BACKGROUND
hybridisation has long been considered as a process reducing genetic diversity through introgression  <cit> .
thus, the effect of hybridisation on species diversification should, if anything, be negative due to the potential reduction in fitness of the hybrids.
however views are changing as many evolutionary radiations have now documented cases in which hybridisation plays a key role in promoting species diversification.
common examples of hybridisation inducing diversification can be found in the galapagos finches  <cit> , hawaiian lapaula crickets  <cit>  and rift lakes cichlids  <cit> .
building on those evidences and others, seehausen proposed that hybridisation may promote speciation and adaptive radiation by generating new genetic and phenotypic variation that can be the target of selection  <cit> .
with more and more cases of hybrid speciation being documented  <cit> , the main question is now shifting towards understanding how evolutionary radiations are facilitated or even catalysed, rather than prevented, by hybridisation  <cit> .
during adaptive radiation, available ecological niches are filled by the diversifying species  <cit> .
in the classical view, this process occurs through specific phenotypic adaptations that allow the constituent species to take advantage of the diversity of ecological niches  <cit> .
hybridisation between incipient species has been proposed as a mechanism that is able to rapidly create new phenotypic combinations suitable for further occupancy of untapped niches  <cit> .
indeed, transgressive segregation can produce individuals with novel phenotypes reaching beyond the possibilities of the parental populations  <cit> .
for example, the complementary action of genes regulating mineral ion uptake allowed hybrids of species of helianthus sunflowers to survive in salt marshes while none of the parent species are salt tolerant  <cit> .
such an event can create new species very rapidly, especially when the newly formed hybrids are ecologically distinct from the two parent species  <cit> .
for instance, as little as  <dig> to  <dig> generations are needed for recombination to no longer reduce the size of parental linkage blocks in helianthus, which is a clear sign of genome stabilisation in the hybrids  <cit> .
this mechanism is nevertheless dependent on the availability of untapped ecological niches.
indeed, the frequency of hybridisation events leading to speciation will decrease after the initial burst of adaptive radiation because of the saturation of the niche space  <cit> .
while the theoretical grounds defining the potential role of hybridisation as an important component of species diversification have been laid, empirical evidence is needed to better illustrate the effects of this factor during adaptive radiations.
several methods exist to identify putative hybridisation events in evolutionary radiations .
discordance between phylogenetic trees inferred with either cytoplasmic  or nuclear dna can suggest the occurrence of hybridisation.
this is possible because phylogenetic trees based on cytoplasmic information will show the evolutionary history of females while nuclear dna will illustrate that of the parental allele that has been fixed.
moreover, a tree-based method  has been proposed to identify putative hybrid taxa and likely parental lineages  <cit> .
while dna sequences of cytoplasmic or nuclear origin are now readily available for a growing number of species, these approaches have only been used scarcely .
given the potential importance of hybridisation for species diversification, there is a need to study new cases such as comprehending more fully the interactions between hybridisation and speciation.
in this context, we study hybridisation patterns in the clownfishes , a monophyletic clade which maintains mutualistic interactions with sea anemones  <cit> .
this behaviour is the key innovation that promoted the adaptive radiation of clownfishes as species segregated among specific combinations of potential host species and habitat, each time matching their phenotype to the environment  <cit> .
while this process of ecological speciation  <cit>  could alone be responsible for the extant diversity of clownfish species, evidence suggests that hybridisation may have occurred during the evolution of the group.
indeed, a recent phylogenetic tree of the clade based on nuclear markers showed several discordant nodes with previous phylogenetic trees reconstructed mostly from mitochondrial markers  <cit> .
moreover, interspecific pairs have been observed in the wild and two species, amphiprion leucokranos and a. thiellei, were likely described from hybrid material  <cit> .
this indicates a potential history of hybridisation in the group.
however, whether it impacted on the diversification of clownfishes is yet unknown.
to resolve this issue, we analyse a dataset of mitochondrial and nuclear dna sequences of clownfish species to assess cyto-nuclear discordances and highlight possible ancient hybridisation events.
furthermore, we use the het approach to identify species of hybrid origin.
finally, we investigate potential hybrids among clownfish individuals showing hybrid phenotypic characteristics and use molecular cloning to assess their status and putatively identify the parental species.
