DNA Barcoding Reveals Association of Glossiphoniidae Species on Endangered Freshwater Turtles in Northeast India.

BACKGROUND: The identification of Glossiphoniidae species is often confusing due to the lack of both morphological and molecular data worldwide. PURPOSE: The study aimed to identify the collected leech specimens from two endangered freshwater turtles, Chitra indica and Pangshura sylhetensis from northeast India. METHODS: We generated DNA barcode sequences and estimated the genetic distances and phylogenetic relationship of the studied taxa with another 14 Glossiphoniidae genera (114 barcode sequences). RESULTS: The high genetic distinctiveness (17.9 to 26.3%) and distinct clustering in Neighbor-Joining (NJ) phylogeny, we identified the studied specimens as Glossiphoniidae sp. We assumed that the studied specimens might be a new member of Glossiphoniidae or an extant species without DNA information. CONCLUSION: Hence, the study recommends further sampling of leeches from the similar host as well as from same and various localities and also generates the molecular data to perceive the exact diversity. The aimed study is also helpful to encourage the awareness and conservation management of freshwater turtles and other threatened animals.

DNA barcoding of fishes from River Diphlu within Kaziranga National Park in northeast India.

DNA barcoding technique has been gaining importance in biodiversity research for its easy and rapid ability of delineating organisms' partial DNA sequences into molecular operational taxonomic units (MOTUs), and identification based on referral sequences from expert identified species. We generated mtCOI barcode sequences from morphologically identified fishes from River Diphlu in northeast India. A portion of this river falls within an important rhinoceros and tiger conservation site, the Kaziranga National Park. Partial mtCOI sequences for 103 fish specimens belonging to six orders, 19 families, 37 genera and 47 a priori identified species, were delineated into 48 MOTUs based on reciprocal monophyly criteria in maximum likelihood and Bayesian tree, and 49 groups by automatic barcode gap discovery (ABGD). Morphological and molecular basis of species identification was congruent for around 80% straightforward cases. We contributed barcodes for eight species which either had no barcodes in databases or are having ambiguous barcodes. We detected four 'near threatened' and two data deficient species as per the IUCN Red List status, besides a few 'least concerned' species. We also observed a wide scope of barcoding studies on fishes from northeast India to cover the endemic species and to resolve the prevailing taxonomic problems.

Morphology and genetic variation in the endangered tortoise Manouria emys: distinct lineages or plastron anomalies?

Anecdotal reports indicate the allopatric populations of two Manouria emys subspecies differ in colour variation and plastron pattern, intimating that these may constitute separate evolutionary lineages. We examined the studied taxa both morphologically and genetically to determine the described morphological differences correlated with genetic divergence. Based on the plastron pattern, the study detected three morphologically different M. emys having their footmarks in northeast India, bordering international boundaries of Bangladesh and Myanmar. Nevertheless, we found shallow genetic divergence in both mtCOI and mtCytb gene segment among the different M. emys morphospecies. This study concludes that the detected plastron anomalies in M. emys do not suggest their distinct genetic lineages.

Pet and turtle: DNA barcoding identified twelve Geoemydid species in northeast India.

Geoemydid turtles are one of the most imperilled fauna on the planet, with nearly half of them are threatened with extinction due to bushmeat crisis, traditional medicine, and the illegal pet trade. Classical taxonomy often fails to identify the pet-kept turtle specimens due to amorphous form, unusual shell colouration owing to poor storage in captivity or intensely tinted for high demanding value. The DNA barcoding technique has evidenced as a supportive tool for accurate species identification in systematics research and discerned the nameless taxa in forensic sciences. We tested the effectiveness of DNA barcoding tools for identifying the pet-kept Geoemydid turtle in northeast India. The 36 generated sequences are readily delineated into 12 Geoemydid species using molecular data. The overall mean genetic distance of the studied Geoemydid turtles dataset is 15.3% and ranges from 3.4% to 22.6% between the species. The NJ, ML and Bayesian phylogeny also resulted monophyletic clustering and discriminated all the studied species. The present study contributes DNA barcode sequences of Geoemydid turtles in the global database and also affirms the on-going illegal pet trade of highly threatened species in northeast India.

DNA Barcoding studies on Thrips in India: Cryptic species and Species complexes.

