Materials and Methods
Sample collection: A total of 32 samples of Tor tor were collected from eight different geographical sites in southern Rajasthan (Chambal River, Rana Pratap Sagar Dam, Bassi Dam, Kali Sindh River, Parwan River, Badi Lake, Daya Dam and Madar Tank; Figure 1). Fin tissues were collected by non-invasive techniques (Wasco et al.,2003) followed by antiseptic treatment of organism before they were reverted to their habitat. The fishes were handled according to the guidelines approved by the Agricultural Laboratory, Udaipur, Rajasthan, India. Caudal fin samples were preserved in 2-ml vials containing 75% ethanol and stored at -20ºC until further processing.
DNA extraction: The DNA extraction, quantification, amplification, and authentication were carried out according to protocol proposed by Crandall et al. (1999). DNA extraction was performed using the phenol-chloroform extraction protocol. DNA quantification was performed by two methods, using a Nano-Drop spectrophotometer (Implen) and agarose gel electrophoresis with HaeIII digested φ174 RF DNA as a molecular marker (Abgene).
Capillary sequencing: The PCR amplicons were amplified using the forward and reverse primer in separate reactions. These amplicons were purified and sequenced in an automated DNA Sequencer (ABI PRISM 310 Genetic Analyzer Applied Biosystems). Sequence Analysis Software Version 5.2 (Applied Biosystems, CA, USA) was used to generate sequence trace files and contiguous read lengths. The standard protocol prescribed by ABI systems for operating ABI310 Genetic Analyzer was followed for sequencing the COI gene of the specimens.
Sequence Analysis: The raw DNA sequences were edited using BioEdit sequence alignment editor version 7.0.5.2. BLASTN program was used to compare the sequences retrieved from the two chromatograms, and fragments showing 100% alignment with no gap or indels (insertion/deletions) were selected. The selected sequence fragments were aligned using ClustalX software. Finally, each sequence was compared using the NCBI BLASTN tool to examine the complete alignment with the partial coding sequence of fish mitochondrial COI gene. The sequences were translated using the online software ORF finder (http://www.ncbi.nlm.nih.gov/gorf/ gorf.html) and aligned using the NCBI BLASTP tool.
Haplotyping and phylogenetic analysis: A haplotype network was constructed using the median-joining network method as implemented in NETWORK (version 4.1.1.1 available at www.fluxus-engineering.com;Bandelt et al. 1999). This method begins by combining the minimum spanning trees (MSTs) within a single network (minimum spanning network, MSN) using an analogous algorithm to that proposed by Excoffier and Smouse (1994). Then, median vectors (which represent missing intermediates haplotypes) are added to the network using the parsimony criterion. Tajima’s D test (Tajima 1989) and Fu’s Fs test (Fu 1997) with 10,000 permutations as implemented in ARLEQUIN (version 3.01; Schneider et al., 2000) were used to test the hypothesis of neutrality of the marker. Negative Tajima’s D-values can indicate selection, but also population bottlenecks or population expansions (Tajima 1989). We used mismatch distributions to evaluate the hypothesis of recent population growth with 99,999 permutations as implemented in ARLEQUIN 3.0.1 (Rogers and Harpending 1992). This distribution is commonly unimodal in populations that have passed through a recent demographic expansion, and is multimodal in stable populations.