sign in sign up
<<
Home , Grooved carpet shell, Squid, Venerupis decussata

Biologia 65/4: 688—696, 2010 Section Zoology DOI: 10.2478/s11756-010-0069-8

Genetic variation and population structure of the carpet shell decussatus along the Tunisian coast inferred from mtDNA and ITS1 sequence analysis

Aicha Gharbi1,2, Noureddine Chatti2,KhaledSaid2 & Alain Van Wormhoudt1

1Station de Biologie Marine du Muséum National d’Histoire Naturelle, BP225, 29900 Concarneau, France; e-mail: [email protected] 2Unité de Recherche: Génétique, Biodiversité et Valorisation des Bioressources, UR03ES09, Institut Supérieur de Biotech- nologie de Monastir, Tunisie

Abstract: Surveys of allozyme polymorphisms in the carpet shell clam Ruditapes decussatus have revealed sharp genetic differentiation of populations. Analysis of population structure in this species has now been extended to include nuclear and mitochondrial genes. A partial sequence of a mitochondrial COI gene and of the internal transcribed spacer region (ITS-1) were used to study haplotype distribution, the pattern of gene flow, and population genetic structure of R. decussatus.The samples were collected from twelve populations from the eastern and western Mediterranean coasts of Tunisia, one from Concarneau and one from Thau. A total of twenty and twenty-one haplotypes were detected in the examined COI and ITS1 regions respectively. The study revealed higher levels of genetic diversity for ITS1 compared to COI. The analysis of haplotype frequency distribution and molecular variation indicated that the majority of the genetic variation was dis- tributed within populations (93% and 86% for COI and ITS1 respectively). No significant differentiation was found among eastern and western groups on either side of the Siculo-Tunisian strait. However, distinct and significant clinal changes in haplotypes frequencies between eastern and western samples were found at the most frequent COI haplotype and at three out of five major ITS1 haplotypes. These results suggest the relative importance of historical processes and contemporary hydrodynamic features on the observed patterns of genetic structure. Key words: Ruditapes decussates; COI; ITS1; Tunisia

Introduction netic variation within and among populations. In the Mediterranean , the shared stocks of the clam R. The carpet shell clam (Ruditapes decussatus L., 1758), decussatus represent important marine fishery resource is a commercially valuable bivalve mollusk and among exploited by all coastal countries. An increasing number the most common clam species found on the market for of studies on marine bivalve genetic structure are avail- human consumption, widely distributed in the Mediter- able, especially concerning exploited species. Knowl- ranean and its adjacent Atlantic waters from the North edge about population structure is among the most im- Sea to the coast of Senegal (Breber 1985). In Tunisia, portant questions to investigate. The resulting informa- this species is distributed along the coastline except the tion can be critical both for the understanding of the Cap Bon and Gulf of Hammamet (Medhioub 1993). Ac- of the species and for better management of cording to fishery statistics (capture production) pub- their stocks. The development of molecular techniques lished by the Food and Agriculture Organisation of that identify nucleotide-level DNA sequence polymor- the United Nations (FAO 2006a) the world’s nomi- phisms among species has created a large source of ge- nal catches of “, cockles and arkshells” in 2006 netic markers which can be used in phylogenetic and was 738,845 tons. Moreover, World pro- population studies. Compared to analysis tech- duction of this species group in 2006 was 4,310,488 niques, analysis of DNA variation is rapidly becom- tons (FAO 2006b). Bivalve species have also important ing the method of choice because DNA is more ther- roles in ecosystems. As filter feeders, they form a signif- mostable than and the higher probability of icant link with primary producers (mainly phytoplank- variation being neutral. DNA-based methods such as ton and bacteria), and also act as calcium and carbon nucleotide sequence analysis, RAPD (random ampli- accumulators, which they use in their shell construc- fication of polymorphic DNA) and RFLP (restriction tion. fragment length polymorphism) analysis are useful ge- Studies on the genetic structure of wild pop- netic techniques for population’s studies. RAPD is a ulations aim at identifying the evolutionary events very fast method that can identify a large number of that have shaped the contemporary distribution of ge- polymorphism, but its major drawback is the poor re-

