Determination of Species Diversity Through Electrophoresis in Family Ocypodidae Along the Coast of Pakistan

Abstract

The family Ocypodidae is a group of intertidal brachyuran crabs found in tropical to temperate seas worldwide. The most recent classification recognises three subfamilies Gelasiminae Miers, 1886, Ocypodinae Rafinesque, 1815; and Ucididae Števčić, 2005. The family Ocypodidae consists of total 125 species among them the ghost crabs contain 21 species and the fiddler crabs consists of 104 species across the coastal areas of world. This family revealed total two sub-families (Gelasiminae Miers, 1886 and Ocypodinae Rafinesque, 1815) and three genera (Austruca Bott, 1973, Tubuca Bott, 1973 and Ocypode Weber, 1795) from the Pakistan coastline. Two genera Austruca and Tubuca belong to sub-family Gelasiminae (fiddler crab) found near the mangrove swamps by constructing their special type of burrows except few sites where no mangrove forest is available (i.e., Sonari). The fiddler crabs construct burrows in muddy beaches except one species (Leptuca pugilator). While genus Ocypode are commonly known as ghost crabs only found along the sandy shores which construct deep and complex burrows for shelter and reproduction. The present study is based on taxonomic study and species diversity of family Ocypodidae along the coastal belt of Pakistan by using the gel electrophoresis Polyacrylamide gel electrophoresis (PAGE) and Agarose gel electrophoresis (AGE). The study was carried out from March 2011 to December 2016. The species were collected randomly from different coast lines of Pakistan: Bhanbhore, Dhabeji, Korangi, Sandspit, Buleji, Sonari, Mubarak village and Sonmiani. Initially total six species have been identified through morphological character by using the available taxonomic keys three from genus Austruca (A. annulipes, A. iranica and A. sindensis), two from genus Tubuca (T. urvillei) and three from genus Ocypode (O. rotundata and O. ceratophthalmus). The collected species showed much intra-specific morphological variations therefore study further approached using the PAGE and AGE electrophoresis for identification of species. then by using the gel electrophoresis (AGE) two more species revealed (Tubuca saherei and Ocypode sanduna). All these species identified well through their morphological characters. The current study was based on their morphological characters showed major difference between species. In fiddler crabs of genus Austruca and Tubuca following xxix morphological characters were used to observe the taxonomic study such as pollex of enlarged cheliped, dactyl of enlarge cheliped, tubercles present of posterior side of enlarged cheliped palm, antero-lateral angles of carapace, front width of carapace, spoon tipped setae, gonopod structure. Whereas, the identification marks of ghost crabs (genus Ocypode) observed were stridulating ridge (number of tubercles and striae present on ridge) found on inner surface of palm of enlarged cheliped, eye stalks with cornea located distally, antero-lateral angles of carapace, gonopod structure, brush of hairs present on ambulatory legs. For the biochemical identification of species vertical slab gel electrophoresis was used to analyse the isozymes and general protein extracted from the species of family Ocypodidae collected from different localities of Pakistan. Total five isozymes selected from identification of species: Carbonate dehydratase, Catalase, amylase, peroxidase and octanol dehydrogenase and two general proteins by using different dyes (Amido black and Coomassie brilliant blue). Total three isozymes carbonate dehydratase, catalase and amylase showed much genetic variation deviate from HW-Equilibrium while the octanol dehydrogenase and peroxidase did not show much variation and mostly found monomorphic structure. Statistical analysis carried out to analyse the difference between the species: expected heterozygosity, observed heterozygosity, percent polymorphic loci, effective number of alleles per locus, Shannon information index, and level of differentiation between the species through wright’s (1943) fixation index (FIS, FIT and FST) and Nei’s (1973) genetic distance and genetic similarity. The A. iranica showed higher level of genetic variation (0.2780±0.244) with higher number of polymorphic loci 15/27 (55.65%) also Shannon information index revealed higher value (0.377±0.344) in Bhambhore site as compared to others. The isozymes catalase, peroxidase and octanol dehydrogenase showed significant value (P<0.001) in HW-Equilibrium. For Nei’s genetic distance species of bhambhore showed higher genetic distance with localities ranges from (38.88% to 64.48%). The Austruca annulipes showed higher value of genetic distance in Korangi population (0.3970±0.1808) and highest percentage in polymorphic loci 19/22 (86.36) along with higher Shannon information index (0.560±0.246). Three isozyme carbonate dehydratase, catalase, peroxidase and octanol dehydrogenase showed significant values xxx (P<0.001) among the three populations of A. annulipes which deviate populations from the HW-equilibrium. The level of genetic differentiation found higher (FST=43.29%) between the three populations of A. annulipes. The Nei’s (1973) genetic distance showed higher genetic distance between Sandspit and Sonari populations (81%). The A. sindensis species showed higher genetic variation (0.3462±0.2257) in