RESULTS
we define from phylogenetic inference eight monophyletic groups of species to facilitate the interpretation of our results .
the clade names are based on classical clownfish taxonomy that describes  <dig> species complexes  <cit> .table  <dig>
clades of clownfishes used in this study
clade name
species
percula
a. ocellaris, a. percula, p. biaculeatus
a. akindynos, a. mccullochi
akallopisos
a. akallopisos, a. perideraion, a. pacificus, a. sandaracinos
ephippium
a. frenatus, a. ephippium, a. rubrocinctus, a. melanopus, a. barberi
polymnus
a. sebae, a. polymnus
clarkii
a. clarkii, a. tricinctus
a. bicinctus, a. omanensis, a. chagosensis, a. latifasciatus,
a. nigripes, a. allardi, a. chrysogaster, a. fuscocaudatus*
the table gives the list of species included in each clade.
a. stands for amphiprion, p. for premnas.
not included are a. chrysopterus and a. latezonatus, which are monospecific lineages and a. leucokranos and a. thiellei, which have likely been described from hybrid specimens.
*exemplars of a. fuscocaudatus have never been sequenced.
we hypothetically placed this species in the indian clade because it is the most parsimonious solution regarding the biogeography of clownfish species  <cit> .
discordance between phylogenetic hypotheses
the two consensus phylogenetic trees that we obtain show an overall good support with only several recent splits showing low posterior probabilities .
major clades appear in both mitochondrial and nuclear phylogenetic trees.
despite the fact that the two datasets show well resolved topologies, the relative position of the main clades is different between the two analyses .
the organisation of the percula clade at the base of the clownfish tree and the position of a. latezonatus is congruent between the two datasets.
however, the australian group and a. chrysopterus intercalate between the polymnus and indian groups in the nuclear dataset.
we identify two mitochondrial lineages in a. sandaracinos with a posterior probability of  <dig>  but both samples are clustered in the nuclear phylogenetic tree.
the ephippium clade, which is the sister group of the clarkii clade in the nuclear phylogenetic tree, becomes sister to the australian clade with the mitochondrial dataset.
this suggest the existence of a hybridisation event at the time of node  <dig> .
other studies showed that a main upward shift in speciation rate occurred at this specific node  <cit> .figure  <dig>
cytonuclear incongruence.
majority-rule consensus tree with all compatible groups for mitochondrial  and nuclear  datasets.
red dots indicates nodes having bayesian posterior probabilities lower than  <dig> .
links are drawn to highlight the topological differences.
colours correspond to species clades  as follows: black: outgroups, turquoise: percula, grey: amphiprion latezonatus and a. chrysopterus , orange: clarkii, blue: akallopisos, rose: australian, green: ephippium, yellow: polymnus, brown: indian.
a coloured legend, which repeats this information for the major clades, is located at the right of the figure.
homoplasy excess test.
panel  shows the distribution of the bootstrap support for each node which showed a homoplasy excess during the removal analysis.
each boxplot shows the null distribution of bootstrap values for the node and the red dot shows the bootstrap value of the node when the taxon of interest  has been removed.
nodes names as given on the left correspond to that of panel  which shows the consensus tree of the nuclear phylogeny.
there, nodes and taxa which showed up as outliers in the analysis have been coloured in orange.
species used as outgroup are shown in grey.
the size of node labels has been altered to facilitate reading.
hybrid signal
we recover an excess of homoplasy at nine nodes of the clownfish phylogenetic tree .
removing either a. ocellaris from bali or a. percula from papua new guinea increases the bs of node  <dig> , which suggests genetic exchange between members of the percula clade.
we find that the “black” specimen of a. clarkii collected in bali likely originated from hybridisation between other members of the clarkii group .
we measure further increase of bs at three different nodes  when individuals of a. chrysopterus from either moorea or the solomon islands are removed.
moreover, the bs of node  <dig> increases when a. akindynos is removed, which suggests several hybridisation events between a. chrysopterus and species of the australian and polymnus groups occurring around the timeframe of node  <dig> and  <dig>  we also find evidence of genetic exchanges between members of the indian group at nodes  <dig>   <dig> and  <dig> .
finally, we identify gene exchange between the two a. sandaracinos individuals at node  <dig> and a. pacificus, but the effect on bootstrap values is relatively small.
identification of recent hybrid parental species
we infer the phylogenetic trees of mitochondrial  and nuclear  markers for all sampled clownfish species augmented with the additional sequences of the supposed hybrid individuals.
the cloning procedure allowed us to separate the two parental bmp <dig> sequences of each hybrid individual analysed .
as expected, we find that the bmp <dig> sequences from those hybrid individuals that were cloned do not cluster by individual species but are separated among parental clades .
we deduced the maternal origin of the putative hybrids with the mitochondrial phylogenetic tree.
we find that both hybrids  <dig> and  <dig> inherited their mitochondrial genes from a. akallopisos.
the plastid sequence of hybrid  <dig> is similar to a. perideraion and a. leucokranos, as already proposed  <cit> , derives from a. chrysopterus.
