Impact of geographical isolation on genetic differentiation in an insular population of the carpet shell Ruditapes decussatus in the Mediterranean Basin.
KEY WORDS: Ruditapes decussatus, genetic differentiation, Kerkennah Island. Tunisia
Studies of genetic structure in marine species continue to increase, especially concerning commercially exploited species (Utter 1991, Thorpe et al. 2000). Some of the main putative roles for genetics in marine fisheries may be summarized as understanding the structuring of populations; identification of stocks (breeding units) and units for conservation; mixed stock analysis; genetic effects on growth rate, survival, disease resistance, or other important parameters; and development of strains for captive breeding. All of these possible uses are potentially relevant to marine species, and, of course, they are not all mutually exclusive; they are not completely discrete and in any practical application there is likely to be a degree of overlap between many of them. For example, the identification of breeding units is likely to entail understanding the structuring of populations and possibly also the identification and separation of mixed stocks (Thorpe et al. 2000).
The carpet shell Ruditapes decussatus (Linnaeus, 1758) was chosen as it is a commercially valuable bivalve mollusc. It is a morphologically and ecologically variable species occurring in a wide variety of habitats over its distribution range, which extends from the North Sea to the coast of Senegal and along the coasts of the whole Mediterranean, reaching as far east as the Red Sea. The carpet clam is a euryhaline species often occurring in marine and coastal lagoon habitats, lives in muddy sand sediments of tidal flats or shallow coastal areas (Parache 1982). It is a gonochoric species with external fertilization and planktonic larvae, although rare juvenile hermaphroditism has been reported (Lucas 1968). Hatchery experiments have shown that the duration of the planktotrophic larval stage is 8-10 days at 25[degrees]C (Borsa et al. 1994). Adult clams are sedentary. Samples of R. decussatus analyzed in this study were collected from either mainland or insular areas.
Marine organisms that inhabit isolated islands are faced with limited adult habitat and with marine currents that advect larvae away from the source population; some species have probably developed mechanisms for population maintenance, but these remain largely unknown at the present time. Thus the problems of recruitment of island resources differ significantly from those of species associated with continental shelf and slope regions, regardless if in boreal, temperate, or tropical regions.
Several early studies held that insular populations appear to have considerable importance for ecology and evolution (Otto & Endler 1989, Futuyma 1998). Insular populations often characterize the total genetic structure on the phylogeography of the species as a whole (Cheylan et al. 1998, Paetkau et al. 1998). As shown in the list of extinct species during the past several decades, a substantial proportion of endangered and vulnerable organisms are insular forms (Frankham 1997). The reasons for their higher extinction risk remain controversial, but recent conservation practice regards loss of genetic variability as one of the driving forces for extinction (Sheridan 1995, Meffe 1996). Species or populations with low genetic variability would be expected to have such reduced ability to cope with environmental change during evolution that they have a shorter evolutionary lifespan (O'Connel & Wright 1997). Although few empirical demonstrations for such associations in wildlife are available, there are many, though indirect, examples from genetics that support this hypothesis (Frankham 1995).
Theoretical models of subdivided finite populations consider local extinction, migration, and mutation rates as determinants of genetic variability (Slatkin 1977, Wade & McCauley 1988, Hedrick & Gilpin 1997). Compared with mainland populations, insular populations tend to have quantitatively lower genetic variability, because topographical isolation limits immigration and the smaller carrying capacity enhances the probability of local extinction through stochastic events (Gilpin & Soule 1986). More detailed analysis, however, is expected to reveal that each insular population possesses its own unique genetic variability according to the geographic properties of the island that can affect patterns of migration and extinction rates. One goal of conservation is to maintain the evolutionary potential of species or populations under natural environmental conditions (Jones et al. 1996). In this context, the genetic status of the population concerned should be assessed for deviation from the above expectation rather than by the detected genetic variation itself.
Allozyme variation in some populations of Ruditapes decussatus was already studied by (Gharbi et al. 2011) who provided evidence of high level of genetic variability in almost all populations and especially in insular population. Indeed, genetic variation and differentiation in populations of the species were previously also investigated by Gharbi et al. (2010), based on the sequencing of a portion of a mitochondrial gene and of the internal transcribed spacer region 1 (ITS1).
Genetic and morphometric variability of some Ruditapes decussatus populations was investigated paying particular attention to the Kerkennah insular population. Specifically, the following questions were posed: (1) Is Kerkennah Island population genetically and morphologically differentiated from mainland populations? (2) Do Kerkennah sample exhibit particular changes in the overall genetic diversity?
MATERIALS AND METHODS
Samples of Ruditapes decussatus used in this study were obtained from nine mainland localities of western and eastern Mediterranean, and from two insular areas (Kneiss Island and Kerkennah Island). The precise geographic origin of each sample and the number of individuals analyzed per population are indicated in Figure 1 and in Table 1.
