Phylogenetic relationship and molecular dating of Indian pangolin (Manis crassicaudata) with other extant pangolin species based on complete cytochrome b mitochondrial gene.

Pangolins are a group of eight endangered mammalian species included in the family Manidae (Order Pholidota) and divided into four each African (Arboreal species; Phataginus tetradactyla, Phataginus tricuspis, Ground dwelling species; Smutsia gigantea and Smutsia temminckii) and Asian (Ground dwelling; Manis crassicaudata, Manis pentadactyla, Manis javanica and Manis culionensis) species. The taxonomy of all the eight extant pangolin species based on molecular genetics studies, remains unresolved and poorly examined. The present study is to address this lacuna by studying the phylogenetic, taxonomic status and molecular dating of Indian pangolin with other six out of eight extant pangolins (Sunda pangolin possibly extinct) based on complete coding region of mitochondrial cytochrome b gene. Overall sequences divergence among pangolins ranged between 0.01 ± 0.01 and 0.26 ± 0.03, where within 'Manis' it ranged between 0.01 ± 0.01 and 0.14 ± 0.03. Cytochrome b sequences based phylogenetic tree revealed, the division of seven pangolin species into two paraphyletic clades of African and Asian species, further these two paraphyletic clades were divided into three well-supported monophyletic clades, first for the genus 'Smutsia' with two African ground pangolins, second for the genus 'Phataginus' with two African arboreal pangolins and third for 'Manis' with three Asian species. Within clade of 'Manis', Chinese pangolin and Malayan pangolin are basal where Indian pangolin is present as a sister clade and furthermore, molecular dating analysis suggested that pangolins diverged from Carnivora at ∼87.2 MYA, followed by the split of Asian pangolins and African pangolins at ∼36.1 MYA and Indian pangolin split from Chinese pangolin and Malayan pangolin at ∼16.7 MYA.

Araniella cucurbitina: the first molecular evidence of a Palearctic species of genus Araniella inhabiting India.

The present study represents first genetic record of single spider species Araniella cucurbitina, genus Araniella from Uttarakhand, India. There are 12 identified species under Araniella genus and they are distributed in Palearctic region. Here, we used known N = 47 (2 from present study and 45 from GenBank) cytochrome oxidase 1 (CO1) sequences of A. cucurbitina representing seven different geographical groups, additionally 23 sequences of eight Araniella species were used for phylogenetic relationship. The CO1 (561 bp) sequences of A. cucurbitina consisted of n = 14 haplotypes, where haplotype 14 (Hap 14) represents Indian species, while all 13 haplotypes (Hap1-13) shared between six A. cucurbitina groups. The overall 'h' and 'π' diversities among seven groups of A. cucurbitina were 0.85291 and 0.00888, respectively, while overall evolutionary divergence was 0.04. The Indian Hap 14, showed minimum sequence divergence (0.02) from Italy and Czech Republic haplotypes (Hap 6), it means it is the closest group compared to others. Evolutionary divergence among eight species of Araniella ranges from 0.003 to 0.114. The maximum likelihood (ML) topology based on 14 haplotypes of A. cucurbitina was divided into two major clades and further two subclades. Furthermore, ML topology between eight species of Araniella was divided into three major clades, where A. cucurbitina and A. proxima clustered together in clade 'A', while six others were present together in clade 'B' and 'C'. This study helps to identify the Indian species from the rest of Araniella species and other cucurbitina population across the world. This study further needs to be on a large scale to know the exact status distribution and molecular phylogeography of this single species of genus Araniella from India.

DNA barcoding as a tool for robust identification of cervids of India and its utility in wildlife forensics.

