C F A U V E L O T

D F R C L E A R Y

S B J M E N K E N

Cécile Fauvelot
email: fauvelot@science.uva.nl

Short-term impact of disturbance on genetic diversity and structure of Indonesian populations of the butterfly Drupadia theda in East Kalimantan

Keywords: bottleneck, disturbance, gene flow, genetic diversity, Lepidoptera, mtDNA

We investigated the short-term impact of disturbance on genetic diversity and structure of the tropical butterfly Drupadia theda Felder (Lepidoptera: Lycaenidae). Populations were sampled from five landscapes in East Kalimantan (Borneo, Indonesia) which were differentially disturbed by selective logging and the 1997/1998 El Niño Southern Oscillation (ENSO)-induced drought and fires. Sampling occurred before (in 1997) and after the forest fires (in 1998, 1999, 2000, and 2004). Drupadia theda populations underwent serious population size reductions following the 1997/1998 ENSO event. For a total of 208 individuals, we sequenced a 509-bp segment of mtDNA containing the control region plus the 5' end of the 12S rDNA gene. Haplotype diversity in D. theda populations ranged from 0.468 to 0.953. Just after the 1997/1998 ENSO event, number of recorded individuals and genetic diversity were very low in D. theda populations sampled in the two severely burned areas and in a small pristine forest fragment that was surrounded by burned forest and thereby affected by drought. Interestingly, higher levels of genetic diversity were observed in logged forest compared to proximate pristine forest. After 1998, the genetic composition within the three ENSO-disturbed areas diverged. In the twice-burned forest, the genetic diversity in 1999 already approached pre-fire levels, while it remained nearly unchanged in proximate onceburned forest. Our data suggest that the 1997/1998 ENSO-induced drought and fires caused massive reductions in the genetic diversity of D. theda and that population recoveries were linked to their geographical position relative to patches of unburned forest (and thus to source populations).

Introduction

Disturbance is often cited as the principal factor regulating species diversity. It has been described as a force, often abrupt and unpredictable, that kills or badly damages organisms and alters the availability of resources [START_REF] Mackey | A re-examination of the expected effects of disturbance on diversity[END_REF]. Different forms of disturbance can have different impacts on biotic assemblages with respect to species richness. In tropical rainforests, the two prevalent forms of disturbance are logging and El Niño Southern Oscillation (ENSO)-induced disturbances. Previous studies have found that, despite often causing severe structural damage, legal logging seldom leads to reductions in species diversity [START_REF] Cannon | Tree species diversity in commercially logged Bornean rain forest[END_REF][START_REF] Willott | Effects of selective logging on the butterflies of a Bornean rainforest[END_REF][START_REF] Lewis | Effects of experimental selective logging on tropical butterflies[END_REF][START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF] and may be considered as an intermediate disturbance (i.e. intermediate in intensity or in frequency). On the contrary, drought and fires induced by ENSO events are a major disturbance because they can dramatically modify natural habitats over very large spatial and temporal scales [START_REF] Holmgren | El Niño effects on the dynamics of terrestrial ecosystems[END_REF][START_REF] Siegert | Increased damage from fires in logged forests during droughts caused by El Nino[END_REF][START_REF] Laurance | Slow burn: the insidious effects of surface fires on tropical forests[END_REF]. Effects of disturbance on tropical species diversity have been investigated in quite a number of organisms, such as flowering plants [START_REF] Van Nieuwstadt | The ecological consequences of logging in the burned forests of East Kalimantan, Indonesia[END_REF][START_REF] Brown | Long-term impacts of logging on forest diversity in Madagascar[END_REF][START_REF] Cleary | Vegetation responses to burning in a rain forest in Borneo[END_REF], birds [START_REF] Thiollay | Influence of selective logging on bird species diversity in a Guianan rain forest[END_REF], mammals [START_REF] Malcolm | Influence of timber extraction routes on Central African small mammals communities, forest structure, and tree diversity[END_REF], and insects [START_REF] Willott | Effects of selective logging on the butterflies of a Bornean rainforest[END_REF][START_REF] Lewis | Effects of experimental selective logging on tropical butterflies[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] Cleary | Changes in rain forest butterfly diversity following major ENSO-induced fires in Borneo[END_REF]Cleary et al . 2004). However, little is known about its effect on genetic diversity within species.

The Indonesian province of East Kalimantan (Borneo) was severely affected by the 1997/1998 ENSO event (more than 5 million hectares burned), which was by far the most severe and widespread recorded event in Borneo [START_REF] Harrison | Repercussions of El Niño: drought causes extinction and the breakdown of mutualism in Borneo. Proceedings of the Royal Society of London[END_REF][START_REF] Siegert | Increased damage from fires in logged forests during droughts caused by El Nino[END_REF]. Butterflies in Borneo have previously been extensively studied in terms of community responses to both logging and ENSO-induced droughts and fires [START_REF] Willott | Effects of selective logging on the butterflies of a Bornean rainforest[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] Hamer | Ecology of butterflies in natural and selectively logged forests of northern Borneo: the importance of habitat heterogeneity[END_REF][START_REF] Cleary | Changes in rain forest butterfly diversity following major ENSO-induced fires in Borneo[END_REF]Cleary & Mooers 2004;Cleary et al . 2004). In East Kalimantan, the fires created a mosaic of unburned forests (some of which were affected by drought), isolating local populations of butterfly species and reducing species richness in the affected areas [START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF][START_REF] Cleary | Changes in rain forest butterfly diversity following major ENSO-induced fires in Borneo[END_REF]. Higher levels of species richness were observed in logged forests compared to pristine forest [START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF][START_REF] Cleary | Assessing the use of butterflies as indicators of logging in Borneo at three taxonomic levels[END_REF]. However, we do not know the consequences of such disturbance at the intraspecific level.

Genetic diversity using the mitochondrial DNA control region (CR; also called D-loop) was assessed in Drupadia theda Felder (Lepidoptera: Lyceanidae), a rather common rainforest lycaenid that feeds on a wide range of plant families including Fabaceae, Guttiferae, Meliaceae, Rubiaceae, Meliaceae, Clusiaceae, and Sapindaceae [START_REF] Elliot | The Butterflies of the Malay Peninsula[END_REF][START_REF] Igarashi | The Life Histories of Asian Butterflies[END_REF][START_REF] Harrison | Repercussions of El Niño: drought causes extinction and the breakdown of mutualism in Borneo. Proceedings of the Royal Society of London[END_REF][START_REF] Robinson | Hostplants of the Moth and Butterfly Caterpillars of the Oriental Region[END_REF]K. Fiedler, personal communication). This species can thus be considered a generalist with respect to host plant use. However, D. theda is considered a specialist in terms of myrmecophily as it is obligatorily and specifically associated with Crematogaster ant species [START_REF] Seufert | Life-history diversity and local coexistence of three closely related lycaenid butterflies (Lepidoptera: Lycaenidae) in Malaysian rainforests[END_REF], which often form very large and quite territorial colonies. Egg laying typically occurs in close physical contact with host ants, mostly in small groups, and larvae are attended from the first instar onwards (K. Fiedler, personal communication). The average development time from egg to adult of D. theda is 28.3 days [START_REF] Seufert | Life-history diversity and local coexistence of three closely related lycaenid butterflies (Lepidoptera: Lycaenidae) in Malaysian rainforests[END_REF]. Drupadia theda populations underwent serious population size reductions in East Kalimantan following the 1997/1998 ENSOinduced fires [START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF][START_REF] Cleary | Butterfly response to severe ENSOinduced forest fires in Borneo[END_REF]). These population size changes are expected to leave a genetic footprint, different from the one expected for constant size populations, which can be revealed by DNA sequencing [START_REF] Slatkin | Pairwise comparisons of mitochondrial DNA sequences in stable and exponentially growing populations[END_REF][START_REF] Rogers | Population growth makes waves in the distribution of pairwise genetic differences[END_REF]. Theoretically, population size reductions are predicted to result in a loss of genetic diversity [START_REF] Wright | Evolution in Mendelian populations[END_REF][START_REF] Frankham | Inbreeding and extinction: a threshold effect[END_REF] and an increase of genetic differentiation between populations, due to the increased influence of genetic drift [START_REF] Chakraborty | Bottleneck effects on average heterozygosity and genetic distance with the stepwise mutation model[END_REF]). In addition, small and isolated populations are vulnerable to inbreeding depression [START_REF] Sakai | The population biology of invasive species[END_REF][START_REF] Frankham | Introduction to Conservation Genetics[END_REF], that might reduce their evolutionary potential and increase their risk of extinction [START_REF] Frankham | Inbreeding and extinction: a threshold effect[END_REF][START_REF] England | Effects of intense versus diffuse population bottleneck[END_REF]. Historically documented bottlenecks, such as those resulting from the 1997/1998 ENSO event, provide unique opportunities to study the consequences of such events on genetic variability and the evolutionary potential of natural populations.

We investigated the short-term impact of disturbance on genetic diversity and composition of differentially affected D. theda populations. The analysed populations originated from pristine, logged, and burned forests in different landscapes in East Kalimantan, which were sampled before (in 1997) and after the ENSO event (in 1998, 1999, 2000, and 2004). To our knowledge, this is the first study investigating the short-term impact of logging and ENSO-induced disturbance on the genetic diversity of a tropical rainforest species, with pre-and postdisturbance samples analysed.

Materials and methods

Sampling

Specimens of Drupadia theda were collected during surveys conducted in 1997 (before the fires), 1998, 1999, 2000, and 2004 in the Balikpapan-Samarinda region of East Kalimantan, Indonesian Borneo (Fig. 1). Five differentially disturbed landscapes were sampled. The three landscapes in unburned isolates (I1, I2, I3) and the remaining two in burned forests (B1, B2) were located in the 5.2 million ha of East Kalimantan that changed from a habitat mosaic of primary forest with areas of secondary forest to an area dominated by secondary (burned) forest with only remnant unburned patches [START_REF] Siegert | Increased damage from fires in logged forests during droughts caused by El Nino[END_REF]). All landscapes are described in detail, in terms of location and dominant vegetation, in [START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF]. Briefly, I1 (unlogged primary) and I3 (logged in 1993/1994) landscapes are located in a 108 000 ha large unburned isolate located in part of the ITCI (International Timber Concessions Indonesia) and including the Gunung Meratus Protected Forest Reserve ( c . 30 000 ha). The I2 landscape is located in a small, 3500-ha unburned primary isolate that is all that remains of the Sungai Wain Protected Forest; 6500 ha of the original 10 000 ha burned during the 1997/1998 ENSO event. The once-burned landscape B1 is located in the burned part of the Sungai Wain Nature Reserve and burned for the first time during the 1997/1998 ENSO event. The twice-burned landscape B2 was partially burned during the 1982/1983 ENSO event and severely burned during the 1997/1998 ENSO event; it is located in the Wanariset Samboja Research Forest. None of the burned landscapes have been commercially logged although they may have been subjected to unrecorded illegal logging.

