Aquaculture Research, 2008, 39, 474^481
doi:10.1111/j.1365-2109.2008.01899.x
Genetic analysis of Aequipecten opercularis and
Mimachlamys varia (Bivalvia: Pectinidae) in several Atlantic and Mediterranean localities, revealed by mitochondrial PCR-RFLPs: a preliminary study Mercedes Fernandez-Moreno, Alberto Arias-Perez, Ruth Freire & Jose¢na Me¤ndez Department of Cell and Molecular Biology, Faculty of Sciences, University of A Coruna, A Coruna, Galicia, Spain Correspondence: J Me¤ndez, Department of Cell and Molecular Biology, Faculty of Sciences, University of A Coruna, A Zapateira s/n, 15071 A Coruna, Spain. E-mail: ¢
[email protected]
Abstract Aequipecten opercularis (Queen scallop) and Mimachlamys (Chlamys) varia (Black scallop) are important natural resources occurring in Atlantic and Mediterranean coasts. To develop an optimal sustainable exploitation plan, it is important to study the genetic structure of the di¡erent populations. In this study, we used polymerase chain reaction-restriction fragment length polymorphisms for the determination of the genetic variation and population structure of these two species in di¡erent localities. Ten composite haplotypes were generated for A. opercularis and 15 haplotypes for M. varia. Of these, six and four were unique respectively. The analysis of the distribution of the di¡erent haplotypes between the localities showed no clear evidence of subdivision in A. opercularis, while in M. varia the results indicated that the two localities analysed should be managed as separate stocks.
Keywords: 16S rRNA, 12S rRNA, PCR-RFLPs, population structure, Aequipecten opercularis, Mimachlamys (Chlamys) varia
Introduction Aequipecten opercularis (Queen scallop) and Mimachlamys (Chlamys) varia (Black scallop) are two pectinid species belonging to the subfamily Chlamydinae. Both species are characterized by circular shells and £attened valves. Their bodies are soft and they have a single central adductor muscle. These species are
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hermaphrodites. They have a similar distribution and are present all along the coast from Norway up to the Mediterranean Sea. Mimachlamys varia is found in the northeast coast of Africa right through to Senegal (Wagner 1991; Brand 2006). Both species are of economic interest; A. opercularis is found in ¢sheries on the Atlantic and Mediterranean coasts and M. varia is found in French Atlantic waters. A total of 13 418 tons of both species were captured in 2005, this being the second most abundant group in the production of scallops after the great scallops (FAO production statistics). Approximately 70% of marine invertebrate species have pelagic larvae, which create a high dispersal capability that may compensate for constraints on adult migration. Marine bivalve molluscs, which include scallops, oysters, mussels and clams, are sessile or sedentary as adults but have high fecundity and pelagic larvae. Consequently, larval transport is expected to play a prominent role in facilitating gene £ow and determining population structure. Gene £ow is highly correlated with dispersal ability (Burton 1983; Shaklee & Bentzen 1998; Bohonak 1999), and the oceanic environment is favourable for the mixing of populations through long-distance dispersal of adults, larvae and gametes. Studies that have surveyed genetic variation in marine bivalves over broad geographic scales have failed to identify population structure in some species (e.g. mussels: Skibinski, Beardmore & Cross 1983; clams: Benzie & Williams 1992; Vadopalas, Leclair & Bentzen 2004). However, genetically di¡erentiated populations over large scales along open coasts have been
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Genetic analysis of two pectinids by PCR-RFLPs M Fernandez-Moreno et al.
