Development and characterization of tri- and tetra-nucleotide polymorphic microsatellite markers for skipjack tuna (Katsuwonus pelamis)

Skipjack tuna (katsuwonus pelamis) (SJT) is the largest tuna fishery in all the major oceans around the world, and the largest marine fishery in Sri Lanka. Knowledge of genetic population structure and effective population size of SJT in the Indian Ocean and other major oceans, however, is still lacking for better management practices and conservation strategies. We developed microsatellite genetic markers using SJT around Sri Lanka in the Indian Ocean, and characterise one tri- and seven tetra-nucleotide microsatellite loci isolated from enriched genomic libraries from SJT, to provide tools for addressing both conservation and fisheries management questions. An analysis of these eight microsatellite markers in two populations of SJT from eastern Sri Lanka (n = 44) and the Maldives Islands (n = 53) showed that all eight microsatellites were polymorphic with an average number of alleles per locus of 11.80 (range 5-27). Expected heterozygosities at marker loci ranged from 0.450 to 0.961. These markers are being used currently to characterise population structure and extent of natural gene flow in SJT populations from the eastern and western Indian Ocean. No significant linkage disequilibrium was detected among any loci pairs

Genetic stock structure and inferred migratory patterns of skipjack tuna (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) in Sri Lankan waters

Tuna are the major marine fishery in Sri Lanka, and yellowfin tuna (YFT) (Thunnus albacares) and skipjack tuna (SJT) (Katsuwonus pelamis) represent 94% of all tuna caught. The tuna catch in Sri Lanka has increased rapidly over recent years and this is true generally for the Indian Ocean. Tuna are a major animal protein source for 20 million people in Sri Lanka, while marine fisheries provide the main income source for most Sri Lankan coastal communities. While the importance of the fishery will require effective stock management practices to be employed, to date no genetic studies have been undertaken to assess wild stock structure in Sri Lankan waters as a basis for developing effective stock management practices for tuna in the future. This thesis undertook such a genetic analysis of Sri Lankan T. albacares and K. pelamis stocks. Samples of both YFT and SJT were collected over four years (2001 - 2004) from seven fishing grounds around Sri Lanka, and also from the Laccadive and Maldive Islands in the western Indian Ocean. Partial mitochondrial DNA (mtDNA) ATPase 6 and 8 genes and nuclear DNA (nDNA) microsatellite variation were examined for relatively large samples of each species to document genetic diversity within and among sampled sites and hence to infer stock structure and dispersal behaviour. Data for YFT showed significant genetic differentiation for mtDNA only among specific sites and hence provided some evidence for spatial genetic structure. Spatial Analysis of Molecular Variance (SAMOVA) analysis suggests that three geographically meaningful YFT groups are present. Specifically, one group comprising a single site on the Sri Lankan west coast, a second group comprising a single site on the east coast and a third group of remaining sites around Sri Lanka and the Maldive Islands. Patterns of variation at nDNA loci in contrast, indicate extensive contemporary gene flow among all sites and reflect very large population sizes. For SJT, both mtDNA and nDNA data showed high levels of genetic differentiation among all sampling sites and hence evidence for extensive spatial genetic heterogeneity. MtDNA data also indicated temporal variation within sites, among years. As for YFT, three distinct SJT groups were identified with SAMOVA; The Maldive Islands in the western Indian Ocean comprising one site, a second group comprising a single site on the east coast and a third group of remaining sites around Sri Lanka and the Laccadive Islands. The mtDNA data analyses indicated two divergent (M^ = 1.85% ) SJT clades were present among the samples at all sample sites. SJT nDNA results support the inference that multiple 'sub populations' co-exist at all sample sites, albeit in different frequencies. It appears that variation in the relative frequencies of each clade per site accounts for much of the observed genetic differentiation among sites while effective populations remain extremely large. Based on combined data sets for management purposes therefore, there is no strong evidence in these data to indicate that more than a single YFT stock is present in Sri Lankan waters. For SJT however, evidence exists for two divergent clades that are admixed but not apparently interbreeding around Sri Lanka. The identity of spawning grounds of these two clades is currently unknown but is likely to be geographically distant from Sri Lanka. Spawning grounds of the two distinct SJT clades should be identified and conserved

Genetic analysis reveals two stocks of skipjack tuna (Katsuwonus pelamis) in the northwestern Indian Ocean

