S A Poulet

Roscoff C Fauvelot

Ae S Planes
email: planes@univ-perp.fr

C Fauvelot

in marine fish: biologically or historically driven patterns?

Understanding origins of present-day genetic structure in marine fish: biologically or historically driven patterns?

Introduction

A central challenge with respect to marine species is to understand patterns of genetic differentiation, if any, in a fluid ecosystem that tends to homogenize populations [START_REF] Palumbi | Genetic divergence, reproductive isolation, and marine speciation[END_REF]. Even for sedentary species, such as coral reef ones, for which adults are often confined to a limited area by their behaviour or the fragmentation of their habitat, the pelagic larval stage allows for migration and gene flow [START_REF] Leis | Larval fish dispersal and the East Pacific Barrier[END_REF]. Such dispersal is not without benefit to animals that inhabit a patchy heterogeneous environment, and larval exchange is assumed, for most species, to be the only mechanism uniting spatially discrete populations [START_REF] Bonhomme | Some evolutionary arguments about what maintains the pelagic interval in reef fishes[END_REF]. In this sense, coral reef fish provide a good model for investigating the impact of the pelagic larval stage on dispersal, since the fragmentation of their habitat restricts exchange to the larval stage that can be accurately estimated at least in time using the microstructure of otoliths [START_REF] Wellington | Planktonic larval duration of one hundred species of Pacific and Atlantic damselfishes (Pomacentridae)[END_REF]. Initial models considered pelagic larvae as drifting propagules that matched oceanic current [START_REF] Roberts | Connectivity and management of Caribbean coral reefs[END_REF]), but recent investigations demonstrated real swimming capabilities or swimming orientation at least for the latest stage [START_REF] Leis | What the pelagic stages of coral reef fishes are doing out in the blue water: daytime field observation of larval behavioral capabilities[END_REF][START_REF] Leis | In situ swimming speeds in the late pelagic larvae of some Indo-Pacific coral-reef fishes[END_REF][START_REF] Stobutzki | Sustained swimming abilities for the late pelagic stages of coral reef fishes[END_REF]. These oriented swimming capabilities have been proposed as mechanisms that would explain retention and self-recruitment in very open areas, such as the Great Barrier Reef [START_REF] Jones | Self-recruitment in a coral reef fish population[END_REF] or the Caribbean [START_REF] Swearer | Larval retention and recruitment in an island population of a coral-reef fish[END_REF].

Dispersal is among the most difficult problems to study empirically in the sea because of the impracticality of direct observations of larval stages. Besides plankton net surveys and/or otolith analysis, which provided significant results on dispersal [START_REF] Leis | Offshore distributional patterns of Hawaiian fish larvae[END_REF][START_REF] Jones | Self-recruitment in a coral reef fish population[END_REF][START_REF] Swearer | Larval retention and recruitment in an island population of a coral-reef fish[END_REF], natural genetic markers have also produced significant results (reviews in [START_REF] Smith | Electrophoretic studies and the stock concept in marine teleosts[END_REF][START_REF] Bohonak | Dispersal, gene flow, and population structure[END_REF]. As early as 1975, Ehrlich suggested that assessment of allozymic variation among geographically remote populations of reef fish is the optimal approach for testing dispersal and migration capabilities of larvae. Allozyme electrophoresis has become a powerful technique to demonstrate genetic variation and has been widely used in coral reef fish surveys [START_REF] Planes | Biogeography and larval dispersal inferred from population genetic analysis[END_REF]. Analyses of genetic structure of coral reef fish populations were initiated in 1982 in Japan [START_REF] Bell | Morphological and genetic variation in Japanese populations of the anemone fish Amphiprion clarkii[END_REF] and in 1984 in Hawaii [START_REF] Shaklee | Genetic variation and population structure in the damselfish, Stegastes fasciolatus, throughout the Hawaiian archipelago[END_REF][START_REF] Shaklee | Genetic variation and population structure in a deep-water snapper, Pristipomoides filamentosus, in the Hawaiian archipelago[END_REF]; both looked at allozyme variations. With the exception of a remote oceanic population, these first studies undertaken in the Pacific did not reveal significant genetic differentiation among populations. The straightforward conclusion and general belief during that period was that coral reef fish populations exchanged genes through the pelagic stage at rates sufficient to homogenize the genetic structure over broad geographical ranges. Since the early 1980s several other studies have been undertaken on coral reef fish and provided divergent outcomes, ranging from panmixis over large areas to local and regional differentiation [START_REF] Ehrlich | The population biology of coral reef fishes[END_REF][START_REF] Bell | Morphological and genetic variation in Japanese populations of the anemone fish Amphiprion clarkii[END_REF][START_REF] Waples | A multispecies approach to the analysis of gene flow in marine shore fishes[END_REF][START_REF] Lacson | Genetic differentiation of bicolour damselfish (Eupomacentrus partitus) populations in Florida Keys[END_REF][START_REF] Lacson | Minimal genetic variation among samples of 6 species of coral reef fishes collected at La Parguera, Puerto Rico and Discovery Bay, Jamaica[END_REF][START_REF] Planes | Genetic differentiation in relation to restricted larval dispersal of the convict surgeon fish, Acanthurus triostegus, in French Polynesia[END_REF][START_REF] Planes | Genetic structure of Dascyllus aruanus populations in French Polynesia[END_REF][START_REF] Planes | Evidence of limited gene flow in three coral reef fish species in the lagoon of New Caledonia[END_REF][START_REF] Doherty | Acanthochromis polyacanthus, a fish lacking larval dispersal, has genetically differentiated populations at local and regional scales on the Great Barrier Reef[END_REF][START_REF] Doherty | Gene flow and larval duration in seven species of fish from the Great Barrier Reef[END_REF]. The origin of such discrepancies is probably manifold.

Detectable allelic frequencies can be used to estimate levels of migration if it can be assumed that these frequencies reflect a balance between the opposing forces of migration (gene flow) and random divergence of allelic frequencies (genetic drift). Of course, other forces, notably natural selection and historical contact, can influence allelic frequencies, and the relative importance of these forces in natural populations has been extremely difficult to evaluate directly. While this theory is silent about allopatric populations with identical genetic profiles [START_REF] Shaklee | Speciation and evolution of marine species studied by the electrophoretic analysis of proteins[END_REF], differentiation provides unequivocal evidence of reproductive isolation. The main problem when trying to summarize genetic data relating to larval dispersal is to be able to exclude all other factors likely to affect the genetic structure of populations, such as historical events. Historical factors may conflict with the current situation in areas affected by sea-level changes during the last glaciation events, creating reductions in the effective population size when suitable habitat disappeared. Such a hypothesis has been proposed to explain some contradictions between level of genetic differentiation and dispersal capabilities, in comparison of two species in French Polynesia [START_REF] Planes | Genetic structure of Dascyllus aruanus populations in French Polynesia[END_REF]). The species (Dascyllus aruanus) with reduced oceanic larval stage (i.e. dispersal stage) exhibited more or less genetic homogeneity within French Polynesia, while the other species (Acanthurus triostegus), with longer pelagic phase, showed genetically structured populations within the same region [START_REF] Planes | Genetic differentiation in relation to restricted larval dispersal of the convict surgeon fish, Acanthurus triostegus, in French Polynesia[END_REF]. Such antagonistic results initiated the hypothesis tested in the present work, since the absence of common sampling protocols and the variety of geographical scales surveyed make it impossible to identify any general trends from the data available in the literature.

