Abstract: A detailed investigation was carried out to determine the genetic structure and haplotype diversity of Malaysian horseshoe crab (Tachypleus gigas [Muller, 1785]) distributed along the west coast of Peninsular Malaysia. Mitochondrial DNA (AT rich region = 369 bp) analysis showed that T. gigas had 13 haplotypes along the Malaysian west coast of which 4 were unique to Selangor samples while 3 were unique to Johor sample and 1 each were unique to other two stations respectively. Highest haplotype diversity (h) was observed among the Selangor samples (0.873±0.071) followed by Langkawi, Johor and Kedah samples with 0.833±0.222, 0.752±0.066 and 0.733±0.155 values, respectively. Over all haplotype diversity of T. gigas in west coast of Malaysia was observed to be 0.797±0.129. Pair wise haplotype frequency (FST) value were statistically significant (p<0.05) for all the groups except for Langkawi/Kedah samples indicating higher gene flow (Lower haplotype diversity) among these two populations. Average nucleotide diversity (π) was higher in Selangor samples (0.0083±0.001) followed by Johor (0.0063±0.0011) and it was almost similar in Langkawi (0.0045±0.0012) and Kedah (0.0040±0.0008) samples which indicated higher polymorphic sites in Selangor and Johor samples while it was lower in Langkawi and Kedah samples. In addition phylogenetic analysis clearly clustered T. gigas samples from T. tridentatus samples indicating good phylogenetic signals in mtDNA AT rich region. Overall, findings from this study have important implications for proper management and conservation of this living fossil along the west coast of Peninsular Malaysia.
INTRODUCTION
Horseshoe crabs are one of the interesting groups of organism maintaining their genetic structure virtually unchanged over millions of years (Kamaruzzaman et al., 2011; John et al., 2010b). Their limited distribution, restricted gene flow in the population and higher stress tolerance ability would have played a key role in their emergence over time (Smith et al., 2009a; Smith et al., 2009b). Tachypleus gigasis one of the four extant species of horseshoe crab found in shallow water in South East Asia at depths of up to 40 m (130 ft). In Malaysia, their distribution was recorded in both the coasts including Borneo island (Kassim et al., 2008; Smith et al., 2009c; Smith et al., 2009d). Members of this species generally inhabit a cove or bay that is protected from surf (Almendral and Schoppe, 2005). Same like T. tridentatus, they molt at least 13 times (for male) and 14 times (for female) as they grow from the larval stage to sexual maturity. This period exceeds 10 years for captive breeding from an egg (Harada, 2003).
In recent decades, various molecular tools have been widely used in variety of generic diversity studies, population structure prediction including their phylogeny and genetic distance analysis. Among which mitochondrial DNA (mtDNA) sequencing analysis is widely being used for a range of molecular level predictions (John et al., 2010a). It was also evident from recent studies that molecular genetic analysis is a powerful tool for investigating genetic differentiation within a population, genetic structure and diversity throughout the history of a population (Ward et al., 2005). Analysis of populations of the American horseshoe crab (Limulus polyphemus) along the eastern coast of North America using several genetic markers detected a major genetic “break” between the northern and southern populations along the Florida state by using allozyme (Giribet et al., 2001), mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis (Pierce et al., 2000) and DNA microsatellite (King and Eackles, 2004). Sequence analyses of mitochondrial Cytochrome C Oxidase subunitI (COI) suggested that there has been limited gene flow between these populations (Pierce et al., 2000).
The mtDNA AT-rich region is a highly variable, non coding region that is useful for phylogeographic studies and population genetic surveys, although the high AT content poses technical and analytical problems (Vila and Bjorklund, 2004). Research had been carried out to predict the population structure of tri spine horseshoe crab (T. tridentatus) using mtDNA AT rich region as a marker gene (Yang et al., 2007). Due to the lack of genetic information on T. gigas population along the Malaysian coast, present work was initiated to investigate genetic structure and genetic diversity of Malaysian horseshoe crab (T. gigas) using mtDNA AT rich region as a reference gene.
