BACKGROUND
acoustic communication provides a good model for studying the evolution of behaviour.
generally speaking, acoustic signals used in mate choice and mate recognition can play a role in speciation for sound producing taxa such as anurans  <cit> , insects  <cit>  and birds  <cit> .
variation in acoustic signals can act as pre-zygotic isolating mechanisms  <cit> , the receivers being the selective force on the evolution of the signal  <cit> .
the acoustic signal is typically composed of both temporal and spectral components, which may evolve at different rates  <cit> .
examining these patterns of acoustic variation among species may be useful for testing the evolutionary history of the characters  <cit> .
among the sound-producing fish, the coral reef damselfishes  are one of the most intensely studied families with sound production being documented for more than  <dig> species, belonging to seven different genera  <cit> .
within this large and diverse fish family, clownfishes are colorful coral reef fishes well known for their mutualistic relationship with tropical sea anemones that host them  <cit> .
recent phylogenetic investigations using both morphological and molecular characters supported the monophyly of the clownfishes belonging to the genera amphiprion and premnas  <cit> .
more specifically, on the basis of  <dig>  species and three mitochondrial genes, santini and polacco  <cit>  proposed a first hypothesis concerning the lifestyle and origin of the ancestral clownfish.
they suggested it was a slender-bodied animal with a rounded caudal fin.
however, their interpretation of the evolutionary history of this group still remains poorly explained.
in clownfishes, groups are composed of a breeding pair and between zero to four non-breeders, depending on species and size of host  <cit> .
within each group, the sex is controlled socially and there is a size-based dominance hierarchy: the breeding female is the largest individual, the breeding male is the second largest and the non-breeders get progressively smaller as the hierarchy descends  <cit> .
the size hierarchy forms a queue to attain dominant status; individuals only ascend in rank when a higher rank individual disappears, and the smallest fish in the group is always the most recent recruit  <cit> .
clownfishes are prolific callers, producing sounds during interactions among group members  <cit> .
in such a system, sounds are not used for mate attraction.
however, acoustic signals might confer a higher probability of attaining breeding status.
dominant frequency and pulse duration of the calls being morphologically determined signals related to fish size  <cit> , sounds seem to be important for living in social group because the hierarchy determines which fish can have access to reproduction  <cit> .
agonistic interactions are involved in daily behavior  <cit>  and sounds are known to be associated with them  <cit> , suggesting how important acoustic communication is in clownfish group.
so, the question arises as to whether this behaviour is also important in speciation process.
to find out, the first step was to compare the calls of closely related clownfish species in order to evaluate the variation in call characters .
amphiprion species produce the same kind of broadband-pulsed sounds during agonistic interactions  <cit> , meaning the differences should not be at the level of the biomechanics.
although sounds are all produced by snapping jaws  <cit> , other characteristics could display variation among species.
moreover, dominant frequency and pulse duration are known to be size-related acoustic signals in clownfishes  <cit> .
we predict that if these characters were important in the taxon diversification, the different species would have evolved different relationships between dominant frequency and fish size, and between pulse duration and fish size.
overlap in other characters such as pulse period or number of pulses in a call would not be observable in case of diversifying character of the taxa.
this study analysed the sounds of  <dig> different clownfish species.
the aim was to test the hypothesis that acoustic features can help to evaluate the potential role of acoustic communication as a driving force in the evolution of clownfishes.