recognized (e.g. scallops and oysters: Reeb & Avise 1990; Beaumont & Zouros 1991; Heipel, Bishop & Brand1999; Arnaud, Monteforte, Galtier, Bonhomme & Blanc 2000; Launey, Ledu, Boudry, Bonhomme & Naciri-Graven 2002), along with small-scale genetic di¡erentiation (e.g. oysters: Buroker 1983; mussels: Ridgway 2001; clams: Luttikhuizen, Drent, Delden & Piersma 2003). Restoration of shell¢sh populations is becoming an increasingly common practice worldwide, as natural ¢sheries succumb to pressures of over-harvesting, habitat loss or degradation and challenges from invasive competitors and pathogens. Because the typical restoration project involves hatchery propagation of stocks for outplanting, some genetic impact on the recipient (wild) population is inevitable if the planted stocks survive and reproduce (Ga¡ney 2006). The ¢rst step in starting any aquaculture action is to know the genetic structure of the cultured or exploited populations (Heipel et al. 1999). In economically important species such as A. opercularis and M. varia, it is necessary to assess whether indeed the localities are di¡erent or whether they constitute a panmictic population. Nevertheless, studies of genetic variation in both species are scarce. Beaumont (1982) analysed the geographic variation in A. opercularis using three allozyme loci in Atlantic localities. Intraspeci¢c variation of A. opercularis and M. varia including Atlantic and Mediterranean localities was assessed by Lopez-Pinon, Insua and Me¤ndez (2002) using analysis of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the ITS region, but only to obtain molecular markers for species identi¢cation. Mitochondrial DNA (mtDNA) is generally a circular, double-stranded DNA molecule that is highly variable in its sequence but conservative in its gene content (Wolstenholme 1992). In Bivalvia, this genome is highly rearranged among di¡erent groups and some species have lost or gained additional gene copies (La Roche, Snyder, Cook, Fuller & Zouros1990; Ho¡mann, Boore & Brown1992; Kim, Je & Park1999). Moreover, some bivalves have a special model of mtDNA inheritance termed Doubly Uniparental Inheritance (DUI) (Zouros, Ball, Saavedra & Freeman 1994a, b). The presence of DUI has not been detected in scallops (Nagashima, Sato, Kawamata, Nakamura & Ohta 2005). Mitochondrial DNA has been useful in determining the genetic population structure of scallop species (Wilding, Beaumont & Latchford 1997; Heipel et al. 1999; Kong, Yu, Liu & Chen 2003; Nagashima
et al. 2005; Mahidol, Na-Nakorn, Sukmanomon, Taniguchi & Nguyen 2007), and it has been a successful marker in evolutionary studies among pectinidae (Canapa, Marota, Rollo & Olmo 1999; Canapa, Barucca, Marinelli & Olmo 2000; Matsumoto & Hayami 2000) or within the rest of the bivalves (Giribet & Wheeler 2002). PCR-RFLPs are a useful technique for systematic studies, species identi¢cation and in population analysis (Cronin, Spearman, Patton & Bickham 1993; Hall & Nawrocki 1995). In the present study, we used PCR-RFLPs of mtDNA in A. opercularis and M. varia from several localities to assess the level of genetic di¡erentiation of these species. The information obtained will provide a useful insight into the management practices of scallop resources.
Materials and methods Sample collection The samples were collected by dredge, 184 individuals of A. opercularis from four localities in Spain and one in Ireland, and 69 individuals of M. varia from two Spanish localities (Fig.1).
Figure 1 Map showing the localities analysed, corresponding toAequipecten opercularis and Mimachlamys varia along the Atlantic and Mediterranean coasts. Aequipecten opercularis from El Grove (Gr), Cambados (Ca), San Simon-Rande (SS) and Fuengirola (Fu) in Spain and Antrim (An) in Ireland. Mimachlamys varia from El Grove (Gr) and Fuengirola (Fu), both in Spain.
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DNA extraction, PCR and sequencing DNA was extracted from 20 mg of adductor muscle using the method described by Winnepenninckx, Blackeljau and Wachter (1993) based on extraction with CTAB bu¡er. Polymerase chain reactions were performed using universal primers. For the 16S rRNA, the primers 16Sar and 16Sbr (Palumbi 1996) were chosen because they allowed a reliable ampli¢cation of an internal region of 16S rRNA, matching domains IVand V. In the case of 12S rRNA, the primers selected were designed by Barucca, Olmo, Schiaparelli and Canapa (2004) and they were used to amplify an internal region of this gene corresponding to domain III. Polymerase chain reaction was performed in 25 mL of a solution containing 1ng mL 1 DNA, 10 mM TrisHCl,50 mM KCl pH 8.3,1.5 mM MgCl2, 2.5 mM dNTPs, 1U of Taq DNA polymerase and 1 mM of each primer. The PCR pro¢le consisted of one initial denaturation cycle of 3 min at 94 1C, followed by 35 cycles at 94 1C for 20 s, at 51 1C for 16S rRNA and 50 1C for the 12S rRNA for 20 s and at 72 1C for 45 s. A ¢nal extension was carried out at 72 1C for 5 min. Sequencing was performed at the sequencing laboratory in the University of A Coruna (Spain), using the Thermo SequenaseTM CyTM5 dye terminator cycle sequencing kit (Amersham Biosciences, Piscataway, NJ, USA) in an automatic capillary DNA sequencer (CEQTM 8000 Genetic Analysis System, Beckman Coulter UK, High Wycombe, UK). Data analysis Alignments were performed with CLUSTAL X program (Thompson, Gibson, Plewniak, Jeanmongin & Higgins 1997) using default parameters. Genetic distances (d) were calculated according to Tamura’s three-parameter model (Tamura 1992). Phylogenetic trees were constructed using MEGA software, version 3.1 (Kumar,Tamura & Nei 2004). PCR-RFLPs The ampli¢cation of the 16S rRNA gene was used to develop PCR-RFLPs. Di¡erent endonucleases were selected: Acs I, Alu I, Hae III and Hinf I forA. opercularis and Acs I, Alu I, Cfo I, Dra I, Hae III and Sty I for M. varia. They were used in trial screenings to assess variability. Restriction reactions were performed in a 20 mL volume containing 5 mL of PCR product, 1 reaction bu¡er and 3 U of endonuclease and incubated at the appropriate temperature overnight.
