised 12
individuals, all from Madagascar, clade B comprised 7
individuals, also all from Madagascar, whereas clade C
comprised the remaining 51 individuals from New Cale-
donia, Okinawa and the Philippines plus one previously
published sequence (from the complete mitochondrial
genome 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 that
these three clades represent distinct species
We obtained nuclear internal transcribed spacer 2 (ITS2)
sequences from all individuals sampled, and analyzed
them together with the single published Stylophora ITS2
sequence available from GenBank (also from a Stylophora
pistillata 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 a
“regular” single-copy nuclear marker, with each indivi-
dual harboring either one or two sequence types. More-
over, most ITS2 sequences types found co-occurring in
some individuals were also observed occurring alone in
other coral colonies, suggesting that these sequence types
were allelic and segregated in a Mendelian fashion: for
this reason we decided to call “heterozygotes” all indivi-
duals found to possess two different ITS2 types, even
though we could not be totally sure that all ITS2
sequences obtained were really allelic (in the closely
related genus Pocillopora, for instance, three ITS2
sequences 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 these
clades were conspecific and which ones belonged to dif-
ferent species, curves were added connecting sequences
found co-occurring in heterozygous individuals, thereby
converting the ITS2 tree into a haploweb [9]. This
approach revealed three distinct pools of ITS2
sequences, corresponding to the three clades obtained
from mitochondrial DNA. The monophyly of clades A
and B in our ITS2 dataset received very strong bootstrap
support using all three methods, whereas the monophyly
of clade C was only weakly supported using maximum
likelihood (<50% bootstrap support) and not at all sup-
ported using neighbor-joining and parsimony. None of
the 43 heterozygous individuals in our dataset contained
ITS2 sequences from two different clades: therefore,
clades A, B and C correspond to three distinct species
according to the mutual allelic exclusivity criterion
[37,9].
Phylogenetic analysis of nuclear ITS1 sequences reveals
the existence of a fourth species of Stylophora in the Red
Sea
Even though the ITS2 of scleractinian corals is generally
easier to align and more informative than ITS1 [35] and is
therefore more commonly used, the vast majority of ITS
sequences available to date for the genus Stylophora are
ITS1 sequences. In order to compare the results of our
study with those previously published data, the ITS1
regions of 14 representative individuals from our molecu-
larly defined species A, B and C were sequenced and
aligned with sequences available in GenBank. Alignment
was indeed markedly more uncertain for ITS1 than for
ITS2, and the resulting alignment was rather short (345
positions including many gaps, vs. 700 positions for ITS2
and 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 of
ITS1 sequences: the monophyly of species B was very
strongly supported (>98% bootstrap support using all
three methods), whereas the monophyly of species C
received weaker bootstrap support and the monophyly of
species A was very weakly supported. All previously pub-
lished ITS1 sequences from Australia, Malaysia, the
South China Sea, Taiwan and Japan turned out to belong
to species C, whereas all published Stylophora sequences
from the Red Sea fell in a well supported fourth clade D
(>90% bootstrap support using maximum likelihood and
neighbor-joining) that can be considered a distinct spe-
cies following the criterion of reciprocal monophyly.
Discussion
Haplowebs are useful tools to delineate species
The present study confirms the usefulness of our
recently proposed haploweb approach to deal with
sequences of nuclear markers [9]. Whereas the corre-
sponding phylogenetic tree supported the delineation of
clades A and B but revealed a large number of clades
among our sequences from the Pacific Ocean, haploweb
analysis (Figure 4) showed that these various Pacific
Ocean clades are actually conspecific (since many het-
erozygous individuals harbor sequences from two differ-
ent clades). The morphology of species C was only very
weakly supported in the maximum-likelihood phylogeny
(with a bootstrap value of only 43%) and not at all sup-
ported using neighbor-joining and parsimony: therefore,
this species would probably not have been detected in
our ITS2 data if we had not taken into account the
information provided by the co-occurrence of
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0.01
04NC130
Pocillopora meandrina (02Oahu18)
04NC436
04NC071
EU400214 (Taiwan)
07Mad157
Seriatopora Cluster 3 (04NC360)
04NC145
04Oki195
04NC365
04NC452
Pocillopora damicornis (02Oahu08)
07Mad160
04NC440
04NC282
04NC232
04NC253
04NC141
Seriatopora Cluster 2 (04Oki112)
07Mad159
07Mad189
04NC439
07Mad172
04NC199
04NC336
04NC139
04NC379
04NC152
04NC237
04NC131
04NC087
04Oki140
07Mad088
07Mad071
04NC189
07Mad151
04NC170
Pocillopora eydouxi (02Oahu32)
Seriatopora Cluster 1 (04NC432)
07Mad086
04NC132
07Mad082
04NC024
04NC233
07Mad161
04NC289
04NC101
04NC105
04NC251
07Mad150
07Mad079
04NC434
04NC323
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
93/100/100
100/100/100
100/100/100
100/100/100
83/52/98
100/100/100
100/100/100
100/100/100
100/100/100
A
B
C
Figure 3 Phylogenetic tree of mitochondrial DNA sequences (ORF and CR combined). Outgroup sequences are from [6] and [7], whereas
one ingroup sequence comes from the complete mitochondrial genome of one Stylophora pistillata individual from the Penghu Islands near
Taiwan [34]. This haplotree was generated with PhyML using the TPM3uf+G model suggested by jModelTest. Bootstrap values obtained using
maximum likelihood, neighbor-joining and parsimony (1000 replicates each) are displayed next to each node. The three main clades are
delineated with brackets and labeled A, B and C.