Dhabeji population with higher percentage of Polymorphic loci 15/21 (71.43%) and higher value of Shannon information index (0.484±0.315). Catalase, amylase and octanol dehydrogenase isozymes showed significant value which deviate from HW equilibrium. Also, the level of genetic differentiation found higher (FST=55.57%) between the six populations of A. sindensis. Nei’s genetic distance shows very high genetic distance between the populations of A. sindensis (95%) between Sonari and Bhambhore while 80% between Sonmiani and Korangi which is very much high. T. urvillei showed higher value of genetic variation in Sonari population (0.3327±0.2295) and higher rate of percent polymorphic loci in Sandspit and Sonari 18/26 (69.23%) Shannon information index showed higher value in Sonari population (0.478±0.331). Three isozymes deviate from HW-equilibrium carbonate dehydratase, catalase and octanol dehydratase showing significant value (P<0.001). The level of genetic differentiation between three populations of T. urvillei showed higher rate (FST=42.16%). The Nei’s genetic distance showed Korangi population distinct from the other two populations with 52.97%. O. rotundata showed higher value of genetic variation (0.2889±0.2484) with higher value of percent polymorphic loci 17/28 (60.71%) with Shannon information index (0.403±0.343). total 23 loci showed significant value (P<0.001) which deviate from HW-Equilibrium. The level of genetic differentiation was also higher between the three populations of O. rotundata (FST=43.77%). The Nei’s genetic distance showed higher value of genetic distance (45%) between Sonari and other two populations. O. ceratophthalmus showed higher genetic variation in Sonari population (0.3300±0.2377) wither percent of polymorphic loci 18/26 (69.23%) and higher value of Shannon information index (0.460±0.327). peroxidase, amylase and octanol dehydrogenase enzymes showed significant value between three populations which concludes that these populations are not in HW equilibrium. The degree of genetic differentiation is also very high (FST=0.6061). The Nei’s (1973) genetic distance (97%) showed that Sonmiani population is more distant from other two populations. xxxi The overall biochemical analysis showed that inter-specific and intra-specific variation very much higher along with level of genetic differentiation (FST>40%) and Nei’s (1973) genetic distance (D>50%). The isozymes carbonated dehydrogenase, catalase and Amylase found polymorphic in all loci during inter-specific and intra-specific analysis. The Shannon information index also found higher in all the loci (I>2.0). In fiddler crab species Korangi site tend to show higher variation with relation to percent polymorphic loci (40-75%) while ghost crabs showed higher percentage of polymorphic loci in Sonari (64%-71%). Isozyme electrophoresis resulted with much variation between and among the species which needed further DNA analysis for the confirmation. Total seven genes utilized for phylogenetic analysis of crabs belong to family Ocypodidae from Pakistan coast areas. Four genes from mitochondrial DNA: 12S rRNA gene, 16S rRNA gene, cytochrome oxidase I (COI) and cytochrome b (cytb), while, three from nuclear genome: 28S rRNA, Histone 3 (H3) and Enloase (En). Among them four genes are protein coding genes and three ribosomal RNA coding genes. The COI, Cytb and 12S rRNA showed higher genetic variability and genetic distance as compared to 16S rRNA genes. While the histone protein gene and 28S rRNA also showed higher genetic variability among the species as compared with enolase protein coding genes. The overall all seven genes yielded two new species T. saherei and O. sanduna. These species are well supported bootstrap value (>70%) in COI, 12S, 28S, histone 3 and enolase gene fragments by constructing an individual phylogenetic analysis. The phylogenetic trees were constructed through Neighbor Joining (NJ), Maximum Likelihood tree (ML), Maximum parsimony Tree (MP) and Bayesian Inferences (BI). Also, higher nucleotide divergence have been observed between the species ranges from 0.44% to 10.7% (16S rRNA), 2.13% to 15.9% (12S rRNA), 2.6% to 15.2% (COI), 4.9% to 22.3% (Cyt b), 1.4% to 2.8% (28S rRNA), 1.3% 3.9% histone 3) and 1.0% to 4.2% which support the species very well. The two genera Austruca and Tubuca are commonly inhabitant of mangrove and estuaries of Pakistan coastline while genus Ocypode commonly found at sandy beaches of Pakistan. These all three genera belong the family Ocypodidae and can be identified through habitat distribution, feeding habit and morphological characters. In recent xxxii years, detailed molecular study revealed new species within this family especially in genus Austruca. In the present study phylogenetic tree supports the morphological differences and biochemical differences and gave well supported clads and sub-clads. A total of 8 species belong to three genera (Austruca, Tubuca and Ocypode) are recorded from the Pakistan coastal areas. Two genera (Tubuca and Ocypode) yielded two new species which is well supported by morphological, biochemical and molecular data analysis. T. saherei and O. sanduna found as native species from Sandspit area but O. sanduna can also be found from Sonmianisandy coast. All the identified new species need more work on their biology such as reproduction, larval distribution and protein characterization to understand completely.