3.1 Molecular genetic diversity
We obtained 581 bp COI gene sequence from 161 biological samples, of these 319 sites were constant and 34 were variable. We detected 26 distinct COI mtDNA haplotypes with haplotype diversity (Hd) 0.6614, which occupied 5.852% of all analyzed sites, including 20 singletons and 14 parsimoniously informative sites (GenBank accession numbers MZ262422-MZ262447). It was also observed that there were 25 transitions, 13 transversions, or inversions/deletions. The estimated Transition/Transversion bias (R) was 1.94. The frequencies of nucleotide were A =9.17%, T = 42.92%, C = 24.56%, and G = 23.36%. For estimating ML values, a tree topology was automatically computed. The maximum Log-likelihood for this computation was -746.630. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated (complete deletion option). Total 161 individual sequences were categorized into three populations as an Indian, Malaysian, and South China of T. gigas. Pairwise F-statistic distance (FST) between South China-India was 0.708; Malaysia-India was 0.608, and South China-Malaysia was 0.136. We also confirmed the intraspecies genetic distance, ranging from 0.00 to 0.05, while inter-species distance varied from 0.06 to 0.08 for the species under study. The highest distance was observed between T. gigas and L. polyphemus.
3.2 Genetic variation and haplotype diversity
To estimates the diversity indices, we used only the COI gene for molecular analysis. We detected two major haplotypes (H2 and H3) distributed in all sampling sites, while some local haplotypes were specific to the particular sites. Molecular diversity index such as haplotype (h) and nucleotide (π) diversity was estimated among the horseshoe crab’s population in the Bay of Bengal. Nucleotide (π) and haplotype (h) diversity were found 0.00 to 0.00059 and 0.00 to 0.534, respectively. Overall nucleotide diversity and haplotype diversity in the Indian horseshoe crab population in the Bay of Bengal was 0.00265 and 0.578, respectively. We compared mtDNA genetic variation in the Indian horseshoe crab population in the Bay of Bengal with other Tachypleus gigas (South Chinese and Malaysian) populations across its distribution range. We found that Indian horseshoe crab has a comparatively lower level of genetic variation (h=4, s=3, mean pairwise difference =1.53) than the well-connected populations of South China and Malaysia.
3.3 Historical demography
The selective neutrality test was taken to confirm population expansion Fu and Li’s, Tajima’sD, and Fu’s statistics. The whole data set was included for this analysis. This analysis reveals that Tajima’s D was –2.27032 (P <0.01), Fu and Li’s was–4.736415 (P <0.02), while Fu’s Fs value was –22.25092(P <0.05). Our data suggest that the T. gigas has experienced a sudden demographic expansion. In the mismatch distribution, a smooth and unimodal curve (Fig. 2) was observed among the fourteen geographic populations. The analysis of mismatch distribution was consistent with Fu’s Fs statistical test for the population expansion model.
3.4 Phylogenetic analysis
Based on the partial sequence mtDNA COI gene, the phylogenetic tree was established. The best model was also considered as GTR+I from the JModel test. Only T. gigas species were included in the time of phylogenetic analysis. 17 unique haplotypes were obtained from 152 samples. These 17 haplotypes and ten sequences of T. gigas from NCBI GenBank were included for phylogenetic analysis. Interestingly our sample data categorized into two major subpopulations. 11 haplotypes fall under one subpopulation and the remaining six were in another group. In contrast, these were believed a sister group relationship with each other. Each phylogenetic partition was significantly different from one another (P<0.01). Its overall analysis yielded constant phylogeny. In our study, open and highly supported the connection between Tachypleus gigas and Tachypleus tridentatus was observed than the C. rotundicauda, which showed sister taxon relationship, i.e., monophyletic genus Tachypleus. It was also strongly supported for Tachypleinae  between Tachypleus and C. rotundicauda to establishing phylogenetic clade as a monophyletic relationship.
3.5. Divergence time estimates
We obtained 150-130 Mya divergence between Limulidae-Tachypleinae. The diversification of Asian horseshoe crab Tachypleinae occurred during 50 Mya (22–90 Mya), and Tachypleus genus was originated in 36 Mya (14–65), while the internal split of the T. gigas across the South China-Indian was dated to 6 Mya (2–15), shown in Fig. 2.