Genetic diversity and population differentiation of small giant clam Tridacna maxima in Comoros islands assessed by microsatellite markers
© The Author(s) 2016
Received: 25 May 2016
Accepted: 11 October 2016
Published: 22 October 2016
Small giant clam, Tridacna maxima, widely distributed from French Polynesia to East Africa, has faced population declines due to over-exploitation. Comoros islands are an important biogeographic region due to potential richness of marine species, but no relevant information is available. In order to facilitate devising effective conservation management plan for T. maxima, nine microsatellite markers were used to survey genetic diversity and population differentiation of 72 specimens collected from three Comoros islands, Grande Comore, Moheli and Anjouan. A total of 51 alleles were detected ranged from 2 to 8 per locus. Observed and expected heterozygosity varied from 0.260 to 0.790 and from 0.542 to 0.830, respectively. All populations have high genetic diversity, especially the population in Moheli, a protected area, has higher genetic diversity than the others. Significant heterozygote deficiencies were recorded, and null alleles were probably the main factor leading to these deficits. F ST value indicated medium genetic differentiation among the populations. Although significant, AMOVA revealed 48.9 % of genetic variation within individuals and only a small variation of 8.9 % was found between populations. Gene flow was high (Nm = 12.40) between Grande Comore and Moheli, while lower (Nm = 1.80) between Grande Comore and Anjouan, explaining geographic barriers to genetic exchanges might exist in these two islands. Global gene flow analysis (Nm = 5.50) showed that larval dispersal is enough to move between the islands. The high genetic diversity and medium population differentiation revealed in the present study offer useful information on genetic conservation of small giant clams.
KeywordsTridacna maxima Comoros islands Genetic diversity Population differentiation Gene flow Marine protected areas
The giant clam subfamily Tridacninae (Schneider and Foighil 1999) is the most widespread of the bivalves and is distributed throughout the Red sea and Indo-Pacific Ocean, from French Polynesia to East Africa (bin Othman et al. 2010). There are currently eight species from the genus Tridacna in the world: Tridacna. gigas (Linnaeus, 1758), T. maxima (Röding, 1798), T. crocea (Lamarck, 1819), T. squamosa (Lamarck, 1819), T. derasa (Röding, 1798), T. tevoroa (Lucas, Ledua and Braley, 1991), T. rosewateri (Sirenko and Scarlato, 1991), T. costata (Roa-Quiaoit, Kochzius, Jantzen, Zibdah and Richter, 2008) (bin Othman et al. 2010). Recently, T. noae was separated from T. maxima by their genetic and morphological description (Su et al. 2014). Among these bivalves, T. maxima has commonly the largest distribution range (Lucas 1988). All those giant clams are settled on the coral reef in shallow water and live in symbiotic photosynthetic with xanthophyllae algae (genus Symbiodinium) that grow in the mantle tissues (Soo and Todd 2014).
Like other marine bivalves, small giant clam species (T. maxima) are sedentary as adults, reproduce by broadcast spawning with high fecundity (>106 eggs per female), and have pelagic larval dispersal about 9 days (Lucas 1988). Based on these aspects, population genetics studies can provide more information about the ecological interactions, larval dispersal, distribution patterns, as well as evolution of the species. To date, most of studies have been conducted on T. maxima about spawning (Lucas 1994; Soo and Todd 2014), larval and post-larval development (Jameson 1976), and growth (Hart et al. 1998; Smith 2011; Toonen et al. 2011). Whereas only a few studies have been done on genetic diversity and genetic structure of T. maxima. Indeed, genetic variations studies using allozyme analysis (Campbell et al. 1975; Laurent et al. 2002) and, recently, mitochondrial markers (Nuryanto and Kochzius 2009), have provided information on highly genetic variability, larval dispersal and also the connectivity of different sites of Indo-Pacific Ocean that can be explained by marine currents or geographic isolation (Benzie and Williams 1992a, b).
Small giant clam is listed in Appendix II of CITES (United Nations Convention on International Trade in Endangered Species of Wild Fauna and Flora) and classified as lower risk conservation dependent on the IUCN (International Union for Conservation of Nature) Red List of Threatened Species. This status indicates that the population densities have declined in a large geographical region by their overexploitation and the degradation of their natural habitat (Lucas 1994; bin Othman et al. 2010; Hui et al. 2011). It seems to be still abundant according to the population densities data in some part of countries (Australia, up to 3.83 × 101 individuals per m2 and French Polynesia, 5.84 per m2, for instance, see bin Othman et al. 2010). Therefore, it is crucial to intensify the conservation efforts of marine biodiversity as well as to preserve the natural marine species for sustainable development.