RESULTS
interspecific differences in sounds
sounds were produced by all the  <dig> species during aggressive interactions.
the call of each species consisted of short pulses emitted alone or in series, and in a relatively narrow band of low frequencies .
pulse duration and dominant frequency were highly related to fish size across species .
the more fish size increased, the more pulse duration increased , and the more dominant frequency decreased .
to determine whether these size-related acoustic features evolved in a similar way among species, five groups of individuals were analysed .
the inter-species comparison using fish size as a covariate showed that pulse duration  and dominant frequency  did not differ among species, with all the  <dig> species being situated on the same slope.
thereby, variation among species in both acoustic features was clearly explained by size dimorphism between clownfish species.
all recordings were made at 26°c.
species are presented in ascending size order
n, number of recorded individuals per species with  <dig> sounds analysed per individual.
thus, the standard deviation was sometimes calculated from  <dig> measurements  or from  <dig> measurements when only one specimen per species was recorded.
some differences between species still remained for the frequency-size and duration-size relationships even after removing the effect of body size .
however, this observation needs to be carefully interpreted because deeper attention to pairwise comparisons revealed that pulse duration/body size relationship  and dominant frequency/body size relationship  were not significantly different between individuals having similar body size  and belonging to five different species .
the number of pulses broadly overlapped between species , although there were some differences .
pairwise comparisons showed a few species were significantly different : a. ocellaris and a. nigripes, a. ocellaris and a. clarkii, a. ocellaris and a. akallopisos.
pulse period displayed the most variation among species , but considerable overlap in pairwise comparisons showed that several species were similar .
morphology and sounds
deeper attention was given to three species having different teeth shape.
sound comparisons of these three species based on three specimens having the same size  revealed that the dominant frequency of a. akallopisos, a. ocellaris and a. frenatus was not significantly different  and was respectively  <dig>   <dig> and  <dig> hz.
moreover, the acoustic waveform and the power spectrum exhibited the same pattern despite the different types of teeth: rectangular and incisiform in a. akallopisos, conical and caniniform in a. frenatus and spatulate in a. ocellaris .
discussion
this study is one of the first comparisons of acoustic characteristics in a sizeable number of closely related species of fishes .
the most important insight was found at the level of the relationship between fish size and both dominant frequency and pulse duration.
these kinds of relationships were already well known in fishes and have been found in numerous species from different taxa  <cit> .
in the present study and for the first time, it appears that these relationships are observed at a higher taxonomic level in the case of clownfishes  since dominant frequency and pulse duration are strongly predicted by body size among the  <dig> different species.
it clearly shows all clownfish species use the same mechanism of vocalization  <cit> , which has remained largely conserved throughout evolution.
moreover, different species having different body shape and different types of teeth  but the same size produce sounds that display the same power spectrum and the same oscillogram, demonstrating these morphological variations do not make significant differences in the sounds produced by the jaw teeth snapping.
the size influence highlights that all the fourteen species might have a major overlap at the level of pulse length and dominant frequency.
surprisingly, using the pulse duration/body size and dominant frequency/body size ratios removes the effect of body size at the level of pulse duration , but not at the level of dominant frequency .
regarding the dominant frequency, the ratio continues to be smaller in larger species, but highly reduces the overlap between species.
for example, some amphiprion clarkii, measuring between  <dig> and  <dig> mm in sl, produced a frequency range that was between  <dig> and  <dig> hz, overlapping also the frequency of smaller a. ocellaris .
however, the ratio of dominant frequency/body size was  <dig>  in a. clarkii and  <dig>  in a. ocellaris, clearly distinguishing the species.
therefore, a factor other than body length must be important in determining the absolute frequency values.
the volume of the swimbladder could be the determining factor.
moreover, the data of this last factor are also correlated with fish size.
the question arises whether the fish is able to determine the relationship  between the emitter size and the sound frequency it detects.
if so, it could enable the fish to distinguish whether the emitter is conspecific or not.
in the future, it would be interesting to compare these size-related relationships between amphiprion and other pomacentrids.
comparison with pomacentrus partitus data  <cit>  seems to indicate these fish might also be placed on the same slope.
in this case, we could expect there is an ancestral relationship between dominant frequency and body size, and this relationship is not important in the taxon diversification because it does not change between species.
however, more precise data from other pomacentrids are needed.
variations in sounds are usually considered as pre-zygotic isolating mechanisms leading to speciation  <cit> .