In the present work we used a comparative approach, based on a multispecific analysis, to evaluate the features influencing the genetic structure between populations of coral reef fish and to provide testable hypotheses. Previous work on the basis of a comparative approach in marine fish all focused on testing for and showing a significant relationship between potential larval dispersal and genetic structure [START_REF] Waples | A multispecies approach to the analysis of gene flow in marine shore fishes[END_REF][START_REF] Doherty | Gene flow and larval duration in seven species of fish from the Great Barrier Reef[END_REF][START_REF] Gold | Mitochondrial DNA diversification and population structure in fishes from the Gulf of Mexico and western Atlantic[END_REF]. The present survey was intended to contrast the biological and historical features that mainly affect the genetic structure observed. This is of primary interest when genetic data are to be used for management and conservation, since we need to know whether we are looking at a present-day trend or an ancestral pattern. Regarding the biological features, we contrast species having pelagic eggs (generally linked to longer larval duration) to species with benthic attached eggs (generally associated with shorter larval duration) [START_REF] Thresher | Geographic variability in the ecology of coral reef fishes: evidence, evolution and possible implications[END_REF]. As historical factors, we concentrated on testing the effect of sea-level changes and its consequence for habitats according to species, by comparing species showing stable habitat, whatever the sea level (outer slope inhabitants), to species experiencing removal of their habitat in low sea-level periods, leading to local extinction (lagoonal species).

For the comparative analysis, we collected individuals of eight species showing different biological (reproductive strategy) and ecological (habitat) features within a similar network of isolated islands, providing different spatial scales within French Polynesia. The aim of this study was then to evaluate the level of genetic structure of these species, using allozymes to detect clustering of species showing similar features. We addressed the following questions: (1) Do allozymes show genetic differentiation among geographically isolated benthic populations of coral reef fishes in French Polynesia? (2) Is the amount of genetic differentiation correlated with geographical separation? (3) Are single-species genetic patterns correlated with the type of egg? (4) Does the habitat of adults have an impact on single-species genetic structure?

Materials and methods

Sampling

The geomorphology of French Polynesian islands is of particular interest when investigating larval dispersal between reefs, since the lack of reef continuum and the deep bottom between islands eliminate potential migration by adult movement [START_REF] Duncan | Linear volcanism in French Polynesia[END_REF]. Five isolated islands were sampled in French Polynesia (Fig. 1). One high volcanic island (Moorea) and one atoll (Tetiaroa) in the Society Archipelago were chosen, together with atolls from the Tuamotu Archipelago (Rangiroa, Takapoto and Marutea). Sites were selected according to the access facilities and the geographical distances between islands, in order to provide small (e.g. 60 km between Moorea and Tetiaroa), medium (about 250 km between Moorea and Takapoto), and large spatial scales (up to 1,550 km between Moorea and Marutea). Such distances were specifically chosen to test the relationship between genetic differentiation and geographical distance (cf. question 2, above).

Species were chosen from two different families in order to provide different reproductive strategies, based on egg type [START_REF] Thresher | Geographic variability in the ecology of coral reef fishes: evidence, evolution and possible implications[END_REF] and different habitats, to answer questions 3 and 4. The Chaetodontidae family provided species spawning pelagic eggs. Spawning in butterflyfishes involves large groups, with both sexes well represented [START_REF] Allen | A guide to angelfishes and butterflyfishes[END_REF]. Eggs hatch in the open sea approximately 24 h after their release and larvae spend an average of 45 days in the pelagos before returning to reefs [START_REF] Hourigan | Mid-ocean isolation and the evolution of Hawaiian reef fishes[END_REF]. The second family (Pomacentridae) provided species spawning benthic eggs. Most damselfish species are highly territorial and several days prior to spawning, the male (or in some cases both partners) select the nesting site. Eggs are under care in the nest for a period ranging from 2 to 7 days until hatching [START_REF] Allen | Damselfishes of the world[END_REF]. Larvae are pelagic and remain in the water column about 25 days on average [START_REF] Wellington | Planktonic larval duration of one hundred species of Pacific and Atlantic damselfishes (Pomacentridae)[END_REF].

Within these two families, eight species were selected according to their habitat, with species only found in the lagoon and species only found on the outer slope. Selection of species according to their habitat was based on previous studies of the fish communities [START_REF] Galzin | Structure of fish communities of French Polynesia coral reefs. 1. Spatial scales[END_REF]Galzin, personal communication): Chaetodon citrinellus as the butterflyfish inhabiting lagoons; Chaetodon quadrimaculatus and Forcipiger flavissimus, as butterflyfishes living on the outer slope; Pomacentrus pavo, Chrysiptera glauca and Dascyllus aruanus, as damselfishes inhabiting lagoons; and Chromis xanthura and Plectroglyphidodon dickii, as damselfishes living on the outer slope.

The standard sampling effort was 50 fishes per species per site, but some of the species could not be found or were rare in certain localities (Table 1): Chaetodon citrinellus was not found in Marutea, and Plectroglyphidodon dickii was absent at Moorea and Tetiaroa; Chrysiptera glauca was rare and difficult to collect at Moorea and Tetiaroa. A total of 1,662 individuals were collected either with a speargun or using an anaesthetic solution for smaller fishes, between March 1998 and December 2000.

Isoenzyme analysis

Fish were preserved in ice after collection until the liver and a piece of muscle were removed from each fish and placed separately in liquid nitrogen for long-term conservation. Individual pieces of tissues were homogenized at 4°C in an equal volume of Tris-HCl/EDTA/NADP buffer (pH 6.8). Homogeneous samples were centrifuged at 15,000•g for 30 min at 4°C, and the supernatant was removed and stored in individual labeled Eppendorf at -80°C until all electrophoretic determinations were completed. First, 12 individuals of each species were used in order to design a protocol: only loci with clearly interpretable patterns were scored. The samples were then processed by routine electrophoresis on horizontal starch gel, following [START_REF] Pasteur | Manuel de ge´ne´tique par e´lectrophore`se des prote´ines. Collection technique et documentation. Lavoisier, Paris Pirazzoli PA, Montaggioni L[END_REF]. Alleles at polymorphic loci were assigned numerical designations expressing the mobility of their respective protein products relative to the mobility of the most common allele (designated 100) among the samples. Enzyme names and numbers follow [START_REF] Shaklee | Gene nomenclature for protein-coding loci in fish[END_REF].

Statistical analysis

Allelic and genotypic frequencies for the polymorphic loci were obtained by counting phenotypes directly from the gels using the Genetix 4.0 package [START_REF] Bonhomme | Genetix -logiciel d'analyse des donne´es du groupe de ge´ne´tique des populations de Montpellier, version 1.0[END_REF]) available on: http:// www.univ-montp2.fr/genome-pop/genetix.htm. Observed heterozygosity values were computed for each population from the genotypic frequencies, adding monomorphic loci for multilocus values [START_REF] Nei | Analysis of gene diversity in subdivided populations[END_REF]. Heterozygosity values were assessed with their estimates of variance and standard deviation generated by resampling over loci (available on the Genetix package). Significant differences in multilocus heterozygosity were analyzed by a t-test between species to test hypotheses on the impact of the reproductive strategy and/or the habitat. The fixation index (F is ) was used to represent the Mendelian equilibrium, and single and multilocus deviation from Hardy-Weinberg equilibrium was tested using the Markov chain reaction implemented in Genepop 3.1d [START_REF] Raymond | An exact test for population differentiation[END_REF]. Significance levels for statistical tests were adjusted, for each species separately, according to the sequential Bonferroni [START_REF] Rice | Analyzing tables of statistical tests[END_REF].

Fig. 1. Location of sampling sites of the eight fish species in French Polynesia. Details of sites are described in the text and sampling effort is detailed in Table 1 Wright's standardized variance in allelic frequencies (F st ;[START_REF] Wright | Evolution and the genetics of populations[END_REF]) was computed over all sites and between each pair of sites, following the [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] algorithm available on Genetix. Significant differences between allelic frequencies were tested using the Fisher exact test implemented in Genepop 3.1d. Significance levels for statistical tests were adjusted, for each species separately, according to the sequential Bonferroni [START_REF] Rice | Analyzing tables of statistical tests[END_REF].

Correlation between geographical distance and genetic differentiation (multilocus F st ) was performed over all loci for each species using Mantel's test. We computed the normalized Mantel statistic, r (which takes values from -1 to +1), which was tested for significance through a permutation test at the 0.001 level (i.e. performing 999 random permutations of populations in one of the distance matrices), under the null hypothesis that both matrices are not linearly related. Mantel's tests were computed using the Mantel procedure of the Genetix package. In such correlations, we followed [START_REF] Hutchison | Correlation of pairwise genetic and geographical distance measures: inferring the relative influences of gene flow and drift on the distribution of genetic variability[END_REF] model which directly links F st and geographical distance. This recent approach removed the problems of relating the estimate of regional multilocus F st to gene flow and the subsequent question linked to the genetic equilibrium assumed by the island model.

