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Vikas Kumar, Kailash Chandra, Kaomud Tyagi, Shantanu Kundu, John TD Caleb, Boni Amin Laskar, Devkant Singha, Sumantika Chatterjee, Rajasree Chakraborty
Centre for DNA Taxonomy, Molecular Systematics Division, Zoological Survey of India, New Alipore-53, Kolkata, IndiaCorresponding author: Vikas Kumar (e-mail: [email protected])
Abstract:The Zoological Survey of India (ZSI), since its inception in 1916, has been maintaining several type materials in its National Zoological Collections at headquarter Kolkata as well asin different regional museums. The survey is the premier institute on faunal research having over 5 million specimens including more than 17000 type specimens from protozoato mammal from India and from more than 60 other countries. Integration of molecular data with morphology is one of the major mandate for ZSI. As of now, over 2000 DNAbarcodes for a number of species falling in different groups like economically important Insects, indicator species of Lepidoptera, freshwater fishes, reptiles, and wildlife seizureshave been contributed to GenBank and BOLD. The study of barcode data from various groups resolved several problems of their identification, taxonomy and distribution. Toname a few, in lower groups of animal, we detected alien insect pest species (Thrips parvispinus), detected cryptic diversity in insect order Thysanoptera (for species complexes,Frankliniella schultzei and Thrips palmi), detected host specific diversity in Hemiptera (Helopeltis theivora). In Araneae, we recorded for the first time from India the speciesbelonging to the genera Neriene and Psechrus. In higher animal, we first time recorded the distribution of a fish of the subfamily Gobionellinae, order Perciformes from northeastIndia. We further detected three non-native species of turtles and tortoises from northeast India (Chitra chitra, Cyclemys fusca and Amyda ornata) along with the detection ofNilssonia nigricans from wild habitat which is though categorized as Extinct in Wild in the IUCN Red List. DNA barcoding has to go a long way for covering all the extant species toachieve its real advantage in biodiversity research and conservation. Our initial success from Indian region motivate towards future collaborative endeavour for barcoding andgenerating mitogenomes from the archival specimens.
Funding Sources/AcknowledgementsThe authors are thankful to the Director, Zoological Survey of India, Kolkata, for providing necessaryfacilities. The authors (VK,KT & BAL) acknowledge the core funding of ZSI, Ministry of EnvironmentForest and Climate Change (MoEF&CC), Govt. of India. The author (SK) acknowledge the DST-SERBNational Post-Doctoral fellowship (F. No. PDF/2015/000302). The author (JC & SC) acknowledge thefellowship of National Mission on Himalayan Studies.
The ZSI with its 16 regional centres and 5 DNA barcoding laboratory facilities, generated and contributed thousands of DNA barcode data of various faunal groups in the global database to accelerate the taxonomic research. We have surveyed the faunal systems from marine ecosystem to the alpine region including protected areas, captivity and commercialized markets. The current approaches adjudicated to conserve the DNA of many highly threatened or endemic taxa from Indian zoogeography under ‘National Faunal Genome Resources’ program.
ReferencesTyagi, K., Kumar, V., Singha, D., Chandra, K., Laskar, B.A., Kundu, S., Chakraborty, R., Chatterjee,
S. (2017) DNA Barcoding studies on Thrips in India: Cryptic species, Species complexes.Scientific Reports, 4898, 7:1-14. Doi:10.1038/s41598-017-05112-7
Laskar BA, Kumar V, Darshan A, Kundu S, Das DN. DNA barcoding of Amblyceps congeners(Siluriformes: Amblycipitidae) from Brahmaputra drainage in northeast India. MitochondrialDNA. DOI: 10.3109/24701394.2016.1174223.
Laskar BA, Kumar V, Kundu S, Tyagi K, Singha D, Chakraborty R, Chatterjee S, Saha S. DNAbarcoding of Gobiid fishes (Perciformes: Gobiidae) from eastern and northeastern India withnew record of a Gobionellinae species for the region. Mitochondrial DNA. doi:10.3109/24701394.2016.1143470.
Kundu S, Kumar V, Laskar BA, Chandra K, Tyagi K. Mitochondrial DNA effectively detects non-native Testudines: Invisible wildlife trade in northeast India. GENE Reports.doi:10.1016/j.genrep.2016.02.002.