moreover, in the mitochondrial tree, a. leucokranos clusters with a. chrysopterus sampled from the solomon islands, the origin of a. leucokranos .
the maternal origin of the four hybrid individuals is highly supported in the phylogenetic tree with posterior probabilities of  <dig> on the respective ancestral nodes .
the nuclear tree confirms those results and each individual has one of the cloned sequence that clusters with the same species as in the mitochondrial tree .
the position of the other clone provides information on the paternal origin of the hybrids.
a. leucokranos clusters with high posterior probability with a. sandaracinos and hybrid  <dig> and  <dig> both cluster with a. perideraion.
the other hybrid individual  clusters with any of a. pacificus, a. sandaracinos and a. akallopisos, but low posterior probabilities forbid any definitive conclusion.
nevertheless, we do not find clear evidence of recombination events amongst the cloned sequences, suggesting that we sequenced individuals deriving from recent hybridisation events.figure  <dig>
mitochondrial phylogeny of hybrid individuals
.
majority-rule consensus tree with all compatible groups for mitochondrial sequences with the inclusion of hybrid specimens in the analysis.
panel  shows a close-up on the topological position of hybrid specimens  <dig> to  <dig> and panel  of a. leucokranos.
the topology of the rest of the phylogeny  is as in figure  <dig>
nuclear phylogeny of hybrid cloned sequences.
majority-rule consensus tree with all compatible groups for nuclear sequences with the inclusion of hybrid specimens in the analysis.
the two panels  are close-ups on the topological position of the cloned sequences.
the topology of the rest of the phylogeny  is as in figure  <dig>
distribution of potentially hybridising species
.
species distribution following  <cit> , of species likely implicated in hybridisation events.
the localities of the sequenced hybrid individuals are shown by yellow dots or area when known.
the precise geographical origin of the hybrid  <dig> individual is unknown, the question mark sign indicates the general area where it was collected.
discussion
our results show instances of cyto-nuclear incongruence in the clownfish phylogenetic tree, which suggests several hybridisation events.
the most ancient of those events occurred in the same time frame as a node that was identified twice independently as the highest increase in diversification rate happening in the damselfish family  <cit> .
this evidence suggests a relationship between this ancient hybridisation event and the main burst of diversification of the adaptive radiation of clownfishes.
moreover, we identify nodes with an excess of homoplasy that can be attributed to more recent hybridisation events.
finally, we identify the parental lineages of four hybrid individuals, showing that hybridisation still occurs within clownfishes.
this evidence suggests that the diversification of the clownfishes has been likely fuelled by hybridisation events that created novel phenotypes and eventually new species in a manner similar to the well-known east african cichlids.
past hybridisation
we reconstruct separately the phylogenetic trees of the mitochondrial  and nuclear  genomes.
most nodes show high posterior probabilities, yet, several shallow nodes of our mitochondrial and nuclear trees are only weakly supported.
this can originate from the lack of phylogenetic informative sites between closely related species.
previous studies of phylogenetic relationships of the pomacentridae inferred by mitochondrial or nuclear markers, all support the monophyly of the clownfish as well as the existence of the main clownfish clades  <cit> .
the topology obtained with the mitochondrial data corresponds to these published phylogenetic trees.
we find several deep cytonuclear discordances in the clownfish.
the relative position of the ephippium clade changes radically between the two datasets, which suggests the occurrence of hybridisation in the timeframe of node  <dig> .
while this node is not basal to all clownfishes, it is ancestral to  <dig> of the  <dig> species .
this clade represents a major shift in diversification rate with a  <dig> -fold increase compared to the diversification rate of pomacentridae  <cit> .
the pattern of concurrent increase in rate of diversification and occurrence of hybridisation fits into the framework of the syngameon hypothesis  <cit> , where hybridisation events preludes an acceleration of ecological differentiation and diversification rate.
moreover, this result is robust regarding node support, as the relationship between the clarkii and the ephippium clade is itself highly supported .
we find paraphyletic mitochondrial genotypes in a. sandaracinos.
the fact that the two a. sandaracinos individuals cluster with high support in the nuclear dataset suggests a recent hybridisation event implying a mitochondrial genotype that belongs to an unknown lineage of the akallopisos clade.
results of the het suggest the ongoing occurrence of genetic exchanges between a. sandaracinos and a. pacificus .
the two species are phenotypically very similar and it is possible that a third cryptic species having the mitochondrial profile of the a. sandaracinos sample from papua new guinea exists.
the remaining topological inconsistencies of the polymnus clade are not well supported as shown by the bayesian posterior probabilities of their respective nodes on the mitochondrial phylogeny.
this discrepancy may be artifactual and is thus not further discussed.