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0.01
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
04NC189b
04NC187
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
04NC324a
04NC365b
05Phil53
04NC253b
04NC024a
04NC233a
04NC152b
07Mad160a
04NC131
04Oki136a
07Mad151b
04NC139a
04NC132a
04NC237b
04NC324b
04NC373
04NC145a
04NC232
04NC379a
07Mad079
100/100/99
100/100/99
43/-/-
99.3/100/100
99.5/100/98
82/83/60
82/89/83
99.6/100/97
79/58/-
80/83/72
75/83/-
A
B
C
Figure 4 Haploweb of ITS2 sequences. This graph was derived from a phylogenetic tree (obtained with PhyML using the TIM2+G model
suggested by jModelTest) by drawing curves connecting haplotypes found co-occurring in heterozygous individuals [9]. The sequence of one
Stylophora pistillata individual from the Penghu Islands near Taiwan [35] is included in the figure, and bootstrap values obtained using maximum
likelihood, neighbor-joining and parsimony (1000 replicates each) are displayed next to each node. The position of the root was inferred from
the mitochondrial DNA phylogeny (Figure 3). This approach delineated three reproductively isolated pools of Stylophora sequences, labeled A, B
and C as each of these groups corresponded to one of the mitochondrial clades of Figure 3.
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phylogenetically distant alleles in some individuals. In
contrast, haploweb analysis delineated three groups of
alleles among our ITS2 sequences, a result perfectly
congruent with the mitochondrial phylogeny obtained
from the same set of individuals.
Unlike in our previous article [9], the haploweb pre-
sented here was built on a tree rather than a network.
Indeed, tree-based haplowebs are more straightforward
to draw than their network-based counterparts, and are
also more informative since they display the genotype of
each sequenced individual. However, precisely due to
their larger information content, tree-based haplowebs
tend to become messy when dealing with large datasets
and/or non-monophyletic species: in such cases, net-
work-based haplowebs often turn out to be faster to
draw and easier to interpret than tree-based ones.
Are molecularly delimited species of Stylophora
congruent with morphology?
Most of the 12 individuals from species A displayed the
characteristic morphology of S. madagascarensis (some
examples are shown on Figure 6): “colonies have thin
D
0.05
AF416161 Stylophora pistillata brown Heron Island
AJ619830 Stylophora pistillata Red Sea
07Mad157a Madagascar
04Oki140 Okinawa
AF416224 Stylophora pistillata pink Sesoko
AJ619838 Stylophora pistillata Red Sea
AF416170 Stylophora pistillata brown One Tree Island
07Mad150 Madagascar
AJ865618 Stylophora pistillata Red Sea
AF416150 Stylophora pistillata pink Akajima
AF416229 Stylophora pistillata brown Terengganu
AF416151 Stylophora pistillata pink Akajima
AF416147 Stylophora pistillata brown Akajima
AF416194 Stylophora pistillata brown Moulter Cay
AF416222 Stylophora pistillata pink Sesoko
AJ619837 Stylophora pistillata Red Sea
AF416219 Stylophora pistillata brown Sesoko
AF416162 Stylophora pistillata pink Heron Island
AJ619844 Stylophora pistillata Red Sea
AJ865621 Stylophora pistillata Red Sea
AJ865631 Stylophora pistillata Red Sea
AJ619831 Stylophora pistillata Red Sea
AF416202 Stylophora pistillata pink Moulter Cay
AF416227 Stylophora pistillata brown Terengganu
AJ619841 Stylophora pistillata Red Sea
07Mad160a Madagascar
AF416182 Stylophora pistillata brown Raine Island
AJ619817 Stylophora pistillata Red Sea
AF416232 Stylophora pistillata brown Terengganu
AJ619811 Stylophora pistillata Red Sea
AF416193 Stylophora pistillata brown Raine Island
AF416231 Stylophora pistillata brown Terengganu
AF416201 Stylophora pistillata brown Moulter Cay
AF416173 Stylophora pistillata brown One Tree Island
AF416196 Stylophora pistillata brown Moulter Cay
AJ619829 Stylophora pistillata Red Sea
AJ619850 Stylophora pistillata Red Sea
AF416156 Stylophora pistillata brown Heron Island
AJ619820 Stylophora pistillata Red Sea
AJ865608 Stylophora pistillata Red Sea