In addition to correlation between pairwise F st values and geographical distances that only consider isolation by distance, we visualized the relationships among sites, based on the F st values, using a multi-dimensional scaling analysis (MDS) as suggested by [START_REF] Lessa | Multidimensional analysis of geographic genetic structure[END_REF]. MDS analysis uses distances between objects (pairwise population F st values in the present approach) and approximates those distances in a reduced number of dimensions through an iterative fitting procedure. A two-dimensional plot fitted to pairwise F st values between sites was created for each species in XLStat v.4.3 (available on http://xlstat.com). The goodness of fit between the fitted and observed distances was measured by a stress test, with a zero stress value indicating a perfect fit between the fitted and observed distances [START_REF] Kruskal | Multidimensional scaling[END_REF]. Such a procedure was used to search for any structure between sites that has no direct link with the geographical distances, such as oceanic currents and geomorphology.

The last set of analyses compared results for the different species according to the hypothesis on their reproductive strategy or their habitat. To evaluate the level of correlation between genetic patterns exhibited by the different species, pairwise F st values for each species were computed and Mantel's test was performed among species using the Mantel software (available on http:// life.bio.sunysb.edu/morph). F st values within each matrix were first standardized before computing the sum of the cross product; the result is divided by (n-1) where n is the number of pairs of distances compared in the computation [START_REF] Smouse | Multiple regression and correlation extensions of the Mantel test of matrix correspondence[END_REF]). The Mantel statistic, r, is then equivalent to the computation of a Pearson correlation coefficient. Probabilities of this statistic were then computed by random permutations, using 10,000 permutations. Finally, in order to represent biological distances between species, a matrix of the Bray-Curtis dissimilarity coefficient (D) was calculated (where D=1-S, and S is the Bray-Curtis similarity coefficient) from pairwise F st values, using Progiciel R [START_REF] Legendre | Le progiciel R: analyse multidimentionelle, analyse spatiale[END_REF]. The relationship among species was investigated using an MDS approach. A two-dimensional plot fitted to the Bray-Curtis dissimilarity coefficient (D) between species was created in XLStat v.4.3. The goodness of fit between the fitted and observed distances was measured by a stress test [START_REF] Kruskal | Multidimensional scaling[END_REF].

Results

Genetic variation within species

A total of 39 loci were screened and the number of loci showing activity varied from 20 (for Dascyllus aruanus) to 27 (for Chaetodon quadrimaculatus and Plectroglyphidodon dickii) (Tables 2,3). Protocols and details are available from the authors. The number of polymorphic loci varied between 8 and 14 when considering a frequency £ 0.95 for the most common allele, and between 9 and 18 when considering a frequency £ 0.99 for the most common allele. Overall population multilocus heterozygosity varied from 0.0785 for Chaetodon quadrimaculatus to 0.1557 for Dascyllus aruanus.

All polymorphic loci were tested for deviations from Hardy-Weinberg equilibrium within each site. For all species, 12 single loci (out of 479 tested) deviated from the equilibrium predictions (P<0.05), but once the probability level was corrected according to a sequential Bonferroni (distinct correction for each species), none of the deviations remained significant. Multilocus F is values, analyzed separately for each site within each species, ranged between -0.0532 (in Marutea for Plectroglyphidodon dickii) and 0.1635 (in Takapoto for Pomacentrus pavo). No species at any site showed significant multilocus deviation from Hardy-Weinberg equilibrium. This result presumes that, for each species, each sample from one site can be attributed to individuals from the same population and that each site provides a good estimation of the population.

With the exception of Plectroglyphidodon dickii, exact tests computed over all samples within each species showed significant divergence in allelic frequencies for all species in French Polynesia ( values ranged from 0 for Plectroglyphidodon dickii to 0.0287 for Chaetodon quadrimaculatus, with an average value of 0.0163 over all eight species. For single-locus tests, the percentage of loci showing significant divergence ranged from 15% for Chrysiptera glauca to 62% for Dascyllus aruanus, with an average of 28% over all species (excluding P. dickii which showed no significant divergence). Altogether, about one polymorphic locus in three contributed to significant divergence among populations.

M M 0 M 0.093 M -0.032 M M ADH* H obs - - - - - - - - - 0.020 M M 0.020 M F is - - - - - - - - - 0 M M 0 M CK* H obs M M M M M M M M M M M M 0.020 M F is M M M M M M M M M M M M 0 M Est1* H obs - - - - 0.
M -0.010 -0.010 M 0 M -0.011 M 0 0 0 M -0.011 M GPD-2* H obs - - - - - - - - - M M 0.04 M M F is - - - - - - - - - M M -0.005 M M GPI-
Considering each species separately, the highest pairwise multilocus F st value was found in Chaetodon quadrimaculatus between Tetiaroa and Moorea (Table 4, F st =0.0536, P=0.0070). Chromis xanthura and Plectroglyphidodon dickii, two species with benthic eggs and inhabiting the outer slope, both showed low F st values, with no significant divergence among any pairs of populations tested. Significant divergence and higher F st values did not show any obvious pattern in relation to the distance of site separation, except for Forcipiger flavissimus, which showed higher F st values and signifi-cant divergences for the most geographically distant population pairs. Finally, relationships computed between F st values and geographical distances were not significant for any of the species analyzed (Table 4). The normalized Mantel statistic, r, obtained from a Mantel's test between genetic and geographical distances, ranged from -0.429 (P=0.840) for Chromis xanthura to 0.571 (P=0.146) for Forcipiger flavissimus. Some species (Chaetodon citrinellus, Chaetodon quadrimaculatus and Dascyllus aruanus) even exhibited highest F st values for the comparison of the two sites separated by the smallest distances (Moorea-Tetiaroa).

Graphs of MDS analysis using pairwise multilocus F st values did not reveal any congruent pattern among the six species analyzed (Fig. 2). Only six species of the eight could be analyzed, because the two remaining (Chrysiptera glauca and Plectroglyphidodon dickii) had insufficient sites to provide enough data for MDS computations. Forcipiger flavissimus showed a distribution of island populations in accordance with geographical distances and archipelago structure. The first axis differentiated closest sites (Moorea, Tetiaroa, Rangiroa and Takapoto) from the furthest site, Marutea; the second axis segregated populations of the Society Archipelago (Moorea and Tetiaroa) from populations of the Tuamotu (Rangiroa and Takapoto)

F is M M M M 0 M M M M -0.065 0 -0.021 -0.011 0 PEPB-1* H obs M M M M M M M M M M M 0.020 M 0.026 F is M M M M M M M M M M M 0 M 0 PEPB-
M M M M M M 0.021 M M - - - - - F is M M M M M M 0 M M - - - - -
M M M M 0.020 M 0.021 M M M M M 0.020 M F is M M M M 0 M 0 M M M M M 0 M SOD-2* H obs - - - - 0.020 0.020 0.021 M M - - - - - F is - - - - 0 0 0 M M - - - - -
Archipelago. However, such structure was uniquely observed for a single species. Overall, some species, such as Forcipiger flavissimus, Chaetodon citrinellus, and Chromis xanthura, showed one isolated island (Marutea, Tetiaroa and Rangiroa, respectively) relative to all other sites remaining together, while the other species had a more spread out distribution of islands. No generalization could be made relative to the historical origin, the geomorphological structure of the islands, or even their

Genetic variation among species

Species with benthic eggs showed significantly higher multilocus heterozygosity than species with pelagic eggs (H benthic =0.117±0.025 versus H pelagic =0.096±0.028; t=2.188, P=0.0358). Species inhabiting lagoons exhibited significantly higher multilocus heterozygosity levels than species found on the outer slope (H lagoon = 0.121±0.032 versus H slope =0.097±0.019; t=2.725, P=0.0102).