c 2010 Institute of Zoology, Slovak Academy of Sciences Genetic variation of Ruditapes decussates 689 producibility of the results obtained. PCR-RFLP analy- sisissimplerandlessexpensive than conventional DNA nucleotide sequence analysis and better suited for large- scale genetic surveys requiring a large number of spec- imens. However, this technique detects differences in DNA sequences only when they are present at the spe- cific recognition site of the corresponding endonuclease. In this work, we have chosen conventional sequencing of PCR products, a relatively costly approach but allowed us a detailed comparison of individual sequences. It has been proposed that benthic marine species with pelagic larvae have population genetic variation reflecting the dispersal capacity of larvae. Most of them are thought to have little genetic structure. Consider- able work has been conducted to test this hypothesis on many species, including , , , gastropods, , oysters, ophiuroids, anos- tracans and many other with planktonic larvae (Yuan et al. 2009 and references therein). Mitochondrial DNA sequences (including 16S rDNA and COI) were used for many of these studies. In the bivalve R. decussatus, this hypothesis has not been tested. Since the 1980s, many papers have been published on the population genetic of the carpet shell clam R. Fig. 1. Location of the twelve Ruditapes decussatus populations decussatus. In all papers, allozyme variation was used sampled from Tunisia. to characterize genetic variation among natural popu- lations covering its distribution area (Jarne et al. 1988; (S-T) Strait in phylogeographical structuring of marine Borsa et al. 1994; Worms & Pasteur 1982; Borsa & species. To explain the concordance of patterns of ge- Thiriot-Quiévreux 1990; Passamonti et al. 1997; Jor- netic differentiation observed in these marine species, daens et al. 2000).We believed that no studies which a hypothesis has been proposed wherein eastern and analysed DNA sequence information for comparing in- western Mediterranean populations have been isolated traspecific phylogeography have been conducted on bi- due to sea level changes during glacial maxima and valve R. decussatus. Most of PCR-based methods for R. subsequently reconnected, possibly several times, in the decussatus have focused on phylogenetic studies of the past. Ruditapes genus (Fernandez et al. 2000, 2001, 2002a, We recently revealed a certain genetic differenti- b; Passamonti et al. 1998; Canapa et al. 1996, 2003; ation among R. decussatus populations collected from Kappner & Bieler 2006; Cordero et al. 2008). eleven localities in the eastern and western Mediter- DNA-level markers could validate and better elu- ranean coasts of Tunisia, using allozyme electrophoresis cidate gene flow patterns based on allozyme data. The as genetic markers (A. Gharbi et al., in litt.). The aim variations at the DNA level come from base alterations of the present study was to establish the extend of gene that may be found in both coding and non-coding re- flow and to approximate the detailed levels of genetic gions of the genome. These variations are more encom- structure of R. decussatus populations, using nucleotide passing than the changes covered by allozyme analysis, sequence analysis of the first subunit of the cytochrome which are confined to coding regions of the genome. c oxidase (COI) region of mitochondrial DNA (mtDNA) The marine environment holds many peculiarities, and the internal transcribed spacer region (ITS-1) be- which, in conjunction with other life history character- tween 5.8S and 18S ribosomal DNA genes. We believe istics of marine , hints at genetic differenti- this study represents the first survey of sequence vari- ation within apparent panmictic populations. In fact, ation at COI and ITS-1 among geographically distant the marine environment shows much more heterogene- populations of bivalve R. decussatus, at least in Tunisia ity owing to the influence of climate, hydrodynamics scale. and topography on natural barriers, which affects dis- persal (Cowen et al. 2000). Genetic structuring is en- Material and methods hanced furthermore by certain biological traits, such Samples and DNA extraction as sex-dependent migration, which can counteract dis- Ruditapes decussatus samples were collected from eleven lo- persal and gene flow. Hence, numerous marine species cations originating from coastal lagoon and marine sites maintain a genetic structure despite a great potential in Tunisia (Fig. 1). Samples from Concarneau (Brittany) for dispersal (Shaw et al. 1999; Maes & Volckaert 2002). and from Thau (French south) were included in this study Studies on several fishes, molluscs and to make comparisons between Mediterranean and Atlantic have suggested the importance of the Siculo-Tunisian samples. Live specimens were shipped to the laboratory 690 A. Gharbi et al. where they were maintained in aqua until labial palpes tis- as different haplotypes. Analyses were limited to reliably sue was removed and preserved in 90% ethanol prior to DNA aligned regions from the data set and regions that could not extraction and sequence analysis. All individuals, except the be unambiguously aligned were excluded from analysis. The Thau and Elouamer specimens, were surveyed in a previ- data sets for both COI and ITS1 genes were analysed inde- ous allozyme study. Total genomic DNA was isolated from pendently. The alignment of COI sequences was confirmed approximately 20 mg labial palpes tissue using the CTAB by translating the aligned DNA sequences into amino acid. DNA Extraction Protocol (Doyle & Doyle 1987). Data analyses were