Specimens of D. theda were sampled along 300-m transects as described in [START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF]. Individuals were immediately killed, after which three legs were removed and stored in 96% ethanol. Each individual was assigned a unique code that was recorded on the tube containing the legs and the papillote with the butterfly. Voucher specimens were preserved (using silica gel) and have been deposited in the Zoological Museum of the University of Amsterdam (ZMA), the Netherlands.

Molecular analysis

Genomic DNA was isolated from one leg of each individual using a cetyltrimethyl ammonium bromide (CTAB) protocol [START_REF] Hillis | Molecular Systematics[END_REF]. Amplifications of the mtDNA control region (CR) plus a fragment of the 5 ′ end of the 12S rDNA gene were conducted using primers SeqLepMet and LepAT2B [START_REF] Vila | The utility of the neglected mitochondrial control region for evolutionary studies in Lepidoptera (Insecta)[END_REF]. Polymerase chain reaction (PCR) was performed in 25 µ L containing 1 × PCR buffer (HT Biotechnology), 3 m m MgCl 2 , 0.12 m m of each dNTP, 0.5 mg/mL bovine serum albumin, 0.05 µ m of each primer, 0.4 U Taq polymerase (HT Biotechnology), and approximately 20 ng DNA. PCRs were carried out in PTC 100 Thermocycler (MJ Research): 2-min denaturation at 94 ° C; 35 cycles of 1 min at 94 ° C, 1 min at 54 ° C, and 1 min at 65 ° C; and a final extension of 5 min at 65 ° C. Amplified fragments were purified with the Boom procedure [START_REF] Boom | Rapid and simple method for purification of nucleic acids[END_REF]) and cycle sequenced in both directions with either SeqLepMet or LepAT2B using BigDye Terminator sequencing kit (Applied Biosystems). Sequences were run on an ABI 3100 Genetic Analyser (Applied Biosystems). Forward and reverse sequences were compared for each individual in bioedit [START_REF] Hall | bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]) and a single sequence was defined for each individual. Individual sequences were then aligned by eye in genedoc [START_REF] Nicholas | GENEDOC: a tool for editing and annotating multiple sequence alignments[END_REF].

Data analysis

Haplotypes were identified and their relative frequency within populations calculated using arlequin version 2.000 [START_REF] Schneider | ARLEQUIN version 2.000: A software for population genetics data analysis[END_REF]. A Minimum spanning network displaying evolutionary relationships between haplotypes was obtained in arlequin and drawn by hand in adobe ® illustrator ® 9.0 (Adobe Systems Inc.). Pairwise distances between haplotypes or groups of haplotypes were estimated according to the uncorrected p distance using mega version 2.1 [START_REF] Kumar | MEGA: Molecular Evolutionary Genetics Analysis[END_REF]. For further analysis, indels were coded as substitutions, and dinucleotide indels situated in the [TA] n -like stretch were coded as single base substitutions (see Results).

Genetic diversity within populations was measured as haplotype diversity ( Hd , Nei 1987), nucleotide diversity ( π , Nei 1987), and mean number of pairwise nucleotide differences between individuals ( k , Tajima 1983) using dnasp 4.0 [START_REF] Rozas | dnasp, DNA polymorphism analyses by the coalescent and other methods[END_REF]). Tajima's D test [START_REF] Tajima | Statistical method for testing the neutral mutation hypothesis by DNA polymorphism[END_REF]) was conducted within each sample separately as well as within regions per year (by pooling samples), viz., Gunung Meratus (I1 + I3) in 2000, and Sungai Wain (B1 + I2) in 1998, and in 2000. Observed haplotype diversities were compared to the empirical distribution of this index generated using coalescent simulations in order to conduct the haplotype diversity test ( H test; [START_REF] Depaulis | Neutrality tests based on the distribution of haplotypes under an infinite-site model[END_REF], performed in dnasp . The computer simulations were based on the coalescent process for a neutral infinite-sites model under the assumption of a large constant population size [START_REF] Hudson | Gene genealogies and the coalescent process[END_REF]. dnasp generates the empirical distribution of Hd , obtained from 10 000 simulations under the neutral coalescent process and given the number of segregating sites observed in each sampled population (the model therefore considers that the number of segregating sites is fixed and that mutations are uniformly distributed, at random, along lineages). dnasp thus estimates the probability of obtaining lower or higher values of Hd , computed from the simulation ( Hd sim ), than the one observed in each sample ( Hd obs ). These simulations were conducted within each sampled population and within the regions, pooling samples as described above. 1997, 1998, 1999, 2000, and 2004. Analyses of the spatial and temporal genetic population structure were performed with arlequin . Genetic divergences between pairwise samples were estimated using Φ statistics ( Φ ST based on haplotype frequencies and molecular divergence, using pairwise differences). P values were obtained using a nonparametric permutation procedure with 10 000 permutations [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data[END_REF]) and the level of significance was adjusted according to the sequential Bonferroni correction for α = 0.05 [START_REF] Rice | Analysing tables of statistical tests[END_REF]. The matrix of pairwise Φ ST was visualized by nonmetric multidimensional scaling (MDS) using the program xlstat ® (Addinsoft SARL). The fit of the data in two dimensions was measured with a stress factor [START_REF] Kruskal | Multidimensional Scaling[END_REF]. The spatial distribution of genetic variation was examined using an analysis of molecular variance ( amova ; [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data[END_REF]. amova s were performed to test the null hypothesis that genetic variation was not associated (i) with temporal variation within sampled landscapes and (ii) with spatial structure in 2000 according to three regions: Gunung Meratus (I1 + I3), Sungai Wain (B1 + I2), and Wanariset (B2).

Results

Sequence analysis

A total of 208 individuals of Drupadia theda sampled from 11 spatial and temporal samples were analysed (Table 1). The percentage of individuals analysed compared to the number of recorded individuals during surveys varied from 4% to 63%. The amplified mtDNA fragment, varying from 502 to 509 bp, contained the control region (CR; 390-397 bp) plus a fragment of 112 bp of the 5 ′ end of the 12S rDNA gene. Sequence length variations observed among individuals within the CR originated from length polymorphism within region A (the poly thymidine stretch at the 5 ′ end of the control region), within region D (the [TA] nlike stretch), and within the homopolymer described in [START_REF] Vila | The utility of the neglected mitochondrial control region for evolutionary studies in Lepidoptera (Insecta)[END_REF] (Table 2). Because length polymorphism observed in region D originated from dinucleotide indels ([TA] indels, bp 96 -101, Table 2), each of these dinucleotide indels were treated as a single base substitution. Among the 208 individuals sequenced, we identified 42 different haplotypes, distinguished by 39 polymorphic sites, from which eight corresponded to indels and 15 were singletons (Table 2). Of the 39 polymorphic sites, five were found in the 12S gene and only one site in the CR presented multiple substitutions (bp 283). Among haplotypes, the overall mean uncorrected sequence divergence was 1.31% and haplotype pairwise divergence ranged from 0.2 to 3.16%. Considered over all individuals, adenine and thymine contents were on average 49.1 and 43.6%, respectively. Haplotype diversity was 0.882, nucleotide diversity was 0.0094, and the mean number of pairwise differences between two individuals for the entire data set was 4.75.

The minimum spanning network revealed a subdivision of the 42 haplotypes into three groups (Fig. 2). Groups 1 and 2 were 1.5 ± 0.4% divergent, and groups 1 and 3 were 2.5 ± 0.6% (average uncorrected p distance). The haplotypes from group 3 (H6, H8, H9, and H11) were exclusively found in seven individuals from the Gunung Meratus region (six individuals from I3-00 and one from I1-00; Table 3). Haplotypes H8, H9, and H11 were all length variants of H6. In order to verify if these seven individuals could belong to a cryptic species (these individuals were not morphologically distinct from the others based on their wing patterns), we sequenced a 757-bp fragment of the mtDNA cytochrome oxydase subunit 1 region (COI) for two individuals carrying haplotype H6, for one individual carrying haplotype H1 (the most common haplotype), and for one individual of Drupadia ravindra (closely related to Table 1 Sampling information. The total number of recorded Drupadia theda as well as the number of sequenced D. theda is given within each population. Ind./transect is the adjusted number of individuals recorded per transect and per day. Population names are given according to the sampled landscape (see Fig. 1) and the year of sampling. The forest status refers to the degree of disturbance of the landscape at the time of sampling. For example, B1 was sampled in an originally pristine forest that burned during 1997/1998 ENSO D. theda ). We used primers C1-J-2183 and TL2-N-3014 [START_REF] Simon | Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers[END_REF]) and the protocol described in [START_REF] Vila | The utility of the neglected mitochondrial control region for evolutionary studies in Lepidoptera (Insecta)[END_REF]. A sequence divergence of 1.45% was observed at COI between individuals of H6 and H1. In comparison, a sequence divergence of 6.87% was found between COI sequences of D. theda (haplotype H1) and D. ravindra , and a sequence divergence of 6.47% was found between COI sequences of haplotype H6 and D. ravindra . As these results (morphologically and genetically) were not final evidence that individuals carrying H6 were belonging to a cryptic species, we kept these seven individuals in the data set for further analyses to avoid reducing sample sizes. Nevertheless, it should be noted that removing the seven group 3 individuals did not significantly change the results. 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 1 1 4 5 7 8 9 9 9 9 9 0 0 0 8 8 8 9 0 1 1 2 5 5 7 8 8 9 0 0 2 3 3 8 8 9 9 0 4 2 8 0 1 1 0 1 6 7 8 9 0 1 3 0 6 8 2 4 7 8 8 0 2 0 2 3 2 2 5 9 1 3 2 8 5 6 5 3 Haplotypes H1 and H2 (length variants) were carried by 44% of the individuals from all sampled landscapes. Seven haplotypes were shared in at least two regions (Gunung Meratus, Sungai Wain or Wanariset), representing 66% of the individuals, while the rest of the individuals carried haplotypes that were restricted to a particular region (i.e. 35 haplotypes; see Table 3). Within landscapes, the number of haplotypes ranged from one (in B2-98) to 15 (in B2-97). Sample B2-98 was excluded from further analysis because only two individuals were collected in the Wanariset forest in 1998. However, it should be noted that this low sample size was the consequence of the low abundance of butterflies observed in this landscape in 1998 (0.08 individuals vs. nearly one individual per transect per day in 1997, with similar sampling intensity; see Table 1).

H1 -A G T C A A T A T A --G T A T A T A A T C A G T A A A G T T A

Genetic diversity and neutrality tests

Within landscapes, haplotype diversity ranged from 0.468 (for I2-00) to 0.953 in B2-97 (Table 4). The highest values of gene diversity were found in B2-97 and I3-00, followed by I1-00 and B2 sampled after 1997, and the lowest values were found in Sungai Wain, both in 1998 and 2000 (Fig. 3). Haplotype diversity was not correlated with the mean number of individuals per transect per day (Spearman's coefficient = 0.394, P = 0.260; Fig. 4) nor the numbers of recorded individuals within landscapes (Spearman's coefficient = 0.382, P = 0.276), nor the sample size (Spearman's coefficient = 0.526, P = 0.123). Samples B1-98 and I2-00 were composed of individuals carrying haplotype H1 (the most common one) and length variants associated with H1 (i.e. H2, H21, and H26).