Morphological A nalysis
A total of 340 specimens of Ruditapes decussatus were collected from 11 localities in Tunisian waters (Table 1). They have a variety of shapes and colors. Five morphometric parameters of shell valves were measured, including the width (greatest anteroposterior dimension), height (greatest dorsoventral dimension), the thickness of the tightly closed animal and two angles (see Fig. 2). All measurements were made by one person, the writer, with a sliding caliper reading directly to millimeters and by a vernier to 0.1 mm. The valves were also weighed on a balance sensitive to 0.01 g.
Two categories of studies were carried out: the first study was based on analysis of both variances and means of morphological characters using analysis of variance, Fisher's Fand least significant difference tests, and Student's /-test; the second study were submitted to multivariate analysis, the principle component analysis (PCA), all analysis using STATISTICA program.
The electrophoretic analysis was undertaken also for 346 specimens from 11 localities, on the eastern and western Mediterranean coasts of Tunisia, to establish the extent of gene flow and levels of genetic differentiation across the known Siculo-Tunisian region. Gene products for 15 presumptive enzyme loci were analyzed (Hk-1, Mod-1, Mod-2, Est-3, Est-14, Sod-1, Idh-1, Mpi-1, Lap-1, Pgm-1, Pgm-2, Mdh-1, Mdh-2, Gpi-1, and Aat-1). The buffer systems used, electrophoretic procedures, and loci and allele designations were describe in Gharbi et al. (2011).
Genotypic and allelic frequencies were determined by direct counts from allozyme phenotypes, and the resulting data were analyzed by various statistical methods to describe the genetic structure of the Ruditapes decussatus populations. All genetic variability and genetic distance measures were calculated using the computer programs GENETIX 4.03 (Belkhir et al. 2001) and GENEPOP 3.4 (Raymond & Rousset 2003). An estimation of phenetic relationships among populations was obtained by generating a phenogram of all samples by means of the unweighted pair-group method with arithmetic averaging (UPGMA) based on the matrix of Nei's unbiased genetic distances (Sneath & Sokal 1973).
Cytochrome c Oxidase Subunit I and ITS1 Analysis
Sequences analysis was performed respectively on 561 bp of the cytochrome c oxidase subunit I (COI) of mitochondrial DNA (mtDNA) and on 597 bp of ITS1 gene amplified by the polymerase chain reaction (PCR). Amplification conditions were described in Gharbi et al. (2010).
Data analyses were performed using ARLEQUIN 3.0 (Excoffier et al. 2005). Intrapopulation diversity was quantified by estimating gene diversity (h) (based on the frequency of haplotypes within a population), and nucleotide diversity (7t) (as the average number of nucleotide substitutions per site for the sequences sampled). To estimate the amount of gene flow between populations and to test for the geographic structuring of collections, the pairwise genetic distances of fixation index ([F.sub.ST]) was calculated with analysis of molecular variance in Arlequin.
Detectable levels of genetic variation often depend on the sensitivity of molecular markers examined. Microsatellite DNA is effective in detecting variation in some species with low levels of either allozyme or mtDNA polymorphism (Brunner et al. 1998).
In this study, a published expressed sequence tag (EST) database for Ruditapes decussatus (Tanguy et al. 2008) was used to search microsatellites. A total of 4,640 target EST of R. decussatus were downloaded from the NCBI-EST database and subject to bioinformatic analyses. All the EST were screened for potential microsatellites by using the software "Tandem Repeat Finder" (Benson 1999). A total of EST or genes were chosen for pilot tests for primer design, locus amplification, and polymorphism. Twenty-one of these EST contained microsatellites inside were maintained. Software "Primer 3" (http://www.genome.wi.mit.edu/cgi-bin/primer/) was used to design primers for the amplification of repeat regions of interest across the flanking regions.
After PCR amplification and polymorphism test for microsatellites, seven of the 21 EST-microsatellites were found to be polymorphic in Ruditapes decussatus populations. In this study five microsatellites loci were only genotyped (from dec-1 to dec-5; Table 2).
Amplification reactions were performed in 25 [micro]l volumes containing pure Taq Ready-to-go PCR beads from Amersham Biosciences, 2 of DNA extract and 1 [micro]l from each primer (with the forward primer fluorescently labeled). A precycling denaturation step of 3 min at 94[degrees]C was followed by 45 cycles of 94[degrees]C denaturation for 1minute, 53[degrees]C annealing for lmin, and 72[degrees]C extension for 1minute, followed by a final extension step of 72[degrees]C for 10 min. Primer sequences, type of microsatellite repeats, and PCR conditions for polymorphic microsatellites loci are described in Table 2. PCR products were run on an ABI PrismTM 310 automated sequencer and analyzed with the GeneMapper Software Version 4.0 to provide alleles calls.
The analysis of variance and Fisher's least significant difference test for several examined characters revealed significant average differences (P < 0.05) among sampling sites, leading to the rejection of the null hypothesis of "no heterogeneity". Significant differences in variances and means for valves' weight, width, height, and thickness were revealed. Samples from Kerkennah Island and from Monastir were different from others samples, they have larger shells than the others (Fig. 3).