hybridisation and adaptive radiation
our results highlight the existence of a main hybridisation event at the base of the amphiprion crown group .
this node has been previously linked with a burst of diversification rate in the clownfish clade  <cit>  and may also be mainly responsible for the increase in rate of morphological evolution found in the group  <cit> .
in contrary, we did not detect traces of ancient hybridisation in the percula group which is species poor relative to the amphiprion crown group.
one recent hybridisation event likely occurred around node  <dig>  between a. percula and a. ocellaris.
the distribution of those two species is mostly allopatric, but they share a common boundary and may overlap at some locations  <cit>  possibly leading to gene exchange.
yet, despite similar behaviour and ecology, the species of the percula clade seemingly failed to radiate  <cit> .
the fact that the clade deriving from a major hybridisation event is the only one to show patterns of rapid speciation supports the view that hybridisation can promote diversification during adaptive radiation.
actually, hybridisation appears to be one of the key factors explaining the adaptive radiation of the clownfishes.
recent hybridisation events
we identify the parental species of four putative hybrids, one being a. leucokranos, which is currently described as a species.
since their taxonomic descriptions, the species status of a. leucokranos and a. thiellei has been questioned  <cit> .
indeed, heterospecific pairs have been observed in the wild and fish with similar phenotype can be produced in aquaria by hybrid crosses  <cit> .
nevertheless, evidence of a. leucokranos pairs also exist  <cit> , tentatively proposing that we may be witnessing hybrid speciation in progress.
yet, demonstrating this ongoing process would require more data than available here.
it is thus more parsimonious to conclude at the moment, that a. leucokranos is an invalid species.
we separate bmp <dig> sequences of each parental chromosome through molecular cloning and identify the putative parental species of a. leucokranos as a. chrysopterus and a. sandaracinos.
this is consistent with field observations  <cit> .
the hybrid individuals that we sequenced are all likely recent hybrids  because the two clones can always be clearly separated and cluster with different putative parental taxa.
this suggests that genomic rearrangement occurring during reproductive events did not yet occur.
the special social structure of clownfish may put some behavioural constraints on hybridisation.
indeed, all clownfish species are protandrous hermaphrodites and live in social groups with size-based dominance hierarchy.
the female is the largest and dominant individual, the male the second largest, and usually smaller non-breeding individuals follow, decreasing in size relative to their hierarchical position  <cit> .
in certain cases  <cit> , size differentiation allows smaller species to cohabit with a larger species in the same anemone.
while reproduction of the smallest species is largely suppressed by the presence of the largest clownfish  <cit> , species hybridisation cannot be fully excluded.
in such instance, the size-based hierarchy ensure that the female of a hybrid pair will most probably always be the largest individual.
this is what we observe in a. leucokranos.
the parental origin of the three other hybrids does not allow for a definitive conclusion regarding this behavioural constraint.
indeed, the size difference between species of the akallopisos group is small and cannot be used as a diagnostic feature separating a. akalloposis, a. pacificus and a. perideraion  <cit> .
it is thus likely that the behavioural constraint on hybridisation between those species is going to be weaker than between species with large size difference.
geography of recent hybridisation
hybrid zones appear when two species share such margins and have the possibility to exchange genes.
at the range margins, the probability of encountering a heterospecific mate is higher than that of meeting an individual of its own species whenever an individual moves into the range of the other species  <cit> .
this will increase the probability of hybridisation events.
as expected, a. leucokranos is distributed at the edge of the two parental distributions and hybrids  <dig> and  <dig> were found at the eastern edge of the range of a. akallopisos .
further genetic sampling would be necessary to better define those potential hybrid zones.
this would be of particular interest given the evolutionary significance of hybridisation in animals, as exemplified by the importance of exogenous selection on contemporary hybrid zones  <cit> .
CONCLUSIONS
hybridisation can impact speciation in many ways.
our analyses show that in the case of the adaptive radiation of the clownfishes, hybridisation events are linked with a burst in diversification rate.
we observe that hybridisation has occurred throughout the evolutionary history of the group and is still happening.
further studies are required to measure the extant of ongoing hybrid speciation, for example in a. leucokranos.
there, field observations coupled with experiments will allow to identify the conditions in which hybrids have a better fitness than their parents, illustrating the thight relationship linking species ecology and evolutionary processes.