Within the population from the burned Wanariset forest (B2), the genetic diversity remained more or less stable through time (with the exception of the first postfire sampling in 1998 where only two individuals carrying identical haplotypes were sampled), but the haplotype composition changed. Apart from H5 (a length variant of H3), the most common haplotypes in B2 in 1997 (and overall landscapes; H1, H2, H3, and H4) have been observed in the population after the fires. In contrast, the rare and endemic haplotypes from 1997 were not found later in samples from the same landscape (see Table 3). Interestingly, in B2, a number of haplotypes, which were not encountered in 1997 and in any other landscapes, were observed in 1999, 2000, and 2004 (Table 3). These haplotypes might 3 for haplotype distribution). Each circle corresponds to one haplotype; each line between two haplotypes corresponds to one mutation (see Table 2 for reference). A small black circle indicates a missing, one-mutational step haplotype.

Population structure

Over all pairwise comparisons, Φ ST estimates ranged from negative to 0.654 (Table 5). Within regions at time t, none of the pairwise Φ ST were significant after sequential Bonferroni adjustment of the significance level (within Gunung Meratus in 2000, Φ ST = 0.092, P = 0.018; within Sungai Wain in 1998, Φ ST = -0.152, P = 0.883; in 2000, Φ ST = 0.015, P = 0.302). In 2000, 32.63% of the total variance was attributed to variation among the five landscapes (global Φ ST = 0.317, P < 0.001; Table 6). However, the genetic variation in 2000 did not appear significantly associated with the three regions, Gunung Meratus, Sungai Wain and Wanariset (among group genetic variance = 0.909, P = 0.063). After Bonferroni corrections, the Wanariset population sampled in 1997 (B2-97) did not appear significantly different from Gunung Meratus populations (I1 & I3) sampled in 2000, but the P values were lower than 0.01 (Φ ST = 0.118, P = 0.005, and Φ ST = 0.109, P = 0.009, respectively; Table 5). Within Wanariset, genetic divergences between years appeared significant between 2000 and 2004 (Φ ST = 0.222, P = 0.001), but not significant after Bonferroni correction between 1997 and 1999 (Φ ST = 0.102, P = 0.030), and between 1999 and 2000 (Φ ST = 0.075, P = 0.062). Also, no significant genetic divergence was observed within Sungai Wain landscapes between 1998 and 2000 (all P > 0.05). Interestingly, no significant divergence was observed in Wanariset between 1997 and 2004 samples (Φ ST = 0.045, P = 0.091). Amongst Wanariset (B2) and the two sampled landscapes from Sungai Wain (B1 and I2), 9.59% of the genetic variance was attributed to temporal variation among years within landscapes (P = 0.001; Table 6).

The genetic divergences found amongst D. theda samples (with the exception of B2-98) are visualized with pairwise Φ ST estimates using a two-dimensional non-metric MDS in Fig. 5. This method gives a good representation of the data with a stress value of 0.019. The MDS graphically illustrates the evolution of patterns of genetic divergences within as well as between populations. It depicts the increase of genetic divergence between B1 and I2 from 1998 to 2000, and the decline of genetic divergence between Wanariset (B2) and Sungai Wain (B1 + I2) from 1997 to 2000. It also illustrates the temporal evolution of genetic composition within Wanariset. [START_REF] Nei | Molecular Evolutionary Genetics[END_REF]; S, number of polymorphic sites; π, nucleotide diversity [START_REF] Nei | Molecular Evolutionary Genetics[END_REF]; and k, mean number of pairwise nucleotide differences between individuals [START_REF] Tajima | Evolutionary relationship of DNA sequences in finite populations[END_REF]. For Tajima's D test [START_REF] Tajima | Statistical method for testing the neutral mutation hypothesis by DNA polymorphism[END_REF], the value of D in reported. For the H test [START_REF] Depaulis | Neutrality tests based on the distribution of haplotypes under an infinite-site model[END_REF]

Discussion

mtDNA control region sequence polymorphism in Drupadia theda

Amongst 208 Drupadia theda individuals and the 509-bp mtDNA fragment analysed (CR+ 12S 5′ end), we identified 42 haplotypes, including alignment gaps; the overall nucleotide diversity was 0.0069. When analysing the same mtDNA region as assessed by [START_REF] Vila | The utility of the neglected mitochondrial control region for evolutionary studies in Lepidoptera (Insecta)[END_REF], i.e. exclusively the CR 5′ end prior to the homopolymer, we found a much higher nucleotide diversity in D. theda (325 bp; π = 0.011) than in the two nymphalid butterflies Erebia triaria (361 bp; π = 0.0044) and Erebia palarica (354 bp; π = 0.0005) [START_REF] Vila | The utility of the neglected mitochondrial control region for evolutionary studies in Lepidoptera (Insecta)[END_REF]). Among the 42 haplotypes, one group of haplotypes was highly divergent compared to the rest of the haplotypes (group 3; H6, H8, H9, and H11, see Fig. 2). The seven individuals carrying these divergent haplotypes might possess an ancient mitochondrial variant preserved in the Gunung Meratus region, belong to a cryptic species, or be of hybrid origin. The analyses of the COI sequences revealed that the sequence divergence between related species (Drupadia theda and Drupadia ravindra) was nearly fivefold the divergence found between individuals carrying haplotypes of each of groups 1 and 3. Moreover, we did not find evidence of wing pattern differences between the individuals belonging to different groups. Therefore, the lack of evidence for the occurrence of cryptic species or hybrids leaded us to maintain all sequenced haplotypes in the data set. Further investigations on male genitalia and other genetic markers will be needed to be more conclusive about the origin of this divergent group of haplotypes. Although the utility of the mtDNA CR for species-level phylogenies and population-level studies in insects remains controversial [START_REF] Taylor | The lepidopteran mitochondrial control region: structure and evolution[END_REF][START_REF] Zhang | Insect mitochondrial control region: a review of its structure, evolution, and usefulness in evolutionary studies[END_REF][START_REF] Caterino | The current state of insect molecular systematics: a thriving tower of Babel[END_REF], our results on sequence Although numbers of D. theda recorded in 1997 in Wanariset per transect and per day were two to five times lower than reported in Gunung Meratus landscapes, levels of genetic diversity were comparable in these forests. In 1998, a massive population size reduction was observed in Wanariset, as the number of individuals recorded per transect and per day decreased by a factor of 10. This major bottleneck was obviously linked with the 1997/1998 ENSO-induced fires [START_REF] Cleary | Butterfly response to severe ENSOinduced forest fires in Borneo[END_REF]. Indeed, during the 1997/1998 winter, the Wanariset forest almost entirely (> 90%) burned [START_REF] Cleary | Butterfly response to severe ENSOinduced forest fires in Borneo[END_REF]). The D. theda that survived the fires were exclusively restricted to a c. 50-ha unburned forest fragment (namely W03 in [START_REF] Cleary | Butterfly response to severe ENSOinduced forest fires in Borneo[END_REF].

The Sungai Wain landscapes (B1 and I2) have been sampled in an originally pristine and protected forest (the Sungai Wain Nature Reserve) of 10 000 ha of lowland mixed dipterocarp rainforest with extensive swamp areas in low lying areas, similar to what was observed in Gunung Meratus protected forest. Despite its protected status, the forest has suffered considerable encroachment from adjacent human settlements, and fires early in 1998 destroyed about two-thirds of the pristine forest so that by mid 1998 only about 3500 ha of pristine forest remained. In 1998, both sampled landscapes B1 and I2 showed very low levels of genetic diversity combined with low number of recorded individuals. In 2000, population sizes increased while the levels of genetic diversity remained lower than in the pristine forest located in a large unburned area (I1). Because we lack temporal data from 1997, we do not have evidence that the low levels of genetic diversity originated from bottlenecks. However, because (i) the two landscapes were sampled in an originally pristine and protected forest, (ii) D. theda densities in Sungai Wain forest were much lower than in Gunung Meratus pristine forest, (iii) a population crash was observed in Wanariset following 1997/1998 ENSO-induced fires, and (iv) a population size increase was observed between 1998 and 2000 in both B1 and I2, the low levels of genetic diversity observed in 1998 probably result from population bottlenecks following population crashes due to the 1997/1998 ENSO-induced disturbance.

The similar, low genetic diversity values observed in B1 and I2 further suggest that the drought associated with the fires had as negative an impact on the genetic diversity of D. theda as the fire itself. Indeed, while I2 was located in a small unburned isolated pristine forest, it was surrounded by burned forest. The ENSO-induced drought detrimentally impacted available water and trophic resources [START_REF] Cleary | Butterfly response to severe ENSOinduced forest fires in Borneo[END_REF]. Such effects are known to have caused local extinctions of several fig wasp species in an unburned forest isolate in northern Borneo due to disruption of their primary food resources [START_REF] Harrison | Repercussions of El Niño: drought causes extinction and the breakdown of mutualism in Borneo. Proceedings of the Royal Society of London[END_REF]. This result is further in agreement with the impact of drought on species richness [START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF][START_REF] Cleary | Changes in rain forest butterfly diversity following major ENSO-induced fires in Borneo[END_REF]Cleary et al. 2004). Yet, it is still not clear if the drought affected the butterflies directly, or indirectly, through its host plants or mutualist ants. The latter was the case with most UK populations of the lycaenid Polyommatus bellargus that were affected by severe bottlenecks in the late 1970s when a drought caused its unique host plant (Hippocrepis comosa) to wilt [START_REF] Harper | Microsatellite markers to assess the influence of population size, isolation and demo-graphic change on the genetic structure of the UK butterfly Polyommatus bellargus[END_REF]. Nevertheless, since D. theda feeds on a variety of host plants, it is unlikely that the drought affected all the species host plants. Still, D. theda population bottlenecks could have been caused by population size reductions (or complete disappearance) of its mutualist ants of the genus Crematogaster (morphospecies A and B; [START_REF] Seufert | Life-history diversity and local coexistence of three closely related lycaenid butterflies (Lepidoptera: Lycaenidae) in Malaysian rainforests[END_REF] which could be associated with humid soil conditions, similar to Lasius niger, the host ant of the lycaenid Plebejus argus [START_REF] Seymour | Dispersal of the lycaenid Plebejus argus in response to patches of its mutualist ant Lasius niger[END_REF]. Our results on genetic diversity in pre-and postdisturbed populations suggest that the ENSO-related population bottlenecks, together with genetic drift in small populations (possibly in combination with selection and variance in reproductive success), resulted in massive losses of haplotypes, leading to genetic compositions with one or two highly frequent haplotypes and a few rare haplotypes.

Interestingly, the two samples showing the highest haplotype diversity values were nonpristine; I3-00 had been logged in 1993/94, and B2-97 partially burned during the 1982/83 ENSO event. Moreover within these two samples, a significant deviation from neutrality was detected as higher haplotype diversity than observed under the hypothesis of neutrality and stable population size. These higher values may originate from sample size bias or reflect nonequilibrium conditions within these two previously disturbed forests. Interestingly, higher levels of species diversity were found in the same logged forest than the proximate pristine forest of Gunung Meratus [START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF]. The high genetic diversity found in the logged forest may thus be suggestive of similar processes acting on genetic diversity and species diversity. However, because of the lack of replicates (relative to both species and logged areas), more investigations are needed to conclude that a species-genetic diversity correlation [START_REF] Vellend | Island biogeography of genes and species[END_REF]) is actually occurring in disturbed environments.