AJ865625 Stylophora pistillata Red Sea
AF416209 Stylophora pistillata pink Sabah
AF416154 Stylophora pistillata pink Akajima
AF416181 Stylophora pistillata brown Raine Island
AF416175 Stylophora pistillata pink One Tree Island
AF416210 Stylophora pistillata pink Sabah
AJ619840 Stylophora pistillata Red Sea
AJ619832 Stylophora pistillata Red Sea
AF416164 Stylophora pistillata pink Heron Island
AJ865633 Stylophora pistillata Red Sea
AF416187 Stylophora pistillata brown Raine Island
AJ619808 Stylophora pistillata Red Sea
AF416199 Stylophora pistillata brown Moulter Cay
AF416172 Stylophora pistillata brown One Tree Island
AJ619816 Stylophora pistillata Red Sea
AJ619814 Stylophora pistillata Red Sea
AF416171 Stylophora pistillata brown One Tree Island
AF416179 Stylophora pistillata pink One Tree Island
AF416176 Stylophora pistillata pink One Tree Island
AJ865630 Stylophora pistillata Red Sea
AF416183 Stylophora pistillata brown Raine Island
AF416212 Stylophora pistillata pink Sabah
AF416228 Stylophora pistillata brown Terengganu
AJ865614 Stylophora pistillata Red Sea
AF416146 Stylophora pistillata brown Akajima
04NC101 New Caledonia
AF416216 Stylophora pistillata brown Sesoko
AF416206 Stylophora pistillata brown Sabah
07Mad160b Madagascar
AJ619845 Stylophora pistillata Red Sea
AJ865605 Stylophora pistillata Red Sea
AJ619833 Stylophora pistillata Red Sea
07Mad082 Madagascar
AF416178 Stylophora pistillata pink One Tree Island
AF416223 Stylophora pistillata pink Sesoko
AJ619825 Stylophora pistillata Red Sea
AF416163 Stylophora pistillata pink Heron Island
AF416157 Stylophora pistillata brown Heron Island
AF416155 Stylophora pistillata pink Akajima
AJ619839 Stylophora pistillata Red Sea
AF416217 Stylophora pistillata brown Sesoko
AF038909 Stylophora pistillata One Tree Island
AJ619848 Stylophora pistillata Red Sea
AJ865616 Stylophora pistillata Red Sea
07Mad073a Madagascar
AF416159 Stylophora pistillata brown Heron Island
04NC140a New Caledonia
HM013855 Seriatopora hystrix Xisha Islands
AJ619852 Stylophora pistillata Red Sea
AF416218 Stylophora pistillata brown Sesoko
AF416204 Stylophora pistillata brown Sabah
AJ619846 Stylophora pistillata Red Sea
AJ619819 Stylophora pistillata Red Sea
AJ619828 Stylophora pistillata Red Sea
AJ619810 Stylophora pistillata Red Sea
AJ619818 Stylophora pistillata Red Sea
AJ619826 Stylophora pistillata Red Sea
AJ619835 Stylophora pistillata Red Sea
AJ619806 Stylophora pistillata Red Sea
AF038908 Stylophora pistillata One Tree Island
AJ865620 Stylophora pistillata Red Sea
05Phil53 Philippines
AF416160 Stylophora pistillata brown Heron Island
AF038907 Stylophora pistillata One Tree Island
AF416177 Stylophora pistillata pink One Tree Island
AF416168 Stylophora pistillata brown One Tree Island
AJ865617 Stylophora pistillata Red Sea
AF416174 Stylophora pistillata pink One Tree Island
AJ865626 Stylophora pistillata Red Sea
AF416208 Stylophora pistillata brown Sabah
04NC140b New Caledonia
AJ865623 Stylophora pistillata Red Sea
AF416189 Stylophora pistillata pink Raine Island
AF416220 Stylophora pistillata brown Sesoko
AJ865624 Stylophora pistillata Red Sea
AF416184 Stylophora pistillata brown Raine Island
AJ865627 Stylophora pistillata Red Sea
AJ619822 Stylophora pistillata Red Sea
AF416167 Stylophora pistillata pink Heron Island
AJ619842 Stylophora pistillata Red Sea
AJ865622 Stylophora pistillata Red Sea
HM013847 Pocillopora damicornis Xisha Islands
AY722795 Stylophora pistillata Penghu Island
AJ619824 Stylophora pistillata Red Sea
AF416226 Stylophora pistillata pink Sesoko
AJ865607 Stylophora pistillata Red Sea
AJ619805 Stylophora pistillata Red Sea
AJ619807 Stylophora pistillata Red Sea
AF416213 Stylophora pistillata pink Sabah
AJ865606 Stylophora pistillata Red Sea
AF416207 Stylophora pistillata brown Sabah
AJ619827 Stylophora pistillata Red Sea
AF416203 Stylophora pistillata brown Sabah
07Mad157b Madagascar
AJ619836 Stylophora pistillata Red Sea
07Mad073b Madagascar
AF416192 Stylophora pistillata brown Raine Island
AJ619812 Stylophora pistillata Red Sea
07Mad070 Madagascar
04NC105 New Caledonia