 hybridisation has long been considered as a process reducing genetic diversity through introgression. thus, the effect of hybridisation on species diversification should, if anything, be negative due to the potential reduction in fitness of the hybrids. however views are changing as many evolutionary radiations have now documented cases in which hybridisation plays a key role in promoting species diversification. common examples of hybridisation inducing diversification can be found in the galapagos finches, hawaiian lapaula crickets and rift lakes cichlids. building on those evidences and others, seehausen proposed that hybridisation may promote speciation and adaptive radiation by generating new genetic and phenotypic variation that can be the target of selection. with more and more cases of hybrid speciation being documented, the main question is now shifting towards understanding how evolutionary radiations are facilitated or even catalysed, rather than prevented, by hybridisation. during adaptive radiation, available ecological niches are filled by the diversifying species. in the classical view, this process occurs through specific phenotypic adaptations that allow the constituent species to take advantage of the diversity of ecological niches. hybridisation between incipient species has been proposed as a mechanism that is able to rapidly create new phenotypic combinations suitable for further occupancy of untapped niches. indeed, transgressive segregation can produce individuals with novel phenotypes reaching beyond the possibilities of the parental populations. for example, the complementary action of genes regulating mineral ion uptake allowed hybrids of species of helianthus sunflowers to survive in salt marshes while none of the parent species are salt tolerant. such an event can create new species very rapidly, especially when the newly formed hybrids are ecologically distinct from the two parent species. for instance, as little as to generations are needed for recombination to no longer reduce the size of parental linkage blocks in helianthus, which is a clear sign of genome stabilisation in the hybrids. this mechanism is nevertheless dependent on the availability of untapped ecological niches. indeed, the frequency of hybridisation events leading to speciation will decrease after the initial burst of adaptive radiation because of the saturation of the niche space. while the theoretical grounds defining the potential role of hybridisation as an important component of species diversification have been laid, empirical evidence is needed to better illustrate the effects of this factor during adaptive radiations. several methods exist to identify putative hybridisation events in evolutionary radiations. discordance between phylogenetic trees inferred with either cytoplasmic or nuclear dna can suggest the occurrence of hybridisation. this is possible because phylogenetic trees based on cytoplasmic information will show the evolutionary history of females while nuclear dna will illustrate that of the parental allele that has been fixed. moreover, a tree-based method has been proposed to identify putative hybrid taxa and likely parental lineages. while dna sequences of cytoplasmic or nuclear origin are now readily available for a growing number of species, these approaches have only been used scarcely. given the potential importance of hybridisation for species diversification, there is a need to study new cases such as comprehending more fully the interactions between hybridisation and speciation. in this context, we study hybridisation patterns in the clownfishes, a monophyletic clade which maintains mutualistic interactions with sea anemones. this behaviour is the key innovation that promoted the adaptive radiation of clownfishes as species segregated among specific combinations of potential host species and habitat, each time matching their phenotype to the environment. while this process of ecological speciation could alone be responsible for the extant diversity of clownfish species, evidence suggests that hybridisation may have occurred during the evolution of the group. indeed, a recent phylogenetic tree of the clade based on nuclear markers showed several discordant nodes with previous phylogenetic trees reconstructed mostly from mitochondrial markers. moreover, interspecific pairs have been observed in the wild and two species, amphiprion leucokranos and a. thiellei, were likely described from hybrid material. this indicates a potential history of hybridisation in the group. however, whether it impacted on the diversification of clownfishes is yet unknown. to resolve this issue, we analyse a dataset of mitochondrial and nuclear dna sequences of clownfish species to assess cyto-nuclear discordances and highlight possible ancient hybridisation events. furthermore, we use the het approach to identify species of hybrid origin. finally, we investigate potential hybrids among clownfish individuals showing hybrid phenotypic characteristics and use molecular cloning to assess their status and putatively identify the parental species.  we define from phylogenetic inference eight monophyletic groups of species to facilitate the interpretation of our results. the clade names are based on classical clownfish taxonomy that describes species complexes.table clades of clownfishes used in this study clade name species percula a. ocellaris, a. percula, p. biaculeatus a. akindynos, a. mccullochi akallopisos a. akallopisos, a. perideraion, a. pacificus, a. sandaracinos ephippium a. frenatus, a. ephippium, a. rubrocinctus, a. melanopus, a. barberi polymnus a. sebae, a. polymnus clarkii a. clarkii, a. tricinctus a. bicinctus, a. omanensis, a. chagosensis, a. latifasciatus, a. nigripes, a. allardi, a. chrysogaster, a. fuscocaudatus* the table gives the list of species included in each clade. a. stands for amphiprion, p. for premnas. not included are a. chrysopterus and a. latezonatus, which are monospecific lineages and a. leucokranos and a. thiellei, which have likely been described from hybrid