in amphiprion species, acoustic properties can contribute to the differentiation of species because some of them showed differences in at least one of the acoustic characteristics recorded.
however, the significant overlap in acoustic data due in part to the conserved mechanism of sound production supports the acoustic communication cannot be considered as the unique isolating barrier and does not seem to be the main driving force in the evolution of clownfishes.
the fact that all species have the same biomechanics implies these fishes do not have many possibilities to develop variations in their calls: they can differ in dominant frequency and pulse duration through their body size, in pulse period and in number of pulses in a call.
the alternative hypothesis would be some differences in the calls are a by-product of the size variation.
body size as a trait of natural selection has already been demonstrated in the speciation event of some stickleback species in which this difference is thought to be an adaptation to alternative foraging habitats  <cit> .
in damselfishes, evolutionary change in body size  could be assumed as a relatively common phenomenon  <cit>  and could therefore be involved in the isolation of some amphiprion species.
from the proximal cause point of view, the size appears really important in maintaining the hierarchy existing within groups.
in amphiprion akallopisos, all aggressive interactions  appear to be preferentially directed towards individuals adjacent in ranks  <cit> .
in amphiprion percula, rank was the only factor associated with the probability of mortality; low-rank individuals suffer from a higher mortality rate than high-rank individuals.
the most likely explanation for this pattern is competition for rank  <cit> , preventing smaller fish from having access to reproduction.
it means that acoustic communication can be an important factor for mating access.
from the ultimate cause point of view, competition for the limited anemone resource may have resulted in niche partitioning through specialization for different anemone species  <cit> , most clownfish species remaining in close contact with their hosts and rarely interact with other species on the reef  <cit> .
however, some clownfish species appear to partition the anemone resource with other species by having a refuge in size  <cit> : small a. sandaracinos or a. leucokranos cohabit with a. chrysopterus in the region of madang , while small a. perideraion use the same host sea anemone as a. clarkii in the region of okinawa .
in both cases, the different sizes of cohabiting species imply they possess clear differences in their acoustic repertoire , size-related call characteristics such as main frequency and pulse length being a by-product of the evolutionary trait.
in the japanese heterospecific groups, small a. perideraion are not considered as competitors and should receive less aggressive attention from larger congener a. clarkii.
although a. clarkii suppresses the growth and reproduction of a. perideraion  <cit> , subadult a. perideraion are able to mature in heterospecific groups, and change to female when they are the largest among conspecific members.
this suggests that a. perideraion in heterospecific groups prepare for reproduction before the disappearance or emigration of larger a. clarkii.
thus, they adopt a mating strategy that involves waiting for vacated breeding posts because of their low mobility and a low host density  <cit> .
due to the relative simplicity of many central and peripheral vocal mechanisms, fish typically lack the ability to produce complex and dynamic, frequency-modulated calls  <cit> .
vocal differences among fish species are usually due to variations in temporal patterning  <cit> .
pulse period has been shown to be the most important acoustic feature involved in species recognition in pomacentrids  <cit> .
divergence in this character seems to be sufficient to drive pre-zygotic isolation  <cit> , because differences in the calling characteristics are able to prevent the signaller to be considered as a competitor.
myrberg et al.
<cit>  conducted playback experiments to test the responsiveness of different stegastes species.
although sounds of each species were able to elicit responses of all the other species, males significantly more responded to sounds of their own species than to sounds from congeners.
interestingly, species that cohabit individual sea anemones  present a completely different pulse period.
as previously stated, non-overlap in this character may have been important in the taxon diversification.
however, pulse period is not systematically significantly different among sympatric species: a. clarkii, a. frenatus and a. ocellaris have the same pulse period range  while living in sympatry on the fringing reef around sesoko island  <cit> .
these three species inhabit different host species, being heteractis crispa for a. clarkii, entacmaea quadricolor for a. frenatus and stichodactyla gigantea for a. ocellaris  <cit> , which suggests overlap in pulse period among these species is of minor importance.
