Genetic Variability of Tachypleus gigas (Muller), horseshoe crab from Johor, inferred by cytochrome oxidase subunit 1 gene

In Malaysia, horseshoe crab Tachypleus gigas (Muller) research on abundance and morphology have been done but little information on phylogeny and genetic. This study was conducted to investigate the variability and structure of population in Mersing and Sedili in East Coast while Johor Bahru, Pontian, Batu Pahat and Muar in West Coast. The results of sequencing analysis of 49 samples, there are only 7 different sequences. We can note, sequence or haplotype TG26 only exist in the east coast locations (Mersing and Tanjung Sedili), while TG4 haplotype shared by all locations. The constructed phylogram clearly indicated that the horseshoe crabs are closely related. Among the sampled collected, 7 haplotypes as well as with T.tidentitus sample as an out group clearly clustered T.gigas in separate branch indicating high phylogenetic cues in mtDNA COI gene. A total of 49 individuals of adult horseshoe crab, T. gigas were collected from 6 different shorelines in Johor, Peninsular Malaysia, namely Mersing, Tanjung Sedili, Johor Bahru, Pontian, Batu Pahat and Muar.  Live samples for each population were collected from the fishermen and were brought back to the laboratory. A gills clip of the horseshoe crab T. gigas was extracted using DNeasy blood and tissue kit (Qiagen, Germany). The partial cyt b gene was amplified by a pair of primers, L14841(5'-AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA-3') and H15149 (5'-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3'); [9].  Thermocyclic condition for PCR included the initial denaturation at 94⁰C for 15 sec, annealing at 45⁰C for 15 sec, and extension at 72⁰C for 10 sec, with a final extension at 72⁰C for 10 min, followed by indefinite hold at 4⁰C. Final PCR products were sent to FirstBase Laboratories Sdn Bhd for sequencing. Sequences was align using Clustal application of MEGA 6.0 software. Genetic structure analysis is carried out using MEGA6.0, DNsap and Arlequin. Detail of the sequences used to generate the phylogram. The highest number of T. gigas  found in Pontian(22) meanwhile 5 in Mersing, 4 Muar, 5 Batu Pahat, 2 Johor Bahru and 8 Tanjung Sedili, Kota Tinggi. Based to analysis, indicated there are 7 of haplotypes was found. Among these haplotypes 1 (TT26) is unique to locations on the east coast of peninsular Malaysia (Mersing & Tg Sedili). Neighbor-joining (NJ) phylogram (fig. 1)showing the relationship among haplotypes of T. gigas. The numbers at each node represents the bootstrap percentage values based on 1000 pseudo replications for NJ/MP analyses. T. tridentatus (TT) used as an out group was clearly clustered in separate branch proves the reliability of the constructed phylogram. The constructed phylogram clearly indicated that horseshoe crabs are closely related. Among the sampled collected, 7 haplotypes as well as with T.tridentatus sample as an out group clearly clustered T.gigas in separate branch indicating high phylogenetic cues in mtDNA COI gene.  The partial cyt b sequences in the phylogram constructed clearly showed that T. gigas (Mersing and Sedili) location on the east coast  of Peninsular Malaysia contain haplotypes 1 (TG26) which unique to this location that not exist in sequence represent by T. gigas in West coast of Peninsular Malaysia (Pontian, Batu Pahat, Muar and Johor Bahru). But further studies need to be conducted to prove this concept by analysing fully cyt b gene or other DNA markers.