MATERIALS AND METHODS
Study site and sample collection: A total of 4 sampling sites were identified along the west coast of Peninsular Malaysia and 39 samples were collected from the nesting beaches of T. gigas (Johor = 18; Selangor = 11; Langkawi = 4 and Kedah = 6) during 2008 (Fig. 1). Samples were immediately iced prior to laboratory analysis. Sampling sites were geographically divided into 3 distinct regions, 1. South Peninsular Malaysia (SPM-Johor site), 2. West Peninsular Malaysia (WPM-Selangor site) and 3. North Peninsular Malaysia (NPM-Langkawi and Kedah sampling sites) (Table 1). Samples were dissected out using sterilized scissors and 2x2cm soft muscle tissue were excised and preserved in 95% ethanol (John et al., 2010a).
DNA extraction, PCR and DNA sequencing: Salting out procedure was adopted for precise and quick DNA isolation from horseshoe crab samples (John et al., 2010b). The complete AT rich region of mtDNA was amplified by a pair of primers, Hb-trna (5’-GAGCCCAATAGCTTAAATTAGCTTA-3’) and Hb-12S (5’- GTCTAACCGCGGTAGCTGGCAC-3’) (Yang et al., 2007). Amplification reaction was conducted in 50 μL buffer supplied with the enzyme and under the conditions recommended by the manufacturer (Invitrogen, Germany). Each 50 μL volume contained 50 mM KCl, 10 mM Tris (pH 9), 3 mM MgCl2 0.2 mM each dNTP, 0.04 mM each primer, 0.033 units of Tag polymerase, 1 μL DMSO and 50 ng of mtDNA.
| Fig. 1: | Location of the sampling area |
| Table 1: | Detailed information of the sampling location and geographical information |
| SPM: South peninsular Malaysia, WPM: West Peninsular Malaysia, NPM: North Peninsular Malaysia | |
The thermocyclic conditions for PCR included the initial denaturation at 94°C for 1 min, five cycles of 94°C for 30 sec, annealing at 45°C for 40 sec and extension at 72°C for 1 min, with a final extension at 72°C for 10 min, followed by indefinite hold at 4°C. Following PCR, about 10 μL of PCR product with 2 μL of bromo thymol blue were added to 2% agarose gel, prepared with 2.5 μL of 1% Ethidium bromide and electrophorized at 90 V until the dye moved for 6 cm in the gel. The gel was moved to gel doc system for viewing the amplicons with the aid of UV trans-illuminator. Final PCR product was sequenced using ABI 3730xl sequencer and obtained chromatogram was edited via ABI sequence scanner software 1.0v.
Sequence and population-genetic analyses: All data analyses were analyzed using Arlequin 3.0v for a Macintosh platform (Excoffier and Schneider, 2005). Unique haplotypes and all transitions and transversions were counted. Haplotype diversity (h), nucleotide diversity (Shimatani, 1999) and their standard errors were calculated. Pairwise F- statistics (FST) were calculated as genetic distances based on pair wise differences between populations using DnaSP software 4.50.3v (Rozas et al., 2003). An indirect estimate of gene flow was calculated based on the Eq. 1:
| (1) |
where, Ne is the effective number of females and m is the migration rate. Percentage of AT was calculated using Bio edit software (Hall, 1999) and Transition:Transversion (Ti:Tv) ratio was calculated using MEGA 4.0 (Tamura et al., 2007).
RESULTS AND DISCUSSION
Data analysis proved that the AT rich region undergoes mainly transition mutation (Ti) and the samples from west coast of Malaysia had average Ti: Tv ratio of 2:0. The observed mean AT content in the controlled region (mtDNA AT rich region) was 86.37% in the T. gigas samples. Highest haplotype diversity (h) was observed among Selangor samples (0.873±0.071) followed by Langkawi, Johor and Kedah samples with 0.833±0.222, 0.752±0.066 and 0.733±0.155 values respectively. Over all haplotype diversity of T. gigas in west coast of Malaysia was observed to be 0.797±0.129 (Table 2). This observation suggested that the Selangor and Langkawi population of this species might have been formed recently and that the dispersal rate has been relatively low, leading to the formation of genetically distinct populations. Mean nucleotide diversity (π) was higher in Selangor samples (0.0083±0.001) followed by Johor (0.0063±0.0011) and it was almost similar in Langkawi (0.0045±0.0012) and Kedah (0.0040±0.0008) samples which indicates higher polymorphic sites in Selangor and Johor successfully used as a molecular marker for species identification and for determination of population genetic structure in a wide variety of aquatic taxa (Ward et al., 2005; Thorpe et al., 2000; Quan et al., 2001). It is also to be noted that MtDNA gives a better estimate of genetic differentiation than nuclear markers since it is approximately four fold more sensitive (Lorenz et al., 2005).