performed using ARLEQUIN 3.0 Gonadal tissues were specifically avoided to prevent (Excoffier et al. 2005). Intrapopulation diversity was anal- comparisons of nonorthologous sequences due to the actual ysed by estimating gene diversity (h), the probability that or potential presence of doubly uniparental inheritance of two randomly chosen haplotypes are different, and nu- mtDNA in some bivalve taxa (Skibinski et al. 1994; Zouros cleotide diversity (π), the probability that two randomly et al. 1994). DNA concentrations were spectrophotometri- chosen homologous nucleotides are different (Tajima 1983; cally estimated. Nei 1987). The total number of nucleotide differences and percent sequence divergence values were calculated between DNA sequence production each pair of haplotypes. COI To test for the geographic structuring of collections, A 561-bp fragment of the cytochrome c oxidase subunit I we calculated the pairwise genetic distances (FST) and (COI) of mtDNA was amplified from genomic DNA using their statistical significance by performing 10,000 permu- the PCR. The amplification primers, designed by (Folmer tations among the individuals between populations. We et al. 1994), were: LCOI-490: 5’ att-att-cag-acc-caa-tca-taa- also performed a hierarchical analysis of molecular vari- ... aga-tat-tgg 3’ and HCOI-900: 5’ tgt-agg-aat-agc-aat-aat- ance (AMOVA; Excoffier et al. 1992) pooling the popula- aaa-agt-tac 3’. tions in western and eastern Mediterranean groups. This Amplification reactions were performed in 25 ml vol- analysis provided the correlation of DNA haplotype diver- umes containing pure Taq Ready-to-go PCR beads from sity at different levels of hierarchical subdivision in the µ µ Amersham Biosciences, 2 lofDNAextractand1 lfrom form of three variance components: between groups; within each primer. groups; and within localities. The test enabled us to deter- Amplifications were carried out on a Biometra mine changes in allocation of the components of variance, TGRADIENT Thermal Cycler using these conditions: pre- ◦ ◦ depending on different geographic grouping schemes. Re- cycling denaturation at 94 Cfor3min;45cyclesof94C lationships among observed haplotypes were inferred with denaturing for 1 min, 52 ◦C annealing for 1 min, 72 ◦Cex- ◦ network 4.5.1.0 (Fluxus Technology Ltd, at www.fluxus- tension for 1 min and a postcycling extension at 72 C for 10 .com) using the median-joining method (Bandelt min. The size and the quality of PCR products were visu- et al. 1999). alised on 1.2% agarose gels (stained with ethidium bromide) Clinal variation in haplotypes frequencies (i.e., mono- and viewed under ultraviolet light. Amplified PCR products tonic changes in haplotype frequencies with distance) was were purified with the wizard SV Gel and PCR Clean-Up investigated at each major haplotype. The significance of System purification kit (promega) according to the manu- this pattern of variation was tested by correlation coeffi- facturer’s instructions and directly sequenced using the Big cients. Haplotypes frequencies were plotted to geographi- Dye TM terminator cycle sequencing chemistry following cal distances, measured as the shortest coastal distance be- the manufacturer’s protocol. The sequences were recorded TM tween all samples and the approximate centre of the Siculo- with an ABI Prism 310 automated sequencer. Tunisian Strait. ITS-1 The ITS regions are non coding regions in the rDNA re- Results peat unit that are variable enough to detect intraspecific variation for populations genetic studies. The internal tran- scribed spacer region ITS-1 is flanked by the 18S rDNA and The two DNA fragments could be amplified successfully 5.8S rDNA gene. Sequence analysis was performed on 597 from all R. decussatus specimens. bp of ITS-1 gene amplified by the polymerase chain reac- tion (PCR). The primers were: SPI 5’-cacaccgcccgtcgctacta COI 3’ and SP 3R 5’-tcgacscacgagccragtgatc-3’. PCR reaction Nucleotide sequences of 561 bp in length were ob- components, thermal cycling conditions and amplicon pu- tained from the mitochondrial DNA cytochrome c oxi- rification were similar to that described for COI with the dase subunit I (COI) gene for 170 Ruditapes decussatus exception that the annealing temperature was 58 ◦C. specimens representing twelve geographical populations As the ITS1 region, is repeated many times within an individual midge, these PCRs will contain the products of from Tunisia and two other populations from Thau and many different ITS repeats that are potentially heteroge- Concarneau. neous. To verify heterogeneity, PCR products were cloned. The nucleotide composition of the COI sequences Eight clones were examined per individual sample and plas- averaged 15% C. 37% T. 23% A and 23% G. Twenty pu- mids from single colonies were purified and sequenced using tative haplotypes (H1–H20) resulting from 20 variable forward and reverse primers. sites (including eighteen transitions and two transver- Data analysis sions) were detected. The number of haplotypes per site ranged from 2 to 6. The frequency of H1 was higher than All sequences were aligned using the multiple-alignment pro- gramme CLUSTAL W (Thompson et al. 1994) with adjust- 70% in R. decussatus sample and was the only haplo- ments made by eye, to determine the genotypes (haplotypes) types shared by all populations while the majority of of the COI and ITS1. When homologous sequences differed the other haplotypes (65%) were unique. The H5 was by one or more nucleotides, the sequences were considered present, although with low frequencies, in five of the six Genetic variation of Ruditapes decussates 691