Genetic recovery following bottlenecks

A particularly interesting result from the short-term impact of droughts and fires on D. theda genetic diversity concerns the differences observed in genetic diversity patterns following the bottlenecks. Two years after the fires, levels of genetic diversity within Sungai Wain did not significantly increase, despite observed population expansions. On the contrary, in Wanariset, the number of individuals recorded in 1999 was similar to that in 1997, and levels of genetic diversity were reaching pre-ENSO levels. From the 40 recorded individuals in 1999, 39 were observed along the transect situated in the unburned fragment (W03 described in [START_REF] Cleary | Butterfly response to severe ENSOinduced forest fires in Borneo[END_REF]. In 2000, high population densities were observed in Wanariset, although the recorded D. theda were no longer restricted to the W03 pristine fragment but were also recorded in high densities along transects in partially burned forest (e.g. W05 in [START_REF] Cleary | Butterfly response to severe ENSOinduced forest fires in Borneo[END_REF]).

In 1999, 2000, and 2004, new haplotypes were encountered in Wanariset, causing genetic diversity to rise. These new haplotypes most likely originated from incoming migrants because (i) none of these haplotypes were encountered in 1997 despite a large sampling effort (50% of recorded individuals were randomly sequenced), (ii) the same sampling method was used during each sampling event to avoid sampling bias [START_REF] Cleary | An examination of scale of assessment, logging and ENSO-induced fires on butterfly diversity in Borneo[END_REF], and (iii) the sampling intensity conducted in the following years was not higher than in 1997 (50%, 37.5%, and 10.4% in 1998, 1999, and 2000, respectively). The rapid recovery of the genetic diversity of D. theda in the Wanariset population is thereby certainly due to an influx of genetic diversity through migration.

Conversely, low levels of genetic diversity observed in Sungai Wain populations in 2000 may originate from sampling bias or the lack of incoming migrants with new haplotypes. However, the latter explanation is more likely given that similar sampling efforts were conducted in Sungai Wain and Wanariset in 2000 (11.5 and 10%, respectively, of recorded individuals were randomly sequenced). Because (i) Wanariset and Sungai Wain forests were similarly affected by the 1997/1998 ENSO-induced disturbance in terms of burning (Cleary et al. 2004) and (ii) small pristine fragments remained unburned in both forests (although much smaller in Wanariset), the faster recovery rate in the Wanariset population than in Sungai Wain populations may be due to their different proximities to larger patches of unburned forest. In particular, the Wanariset forest is contiguous with the large (71 905 ha) Bukit Suharto Protected Forest Reserve to the north. Although most (93%) of this reserve also burned during the 1997/ 1998 ENSO event, an estimated 4800 ha escaped conflagration [START_REF] Hoffmann | Fire damage in East Kalimantan in 1997/98 related to land use and vegetation classes: satellite radar inventory results and proposals for further actions[END_REF]) and probably thus served as a source of migrants for the Wanariset population. The Sungai Wain forest, on the contrary, is isolated from proximate forest by the bay of Balikpapan and Balikpapan city itself to the west and south, and by various roads and settlements, as well as twice-burned forest (including Wanariset) to the north and east (see Fig. 1). Thereby, the D. theda population expansion observed in I2 was not sufficient to raise genetic diversity levels in this isolated forest.

High levels of genetic diversity in documented bottlenecked populations are not uncommon and originate from sufficient allelic richness immediately after the bottlenecks [START_REF] Brookes | Genetic analysis of founder bottlenecks in the rare British butterfly Plebejus argus[END_REF][START_REF] Queney | Absence of a genetic bottleneck in a wild rabbit (Oryctolagus cuniculus) population exposed to a severe viral epizootic[END_REF][START_REF] Lefèvre | Impact of founder population, drift and selection on the genetic diversity of a recently translocated tree population[END_REF] or from new haplotypes arriving via postbottleneck immigrations [START_REF] Hansson | Increase of genetic variation over time in a recently founded population of great reed warblers (Acrocephalus arundinaceus) revealed by microsatellites and DNA fingerprinting[END_REF][START_REF] Keller | Immigration and the ephemerality of a natural population bottleneck: evidence from molecular markers[END_REF][START_REF] Whitehouse | Post-bottlenecked genetic diversity of elephant populations in South Africa, revealed using microsatellite analysis[END_REF][START_REF] Colson | Rapid recovery of genetic diversity of dogwhelk (Nucella lapillus L.) populations after local extinction and colonization contradicts predictions from life-history characteristics[END_REF][START_REF] Takami | Genetic diversity and structure of urban populations of Pieris butterflies assessed using amplified fragment length polymorphism[END_REF]). In the latter case, the speed at which the recovery of the genetic diversity occurs depends on the dispersal capacities of the species, and whether the immigrants come from one (propagule pool) or more (migrant pool) source populations. Our study suggests that the degree of geographic isolation of the local population relative to source populations is as well a key factor in driving the recovery of genetic variation following bottlenecks in natural populations.

Fig. 1

 1 Fig. 1 Map of the research area, located in the Balikpapan-Samarinda region of East Kalimantan (Indonesian Borneo) showing location of landscapes in which individuals of Drupadia theda were sampled. Black rectangles represent burned landscapes; white rectangles represent unburned isolates. I1 and I3 landscapes are located in the Gunung Meratus region and were sampled in 2000. B1 and I2 landscapes are located in the Sungai Wain region and were sampled in 1998 and in 2000. B2 is located in the Wanariset region and was sampled in1997, 1998, 1999, 2000, and 2004.

  A A A C T G H2 . . . . . . . . . --. . . . . . . . . . . . . . . . . . . . . . . . . . . . H3 . . . . T G . . . --. . . . G . . . . . . . . A . . . . . C . . . . . . . A H4 . . . . T G . . . --. . . . G . . . G . . . . A . . . . . C . . . . . . . A H5 . . . . T G . . . . . . . . . G . . . . . . . . A . . . . . C . . . . . . . A H6 . . A A T . . . . --. . A . . . . . . G . A . . . . . . A C . . -. G . . A H7 . . . . T G . . . . . . . . . G . . . G . . . . A . . . . . C . . . . . . . A H8 . . A A T . . ----. . A . . . . . . G . A . . . . . . A C . . -. G . . A H9 . . A A T . . . . . . . . A . . . . . . G . A . . . . . . A C . . -. G . . A H10 . . . . T . . . . --. . . . G . . . . . . . . A . . . . A . . . . . . . . A H11 T . A A T . . . . --. . A . . . . . . G . A . . . . . . A C . . -. G . . A H12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . H13 . . . . T G . . . . . . . . . G . . . . . . . . A . . . . . . . . . . . . . A H14 . . . . T . . . . . . . . . . G . . . . . . . . A . . . . A . . . . . . . . A H15 . . . . T G . . . . . . . . . . . . . G . . . . A . . . . . C . . . . . . . A H16 . . . . T G . . . --. . . . . A . . . . . . . A . . . . . C . . . . . . . A H17 . C . . T G . . . . . . . . . . . . . . . . . . A . . . . . C . . . . . . . A H18 . . . . T G . . . . . . . . . . . . G . . . . . A . . . . . C . . . . . . . A H19 T . . . T G . . . --. . . . . . . . . . . . . A . . . . . C . . . . . . . A H20 T . . . T G . . . . . . . . . . . . . . . . . . A . . . . . C . . . . . . . A H21 . . . . . . . . . . . T A . . . . . . . . . . . . . . . . . . . . . . . . . . H22 . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . H23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . H24 . . . . . . . . . . . . . . . G . . . . . . . . A . . . . . . . . . . . . . . H25 . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . H26 T . . . . . . . . --. . . . . . . . . . . . . . . . . . . . . . . . . . . . H27 . . . . T G . . . --. . . . G . . . G . A . . A . . . . . C . . . . . . . A H28 T . . . T G . . . . . . . . . G . . . G . . . . A . . . . . C . . . . . . . A H29 . . . . . . . . . --. . . . . . . . . . . . . . A T . . . . . . . . . . . . H30 . . . . . . . . . --. . . . . . . . . . . . . . . G . . . . . . . . . . . . H31 . . . . T G . . . --. . . . G . . . . . . . T A . . . . . C . . . . . . . A H32 . . . . . . . . . --. . . . . . . . . . . . . . . . . . . . . . . . . . C . H33 . . . . T G . . . . . . . . . G . . . G . . . . A . . . . . C . . . G . . . A H34 . . . . T . . . . --. . . . . . . . . . . . . . . . . . . C . . . . . . . . H35 . . . . T G . . . . . T A . . G . . . . . . . . A . . G . . . . . . . . . . A H36 T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . H37 . . . . . . . . . --. . . C . . . . . . . . . . . . . . . . . . . . . . . . H38 . . . . . . . . . --. . . . . . . . . . . . . . . . . T . . . . . . . . . . H39 . . . . T . . . . --. . . . . . . . . . . . . . . . . . . . . . . . . . . . H40 . . . . . . C . . --. . . . . . . . . . . . . . . . . . . . . . . . . . . . H41 . . . . T G . . . . . . . . . G . . . . . . . . A . . G . . . . . . . . . . A H42 . . . . . . . . . --. . . . . . . . . . . . . A . . . . . . . . . . . . . .

Fig. 2

 2 Fig.2Minimum-spanning network displaying evolutionary relationships among the 42 Drupadia theda mtDNA haplotypes (see Table3for haplotype distribution). Each circle corresponds to one haplotype; each line between two haplotypes corresponds to one mutation (see Table2for reference). A small black circle indicates a missing, one-mutational step haplotype.

Fig. 3

 3 Fig.3Genetic diversity (presented as haplotype diversity, Hd, vs. nucleotide diversity, π) within Drupadia theda sampled populations.

Fig. 4

 4 Fig. 4 Relation between the observed haplotype diversity and the number of individuals of Drupadia theda recorded per transect and per day within each sample (Spearman's coefficient = 0.394, P = 0.260).

Table 2

 2 Sequence differences amongst the 42 haplotypes found in Drupadia theda populations. Dots indicate identical bases. The entire sequence of haplotype H1 was deposited in GenBank, Accession no. DQ350438

	mtDNA control region

Table 4

 4 Genetic diversity and results of neutrality tests in populations of Drupadia theda. N, number of individuals; Hd, haplotype diversity

  , the 95% confidence interval (CI) of simulated values of haplotype diversity based on the number of polymorphic sites under neutrality is reported, as well as the probability that Hd sim < Hd obs (see Material and methods). Significant departures from neutral expectations are indicated in bold Non-metric multidimensional scaling ordination of Drupadia theda populations based on Φ ST values estimated between samples using both haplotype frequencies and molecular divergence of mtDNA sequences. Samples from the same landscape are connected by a line. Dash lines encircle regions.