AF416195 Stylophora pistillata brown Moulter Cay
AJ865604 Stylophora pistillata Red Sea
HM013856 Stylophora pistillata Xisha Islands
AJ619843 Stylophora pistillata Red Sea
AJ619834 Stylophora pistillata Red Sea
07Mad188a Madagascar
AF416152 Stylophora pistillata pink Akajima
AF416186 Stylophora pistillata brown Raine Island
AJ865628 Stylophora pistillata Red Sea
AF416205 Stylophora pistillata brown Sabah
AF416149 Stylophora pistillata brown Akajima
AJ619821 Stylophora pistillata Red Sea
07Mad188b Madagascar
AJ619813 Stylophora pistillata Red Sea
AJ619847 Stylophora pistillata Red Sea
AJ619809 Stylophora pistillata Red Sea
AJ865615 Stylophora pistillata Red Sea
AF416225 Stylophora pistillata pink Sesoko
AF416190 Stylophora pistillata brown Raine Island
AJ865610 Stylophora pistillata Red Sea
AF416198 Stylophora pistillata brown Moulter Cay
AJ865629 Stylophora pistillata Red Sea
AF416221 Stylophora pistillata pink Sesoko
AF416145 Stylophora pistillata brown Akajima
AJ865611 Stylophora pistillata Red Sea
AF416169 Stylophora pistillata brown One Tree Island
AF416148 Stylophora pistillata brown Akajima
AF416214 Stylophora pistillata pink Sabah
AJ865613 Stylophora pistillata Red Sea
AJ865609 Stylophora pistillata Red Sea
AF416158 Stylophora pistillata brown Heron Island
AJ865619 Stylophora pistillata Red Sea
AF416165 Stylophora pistillata pink Heron Island
AF416191 Stylophora pistillata brown Raine Island
AJ619815 Stylophora pistillata Red Sea
AF416215 Stylophora pistillata brown Sesoko
AF416153 Stylophora pistillata pink Akajima
07Mad071 Madagascar
AF416185 Stylophora pistillata pink Raine Island
05Phil19 Philippines
AF416200 Stylophora pistillata brown Moulter Cay
AJ619851 Stylophora pistillata Red Sea
AJ619849 Stylophora pistillata Red Sea
AJ865632 Stylophora pistillata Red Sea
AF416211 Stylophora pistillata pink Sabah
AF416188 Stylophora pistillata brown Raine Island
AF416197 Stylophora pistillata brown Moulter Cay
AJ865612 Stylophora pistillata Red Sea
AF416230 Stylophora pistillata brown Terengganu
AJ619823 Stylophora pistillata Red Sea
AF416166 Stylophora pistillata pink Heron Island
AF416144 Stylophora pistillata brown Akajima
91.2/99/79
69.3/71/18
94.4/90/-
29.7/-/-
98.0/99/66
34.5/-/76
98.7/99/98
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 C,
data from published articles [69,12,35,13] and from an unpublished study by Feng You and Hui Huang [GenBank: HM013847, HM013855,
HM013856] were included in the haplotree (generated with PhyML using the TPM2+G model suggested by jModelTest).
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(up to 5 mm diameter), straight compact branches. Cor-
allites are crowded and uniformly spaced on branch
sides and ends. They have a slight development of
hoods towards branch ends. They have small style-like
columellae and six primary septa which are fused with
the columellae. The coenosteum is covered with fine
spicules. Tentacles are not extended during the day.
Colour: Uniform tan, sometimes with pinkish branch
bases.” [38]. Since Veron’s holotype was collected from
the very same location in Madagascar where we con-
ducted our sampling ("approximately 4 m depth, Tuléar,
south-west Madagascar” [38]), there is little doubt that
species A and S. madagascarensis are conspecific.
According to Veron’s description this species was
“recorded only from shallow reef environments exposed
to some wave action and, in south-west Madagascar, in
shallow sheltered lagoons” [38]. In contrast, we found
this species down to a depth of 30 meters, where it dis-
played less compact and thicker branches compared
with the typical S. madagacarensis morphology (Figure
7): hence, S. madagascarensis appears to be more ecolo-
gically widespread and morphologically variable than
previously thought.
All 7 individuals of species B from Madagascar had
flattened stout branches (Figure 8). This corresponds
unambiguously to the morphological description of S.
mordax, but since S. mordax was originally described
from Fiji in the Pacific Ocean it is dubious whether this
name may be assigned to species B.
