Forensically informative nucleotide sequencing (FINS) for species and subspecies of genus Prionailurus (Mammalia: Carnivora: Felidae) through mitochondrial genes (12SrRNA and cytochrome b) by using old taxidermy samples

Abstract Genus Prionailurus comprises four species, i.e. Leopard cat, Rusty spotted cat, Fishing cat, Flat-headed cat, listed under IUCN as threatened species except P. bengalensis. In U.S.A., P. bengalensis is listed as Endangered. Subspecies of P. bengalensis, i.e. iriomotensis is listed as Critically Endangered under IUCN since 2008. The present study describes the use of two markers 12SrRNA and cytochrome b genes to differentiate the three species and three subspecies of Prionailurus. Old taxidermy samples (three skin samples) of P. viverrinus and P.b. horsfieldi were used from India for the study. The study done by using DNAsp v5, MEGA 6.0, and Network 5.0.0.1, proved that both gene markers are useful for differentiating the species and subspecies of Prionailurus. This study is also the first study to present forensically informative nucleotide sequence (FINS) for three species and three subspecies of Prionailurus.

Mitochondrial 12S rRNA and cytochrome b genes have been applied to understand the interspecies and intraspecies genetic diversity. In the present study, two skin samples were used belonging to P.b. horsfieldi and P. viverrinus for amplification of genes, i.e. 12SrRNA and cytochrome b and of other species and subspecies of the genus Prionailurus were used from NCBI. Since the gene sequences (12SrRNA and cytochrome b) of only two subspecies, i.e. P.b. iriomotensis and P.b. euptilurus are available at NCBI thus the present study is confined to three subspecies of P. bengalensis, i.e. P.b. horsfieldi, P.b. euptilurus, and P.b. iriomotensis. This study is a first attempt of molecular characterization of three species out of four known species of genus Prionailurus, i.e. P. bengalensis, P. viverrinus, and P. planiceps and (as no nucleotide database is available for P. rubiginosus) and four subspecies P.b. iriomotensis and P.b. euptilurus of P. bengalensis horsfieldi and P.b. bengalensis.
This study is useful in wildlife forensic for species identification and enforcement of law and regulations by providing strong scientific proofs and for their status based on forensically informative nucleotide sequencing (FINS) (Bartlett and Davidson 1992). The technique combines DNA sequencing and phylogenetic analysis. The author discussed the genetic difference between three subspecies of P.b. horsfieldi, P.b. iriomotensis, and P.b. euptilurus hence the origin of the seized wildlife parts and products.

Sample collection
In this study, taxidermy skin samples were used. These taxidermy samples (0.5 cm Â 0.5 cm) were collected from Mammals section of Zoological Survey of India, Kolkata (Table 1). Additionally, 12s rRNA and cytochrome b available sequences of small cats of genus Prionailurus were obtained from GenBank (Table 1).

DNA isolation, PCR amplification, and sequencing
After washing with MilliQ water and cleaning, the skin samples were hydrated before digestion by incubating the dried skin sample for 24 h in 1 ml TE solution (Tris 10 mM and EDTA 1 mM, pH 7.6) (Barros and Morgante 2007). After 24 h of hydration, the DNA was isolated from skin sample using HiPur A TM Forensic Sample Genomic DNA Purification Kit (HIMEDIA).
12s rRNA sequences were amplified using a set of primer pair, L1091 and H1478, and a primer set of L14841 and H15149 was used to amplify cytochrome b gene (Kocher et al. 1989). The PCR reaction was performed in Q-cycler, Quanta Biotech (Surrey, UK), in a total volume of 25 ml of reaction mixture (10Â PCR-with MgCl 2 , 2.5 ml; 10 mM dNTP's, 2.5 ml; 5 pmol primer, 0.45 ml each; 15 ng of DNA template; 1.5 U Taq enzyme). Polymerase chain reaction consisted of initial denaturation of 94 C for four minutes and each cycle of denaturation for 1 min at 94 C, hybridization for 1 min at 55 C (50 C for cytochrome b) and extension for 1 min at 72 C followed by final elongation for 10 min at 72 C. The cycle was repeated for 35 times. The PCR products were sequenced using ABI's AmpliTaq FS dye terminator cycle sequencing chemistry on an automated ABI 3100 Genetic Analyser. All experiments were performed in a PCR workstation (Bangalore GeNei TM ). Negative controls were used in all DNA extraction and PCR amplification to control for potential contamination. 12SrRNA gene and cytochrome b gene sequences thus generated are submitted to NCBI after conducting sequence alignment by Bioedit and by checking their similarity with species of genus Prionailurus.