		N	H d	S	π	k	Tajima's D	95% CI Hd sim	P value
	I1-00	24	0.888	20	0.0072	6.638	-1.170	0.670-0.945	0.605
	I3-00	22	0.948	19	0.0141	7.147	1.373	0.666-0.943	0.976
	Gunung Meratus-00	46	0.923	22	0.0110	5.587	0.380	0.657-0.930	0.948
	B1-98	11	0.564	2	0.0012	0.618	-0.290	0.181-0.727	0.628
	I2-98	19	0.526	2	0.0011	0.561	-0.045	0.105-0.678	0.638
	Sungai Wain-98	30	0.524	3	0.0011	0.568	-0.589	0.186-0.720	0.484
	B1-00	24	0.703	12	0.0042	2.120	-1.168	0.539-0.905	0.155
	I2-00	20	0.468	2	0.0011	0.542	-0.090	0.100-0.678	0.467
	Sungai Wain-00	44	0.597	13	0.0028	1.416	-1.613	0.510-0.892	0.058
	B2-97	24	0.953	15	0.0092	4.678	0.327	0.550-0.927	0.999
	B2-99	15	0.810	12	0.0100	5.086	1.473	0.561-0.933	0.402
	B2-00	25	0.863	15	0.0083	4.187	0.189	0.576-0.926	0.598
	B2-04	22	0.788	10	0.0062	3.355	0.760	0.463-0.904	0.481
						Fig. 5

Table 5

 5 Pairwise Φ ST estimates between Drupadia theda populations (above the diagonal). Significance levels are indicated below the diagonal as *** if significant after adjustment of the significance level α = 0.05 according to the sequential Bonferroni correction (corresponding Φ ST values are indicated in bold), ** for P < 0.01, * for P < 0.05, and NS for P > 0.05

Table 6

 6 Results of amova for among-population differences and population structure in relation to the spatial (according to geographic regions) and temporal (according to landscapes) grouping. The analysis partitions total molecular variance into different components, whose significance was obtained by randomization after 10 000 permutations of the mtDNA control region in D. theda suggest that this molecular marker possesses a sufficient amount of polymorphism to conduct population genetic surveys at the spatial scales studied, in line with the results of the[START_REF] Vila | The utility of the neglected mitochondrial control region for evolutionary studies in Lepidoptera (Insecta)[END_REF] study on various species of Lepidoptera.

			Gunung Meratus	Sungai Wain			Wanariset
			I1-00	I3-00	B1-98	B1-00	I2-98	I2-00	B2-97	B2-99	B2-00	B2-04
	G.	I1-00		0.092	0.604	0.532	0.643	0.653	0.118	0.219	0.294	0.089
	Meratus	I3-00	*		0.370	0.341	0.423	0.435	0.109	0.161	0.173	0.158
	S. Wain	B1-98	***	***		-0.015	-0.052	-0.053	0.413	0.317	0.226	0.602
		B1-00	***	***	NS		0.003	0.015	0.334	0.255	0.148	0.513
		I2-98	***	***	NS	NS		-0.043	0.458	0.387	0.259	0.642
		I2-00	***	***	NS	NS	NS		0.470	0.401	0.275	0.654
	Wanariset	B2-97	**	**	***	***	***	***		0.102	0.108	0.045
		B2-99	***	**	***	***	***	***	*		0.075	0.126
		B2-00	***	***	**	**	***	***	*			0.222
		B2-04	***	***	***	***	***	***	NS	*	***
									% of variance
	Population structure tested			d.f.	Variance components	explained	P value
	2000 only, 5 landscapes (I1, I2, I3, B1, B2), no grouping
	Among populations			4	Va		0.874	32.63		< 0.0001
	Within populations			110	Vb		1.788	67.17		-
	2000 only, 5 landscapes (I1, I2, I3, B1, B2), 3 groups (Gunung Meratus, Sungai Wain, Wanariset)
	Among groups			2	Va		0.909	32.06		0.063
	Among populations within groups		2	Vb		0.138	4.88		0.017
	Within populations			110	Vc		1.788	63.06		< 0.0001
	Temporal: 3 landscapes (I2, B1, B2), 3 groups (Gunung Meratus, Sungai Wain, Wanariset)
	Among groups			2	Va		0.609	27.57		0.023
	Among populations within groups		6	Vb		0.212	9.59		0.001
	Within populations			153	Vc		1.389	62.84		< 0.0001

variability

© 2006 Blackwell Publishing Ltd, Molecular Ecology , 15, 2069-2081

© 2006 Blackwell Publishing Ltd, Molecular Ecology, 15, 2069-2081

C . F A U V E L O T , D . F . R . C L E A R Y and S . B . J . M E N K E N © 2006 Blackwell Publishing Ltd, Molecular Ecology, 15, 2069-2081

C . F A U V E L O T , D . F . R . C L E A R Y and S . B . J . M E N K E N © 2006 Blackwell Publishing Ltd, Molecular Ecology, 15, 2069-2081

Acknowledgements

All field assistants in Kalimantan are thanked for their help. We further thank W. van Ginkel for sorting all the materials and P. Kuperus, B. Voetdijk, and Y. Jimenez for laboratory assistance. We thank M. Vila for her valuable advice concerning PCR amplifications and J.A.J. Breeuwer, G. Lecaillon, and K.T.C.A. Peijnenburg who contributed to this work through insightful discussions. S. Planes, B. Emerson, and four anonymous reviewers provided helpful comments on earlier versions of the manuscript. This work was supported by grant 895.100.029 of the Netherlands Foundation for the Advancement of Tropical Research (NWO-WOTRO), within the Priority Programme 'Biodiversity in Disturbed Ecosystems'.

represent postdisturbance immigration or rare haplotypes that were not encountered in B2-97.

Tajima's D neutrality test revealed no significant deviation from neutral expectations in any sample (Table 4, all P > 0.05). H test revealed significantly higher values of haplotype diversity in I3-00, and in B2-97 compared to those expected under the neutral coalescent process (Table 4). The analysis of sequence polymorphism using both Tajima's D test and Depaulis & Veuille's H test did not reveal a significant departure from neutral expectations in any of the disturbed landscapes after the 1997/1998 ENSO-induced drought and fires.


Ce ´cile Fauvelot
email: cecile.fauvelot@ird.fr

Federica Costantini

• Massimiliano

Marco Abbiati

T C Reusch

Á F Fauvelot

Á M Costantini

Abbiati

C Fauvelot

M Virgilio

Do artificial structures alter marine invertebrate genetic makeup?

Human-made structures are increasingly built in marine coastal habitats for a variety of purposes. Offshore oil and gas production platforms are among the largest examples. Yet, biological effects of these increasing density artificial substrata are under evaluated. The objective of our study is to investigate the possible role of offshore platforms in modifying the genetic composition of populations of natural rocky shores species. The serpulid Pomatoceros triqueter was used as a model, and genetic variation was assessed using a 419 bp fragment of the mtDNA COI gene in samples collected on eleven offshore gas platforms, on one coastal buoy on the sandy shore and in four sites located on natural rocky shores in the Adriatic Sea. Deep phylogenetic lineages were uncovered over all samples. Nucleotide diversity and mean number of pairwise differences among haplotypes were significantly smaller in offshore platform samples compared to rocky shores samples. No significant genetic structure was observed over all samples. We found direct evidence of lower genetic diversity on platforms confirming that, although artificial structures attract and support species typical of hard bottoms, they are not analogues of natural rocky habitats.

Introduction

Disturbances related to human activities (e.g. habitat loss and fragmentation, global environmental change, overexploitation and other effects due to fishing, pollution and tourism) are worldwide recognized as a major threat to marine ecosystems [START_REF] Lotze | Depletion, degradation, and recovery potential of estuaries and coastal seas[END_REF][START_REF] Worm | Impacts of biodiversity loss on ocean ecosystem services[END_REF][START_REF] Airoldi | Loss, status and trends for coastal marine habitats of Europe[END_REF]. Among recognized anthropogenic pressures, the impact of artificial structures introduction has received increased attention in the recent decades (see Bulleri and Chapman 2010 for a review).

Artificial hard structures are most often built in sandy areas that, as a result, generate unnatural changes in species composition, abundance and diversity of native soft-bottom assemblages [START_REF] Connell | Urban structures as marine habitats: an experimental comparison of the composition and abundance of subtidal epibiota among pilings, pontoons and rocky reefs[END_REF][START_REF] Martin | Ecological impact of coastal defence structures on sediment and mobile fauna: evaluating and forecasting consequences of unavoidable modifications of native habitats[END_REF][START_REF] Airoldi | Impact of recreational harvesting on assemblages in artificial rocky habitats[END_REF][START_REF] Bulleri | Role of recruitment in causing differences between intertidal assemblages on seawalls and rocky shores[END_REF][START_REF] Moschella | Low-crested coastal defense structures as artificial habitats for marine life: using ecological criteria in design[END_REF][START_REF] Bulleri | The introduction of coastal infrastructure as a driver of change in marine environments[END_REF]. Besides these modifications at the community and species level, artificial structures also play a role at the population level, providing new colonizing substrata [START_REF] Page | Exotic invertebrate species on offshore oil platforms[END_REF][START_REF] Fauvelot | Lower genetic diversity in the limpet Patella caerulea on urban coastal structures compared to natural rocky habitats[END_REF]. Indeed, genetic diversity on these artificial structures may be inclined to deep changes, as recently observed for the limpet Patella caerulea collected on breakwaters [START_REF] Fauvelot | Lower genetic diversity in the limpet Patella caerulea on urban coastal structures compared to natural rocky habitats[END_REF]). Since genetic diversity can affect the species productivity, population growth and stability, as well as inter-specific interaction within community, and ecosystem-level processes [START_REF] Hughes | Ecological consequences of genetic diversity[END_REF]), this parameter is crucial for species survival in highly variable environments or those subject to rapid anthropogenic changes [START_REF] Reusch | Ecosystem recovery after climatic extremes enhanced by genotypic diversity[END_REF]. In addition, since all benthic organisms directly associated with artificial structures rely on a pelagic larval phase to disperse and colonize new habitats, artificial structures may enhance connectivity among differentially adapted populations, (i.e. artificial structures may serve as ''stepping stones'' for hard-bottom species) which may ultimately lead to negative effects of biogenic homogenization (see [START_REF] Olden | Ecological and evolutionary consequences of biotic homogenization[END_REF] for a review).

Offshore oil and gas production platforms are among the largest artificial structures in the marine environment. They are associated with several environmental issues, related to their installation, daily shipping movements, extraction activities, maintenance and final decommissioning [START_REF] Grant | Toxicity of sediments from around a North Sea oil platform: are metals or hydrocarbons responsible for ecological impacts?[END_REF][START_REF] Fabi | Evolution of the fish assemblage around a gas platform in the northern Adriatic Sea[END_REF][START_REF] Page | Effects of an offshore oil platform on the distribution and abundance of commercially important crab species[END_REF][START_REF] Page | Exotic invertebrate species on offshore oil platforms[END_REF][START_REF] Bomkamp | Role of food subsidies and habitat structure in influencing benthic communities of shell mounds at sites of existing and former offshore oil platforms[END_REF][START_REF] Terlizzi | Effects of offshore platforms on soft-bottom macro-benthic assemblages: a case study in a Mediterranean gas field[END_REF]). The north Adriatic Sea is one of the major fields for extraction of natural gas in the Mediterranean basin [START_REF] Teatini | Water-gas dynamics and coastal land subsidence over Chioggia Mare field, northern Adriatic Sea[END_REF]. Since 1950, more than 100 platforms (supplying over 70 % of the total Mediterranean gas production) have been built over an area of 6,400 km 2 . These structures constitute most of the hard substrate in this sandy region and provide suitable habitat for the development of massive mussel (Mytilus galloprovincialis) beds, which are commercially exploited on permit from the petroleum companies [START_REF] Fabi | Evolution of the fish assemblage around a gas platform in the northern Adriatic Sea[END_REF].