Sample collection and DNA extraction
Sample details of T. maxima. For each sampling location, geographical coordinates, number (n) of individuals, shell length (L) and collection time are shown
Sample locality (abbreviation used)
From 11°23′S and 43°17′E to 11°29′S and 43°24′E
16.85 ± 4.34
From 12°22′S and 43°44′E to 12°22′S and 43°52′E
17.08 ± 3.68
12°05′S and 44°25′E
18.80 ± 5.50
Adductor muscles were taken, rinsed and preserved in 95 % ethanol until DNA preparation. Genomic DNA was extracted following the protocol described by Zhan et al. (2009). The DNA was checked on 1 % agarose gel and the concentration was determined for each sample using NanoView spectrophotometer, afterwards stored at −20 °C prior to genetic analysis performed.
SSR amplification and genotyping
Respective sequences of nine microsatellite loci of T. maxima developed by Grulois et al. (2014) using in our study
Primer sequence 5′-3′
For each marker, allele number (Na), allele frequency, observed heterozygosity (H O ), expected heterozygosity (H E ), Nei’s unbiased genetic distance and genetic similarity between populations were calculated using POPGENE 1.32 (Yeh et al. 1999). Allele richness (A R ) was carried out using FSTAT 2.9.3 (Goudet 2001). Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium were conducted using or GENEPOP 4.2 program (Rousset 2008). Sequential Bonferroni correction was conducted to adjust the significant level (Holm 1979; Rice 1989). The presence of null allele was detected using MICOR-CHECKER 2.2.3 (Van Oosterhout et al. 2004). F-statistics (F IS , F ST and F IT ) and gene flow (Nm) were calculated using GENETIX 4.05. Hierarchical Analysis of Molecular Variance (AMOVA) was conducted with ARLEQUIN 3.5 (Excoffier and Lischer 2010) to investigate regional population differentiation. Cluster analysis was performed to construct dendrogram using the unweighted pair group method average (UPGMA) by MEGA 6.06.
Among 72 individuals, a total of 51 alleles were detected. The alleles number per locus ranged from 2 to 8 (mean = 5.6). Overall, Mo specimens showed the highest H O and H E , 0.460 and 0.715, respectively. While Gc had the lowest value of H O and H E , 0.320 and 0.695, respectively (Table 4). Specimens from Mo revealed the highest mean value of Allelic richness (A R = 5.262).
Significant deviations from HWE (P < 0.05) were detected in 21 cases of the 27 locus-population combination after Sequential Bonferroni correction (Table 2). Null alleles decreased the number of significant deviations from HWE from 21 to 12 locus-population. Linkage disequilibrium was significant in only 4 out of 36 pairwise comparisons at the P < 0.05 level (Tm23637 vs Tm23670; Tm20025 vs Tm25349; Tm23637 vs Tm25349 and Tm24224 vs Tm25349), indicating virtually no linkage among loci.
Pairwise F st values (below diagonal) and number of migrants per generation (above diagonal) among three populations of T. maxima
The distance matrix showed that populations from Gc and Mo had the smallest genetic distance (0.120) and the highest genetic similarity (0.885) values, whereas Gc and An populations indicated the highest genetic distance (0.480) and the smallest genetic similarity (0.620) (Table 6). Furthermore, the unweighted pair group method average dendrogram revealed that Gc and Mo populations clustered together and An population formed one group.
Genetic diversity and deviation from HWE
Previous studies have indicated a high level of genetic diversity on T. maxima in Indo-Pacific Ocean (Ayala et al. 1973; Campbell et al. 1975; Nuryanto and Kochzius 2009) and other species on Tridacnidae family (Kochzius and Nuryanto 2008; DeBoer and Barber 2010; Hui et al. 2011). This present study shows a high level of genetic diversity for the small giant clams (H E = 0.699–0.714) within populations. Grulois et al. (2014) made the first attempt to investigate the genetic diversity of T. maxima using microsatellite markers, and observed a high value of expected heterozygosity (H E = 0.591–0.935) in New Caledonia. Comparing these two populations of T. maxima, one from Comoros islands in West Indian Ocean showed lower genetic diversity than the population from New Caledonia in Pacific Ocean. Vicariance process due to Pleistocene sea level fluctuation might be the main factor to affect the genetic diversity among populations of indo-Pacific Ocean (Williams and Benzie 1998; Carpenter et al. 2011). Oceanographic conditions and limited larval dispersal distance could be also an important factors to explain the genetic variability of populations (Froukh and Kochzius 2007).