Analysis of genetic differentiation (F st values) relative to reproductive strategy or habitat did not reveal any significant difference. Mantel's test performed for comparison of F st values between species did not show any significant association of species with similar biological features (Table 5). Plectroglyphidodon dickii and Chrysiptera glauca were excluded from these comparisons since only three samples were collected and they could not be compared (cf. Table 1). All populations of Marutea were excluded in correlations comparing Chaetodon citrinellus with other species, because of missing samples (cf. Table 1). The normalized Mantel statistic, r, ranged from -0.412 (P=0.0775) between Chaetodon citrinellus and Chromis xanthura to 0.733 (P=0.0678) between Chaetodon citrinellus and Dascyllus aruanus, but none of these correlations was significant.

Two MDS analyses were made: one using six species (without Plectroglyphidodon dickii and Chrysiptera glauca) and omitting Marutea data, and a second using only five species (excluding Chaetodon citrinellus) with all locations (Table 6; Fig. 3). Prior to computing the MDS, we estimated Bray-Curtis distances based on the dissimilarity between species in the F st -values distribution. These distances ranged from 0.1555 between Dascyllus aruanus and Chaetodon quadrimaculatus to 0.5925 for Pomacentrus pavo and Chaetodon citrinellus, omitting Marutea from the analysis. After excluding Chaetodon citrinellus and reconsidering all sites, distances appeared similar to the previous estimates (t=0.273; P=0.791), suggesting that most of the divergence in genetic structure between species was due to F st values observed between the closest islands (Moorea, Tetiaroa, Rangiroa and Takapoto). Finally, we did ANOVAs on Bray-Curtis distances to test for variation in the genetic structure between species showing different reproductive strategies and/or habitats. None of the factors (reproductive strategy and/or habitat) had significant influence on the genetic structure (P>0.8 for all ANOVAs on both data sets of Table 6 and tested for reproductive strategy and habitat, respectively).

The MDS graph computed from Bray-Curtis distances of six species, excluding all Marutea comparisons, segregated Chaetodontidae from Pomacentridae (Fig. 3A). However, this pattern was not maintained when excluding one species and introducing Marutea samples (Fig. 3B). Neither of the MDS graphs reveals any structure related to reproductive strategy and/or habitat.

Discussion and conclusions

The allozyme survey conducted on eight species sampled at similar sites within French Polynesia showed significant divergences between populations (except for Plectroglyphidodon dickii), with a similar range of multilocus F st values (from 0.0114 to 0.0287). The genetic structure of each of the eight species was not significantly correlated with the geographical distance between sites. Such a standardized sampling protocol provided a unique test for the impact of reproductive strategy and habitat on the genetic structure of isolated island populations, as well as the relationship with geographical distances.

Regarding the main questions addressed, we found no impact of reproductive strategy and/or habitat on genetic structure, whereas heterozygosity showed significant differences.

Variation in genetic diversity

Gene flow and genetic drift should equally affect neutral loci, whereas selection is more likely to be locus specific [START_REF] Lewontin | Distribution of gene frequency as a test of the theory of selective neutrality of polymorphisms[END_REF]. The role of selection in maintaining genetic polymorphisms at allozyme loci has been reported in diverse marine species [START_REF] Koehn | Maintenance of an aminopeptidase allele frequency cline by natural selection[END_REF][START_REF] Pogson | Genetic population structure and gene flow in the Atlantic cod Gadus morhua: a comparison of allozyme and nuclear RFLP loci[END_REF][START_REF] Powers | Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: a multidisciplinary approach to address the million-year saga of a small fish[END_REF][START_REF] Lemaire | Do discrepancies between microsatellite and allozyme variation reveal differential selection between sea and lagoon in sea bass (Dicentrarchus labrax)[END_REF]. However, such a force has been found to act on allelic frequencies depending on heterogeneous environmental conditions (mostly through temperature and/or salinity gradients). Moreover, assumptions necessary to infer gene flow from allozyme-frequency data (i.e., effective neutrality, weak mutation rate, approximate migration-drift equilibrium) seem in general to be valid [START_REF] Baer | Among-locus variation in F st : fish, allozymes and the Lewontin-Krakauer test revisited[END_REF]. Our data did not demonstrate evidence of a locus-specific process, such as a Hardy-Weinberg disequilibrium.

While not conclusive regarding selection, we considered that migration and genetic drift were the two main evolutionary forces acting on allelic frequencies of allozyme loci.

In the present work we found significant difference in heterozygosity, with species laying benthic eggs and/or inhabiting lagoons showing higher values than species laying pelagic eggs and/or occurring on the outer slope. Considering the neutral theory of evolution [START_REF] Kimura | The neutral theory of molecular evolution[END_REF][START_REF] Nei | Extent of protein polymorphism and the neutral mutation theory[END_REF], heterozygosity is a direct function of the effective population size (N e ) and the mutation rate (l=10 -6 for allozymes; [START_REF] Voelker | Spontaneous allozyme mutations in Drosophila melanogaster: rate of occurrence and nature of the mutants[END_REF]) according to the model H=4N e l/(4N e l+1), with mutation rate being related to the time and the number Fig. 3A,B. Multidimensional scaling plots of species based on Bray-Curtis dissimilarity coefficient (D) between pairs of species. Plectroglyphidodon dickii and Chrysiptera glauca are not included because of their smaller sample size. Squares represent species with benthic eggs; circles represent species with pelagic eggs. Solid symbols represent species living on the outer slope; open symbols show species inhabiting lagoons. A The MDS including Chaetodon citrinellus in the analysis, but without Marutea populations for all the other species. B The MDS including Marutea in the pairwise F st matrices used to compute the Bray-Curtis dissimilarity coefficient, but excluding Chaetodon citrinellus of generations. Regarding the present survey, Pomacentridae and Chaetodontidae species occurring on the outer slope have experienced more stable effective population size and may be derived from much older origins than species inhabiting lagoons that were dry during each sea-level transgression. Therefore, in theory, we expect species from the outer slope to show higher heterozygosity than species inhabiting lagoons, because they did not experience local extinction or bottlenecks during low sea level.

Regarding fecundity, species laying pelagic eggs are usually the ones showing higher individual fecundity compared to benthic spawners [START_REF] Thresher | Geographic variability in the ecology of coral reef fishes: evidence, evolution and possible implications[END_REF]. Species showing high individual fecundity being subject to strong variability in reproductive success [START_REF] Hedgecock | Temporal and spatial genetic structure of marine animal populations in the California Current[END_REF], such as marine fish, will therefore express limited effective population size, because a limited number of adults can provide much of the new generation of offspring. This process has been proposed as the origin of genetic drift signal (i.e., chaotic patchiness or microgeographic differentiation) in marine organisms [START_REF] Johnson | Pattern beneath the chaos: the effect of recruitment on genetic patchiness in an intertidal limpet[END_REF]. Overall, under the neutral theory of evolution, the characteristics of coral reef fish would lead to the prediction of higher heterozygosity in species laying benthic eggs and occurring on the outer slope, although our results are somewhat different.

Several other hypotheses, mostly based on selectionistic models, have also been proposed to predict differences in genetic diversity among species. Various models have been described based on: stability in environmental heterogeneity [START_REF] Levins | Evolution in changing environments[END_REF]; time of divergence between species (Soule´1972); size and mobility variation (Selander and Kaufman 1973); predictability of trophic resources [START_REF] Valentine | Genetic variation in Frieleia halli, a deep-sea brachiopod[END_REF]; and ecological heterogeneity between habitats and niche specialist or generalist species [START_REF] Nevo | Genetic variation in natural populations: patterns and theory[END_REF][START_REF] Smith | Genetic variation in marine teleosts: high variability in habitat specialists and low variability in habitat generalists[END_REF]. Most of the previous models gave a single causative explanation for the variation in genetic diversity. When applied to our data, we can always propose some hypothesis that will fit with previous models, such as species inhabiting lagoons experiencing more heterogeneous environments compared to the outer slope, or species with benthic eggs being less mobile than species laying pelagic eggs.