Table 1. Genetic diversity of COI and ITS1 sequences for R. decussatus populations.

Population COI ITS1

Nh π(SD) N h π(SD)

Concarneau 10 0.511 (0.164) 0.0010 (0.0010) 11 0.545 (0.072) 0.0009 (0.0009) Thau 8 0.643 (0.184) 0.0023 (0.0017) 14 0.923 (0.043) 0.0031 (0.0021) Mzjemil 10 0.377 (0.181) 0.0007 (0.0008) 15 0.790 (0.051) 0.0017 (0.0014) Faroua 12 0.1667 (0.137) 0.0003 (0.0004) 10 0.866 (0.085) 0.0025 (0.0019) Tunis 11 0.318 (0.163) 0.0006 (0.0007) 17 0.698 (0.075) 0.0014 (0.0012) Monastir 16 0.177 (0.106) 0.0003 (0.0004) 14 0.802 (0.068) 0.0020 (0.0015) sfax 10 0.644 (0.101) 0.0013 (0.0011) 13 0.384 (0.132) 0.0013 (0.0011) Kerkennah 15 0.666 (0.100) 0.0023 (0.0017) 11 0.563 (0.134) 0.0016 (0.0013) Kneiss 12 0.757 (0.122) 0.0020 (0.0014) 9 0.805 (0.111) 0.0024 (0.0018) Akarit 10 0.377 (0.181) 0.0007 (0.0008) 20 0.671 (0.091) 0.0017 (0.0013) Elouamer 11 0.562 (0.134) 0.0011 (0.0010) 12 0.560 (0.154) 0.0022 (0.0016) Galala 9 0.583 (0.183) 0.0011 (0.0011) 14 0.494 (0.087) 0.0016 (0.0013) Bougrara 11 0.618 (0.164) 0.0015 (0.0013) 10 0.933 (0.077) 0.0056 (0.0035) Biben 18 0.405 (0.142) 0.0008 (0.0008) 17 0.661 (0.125) 0.0024 (0.0017)

Mean 0.486 (0.198) 0.0011 (0.0009) 0.700 (0.177) 0.0021 (0.0018)

Explanations: Sample size (N), haplotype diversity (h), nucleotide diversity (π) and standard deviation (SD).

Table 2. Pairwise FST values between populations of R. decussatus based on cytochrome C oxidase subunit I (COI) sequences (below diagonal) and on ITS1 sequences (above diagonal). Significant values are indicated in bold estimates (5% level).

Mona- Ker- Elou- Boug- Con- Mzjemil Faroua Tunis stir Sfax kennah Kneiss Akarit amer Galala rara Biben carneau Thau

Mzjemil – –0.0257 –0.004 –0.0517 0.2086 0.1143 –0.0256 0.0858 0.1734 0.1702 0.0598 0.12365 0.0903 –0.0161 Faroua –0.0463 – 0.0342 0.0195 0.1194 0.0063 –0.0753 0.0247 0.0421 0.0371 –0.0227 0.0371 0.1101 –0.0535 Tunis –0.0426 –0.0578 – 0.0357 0.1422 0.2465 –0.0146 0.0411 0.3075 0.2782 0.1535 0.2719 0.2742 0.0564 Monastir 0.0540 –0.0201 0.0255 – 0.3037 0.1790 0.0354 0.1579 0.2226 0.2435 0.0852 0.1565 0.1076 –0.0064 Sfax 0.2381 0.2687 0.1840 0.2896 – 0.2679 0.337 0.0282 0.344 0.2367 0.2453 0.3506 0.4726 0.218 Kerkennah 0.1368 0.1678 0.1048 0.2386 0.1052 – 0.0631 0.1764 –0.0599 –0.0711 0.0453 –0.0253 0.0932 0.0777 Kneiss 0.1171 0.1468 0.0779 0.1791 –0.0271 0.0301 – –0.0239 0.1289 0.0821 0.0209 0.1238 0.1904 –0.0110 Akarit –0.0493 –0.0482 –0.0696 0.0304 0.0873 0.0768 0.0205 – 0.2368 0.1799 0.1277 0.2376 0.3022 0.0941 Elouamer 0.109 0.1298 0.0640 0.1568 0.0649 –0.048 0.0078 0.0210 – –0.0299 0.0411 –0.0321 0.1308 0.0927 Galala 0.0274 0.0551 –0.0057 0.0901 0.0628 0.0267 –0.0093 –0.0278 0.0043 – 0.0868 0.0355 0.2009 0.1262 Bougrara 0.0365 0.0638 0.0058 0.0855 0.0221 –0.0104 –0.0441 –0.0355 –0.0514 –0.0439 – 0.0329 0.1193 –0.0071 Biben –0.0376 –0.0103 –0.0316 0.0285 0.1159 0.0973 0.0549 –0.0519 0.0457 –0.0248 –0.0163 – 0.0315 0.0598 Concarneau 0.0396 0.0652 0.0090 0.1142 0.1111 0.0501 0.0547 0.0204 0.0101 0.0304 –0.019 –0.0004 – 0.0954 Thau 0.0539 0.0852 0.0128 0.1068 –0.0298 –0.0084 –0.0532 –0.0406 –0.0186 –0.0760 –0.0664 –0.0336 –0.023 – eastern populations and was absent in western popula- The hierarchical AMOVA analysis revealed that tions. But was not strong enough to affect the AMOVA more than 90% of the variance was observed within analyses. the samples, and no significant variation could be at- The haplotypes differed from one another by 1–5 tributed to variation between groups (Table 3). mutations, and had pairwise sequence divergence values The parsimony network of the observed 20 hap- ranging from 0.178 to 0.9%. This finding suggests that lotypes characterized by a star genealogy is shown in the mitochondrial genome in R. decussatus appears to Fig. 2A with haplotype abundance in circle size. In evolve at a slow rate. the network, most frequent haplotype H1 was apparent Within locality, genetic diversity was estimated in with high frequency and wide geographical distribution, terms of haplotype diversity (H) and nucleotide diver- from which other infrequent haplotypes were radiated sity (π) (Table 1). Both diversity indices were highest with a single substitution (except H8), suggesting that in Kerkennah followed by Thau. Of the 10 informa- major haplotype was old enough to be considered a tool tive sites, two were at the first position of the codon of population genetics. and 8 were at the third. The high percentage of third Correlation between major haplotype (H1) fre- position substitutions provides assurances that the re- quency and geographical distances showed significant gion sequenced is not a nuclear pseudogene. Transla- correlation (r = –0.65; P < 0.05) (Fig. 3). tion of codons into amino acids indicated that two of the mutations did not result in synonymous substitu- ITS-1 tions. The total aligned data matrix for the R. decussatus As shown in Table 2, Fst values were generally low, species including indels was 597 base pair, and has been suggesting small genetic differentiation between sam- determined in 187 specimens. ples. Analysis of the cloned ITS1 PCR products showed 692 A. Gharbi et al.