Tyagi, K., Kumar, V., Singha, D. and Chrakraborty, R. Morphological and DNA BarcodingEvidence for Invasive Pest thrips, Thrips parvispinus (Thripidae:Thysanoptera), newlyrecorded from India. Journal of Insect Science, 15(1): 1-4. DOI: 10.1093/jisesa/iev087
Banerjee, D., Kumar, V., Maity, A., Ghosh, B., Tyagi, K., Singha, D., Kundu, S., Laskar, B. A.,Naskar, A. &Rath, S. 2015. Identification through DNA barcoding of Tabanidae (Diptera)vectors of surra disease in India. Acta Tropica, 150: 52-58.
Kundu, S., Laskar, B.A., Venkataraman, K., Banerjee, D. & Kumar, V. 2015. DNA barcoding ofNilsssonia congeners corroborates existence of wild N. nigricans in Northeast India.Mitochondrial DNA, 1-4.
Materials and methods:Survey the samples with prior permission from wildlife authority
where required. Molecular biology grade chemicals, Commercialized Kits and instrumentation facilities.
Introduction:
Figure 1: Glimpse of Indian zoogeography and collection localities of several faunal systems.
Results and discussion:
The study detected cryptic diversity, species complexes and alienspecies of Thrips, an economically important species from India.
The study demonstrated the presence of cryptic species inChrysops dispar and resolved the Tabanus striatus speciescomplex using DNA barcode.
The DNA data clearly segregated the studied spider species andidentified many new species to science and new record to India.
The aimed study detected three non-native and one ‘Extinct in theWild’ species of turtles and tortoises from northeast India.
The DNA data clearly separated the studied fish species and firstrecord of Gobionellinae in Indian waters, resolve the speciescomplexes in Amblyceps and Tor species.
The generated DNA data identified the confiscated, amorphouswildlife materials seized from poachers hand by similarity searchengine (BLASTn and BOLD-IDs) and helps to the court of law.
The DNA barcode data identified the freshwater sponge diversityfrom urban ponds, identified sea food (crab & prawn) and detectedthe contamination of Maxillopoda species.
Table 1: DNA barcoding data of various faunal systems from India
Faunal system Order No. of DNA Barcodes
Thrips Thysanoptera 604
Butterflies & Moths Lepidoptera 499
Horse flies & deer flies Diptera 59
Spiders Araneae 664
Turtles Testudines 40
Fishes Cypriniformes , Siluriformes, Perciformes, Mugiliformes etc.
441
Confiscated Wildlife samples (Mammals, Birds, Retiles etc.)
Carnivora, Perissodactyla, Artiodactyla, Squamata, Testudines
etc.
25
Other invertebrates Hymenoptera, Coleotera, Spongillida, Decapoda etc.
120
Tropical
Temperate
Alpine
Aquatic
The bi-directional chromatograms were checked through ABI Sequence Scanner, DNA Baser, Chromas, BLASTn, ORF Finder etc.
The bioinformatics softwares and online server were used:Multiple and Pairwise alignment: MEGA, BioEdit, and ClustalX etc.Genetic divergence: MEGA, and PAUP etc.Phylogeny: MEGA, PAUP, and MrBayes etc.Population genetics and Haplotyping: BEAST, DnaSP, and PopART etc.Multiple species delimitation methods (BIN, ABGD, GMYC and bPTP).
Process the specimens under Leica microscope and vouchered with unique identification numbers.
The morphological measurements were acquired by adopting appropriate methods specific to the studied groups.
Morphological data was assembled and comparative studies were perfomed with the type specimens or other sister species to validate the species identity
DNA isolation from different biological samples (Blood, Tissue, Saliva, Hair etc.) and checked in Agarose Gel Electrophoresis.
The DNA barcode fragments (mtCOI~650bp) were amplified by specific primer pairs through PCR.
The PCR products were checked in Agarose Gel Electrophoresis and cleaned by commercialized purification Kits.
Bi-directional Cycle Sequencing were performed by Forward and Reverse primer separately in Thermal Cycler.
The DNA sequences were generated by using 48 capillary ABI-3730 DNA Analyzer in the in-house facilities at ZSI, Kolkata.
Conclusion: The success of DNA barcoding in systematics studies andits countless applications in biodiversity research and conservationactivities, we are motivated to established long term collaboration withthe global DNA barcoding networks to asses the Indian faunalbiodiversity as well as the reexamine the oldest archives in NationalZoological collections at Zoological Survey of India.
Barcoding fauna of India: An initiative byZoological Survey of India
In-silico analysis:
Classical taxonomy:
Molecular taxonomy:
Figure 2: The adopting DNA barcoding approaches for systematicsresearch by genetic divergence, phylogeny and estimated (molecularoperational taxonomic units (MOTUs).
Ministry of Environment Forest& Climate Change