DNA barcoding has become a popular method of choice for identification of specimen based on molecular techniques. Here, we present preliminary findings on generating robust DNA barcode library of Cervids of India. The dataset comprising the DNA barcode library of seven deer species included in the genus Cervus, Axis and Muntiacus classified under family Cervidae. Mitochondrial Cytochrome C Oxidase subunit I gene of ca. 710 bp accepted widely as DNA barcode region, was used for generating species specific signature from 31 known samples of seven Indian deer species. Expectedly, the NJ tree clustered three genera i.e. Cervus, Axis and Muntiacus of Cervids of India into three clades. Further, the intra- and interspecies distances based on Kimura 2 parameter model also supported the results. The average intra- and interspecies sequence divergence were 0.011 (±0.09) and 0.65 (±0.14), respectively. The present study has exhibited that DNA barcoding has discriminating power to delineate boundaries among the closely related species. The data generated are of high importance to the law enforcement agencies in effective identification of species in wildlife offence cases. The similar approach can be utilized for generating DNA barcodes for other Indian mammals for making effective management and conservation action decisions.

Inferring the molecular affinity of Indian pangolin with extant Manidae species based on mitochondrial genes: a wildlife forensic perspective.

Pangolins are the world`s most trafficked mammalian species classified under family Manidae and face severe threat of extinction, largely due to the illicit trade of its parts and products, especially scales, in international markets. Pangolin scales are believed to be used in Traditional Chinese Medicines (TCM) and meat is used as delicacies in restaurants. Of the eight extant species of pangolin, morphological discrimination is easy but the situation becomes precarious once the scales and meat samples are seized and it is difficult to identify species based on morphology in such cases. However, wildlife DNA forensics has played an instrumental role in the identification of species from such type of materials. The present study investigated that three mitochondrial genes (Cyt b, 16S rRNA, and 12S rRNA) clearly showed the variation among seven extant pangolin species (Manis culionensis; possibly extinct), whereas, maximum variation was obtained in cytochrome b when compared to another two mitochondrial genes. The present study revealed that obtained SNPs based on short sequence length (Intervals) within the three mitochondrial genes will be helpful to design the short molecular marker and species-specific probe that is used in wildlife forensic for identifying pangolin species from the degraded sample. We also advocate using more than one molecular marker for species discrimination so as to minimize any false identification of the mammal's species reported in the trade. Furthermore, data generated from the study would help in strengthening the DNA database of Indian pangolin species.

Wildlife forensics in battle against veneration frauds in Uttarakhand, India: identification of protected Indian monitor lizard in items available in the local market under the name of Hatha Jodi.

Certain articles of worship are commonly sold in Uttarakhand, India by the name Hatha Jodi, a root of a rare plant found only in a few parts of central India. The present work provides genetic proof that the Hatha Jodi sold in three local markets of Uttarakhand contained material from the Varanus species, species protected under the Indian Wildlife (Protection) Act, 1972. A total of eight samples were bought, two each from the local markets in Haridwar and Rishikesh, three from Dehradun and one from an online source (Amazon). The initial inspection confirmed that two of the samples were made of plastic material. Therefore only the other six samples were subjected to DNA analysis. DNA sequences were successfully obtained and matched with reference sequences available in NCBI Genbank database through BLAST search tool for species identification. All the six samples matched 100% with the Indian monitor lizard. The findings indicate how commercialization and the wildlife trade are playing a role in decline of the population of the Indian monitor lizard. If strong protection measures are not taken as soon as possible, the Indian monitor lizards will go Extinct very soon. Therefore, we suggest that the Government and Wildlife enforcement agencies take serious action against the illegal articles available in the local markets of Uttarakhand under the name Hatha Jodi. Further, the government needs to take legal action against offenders in other states in which the product is available for sale.

The preliminary molecular study of four skink species in Rajaji Tiger Reserve (RTR), Uttarakhand, using 12S rRNA mitochondrial locus.