Table 3. Hierarchical analysis of molecular variance (AMOVA) for the COI and ITS1 sequences of R. decussatus from Tunisia.

Source of variation Degrees Sum of Variance Percentagage Source of variation of freedom squares components of variation Fixation indices P-value

COI Among groups 1 0.760 0.01161 Va 4.56 FCT = 0.0456 0.087 Among populations within groups 10 3.603 0.00717 Vb 2.07 FSC = 0.0217 0.102 Within populations 133 27.437 0.26552 Vc 93.37 FST = 0.066 0.019

Total 144 31.800 0.28521 ITS1 Among groups 1 1.689 0.01168 Va 3.01 FCT = 0.030 0.187 Among populations within groups 10 9.111 0.04255 Vb 10.67 FSC = 0.110 <0.001 Within populations 150 51.321 0.33764 Vc 86.32 FST = 0.136 <0.001 Total 161 62.122 0.39187

Explanations: Analyses are presented for pooled populations in western and eastern Mediterranean groups. The eastern group cor- responds to populations on the eastern side of the Siculo–Tunisian strait and the western group corresponds to populations on the western side. Va, Vb and Vc are the associate covariance components. FCT,FSC and FST are the F-statistics.

Fig. 2. Median-joining networks constructed using COI (A) and ITS1 (B) haplotypes data for R. decussatus. The sizes of circles are proportional to Haplotype frequency. Perpendicular tick marks on the lines joining haplotypes (drawn by hand) represent the number position of nucleotide substitutions. The red circles represent theoretical median vectors (mv) introduced by the network software. an absence of intra-individual variation of ITS1. A com- The median network of the observed 21 haplotypes parison with GenBank database sequences indicated characterized by a complex star genealogy is shown in that the whole sequenced region corresponds to a par- Fig. 2B and has more than one dominant central hap- tial sequence of the 18S subunit, and of the ITS 1 re- lotype. gion, but all 597 nucleotides were considered in the Likewise to COI, the analysis of the partitioning analyses. of the haplotype diversity indicated that the majority Comparing all R. decussatus populations, 20 vari- (86.3%) of the genetic variation was distributed within able sites (3.3%) were identified. In addition to nu- population (Table 3). cleotide substitutions, nine indels were observed. A Regression analysis between most frequent haplo- total of 21 different haplotypes (H1–H21) was found type frequencies and distance showed a clinal variation among all samples. Only haplotype 2 (present in 33% at three out of five of most common ITS1 haplotypes. of the individuals analysed) was shared among all pop- H1 (r = –0.67; P < 0.05) and H7 (r = –0.71; P < 0.05) ulations. showed a significant decrease in frequencies on either The haplotypes differed from one another by 1–4 side of the S-T strait, while H2 (r = 0.66; P < 0.05) mutations (indels excluded), and had pairwise sequence displayed cline of opposite directions at both sides (in- divergence values ranging from 0.17 to 0.68 %. creased in frequency with distance) (Fig. 3). A cline Compared to COI, genetic diversity indices of was also detected at H6 but was not significant (data ITS1 were high. The overall π–value was 0.0021 and not shown). This result is concordant with the clinal the within population π values ranged from 0.0009 to variation observed for some isozyme loci (A. Gharbi et 0.0056 (Table 1). Haplotype diversity values ranged al., in litt.). from 0.384 to 0.933 (Table 1). Intraspecific DNA sequence variation in ribosomal Pairwise FST values were significantly different DNA (ITS-1) and mitochondrial DNA (COI) regions from zero (P < 0.05) in 35 (38%) of the 91 compar- among geographical populations of R. decussatus was isons including all populations (Table 2). relatively congruent. The continuity identified between Genetic variation of Ruditapes decussates 693

H1-ITS1 H2-ITS 0.40 0.70 P < 0.05 P < 0.05 0.35 0.60

0.30 y 0.50 0.25 0.40 0.20 0.30 0.15

0.10 0.20 Haplotype frequency

0.05 Haplotype frequenc 0.10

0.00 0.00 -300 -200 -100 0 100 200 300 400 500 600 700 -300 -200 -100 0 100 200 300 400 500 600 700 Geographic distance (Km) Geographic distance (Km)

H7-ITS1 H1-COI 0.30 1.00 P < 0.05 0.90 P < 0.05 0.25 y 0.80

0.20 0.70 0.60 requenc 0.15 f 0.50 0.40 ype 0.10 t o

l 0.30 ap

Haplotype frequency 0.20

0.05 H 0.10 0.00 0.00 -300 -200 -100 0 100 200 300 400 500 600 700 -300 -200 -100 0 100 200 300 400 500 600 700

Geographic distance (Km) Geographic distance (Km)