According to correlation matrix (data not shown), the most important discriminative character in distinguishing between the samples was width, which contributed to defining the first PC A axis (axis 1). Both angle alpha and angle beta defined the second and the third axes (Fig. 4).
The PCA of Ruditapes decussatus variables yielded three initial factorial axes, accounting for 63.56%, 18.78%, and 17.62% of total variance, respectively. Hence, the three axes were chosen for the analyses to expressing variation; however, here we have only plotted two PCA axes. Transforming and plotting the expression data in a two-dimensional graph resulted in a relatively distinction among the 11 studied localities (Fig. 4). The Kerkennah island sample, which projected onto the positive side of axis 1, was the most extreme sample along PCA axis 1 and seems to be different from all others samples.
Of the 15 electrophoretic loci analyzed, 6 (40%) were monomorphic and fixed for the same allele in all samples, whereas the remaining 9 (60%) loci were found to be polymorphic showing from 3 (Mdh-2) to 7 alleles (Gpi and Mpi) (Gharbi et al. 2011).
Quantitative parameters of the genetic variability, heterozygosity (H), proportion of polymorphic loci at the 95% criterion (P95) and the mean number of alleles per locus (A), showed a high genetic variation among all samples (Table 3). The observed heterozygosity (Ho) values ranged from 0.24 to 0.36 and were the highest in the Kerkennah Island sample. Positive multilocus estimates of the fixation index (FIS) values at almost all populations indicated significant heterozygote deficiencies (Table 3).
Pairwise Fsx values are given in Table 4 and were significantly different from 0 (P < 0.05) in 30 (54%) of the 55 comparisons. After the sequential Bonferroni correction, 23 of the pairwise comparisons remained statistically significant. All significant [F.sub.ST] values were found between populations belonging to different regions (western and eastern regions) and between the insular population of Kerkennah and almost all the remaining populations.
Clams from these other 10 populations, however, evidently do not constitute a single homogeneous population. The genetic relationships among the samples studied are presented in Figure 5. The UPGMA clustering procedure revealed two main clusters in the phenogram constructed on the basis of the matrix of Nei's unbiased genetic distances. The first cluster includes the related samples (Faroua, Mzjemil, and Tunis) belonging to western Mediterranean basin; however, the second cluster includes remaining samples from the eastern Mediterranean one. The Kerkennah Island sample is genetically distinct from the samples belonging to the same geographic region, and it is located in a separate branch.
Cytochrome c Oxidase Suhunit I and IT SI Analysis
Twenty putative haplotypes (H1-H20) resulting from 20 variable sites were detected from the mtDNA COI gene. The number of haplotypes per site ranged from 2 to 6. The frequency of H1 was higher than 70% in Ruditapes decussatus sample and was the only haplotype shared by all populations, whereas the majority of the other haplotypes (65%) were unique. Within locality, genetic diversity was estimated in terms of haplotype diversity (h) and nucleotide diversity (7t) (Table 3). Both diversity indices were highest in Kerkennah and Kneiss Islands. As shown in Table 5, [F.sub.ST] values were generally low, suggesting small genetic differentiation between samples.
A total of 21 different haplotypes (H1-H21) were found among all samples for ITS]. The haplotypes differed from one another by 1-4 mutations (indels excluded), and had pairwise sequence divergence values ranging from 0.17% to 0.68%. Compared with COI, genetic diversity indices of ITS1 were high. The overall n value was 0.0021 and the within population [pi] values ranged from 0.0013 to 0.0056 (Table 3). Haplotype diversity values ranged from 0.384 to 0.933 (Table 3).
Pairwise [F.sub.ST] values were significantly different from 0 (P < 0.05) in 21 (38%) of the 55 comparisons including all populations (Table 5).
The most common haplotypes within populations described in this article have been submitted to the GenBank data library under accession nos KC149953-KC149960 and HQ179957-HQ179965 for COI and ITS1, respectively.
The five analyzed loci were found to be polymorphic showing from 6 (dec-5) to 12 alleles (dec-1). The mean expected heterozygosity (He) and Ho of these polymorphic loci are given in Table 6 and ranged from 0.58 to 0.66 and from 0.33 to 0.50, respectively. Higher heterozygosities and number of alleles were observed for Kerkennah island sample. When the frequencies and distributions of the alleles and genotypes were compared under the Hardy-Weinberg equilibrium expectation for an ideal population (random mating, no mutation, no drift, and no migration), all loci showed heterozygote deficits (occurs when there are more homozygotes than expected under HardyWeinberg equilibrium) in all samples. Likewise, the multilocus test by population showed departure from panmixia in all populations with a significant (P <0.05) heterozygote deficiency (Table 6). The between pairs of population [F.sub.ST] estimates are given in Table 7. All pairwise comparisons are presented for these populations. [F.sub.ST] values varied between -0.002 and 0.054. Among 15 comparisons, 8 (53%) were significantly different from 0 (P < 0.05) and after the sequential Bonferroni correction, only two of the pairwise comparisons remained statistically significant. These significant [F.sub.ST] concerned the comparisons between the insular population of Kerkennah and almost all the remaining populations.