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The resulting fragments were resolved after electrophoresis on 2.5% agarose gels in 1 TAE (40 mM Tris-acetate,1mM EDTA, pH 8.0). Haplotypic data were analysed using ARLEQUIN, version 3.0 (Exco⁄er, Laval & Schneider 2005) to estimate the frequencies and gene diversity (Nei 1987) among samples. ARLEQUIN was also used to assess genetic di¡erentiation by several procedures: the analysis of molecular variation (AMOVA) method of Exco⁄er, Smouse and Quatro (1992), conventional pairwise FST computed from haplotype frequencies and an exact test of population di¡erentiation (Raymond & Rousset 1995). The correlation between genetic distances (measured as pairwise FST) and geographic distances was also studied using the Mantel test following the procedure of Smouse, Long and Sokal (1986). The signi¢cance of these procedures was assessed by 10 000 permutations.
Results Two mitochondrial regions, partial 16S rRNA and 12S rRNA genes, were chosen for this work. A preliminary study was performed to analyse sequence variability of both regions. PCR ampli¢cations in at least 30 individuals from each locality yielded a single product in all cases. The length of the region ampli¢ed was 482 nucleotides for A. opercularis and 566 nucleotides for M. varia in the case of 16S rRNA, and 432 bp forA. opercularis and 440 bp for M. varia in the case of12S rRNA, excluding the primers. The nucleotide sequences of these genes from two individuals of each locality showed few di¡erences among individuals, di¡ering at most by one substitution and/or by one bp indel. The 16S rRNA gene showed more variability than the 12S rRNA gene; therefore, the 16S rRNA gene was selected for PCR-RFLP analysis. Phylogenetic analysis carried out with the 16S rRNA and 12S rRNA sequences, both separately and in combination, with other species belonging to the same family selected from the GenBank was also included. They showed a topology in accordance with other molecular studies, with both nuclear and mitochondrial sequences (Canapa et al. 2000; Steiner & Hammer 2000; Insua, Lopez-Pinon, Freire & Me¤ndez 2003; Wang, Bao, Li, Zhang & Hu 2007) (data not shown).
PCR-RFLPs of A. opercularis After digestion of the mtDNA of 184 individuals from A. opercularis, 10 composite haplotypes were
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Genetic analysis of two pectinids by PCR-RFLPs M Fernandez-Moreno et al.
Table 1 Relative composite haplotype frequencies in Aequipecten opercularis from digestion of 16S rRNA
Haplotype
An (N 5 52)
Ca (N 5 33)
Fu (N 5 34)
Gr (N 5 33)
SS (N 5 31)
hap1 hap5 hap6 hap10 hap7 hap8 hap9 hap2 hap3 hap4
0.865 0.096 0.019 0.019 0 0 0 0 0 0
0.848 0 0 0 0.091 0.030 0.030 0 0 0
1 0 0 0 0 0 0 0 0 0
0.879 0 0 0 0 0 0 0.061 0.030 0.030
1 0 0 0 0 0 0 0 0 0
Composite haplotypes carried by single individuals. N, sample size; An, Antrim; Ca, Cambados; Fu, Fuengirola; Gr, El Grove; SS, San Simon-Rande.