Fig. 3. Geographic variation of most frequent haplotypes frequencies of ITS1 and COI H1-ITS1, H7-ITS1 and H1-COI showed a significant decrease in frequencies on either side of the S-T strait, while H2-ITS1 displayed cline of opposites directions at both sides. Geographical distances, are measured as the shortest coastal distance between all samples and the approximate centre of the Siculo-Tunisian Strait. — Indicate the position of the Siculo-Tunisian Strait. northern and southern populations in the ITS-1 region type relationship network, obtained for COI is charac- was observed in the COI sequences. terized by a star-shaped genealogy centered on one ge- ographically widespread haplotype. This phylogenetic Discussion pattern is commonly interpreted as a signature of a re- cent population expansion following a population bot- The present study was conducted to determine the phy- tleneck (Slatkin & Hudson 1991). A possible decline logeographic structure among populations of the bi- of population sizes caused by overexploitation can also valve R. decussatus using the cytochrome c oxidase explain the low genetic diversity. Also, methodological subunit I (COI) region of mtDNA and nuclear riboso- factors such as sample bias or sampling inadequacies mal DNA internal transcribed spacer region (ITS- 1). may explain the low genetic variability of R. decussa- To our knowledge, this study represents the first ef- tus samples. fort to characterize genetic population structure in this Molecular variance analysis (AMOVA) showed no species using DNA-based markers. The results demon- differentiation among the pooled groups on either side strate that DNA sequence polymorphism in the COI of the Siculo-Tunisian strait. The same pattern of ge- and ITS-1 regions were effective in the characterization netic structure was reported for other species in the of intraspecific phylogeographic structure among R. de- same geographical region (Arcuelo et al. 2003; Zardoya cussatus populations. et al. 2004). In contrast, some genetic studies on other Estimates of genetic diversity recorded for ITS1 species showed that the Siculo-Tunisian (S–T) strait were higher than those for COI. This low polymorphism is an important genetic boundary between the east- in COI sequences could be attributed to its haploid ern and western Mediterranean basins (Bahri-Sfar et nature and maternal inheritance. However, ITS1 is a al. 2000; Nikula & Vainola 2003; Arnaud-Haond et al. non-coding region and not submit to selection effect. 2007; Zitari-Chatti et al. 2008) and have highlighted the Moreover, values of haplotype and nucleotide diversi- importance of climatic fluctuations during later Pleis- ties of mtDNA are lower than those reported for other tocene glacial period as an evolutionary force shaping bivalves (Nikula & Vainola 2003; Cho et al. 2007; Kat- population structure in this region. sares et al. 2008; Yuan et al. 2009). There are several The present genetic structure pattern obtained for possible explanations for such reduced genetic diversity, mtDNA, appears at first sight, not coincident with the which could be a result of population life history traits previously reported allozyme differentiation detected or related to the current population size: The haplo- for R. decussatus populations. There are many exam- 694 A. Gharbi et al. ples where nuclear and mitochondrial markers do not present study, populations of R. decussatus had many show the same pattern of genetic structure (Grosberg kinds of rare and unique haplotypes (65% and 76% & Cunningham 2001). In some cases this is because of COI and ITS1 haplotype, respectively, were found the nuclear markers, in particular many allozyme loci, only in one locality) suggesting geographic restriction are affected by selective pressures, whereas mitochon- in their distribution and a recent population expansion drial markers are apparently neutral and therefore bet- following a population bottleneck. ter represent true patterns of gene flow. Another reason The important presence of unique haplotype might for differing patterns of genetic structure is that nuclear have major consequences on the population genetic markers take longer to reach an equilibrium between structure and influence on genetic differentiation among drift and migration than mitochondrial markers (Neigel populations. 1994). For example, allozyme variation has sometimes Moreover, if we observe the distribution of rare and been shown to reflect patterns that are not consistent unique haplotypes on both sides of the Siculo-Tunisia with present day currents, but may instead re- strait, we note that they are characteristic of one or flect past patterns of dispersal along ocean currents that the other of the two basins. High frequencies of rare are now non-existent (Benzie & Williams 1995, 1997). and unique haplotypes could then reflect the effects of a Equally, nuclear genes, represented by allozymes, nu- recent demographic expansion which would mask a real clear sequences and morphological differences, may still ancient and significant genetic differentiation displayed be showing some evidence of past historical barriers to by the two basins. dispersal which have not yet come to equilibrium with Furthermore, in our present study, a significant cli- present-day gene flow. Thus, mitochondrial and nuclear nal variation in one major haplotype frequency of mt genes might be expected to differ in their resolution of DNA(H1) and at three out of five most frequent hap- past and present gene flow because of differences in the lotypes frequencies of ITS1 (H1, H2 and H7) occurs rates at which they come to genetic equilibrium. between the eastern and western populations (Fig. 3) There is still only a limited number of broadscale with a phylogeographic midpoint that coincide with S- (S-T) strait studies using both nuclear and mtDNA T strait. genes, but in some cases mitochondrial and nuclear The most commonly proposed causes of clinal pat- variation show the same pattern of population struc- terns in gene frequencies are selection across an en- ture. For instance in the Engraulis encrasico- vironmental gradient (e.g., Koehn et al. 1980), ran- lus (L., 1758) and in the Penaeus keraturus (Forssk˚al, dom genetic drift with isolation-by distance effects (e.g., 1775), genetic differentiation was found using both al- Maes et al. 2002; Wirth & Bernatchez 2001), and sec- lozymes and mtDNA sequences (Borsa 2002; Nikula & ondary contact and introgression between previously Vainola 2003; Zitari et al. 2008, 2009). This suggests isolated and genetically divergent populations (e.g., that present day gene flow is still very low between two Pérez-Losada et al. 1999, 2002). Although selection may basins, possibly because of reduced population size re- at least partly explain patterns in genetic variation in sulting from overfishing. several marine species in this area, it is unlikely that Populations of the on the other hand do not this factor can be proposed to account for the clinal show the same pattern of genetic structure using nu- patterns observed in the current study: the haplotypes clear and mitochondrial markers. Blanquer et al. (1992) clines are not associated with environmental gradients studied allozyme variation at 17 loci in 10 samples across the region sampled, moreover, there is little evi- from the Mediterranean to the Kattegat. No popula- dence for direct selection on mtDNA and likely for ITS1 tion structure was apparent. However, some differences which is a non coding region. Mantel test on the cor- have been found in mtDNA in turbot from the eastern relation between FST/(1 – FST) and geographical dis- and western Mediterranean (Suzuki et al. 2004). tance matrices was not significant (P > 0.05 for either of The present genetic structure pattern obtained for the markers), suggesting that the observed clinal hap- mtDNA and ITS1 showed no differentiation among the lotypes frequency is the result of the action of other pooled groups on either side of the Siculo-Tunisian factors. strait. At first sight, these results could appear to be Theoretical and experimental studies suggest that compatible with a genetic relatedness among localities. the interpretation of clinal variation is aided by histor- This