CONCLUSIONS
we predicted that no-overlap in different acoustic features would drive the taxon diversification.
however, results surprisingly showed significant overlap in some acoustic features  in amphiprion species.
it is the first case for which so many different species can be placed on the same slope, giving the opportunity to use the dominant frequency and pulse duration for assessing fish size.
this set of observations highlights 1) the use of a highly conservative mechanism, 2) the important role of body size in clownfish ways of life and 3) that this character is not important in this taxon diversification because all the clownfish species maintained the same relationships between fish size and both dominant frequency and pulse duration.
however, in some case, the refuge in size could be a way to access to diversification.
significant overlap in sonic features could also be due to the fact that sounds are not produced to find mate, but to defend mate access, which restricts the constraints of diversification.
we conclude that sounds do not appear to be the main driving force in the diversification of clownfishes.
however, differences in the pulse period between cohabiting species showed that, in some case, sounds can help to differentiate the species, to prevent competition between cohabiting species and to promote the diversification of taxa.

 acoustic communication provides a good model for studying the evolution of behaviour. generally speaking, acoustic signals used in mate choice and mate recognition can play a role in speciation for sound producing taxa such as anurans, insects and birds. variation in acoustic signals can act as pre-zygotic isolating mechanisms, the receivers being the selective force on the evolution of the signal. the acoustic signal is typically composed of both temporal and spectral components, which may evolve at different rates. examining these patterns of acoustic variation among species may be useful for testing the evolutionary history of the characters. among the sound-producing fish, the coral reef damselfishes are one of the most intensely studied families with sound production being documented for more than species, belonging to seven different genera. within this large and diverse fish family, clownfishes are colorful coral reef fishes well known for their mutualistic relationship with tropical sea anemones that host them. recent phylogenetic investigations using both morphological and molecular characters supported the monophyly of the clownfishes belonging to the genera amphiprion and premnas. more specifically, on the basis of species and three mitochondrial genes, santini and polacco proposed a first hypothesis concerning the lifestyle and origin of the ancestral clownfish. they suggested it was a slender-bodied animal with a rounded caudal fin. however, their interpretation of the evolutionary history of this group still remains poorly explained. in clownfishes, groups are composed of a breeding pair and between zero to four non-breeders, depending on species and size of host. within each group, the sex is controlled socially and there is a size-based dominance hierarchy: the breeding female is the largest individual, the breeding male is the second largest and the non-breeders get progressively smaller as the hierarchy descends. the size hierarchy forms a queue to attain dominant status; individuals only ascend in rank when a higher rank individual disappears, and the smallest fish in the group is always the most recent recruit. clownfishes are prolific callers, producing sounds during interactions among group members. in such a system, sounds are not used for mate attraction. however, acoustic signals might confer a higher probability of attaining breeding status. dominant frequency and pulse duration of the calls being morphologically determined signals related to fish size, sounds seem to be important for living in social group because the hierarchy determines which fish can have access to reproduction. agonistic interactions are involved in daily behavior and sounds are known to be associated with them, suggesting how important acoustic communication is in clownfish group. so, the question arises as to whether this behaviour is also important in speciation process. to find out, the first step was to compare the calls of closely related clownfish species in order to evaluate the variation in call characters. amphiprion species produce the same kind of broadband-pulsed sounds during agonistic interactions, meaning the differences should not be at the level of the biomechanics. although sounds are all produced by snapping jaws, other characteristics could display variation among species. moreover, dominant frequency and pulse duration are known to be size-related acoustic signals in clownfishes. we predict that if these characters were important in the taxon diversification, the different species would have evolved different relationships between dominant frequency and fish size, and between pulse duration and fish size. overlap in other characters such as pulse period or number of pulses in a call would not be observable in case of diversifying character of the taxa. this study analysed the sounds of different clownfish species. the aim was to test the hypothesis that acoustic features can help to evaluate the potential role of acoustic communication as a driving force in the evolution of clownfishes.  