specimens. *exemplars of a. fuscocaudatus have never been sequenced. we hypothetically placed this species in the indian clade because it is the most parsimonious solution regarding the biogeography of clownfish species. discordance between phylogenetic hypotheses the two consensus phylogenetic trees that we obtain show an overall good support with only several recent splits showing low posterior probabilities. major clades appear in both mitochondrial and nuclear phylogenetic trees. despite the fact that the two datasets show well resolved topologies, the relative position of the main clades is different between the two analyses. the organisation of the percula clade at the base of the clownfish tree and the position of a. latezonatus is congruent between the two datasets. however, the australian group and a. chrysopterus intercalate between the polymnus and indian groups in the nuclear dataset. we identify two mitochondrial lineages in a. sandaracinos with a posterior probability of but both samples are clustered in the nuclear phylogenetic tree. the ephippium clade, which is the sister group of the clarkii clade in the nuclear phylogenetic tree, becomes sister to the australian clade with the mitochondrial dataset. this suggest the existence of a hybridisation event at the time of node. other studies showed that a main upward shift in speciation rate occurred at this specific node.figure cytonuclear incongruence. majority-rule consensus tree with all compatible groups for mitochondrial and nuclear datasets. red dots indicates nodes having bayesian posterior probabilities lower than. links are drawn to highlight the topological differences. colours correspond to species clades as follows: black: outgroups, turquoise: percula, grey: amphiprion latezonatus and a. chrysopterus, orange: clarkii, blue: akallopisos, rose: australian, green: ephippium, yellow: polymnus, brown: indian. a coloured legend, which repeats this information for the major clades, is located at the right of the figure. homoplasy excess test. panel shows the distribution of the bootstrap support for each node which showed a homoplasy excess during the removal analysis. each boxplot shows the null distribution of bootstrap values for the node and the red dot shows the bootstrap value of the node when the taxon of interest has been removed. nodes names as given on the left correspond to that of panel which shows the consensus tree of the nuclear phylogeny. there, nodes and taxa which showed up as outliers in the analysis have been coloured in orange. species used as outgroup are shown in grey. the size of node labels has been altered to facilitate reading. hybrid signal we recover an excess of homoplasy at nine nodes of the clownfish phylogenetic tree. removing either a. ocellaris from bali or a. percula from papua new guinea increases the bs of node, which suggests genetic exchange between members of the percula clade. we find that the “black” specimen of a. clarkii collected in bali likely originated from hybridisation between other members of the clarkii group. we measure further increase of bs at three different nodes when individuals of a. chrysopterus from either moorea or the solomon islands are removed. moreover, the bs of node increases when a. akindynos is removed, which suggests several hybridisation events between a. chrysopterus and species of the australian and polymnus groups occurring around the timeframe of node and we also find evidence of genetic exchanges between members of the indian group at nodes and. finally, we identify gene exchange between the two a. sandaracinos individuals at node and a. pacificus, but the effect on bootstrap values is relatively small. identification of recent hybrid parental species we infer the phylogenetic trees of mitochondrial and nuclear markers for all sampled clownfish species augmented with the additional sequences of the supposed hybrid individuals. the cloning procedure allowed us to separate the two parental bmp sequences of each hybrid individual analysed. as expected, we find that the bmp sequences from those hybrid individuals that were cloned do not cluster by individual species but are separated among parental clades. we deduced the maternal origin of the putative hybrids with the mitochondrial phylogenetic tree. we find that both hybrids and inherited their mitochondrial genes from a. akallopisos. the plastid sequence of hybrid is similar to a. perideraion and a. leucokranos, as already proposed, derives from a. chrysopterus. moreover, in the mitochondrial tree, a. leucokranos clusters with a. chrysopterus sampled from the solomon islands, the origin of a. leucokranos. the maternal origin of the four hybrid individuals is highly supported in the phylogenetic tree with posterior probabilities of on the respective ancestral nodes. the nuclear tree confirms those results and each individual has one of the cloned sequence that clusters with the same species as in the mitochondrial tree. the position of the other clone provides information on the paternal origin of the hybrids. a. leucokranos clusters with high posterior probability with a. sandaracinos and hybrid and both cluster with a. perideraion. the other hybrid individual clusters with any of a. pacificus, a. sandaracinos and a. akallopisos, but low posterior probabilities forbid any definitive conclusion. nevertheless, we do not find clear evidence of recombination events amongst the cloned sequences, suggesting that we sequenced individuals deriving from recent hybridisation events.figure mitochondrial phylogeny of hybrid individuals. majority-rule consensus tree with all compatible groups for mitochondrial sequences with the inclusion of hybrid specimens in the analysis. panel shows a close-up on the topological position of hybrid specimens to and panel of a. leucokranos. the topology of the rest of the phylogeny is as in figure  nuclear phylogeny of hybrid cloned sequences. majority-rule consensus tree with all compatible groups for nuclear sequences with the inclusion of hybrid specimens in the analysis. the