resulted in the subsequent FST and AMOVA anal- ical considerations (Gallant et al. 1993; Quesada et al. ysis (Tables 2, 3). The genetics of marine populations 1995). For instance, mitochondrial DNA analysis pro- with pelagic larval development has often been char- vides a historical perspective because it permits the acterized by low genetic differentiation among popu- construction of a of haplotypes. If this lations, a pattern driven by high dispersal capabilities tree has a deep phylogenetic split, which separates hap- and large scale oceanic mixing (Reichow & Smith 2001; lotypes occurring at high frequency on either side of the Rivera et al. 2004). However, in the shellfish complex, cline, secondary contact is favored (Avise 1994). The the existence of both the planktonic larval stage and the connection between the eastern and western Mediter- sessile adult stage makes it difficult to predict genetic ranean Sea is known to have been closed in the region population structure and level of gene flow. of the S-T Strait several times during glacial periods Hierarchical AMOVA as well as pairwise FST com- in the Pleistocene (Thiede 1978) sometimes associated putations were based on haplotype frequencies. In the with substantial environmental changes to the Mediter- Genetic variation of Ruditapes decussates 695 ranean Sea. Although there is no evidence for the histor- parallel ocean circulation. Mar. Biol. 123: 781–787. DOI ical persistence of R. decussatus in either water body, 10.1007/BF00349121 Benzie J.A.H. & Williams S.T. 1997. Genetic structure of giant it is conceivable that this species may have been subdi- clam (Tridacna maxima) populations in the West Pacific is vided into isolated populations during the most recent not consistent with dispersal by present-day ocean currents. ice-ages. 51: 768–783. The water circulation in the Siculo-Tunisian Strait Blanquer A., Alayse J.P., Berrada-Rkhami O. & Berrebi P. 1992. Allozyme variation in turbot (Psetta maxima) and brill is characterized by a unidirectional east-south-east flow (Scophthalmus rhombus) (Osteichthyes. Pleuronectiformes. of pelagic currents which go round Cape Bon and Scophthalmidae) throughout their range in . J. Fish leave the coasts of Tunisia at the latitude of Kelibia, Biol. 41: 725–736. DOI 10.11118j.1095-8649.1992.tb02702.x Borsa P. 2002. Allozyme, mitochondrial-DNA, and morpho- while eastern Mediterranean waters stay in the Lybico- metric variability indicate cryptic species of anchovy (En- Tunisian Gulf (Pinardi & Masetti 2000). It is possible graulis encrasicolus). Biol. J. Linn. Soc. 75: 261–269. DOI that the pattern of genetic variation observed in the 10.1046/j.1095-8312.2002.0018.x carpet shell clam reflects present-day processes in that Borsa P. & Thiriot-Quiévreux C. 1990. Karyological and al- lozymic characterization of Ruditapes philippinarurn, R. au- such currents could have permitted sufficient gene flow, reus and R. decussatus (: ). Aquaculture at least in one direction from western to eastern re- 90: 209–227. gion, preventing the accumulation of substantial genetic Borsa P., Jarne P., Belkhir K. & Bonhomme F. 1994. Genetic structure of the palourde Ruditapes decussatus L. in the differentiation unless the hydrographic regime was an- Mediterranean, pp. 103–113. In: Beaumont A.R. (ed.), Genet- cient. Moreover, the Nm estimates (number of female ics and Evolution of Aquatic Organisms, Chapman & Hall, migrants per generation for COI, Nm ≈ 7; and number London. of migrants for ITS1, Nm ≈ 2) among the R. decussatus Breber P. 1985. On-growing of the carpet shell clam Ruditapes decussatus two years’ experience in Venice lagoon. Aquacul- samples largely exceed those generally considered suf- ture 44: 51–56. ficient to prevent subpopulation structuring (Slatkin & Canapa A., Marota I., Rollo F. & Olmo E. 1996. Phylogenetic Barton 1989; Mills & Allendorf 1996). These observa- analysis of Veneridae (Bivalvia): comparison of molecular and paleontological data. J. Mol. Evol. 43: 517–522. DOI tions could be explained by the fact that the life cycle 10.1007/BF02337522 of R. decussatus (After spawning and external fertiliza- Canapa A., Schiaparelli S., Marota I. & Barucca M. 2003. Molec- tion, developing larvae spend a variable period of time ular data from the 16S rRNA gene for the phylogeny of Vene- 142: as part of the , which can be passively drifted ridae (: Bivalvia). Mar. Biol. 1125–1130. DOI 10.1007/s00227-003-1048-1 by water currents, often over considerable distances) Cho E.S., Jung C.G., Sohn S.G., Kim C.W. & Han S.J. 2007. should facilitate gene flow, it is therefore feasible that Population genetic structure of the ark shell Scapharca the present clinal gene frequencies between these areas broughtonii Schrenck from Korea, China, and Russia based on COI gene sequences. Mar. Biotechnol. 9: 203–216. DOI result from secondary contact between both formerly 10.1007/s10126–006–6057–x separated group with a contact zone between the Gulf Cordero D., Pena J.B. & Saavedra C. 2008. Polymorphisms at of Tunis and the Gulf of Hammamet. three introns in the manila clam (Ruditapes philippinarum) and the clam (R. decussatus). J. Shellfish Res. 27: 301–306. Acknowledgements Cowen R.K., Lwiza K.M.M., Sponaugle S., Paris C.B. & Ol- son D.B. 2000. Connectivity of marine populations: Open or closed? Science 287: 857–859. DOI 10.1126/science.287.5454. The authors thank all people contributed to R. decussatus 857 sampling. We are grateful to the reviewers for helpful com- Doyle J.J. & Doyle J.L. 1987. A rapid DNA isolation procedure ments on the manuscript. for small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11–15. Excoffier L., Laval G. & Schneider S. 2005. ARLEQUIN version References 3.0– an integrated software package for population genetics data analysis. Evol. Bioinform. Online 1: 47–50. Excoffier L., Smouse P.E. & Quattro J.M. 1992. Analysis of Arculeo M., Lo Brutto S., Sirna-Terranova M., Maggio T., Can- molecular variance inferred from metric distances among nizzaro L. & Parrinello N. 2003. The stock genetic structure DNA haplotypes: application to human mitochondrial DNA of two Sparidae species. Diplodus vulgaris and Lithognathus restriction data. Genetics 131: 479–491. 63: mormyrus in the Mediterranean Sea. Fish. Res. 339–347. Fernández A., García T., Asensio L., Rodríguez M.Á., González DOI 10.1016/S0165-7836(03)00102-4 I., Céspedes A., Hernández P.E. & Martín R. 2000. Identi- Arnaud-Haond S., Migliaccio M., Diaz-Almela E., Teixeira S., fication of the clam species Ruditapes decussatus (Grooved Van de Vliet M.S., Alberto F., Procaccini G., Duarte C.M. & Carpet Shell), pul lastra (Pullet Carpet Shell) and Serrao E.A. 2007. Vicariance patterns in the Mediterranean Ruditapes philippinarum (Japanese Carpet Shell) by PCR- Sea: east-west cleavage and low dispersal in the endemic Sea- RFLP.J.Agric.FoodChem.48: 3336–3341. grass Posidonia Oceanica. J. Biogeogr. 34: 963–976. Fernández A., García T., Asensio L., Rodríguez M.Á., González Avise J.C. 1994. Molecular markers, and evolu- I., Hernández P.E. & Martín R. 2001. PCR-RFLP analysis tion. Chapman & Hall, London, 532 pp. of the internal transcribed spacer (ITS) region for identi- Bahri-Sfar L., Lemaire C., Ben Hassine O.K. & Bonhomme F. fication of 3 clam species. J. Food Sci. 66: 657–661. DOI 2000. Fragmentation of sea-bass populations in the western 10.1111/j.1365-2621.2001.tb04617.x and eastern Mediterranean as revealed by microsatellite poly- Fernández A., García T., González I. Asensio L., Rodríguez morphism. Proc. R. Soc. Lond. B Biol. Sci. 267: 929–935. M.Á., Hernández P.E. & Martín R. 2002a. Polymerase chain DOI 10.1098/rspb.2000.1092 reaction-restriction fragment length polymorphism analysis Bandelt H.J., Forster P. & R¨ohl A. 1999. Median-joining networks of a 16S rRNA gene fragment for authentification of four clam for inferring intraspecific phylogenies. Mol. Biol. Evol. 16: species. J. Food Protect. 65: 692–695. 37–48. Fernández A., García T., Asensio L., Rodríguez M.Á., González Benzie J.A.H. & Williams S.T. 1995. Gene flow among gi- I., Lobo E., Hernández P.E. & Martín R. 2002b. Genetic dif- ant clam (Tridacna gigas) populations in Pacific does not ferentiation between the clam species Ruditapes decussatus 696 A. Gharbi et al.