Despite the extent of these offshore activities in the North Adriatic Sea, few ecological impact studies have been carried out, and these have focussed on possible changes in the physiology, composition and structure of benthic and pelagic assemblages associated with the presence of offshore platforms [START_REF] Relini | The macrofouling on offshore platforms at Ravenna[END_REF][START_REF] Crema | Effect of submerged structures on the diversity of macrozoobenthos in the northern Adriatic Sea[END_REF][START_REF] Ponti | Drilling platforms as artificial reefs: distribution of macrobenthic assemblages of the ''Paguro'' wreck (northern Adriatic Sea)[END_REF][START_REF] Fabi | Effects on fish community induced by installation of two gas platforms in the Adriatic Sea[END_REF][START_REF] Consoli | Fish diversity associated with gas platforms: evaluation of two underwater visual census techniques[END_REF][START_REF] Gorbi | An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic sea[END_REF][START_REF] Gorbi | Biological effects of diethylene glycol (DEG) and produced waters (PWs) released from offshore activities: a multi-biomarker approach with the sea bass Dicentrarchus labrax[END_REF]. However, so far, no study has investigated the genetic consequences of the platforms colonization on populations of natural rocky shores species. The goal of our study was to examine the potential consequences of the presence of offshore platforms on the genetic diversity and structure of natural rocky shore organisms. During a preliminary survey conducted in July 2005, benthic organisms have been collected and identified from three offshore platforms in order to select several target species as model organisms. The primary objectives of this survey were to identify the fauna that could be sampled on the legs of the offshore platforms, and from these, select the ones that could be sampled in sufficient numbers to conduct reliable analysis. Among macrobenthic organisms, we found a majority of mussels (Mytilus galloprovincialis), serpulids (Pomatoceros triqueter and in lower densities Serpula vermicularis), oysters (Ostrea edulis and Crassostrea gigas), and in lower densities barnacles, brittle stars and errant polychaetes (Nereididae). A detailed description of the platform fauna at Ravenna can be found in [START_REF] Relini | The macrofouling on offshore platforms at Ravenna[END_REF] and in [START_REF] Ponti | Drilling platforms as artificial reefs: distribution of macrobenthic assemblages of the ''Paguro'' wreck (northern Adriatic Sea)[END_REF]. Due to health and safety regulations, we were not authorized to sample benthic organisms from the platforms ourselves, and we had to rely on what was sampled by the professional divers harvesting mussels on the legs of platforms. We chose to discard Mytilus galloprovincialis as a target species to avoid a possible harvesting impact on genetic diversity, and thus focused our sampling effort and genetic analyses on the second most common species on the platforms, Pomatoceros triqueter (Linnaeus, 1767), a benthic worm typical of the fouling community [START_REF] Crisp | Factors influencing the settlement of marine invertebrate larvae[END_REF].

The serpulid genus Pomatoceros are sessile, tubicolous and filter-feeders, occurring on a variety of substrata at depths ranging from low water intertidal into the shallow benthic, with a wide geographical distribution from cold temperate latitudes to the equator in the northern hemisphere [START_REF] Crisp | Polymorphism in Pomatoceros[END_REF]). Pomatoceros spp. have feeding, planktonic larvae [START_REF] Kupriyanova | Live history evolution in Serpulimorph polychaetes: a phylogenetic analysis[END_REF] and are capable of reproducing throughout the year [START_REF] Castric-Fey | Contribution a `l'e ´tude de la sexualite ´chez Pomatoceros lamarckii et Pomatoceros triqueter en baie de Concarneau (Sud Finiste `re)[END_REF]. The larval duration of Pomatoceros triqueter varies between 15 days and 4 weeks, depending on the temperature, as for its sister species Pomatoceros lamarckii [START_REF] Castric-Fey | Contribution a `l'e ´tude de la sexualite ´chez Pomatoceros lamarckii et Pomatoceros triqueter en baie de Concarneau (Sud Finiste `re)[END_REF]Hayward and Ryland 1995;[START_REF] Cotter | Recruitment patterns of serpulids (Annelida: Polychaeta) in Bantry Bay, Ireland[END_REF]. P. triqueter and P. lamarckii (co-occurring in the Mediterranean Sea, though not encountered on the platforms, Ponti et al. 2002, personal observations) can be distinguished morphologically by differences in their operculum structure; in P. triqueter, the operculum is convex, cone-shaped and obliquely mounted on the peduncle, whereas in P. lamarckii, the operculum is concave, cup-shaped and mounted centrally on its stalk [START_REF] Zibrowius | Etude morphologique, syste ´matique et e ´cologique des serpulidae (Annelida, Polychaeta) de la re ´gion de Marseille[END_REF].

In this study, we investigated the genetic diversity and structure of Pomatoceros triqueter collected on eleven offshore gas platforms, on one costal buoy and in four sites located on natural rocky shores in the Adriatic Sea. We specifically tested (a) whether the genetic diversity of populations on platforms was reduced compared to populations on natural rocky shores, as could be expected from the recent founding of the platforms, or alternatively enhanced if the platform populations act as reservoir of genetic diversity and (b) for differences in the genetic structuring of populations between platform and natural shores.

Materials and methods

Sampling

The sampling was conducted in the Adriatic Sea, on natural rocky shores, and more intensively on gas platforms. Sampling was conducted between May and August 2006. Platforms have been selected according to their location (in order to cover the range of platforms inter-distances) and the possibility to sample with vessels. Sampling was completed by the use of the vessels from the Cooperative Society 'La Romagnola' which conducted daily sampling on the platforms for mussels harvesting. In total, ten effective platforms have been sampled (Agostino-A, Amelia-D, Anemone-B, Angelina, Antonella, Armida, Cervia-K, Garibaldi-C, Porto Corsini Est-80bis and Porto Corsini C), as well as the wreck platform Paguro (Fig. 1). For each platform, mussel beds collected by the professional divers were brought to the laboratory for sorting, and Pomatoceros triqueter tubes were detached from mussel shells.

Along the west shoreline of the Adriatic Sea, natural hard-bottom habitats, represented by isolated rocky promontories, interrupt large sandy coasts extending between the Friuli-Venezia Giulia and Puglia regions (NE and SE of Italy, respectively). The largest of these rocky promontories are Sistiana/Miramare (Friuli-Venezia Giulia), Gabicce (Emilia-Romagna), Conero (Marche) and Gargano (Puglia). Sampling along the Adriatic coasts was conducted along the sea shore by SCUBA diving in Trieste (Miramare), Tegnue di Chioggia, Ancona (Conero), Brindisi, Taranto and in Zaglav (on the Croatian island of Dugi Otok) where Pomatoceros triqueter were found on rocks (Fig. 1), except in Tegnue di Chioggia where P. triqueter were found on buoys delimiting the marine protected area. In Ancona, only Pomatoceros lamarckii was encountered and sampled. In Brindisi, the sampling consists mostly of P. lamarckii so that only few P. triqueter could be analysed.

Molecular analysis

Immediately after sampling, alive individual worms were placed at 4 °C for at least 3 h in order to facilitate the extraction of the worm body from its calcareous tube without damaging the body. Each collected worm was examined alive under binocular microscope for species identification and operculum shape notification. Each worm was then stored in individual Eppendorf tubes in 80 % EtOH and kept at -20 °C.

Genomic DNA was isolated from the caudal extremity of 11-29 individuals per site, using a cetyltrimethyl ammonium bromide (CTAB) protocol [START_REF] Hillis | Molecular systematics. Sinauer, Sunderland[END_REF]. Specifically, each tissue sample was digested for 2 h at 65 °C in 400 ll of 2X CTAB solution containing 3 U of Proteinase K. Genomic DNA was extracted using Chloroform-Isoamyl Alcohol (24:1) and precipitated in 100 % EtOH.

Like all non-model organisms, there were no markers available in the literature for Pomatoceros triqueter. We choose to test several universal primers amplifying mtDNA genes due to the development cost of microsatellites. Three genes could successfully be amplified in P. triqueter: COI, 12S and 16S (details available on request). We chose to conduct further genetic analysis using COI since this marker happened to be useful in revealing the genetic structure in two polychaete tubeworms [START_REF] Jolly | Comparative phylogeography of two coastal polychaete tubeworms in the Northeast Atlantic supports shared history and vicariant events[END_REF]. Furthermore, the COI gene is more polymorphic than the 16S and 12S genes in annelids, both conserved within species [START_REF] Jolly | Comparative phylogeography of two coastal polychaete tubeworms in the Northeast Atlantic supports shared history and vicariant events[END_REF][START_REF] Halanych | A review of molecular markers used for Annelid phylogenetics[END_REF]. A 489-bp fragment of the mitochondrial DNA cytochrome oxidase I gene was amplified using the primers PtCOI-F (5 0 -GCT TGA GCC GGA ACC TGT GG-3 0 ) and PtCOI-R (5 0 -CCC CCA GCT AAT ACA GGA AC-3 0 ), which were specifically designed for P. triqueter based on several sequences obtained using LCO1490 and HCO2198 [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF]. Each 25 ll PCR amplification contained 1X PCR buffer, 2 mM MgCl 2 , 0.08 mM of each dNTP, 3 lg of BSA (Bovine Serum Albumin), 0.2 lM of each PtCOI-F and PtCOI-R primer, 0.25 U of Taq polymerase and about 30 ng DNA. Amplified fragments were purified using the ExoSAP-IT kit (USB) following the manufacturer recommendations and cycle sequenced in both directions using Big Dye Terminator sequencing kit (Applied Biosystems). Sequences were run on an ABI 310 Genetic Analyser (Applied Biosystems). Forward and reverse sequences were compared for about 10 % of the individuals in BioEdit [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. Then, since forward and reverse sequences gave identical results, further sequences were obtained using only the forward primer (PtCOI-F). Individual sequences were then aligned by eye in GeneDoc [START_REF] Nicholas | GeneDoc: a tool for editing and annotating multiple sequence alignments[END_REF].

