Pradosh Mahadani

The Species Dilemma of Northeast Indian Mahseer (Actinopterygii: Cyprinidae): DNA Barcoding in Clarifying the Riddle

Background The taxonomic validity of Northeast Indian endemic Mahseer species, Tor progeneius and Neolissochilus hexastichus, has been argued repeatedly. This is mainly due to disagreements in recognizing the species based on morphological characters. Consequently, both the species have been concealed for many decades. DNA barcoding has become a promising and an independent technique for accurate species level identification. Therefore, utilization of such technique in association with the traditional morphotaxonomic description can resolve the species dilemma of this important group of sport fishes. Methodology/Principal Findings Altogether, 28 mahseer specimens including paratypes were studied from different locations in Northeast India, and 24 morphometric characters were measured invariably. The Principal Component Analysis with morphometric data revealed five distinct groups of sample that were taxonomically categorized into 4 species, viz., Tor putitora, T. progeneius, Neolissochilus hexagonolepis and N. hexastichus. Analysis with a dataset of 76 DNA barcode sequences of different mahseer species exhibited that the queries of T. putitora and N. hexagonolepis clustered cohesively with the respective conspecific database sequences maintaining 0.8% maximum K2P divergence. The closest congeneric divergence was 3 times higher than the mean conspecific divergence and was considered as barcode gap. The maximum divergence among the samples of T. progeneius and T. putitora was 0.8% that was much below the barcode gap, indicating them being synonymous. The query sequences of N. hexastichus invariably formed a discrete and a congeneric clade with the database sequences and maintained the interspecific divergence that supported its distinct species status. Notably, N. hexastichus was encountered in a single site and seemed to be under threat. Conclusion This study substantiated the identification of N. hexastichus to be a true species, and tentatively regarded T. progeneius to be a synonym of T. putitora. It would guide the conservationists to initiate priority conservation of N. hexastichus and T. putitora.

Identification of ethnomedicinal plants (Rauvolfioideae: Apocynaceae) through DNA barcoding from northeast India

Background: DNA barcode-based molecular characterization is in practice for plants, but yet lacks total agreement considering the selection of marker. Plant species of subfamily Rauvolfioideae have long been used as herbal medicine by the majority of tribal people in Northeast (NE) India and at present holds mass effect on the society. Hence, there is an urgent need of correct taxonomic inventorization vis-à-vis species level molecular characterization of important medicinal plants. Objective: To test the efficiency of matK in species delineation like DNA barcoding in Rauvolfiadae (Apocynaceae). Materials and Methods: In this study, the core DNA barcode matK and trnH-psbA sequences are examined for differentiation of selected ethnomedicinal plants of Apocynaceae. DNA from young leaves of selected species was isolated, and matK gene (~800 bp) and trnH-psbA spacer (~450 bp) of Chloroplast DNA was amplified for species level identification. Results: The ~758 bp matK sequence in comparison to the trnH-psbA showed easy amplification, alignment, and high level of discrimination value among the medicinal Rauvolfioidae species. Intergenic spacer trnH-psbA is also exhibited persistent problem in obtaining constant bidirectional sequences. Partial matK sequences exhibited 3 indels in multiple of 3 at 5 end. Evidently, generated matK sequences are clustered cohesively, with their conspecific Genbank sequences. However, repeat structures with AT-rich regions, possessing indels in multiple of 3, could be utilized as qualitative molecular markers in further studies both at the intra-specific and shallow inter-specific levels like the intergenic spacers of CpDNA. Conclusion: matK sequence information could help in correct species identification for medicinal plants of Rauvolfioideae.

Bioinformatics Tools in Marine DNA Barcoding

Information science has been applied to manage the information generated in molecular biology to produce the field called Bioinformatics. Application of bioinformatics in several ways is an integral part of DNA barcode research. Since the origin of the DNA barcode concept, three major criteria were set up to test the efficiency of barcode regions. These are (i) Universal Primers to amplify the barcode region (ii) Calculation of Barcode gap (intra and interspecies distance) (iii) Species resolution power. For evaluation of these criteria, DNA barcode community is applying bioinformatics tools and algorithms in primer design, distance calculation, phylogenetic analysis, etc. DNA Barcoding produces a huge amount of Cytochrome c oxidase subunit I (COI) sequences information for species identification from animal kingdom of marine biodiversity. Similarly, Consortium of Barcode of Life (CBOL) plant group proposed plastid genes rbcL and matK either singly or in combination as the standard DNA barcode for plants (CBOL Plant Working Group 2009). ITS of the nuclear ribosomal RNA standardized as the universal barcode marker for fungi. Bioinformatics play a major role in storing of DNA barcode information, and it is easy to retrieve from the database. Software-based sequence quality assurance is main starting and check point for the production of barcode sequences. In this chapter, we discussed different tools and methods of bioinformatics and their proper utilization.