(grooved carpet shell) and Venerupis pul lastra (pullet carpet Cephalopoda) from the NE Atlantic and Mediterranean. shell) by PCR-SSCP analysis. J. Sci. Food Agric. 82: 881– Heredity 83: 280–289. DOI 10.1038/sj.hdy.6885520 885. DOI 10.1002/jsfa.1117 Pinardi N. & Masetti E. 2000. Variability of the large scale gen- Folmer O., Black M., Hoeh W., Lutz R. & Vrijenhoek R. 1994. eral circulation of the Mediterranean Sea from observations DNA primers for amplification of mitochondrial cytochrome and modelling: a review. Palaeogeogr. Palaeoclim. Palaeoecol. c oxidase subunit I from diverse metazoan invertebrates. Mol. 158: 153–173. Mar. Biol. Biotech. 3: 294–299. Quesada H., Beynon C.M. & Skibinski D.O.F. 1995. A mito- Food and Agriculture Organization of the United Nations (FAO) chondrial DNA discontinuity in the Mytilus gallo- 2006a. FAO Yearbook of Statistics: Summary Ta- provincialis Lmk: Pleistocene vicariance biogeography and bles. Fish, Crustaceans, Mollusks, etc.Capture Production by secondary intergradation. Mol. Biol. Evol. 12: 521–524. Groups of Species. ftp://ftp.fao.org/fi/stat/summary/a1d. Reichow D. & Smith M.J. 2001. Microsatellites reveal high levels pdf (accessed 15.09.2009). of gene flow among populations of the California Food and Agriculture Organization of the United Nations opalescens.Mol.Ecol.10: 1101–1109. DOI 10.1046/j.1365- (FAO) 2006b. FAO Yearbook of Fishery Statistics: Sum- 294X.2001.01257.x mary Tables. World aquaculture production by species Rivera M.A.J., Kelley C.D. & Roderick G.K. 2004. Subtle popu- groups. ftp://ftp.fao.org/fi/stat/summary/b-1.pdf (accessed lation genetic structure in the Hawaiian , Epinephelus 15.09.2009). quernus (Serranidae) as revealed by mitochondrial DNA anal- 81: Gallant S.L., Preziosi R.F. & Fairbairn D.J. 1993. Clinal variation ysis. Biol. J. Linn. Soc. 449–468. DOI 10.1111/j.1095- in eastern populations of the waterstrider Aquarius remigis: 8312.2003.00304.x gradual intergradation or discontinuity? Evolution 47: 957– Shaw P.W., Pierce G.J. & Boyle P.R. 1999. Subtle population 964. structuring within a highly vagile marine , the Grosberg R.K. & Cunningham C.W. 2001. Genetic structure in veined squid Loligo forbesi, demonstrated with microsatellite 8: the sea: from populations to communities, pp. 61–84. In: Bert- DNA markers. Mol. Ecol. 407–417. DOI 10.1046/j.1365- ness M.D., Hay M.E. & Gaines S.D. (eds), Marine Commu- 294X.1999.00588.x nity Ecology, Sinauer, Sunderland, MA. Skibinski D.O.F., Gallagher C. & Beynon C.M. 1994. Sex lim- ited mitochondrial DNA transmission in the marine mussel Jarne P., Berrebi P. & Guelorget O. 1988: Variabilité génétique Mytilus edulis. Genetics 138: 801–809. et morphométrique de cinq populations de la palourde Rudi- Slatkin M. & Barton N.H. 1989. A comparison of three indirect tapes decussatus. Oceanol. Acta. 11: 401–407. methods for estimating average levels of gene flow. Evol. Int. Jordaens K., De Wolf H., Willems T., Van Dongen S., Brito C., J. Org. Evol. 43: 1349–1368. Frias Martins A.M. & Backeljau T. 2000. Loss of genetic vari- Slatkin M. & Hudson R.R. 1991. Pairwise comparisons of mito- ation in a strongly isolated Azorean population of the edible chondrial DNA sequences in stable and exponentially growing clam, Tapes decussates. J. Shellfish Res. 19: 29–34. populations. Genetics 129: 555–562. Kappner I. & Bieler R. 2006. Phylogeny of venus clams (Bi- Suzuki N., Nishida M., Yoseda K., Ustundag C., Sahin T. & valvia: Venerinae) as inferred from nuclear and mitochon- 40: Amaoka K. 2004. Phylogeographic relationships within the drial gene sequences. Mol. Phylogenet. Evol. 317–331. Mediterranean turbot inferred by mitochondrial DNA haplo- DOI 10.1016/j.ympev.2006.02.006 type variation. J. Fish Biol. 65: 580–585. DOI 10.1111/j.0022- Katsares V., Tsiora A., Galinou-Mitsoudi S. & Imsiridou A. 2008. 1112.2004.00433.x Genetic structure of the endangered species Pinna nobilis Tajima F. 1983. Evolutionary relationship of DNA sequences in (Mollusca: Bivalvia) inferred from mtDNA sequences. Biolo- finite populations. Genetics 105: 437–460. 63: gia 412–417. DOI 10.2478/s11756-008-0061-8 Thiede J. 1978. A glacial Mediterranean. Nature 276: 680–683. Koehn R.K., Newell R.I.E. & Immermann F. 1980. Maintenance DOI 10.1038/276680a0 of an arninopeptidase allele frequency cline by natural selec- Thompson J.D., Higgins D.G. & Gibson T.J. 1994. CLUSTAL tion. Proc. Natl. Acad. Sci. USA 77: 5385–5389. W: improving the sensitivity of progressive multiple sequence Maes G.E. & Volckaert F.A.M. 2002. Clinal genetic variation and weighting. position-specific gap penalties and weight metric isolation by distance in the European Anguilla Anguilla choices. Nucleic Acids Res. 22: 4673–4680. (L.). Biol. J. Linn. Soc. 77: 509–521. DOI 10.1046/j.1095– Wirth T. & Bernatchez L. 2001. Genetic evidence against pan- 8312.2002.00124.x mixia in the European eel. Nature 409: 1037–1040. DOI Medhioub M.N. 1993. La conchyliculture en Tunisie. Pro- 10.1038/35059079 jet Tunis/92/002. Direction Générale de la P˛eche et de Worms J. & Pasteur N. 1982. Polymorphisme biochimique de l’Aquaculture. PNUD/FAO, 83 pp. la palourde, ,deI’étangduPrévost Mills L.S. & Allendorf F.W. 1996. The one-migrant-per-generation (France). Oceanol. Acta 5: 395–397. rule in conservation and management. Conserv. Biol. 10: Yuan T., He M. & Huang L. 2009. Intraspecific genetic variation 1509–1518. DOI 10.1016/j.1523-1739.1996.10061509.x in mitochondrial 16S rRNA and COI genes in domestic and Nei M. 1987. Molecular Evolutionary Genetics. Columbia Uni- wild populations of Huaguizhikong Chlamys nobilis versity Press, New York, 512 pp. Reeve. Aquaculture 289: 19–25. Neigel J.E. 1994. Analysis of rapidly evolving molecules and DNA Zardoya A., Castilho R., Grande C., Favre-Krey L., Caetano S., sequence variants: Alternative approaches for detecting ge- Marcato S., Krey G. & Patarnello T. 2004. Differential popu- netic structure in marine populations. CalCOFI Rep. 35: 82– lation structuring of two closely related fish species, the mack- 89. erel (Scomber scombrus)andthechubmackerel(Scomber Nikula R. & Vainola R. 2003. Phylogeography of Cerastoderma japonicus) in the Mediterranean Sea. Mol. Ecol. 13: 1785– glaucum (Bivalvia: Cardiidae) across Europe: a major break 1798. DOI 10.1111/j.1365-294X.2004.02198.x in the Eastern Mediterranean. Mar. Biol. 143: 339–350. DOI Zitari-Chatti R., Chatti N., Elouaer A. & Said K. 2008. Genetic 10.1007/s00227-003-1088-6 variation and population structure of the caramote Passamonti M., Mantovani B. & Scali V. 1997. Allozymic car- Penaeus kerathurus (Forsk¨al) from the eastern and western 39: acterisation and genetic relationships among four species of Mediterranean coasts in Tunisia. Aquat. Res. 70–76. Tapetinae. Ital. J. Zool. 64: 117–124. Zitari-Chatti R., Chatti N., Fulgione D., Caiazza I., Aprea G., Passamonti M., Montavani B. & Scali V. 1998. Characterization El Ouaer A., Said K. & Capriglione T. 2009. Mitochondrial of a highly repeated DNA family in Tapetinae species (Mol- DNA variation in the caramote prawn Penaeus (Melicertus) lusca Bivalvia: Veneridae). Zool. Sci. 15: 599–605. kerathurus across a transition zone in the Mediterranean Sea. Genetica 136: 439–447. Pérez-Losada M., Guerra A., Carvalho G.R., Sanjuan A. & Shaw P. W. 2002. Extensive population subdivision of the cuttlefish Zouros E., Ball A.O., Saavedra C. & Freeman K.R. 1994. An unusual type of mitochondrial DNA inheritance in the blue Sepia officinalis (Mollusca: Cephalopoda) around the Iberian 91: Peninsula indicated by microsatellite DNA variation. Hered- mussel Mytilus. Proc. Natl. Acad. Sci. USA 7463–7467. ity 89: 417–424. DOI 10.1038/sj.hdy.6800160 Received October 6, 2009 Pérez-Losada M., Guerra A. & Sanjuan A. 1999. Allozyme differentiation in the cuttlefish Sepia officinalis (Mollusca: Accepted March 30, 2010