Overall, it is difficult to find any clear origin that would explain the variation in heterozygosity, even when using a comparative analysis of several species collected according to standardized sampling protocol. Therefore, a significant difference in heterozygosity cannot be used to understand ecological and/or biological features. In addition, the heterozygosity encountered in a previous survey of Dascyllus aruanus [START_REF] Planes | Genetic structure of Dascyllus aruanus populations in French Polynesia[END_REF] was very low compared to our present value (0.065 versus 0.1557), because some monomorphic loci were not scored in the present survey. Therefore, we arrive at the problem of how comparative genetic surveys can be related to heterozygosity and how we need to standardize sampling and genetic protocols [START_REF] Planes | Genetic diversity and dispersal capabilities in marine fish[END_REF].

Variation in the genetic structure Over all populations, multilocus F st values computed for the eight species of this survey showed concordant ranges with values usually obtained for species with high dispersal capabilities on the same spatial scale (except Plectroglyphidodon dickii). The present research strategy was developed from results of previous genetic surveys that examined Acanthurus triostegus [START_REF] Planes | Genetic differentiation in relation to restricted larval dispersal of the convict surgeon fish, Acanthurus triostegus, in French Polynesia[END_REF] and Dascyllus aruanus [START_REF] Planes | Genetic structure of Dascyllus aruanus populations in French Polynesia[END_REF] in French Polynesia. Both species showed significant genetic differences among populations. Dascyllus aruanus populations appeared almost homogeneous across Polynesia (minor genetic differences could be detected only when comparing remote archipelagoes; overall F st =0.01). In contrast, Acanthurus triostegus populations were highly structured, with large genetic differences between islands (overall F st =0.09) and the genetic structure was concordant with the major oceanic currents in the area. Comparison of results obtained for Dascyllus aruanus and Acanthurus triostegus did not fit the idea that species with long pelagic larval stages exhibit greater dispersal and consequently show higher gene flow between populations [START_REF] Waples | A multispecies approach to the analysis of gene flow in marine shore fishes[END_REF][START_REF] Doherty | Gene flow and larval duration in seven species of fish from the Great Barrier Reef[END_REF]. In fact, Dascyllus aruanus larvae spend about 25 days in the ocean, whereas Acanthurus triostegus larvae remain in the plankton for 60 days. At that time, it was proposed that this contradiction (species with longer larval duration showing restricted gene flow) was due to historical factors. During the last glaciations, 15 to 20 thousand years BP [START_REF] Bard | Deglacial sea level record from Tahiti corals and the timing of global meltwater discharge[END_REF], sea level was 120 m below present-day sea level, and lagoons of classic Darwinian atolls that are 50-70 m deep at the most were completely dry. Because Dascyllus aruanus only inhabits lagoons, and such habitats were not available in French Polynesia during the last glaciations, the authors proposed that the species became extinct in this region during this period. Therefore, the present population is a result of recent recolonizations, occurring when the present sea level was reached (5 to 8 thousand years BP; [START_REF] Bard | Deglacial sea level record from Tahiti corals and the timing of global meltwater discharge[END_REF]. Consequently, there has not been enough generations for the appearance of genetic structure due to genetic drift. In contrast, Acanthurus triostegus is more ubiquitous and inhabits lagoons as well as oceanic slopes. Because this species could be maintained on oceanic slopes when the sea level was lower, the structure of Acanthurus triostegus populations is older and expresses longer evolution more likely to show a genetic differentiation due to long-term genetic drift together with limited gene flow. These studies suggested that oceanic currents, as well as historical events, affect the population structure between oceanic islands. In these interpretations, historical factors played a more crucial role than the pelagic larval duration and this suggests that the present-day genetic structure cannot be directly interpreted as a pattern of dispersal.

The present protocol intended to confirm these hypotheses by comparing the genetic structure of species showing different reproductive strategies and different habitats. The reproductive strategy was used to represent the potential biological dispersal, since species with benthic eggs exhibit statistically much shorter pelagic periods compared to species that lay pelagic eggs [START_REF] Thresher | Geographic variability in the ecology of coral reef fishes: evidence, evolution and possible implications[END_REF]. Furthermore, benthic eggs themselves do not participate in the dispersal phase, which reduces potential dispersal even more. The difference in habitats indirectly tested for the impact of historical factors [START_REF] Nelson | Phylogeographic structure of false clownfish, Amphiprion ocellaris, explained by sea level changes on the Sunda shelf[END_REF][START_REF] Hickerson | Post-glacial population history and genetic structure of the northern clingfish (Gobiesox maeandricus), revealed from mtDNA analysis[END_REF], since we assumed that species restricted to lagoonal habitats experienced severe changes in the effective population size with potential extinction and recolonization in some lagoons following sea-level changes during the Holocene (Pirazzoli and Montaggioni 1988; [START_REF] Bard | Deglacial sea level record from Tahiti corals and the timing of global meltwater discharge[END_REF]. Such a test can only be carried out using comparative approaches, since it requires standardized protocol and we cannot use published data conducted in different areas, with different protocols [START_REF] Waples | A multispecies approach to the analysis of gene flow in marine shore fishes[END_REF][START_REF] Doherty | Gene flow and larval duration in seven species of fish from the Great Barrier Reef[END_REF][START_REF] Gold | Mitochondrial DNA diversification and population structure in fishes from the Gulf of Mexico and western Atlantic[END_REF]. Genetic structure of the eight species did not appear significantly related either to the reproductive strategy or to the habitat preference (cf. t-test on global multilocus F st and MDS analyses) and none of the species showed a significant relationship between genetic structure and geographical distance. Overall, the genetic structure of each species appeared variably determined by the genetic difference among closest and/or most-distant islands (except for Chaetodon citrinellus) emphasizing some chaotic patterns among species, with variations in genetic structure occurring over large spatial scales, as well as small ones, within and among species. Such spatially random genetic differentiation accommodates Wright's island model in the sense that each individual issued from one population is equally likely to move to any other population [START_REF] Wright | Evolution in Mendelian populations[END_REF]. In such a model, no relationship between the geographical distance and genetic differentiation is expected. The genetic similarity between islands (i.e., populations) will be a function of the regularity of the migrations, assuming that they are very infrequent in time. Biologically, such a model requires that populations be mostly self-recruiting and that pelagic larval flux between islands be very rare, so that it cannot counterbalance genetic drift (a very slow process when acting on large effective population sizes). These features have been partly demonstrated in coral reef fish in which self-recruitment has been observed [START_REF] Jones | Self-recruitment in a coral reef fish population[END_REF][START_REF] Swearer | Larval retention and recruitment in an island population of a coral-reef fish[END_REF], and where larval fluxes appear to be unpredictable and largely chaotic [START_REF] Dixon | Episodic fluctuations in larval supply[END_REF]). The island model satisfies most of our genetic results but not the previous data on Acanthurus triostegus, which demonstrated a significant relationship between genetic differentiation and geographical distance favoring isolation by distance [START_REF] Planes | A genetic metapopulation model for reef fishes in oceanic islands: the case of the surgeonfish, Acanthurus triostegus[END_REF].

The genetic structure of two species was notable. Plectroglyphidodon dickii stands out from other species, appearing homogeneous between the three sampled populations that are separated by more than 1,000 km, even if it shows a larval duration of about 27 days [START_REF] Wellington | Planktonic larval duration of one hundred species of Pacific and Atlantic damselfishes (Pomacentridae)[END_REF], similar to most Poma-centridae analyzed. While showing genetic homogeneity over a large spatial scale, this species appeared absent (or very rare) in islands of the Society Archipelago such as Tetiaroa or Moorea sampled in the present survey, as well as in other islands not sampled [START_REF] Galzin | Structure of fish communities of French Polynesia coral reefs. 1. Spatial scales[END_REF]Galzin personal communication). Such absence (or rarity) in some islands, independently of the presence of suitable habitats [START_REF] Galzin | Structure of fish communities of French Polynesia coral reefs. 1. Spatial scales[END_REF], suggests that migration is a rare event in the insular model we investigated.