Skinks are present under the Scincidae family, widely distributed species in Indian subcontinent. Uttarakhand is one of the hotspot where number of identified and unidentified skink species reported. Herein, we first time provided the 12S rRNA genetic reference database of four skink species, i.e. Eutropis macularia, Eutropis carinata, Asymblepharus himalayanus and Lygosoma punctata, in Rajaji Tiger Reserve (RTR), Uttarakhand (India). The identified four species belong to three different genera, where Eutropis carinata and Asymblepharus himalayanus listed Least Concern and Vulnerable in IUCN, respectively. Here, we collected tissue samples of four different skink species from Rajaji Tiger Reserve during field survey. After successful laboratory procedure, we compared obtained sequences with publically available genetic database and we observed four sequences matched with respective species. Furthermore, the evolutionary sequence divergence result revealed that the Eutropis carinata and Eutropis macularia are close to each other with 0.11 genetic distance. The present study indicates that the exact number and population distribution of skink species are unidentified; therefore, herein we suggest the proper screening of Uttarakhand population around should be investigated, further genetic study in combination with a good sampling strategy to investigate species biology and status for conservation program.

Resolving the phylogenetic status and taxonomic relationships of the Hangul (Cervus elaphus hanglu) in the family Cervidae.

The Hangul (Cervus elaphus hanglu) is a "Least Concern" deer species, and it is the only survivor of the Red Deer group in the Indian subcontinent. The phylogenetic status of the Hangul relative to the other members of the family Cervidae is not known because sequence data are not available in public databases. Therefore, this study was carried out to determine the phylogenetic status and delineate the genetic boundaries of the Hangul with respect to the other Red Deer subspecies on the basis of cytochrome b gene sequence data (ca 421 bp). There are three major monophyletic groups of the Red Deer in the phylogenetic tree, which are referred to as the western (Hap-01 to Hap-10), eastern (Hap-11 to Hap-20) and tarim (Hap-21 to Hap-25) groups. The overall haplotype diversity and per-site nucleotide diversity were 0.9771 (±0.0523) and 0.0388 (±0.00261), respectively. In the phylogenetic tree, the Hangul clustered with the tarim group (Yarkand and Bactrian Red Deer) with a strong bootstrap support (92%) and was found to be genetically closer to the Bactrian Red Deer than to the Yarkand Red Deer. Our molecular analysis supported the idea that the Hangul diverged from the Bactrian Red Deer and migrated to India from Tajikistan approximately 1.2 MYA.

Forensically informative nucleotide sequencing (FINS) for the first time authentication of Indian Varanus species: implication in wildlife forensics and conservation.

Monitor lizards are Varanus species widely distributed, endangered reptile in the IUCN red data list. In India, based on the morphological and ecological characteristic, it is divided into four species viz. Bengal monitor lizard, Yellow monitor lizard, Desert monitor lizard and Water monitor lizard. These four species listed as Schedule I species in Indian Wildlife (Protection) Act 1972. This paper first attempt to present Forensically Informative Nucleotide Sequencing (FINS) for the Indian Varanus based on three mitochondrial genes. The molecular framework will be useful for the identification of Indian Varanus species and trade products derived from monitors and as such, have important applications for wildlife management and conservation. Here, we used known 14 individual skin pieces of four species of monitor lizards; the partial fragment of three mitochondrial genes (Cyt b, 12S rRNA, and 16S rRNA) were amplified for genetic study. In Cyt b, 12S rRNA and 16s rRNA, we observed, 5, 5 and 4 Haplotypes; 71, 69, and 43 Variables sites; 90, 89, and 50 Parsimony Informative sites within four species of Indian monitor lizards, respectively. Despite it, the nucleotide composition was T 26.4, C 32.8, A 29.2 and G11.6; T 18.8, C 29.7, A 34.0 and G 17.5; T 21.7, C 27.3, A 32.5 and G 18.5 in Cyt b, 12S rRNA and 16S rRNA, respectively. The neighbor joining phylogenetic tree and maximum parsimony tree of three mitochondrial genes, showed similar results and reveal that, there are two major clades are present in Indian monitor lizards.