Allele number (N A ), observed heterozygoty (H O ), expected heterozygoty (H E ), allelic richness (A R ), population inbreeding coefficient (F IS ) and Hardy–Weinberg equilibrium (HWE)
Grande Comore (Gc)
HWE (P value)
HWE (P value)
HWE (P value)
Genetic differentiation among populations
AMOVA analysis for three populations of T. maxima
Source of variation
Sum of squares
Percentage of variation
Among individuals within populations
Nei’s unbiased genetic similarity (above diagonal) and genetic distance (below diagonal)
Implication for conservation
Tridacnid species are listed in Appendix II of CITES and are classified as vulnerable on the IUCN Red List of Threatened Species due to their extreme exploitation for the food and marine ornamental trade. According to the CITES data, international trade of Giant clams non-captive bred increased from about 40,000 to 100,000 individuals between 1993 and 2001 (Wabnitz et al. 2003). In Comoros islands, despite the existence of the legislations for marine resources, T. maxima were especially exploited for food and their big shells were used for different purposes such as ornamental objects. Furthermore, over-exploitation, pollution, reef degradation by trampling or destructive fishing practices, and coral bleaching event due to rising sea temperature by El-nino event in 1997/1998 are likely to lead negative effects (ASCLME 2012). Therefore, one protected area (Moheli Marine Park) covering a total area of 403.6 km2 was created in Comoros to ensure the sustainable use of living marine resources (Beudard 2003).
In our study, the genetic diversity in the three islands showed that population from Mo (H E = 0.714; A R = 5.26) is higher than Gc and An (H E = 0.694; A R = 4.720, H E = 0.699; A R = 4.75), respectively. The protection of the area could be the major factor to explain high genetic diversity in Mo population. As there is a Marine national park in Moheli (Beudard 2003), species in the island benefit from its protection. Compared to the others islands where there are not restricted of any specific protection, Moheli is genetically more diverse, which can play an important role for allele distribution in the others islands. Therefore, Moheli Marine Park is most probably insufficient for the protection of T. maxima. While they have a larval dispersal time about 9 days, specimens of T. maxima are able to travel a distance about 500 km. Even though the populations between Gc and An showed low larval dispersal, it is possible to detect a connectivity for populations among the three islands from Comoros because of their small scale area. Therefore, further studies based on oceanographic barriers and ecological barriers in addition to genetic data are more important to understand the marine organism movements and connectivity between the islands. The genetic diversity and population differentiation of T. maxima can offer useful information to establish an effective plan for conservation management.
NAM carried out the experimental work, data analysis and interpretation of all genetic data, and drafted the manuscript. QY and LP assisted the experimental work and data analysis. MIC collected the samples of T. maxima. SW, XH and ZB have reviewed and have involved in drafting the manuscript. All authors read and approved the final manuscript.
We gratefully thank the Ministry of Environment of Comoros and the Faculty of Sciences and Technology, University of Comoros especially the LBEPA (Laboratoire de Biologie, Ecologie et Physiologie Animales) who provided the CITES export permit (No. 003/KM/15/DNEF) for expedition of T. maxima. Thanks to the staff of Marine Park of Moheli and fishermen of different islands for their help to obtain the samples. We would also to thank Athoumani Artadji for help to draw the map and the anonymous reviewers for their valuable comments and corrections. This work was supported by the Earmarked Fund for Modern Agro-Industry Technology Research System (CARS-48), Seed Improvement Project of Shandong Province and the National Infrastructure of Fishery Germplasm Resources.
The authors declare that they have no competing interests.
Ethics, consent and permissions
All the procedures involved in handling and treatment of small giant clams during this study were approved by the Ocean University of China Institutional Animal Care and Use Committee (OUC-IACUC) prior to the initiation of the study.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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