Data analysis

Haplotypes were identified, and their relative frequency within samples estimated using Arlequin version 3.11 [START_REF] Excoffier | Arlequin ver. 3.0: an integrated software package for population genetics data analysis[END_REF]. Genetic diversity within populations was estimated as haplotype diversity (Hd, Nei 1987), nucleotide diversity (p, [START_REF] Nei | Molecular evolutionary genetics[END_REF]) and mean number of pairwise nucleotide differences between individuals (k, Tajima 1983) using DnaSP 4.0 [START_REF] Librado | DnaSP v5: a software for comprehensive analysis of DNA polymorphism data[END_REF]. Significant differences in genetic diversity (Hd, p and k) between natural versus artificial habitats were tested using a Wilcoxon test. Phylogenetic relationships among haplotypes were constructed using Bayesian analyses implemented in MrBayes 3.1 (Huelsenbeck and Ronquist 2001) and employed a cold chain and three incrementally heated chains with T = 0.1. Starting trees for each chain were random, and the default values of MrBayes were chosen for all settings (including prior distributions). Each metropolis-coupled Markov chain Monte Carlo (MCMC) was run for 30 million generations, with trees sampled every 5,000 generations, and discarded the first 20 million generations (4,000 trees). By this time, the chains had converged to stable likelihood values\0.01. Posterior probabilities (PP) were used to assess clade supports. Analyses were run using the evolutionary model selected by the Akaike information criterion of MrModeltest (Nylander 2004). Pomatoceros larmarkii was used as outgroup: two specimens were sampled in Ancona (Marche, Italy, Adriatic Sea), and sequences were obtained using primers LCO1490 and HCO2198 as described above. Genetic divergences between supported monophyletic clades were estimated in Mega version 4 [START_REF] Tamura | MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0[END_REF]) using the mean net nucleotide divergence [START_REF] Nei | Mathematical model for studying genetic variation in terms of restriction endonucleases[END_REF], defined as d xy -0.5 (d x ? d y ), that subtracts the average 'within-group' divergence from the observed 'between-group' estimate and p-distance model.

A minimum spanning network (MSN) displaying evolutionary relationships between mtCOI haplotypes was constructed in TCS version 1.13 [START_REF] Clement | TCS: a computer program to estimate gene genealogies[END_REF] to infer the most parsimonious branch connections at the 95 % confidence level between haplotype pairs. The MSN was then redrawn by hand in Adobe Ò Illustrator Ò 9.0 (Adobe Systems Incorporated).

Data analyses of spatial genetic structure among populations were performed using Arlequin. Genetic divergences between pairwise samples were estimated using U-Statistics (U ST based on haplotype frequencies and molecular divergence using pairwise differences). P values were obtained using a non-parametric permutation procedure with 10,000 permutations. In order to examine the partition of the genetic variance among samples based on the type of habitat or geographical locations, analyses of molecular variance (AMOVA) [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data[END_REF] were conducted in Arlequin.

Results

The amplification of a fragment of the mtDNA cytochrome oxidase I gene in a total of 350 individuals of Pomatoceros triqueter from 16 geographical samples resulted in fragments of 419 bp that could unambiguously be aligned and analysed. Among the 350 individuals sequenced, we identified 160 different haplotypes (GenBank accession numbers JX308620-JX308779), distinguished by 136 polymorphic sites and a total number of 162 mutations (i.e. some nucleotide positions carried more than two allelic states). Among the 16 sample sites, the number of haplotypes per sample ranged between 11 and 21 haplotypes. From the 160 observed haplotypes, only 21 were found in at least two individuals, and the 139 remaining haplotypes were singletons (i.e. haplotypes carried by only one individual, Table 1).

Over all the 16 samples, haplotype diversity ranged between 0.89 and 1, nucleotide diversity between 0.020 and 0.048 and mean number of pairwise differences between 9.05 and 20.16. Excluding TEG for the comparisons (as it was neither a platform or a rocky shore sample), all three genetic diversity estimates were significantly different among samples when comparing offshore platform against rocky shores samples (mean Hd platforms = 0.942 ± 0.023, mean Hd natural = 0.982 ± 0.019, P = 0.006; mean p platforms = 0.0245 ± 0.002, mean p natural = 0.0364 ± 0.010, P = 0.045; mean k platforms = 10.27 ± 0.88, mean k natural = 15.24 ± 4.05, P = 0.045, Fig. 2).

The Bayesian tree (Fig. 3) revealed two highly supported and reciprocally monophyletic clades (both with 100 % posterior probability). Clade 2 was only represented by two haplotypes that were found in Taranto (H125) and Trieste (H144). Clade 1 included a number of supported subclades (PP [ 95 %) as well as paraphyletic haplotypes. The mean net divergence between the two main P. triqueter clades was 12.40 ± 1.46 % and 20.92 ± 1.73 % between P. triqueter and P. lamarkii.

The minimum spanning network (MSN) showing the most parsimonious branch connections at the 95 % level also revealed the presence of two networks (Fig. 4) that could not be connected since haplotypes 125 and 144 were too divergent from the rest of the haplotypes (69 substitutions out of 419 bp). The bigger network showed a partition in two groups that were connected by haplotypes exclusively found in Taranto (H127, H128 and H129). Apart from these haplotypes, the two sub-networks were connected by a minimum of 13 mutation steps. The haplotypes found within the sub-networks were not specific of any geographical location, and all three sub-networks were No significant genetic structure was observed overall samples (U ST = 0.0177, P = 0.073), with 1.77 % of the total variance being found among the 16 samples. The pairwise U ST estimates among samples ranged from 0 to 0.223 (P = 0.004) between Tegnue di Chioggia and Garibaldi platform, but none of the pairwise comparisons were significant after adjustment of the significance level a = 0.05 according to the sequential Bonferroni correction (data not shown). The AMOVAs conducted revealed no significant partition of the variance when considering offshore platforms versus natural substrates or north versus south Adriatic samples (P [ 0.05, data not shown).

H 3 2 3 4 1 2 1 3 3 2 2 3 1 2 2 3 1 H 4 1 1 1 2 1 3 1 1 3 1 1 1 1 7 H 5 1 1 1 1 1 2 1 1 4 1 3 H 6 1 1 1 1 1 1 6 H7 1 2 1 1 1 6 H8 1 1 1 1 4 H9 1 2 1 4 H10 1 1 1 1 4 H11 1 1 1 3 H12 1 1 1 3 H13 1 1 2 H14 1 1 2 H15 1 1 2 H16 1 1 2 H17 1 1 2 H18 1 1 2 H19 1 1 2 H20 1 1 2 H21 1 1 2 H22-H28 7

Discussion

Deep mitochondrial phylogeny or evidence of cryptic species?

The genetic divergence observed between Pomatoceros triqueter and its closely related species Pomatoceros lamarckii, also present in the Adriatic Sea, was about 20 %. These two species are well differentiated on the basis of the operculum morphology [START_REF] Zibrowius | Etude morphologique, syste ´matique et e ´cologique des serpulidae (Annelida, Polychaeta) de la re ´gion de Marseille[END_REF]), their karyotype [START_REF] Dixon | Karyotypic differences between two species of Pomatoceros, P. triqueter and P. lamarckii (Polychaeta: Serpulidae)[END_REF]) and genetics [START_REF] Ekaratne | Separation of two Atlantic species of Pomatoceros, P. triqueter and P. lamarckii (Annelida: Serpulidae) by means of biochemical genetics[END_REF][START_REF] Crisp | Polymorphism in Pomatoceros[END_REF]. The genetic analyses conducted on a fragment of the mtDNA COI gene revealed the presence of two deep (12 % divergent) monophyletic clades in P. triqueter within the Adriatic Sea. No morphological differences were observed between the individuals belonging to the two deep monophyletic clades. Shape differences of the operculum were observed among all 350 individuals but fell in those described in the literature [START_REF] Zibrowius | Etude morphologique, syste ´matique et e ´cologique des serpulidae (Annelida, Polychaeta) de la re ´gion de Marseille[END_REF][START_REF] Dixon | Karyotypic differences between two species of Pomatoceros, P. triqueter and P. lamarckii (Polychaeta: Serpulidae)[END_REF] and were not related to specific clades. The sympatric occurrence of highly divergent mitochondrial clades has already been observed in other polychaete tubeworms in European seas, with similarly high net genetic divergences among clades (16.4 % divergence between two sympatric clades of Pectinaria koreni and 16 % divergence between two sympatric clades of Owenia fusiformis; [START_REF] Jolly | Comparative phylogeography of two coastal polychaete tubeworms in the Northeast Atlantic supports shared history and vicariant events[END_REF]. Therefore, we considered the two uncovered monophyletic clades as belonging to Pomatoceros triqueter and thus rejected the occurrence of a possible cryptic species in our data set.

The phylogenetic analysis also recovered divergent groups of haplotypes, which formed a well-supported clade (Bayesian analysis: PP = 100 %, mean of 12 % genetic divergence, two subnetworks in the MSN). The occurrence of divergent groups of sequences within Pomatoceros triqueter may be attributed to different glacial relicts, resulting from geographical isolation of P. triqueter populations during lowered sea levels followed by a recent colonization from different ancestral populations located in the Mediterranean Sea, as observed for numerous marine organisms [START_REF] Lee | Global phylogeography of a cryptic copepod species complex and reproductive isolation between genetically proximate ''populations[END_REF][START_REF] Schroth | Speciation and phylogeography in the cosmopolitan marine moon jelly, Aurelia sp[END_REF][START_REF] Goetze | Cryptic speciation on the high seas; global phylogenetics of the copepod family Eucalanidae[END_REF][START_REF] Peijnenburg | Phylogeography of the planktonic chaetognath Sagitta setosa reveals isolation in European seas[END_REF]). Investigating the phylogeographic patterns of P. triqueter at the European scale may permit identifying the locations from which relic haplotypes arose.

May the introduction of platforms enhanced genetic connectivity?

The absence of significant pairwise comparisons and significant structure among various groups of samples may have several explanations. The first one is that the mtCOI gene may not be the best molecular marker to reveal potential genetic structure among Pomatoceros triqueter samples due to a high number of singletons and low number of shared haplotypes. The way to test for this hypothesis would be to use additional polymorphic nuclear markers such as microsatellites loci in order to test for Haplotypes are coded according to Table 1 significant differences among samples. However, we believe that if there would have been a significant partition of the genetic variation, mtDNA COI haplotypes would not appear randomly spatially distributed. A second explanation for the lack of significant structuring of the samples would be that Pomatoceros triqueter populations were genetically connected prior the introduction of the platforms. This could be the result of a rather long larval duration of P. triqueter (about 3 weeks), which would allow sufficient gene flow among samples to homogenize genetic structure within the Adriatic Sea. Similar patterns have been observed (using more fast evolving nuclear markers) in the same area for the limpet Patella caerulea [START_REF] Fauvelot | Lower genetic diversity in the limpet Patella caerulea on urban coastal structures compared to natural rocky habitats[END_REF]), a species inhabiting natural rocky shores as well as artificial substrates along the shore (e.g. breakwaters). Alternatively, the observed genetic structure at the mtCOI may depict the footprint of the recent colonization of the Adriatic Sea after the Last Glacial Maximum (LGM). Indeed, during the LGM (about 18,000 ya), the sea level was about 100 m below the actual mean water level, and most of the Adriatic Sea bed was dried [START_REF] Dondi | Recent geological evolution of the Adriatic Sea[END_REF][START_REF] Thiede | A glacial mediterranean[END_REF]. The sea water invaded the Adriatic during the last 10,000 years, and the colonization by the marine flora and fauna is very recent. Genetic similarities in Adriatic samples of P. triqueter may reflect past founder effects linked with the colonization of the Adriatic Sea after the Pleistocene glaciation. Indeed, several studies have recently stressed the relevance of palaeoecological events in determining the genetic patterns in marine populations (e.g. [START_REF] Fauvelot | Reductions in the mitochondrial DNA diversity of coral reef fish provide evidence of population bottlenecks resulting from Holocene sea-level change[END_REF][START_REF] Wilson | Genetic signature of recent glaciations on populations of a near-shore marine fish species (Syngnathus leptorhynchus)[END_REF][START_REF] Jevreyb | Pleistocene isolation and recent gene flow in Haliotis asinina, an Indo-Pacific vetigastropod with limited dispersal capacity[END_REF][START_REF] Virgilio | Deep phylogenetic splits and disjunct haplotype distribution suggest cryptic speciation in Hediste diversicolor (Polychaeta: Nereididae)[END_REF]. Interestingly, intermediate haplotypes between the two observed sub-networks are only located at the Taranto locality, suggesting that this part of Italy may have been a refuge for the Adriatic fauna during lowered sea levels.