Data analysis
Sequences were visualized and edited using Chromas 1.6 (Technelysium Pty Ltd., South Brisbane, Australia).
To crosscheck, quarry sequences were compared using GenBank BLAST (http://www.ncbi.nlm.nih.gov/BLAST). CLUSTAL W was used to compare DNA sequence data implemented in BioEdit v 7.0.9.0 software (Hall 1990) with outgroup Canis lupus lupus (AM711902.1). All sequences were proof read and analysed by using MEGA6.0 (Tamura et al. 2011) and were aligned by using ClustalW (Thompson et al. 1994). MEGA6.0 was used for finding the conserved, variable, parsimony informative and singleton sites as well as for phylogeny construction. Two methods were used for phylogeny construction: (i) maximum likelihood and (ii) neighbour joining with Kimura 2 Parameter (Pevsner 2009). All trees were subjected to bootstrap analysis with 1000 replicates to get bootstrap value support. The complete mitochondrial genome of Cunis lupus lupus was retrieved from NCBI database to be used as outgroup.

Results and discussion
12Sr RNA gene was amplified from extracted DNA of P. viverrinus, P.b. bengalensis, and P.b. horsfieldi. After sequencing, the unambiguous lengths of 12S rRNA (ca. 377 bp) were obtained and the same was tried for cytochrome b. The BLAST result indicated that the obtained sequences matched 99-100% with their respective species. In 12srRNA with 377 bp, the author observed five haplotypes, 312 conserved sites, 59 variable sites, 17 parsimony informative sites, and 41 singleton sites and in cytochrome b with 1100 bp, the author observed seven haplotypes; 816 conserved sites, 284 variable sites, 84 parsimony informative sites, and 200 singleton sites.

Cytochrome b gene analysis
Genetic analysis was done by MEGA 6 with seven sequences, ca. 1100 bp with Canis lupus lupus as outgroup inferred 284 variable/polymorphic (segregating) sites and seven haplotypes.
Phylogenetic study by using maximum likelihood and neighbour joining algorithms (MEGA 6) produced congruent trees (Figure 2(A,B)) with three clades where all subspecies sequences of P. bengalensis, i.e. irimotensis, euptilurus, and bengalensis form one clade, whereas P. viverrinus and P. planiceps were present in clade B and clade C, respectively, with 100% bootstrap value.
Among the species and subspecies examined in the present study, it was noted that the iriomote cat is a subspecies of P. bengalensis and not a distinct species based on genetic and evolutionary analysis and P.b. iriomotensis and P.b. euptilurus, i.e. Amur leopard cat, shared similar haplotype of 12SrRNA, but P.b. horsfieldi has distinct haplotype for 12SrRNA gene.
Amur Leopard Cat P.b. euptilurus was earlier proposed as a distinct species based on morphological differences from  southeast Asian specimens, but Chinese specimens have been shown to be similar to those from southeast Asia (Wozencraft 2005). In a molecular study done by Masuda and Yoshida (1995), a clear distinction was noted between northern populations from Tsushima, Korea, Siberia, China and Taiwan and south east Asian populations. If these genetic differences indicate a specific distinction, P.b. euptilurus may yet be a valid species. The Iriomote cat (P.b. iriomotensis) from Japan's Iriomote island was also originally described as a distinct species based on morphology (Imaizumi 1967), but based on mt DNA analysis, it is now considered a subspecies of Leopard Cat (Masuda and Yoshida 1995;Johnson et al. 1999;Wozencraft 2005). The present study also reveals that the Iriomote cat is a subspecies of P. bengalensis and genetically similar to P.b. euptilurus. It has been classified as Critically Endangered by IUCN since 2008, as the population size is fewer than 250 thus there is need to apply conservation measures. Recent genetic analysis (Luo et al. 2014) suggests species-level distinction between the Indochinese and Sundaic populations of the Leopard Cat. DNA sequence, 12SrRNA, and cytochrome b comparisons were used by Janczewski et al. (1995) to infer phylogenetic relationships among 17 Felid species and supports the use of the two genes for genetically differentiating the species and subspecies for small cats. According to Janczewski et al. (1995), cytochrome b sequences appear to accumulate differences rapidly and then stabilize, whereas 12SrRNA differences with lower number of changes per variable sites seem to accumulate more gradually but steadily.

Conclusions
Cytochrome b is potentially good to determine the genetic difference between the two subspecies of P. bengalensis, i.e. iriomotensis and euptilurus and species of Prionailurus hence in providing the strong scientific proof for identification of the subspecies. However, 12SrRNA is effective to differentiate P. bengalensis horsfieldi from P.b. iriomotensis and P.b. euptilurus.