interspecific differences in sounds sounds were produced by all the species during aggressive interactions. the call of each species consisted of short pulses emitted alone or in series, and in a relatively narrow band of low frequencies. pulse duration and dominant frequency were highly related to fish size across species. the more fish size increased, the more pulse duration increased, and the more dominant frequency decreased. to determine whether these size-related acoustic features evolved in a similar way among species, five groups of individuals were analysed. the inter-species comparison using fish size as a covariate showed that pulse duration and dominant frequency did not differ among species, with all the species being situated on the same slope. thereby, variation among species in both acoustic features was clearly explained by size dimorphism between clownfish species. all recordings were made at 26°c. species are presented in ascending size order n, number of recorded individuals per species with sounds analysed per individual. thus, the standard deviation was sometimes calculated from measurements or from measurements when only one specimen per species was recorded. some differences between species still remained for the frequency-size and duration-size relationships even after removing the effect of body size. however, this observation needs to be carefully interpreted because deeper attention to pairwise comparisons revealed that pulse duration/body size relationship and dominant frequency/body size relationship were not significantly different between individuals having similar body size and belonging to five different species. the number of pulses broadly overlapped between species, although there were some differences. pairwise comparisons showed a few species were significantly different: a. ocellaris and a. nigripes, a. ocellaris and a. clarkii, a. ocellaris and a. akallopisos. pulse period displayed the most variation among species, but considerable overlap in pairwise comparisons showed that several species were similar. morphology and sounds deeper attention was given to three species having different teeth shape. sound comparisons of these three species based on three specimens having the same size revealed that the dominant frequency of a. akallopisos, a. ocellaris and a. frenatus was not significantly different and was respectively and hz. moreover, the acoustic waveform and the power spectrum exhibited the same pattern despite the different types of teeth: rectangular and incisiform in a. akallopisos, conical and caniniform in a. frenatus and spatulate in a. ocellaris. discussion this study is one of the first comparisons of acoustic characteristics in a sizeable number of closely related species of fishes. the most important insight was found at the level of the relationship between fish size and both dominant frequency and pulse duration. these kinds of relationships were already well known in fishes and have been found in numerous species from different taxa. in the present study and for the first time, it appears that these relationships are observed at a higher taxonomic level in the case of clownfishes since dominant frequency and pulse duration are strongly predicted by body size among the different species. it clearly shows all clownfish species use the same mechanism of vocalization, which has remained largely conserved throughout evolution. moreover, different species having different body shape and different types of teeth but the same size produce sounds that display the same power spectrum and the same oscillogram, demonstrating these morphological variations do not make significant differences in the sounds produced by the jaw teeth snapping. the size influence highlights that all the fourteen species might have a major overlap at the level of pulse length and dominant frequency. surprisingly, using the pulse duration/body size and dominant frequency/body size ratios removes the effect of body size at the level of pulse duration, but not at the level of dominant frequency. regarding the dominant frequency, the ratio continues to be smaller in larger species, but highly reduces the overlap between species. for example, some amphiprion clarkii, measuring between and mm in sl, produced a frequency range that was between and hz, overlapping also the frequency of smaller a. ocellaris. however, the ratio of dominant frequency/body size was in a. clarkii and in a. ocellaris, clearly distinguishing the species. therefore, a factor other than body length must be important in determining the absolute frequency values. the volume of the swimbladder could be the determining factor. moreover, the data of this last factor are also correlated with fish size. the question arises whether the fish is able to determine the relationship between the emitter size and the sound frequency it detects. if so, it could enable the fish to distinguish whether the emitter is conspecific or not. in the future, it would be interesting to compare these size-related relationships between amphiprion and other pomacentrids. comparison with pomacentrus partitus data seems to indicate these fish might also be placed on the same slope. in this case, we could expect there is an ancestral relationship between dominant frequency and body size, and this relationship is not important in the taxon diversification because it does not change between species. however, more precise data from other pomacentrids are needed. variations in sounds are usually considered as pre-zygotic isolating mechanisms leading to speciation. in amphiprion species, acoustic properties can contribute to the differentiation of species because some of them showed differences in at least one of the acoustic characteristics recorded. however, the significant overlap in acoustic data due in part to the conserved mechanism of sound production supports the acoustic communication cannot be considered as the unique isolating barrier and does not seem to be the main driving force in the evolution of clownfishes. the fact that all species have the same biomechanics implies these fishes do