A last explanation would be that platforms act as stepping stones for hard-bottom species, and thus the introduction of platforms has created corridors among former discrete natural populations. Because we did not have samples from natural rocky shores prior the introduction of all artificial structures nowadays found in the Adriatic Sea, it is impossible to conclude that artificial structures are responsible for the genetic connectivity currently observed for both Pomatoceros triqueter and Patella caerulea [START_REF] Fauvelot | Lower genetic diversity in the limpet Patella caerulea on urban coastal structures compared to natural rocky habitats[END_REF]. A possible way to test for genetic connectivity increase due to the platforms would be to conduct a study on P. triqueter genetic structure in an area where only discrete populations occur on natural shores Circles represent haplotypes with their corresponding number as in Table 1, while small black circles represent missing haplotypes within the network (i.e. haplotypes not encountered within the samples). Each line linking two circles represents one base pair substitution along the 419-bp length-analysed sequence. The locations of the haplotypes are indicated with color code as in the legend (color figure online) and where no artificial structures may connect them, in order to estimate the dispersal kernel of P. triqueter in none impacted environments. Unfortunately, such areas likely do not exist anymore [START_REF] Airoldi | Loss, status and trends for coastal marine habitats of Europe[END_REF]. Do the platforms affect the genetic diversity of P. triqueter? Significantly smaller genetic diversity was observed in Pomatoceros triqueter samples located on the platforms as compared to natural rocky shore samples. Lower genetic diversity levels on artificial urban structures as compared to natural rocky shores have already been observed in another invertebrate sampled in the same area, Patella caerulea [START_REF] Fauvelot | Lower genetic diversity in the limpet Patella caerulea on urban coastal structures compared to natural rocky habitats[END_REF]. It has been evidenced that artificial structures support assemblages that differ significantly in composition, structure, reproductive output, patterns of recruitment and population dynamics from assemblages on nearby natural rocky habitats (see Bulleri and Chapman 2010 for a review) suggesting important functional and ecological differences between artificial and natural habitats. At the population level, the propagule pressure [START_REF] Lockwood | The role of propagule pressure in explaining species invasions[END_REF]) through small inoculum size (i.e. the number of viable settlers) creates a filter from the amount of genetic diversity found in surrounding natural populations, further causing genetic diversity to decrease while maintaining genetic homogeneity between artificial and natural populations [START_REF] Roman | Paradox lost: genetic diversity and the success of aquatic invasions[END_REF]. Our results support this hypothesis.

Lower levels of haplotype diversity have also already been found on meiobenthic harpaticoids (Copepoda) populations living near platforms (\50 m) as compared to distant populations ([3 km) in the Gulf of Mexico [START_REF] Street | Loss of genetic diversity in Harpacticoida near offshore platforms[END_REF]. In this study, the low genetic diversity in samples near platforms was likely attributed to high concentrations of potentially toxic substances (hydrocarbons and trace metals) in the sand content, but no alternative hypothesis was proposed. Reductions in genetic diversity of Pomatoceros triqueter populations on platforms could similarly be attributed to extraction activities (i.e. pollution, [START_REF] Gorbi | An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic sea[END_REF], and/or periodical removal of mussel beds (for commercial mussel exploitation and cleaning of fouling) on which P. triqueter grow, creating local bottlenecks [START_REF] Ekaratne | Separation of two Atlantic species of Pomatoceros, P. triqueter and P. lamarckii (Annelida: Serpulidae) by means of biochemical genetics[END_REF]. Eventually, the lower genetic diversity on artificial substrata as compared to natural ones may not be a result of a single factor but could likely be the consequence of a combination of these various processes.

Conclusion

All together, our results suggest that the phylogeographic patterns observed for Pomatoceros triqueter in the Adriatic Sea based on mtCOI gene likely reflect the interaction between historical events (associated with Pleistocene sea level changes) and contemporary gene flow. The samples collected on platforms showed lower genetic diversity than those collected on the natural rocky shores. Our results contribute to the growing body of evidence showing that although artificial structures attract and support species typical of hard bottoms, they are not analogues of natural rocky habitats (see among others [START_REF] Bulleri | Role of recruitment in causing differences between intertidal assemblages on seawalls and rocky shores[END_REF][START_REF] Glasby | Urban structures as marine habitats[END_REF][START_REF] Moreira | Seawalls do not sustain viable populations of limpets[END_REF][START_REF] Moschella | Low-crested coastal defense structures as artificial habitats for marine life: using ecological criteria in design[END_REF][START_REF] Fauvelot | Lower genetic diversity in the limpet Patella caerulea on urban coastal structures compared to natural rocky habitats[END_REF]). Our study calls for additional investigations related to the local functional and ecological processes that act on populations in a fragile marine ecosystem increasingly impacted by anthropogenic activities.

Fig. 1

 1 Fig. 1 Location of sampling sites (A) along the coasts of Italy (Tegnue di Chioggia, Trieste, Brindisi, Taranto) and Croatia (Zaglav on the island of Dugi Otok) and (B) from eleven offshore platforms off the coast of Ravenna (AGO Agostino-A, AME Amelia-D, ANE Anemone-B, ANG Angelina, ANT Antonella, ARM Armida, CER Cervia-K, GAR Garibaldi-C, PC80 Porto Corsini Est-80bis, PCC Porto Corsini C, PAG Paguro)

Fig. 2 Fig. 3

 23 Fig.2Average haplotype diversity, nucleotide diversity and mean number of pairwise differences among mtCOI sequences of Pomatoceros triqueter over all platform samples (P) and natural rocky shore samples (N). *P \ 0.05 (Wilcoxon test)

Fig. 4

 4 Fig. 4 Minimum spanning network illustrating the phylogenic relationships among the 160 mtCOI haplotypes of Pomatoceros triqueter.Circles represent haplotypes with their corresponding number as in Table1, while small black circles represent missing haplotypes within

Table 1

 1 Spatial distribution of the Pomatoceros triqueter mtCOI haplotypes and genetic diversity per sample

		AGO AME ANE ANG ANT ARM CER GAR PAG PC80 PCC BRI	TAR TEG TRI	ZAG Total
	H 1	5	4	3	5	3	6	2	4	4	5	1	1	2	6	2	5	5 8
	H 2	2	4	2	6	4	2	5	6	3	1	4		1	2		2	4 4

  BRI Brindisi, TAR Taranto, TEG Tegnue di Chioggia, TRI Trieste and ZAG Zaglav, N number of individuals, H number of haplotypes, Hd haplotype diversity, p nucleotide diversity, k mean number of pairwise differences sympatric. The MSN revealed a star-like structure within each sub-networks, with central haplotypes shared among several samples and singletons diverging by only few substitutions from the central haplotypes.

																		7
	H29-H37		9															9
	H38-H49			12														12
	H50-H57				8													8
	H58-H66					9												9
	H67-H75						9											9
	H76-H82							7										7
	H83-H88								6									6
	H89-H96									8								8
	H97-H104										8							8
	H105-H117											13						13
	H118-H121												4					4
	H122-H130													9				9
	H131-H137														7			7
	H138-H146															9		9
	H147-H160																14	14
	N	17	27	27	25	25	23	24	25	23	20	26	11	16	18	14	29	350
	H	11	19	21	15	19	16	15	14	17	13	20	11	14	12	13	20	160
	Hd	0.912 0.957 0.972 0.913 0.967 0.933 0.938 0.917 0.96	0.926 0.969 1	0.983 0.895 0.989 0.956 0.945
	p	0.023 0.026 0.024 0.026 0.027 0.022 0.025 0.024 0.026 0.021 0.026 0.028 0.04	0.022 0.048 0.029 0.027
	k	9.69	10.93 10.04 11.04 11.31 9.05	10.64 9.96	10.71 8.61	11.03 11.71 16.94 9.111 20.17 12.15 11.34
	Abbreviations of sampling sites as on Fig. 1;

Mar Biol (2012) 159:2797-2807

MarBiol (2012Biol ( ) 159:2797Biol ( -2807

Acknowledgments This work was supported by the EU project EUMAR (European Marine Genetic Biodiversity-EVK3CT2001-00048) and by the AGIP funded research project BIOMARE (Project for the development of methodologies for monitoring the impact of off-shore structures in the northern Adriatic). We thank M. Pierpaoli for assistance in the lab. We are grateful to the Cooperative Society 'La Romagnola' (Marina di Ravenna) and to all the people that helped collecting samples.


Unexpected high densities of the hybrid coral Acropora prolifera (Lamarck 1816) in Guadeloupe Island, Lesser Antilles

A Japaud, C Fauvelot, C Bouchon

Unexpected high densities of the hybrid coral Acropora prolifera (Lamarck 1816) in Guadeloupe Island, Lesser Antilles Since the 1980s, coral populations of Acropora palmata (Lamarck 1816) and A. cervicornis (Lamarck 1816) have dramatically declined in the Caribbean and have been consequently classified as critically endangered since 2008 (IUCN).

Acropora palmata and A. cervicornis can form a viable F1 offspring previously described as A. prolifera by Lamarck [START_REF] Vollmer | Hybridization and the evolution of reef coral diversity[END_REF]. Few isolated colonies of that morphotype have punctually been reported on Lesser Antilles reefs in the previous decades. In January 2013, high densities of morphologically identified A. prolifera colonies were observed in the Bay of Grand Cul-de-Sac Marin (16°21¢24.19 †N; 61°35¢35.12 †W, Guadeloupe Island, Lesser Antilles), whereas no colonies were found at this exact same site prospected in May 2011 (Fig. 1). Genetic microsatellite analyses (unpublished data) verified the hybrid nature of the colonies and suggest that they may constitute a single clone. However, in other Caribbean sites, [START_REF] Fogarty | Reproductive isolation and hybridization dynamics in threatened Caribbean acroporid corals[END_REF] found high genotypic diversity for that hybrid.

As a consequence of decreased parental species' density, eggs likely float unfertilized, increasing the probability of heterospecific sperm encounters and thus hybrid embryo formation [START_REF] Fogarty | Weak prezygotic isolating mechanisms in threatened Caribbean Acropora corals[END_REF]).