Population genetic structure of Penaeus merguiensis in Thailand based on nuclear DNA variation
Introduction
Penaeid prawns are distributed worldwide in tropical to temperate latitudes (Holthuis, 1980). One hundred twelve species of Penaeids are known to occur in the Western Central Pacific. Among these, the genus Penaeus is of greatest economical importance, especially Penaeus monodon and Penaeus merguiensis (Carpenter and Niem, 1998). Grey et al. (1983) described species, P. merguiensis (de Man) is very widespread, occurring throughout the Indo-West Pacific region from the Persian Gulf to Thailand, Hong Kong, the Philippines, Indonesia to New Guinea, New Caledonia and northern Australia (Fig. 1). It constitutes the basis of extensive fisheries in Thailand, Australia, New Guinea, Indonesia, the Philippines and to a lesser extent Malaysia, India, Pakistan and the Persian Gulf Dall, 1957, Chaitiamvong and Supongpan, 1992. Distribution of zoeae larvae of P. merguiensis is generally very coastal, as spawning occurs in 5–25 m depths. The life cycle of this species can be completed in captivity, diminishing the dependence on wild-caught broodstock of P. merguiensis and allowing to commercially bred juveniles to sustain production or to develop selective breeding programs. Before attempting such commercial operations, however, it is important to obtain data on genetic diversity of wild stocks for successful broodstock and genetic resources management. Already published surveys of P. monodon populations have exemplified the potential effects of uncontrolled inbreeding and the need to maintain reasonably high levels of effective population size Sodsuk et al., 1992, Klinbunga et al., 1999. Until recently, relatively little was known about the population structure of P. merguiensis. Allozymes were used to examine the population structure in Thailand. Five polymorphic loci out of 26 were screened on samples collected from three geographic regions, and revealed an average heterozygosity (Ho) of 0.067±0.029, suggesting a low level of genetic diversity in this taxon (Sodsuk and Sodsuk, 1999). However, this diversity seems to be higher than reported in other Penaeid species, P. monodon Mulley and Latter, 1980, Benzie et al., 1992, Tam and Chu, 1993, Penaeus japonicus Taniguchi and Han, 1989, Tam and Chu, 1993, Penaeus semisulcatus Mulley and Latter, 1980, Taniguchi and Han, 1989, Tam and Chu, 1993 and P. indicus (Ko, 1984).
The desire to assess the genetic diversity within P. merguiensis stocks using markers with higher levels of variation compared to allozymes was partially met previously by mitochondrial DNA analyses (Hualkasin et al., 2003). In this last study, 26 adult P. merguiensis were collected from eight locations in the Gulf of Thailand and the Andaman Sea and assessed for variations in a 558 bp fragment of COI-DNA (mitochodrial Cytochrome Oxidase subunit I gene). The analysis of sequence divergence revealed the presence of two distinct clades, A and B, with some overlap in their distribution. A phylogenetic and phylogeographic study of marine prawns of the genus Penaeus by Baldwin et al. (1998) revealed a genetic divergence of 8–24% between species by using 558 bp COI sequence analysis. The divergence of 5% between two distinct clades, A and B, in samples referred to P. merguiensis (Hualkasin et al., 2003) is thus the evidence of substantial genetic differentiation. Such pattern can result either from the existence of two cryptic species underlying the so-called P. merguiensis or as a single species with phylogeographic subdivision. However, mtDNA is maternally inherited and does not reflect the mating situation. Therefore to test which possibility is correct, it was required to examine genetic variability at nuclear DNA (nDNA) loci.
This paper focuses on two main issues: the development of DNA markers using three different techniques, microsatellites, direct amplification of length polymorphism (DALP) (Desmarias et al., 1998), and exon-primed intron-crossing PCR (EPIC-PCR) Côrte-Real et al., 1994, Bierne et al., 2000. These markers were then applied to test the existence of significant genetic structuring among five populations of P. merguiensis along the Thailand peninsula.
Section snippets
Sample collection
Samples of wild P. merguiensis were collected from five different geographic regions in Thailand: Trad, (TDE, N=39), Songkhla (SKE, N=40) and Surat Thani (SRE, N=24) located in the Gulf of Thailand, and Satun (STW, N=27) and Trang (TRW, N=33) located in the Andaman Sea. Among these samples, 16 individuals (SKE26, SKE43, SKE92, SKE96, SKEB1, SKEB5, TDE1, TDE2, TDE12, TDE23, SRE30, STW4, STW31, TRW7, TRW11 and TRW15) have previously been examined by mtDNA analyses (Hualkasin et al., 2003) (Fig. 2
Microsatellite polymorphism
From three microsatellite-enriched libraries, 334 clones were screened by dot blot hybridisation with (TAA)10, (CT)15 and (GT)15 probes. Of these clones, 102 were identified as positive. From a total of 67 clones sequenced, seven contained a TAA/ATT, 17 contained a CT/GA and 13 contained a GT/CA. PCR primers were designed for amplifying the five microsatellites (ACT1, ACT15, ACT26, ACT31 and ACT38) by locating the primer in the unique flanking sequence. Polymorphisms at these loci were checked
Discussion
Highly variable markers have been developed that allow the genetic variations in prawn stock to be assessed, but only a few species (Penaeus monodon, P. japonicus, P. vannamei) have been the main target of such research. Knowledge of DNA polymorphism of P. merguiensis in Thailand could be an important contribution to the phylogeographic history of this widespread genus and provide important genetic information for management of this resource. Microsatellites have been isolated from P.
Acknowledgements
We thank Dr. Ausa Chanumphai, Department of Biochemistry, Faculty of Science, Prince of Songkla University, for providing some shrimp specimens. This work was supported by grant number BT-B-06-2B-18-308 from the National Science and Technology Development Agency, Thailand, National Research Council of Thailand (year 2000), the Royal Golden Jubilee Graduate Program from the Thailand Research Fund (PHD/0085/2542), the French Embassy in Thailand and the Graduate School, Prince of Songkla
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