two panels are close-ups on the topological position of the cloned sequences. the topology of the rest of the phylogeny is as in figure  distribution of potentially hybridising species. species distribution following, of species likely implicated in hybridisation events. the localities of the sequenced hybrid individuals are shown by yellow dots or area when known. the precise geographical origin of the hybrid individual is unknown, the question mark sign indicates the general area where it was collected. discussion our results show instances of cyto-nuclear incongruence in the clownfish phylogenetic tree, which suggests several hybridisation events. the most ancient of those events occurred in the same time frame as a node that was identified twice independently as the highest increase in diversification rate happening in the damselfish family. this evidence suggests a relationship between this ancient hybridisation event and the main burst of diversification of the adaptive radiation of clownfishes. moreover, we identify nodes with an excess of homoplasy that can be attributed to more recent hybridisation events. finally, we identify the parental lineages of four hybrid individuals, showing that hybridisation still occurs within clownfishes. this evidence suggests that the diversification of the clownfishes has been likely fuelled by hybridisation events that created novel phenotypes and eventually new species in a manner similar to the well-known east african cichlids. past hybridisation we reconstruct separately the phylogenetic trees of the mitochondrial and nuclear genomes. most nodes show high posterior probabilities, yet, several shallow nodes of our mitochondrial and nuclear trees are only weakly supported. this can originate from the lack of phylogenetic informative sites between closely related species. previous studies of phylogenetic relationships of the pomacentridae inferred by mitochondrial or nuclear markers, all support the monophyly of the clownfish as well as the existence of the main clownfish clades. the topology obtained with the mitochondrial data corresponds to these published phylogenetic trees. we find several deep cytonuclear discordances in the clownfish. the relative position of the ephippium clade changes radically between the two datasets, which suggests the occurrence of hybridisation in the timeframe of node. while this node is not basal to all clownfishes, it is ancestral to of the species. this clade represents a major shift in diversification rate with a -fold increase compared to the diversification rate of pomacentridae. the pattern of concurrent increase in rate of diversification and occurrence of hybridisation fits into the framework of the syngameon hypothesis, where hybridisation events preludes an acceleration of ecological differentiation and diversification rate. moreover, this result is robust regarding node support, as the relationship between the clarkii and the ephippium clade is itself highly supported. we find paraphyletic mitochondrial genotypes in a. sandaracinos. the fact that the two a. sandaracinos individuals cluster with high support in the nuclear dataset suggests a recent hybridisation event implying a mitochondrial genotype that belongs to an unknown lineage of the akallopisos clade. results of the het suggest the ongoing occurrence of genetic exchanges between a. sandaracinos and a. pacificus. the two species are phenotypically very similar and it is possible that a third cryptic species having the mitochondrial profile of the a. sandaracinos sample from papua new guinea exists. the remaining topological inconsistencies of the polymnus clade are not well supported as shown by the bayesian posterior probabilities of their respective nodes on the mitochondrial phylogeny. this discrepancy may be artifactual and is thus not further discussed. hybridisation and adaptive radiation our results highlight the existence of a main hybridisation event at the base of the amphiprion crown group. this node has been previously linked with a burst of diversification rate in the clownfish clade and may also be mainly responsible for the increase in rate of morphological evolution found in the group. in contrary, we did not detect traces of ancient hybridisation in the percula group which is species poor relative to the amphiprion crown group. one recent hybridisation event likely occurred around node between a. percula and a. ocellaris. the distribution of those two species is mostly allopatric, but they share a common boundary and may overlap at some locations possibly leading to gene exchange. yet, despite similar behaviour and ecology, the species of the percula clade seemingly failed to radiate. the fact that the clade deriving from a major hybridisation event is the only one to show patterns of rapid speciation supports the view that hybridisation can promote diversification during adaptive radiation. actually, hybridisation appears to be one of the key factors explaining the adaptive radiation of the clownfishes. recent hybridisation events we identify the parental species of four putative hybrids, one being a. leucokranos, which is currently described as a species. since their taxonomic descriptions, the species status of a. leucokranos and a. thiellei has been questioned. indeed, heterospecific pairs have been observed in the wild and fish with similar phenotype can be produced in aquaria by hybrid crosses. nevertheless, evidence of a. leucokranos pairs also exist, tentatively proposing that we may be witnessing hybrid speciation in progress. yet, demonstrating this ongoing process would require more data than available here. it is thus more parsimonious to conclude at the moment, that a. leucokranos is an invalid species. we separate bmp sequences of each parental chromosome through molecular cloning and identify the putative parental species of a. leucokranos as a. chrysopterus and a. sandaracinos. this is consistent with field observations. the hybrid individuals that we sequenced are all likely recent hybrids because the two clones can always