not have many possibilities to develop variations in their calls: they can differ in dominant frequency and pulse duration through their body size, in pulse period and in number of pulses in a call. the alternative hypothesis would be some differences in the calls are a by-product of the size variation. body size as a trait of natural selection has already been demonstrated in the speciation event of some stickleback species in which this difference is thought to be an adaptation to alternative foraging habitats. in damselfishes, evolutionary change in body size could be assumed as a relatively common phenomenon and could therefore be involved in the isolation of some amphiprion species. from the proximal cause point of view, the size appears really important in maintaining the hierarchy existing within groups. in amphiprion akallopisos, all aggressive interactions appear to be preferentially directed towards individuals adjacent in ranks. in amphiprion percula, rank was the only factor associated with the probability of mortality; low-rank individuals suffer from a higher mortality rate than high-rank individuals. the most likely explanation for this pattern is competition for rank, preventing smaller fish from having access to reproduction. it means that acoustic communication can be an important factor for mating access. from the ultimate cause point of view, competition for the limited anemone resource may have resulted in niche partitioning through specialization for different anemone species, most clownfish species remaining in close contact with their hosts and rarely interact with other species on the reef. however, some clownfish species appear to partition the anemone resource with other species by having a refuge in size: small a. sandaracinos or a. leucokranos cohabit with a. chrysopterus in the region of madang, while small a. perideraion use the same host sea anemone as a. clarkii in the region of okinawa. in both cases, the different sizes of cohabiting species imply they possess clear differences in their acoustic repertoire, size-related call characteristics such as main frequency and pulse length being a by-product of the evolutionary trait. in the japanese heterospecific groups, small a. perideraion are not considered as competitors and should receive less aggressive attention from larger congener a. clarkii. although a. clarkii suppresses the growth and reproduction of a. perideraion, subadult a. perideraion are able to mature in heterospecific groups, and change to female when they are the largest among conspecific members. this suggests that a. perideraion in heterospecific groups prepare for reproduction before the disappearance or emigration of larger a. clarkii. thus, they adopt a mating strategy that involves waiting for vacated breeding posts because of their low mobility and a low host density. due to the relative simplicity of many central and peripheral vocal mechanisms, fish typically lack the ability to produce complex and dynamic, frequency-modulated calls. vocal differences among fish species are usually due to variations in temporal patterning. pulse period has been shown to be the most important acoustic feature involved in species recognition in pomacentrids. divergence in this character seems to be sufficient to drive pre-zygotic isolation, because differences in the calling characteristics are able to prevent the signaller to be considered as a competitor. myrberg et al. conducted playback experiments to test the responsiveness of different stegastes species. although sounds of each species were able to elicit responses of all the other species, males significantly more responded to sounds of their own species than to sounds from congeners. interestingly, species that cohabit individual sea anemones present a completely different pulse period. as previously stated, non-overlap in this character may have been important in the taxon diversification. however, pulse period is not systematically significantly different among sympatric species: a. clarkii, a. frenatus and a. ocellaris have the same pulse period range while living in sympatry on the fringing reef around sesoko island. these three species inhabit different host species, being heteractis crispa for a. clarkii, entacmaea quadricolor for a. frenatus and stichodactyla gigantea for a. ocellaris, which suggests overlap in pulse period among these species is of minor importance.  we predicted that no-overlap in different acoustic features would drive the taxon diversification. however, results surprisingly showed significant overlap in some acoustic features in amphiprion species. it is the first case for which so many different species can be placed on the same slope, giving the opportunity to use the dominant frequency and pulse duration for assessing fish size. this set of observations highlights 1) the use of a highly conservative mechanism, 2) the important role of body size in clownfish ways of life and 3) that this character is not important in this taxon diversification because all the clownfish species maintained the same relationships between fish size and both dominant frequency and pulse duration. however, in some case, the refuge in size could be a way to access to diversification. significant overlap in sonic features could also be due to the fact that sounds are not produced to find mate, but to defend mate access, which restricts the constraints of diversification. we conclude that sounds do not appear to be the main driving force in the diversification of clownfishes. however, differences in the pulse period between cohabiting species showed that, in some case, sounds can help to differentiate the species, to prevent competition between cohabiting species and to promote the diversification of taxa. 
