BackgroundThe Malabar snakehead Channa diplogramma is one of the most enigmatic and least understood species within the family Channidae, which comprise one of the most important groups of freshwater food fish in tropical Asia. Since its description from peninsular India in 1865, it has remained a taxonomic puzzle with many researchers questioning its validity, based on its striking similarity with the South East Asian C. micropeltes. In this study, we assessed the identity of the Malabar snakehead, C. diplogramma, using morphological and molecular genetic analyses, and also evaluated its phylogenetic relationships and evolutionary biogeography.Methodology/Principal FindingsThe morphometric and meristic analysis provided conclusive evidence to separate C. diplogramma and C. micropeltes as two distinct species. Number of caudal fin rays, lateral line scales, scales below lateral line; total vertebrae, pre-anal length and body depth were the most prominent characters that can be used to differentiate both the species. Channa diplogramma also shows several ontogenic color phases during its life history, which is shared with C. micropeltes. Finally, the genetic distance between both species for the partial mitochondrial 16S rRNA and COI sequences is also well above the intra-specific genetic distances of any other channid species compared in this study.Conclusions/SignificanceThe current distribution of C. diplogramma and C. micropeltes is best explained by vicariance. The significant variation in the key taxonomic characters and the results of the molecular marker analysis points towards an allopatric speciation event or vicariant divergence from a common ancestor, which molecular data suggests to have occurred as early as 21.76 million years ago. The resurrection of C. diplogramma from the synonymy of C. micropeltes has hence been confirmed 146 years after its initial description and 134 years after it was synonymised, establishing it is an endemic species of peninsular India and prioritizing its conservation value.

Whole genome sequencing was performed on three samples of four finger threadfin Eleutheronema tetradactylum (KET 25 , KET 29 and KET 30) using illumina NextSeq500 platform using 2 × 150 bp chemistry. 8,390,317, 7,085,775 and 8,461,589 high quality reads were obtained after trimming low quality reads and adapter sequence. These high quality reads obtained were used for de novo assembly and obtained a number of scaffolds. From these scaffolds of vast sequenced data, we were able to identify 60246, 46107 and 60907 Simple Sequence Repeats (SSR) markers in KET 25 , KET 29 and KET 30 respectively, which will be useful in population genetic analysis and other diversity studies in Indian salmon. The gene prediction on assembled scaffolds predicted 31,943 genes for KET 25 ; 26,487 genes for KET 29 and 31,654 genes for KET 30 with average gene size of 458bp, 424bp and 459bp respectively. A total of 30,209, 25,107 and 29,943 genes were annotated against the NCBI Nr database for the samples respectively. E. tetradactylum is a commercially important fish species for many countries. This is the first report on the identification of genomic SSR markers in E. tetradactylum using NGS technology. This study provides an insight of baseline knowledge of the genome sequence of Indian salmon for future studies.

A modified protocol was developed for extracting DNA and direct PCR from tissues of marine invertebrates and finfishes. Protocol represented combination of classical lysis using a modified buffer system comprising KCl, Tris buffer and MgCl 2 . The DNA obtained was quantified and tested by mtDNA-PCR and RAPD. Test results indicated usefulness of this method in studies involving screening of large numbers of samples, such as evolutionary, forensic and population studies.Genomic DNA isolation to perform the polymerase chain reaction by the traditional method is usually time consuming and it involves use of corrosive chemicals, and steps such as organic solvent extraction and alcohol precipitation. Development of fast and effective DNA extraction protocols involving fewer steps would be more useful. Here we propose a two step protocol for cell lysis, DNA extraction; and direct PCR that is found to be fast and more effective than the existing methods in molluscs, crustaceans and finfishes. Since the polymerase chain reaction (PCR) is employed to amplify the DNA there is a very small requirement of the template DNA (which is often less then 50 ng) and the use of specific primers guarantees amplification even if the DNA stock does not show an optical density (OD) value in between 1.8 and 2.0 at 260/280 nm, if there are no PCR hindering substances in the DNA preparation. Moreover the forensic applications of DNA markers have made it necessary to derive a result within a short span of time. This calls for a simple and quick method for DNA preparation which really speeds up the whole process.

Genetic and morphologic variation, haplotype relationships, and structuring of populations within Puntius denisonii and its close related species Puntius chalakkudiensis have been tested using molecular and biometric data, to infer phylogeographic patterns. Sequences of mitochondrial DNA ATPase 8 and 6 genes, and morphometric data, were used to find population structuring. Specimens were collected from 7 locations in the southern region of Western Ghats, a global biodiversity hotspot in India. Biometric analysis revealed apparent heterogeneity in the morphology and color pattern between the species at juvenile and adult stages, and among different geographically separated populations of these species. High values for mean pair wise distances and a high proportion of the total variance attributed to differences between the geographically isolated populations with AMOVA, indicated clear population structuring within these species. Extremely high values for Pair wise F ST and significantly lower Nm values observed among the populations studied, suggested little or no effective gene flow among them. Constructed phylogenies further confirmed a high degree of population structuring within the species, showing local endemism with population specific haplotypes forming a species complex. The present study thus estimates the validity of subpopulations within P. denisonii and P. chalakkudiensis; clarifies the relationships of populations of P. denisonii with that of P. chalakkudiensis, and also indicates the presence of four different independent evolutionary lineages forming cryptic species within P. denisonii. The study further emphasizes the need for a conservation policy to be developed for each population of both species, separately based on MUs (Management Units).

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