Evidence of geographical structuring in the Malaysian Snakehead, Channa striata based on partial segment of the CO1 gene

Channa striata, locally known as “haruan”, is economically important in fisheries and aquaculture industries in several Asian countries. DNA sequencing, based on a partial segment of the Cytochrome oxidase c subunit 1 (CO1) gene, was used to determine genetic variation in C. striata samples from four different populations on the west coast of Peninsular Malaysia. The highest nucleotide and haplotype diversities were observed in the Linggi population (π = 0.0067, h = 0.835), and the lowest in the Timah Tasoh population (π = 0.0008, h = 0.286). Apart from Kajang-Linggi, which was insignificant, FST values were significant (p < 0.05) in all pairwise-population comparisons. Consequently, it is inferred that genetic structuring C. striata populations in this region was largely shaped by a common origin, with secondary influences from geographical factors and isolation.

Forty five samples were collected from two northern sites, Timah Tasoh (Perlis) and Jeniang (Kedah) and two southern Linggi (Negeri Sembilan) and Kajang (Selangor) (Figure 1). With the exception of Kajang, a cultured species of unknown origin, but believed to be from the southern region itself, all were wild populations. Prior to DNA extraction, fin tissues were preserved in TNES-Urea (100 mM Tris-HCL pH 7.5, 125 mM NaCl, 10 mM EDTA pH 7.5, 1% SDS, 3 M Urea), modified according to Valles-Jimenez et al. (2004).
DNA extraction was with an Aquagenomic Kit (Bio-Syntech, Salt Lake City, Utah, USA), according to manufacturer's instructions. A segment of the CO1 mtDNA gene was amplified using a pair of primers -forward primer L6154 (5'-AYC ARC AYY TRT TYT GRT TCT-3') and reverse primer H6556 (5' TGR AAR TGI CGI ACW ACR TA-3') (Telechea et al., 2006). PCR was done in a Peltier thermal cycler (MJ Research Waltham, MA, USA), with the following profile: pre-denaturation at 95°C for 5 min, 30 cycles of denaturation at 94°C, annealing at 50°C and extension at 70°C for 1 min each, followed by final extension at 72°C for 5 min. The PCR products were then purified using Wizard ® SV Gel and a PCR Clean-Up System by Promega (Promega Madison, WI) and sequenced on an ABI3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Sequences were edited using MEGA 4.0 (Tamura et al., 2007) and aligned with Clustal W 1.6, implemented in same software. Arlequin version 3.1 (Schneider et al., 2000) was used for calculating nucleotide and haplotype diversities, haplotype frequency and F ST values. Bonferroni correction was applied, with global significance level at 0.05 to correct for multiple comparison. The relationships among all haplotypes were constructed using the Network program (Bandelt et al., 1999).
Aligned sequences of 364 bp in the mtDNA CO1 gene were obtained. Twelve unique haplotypes were identified from the four populations of 45 individuals. The sequences were all submitted to the Genbank under accession numbers GQ204314, GQ204319, GQ204321, GQ204323, GQ204326, GQ204330, HM192913 and HM192914. The highest nucleotide and haplotype diversities were observed in Linggi (p = 0.007, h = 0.835) and the lowest in Timah Tasoh (p = 0.0008, h = 0.286). Linggi presented 13 polymorphic sites, Jeniang and Kajang 7 each, and Timah Tasoh only one (Table 1).
The minimum spanning-network relationships among all the haplotypes are represented in Figure 1. Except for Timah Tasoh, Hap1, the most common haplotype, was found in the remaining three populations. From this common haplotype, two clades emerged, the northern and the southern. The spanning network showed that geographically close populations, i.e. Timah Tasoh and Jeniang (north) and Kajang and Linggi (south), were also genetically close.
The highest inter-population genetic distance occurred between Linggi and Jeniang, at 0.011, and the lowest between Timah Tasoh and Jeniang, at 0.004 (Table 2). Genetic differentiation, as revealed by pairwise F ST analysis, was insignificant between the Kajang and Linggi populations, and significant between the remaining two ( Table 2). The scatterplot revealed a highly positive correlation of genetic isolation versus geographical distance (Figure 2 thereby fitting the 'isolation by distance' model (Sun et al., 2004). Nevertheless, caution is called for, due to the small sample sizes. Due to geological events, such as river formation (Tzeng et al., 2006), the distribution of freshwater fishes is usually restricted, thereby lowering the chances of free-gene-flow. Thus, fairly low intra-population genetic diversity (p = 0.0008-0.0047, h = 0.286-0.483) was the case, except in the Linggi population (p = 0.0067, h = 0.835). Esa et al. (2008), also through CO1-gene analysis, reported even lower variability indices (p = 0-0.001 and h = 0.186-0.450) in an endangered fish, Tor tambroides, from Sarawak (Malaysian Borneo). Furthermore, although low (0.4%-1.1%), the inter-population distances in C. striata were higher (0.1%-0.3%) than in Tor tambroides. Thus, the indications of prevailing genetic variability advocates initiating management procedures. Nonetheless, as a consequence of marker characteristics, an underestimation of the true levels of genetic variability is possible. Several recent studies employing the bar-coding approach have shown that the CO1 gene is a more useful marker for unambiguous-species identification, due to its genetically conserved withinspecies characteristics (Dasmahapatra and Mallet, 2006;Lara et al., 2009;Valdez-Moreno et al., 2009). Notwithstanding, other markers for population studies, such as microsatellites, or more variable mtDNA markers, such as the D-loop, should also be investigated.
The absence of the common haplotype Hap1 in the Timah Tasoh population is the evidence of its isolation. The presence of a dam could be a likely factor, through its construction possibly changing the original riverine environment into a lacustrine. Notwithstanding, although Hap1 is missing, the main Timah Tasoh haplotype (Hap3) only differed from it by two bases. Thus, the most probable scenario for this population would be a shared origin which evolved independently, due to its isolation. Wang et al. (2004) described the common haplotype as having immense potentiality for producing mutational derivatives. Through being more recently derived, the other haplotypes were unique.
Based on this preliminary analysis, it can be inferred that the genetic structuring of the Peninsular Malaysia C. striata population was largely shaped by a common origin, with secondary influences from geographical factors and isolation. Further studies, with more efficient markers and larger populations, especially from the northern and southern regions, are required to corroborate the findings.