Species C gathered all individuals collected from loca-
tions in the Pacific Ocean. It is characterized by exten-
sive morphological variation, comprising individuals
attributable to S. pistillata and others attributable to S.
Figure 6 Morphology of species A. From top to bottom: colonies
07Mad074 sampled at 5 meters depth, colony 07Mad086 sampled
at 6 meters depth and colony 07Mad170 sampled at 11 meters
depth display the typical morphology of S. madagascarensis (thin,
straight compact branches with small hoods). The green wire on
the pictures of the skeletons has a diameter of 0.9 mm.
Figure 7 Deep-water morphs of species A. Colony 07Mad088
(left) and colony 07Mad097 (right) were both sampled at 30 meters
depth and have thicker, less compact branches compared with
typical S. madagascarensis morphology.
Figure 8 Morphology of species B. From top to bottom: colony
07Mad150 collected at 8 meters depth, colony 07Mad071 collected
at 7 meters depth and colony 07Mad160 collected at 3 meters
depth all display the typical morphology of S. mordax (short,
compressed branches with well-developed hoods). The green wire
on the pictures of the skeletons has a diameter of 0.9 mm.
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mordax, as well as morphotypes somehow intermediary
in shape (Figure 9). Several colonies of this species col-
lected in a very muddy location (Banc des Japonais in
New Caledonia) displayed a peculiar phenotype charac-
terized by extremely elongated thin branches (Figure
10). Published sequences of S. pistillata from Taiwan
(ITS2, ORF) and from Okinawa, Taiwan, the South
China Sea, Malaysia and Australia (ITS1) grouped with
our sequences from species C, further supporting its
putative identification as S. pistillata.
Finally, all published ITS1 sequences from the Red Sea
fell in a distinct well-supported species D. The coral
colonies sequenced were collected at depths of 2-4
meters in the Gulf of Aqaba and reported under the
name S. pistillata [13], but according to another study
the main morphospecies of Stylophora found in shallow
waters in Aqaba is actually S. mordax [21]: therefore,
species D probably comprises a mixture of S. pistillata
and S. mordax morphotypes. Since the type localities of
S. pistillata and S. mordax are both in the Pacific
Ocean, 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 authors
as a synonym of S. pistillata [27]).
What causes the discrepancy between morphological and
molecular species delimitations in Stylophora?
Our study revealed extensive morphological variation
within species A and C: a more detailed genetic investi-
gation using a larger number of variable markers such
as microsatellites will be required to find out whether
these variations are due to phenotypic plasticity or to
underlying intraspecific genetic differences. However,
phenotypic plasticity is well documented in Stylophora
[30,31,28,29] and is therefore likely to be responsible for
at least part of the observed morphological variations.
The occurrence of the S. mordax morphotype in both
species B and C can hardly be explained by intra-speci-
fic variation alone, but may rather result from phenoty-
pic convergence (whereby two non-sister species
independently evolve similar morphologies) and/or mor-
phological stasis (whereby the appearance of the com-
mon ancestor of two sister species is passed on to both
of them). Since the sister-species relationship between B
and C was very strongly supported by all molecular
markers analyzed, morphological stasis appears more
likely than phenotypic convergence to explain the simi-
lar appearance of species B and of some morphs of spe-
cies C.
Even though the data available are not yet sufficient to
pin down completely the causes of the interspecific
morphological similarities and intraspecific phenotypic
variations observed in Stylophora, the observed congru-
ence between nuclear and mitochondrial phylogenies
allows us to reject the hypothesis that hybridization
could be responsible for the discrepancy between mor-
phological and genetic species boundaries in this genus.
This contrasts with previous reports that hybridization
may be rampant in corals (e.g. [39-50]); instead, our
results concur with two recent articles on the closely
related genus Pocillopora [9,11] in suggesting that many
such reports actually result from improper delineation
Figure 9 Morphology of species C. From top to bottom: colony
04NC253 was collected at 19 meters depth and displays the typical
S. mordax morphology; colony 04NC323 was collected at 6 meters
depth and displays the typical S. pistillata morphology; colony
04NC282 was collected at 6 meters depth and is intermediate in
shape between S. mordax and S. pistillata. The distance between
two white lines in the background is 30 mm.
Figure 10 Extremely elongated morph of species C from a very
muddy site in New Caledonia. Colonies 04NC149 (left) and
04NC170 (right) were sampled at 16 and 15 meters depth at the
Banc des Japonais in New Caledonia.
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of species boundaries and not from actual introgression
between distinct genetic entities.