Thrips are one of the major sucking pest and vector of plant viruses causing huge economic loss in agriculture. The accurate identification of thrips is crucial for effective pest management strategies. However, morphology based identification has limitations and warrants integration of molecular data. We attempted the largest DNA barcoding initiative on 370 sequences of 89 thrips morphospecies including 104 novel sequences from 39 morphospecies, including the type specimens of four species. The results of multiple species delimitation methods (BIN, ABGD, GMYC and bPTP) were consistent for 73 species (82%) with their morphological identifications. A total of 107 molecular operational taxonomic units (MOTUs) was recovered for 89 morphospecies by superimposing multiple methods and applying a three level nomenclature system. We detected more than one MOTU in 14 morphospecies indicating to have cryptic diversity including, two major vector species (Frankliniella schultzei and Thrips palmi). However, four morphospecies (Thrips moundi, Thrips carthami, Haplothrips andersi and Haplothrips gowdeyi) showed low genetic distances between them with overlapping in barcode gap that requires further analysis with multiple molecular markers and more specimens from wide geographical areas for better taxonomic judgment. We also presented the advantage of simultaneous use of multiple delimitation methods for detection and identification of cryptic species.

DNA barcoding of Gobiid fishes (Perciformes: Gobiidae) from eastern and northeastern India with new record of a Gobionellinae species for the region.

The study attempted identification of Gobiid fishes from freshwaters in the east and northeast India on a collection of 20 specimens. The DNA barcode data delineated the collected samples into three species clades in the neighbor-joining tree. The results confirmed the identification of five sample sequences belonging to the subfamily Gobionellinae due to cohesive cladding with Awaous congeners. This is a new subfamily record for the northeastern region. Another 15 sample sequences showed conspecific cladding with Glossogobius giuris in the database. Among the 15 sample sequences, 14 sequences cladded with G. giuris sequences of Indian specimens while one sample sequence cladded with G. giuris sequences of South African specimens. This indicated the presence of either a hidden species or a previously synonymized species in the G. giuris complex.

DNA barcoding of Nilssonia congeners corroborates existence of wild N. nigricans in northeast India.

DNA barcode data of soft-shell turtles is limited in global DNA database while it is completely lacking for the highly debated species Nilssonia nigricans. We employed DNA barcoding technique to discriminate the species cluster for Nilssonia congeners, especially for the highly debated N. nigricans from different localities of northeast India. Sampling across the region included a few live specimens from wild, market sold carcass specimens, and a few dry carapaces meant for home decoration purpose. The generated sequences (621 bp of mtCOI) of dry carapaces showed 99-100% homology with the generated sequences of morphologically identified N. nigricans. The COI barcode sequences of N. nigricans (n = 12) showed 3.8% mean genetic divergence with N. hurum (n = 3), 10% with N. gangetica (n = 4), and 9.2% with N. formosa (GenBank sequences). Similarly, the mtCytb sequences of the dry carapace and live specimens of N. nigricans were 99-100% homologous with the conspecific database sequences and formed specific clusters. The inferred Neighbor-Joining (NJ), Maximum Likelihood (ML), and Bayesian (BA) phylogeny based on partial mtCOI gene efficiently discriminated all the congeners of Nilssonia into specific clusters and, therefore, it was helpful to detect the existence of N. nigricans.

Identification through DNA barcoding of Tabanidae (Diptera) vectors of surra disease in India.

Horse flies and deer flies are common names applied to members of the family Tabanidae (Diptera). Tabanid flies are pestiferous and of veterinary and medical importance, with about 244 species in India. They are major vectors of Trypanosoma evansi that causes trypanosomiasis (surra disease). Lack of stable morphological characters, and scarcity of taxonomic expertise, is major impediments for accurate species identification of these important pest and disease vectors. Molecular data, especially DNA barcode data, has been widely used in the identification of Diptera of economic importance. We evaluated the utility of DNA barcode data to discriminate the vectors of surra disease (trypanosomiasis) from India. We used barcode gap and reciprocal monophyly (neighbor-joining and Bayesian tree) criteria to analyze barcode data. A total of 46 specimens belonging to 7 species under four genera in two subfamilies were used for this study. DNA barcode data was not available previously for these species. Analysis revealed that all morphologically identifiable species can be discriminated using DNA barcoding data. Further, our study clearly demonstrated the presence of cryptic species in Chrysops dispar. Moreover, we revealed that closely related species without stable taxonomic distinguishing characters in the "Tabanus striatus species complex" can be discriminated using DNA barcode data.