observed. Of these, six were carried by single individuals; Cambados, El Grove and Antrim samples showed two each. Haplotype 1 was the only common one in the ¢ve localities. The total number of haplotypes per sample was four for Cambados, El Grove and Antrim and one for Fuengirola and San SimonRande. Gene diversity (Nei 1987) was similar for the three ¢rst sites, ranging from 0.229 to 0.278, and zero for the last two sites. Only one composite haplotype was shared among samples, showing a high frequency, ranging from 0.848 in Cambados to 1 in Fuengirola and San Simon-Rande (Table 1). AMOVA results indicated that 96% of the total genetic variation was within-site variability. Variability attributable to variation among the site groupings was negligible as indicated by a negative value. Approximately 4% of the total genetic variability was variation among sites within each grouping. The associated ¢xation indices were FSC 5 0.039, FST 5 0.038 and FCT 5 0.001 with a P-value of 0.047, 0.007 and 0.604 respectively. The pairwise FST values computed from haplotype frequencies were 0 (Fuengirola^San Simon-Rande) to 0.083 (Cambados^Fuengirola) (Table 2). Three comparisons were signi¢cant at Po0.05 (Antrim^ Fuengirola, Cambados^Fuengirola and El Grove^ Fuengirola) but none after sequential Bonferroni correction (Rice 1989). The global test of di¡erentiation among samples was signi¢cant (Po0.001), and the di¡erentiation test between all pairs of samples showed three signi¢cant values (Antrim^Cambados, Antrim^El Grove and Cambados^Fuengirola) at Po0.05 but none after sequential Bonferroni correction (Rice 1989).
Table 2 Genetic di¡erentiation between populations of Aequipecten opercularis
An Ca Fu Gr SS
An
Ca
Fu
Gr
SS
– 0.01444 0.06527 0.0079 0.06107
0.01334 – 0.08319 0.00226 0.07728
0.1265 0.02418 – 0.05631 0
0.02081 0.11825 0.05347 – 0.05134
0.18589 0.12732 NC 0.35894 –
Signi¢cant at Po0.05. Pairwise FST values below the diagonal and P-values of the differentiation test above the diagonal. An, Antrim; Ca, Cambados; Fu, Fuengirola; Gr, El Grove; NC, not calculable; SS, San Simon-Rande.
Table 3 Relative composite haplotype frequencies in Mimachlamys (Chlamys) varia resulting from digestion of the 16S rRNA Haplotype
Fu (N 5 29)
Gr (N 5 40)
hap2 hap12 hap3 hap1 hap9 hap10 hap13 hap6 hap7 hap5 hap4 hap8 hap11
0.379 0.276 0.138 0.103 0.035 0.035 0.035 0 0 0 0 0 0
0 0 0 0 0 0 0 0.5 0.2 0.125 0.1 0.05 0.025
Composite haplotypes carried by single individuals.
N, sample size; Fu, Fuengirola; Gr, El Grove.
Mantel test analysis did not detect a signi¢cant correlation between genetic distance (measured as pairwise FST) and geographic distance (correlation coe⁄cient 5 0.023, P 5 0.461).
PCR-RFLPs of M. varia After digestion of a fragment of M. varia 16S rRNA from two Spanish localities, we could establish 13 composite haplotypes. Of these, four were carried by single individuals (Table 3): one in El Grove and three in Fuengirola. The analysis of the distribution of these haplotypes showed that these two localities were polymorphic and that they did not share any haplotype. The El Grove sample showed six haplotypes and Fuengirola seven, with a gene diversity (Nei 1987) of 0.699 and 0.773 respectively.
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Haplotype frequencies ranged from 0.025 to 0.500 for El Grove and from 0.035 to 0.379 for Fuengirola. The pairwise FST value computed from haplotype frequencies was 0.266, which was signi¢cantly di¡erent from zero (Po0.001). The exact test of sample differentiation based on haplotype frequencies (Raymond & Rousset1995; Goudet, Raymond, de Meeˇs & Rousset 1996) was also signi¢cant (Po0.001).