A new light on the biogeography and biodiversity of
Stylophora in the Indo-Pacific Ocean
Surprisingly, the incongruence between morphological
species delimitations and genetic species boundaries
revealed in our study does not seem to affect the general
picture of the biogeographic distribution of Stylophora
species: with a least three species in the westernmost
part of its area of occurrence versus a single one so far
in the “Coral Triangle”, Stylophora stands confirmed as
a blatant exception to the usual biodiversity pattern
observed in tropical marine invertebrates. The mito-
chondrial and ITS2 phylogenies of Stylophora comprise
only species A, B and C and do not contradict the
topology of the more complete (but less resolved) ITS1
phylogeny: the sister-group relationship of species B and
C is strongly supported by all markers, whereas the root
of the mitochondrial and ITS1 phylogenies fall between
species A (from Madagascar) and species B and C
(respectively from Madagascar and from the Pacific
Ocean). Even though the sister-group of species D could
not be determined unambiguously due to the current
lack 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 is
located in the western Indian Ocean. This hypothesis
will need to be confirmed by analyzing more samples
from key locations in the Red Sea, the Gulf of Aden and
the Indian Ocean, but would explain well the unusual
concentration of the biodiversity of this genus in the
westernmost part of his area of distribution.
While waiting for a global taxonomic revision of the
genus Stylophora, for the sake of taxonomic stability we
recommend that the preliminary results presented here
not be translated yet into nomenclature, but that each
genetically delimited species be provisionally designated
by a letter (i.e., “Stylophora sp. A”, “Stylophora sp. B”,
“Stylophora sp. C” and “Stylophora sp. D”). It is only
when a complete picture of the species boundaries of
Stylophora over its whole area of distribution becomes
available that names will be reliably assigned to each
species: 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, this
species may very well have been described first under
another name in a different location, in which case S.
madagascarensis would become a junior synonym of the
actual name of this species.
Conclusions
Genetic analysis of the coral genus Stylophora reveals
species 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 interspecific
hybridization), the first two seem likely to play a role
but the fourth one is rejected since mitochondrial and
nuclear markers yield congruent species delimitations.
The center of origin of Stylophora appears to be located
in the Indian Ocean, which probably explains why this
genus presents a higher biodiversity in the westernmost
part of its area of distribution than in the “Coral
Triangle”.
Methods
Sample collection and processing
Fragments from 70 Stylophora coral colonies were col-
lected while scuba diving or snorkeling on reefs of New
Caledonia, Okinawa (Japan), Bolinao (Philippines) and
Toliara (Madagascar) between 2004 and 2007. Each col-
ony sampled was photographed underwater and its
depth recorded (Table 1). Coral tissues were preserved
in a buffered guanidium thiocyanate solution [51,52]
and their DNA purified on an ABI Prism 6100 Nucleic
Acid PrepStation.
PCR amplification and sequencing
Three DNA markers previously developed in the closely
related genus Pocillopora [33] were amplified and
sequenced for each individual: the nuclear ribosomal
intergenic transcribed spacer 2 (ITS2), the mitochondrial
ORF and the putative control region (CR). In addition,
new primers were used to amplify the ITS1 region of a
few selected individuals (Table 2). Amplifications were
performed in 25 μl reaction mixes containing 1x Red Taq
buffer, 264 μM dNTP, 5% DMSO, 0.3 μM PCR primers,
0.3 units Red Taq (Sigma), and 10-50 ng DNA. PCR con-
ditions comprised an initial denaturation step of 60 s at
94°C, followed by 40-50 cycles (30 s denaturation at 94°
C, 30 s annealing at 53°C, 75 s elongation at 72°C) and a
final 5-min elongation step at 72°C. PCR products were
sequenced in both directions with the same primers as
for amplification, and chromatograms were assembled
and cleaned using Sequencher 4 (Gene Codes).
Determination of nuclear haplotypes
The ITS2 chromatogram pairs obtained from 43 indivi-
duals contained double peaks, indicating that each of
these individuals harbored two sequence types. Finding
out the sequence types was trivial for 5 individuals
whose chromatograms contained only one double peak.
Furthermore, 21 other chromatogram pairs had numer-
ous double peaks, a situation typical of length-variant
heterozygotes [53,54] that allowed direct deconvolution
of their superposed sequences using the program
CHAMPURU [55] (available online at http://www.mnhn.
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fr/jfflot/champuru). The remaining 17 chromatograms
pairs had several double peaks (at most 9), as expected
from 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 using
SeqPHASE [56] (available online at http://www.mnhn.fr/
jfflot/seqphase) and PHASE [57], but only 7 individuals
were phased unambiguously, i.e., with posterior prob-
abilities equal or nearly equal to 1 (04NC064, 04NC182,
04NC251, 04NC282, 04NC324, 04NC436, 07Mad087).