The species Ruditapes decussatus is characterized by a relatively high inter- and intrapopulation variability in morphological traits. Various ecological factors are identified for their effect on bivalve shell shape: wave impact, trophic conditions, water depth, density, etc. (Seed 1980, Caill-Milly et al. 2012). Other factors such as nature of the sediment have also been proposed for their influence on Venerids' shape. For example, for Gerard (1978), the nature of the sediment is of a great influence on the sharpness of the shell. In this study, globular character was depicted in region with muddy substrates (data not shown).
Specimens from Kerkennah Island show significant distinctive morphological characteristics. They have thicker and larger valves perhaps to avoid predation or could be related to the "island rule". Mainland small animals tend to develop larger body sizes after colonizing islands for ecological strategy (Lomolino 1985, 2005).
Islands are somehow fragile and tend to have fewer species than mainland areas. Therefore, predation pressure on island may be stronger than in mainland areas because the resource base is reduced: specimens with a large body size in Kerkennah Island are in some way a result of the combination of the interspecific competition and predation pressure.
Moreover, from an evolutionary point of view, defense against predator is considered as the most important function of the shell as reminded by Tokeshi et al. (2000). Considering different species of bivalves including a related species Ruditapes variegatus, these authors pointed out that the larger the shell, the more resistant the shell is regarding breakage by predators. For Macoma balthica in the North Sea, the hypothesis of a selective predation of the more globular shells has been proposed by Luttikhuizen et al. (2003).
In this study, the results of the allozyme analyses indicate that genetic variability is relatively high in Ruditapes decussatus. The level of polymorphism was within the range detected in marine invertebrates, whereas the average heterozygosity (Ho = 0.29) was higher than the average 0.15 reported for these organisms (Berger 1983, Buroker 1983, Gallardo et al. 1998). The highest values of heterozygosity were found in the sample from Kerkennah Island. Likewise, the level of genetic variability for COI and ITS 1 markers quantified using h and [pi] was high for the two populations from Kerkennah and Kneiss islands.
Based on the theory (see e.g., Nei et al. 1975) levels of genetic variability in the insular sample of Ruditapes decussatus are expected to be low because this is more subject to the effects of geographic isolation, smaller population size, and founder effect. Nevertheless, results were in some way not congruent with these expectations as sample from Kerkennah island was characterized by levels of percent polymorphism and heterozygosity similar and sometimes higher than those observed in most of mainland populations. This finding is in agreement with the microsatellites data. This is probably because the insular populations inhabit marginal environments characterized by temporal and ecological instability. According to Lewontin (1974), in such environments no particular genotype is favored for long periods and natural populations usually show levels of genetic variability higher than those found in more stable environments. On the basis of these considerations, finding greater genetic variability in insular populations of R. decussatus could indicate that high heterozygosity levels can be preserved after colonization events in marginal populations of invertebrates, unless founder populations are so small that bottleneck effects occur.
The investigated sample from Kneiss Island exhibit genetic variability similar to mainland ones. One of the islands considered here (Kerkennah) is a large island (160 [km.sup.2]), whereas the other (Kneiss) is a tiny island (0.5 [km.sup.2]), and is separated by a relatively short geographic distance from mainland. In general, the magnitude of the stochastic factors that affect insular populations can vary according to island size. Levels of genetic diversity have been related to, among other things, island size, because larger islands can usually sustain higher population sizes, whereas small islands with lower population sizes are frequently associated with inbreeding, genetic bottlenecks, and higher extinction rates. A coastline of ~110 km in the Kerkennah Island could promote sufficient population sizes of Ruditapes decussatus as to avoid significant levels of inbreeding and/or genetic drift.
As deduced from h within a population, the level of genetic variability quantified using [pi] may be explained by the rate of gene flow. We hypothesize that the genetic variability on each island is a product of a dynamic equilibrium maintained by stochastic events, that is, continuous immigration from the mainland population balanced by local extinction. This view is analogous to the theory of island biogeography (MacArthur & Wilson 1967, MacArthur 1972), although we have examined populations rather than communities and focused on genetic variability rather than species richness.
The results illustrate that the geographical properties of islands allow for particular genetic equilibria. When an island is located far from the mainland and other conditions remain the same, the migration rate will be low owing to the isolation by distance, and accordingly the equilibrated genetic variability will be low. Likewise, when an island is small in size, the extinction rate of a haplotype will be high owing to the effects of demographic and genetic stochasticity (Gilpin & Soule 1986), and accordingly the equilibrated genetic variability will be low. Namely, for a given island size, populations on more isolated islands will have less variation, and for a given degree of isolation, populations on smaller islands will have less variation than those on larger islands. Indeed, among the surveyed populations, only the Kerkennah population seems to achieve distinctive morphological characteristics. On islands closer to the mainland and smaller in size, in contrast, genetic equilibrium will attain sooner, without any signs of phenotypic changes (Kneiss Island).