The other notable result arises from the observed genetic structure of Dascyllus aruanus, which is similar to other species analyzed in this study, but differs from previous data [START_REF] Planes | Genetic structure of Dascyllus aruanus populations in French Polynesia[END_REF]. F st values have increased significantly (from 0.0076 in 1990 to 0.0215 in 2000) over a short period of time for the same localities. Temporal change in genetic structure has already been observed in marine fish [START_REF] Lacson | Temporal genetic variation in subpopulations of bicolor damselfish (Stegastes partitus) inhabiting coral reefs in the Florida Keys[END_REF][START_REF] Gold | Temporal stability of nuclear gene (allozymes) and mitochondrial DNA genotype among red drums from the Gulf of Mexico[END_REF] as well as marine invertebrates, and there is a growing belief that such variations arise from variance in reproductive success [START_REF] Hedgecock | Temporal and spatial genetic structure of marine animal populations in the California Current[END_REF][START_REF] Ruzzante | Spatial and temporal variation in the genetic composition of a larval cod (Gadus morhua) aggregation: cohort contribution and genetic stability[END_REF][START_REF] Herbinger | Family relationships and effective population size in a natural cohort of Atlantic cod (Gadus morhua) larvae[END_REF][START_REF] Lenfant | Temporal genetic changes between cohorts in a natural population of a marine fish, Diplodus sargus[END_REF]. Such variance in reproductive success among cohorts was suggested by the chaotic genetic patchiness model that has been proposed to explain genetic variations in new recruits of marine invertebrates (Johnson andBlack 1982, 1984;[START_REF] Watts | Effect of recruitment on genetic patchiness in the urchin Echinometra mathaei in Western Australia[END_REF]). In the present data, multilocus F st values for each species over all sites ranged from 0.0114 to 0.0287; a similar range of values was found between groups of new recruits of sea urchins [START_REF] Watts | Effect of recruitment on genetic patchiness in the urchin Echinometra mathaei in Western Australia[END_REF] or between cohorts of temperate sea bream [START_REF] Lenfant | Temporal genetic changes between cohorts in a natural population of a marine fish, Diplodus sargus[END_REF]. Similarly, the level of genetic variation we observed in coral reef fish throughout French Polynesia could partially reflect variable recruitment within a large panmictic population, rather than genetic differences due to isolation [START_REF] Bernardi | Gene flow at three spatial scales in a coral reef fish, the three-spot dascyllus, Dascyllus trimaculatus[END_REF]). Overall, it appears almost impossible to generate a single theory to explain the genetic structure after having analyzed about ten species through time on the same spatial scale. Such research effort is considerable when considering the total number of species, individuals, and sites surveyed; but even with such effort, we could not elucidate any generalized model. The underlying reasons are to be found in the number of factors potentially affecting the model, as emphasized in some recent surveys [START_REF] Mcglashan | Low level of genetic differentiation among populations of the freshwater fish Hypseleotris compressa (Gobiidae: Eleotridinae): implications for its biology, population connectivity and history[END_REF][START_REF] Riginos | Population subdivision in marine environments: the contributions of biogeography, geographical distance and discontinuous habitat to genetic differentiation in a blennioid fish, Axoclinus nigricaudus[END_REF]. Therefore, it is reasonable to consider that a multifactorial process is driving the genetic structure of populations. It is almost impossible to disentangle and identify which parameters have most effect on the system, assuming that each parameter is likely to affect differently each species, depending on its origin. Finally, when conducting a standardized sampling protocol, it seems that the ecology of the species, the geography of the area and the history of the species do not individually explain the genetic structure of coral reef fish in French Polynesia. However, we have shown that, for most species, French Polynesia cannot be considered as a single panmictic population. The randomly variable genetic structure observed among species fits the idea that coral reef fish species in French Polynesia are likely to follow an island model in which migrations between populations are randomly driven in space and time. Nevertheless, the chaotic genetic pattern found for most species in a context of limited genetic differentiation (all F st values were lower than 3%) may also result from temporal variation in reproductive success, which may drive the small genetic differences observed between locations. Further analysis combining temporal and spatial sampling should now be undertaken to validate definitively the origin of the genetic structure in such an insular model.

( 0 .

 0 95) and P (0.99) are percentages of polymorphic loci within each population. Significant deviation from Hardy-Weinberg equilibrium is denoted by* (P<0.05) following F

Fig. 2 .

 2 Fig. 2. Multidimensional scaling plots of sites computed for each species based on pairwise F st matrices. Plectroglyphidodon dickii and Chrysiptera glauca are missing because the small number of sites (3) did not allow analysis by MDS

Table 4 )

 4 . Multilocus F st

Table 1 .

 1 Sampling effort by species and sites. N total number of individuals of each species collected from all sites; -no individuals

	Fish taxon	Tuamotu Archipelago		Society Archipelago
		Marutea	Rangiroa	Takapoto	Tetiaroa	Moorea	N	Habitat

Table 2 .

 2 Summary (part 1) of the electrophoretic results for each species, for each population, for each locus and over all loci. N number of fishes sampled; H obs observed heterozygosity; F is fixation index[START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF], M monomorphic loci; -no activity of this locus for the species; P (0.95) and P (0.99) are percentages of polymorphic loci within each population. Significant deviation from Hardy-Weinberg equilibrium is denoted by* (P<0.05) following F is values. Ma Marutea, Te Tetiaroa, Mo Moorea, R Rangiroa and Ta Takapoto

	1*
	H obs	0.020	0.100	0.120	0.040	0.060	0.100	0.104	0.102	0.225	0.020 M	0.020	0.020 M
	F is	0.662* -0.029 -0.037 -0.005 -0.014 -0.029 -0.044	0.241 -0.089	0	M	0	0	M
	GPI-2*
	H obs	0.020	0.020	0.040 M	0.040 M	0.021 M	0.041	0.100	0.020	0.020	0.082	0.501
	F is	0	0	-0.010 M	-0.010 M	0	M	-0.011 -0.043	0	0	-0.021 -0.007
	IDH-1*
	H obs	0.460	0.360	0.340	0.420	0.020 M	0.021 M	M	M	M	M	0.020	0.026
	F is	-0.157	0.117 -0.095 -0.011	0	M	0	M	M	M	M	M	0	0
	IDH-2*
	H obs	M	M	M	M	M	0.020	0.042	0.020 M	0.060	0.040	0.100	0.245	0.184
	F is	M	M	M	M	M	0	-0.005	0	M	-0.021 -0.010 -0.029	0.030	0.177
	LDH*

Table 2 .

 2 Contd.

	Locus C. citrinellus			C. quadrimaculatus			F. flavissimus
		Ra	Ta	Te	Mo	Ma	Ra	Ta	Te	Mo	Ma	Ra	Ta	Te	Mo
	MEP-1*
	H obs	0.100	0.080	0.100 M	0.120	0.080	0.042	0.612	0.041 M	0.040	0.020 M	M
	F is	-0.043 -0.032 -0.034 M	-0.054 -0.032	0.486	0.382 -0.011 M	-0.005	0	M	M
	MEP-2*
	H obs	M	M	M	M	0.220	0.280	0.250	0.408	0.143	0.020 M	M	M	M
	F is	M	M	M	M	-0.080 -0.153	0.130 -0.045 -0.047	0	M	M	M	M
	MPI*
	H obs	M	M	M	M	0.040 M	M	M	M	0.140	0.020	0.060	0.041	0.026

Table 3 .

 3 Summary (part 2) of the electrophoretic results for each species, for each population, for each locus and over all loci. N

	Overall
	H obs	0.1254 0.1417 0.1325 0.1292	0.0815 0.0704 0.0802 0.0733 0.0869 0.1000 0.0752 0.0776 0.0996 0.0738
	F is	0.1119 0.1144 0.0510 0.0368	0.0384 0.0540 0.1336 0.1274 -0.0368 0.0445 0.0829 -0.0431 0.0100 0.0489
	P (0.95)	0.4583 0.4583 0.4583 0.4167	0.2963 0.2222 0.2222 0.2593 0.2222 0.4000 0.2800 0.2800 0.3200 0.2000
	P (0.99)	0.5417 0.5833 0.6250 0.4583	0.4074 0.3704 0.6296 0.4074 0.5556 0.5200 0.4400 0.4400 0.7200 0.6000

Table 4 .