be clearly separated and cluster with different putative parental taxa. this suggests that genomic rearrangement occurring during reproductive events did not yet occur. the special social structure of clownfish may put some behavioural constraints on hybridisation. indeed, all clownfish species are protandrous hermaphrodites and live in social groups with size-based dominance hierarchy. the female is the largest and dominant individual, the male the second largest, and usually smaller non-breeding individuals follow, decreasing in size relative to their hierarchical position. in certain cases, size differentiation allows smaller species to cohabit with a larger species in the same anemone. while reproduction of the smallest species is largely suppressed by the presence of the largest clownfish, species hybridisation cannot be fully excluded. in such instance, the size-based hierarchy ensure that the female of a hybrid pair will most probably always be the largest individual. this is what we observe in a. leucokranos. the parental origin of the three other hybrids does not allow for a definitive conclusion regarding this behavioural constraint. indeed, the size difference between species of the akallopisos group is small and cannot be used as a diagnostic feature separating a. akalloposis, a. pacificus and a. perideraion. it is thus likely that the behavioural constraint on hybridisation between those species is going to be weaker than between species with large size difference. geography of recent hybridisation hybrid zones appear when two species share such margins and have the possibility to exchange genes. at the range margins, the probability of encountering a heterospecific mate is higher than that of meeting an individual of its own species whenever an individual moves into the range of the other species. this will increase the probability of hybridisation events. as expected, a. leucokranos is distributed at the edge of the two parental distributions and hybrids and were found at the eastern edge of the range of a. akallopisos. further genetic sampling would be necessary to better define those potential hybrid zones. this would be of particular interest given the evolutionary significance of hybridisation in animals, as exemplified by the importance of exogenous selection on contemporary hybrid zones.  hybridisation can impact speciation in many ways. our analyses show that in the case of the adaptive radiation of the clownfishes, hybridisation events are linked with a burst in diversification rate. we observe that hybridisation has occurred throughout the evolutionary history of the group and is still happening. further studies are required to measure the extant of ongoing hybrid speciation, for example in a. leucokranos. there, field observations coupled with experiments will allow to identify the conditions in which hybrids have a better fitness than their parents, illustrating the thight relationship linking species ecology and evolutionary processes. 
it is increasingly recognised that hybridisation events occurred during the evolutionary history of numerous species, especially during the early stages of adaptive radiation. then, using a tree-based jack-knife method, we identify clownfish species likely stemming from hybridisation events. our results show that consistently with the syngameon hypothesis, hybridisation events are linked with a burst of diversification in the clownfishes. moreover, several recently diverged clownfish lineages likely originated through hybridisation, which indicates that diversification, catalysed by hybridisation events, may still be happening. we study the effect of hybridisation on diversification in the clownfishes, a clade of coral reef fish that diversified through an adaptive radiation process. we generate sequences of three mitochondrial genes to complete an existing dataset of nuclear sequences and document cytonuclear discordance at a node, which shows a drastic increase of diversification rate. the importance of hybridisation during species diversification has long been debated among evolutionary biologists. while two species of clownfish are likely to have been described from hybrid specimens, the occurrence and effect of hybridisation on the clade diversification is yet unknown. finally, we use molecular cloning and identify the putative parental species of four clownfish specimens that display the morphological characteristics of hybrids.
 the importance of hybridisation during species diversification has long been debated among evolutionary biologists. it is increasingly recognised that hybridisation events occurred during the evolutionary history of numerous species, especially during the early stages of adaptive radiation. we study the effect of hybridisation on diversification in the clownfishes, a clade of coral reef fish that diversified through an adaptive radiation process. while two species of clownfish are likely to have been described from hybrid specimens, the occurrence and effect of hybridisation on the clade diversification is yet unknown.  we generate sequences of three mitochondrial genes to complete an existing dataset of nuclear sequences and document cytonuclear discordance at a node, which shows a drastic increase of diversification rate. then, using a tree-based jack-knife method, we identify clownfish species likely stemming from hybridisation events. finally, we use molecular cloning and identify the putative parental species of four clownfish specimens that display the morphological characteristics of hybrids.  our results show that consistently with the syngameon hypothesis, hybridisation events are linked with a burst of diversification in the clownfishes. moreover, several recently diverged clownfish lineages likely originated through hybridisation, which indicates that diversification, catalysed by hybridisation events, may still be happening. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords syngameonspeciationdiversificationcytonuclear discordanceanemonefishissue-copyright-statement© the author 2014 