A total of 13 haplotypes were identified in T. gigas samples from west coast, of which 4 haplotypes were unique to Selangor samples (TG6,7,8 and 9) and 3 each were unique to Johor samples (TG2,4 and 5) and 1 each were unique other 2 stations Langkawi (TG11) Kedah (TG13). This observation showed that SW Malaysian horseshoe crab population had higher haplotype diversity samples while it was lower in Langkawi and Kedah samples. Similar observation was reported by Yang et al. (2007) where he observed T. tridentatus population from closer geographical area of Taiwan coastal waters showed almost similar level of nucleotide diversity. Present study also proved the competence of mtDNA region in various molecular marker studies. Hence it was evident that Mitochondrial DNA (mtDNA) analysis could be which would ultimately lead to restricted gene flow among this population where as NW Malaysian samples had higher gene flow and lesser haplotype diversity.
| Table 2: | Localities and molecular characters in T. gigas mtDNA AT-rich region |
| Sample size (n), Nucleotide content (AT%), Number of substitutions (Ti, transition: Tv, Transversion), Number of haplotypes (Nh), Haplotypes diversity (h) and Nucleotide diversity (π) | |
| Table 3: | Variable sites found in a fragment of AT-rich region of Tachypleus gigas and their distribution in the population |
| Sampling stations IDs, JOH: Johor, SEL: Selangor, LAN: Langkawi, KED: Kedah, TG1- TG13 represents the observed haplotypes in Tachypleus gigas; ‘*’ represents the polymorphic mismatches in mentioned nucleotide position) | |
| Table 4: | Pair wise F-statistic (FST) values of genetic differentiation and migrants per generation (Nem) values of gene flow among populations. FST values are above the diagonal and Nem values are below the diagonal |
| *indicates significant variation (p<0.05) and ns indicates Non significance | |
The overlapping of TG1 and TG3 haplotype in SW Malaysian samples and TG11 and TG12 in haplotype in NW Malaysian samples might be due to the restricted migration of horseshoe crab samples between closest geographical areas (Table 3). This finding was well corresponded with recent study on tri spine horseshoe crab T. tridentatus samples from west Japan water samples (Smith et al., 2009e) where he observed the dispersal rate of lower haplotype divers samples lead to genetically distinct population. Higher polymorphic sites in SW Malaysian samples compared to NW Malaysian samples indicated comparatively faster genetic mutation (Single Nucleotide Polymorphism) in SW Malaysian T. gigas samples.
Population structure and gene flow: The fixation index (FST value) between Johor vs Selangor samples and Langkawi vs Kedah samples were lower with 0.119 (p<0.05) and 0.111 (p>0.05), respectively which indicating higher gene flow between these population. This observation was also proved by migratory rate per generation between populations (Nem) which revealed the higher migratory rate between Johor vs Selangor samples (3.702) and Langkawi vs Kedah samples (4.005) (Table 4). This analysis clearly proved the restricted migration of horseshoe crab samples along the west coast of Malaysia. Similar observations were also recorded in other aquatic organisms (Wong et al., 2011; Van der Kuyl et al., 2005).
CONCLUSION
The results of this study proved the restricted geographical gene flow among the T. gigas population along the west coast of peninsular Malaysia. The genetic data presented also proved that the gene flow between NW and SW Malaysian T. gigas population is very limited. Additional molecular marker studies need to be addressed on this issue. T. gigas population in Malaysian coast is moderately abundant however their population density is constantly being in declining phase along the Malaysian coast resulting from pollution and loss of suitable spawning and feeding ground due to various anthropogenic activities. The genetic structure of local populations provide molecular information that could be for implementing different conservation strategies such as establishing horseshoe crab sanctuaries along the west coast especially in Selangor and Johor area due to higher genetic diversity of T. gigas.
ACKNOWLEDGMENT
Author wish to extend his sincere thanks to ministry of Higher Education Malaysia who funded this project under Fundamental Research Grant Scheme (FRGS) (Project Reference Number: 02-10-07-307FR ).