 4 Multilocus genetic variation of each population pair for each species (F st[START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF]. Distances separating islands of a pair shown in kilometres. Ma

	Marutea, Te Tetiaroa, Mo Moorea, R Rangiroa and Ta Takapoto. Significant differences in allelic frequencies between populations (P<0.05) are followed by their probabilities. Bold	characters show significant differences after the sequential Bonferroni correction (Rice 1989); -not calculated. Normalized Mantel statistic, r, is followed by its probability. Mantel's	tests were not performed for C. glauca and P. dickii because the number (three) of sampled populations was too small	Pelagic eggs Benthic eggs	Pairwise Lagoon Outer slope Lagoon Outer slope	Islands Distances C. citrinellus C. quadrimaculatus F. flavissimus P. pavo C. glauca D. aruanus C. xanthura P. dickii	Ma/Te 1550 -0.0139 (0.0000) 0.0323 (0.0003) 0.0136 -0.0228 (0.0000) 0 -	Ma/Mo 1548 -0.0505 (0.0000) 0.0462 (0.0015) 0.0368 (0.0023) -0.0354 (0.0000) 0.0080 -	Ma/R 1425 -0.0473 (0.0000) 0.0212 (0.0051) 0.0221 0.0042 0.0059 0.0300 0	Ma/Ta 1270 -0.0040 0.0136 0.0042 0.0289 (0.0076) 0.0109 0 0.0002	Ta/Mo 566 0.0132 (0.0256) 0.0273 0.0401 0.0164 (0.0302) -0.0186 (0.0012) 0.0036 -	Ta/Te 520 0.0139 (0.0083) 0.0092 0.0165 0 -0.0125 0.0047 -	R/Mo 365 0.0024 0.0326 (0.0025) 0.0308 0.0446 (0.0075) -0.0185 (0.0035) 0.0263 -	R/Te 310 0.0112 0.0281 (0.0180) 0.0087 0.0298 -0.0309 (0.0000) 0.0385 -	R/Ta 226 0.0011 0.0199 (0.0406) 0 0.0017 0.0192 0.0135 (0.0226) 0.0247 0.0021	Te/Mo 60 0.0269 (0.000) 0.0536 (0.0070) 0.0036 0.0070 (0.0000) -0.0412 (0.0000) 0.0144 (0.0391) -	Overall 0.0114 (0.0000) 0.0287 (0.0000) 0.0208 (0.0000) 0.0162 (0.0000) 0.0178 (0.0000) 0.0215 (0.0000) 0.0145 (0.0000) 0	Mantel's -0.286 (0.705) -0.039 (0.704) 0.571 (0.146) 0.154 (0.461) -0.220 (0.692) -0.429 (0.840)	test

Table 5 .

 5 Correlation between genetic distance matrices (pairwise F st values) for each species pairs. Normalized Mantel statistics are indicated above the diagonal, and probabilities tested by random permutations are given below the diagonal

		C. citrinellus	F. flavissimus	C. quadrimaculatus	P . pavo	D. aruanus	C. xanthura
	C. citrinellus		-0.153	0.565	-0.354	0.733	)0.412
	F. flavissimus	0.6458		0.175	0.567	0.069	)0.398
	C. quadrimaculatus	0.1270	0.3070		0.501	0.543	0.396
	P . pavo	0.7690	0.0520	0.0925		0.280	0.432
	D. aruanus	0.0678	0.4210	0.0740	0.2312		0.021
	C. xanthura	0.0775	0.8670	0.1630	0.1500	0.4796
		C. citrinellus	F. flavissimus	C. quadrimaculatus	P. pavo	D. aruanus	C. xanthura
	C. citrinellus		0.5031	0.4645	0.5925	0.3393	0.5868
	F. flavissimus	-		0.4110	0.3974	0.4725	0.5568
	C. quadrimaculatus	-	0.3395		0.3725	0.1555	0.3141
	P. pavo	-	0.3051	0.2981		0.3835	0.3638
	D. aruanus	-	0.3528	0.2414	0.3148		0.3079
	C. xanthura	-	0.5807	0.3818	0.3963	0.4479

Table 6 .

 6 Bray-Curtis dissimilarity coefficients between each species, computed from comparisons of pairwise F st matrices. Plectroglyphidodon dickii and Chrysiptera glauca were not included in this analysis because sample size was too small. Distance coefficients above the diagonal were calculated for all six species, excluding all Marutea data. Distance coefficients below the diagonal are comparisons of pairwise F st matrices obtained with all pairwise islands, but excluding C. citrinellus, since the Marutea sample was missing for this species

Acknowledgements We thank anonymous reviewers for their helpful comments. Special thanks go to Franc¸ois Bonhomme who greatly contributed through discussion to set up this work. We gratefully acknowledge the assistance in sampling specimens: ReneǴ alzin and Yannick Chancerelle for collecting specimens from Tetiaroa and additional ones from Takapoto; Robert Wan for the facilities he offered in Marutea; Pierre Remoissonet and Yves Leduc for providing SRM facilities in Rangiroa and Takapoto. This study was supported by grants from PNDB (French National Program on Biodiversity) and by the TOTAL-ELF-FINA foundation.


C É C I L E F A U V E L O T

Isolation and characterization of microsatellites in two tropical butterflies, Drupadia theda and Arhopala epimuta (Lepidoptera: Lycaenidae)

Keywords: Arhopala epimuta, Drupadia theda, El Niño Southern Oscillation (ENSO), Kalimantan, Lycaenidae, microsatellites

Isolation and characterization of microsatellites in two

tropical butterflies, Drupadia theda and Arhopala epimuta (Lepidoptera: Lycaenidae)

Wildfires pose a major threat to extant tropical biodiversity [START_REF] Laurance | Slow burn: the insidious effects of surface fires on tropical forests[END_REF]. The province of East Kalimantan (Indonesian Borneo) was heavily affected by the 1997/98 El Niño Southern Oscillation (ENSO) event, the most severe and widespread ever recorded in Borneo [START_REF] Harrison | Repercussions of El Niño: drought causes extinction and the breakdown of mutualism in Borneo[END_REF]. The 97/98 ENSO-induced fires had a major impact on ecosystem dynamics and species diversity [START_REF] Holmgren | El Niño effects on the dynamics of terrestrial ecosystems[END_REF][START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF][START_REF] Laurance | Slow burn: the insidious effects of surface fires on tropical forests[END_REF][START_REF] Cleary | Changes in rain forest butterfly diversity following major ENSO-induced fires in Borneo[END_REF]), but little is known about the effect on genetic diversity. We are currently investigating the genetic composition of two species of butterflies within landscapes differentially affected by ENSO-induced drought and fires. Drupadia theda and Arhopala epimuta are two common lycaenid butterfly species of the tropical rainforests of East Kalimantan. Polymorphic microsatellite markers will make it possible to assess fine-scale population genetic structure within fragmented areas, as well as temporal variation of genetic diversity within disturbed areas.

Genomic DNA was isolated from legs of two individuals per species, using a cetyltrimethyl ammonium bromide (CTAB) protocol [START_REF] Hillis | Molecular Systematics[END_REF]. Microsatellite enrichment was achieved using streptavidin-coated magnetic spheres, according to the protocol described in [START_REF] Boneh | Microsatellites in the bromeliads Tillandsia fasciculata and Guzmania monostachya[END_REF], with minor modifications. The positive colonies were detected using a three-primer polymerase chain reaction (PCR) (PIMA, [START_REF] Lunt | An efficient method for PCR-based isolation of microsatellite arrays (PIMA)[END_REF], with pGEMT-F (TCACTATAGGGCGAATTGGG) and pGEMT-R (CTCAAGCTATGCATCCAAGG) primers (designed according to the pGEM®-T Easy vector sequence) and the biotin-labelled microsatellite sequences. Inserts were reamplified by direct PCR of the colony with pGEMT-F and -R, sequenced using a BigDye Terminator sequencing kit and an ABI PRISM 3100 genetic analyser (Applied Biosystems).