these fishes are also prolific callers whose aggressive sounds seem to play an important role in the social hierarchy. agonistic interactions being involved in daily behaviour suggest how acoustic communication might play an important role in clownfish group. the number of pulses broadly overlaps among species, whereas the pulse period displays the most variation even if it shows overlap among sympatric species. sound comparisons between three species having different types of teeth and body shape do not show differences neither in the acoustic waveform nor in the power spectrum. significant overlap in acoustic features demonstrates that the sound-producing mechanism is highly conservative among species. clownfishes are colorful coral reef fishes living in groups in association with sea anemones throughout the indo-pacific ocean. calls are produced to reach and defend the competition to mate access. sounds were recorded and compared in fourteen clownfish species to evaluate the potential role of acoustic communication as an evolutionary driving force. surprisingly, the relationship between fish size and both dominant frequency and pulse duration is not only species-specific; all the specimens of the species are situated on exactly the same slope, which means the size of any amphiprion can be predicted by both acoustic features. however, differences in the pulse period between cohabiting species show that, in some case, sounds can help to differentiate the species, to prevent competition between cohabiting species and to promote the diversification of taxa. this morphological constraint does not permit a consideration of acoustic communication as the main driving force in the diversification of clownfishes.
 clownfishes are colorful coral reef fishes living in groups in association with sea anemones throughout the indo-pacific ocean. within their small societies, size hierarchy determines which fish have access to reproduction. these fishes are also prolific callers whose aggressive sounds seem to play an important role in the social hierarchy. agonistic interactions being involved in daily behaviour suggest how acoustic communication might play an important role in clownfish group. sounds were recorded and compared in fourteen clownfish species to evaluate the potential role of acoustic communication as an evolutionary driving force.  surprisingly, the relationship between fish size and both dominant frequency and pulse duration is not only species-specific; all the specimens of the species are situated on exactly the same slope, which means the size of any amphiprion can be predicted by both acoustic features. the number of pulses broadly overlaps among species, whereas the pulse period displays the most variation even if it shows overlap among sympatric species. sound comparisons between three species having different types of teeth and body shape do not show differences neither in the acoustic waveform nor in the power spectrum.  significant overlap in acoustic features demonstrates that the sound-producing mechanism is highly conservative among species. differences in the calls of some species are due to size dimorphism and the sound variation might be in this case a by-product. this morphological constraint does not permit a consideration of acoustic communication as the main driving force in the diversification of clownfishes. moreover, calls are not produced to find mate and consequently are less subject to variations due to partner preference, which restricts the constraints of diversification. calls are produced to reach and defend the competition to mate access. however, differences in the pulse period between cohabiting species show that, in some case, sounds can help to differentiate the species, to prevent competition between cohabiting species and to promote the diversification of taxa. pomacentridaeclownfishevolutionbiogeographysound productionsympatryhierarchysizefrequency 