Discussion This study is the ¢rst approximation in the development of molecular markers to assess the genetic structure of Atlantic and Mediterranean populations for A. opercularis and M. varia, two economically important species. Two mitochondrial regions, one fragment of 16S rRNA and another of 12S rRNA, were chosen for this approximation. The sequences obtained revealed a high level of conservation of these mitochondrial regions between the localities of the same species. The PCR-RFLPs for the 16S rRNA fragment were developed in the present study because this region was more variable than 12S rRNA. The 16S rRNA has been reported to be useful when analysing species and populations (Meyer 1994; Garland & Zimmer 2002), and this mitochondrial region has been used by other authors in the study of genetic diversity in di¡erent species of scallops (Kong et al. 2003; Mahidol et al. 2007). Ten and 13 composite haplotypes were observed for A. opercularis and M. varia respectively. Six and four of these are unique, they were only encountered once in all samples and additional sampling is needed to state their ‘private’character. The levels of intrapopulation variation measured as haplotype diversity were 0^0.278 for A. opercularis and 0.699^0.773 for M. varia; these values concur with the results obtained by several authors in other bivalves species using the same or di¡erent technique, i.e. Wilding et al. (1997) and Heipel et al. (1999), using mitochondrial PCR-RFLPs in di¡erent localities of Pecten maximus, reported values ranging from 0.641 to 1 and from 0.50 to 0.89 respectively. Diaz-almela, Boudry, Launey, Bonhomme and Lape¤gue (2004), developing SSCPs of a fragment of 12S rRNA gene in Ostrea edulis, presented an average mitochondrial haplotypic diversity of 0.49. Saavedra and Pena (2004), sequencing a region of the 16S rRNA in individuals belonging to the genus Pecten, reported a haplotype diversity ranging from 0 to 0.857. Taking into account the number of restriction enzymes used in
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most of these studies, and the use of sequencing and SSCPs, the genetic diversity of the mtDNA 16S RNA of M. varia may be considered to be very high (i.e. obtained with only ¢ve enzymes). However, this gene appears to be much less variable in A. opercularis. The pairwise comparisons between samples of A. opercularis, using the FST values or the di¡erentiation test, were not signi¢cant after Bonferroni correction. Remarkably, the comparisons signi¢cant at Po0.05 always involved Antrim or Fuengirola, localities separated by at least 1000 km from the other sites. Furthermore, the Fuengirola population is located in the Mediterranean Sea. Several studies have found a major intraspeci¢c discontinuity in genetic variation around the Straits of Gibraltar in populations of a number of marine species (Sanjuan, Zapata & Alvarez 1994; Quesada, Beynon & Skibinski 1995a; Quesada, Zapata & Alvarez 1995b; Borsa, Blanquer & Berrebi 1997; Rios, Sanz, Saavedra & Pena 2002; Garcia-vazquez, Izquierdo & Perez 2006). This discontinuity has been explained on the basis of the existence of historical, geographical or ecophysical barriers to gene £ow in this area. Several studies localize this break around the Almeria^Oran oceanographic front. Nevertheless, the extent of the genetic discontinuity seems to be somewhat di¡erent depending on the species involved, but at any rate, it is nearly always found that genetic divergence between populations is higher if populations are separated by this front than if they rely on the same side (Quesada et al.1995a, b). However, the AMOVA suggested di¡erences between localities; this may be attributed to the presence of population-speci¢c haplotypes in El Grove and Cambados sites. The global test of di¡erentiation among samples was also highly signi¢cant. The high frequency of population-speci¢c haplotypes observed (although they appeared in few individuals) suggests that current levels of gene £ow between the regions are restricted and that the lack of divergence revealed by the analysis could re£ect historic exchanges between populations. Another possibility to explain the low variability found in these species is that heavy ¢shing has substantially reduced the population size, which could have possibly resulted in a transient bottleneck. If populations were truly isolated, then mutations within haplotypes could result in ‘private’ haplotypes characteristic of a particular population (Slatkin 1985). The smaller gene diversity of Fuengirola and San Simon-Rande could be a sampling artefact, and the digestion of more individuals could reveal more haplotypes.
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Genetic analysis of two pectinids by PCR-RFLPs M Fernandez-Moreno et al.
The results of the statistical analysis of 16 rRNA in M. varia show a clear genetic di¡erentiation between the two localities. The lack of shared haplotypes between El Grove and Fuengirola suggests the absence of or a very low gene £ow between the sites. These populations are more than 1000 km apart, one being located in the Atlantic Ocean and the other in the Mediterranean Sea. The genetic £ow could be a¡ected, as in the case of A. opercularis, by the factors cited above. The present investigation indicates that M. varia localities should be managed as separate stocks to carry out a rational exploitation of the natural resources and to develop management plans. Nevertheless, we have to consider that only one molecular marker was used and that it would be desirable to analyse additional kind of molecular markers. Although the analysis of 16 rRNA of A. opercularis does not provide clear evidence of subdivision among the localities, the results do suggest that the hypothesis of population homogeneity is not applicable. The di¡erences in the haplotype distributions and the relatively high haplotype diversity indicate that these regions might be somewhat isolated and that di¡erent forces might be in£uencing the genetic structure of the localities. A more extensive analysis of additional populations would contribute to the understanding of population structures in this species, as well as to the development of sustainable exploitation plans.
Acknowledgments This work was funded by a grant from the Spanish ‘Ministerio de Ciencia y Tecnolog|¤ a (I1D1I) (AGL2003-07430)’. We are grateful to Dr Guillermo RomaŁn for supplying the samples and to Mr Jose Garc|¤ a Gil and Ms Mar|¤ a Tamayo for their technical assistance.
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