Out of 384 colonies selected after cloning the enriched fragments, 172 positive ones were detected for D. theda and 96 for A. epimuta . Of these, approximately 75% contained a microsatellite sequence for both species. Forty and 29 primer pairs were designed for D. theda and A. epimuta , respectively, using the primer 3 program (Rozen & Skaletsky 1998). Each primer pair was tested using PCRs with a total volume of 10 µ L containing 1 × PCR buffer (HT Biotechnology), 1.25 m m MgCl 2 , 0.1 m m of each dNTP, 0.3 mg/mL bovine serum albumin, 0.2 µ m of each primer, 0.2 U Taq polymerase (HT Biotechnology) and approximately 3 ng Correspondence: C. Fauvelot, Fax: +31(0)20-5257878; E-mail: fauvelot@science.uva.nl DNA. PCRs were carried out in PTC 100 Thermocycler (MJ Research): 3 min denaturation at 94 ° C, 40 cycles of 30 s at 94 ° C, 30 s at 58 ° C and 30 s at 72 ° C, and a final extension of 5 min at 72 ° C. PCR products were run on Spreadex® gels (Elchrom Scientific). After examining patterns on gel, 18 and 20 microsatellites were selected for D. theda and A. epimuta , respectively. PCRs were conducted under the same conditions with the F-primers labelled (IRD700), loaded on a 6.5% acrylamide gel, and run on a Li-Cor 4200 automatic sequencer, with a size standard (50-350 bp, Li-Cor). Of these microsatellite loci, five produced interpretable and repeatable amplification products without PCR optimizations (Table 1). The reasons for abandoning the other microsatellites were multiband patterns, monomorphic loci or loci awaiting optimization of the PCR conditions.

Variability was tested in 51 and 76 individuals of D. theda and A. epimuta , respectively, collected in a primary forest in East Kalimantan. Genotypes and allele sizes were estimated by eye. The program, genetix [START_REF] Belkhir | Montpellier: Laboratoire Génome, Populations , Interactions[END_REF], was used to determine the number of alleles and heterozygosity estimates per locus. Tests for linkage disequilibrium (LD) and deviations from Hardy-Weinberg equilibrium were performed using genepop version 3.4 [START_REF] Raymond | An exact test for population differentiation[END_REF]. For D. theda , three to nine alleles per locus were observed, and eight to 32 for A. epimuta (Table 1). Expected heterozygosity values ranged from 0.129 to 0.833 for D. theda , and 0.514 -0.894 for A. epimuta . No significant LD was found for any pair of loci ( P > 0.01 for all comparisons) for both species. Significant heterozygote deficiencies were found in locus Dt10 for D. theda ( P < 0.01) and in all loci for A. epimuta (all P < 0.001), with the exception of AeH5. Inbreeding, undetected population subdivision (both affecting all loci) and/or the presence of null alleles (locus-specific) might create such deficiencies. The population structure of these two species will be assessed at a finer geographical scale and on more individuals in order to test these possible hypotheses.

The results of cross-species amplification tests of the 10 loci are presented in Table 2. Five individuals each of the following additional species were used, Drupadia ravindra , Arhopala muta , and Arhopala atosia . Cross-species amplifications conducted both within and between genera were successful in 16 out of 20 tests. Between genera, A. epimuta successfully amplified four loci isolated from D. theda . Within genus, D. ravindra successfully amplified three loci isolated for D. theda (viz.,DtC1,DtD10,and DtF6). The five loci isolated for A. epimuta were successfully amplified for both A. muta and A. atosia , with the exception of AeF10 for A. muta . The small number of loci developed for D. theda and A. epimuta (five loci each) is due to the presence of highly conserved flanking regions shared between microsatellite sequences, detected within both species. This phenomenon appears to be a common problem for developing microsatellite markers in Lepidoptera [START_REF] Meglécz | High similarity between flanking regions of different microsatellites detected within each of two species of Lepidoptera: Parnassius apollo and Euphydryas aurinia[END_REF]; see Zhang 2004 for a review). Nevertheless, microsatellites remain markers of choice for assessing fine-scale genetic structure.

Table 1

 1 Primer sequences, repeat motif, annealing temperature (T a ) and variability measures resulting from the amplification of 51 and 76 individuals in Drupadia theda and Arhopala epimuta, respectively, at five microsatellite loci each , number of alleles; H E , expected heterozygosity; H O , observed heterozygosity; *, significant heterozygote deficiency.

	Locus	Primer sequence (5′-3′)	Repeat motif	T a (°C)	Accession no.	N a	H E	H O
	Drupadia theda
	DtC1	F: TCTGTGCGAGACGGTGAGTA	CCAACACCA (7)	58	AY974053	8	0.797	0.882
		R: GAACTTCCTGGACAGCCTGA
	DtD10	F: GGCATGACATTTTTGTGATGTT	CT (12)	58	AY974054	9	0.754	0.529*
		R: TCCTTGTTACTGCCGATTTG
	DtE6	F: GGTACACCTACATAACGATAAAATGC	TC (13)	58	AY974055	3	0.129	0.098
		R: TGACTATGTGTGTAAACTTTGTATGGA
	DtF6	F: GCGGTATCATGATTCAAGGTG	CAGA (7) CGGA (3)	58	AY974056	7	0.705	0.824
		R: AGTCGTTCTTGCAAAATCACTTA
	DtN10	F: GAAATGACGATCTCTACGCAAA	GACGTT (14)	58	AY974057	9	0.833	0.941
		R: TGTTAAGCCAGTTGGTGAGC
	Arhopala epimuta
	AeA12	F: TTTGTCTCAATTGGTTGATTCG	CA (9)	58	AY974048	8	0.733	0.284*
		R: CCGCTTTGAAAATCGGAAAT
	AeB9	F: TACTGCTGCTTAACGGCTGA	CT (12)	58	AY974049	32	0.894	0.581*
		R: CTGCCTGCCTTTACCTCATC
	AeF10	F: CAGGCCTAACGATCCACAGT	GAT (6)	58	AY974050	13	0.684	0.274*
		R: TTGCATAGGCCCTGAAAATC
	AeG5	F: ACGGGAAAAAGTGTCTGACC	TCA (8)	58	AY974051	12	0.514	0.186*
		R: AGGCCTATGTTCAGCAGTGG
	AeH5	F: GCGGTGACGGCTAGATGATA	GAT (7)	58	AY974052	11	0.673	0.693
		R: TGACGTAGATGTGATAAACGTATGC
	N

a

Table 2

 2 Allele size range (bp) of generated fragments resulting from cross-species amplifications at 10 microsatellite loci; -, failure, n. t., not tested

	Locus	Drupadia theda	Drupadia ravindra	Arhopala epimuta	Arhopala atosia	Arhopala muta
	DtC1	136 -199	145 -181	118 -158	n. t.	n. t.
	DtD10	80 -100	80 -86	78 -92	n. t.	n. t.
	DtE6	136 -140	-	138 -140	n. t.	n. t.
	DtF6	99 -123	103 -123	111-131	n. t.	n. t.
	DtN10	150 -254	-	-	n. t.	n. t.
	AeA12	n. t.	n. t.	144 -175	146 -182	120 -204
	AeB9	n. t.	n. t.	114 -256	146 -178	114 -238
	AeF10	n. t.	n. t.	121 -163	121-139	-
	AeG5	n. t.	n. t.	73 -103	80 -98	86 -111
	AeH5	n. t.	n. t.	167 -188	170 -185	179 -185

© 2005 Blackwell Publishing Ltd, Molecular Ecology Notes , 5, 724-726

Acknowledgements

I am grateful to my two collaborators on this project, Steph B. J. Menken and Daniel F. R. Cleary. I thank Peter Kuperus and Betsie Voetdijk for valuable help in the laboratory. This work was supported by grant 895.100.029 of the Netherlands Foundation for the Advancement of Tropical Research (NWO-WOTRO), within the priority program, 'Biodiversity in disturbed ecosystems'.