As inferred from variation in [pi] value, genetic variability observed in the insular population was on average in the same level (ITS 1 marker) and sometimes higher (COI markers) than that in the mainland populations. Given that high genetic variability is associated with decreased vulnerability to extinction, the overall high genetic variability observed in the insular populations of this species implies their lower extinction risk. Ultimately, high genetic variability is deemed to favor species to adapt to changing environmental conditions and respond to selection (O'Connel & Wright 1997). Proximally, high phenotypic variability involving the fixation of adaptive characteristics is involved. Increase of the variation in spawning period caused by elevated genetic variability may also make populations more resistant to diseases and catastrophes.
Genetic Structure and Differentiation
The genetic heterogeneity analysis demonstrates a certain amount of genetic differentiation among local populations of Ruditapes decussatus, with a relatively high level of genetic subdivision. Microsatellite data presented here indicate a certain amount of molecular divergence among R. decussatus populations and are congruent with the results of the molecular investigations (analysis of mitochondrial DNA sequences). Allozyme data show that, at the scale of the study, genetic variation in R. decussatus is distributed into two major population groups according to their geographic origin on either side of the Siculo-Tunisian Strait: the first includes the related three westernmost localities (Mzjemil, Faroua, and Tunis), the second the remaining localities (eastern group). The hierarchical F-statistics analysis showed an among-groups [F.sub.ST]t estimate ([F.sub.ST] = 0.030; P < 0.001) higher than that at the total population level (Gharbi et al. 2011). The results of the allozyme investigations show a pronounced geographical structure of the R. decussatus populations (Table 4). On the other hand, the data suggest that Kerkennah sample is quite different morphologically and genetically from the other 10 populations. It is genetically differentiated and appears to differ more from the other 10 populations than these 10 differ among themselves. The UPGMA phenogram (Fig. 5) depicts this relationship more clearly (allozymes data). In the absence of obvious barriers to gene flow and the substantial migration rate from and to Kerkennah population, as estimated from F-statistics (Nm ranged from 2.6 to 8.7), this genetic differentiation could result from the occurrence of past biogeographical barriers; however, exchanges probably occurred between the island and mainland because of intermittent contacts due to variations in sea level during Pleistocene glaciations. According to Blanc and Cariou (1987), the Kerkennah archipelago has been cut off from the mainland for 13,000 y; geological data show that communications with the Sahelian littoral could have occurred at several times in the past, especially between 40,000 and 80,000 y ago.
In addition to historical events, ecological attributes such as colonization or dispersal abilities should play a role in establishing patterns of genetic differentiation and colonization events. Although, paleo-oceanographic data about colonization of the island are lacking, it seems likely that the Kerkennah population have established sufficiently long ago to accumulate such high genetic differentiation. The continued existence of this genetic discontinuity suggests it has yet to be erased by present-day gene flow or indicates that present-day gene flow was overestimated. Other plausible explanations for the differentiation of Kerkennah population such as selection against migrants cannot be excluded. Selection against migrant larvae may act as a primary barrier to gene flow by reducing the contribution of migrant genes to a population, and it may also have a secondary effect through the evolution of enhanced habitat choice behavior where local adaptation increases fitness of residents relative to migrants.
Moreover, several mechanisms, in fact, can be proposed to explain genetic differentiation of Kerkennah population. The retention of larvae around islands is essential to the maintenance of populations (Leis & Miller 1976). Unfortunately, concurrent physical data are unavailable from this study to test the retention mechanisms proposed by several scientists; however, we emphasize the possibly marine current, which runs along the Coast of Kerkennah Island and that presumably act to trap waters for significant periods of time. Such currents are important in retaining planktonic stages (larvae), increasing their chance to return to their adult coastal habitat, and thus helping to maintain the influx of larvae around the Kerkennah Island. It appears that larval dispersal localized to the island or location of spawning may well account for the genetic differentiation found for Kerkennah population of Ruditapes decussatus.
In summary, in this study a relatively high level of variability for analyzed markers' pattern was detected. High level of genetic variability in marine invertebrates populations is a well-known fact (Berger 1983), especially for bivalves (Jarne et al. 1988, Nikula & Vainola 2003, Katsares et al. 2008, Yuan et al. 2009). Human impacts on insular populations lead to reduction in population size that could easily collapse the genetic variability of a small population. Overexploitation, habitat loss, or the introduction of alien species often promotes fragmentation and reduction of wild populations (Reid & Miller 1989, Sheridan 1995). Attention should be paid not to disturb the original local adaptation in distant larger populations by introducing maladaptive characteristics of alien stocks. Conserving genetic diversity in mainland populations, which are the source of gene flow, is also important for the conservation of insular populations.