Among the 10 remaining heterozygotes, the haplotypes
of 2 individuals (07Mad151, 07Mad189) were deduced
directly from their chromatograms thanks to clear-cut
differences in peak sizes (reflecting either differences in
copy number in ribosomal DNA arrays or differential
amplification during PCR), and the sequences of the 8
others (04NC024, 04NC132, 04NC365, 04NC379,
07Mad073, 07Mad074, 07Mad088, 07Mad157) were
inferred using Clark’s method [58]. Length-variant het-
erozygosity was also observed in the ITS1 chromato-
grams of five individuals, all of which were resolved
using CHAMPURU.
Table 1 Localization and depth of each Stylophora sample analyzed
Sample name Coordinates Depth (m) Sample name Coordinates Depth (m)
04Oki115 (26°12’10"N, 127°19"12"E) n.r. 04NC323 (20°42’39"S, 165°09’14"E) 6
04Oki136 (26°12’10"N, 127°19"12"E) n.r. 04NC324 (20°42’39"S, 165°09’14"E) 6
04Oki140 (26°12’10"N, 127°19"12"E) n.r. 04NC336 (20°35’42"S, 165°10’40"E) 26
04Oki195 (26°14’25"N, 127°27’50"E) n.r. 04NC365 (20°40’12"S, 164°11’19"E) 22
04NC009 (22°26’54"S, 166°22’23"E) 2 04NC373 (20°40’12"S, 164°11’19"E) 15
04NC012 (22°26’54"S, 166°22’23"E) 6 04NC379 (20°40’12"S, 164°11’19"E) 14
04NC024 (22°26’54"S, 166°22’23"E) 1 04NC404 (20°40’20"S, 164°14’53"E) 4
04NC064 (22°38’30"S, 166°34’40"E) 16 04NC434 (20°39’58"S, 164°15’26"E) 25
04NC071 (22°40’54"S, 168°36’24"E) 1 04NC436 (20°41’39"S, 164°14’50"E) 24
04NC087 (22°37’20"S, 166°36’53"E) 5 04NC439 (20°41’39"S, 164°14’50"E) 23
04NC101 (22°37’20"S, 166°36’53"E) 9 04NC440 (20°41’39"S, 164°14’50"E) 23
04NC105 (22°01’01"S, 165°55’10"E) 26 04NC441 (20°41’39"S, 164°14’50"E) 21
04NC130 (22°22’59"S, 167°05’50"E) 1 04NC452 (20°41’39"S, 164°14’50"E) 1
04NC131 (22°22’59"S, 167°05’50"E) 1 04NC455 (20°41’39"S, 164°14’50"E) 1
04NC132 (22°22’59"S, 167°05’50"E) 1 05Phil19 (16°23’46"N, 119°54’03"E) 24
04NC139 (22°22’59"S, 167°05’50"E) 1 05Phil53 (16°26’22"N, 119°56’33"E) 13
04NC140 (22°22’59"S, 167°05’50"E) 1 07Mad070 (23°25’01"S, 43°38’36"E) 8
04NC141 (22°22’59"S, 167°05’50"E) 1 07Mad071 (23°25’01"S, 43°38’36"E) 7
04NC145 (22°22’59"S, 167°05’50"E) 1 07Mad073 (23°25’01"S, 43°38’36"E) 5
04NC149 (22°15’27"S, 166°24’33"E) 16 07Mad074 (23°25’01"S, 43°38’36"E) 5
04NC152 (22°15’27"S, 166°24’33"E) 16 07Mad079 (23°25’01"S, 43°38’36"E) 8
04NC170 (22°15’27"S, 166°24’33"E) 15 07Mad082 ( 23°23’07"S, 43°38’18"E) 8
04NC182 (22°12’31"S, 166°24’55"E) 4 07Mad086 ( 23°23’07"S, 43°38’18"E) 6
04NC187 (22°12’31"S, 166°24’55"E) 4 07Mad087 (23°30’20"S, 43°41’10"E) 30
04NC189 (22°12’31"S, 166°24’55"E) 4 07Mad088 (23°30’20"S, 43°41’10"E) 30
04NC199 (22°18’40"S, 166°27’26"E) 1 07Mad150 (23°23’29"S, 43°37’38"E) 8
04NC230 (20°46’18"S, 165°16’30"E) 14 07Mad151 (23°23’29"S, 43°37’38"E) 8
04NC232 (20°46’18"S, 165°16’30"E) 13 07Mad157 (23°23’29"S, 43°37’38"E) 7
04NC233 (20°46’18"S, 165°16’30"E) 9 07Mad159 (23°23’29"S, 43°37’38"E) 3
04NC237 (20°46’18"S, 165°16’30"E) 7 07Mad160 (23°23’29"S, 43°37’38"E) 3
04NC251 (20°34’59"S, 165°08’11"E) 21 07Mad161 (23°23’29"S, 43°37’38"E) 2
04NC253 (20°34’59"S, 165°08’11"E) 19 07Mad170 (23°22’58"S, 43°38’11"E) 11
04NC266 (20°34’59"S, 165°08’11"E) 7 07Mad172 (23°22’58"S, 43°38’11"E) 11
04NC282 (20°34’59"S, 165°08’11"E) 6 07Mad188 (23°22’58"S, 43°38’11"E) 5
04NC289 (20°34’59"S, 165°08’11"E) 2 07Mad189 (23°22’58"S, 43°38’11"E) 3
n.r. = not recorded
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Phylogenetic analyses and haploweb construction
All haplotype sequences were deposited in public data-
bases [GenBank: JN558840-JN559111]. ORF sequences
were aligned in MEGA5 [59] by taking advantage of
the high degree of conservation of their aminoacid
translations: all sequences from Stylophora were first
aligned by hand as there were only few indels, before
aligning them with outgroup sequences from Pocillo-
pora and Seriatopora using the MEGA5 implementa-
tion of MUSCLE [60]. CR, ITS1 and ITS2 sequences
were aligned using MAFFT’s Q-INS-I option [61,62].