Skipjack (SJT) (Katsuwonus pelamis) is a medium sized, pelagic, highly dispersive tuna species that occurs widely across tropical and subtropical waters. SJT constitute the largest tuna fishery in the Indian Ocean, and are currently managed as a single stock. Patterns of genetic variation in a mtDNA gene and 6 microsatellite loci were examined to test for stock structure in the northwestern Indian Ocean. 324 individuals were sampled from five major fishing grounds around Sri Lanka, and single sites in the Maldive Islands and the Laccadive Islands. Phylogenetic reconstruction of mtDNA revealed two coexisting divergent clades in the region. AMOVA (Analysis of Molecular Variance) of mtDNA data revealed significant genetic differentiation among sites (ΦST = 0.2029, P < 0.0001), also supported by SAMOVA results. AMOVA of microsatellite data also showed significant differentiation among most sampled sites (FST = 0.0256, P<0.001) consistent with the mtDNA pattern. STRUCTURE analysis of the microsatellite data revealed two differentiated stocks. While the both two marker types examined identified two genetic groups, microsatellite analysis indicates that the sampled SJT are likely to represent individuals sourced from discrete breeding grounds that are mixed in feeding grounds in Sri Lankan waters

Mitochondrial DNA sequence analysis from multiple gene fragments reveals genetic heterogeneity of Crassostrea ariakensis in East Asia

The native Asian oyster, Crassostrea ariakensis is one of the most common and important Crassostrea species that occur naturally along the coast of East Asia. Molecular species diagnosis is a prerequisite for population genetic analysis of wild oyster populations because oyster species cannot be discriminated reliably using external morphological characters alone due to character ambiguity. To date there have been few phylogeographic studies of natural edible oyster populations in East Asia, in particular this is true of the common species in Korea C. ariakensis. We therefore assessed the levels and patterns of molecular genetic variation in East Asian wild populations of C. ariakensis from Korea, Japan, and China using DNA sequence analysis of five concatenated mtDNA regions namely; 16S rRNA, cytochrome oxidase I, cytochrome oxidase II, cytochrome oxidase III, and cytochrome b. Two divergent C. ariakensis clades were identified between southern China and remaining sites from the northern region. In addition, hierarchical AMOVA and pairwise UST analyses showed that genetic diversity was discontinuous among wild populations of C. ariakensis in East Asia. Biogeographical and historical sea level changes are discussed as potential factors that may have influenced the genetic heterogeneity of wild C. ariakensis stocks across this region

Impacts of climatic factors on evolution of molecular diversity and the natural distribution of wild stocks of the giant freshwater prawn (Macrobrachium rosenbergii)

Natural distributions of most freshwater taxa are restricted geographically, a pattern that reflects dispersal limitation. Macrobrachium rosenbergii is unusual because it occurs naturally in rivers from near Pakistan in the west, across India and Bangladesh to the Malay Peninsula, and across the Sunda Shelf and Indonesian archipelago to western Java. Individuals cannot tolerate full marine conditions, so dispersal between river drainage basins must occur at limited geographical scales when ecological or climatic factors are favorable. We examined molecular diversity in wild populations of M. rosenbergii across its complete natural range to document patterns of diversity and to relate them to factors that have driven evolution of diversity in this species. We found 3 clades in the mitochondrial deoxyribonucleic acid (mtDNA) data set that corresponded geographically with eastern, central, and western sets of haplotypes that last shared a common ancestor 1 × 106 y ago. The eastern clade was closest to the common ancestor of all 3 clades and to the common ancestor with its congener, Macrobrachium spinipes, distributed east of Huxley's Line. Macrobrachium rosenbergii could have evolved in the western Indonesian archipelago and spread westward during the early to mid-Pleistocene to India and Sri Lanka. Additional groups identified in the nuclear DNA data set in the central and western clades probably indicate secondary contact via dispersal between regions and modern introductions that have mixed nuclear and mtDNA genes. Pleistocene sea-level fluctuations can explain dispersal across the Indonesian archipelago and parts of mainland southeastern Asia via changing river drainage connections in shallow seas on wide continental shelves. At the western end of the modern distribution where continental shelves are smaller, intermittent freshwater plumes from large rivers probably permitted larval dispersal across inshore areas of lowered salinity