We thank all people who contributed to Ruditapes decussatus sampling. We also thank the anonymous reviewer and editor for their constructive comments on the paper.
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AICHA GHARBI, (1,2) * KHALED SAID 2 AND ALAIN VAN WORMHOUDT (1)
(1) Station de Biologie Marine du Museum National d'Histoire Naturelle, BP225, 29900 Concarneau, France; (2) Laboratoire: Genetique, Biodiversite et Valorisation des Bioressources, LR11ES41, Institut Superieur de Biotechnologie de Monastir, Tunisia
* Corresponding author. E-mail: firstname.lastname@example.org
TABLE 1. Number of individuals (N) collected in each locality and for each analysis. Morphometry Allozyme COI ITS1 Microsatellites Population (N) (N) (N) (N) (N) Faroua (Bizerte 35 35 12 10 -- Lagoon) Mzjemil (Bizerte 30 31 10 15 30 Lagoon) Tunis 30 30 11 17 30 Monastir 30 30 22 14 30 Sfax 30 30 10 13 -- Kerkennah Island 30 30 15 11 30 Kneiss Island 35 35 12 9 -- Akarit 30 30 10 20 -- Galala (Bougrara 30 30 9 14 -- Lagoon) Bougrara 30 35 11 10 24 (Bougrara Lagoon) Biben lagoon 30 30 18 17 30 Total 340 346 140 150 174 TABLE 2. Microsatellites loci identified from EST database. Loci Motif Forward Reverse dec-1 TG CGCGGCCATACATTTAATCA CGGCAATAAATCAAACCAGAA dec-2 TGAG ATGCATCCCTCACCTTTGAA TCAGGCAAATCTTAAACATAATCG dec-3 AG TTGAACCCTGGACCTTAGGA TTTATAGTGTCGATTCGCCGTA dec-4 TC CAGCGCATCTGATTCTGGTA CATTCAGTGACGGTCTAATGG dec-5 TCA TGGGATTGCATTTGTTATTCG GTTCGATTGTGACGCCATTA dec-6 AAC CGTTGGAGGAAGTGAATGTC GCTACCACTAGCAAGGCACA dec-7 CAT AAAAGCGAGTGGGCCTTATT CAATCGCAATGGACAATCAC Loci tm Percent GC PCR product size dec-1 61-59 45-38 250 pb dec-2 60-59 45-33 216 pb dec-3 59 50-40 100 pb dec-4 59-58 50-47 264 pb dec-5 60-59 38-15 200 pb dec-6 59 50-55 240 pb dec-7 59 45-45 246 pb TABLE 3. Quantitative parameters of the genetic variability for populations of Ruditapes decussatus sampled. Allozymes Sample location N He Ho [P.sub.95] A [F.sub.is] Mzjemil 31 0.290 0.240 53.330 2.600 0.210 * (Bizerte Lagoon) Faroua 35 0.280 0.240 53.330 2.400 0.157 * (Bizerte Lagoon) Tunis 30 0.280 0.250 53.330 2.600 0.168 * Monastir 30 0.330 0.300 60.000 2.800 0.111 * Sfax 30 0.340 0.350 60.000 2.800 -0.025 Kerkennah 30 0.370 0.360 60.000 2.800 0.112 * Island Kneiss Island 35 0.310 0.280 60.000 2.730 0.141 * Akarit 30 0.320 0.280 60.000 2.800 0.155 * Galala 30 0.340 0.290 60.000 2.800 0.189 * (Bougrara Lagoon) Bougrara 35 0.340 0.310 60.000 2.800 0.124 * (Bougrara Lagoon) Biben 30 0.340 0.320 60.000 2.930 0.121 * Mean 31 0.321 0.292 58.180 2.732 0.131 * COI Sample location N h [pi] Mzjemil 10 0.377 [+ or -] 0.181 0.0007 [+ or -] 0.0008 (Bizerte Lagoon) Faroua 12 0.1667 [+ or -] 0.137 0.0003 [+ or -] 0.0004 (Bizerte Lagoon) Tunis 11 0.318 [+ or -] 0.163 0.0006 [+ or -] 0.0007 Monastir 16 0.177 [+ or -] 0.106 0.0003 [+ or -] 0.0004 Sfax 10 0.644 [+ or -] 0.101 0.0013 [+ or -] 0.0011 Kerkennah 15 0.666 [+ or -] 0.100 0.0023 [+ or -] 0.0017 Island Kneiss Island 12 0.757 [+ or -] 0.122 0.0020 [+ or -] 0.0014 Akarit 10 0.377 [+ or -] 0.181 0.0007 [+ or -] 0.0008 Galala 9 0.583 [+ or -] 0.183 0.0011 [+ or -] 0.0011 (Bougrara Lagoon) Bougrara 11 0.618 [+ or -] 0.164 0.0015 [+ or -] 0.0013 (Bougrara Lagoon) Biben 18 0.405 [+ or -] 0.142 0.0008 [+ or -] 0.0008 Mean 12 0.486 [+ or -] 0.198 0.0011 [+ or -] 0.0009 ITS1 Sample location N h [pi] Mzjemil 15 0.790 [+ or -] 0.051 0.0017 [+ or -] 0.0014 (Bizerte Lagoon) Faroua 10 0.866 [+ or -] 0.085 0.0025 [+ or -] 0.0019 (Bizerte Lagoon) Tunis 17 0.698 [+ or -] 0.075 0.0014 [+ or -] 0.0012 Monastir 14 0.802 [+ or -] 0.068 0.0020 [+ or -] 0.0015 Sfax 13 0.384 [+ or -] 0.132 0.0013 [+ or -] 0.0011 Kerkennah 11 0.563 [+ or -] 0.134 0.0016 [+ or -] 0.0013 Island Kneiss Island 9 0.805 [+ or -] 0.111 0.0024 [+ or -] 0.0018 Akarit 20 0.671 [+ or -] 0.091 0.0017 [+ or -] 0.0013 Galala 14 0.494 [+ or -] 0.087 0.0016 [+ or -] 0.0013 (Bougrara Lagoon) Bougrara 10 0.933 [+ or -] 0.077 0.0056 [+ or -] 0.0035 (Bougrara Lagoon) Biben 17 0.661 [+ or -] 0.125 0.0024 [+ or -] 0.0017 Mean 14 0.700 [+ or -] 0.177 0.0021 [+ or -] 0.0018 N, sample size; He. expected heterozygosity; Ho, observed heterozygosity; [P.sub.95], proportion of polymorphic loci at the 95% criterion; A. mean number of alleles per locus: Fis. multilocus estimates of the fixation index; h, haplotype diversity; [pi], nucleotide diversity. * P < 0.05. TABLE 4. Pairwise comparison of 11 carpet shell clam populations (allozyme data). Population Mzjemil Faroua Tunis Monastir Mzjemil -- 0.013 0.010 0.025 Faroua 0.011 -- 0.007 0.015 Tunis 0.004 -0.002 -- 0.022 Monastir 0.034#@ 0.016@ 0.028#@ -- Sfax 0.039#@ 0.037#@ 0.043#@ 0.006 Kerkenna 0.087#@ 0.094#@ 0.092#@ 0.042#@ Kneiss 0.026#@ 0.011@ 0.010 0.001 Akarit 0.038#@ 0.036#@ 0.015@ 0.012 Galala 0.028#@ 0.025#@ 0.027#@ -0.006 Bougrara 0.045#@ 0.026#@ 0.032#@ -0.001 Biben 0.043#@ 0.029#@ 0.028@ -0.005 Population Sfax Kerkennah kneiss Akarit Mzjemil 0.028 0.060 0.020 0.027 Faroua 0.025 0.061 0.012 0.024 Tunis 0.029 0.060 0.012 0.015 Monastir 0.012 0.035 0.009 0.016 Sfax -- 0.020 0.011 0.023 Kerkenna 0.012 -- 0.039 0.049 Kneiss 0.006 0.041#@ -- 0.012 Akarit 0.025@ 0.059#@ 0.007 -- Galala 0.001 0.028@ -0.002 0.012 Bougrara 0.010 0.037#@ 0.003 0.010 Biben 0.002 0.028@ 0.001 0.005 Population Galala Bougrara Biber Mzjemil 0.023 0.031 0.032 Faroua 0.022 0.020 0.021 Tunis 0.022 0.024 0.022 Monastir 0.010 0.011 0.007 Sfax 0.010 0.015 0.011 Kerkenna 0.029 0.035 0.025 Kneiss 0.011 0.012 0.009 Akarit 0.016 0.015 0.012 Galala -- 0.009 0.008 Bougrara -0.003 -- 0.009 Biben -0.006 -0.001 -- Above the diagonal Nei's (1972) unbiased genetic distances; below the diagonal pairwise [F.sub.ST] (Weir & Cokerham 1984) estimates. Significant values before (italic) and after (bold) sequential Bonferroni adjustment are indicated for [F.sub.ST] values. Note: bold indicated with #. Note: italic indicated with @. Note: Significant values before (italic) and after (bold) indicated with #@. TABLE 5. Pairwise FST values between populations of Ruditapes decussatus based on COI sequences (below diagonal) and on ITS1 sequences (above diagonal). Mzjemil Faroua Tunis Monastir Mzjemil -- -0.0257 -0.004 -0.0517 Faroua -0.0463 -- 0.0342 0.0195 Tunis -0.0426 -0.0578 -- 0.0357 Monastir 0.0540 -0.0201 0.0255 -- Sfax 0.2381 0.2687# 0.1840 0.2896# Kerkennah 0.1368# 0.1678# 0.1048 0.2386# Kneiss 0.1171 0.1468# 0.0779 0.1791# Akarit -0.0493 -0.0482 -0.0696 0.0304 Galala 0.0274 0.0551 -0.0057 0.0901 Bougrara 0.0365 0.0638 0.0058 0.0855 Biben -0.0376 -0.0103 -0.0316 0.0285 Sfax Kerkennah Kneiss Akarit Mzjemil 0.2086# 0.1143# -0.0256 0.0858# Faroua 0.1194 0.0063 -0.0753 0.0247 Tunis 0.1422# 0.2465# -0.0146 0.0411 Monastir 0.3037# 0.1790# 0.0354 0.1579# Sfax -- 0.2679# 0.337 0.0282 Kerkennah 0.1052 -- 0.0631 0.1764# Kneiss -0.0271 0.0301 -- -0.0239 Akarit 0.0873 0.0768 0.0205 -- Galala 0.0628 0.0267 -0.0093 -0.0278 Bougrara 0.0221 -0.0104 -0.0441 -0.0355 Biben 0.1159 0.0973 0.0549 -0.0519 Galala Bougrara Biben Mzjemil 0.1702# 0.0598 0.12365# Faroua 0.0371 -0.0227 0.0371 Tunis 0.2782# 0.1535# 0.2719# Monastir 0.2435# 0.0852 0.1565# Sfax 0.2367 0.2453# 0.3506# Kerkennah -0.0711 0.0453 -0.0253 Kneiss 0.0821 0.0209 0.1238# Akarit 0.1799# 0.1277# 0.2376# Galala -- 0.0868 0.0355 Bougrara -0.0439 -- 0.0329 Biben -0.0248 -0.0163 -- Significant values are indicated in bold estimates (5% level). Note: bold indicated with #. TABLE 6. Quantitative parameters for microsatellites data of the genetic variability for populations of Ruditapes decussatus sampled. Populations Mzjemil Tunis Monastir (n = 30) (n = 30) (n = 30) Fis 0.4271 * 0.2571 * 0.2834 * He 0.5817 0.6193 0.6202 Ho 0.3333 0.4511 0.4444 [P.sub.95] 1.0000 1.0000 1.0000 A 5.3333 5.6667 6.0000 Populations Kerkennah Bougrara Biben (n = 30) (n = 24) (n = 30) Fis 0.2445 * 0.4677 * 0.2842 * He 0.6611 0.6001 0.6054 Ho 0.5011 0.3194 0.4333 [P.sub.95] 1.0000 1.0000 1.0000 A 6.0000 5.0000 5.3333 Multilocus estimates of the [F.sub.ST] within each population are also indicated. * P < 0.05. TABLE 7. Pairwise [F.sub.ST] (Weir & Cokerham 1984) estimates of carpet shell clam populations for microsatellites data. Mzjemil Tunis Monastir Kerkennah Bougrara Biben Mzjemil 0.01603 0.01829 0.03418@ 0.03947@ 0.01403 Tunis -- 0.00859 0.05241#@ 0.04577@ 0.03209@ Monastir -- 0.05433#@ 0.03748@ 0.02427 Kerkennah -- 0.03567@ 0.01895 Bougrara -- 0.00276 Biben -- Significant values before (italic) and after (bold) sequential Bonferroni adjustment are indicated. Note: italic indicated with @. Note: Significant values before (italic) and after (bold) indicated with #@. Figure 5. Phenogram generated by UPGMA cluster analysis based on Nei's (1972) unbiased genetic distances (allozyme data) among Ruditapes decussatus populations. loci Motif Forward Reverse dec-1 TG CGCGGCCATACATTTAATCA CGGCAATAAATCAAACCAGAA dec-2 TGAG ATGCATCCCTCACCTTTGAA TCAGGCAAATCTTAAACATAATCG dee-3 AG TTGAACCCTGGACCTTAGGA TTTATAGTGTCGATTCGCCGTA dec-4 TC CAGCGCATCTGATTCTGGTA CATTCAGTGACGGTCTAATGG dec-5 TCA TGGGATTGCATTTGTTATTCG GTTCGATTGTGACGCCATTA dec-6 AAC CGTTGGAGGAAGTGAATGTC GCTACCACTAGCAAGGCACA dec-7 CAT AAAAGCGAGTGGGCCTTATT CAATCGCAATGGACAATCAC loci tm % GC PGR product size dec-1 61-59 45-38 250 pb dec-2 60-59 45-33 216 pb dee-3 59 50-40 100 pb dec-4 59-58 50-47 264 pb dec-5 60-59 38-45 200 pb dec-6 59 50-55 240 pb dec-7 59 45-45 246 pb
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|Author:||Gharbi, Aicha; Said, Khaled; Van Wormhoudt, Alain|
|Publication:||Journal of Shellfish Research|
|Date:||Dec 1, 2015|
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