Since the two mitochondrial markers ORF and CR
yielded congruent phylogenies, they were concatenated
and only the result of the combined analysis is pre-
sented here. The best suited nucleotide model among
88 possible ones was determined for each dataset fol-
lowing the Bayesian Information Criterion [63] as
implemented in jModelTest [64], and used to perform
maximum-likelihood phylogenetic analyses in PhyML
[65] with 1000 bootstrap replicates [66]. Additional
bootstrap 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 ("haplotrees”) produced by PhyML were
converted into enhanced metafiles (emf) using the pro-
gram FigTree 1.3.1 (available online at http://tree.bio.
ed.ac.uk/software/figtree/), then imported in Microsoft
PowerPoint. The ITS2 haploweb was obtained from
the corresponding haplotree by drawing connections
between haplotypes found co-occurring in heterozy-
gous individuals [9].
Acknowledgements
Thanks to Annie Tillier, Josie Lambourdière and Céline Bonillo (Service de
Systématique Moléculaire, CNRS UMS 2700, MNHN) for assistance with lab
work, and to Eric Folcher, Catherine Geoffray, Jean-Louis Menou and Mark
Vergara for helping with sample collection. Fieldwork in New Caledonia and
Madagascar was financed by grants from the MNHN programs “Structure et
évolution des écosystèmes” et “État et structure phylogénétique de la
biodiversité actuelle et fossile"; thanks to Man Wai Rabenevanana, director of
the Institut Halieutique et des Sciences Marines, for his logistic support in Tuléar
(Toliara). Thanks also to two anonymous reviewers for their useful comments.
This project was part of agreement n°2005/67 between Genoscope and
MNHN on the project ‘Macrophylogeny of life’ directed by Guillaume
Lecointre; support from the Consortium National de Recherche en Génomique
is gratefully acknowledged. This is contribution n°78 from the Courant
Research Center “Geobiology” funded by the German Initiative of Excellence.
Author details
1Courant Research Center “Geobiology”, University of Göttingen,
Goldschmidtstraße 3, 37077 Göttingen, Germany. 2CEA-Institut de
Génomique, GENOSCOPE, Centre National de Séquençage, 2 rue Gaston
Crémieux, CP5706, 91057 Evry Cedex, France. 3UMR UPMC-CNRS-MNHN-IRD
7138, Département Systématique et Évolution, Muséum National d’Histoire
Naturelle, Case Postale 26, 57 rue Cuvier, 75231 Paris Cedex 05, France.
4URBO, Department of Biology, University of Namur, Rue de Bruxelle 61,
5000 Namur, Belgium. 5UMR LOBP, Centre d’Océanologie de Marseille,
Campus de Luminy, Case 901, 13288 Marseille Cedex 09, France. 6UMR LOBP,
Centre IRD de Tahiti, BP 529, 98713 Papeete, French Polynesia. 7Br. Alfred
Shields FSC Marine Station and Biology Department, De La Salle University,
Manila 1004, Philippines. 8Sesoko Station, Tropical Biosphere Research Center,
University of the Ryukyus, Okinawa 3422, Japan. 9UR COREUS, IRD, B.P. A5,
98848 Nouméa, New Caledonia.
Authors’ contributions
JFF carried out fieldwork, DNA extractions and PCR amplifications, analyzed
the results and drafted the manuscript. CC sequenced all PCR products. JB,
LC, WL, YN and CP provided logistic support, participated in fieldwork and
revised the manuscript. ST supervised the study and revised the manuscript,
the final version of which was read and approved by all authors.
Received: 25 March 2011 Accepted: 4 October 2011
Published: 4 October 2011
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doi:10.1186/1472-6785-11-22
Cite this article as: Flot et al.: Incongruence between morphotypes and
genetically delimited species in the coral genus Stylophora: phenotypic
plasticity, morphological convergence, morphological stasis or
interspecific hybridization? BMC Ecology 2011 11:22.
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