Dna extraction

DNA extraction , sequencing and phylogeny

DNA was extracted from the fin clip using PureLink™ Genomic DNA Mini Kit (Invitrogen) following the manufacturer’s protocol. Subsequently, the target mitochondrial cytochrome b (cyt b) gene was amplified by polymerase chain reaction. The partial cyt b gene was amplified using primers L14724 (5’-G AC TTG AAA AAC CAC CGT TG- 3’) and H15915 (5’ -CTC CGA TCT CCG GAT TAC AAG AC- 3’) ( Irwin et al. 1991) with hot start at 95°C for 2 minutes, followed by denaturation at 95°C for 30 seconds; annealing at 52°C for 10 seconds and extension at 60°C for 4 minutes, followed by final hold at 4°C. The denaturation, annealing and extension processes were set for 25 cycles. The PCR products were treated with Exonuclease I (EXO I), and shrimp alkaline phosphatase (SAP) for 15 minutes each at 37°C and 80°C to remove residual primers, followed by sequencing of both direction using the BigDye® Termination Kit and (v.3.1) and an ABI 3500XL Applied Biosystems Genetic Analyzer. The sequences were edited using Sequencer version 4.9 (Gene Codes Corporation, Ann Arbor, MI) and deposited in NCBI GenBank (PP911355).

A BLASTn ( Altschul et al. 1990) search was performed for the newly generated sequence on NCBI BLAST (http://blast.ncbi.nlm.nih.gov) to confirm genus-level identity against published GenBank sequences. The newly generated sequence was aligned with those downloaded from GenBank ( Benson et al. 2017) (Appendix 1) using ClustalW ( Thompson et al., 1994) with default prior settings implemented in MEGA 7.1 ( Kumar et al. 2016). We used Balitora chipkali and B. mysorensis as outgroup taxa. We checked for unexpected stop codons by translating the sequence to amino acids in MEGA 7.1 ( Kumar et al. 2016).

We performed Maximum likelihood (ML) analysis for the dataset (1067 base pairs) using the GUI version of IQTREE ( Nguyen et al. 2015) implemented within the PhyloSuite ( Zhang et al. 2020). The dataset partitioned by codon positions using ModelFinder ( Kalyaanamoorthy et al. 2017) to find the best fit partitions and models of sequence evolution for each partition suggested three different partitions, Partition 1: TNe+G4, Partition 2: HKY+F+I, Partition 3: TIM2+F+G4. Bayesian inference (BI) analysis was carried out using the program MrBayes 3.2.7a ( Ronquist et al. 2012) implemented within PhyloSuite ( Zhang et al. 2020), with default prior settings. The same dataset used for the ML analysis was partitioned by codon positions using PartitionFinder 2.1.1 ( Lanfear et al. 2017) with default settings to find the best-fit model of sequence evolution. PartitionFinder suggested three different partitions, Partition 1: SYM+G, Partition 2: HKY+I, Partition 3: GTR+G. Two separate runs were set up with four Markov chains each initiated from random trees and allowed to run for 10 million generations, sampling every 1000 generations. Analyses were terminated when the standard deviation of split frequencies was less than 0.001, the first 25% of trees were discarded as burn-in, and trees were constructed under 50% majority consensus rule. We obtained the Effective Sample Size values using TRACER 1.6 ( Rambaut et al. 2018), and confirmed values above 200 for the priors. Support for the internal branches for the ML and BI was quantified using 1000 pseudoreplicates (ultrafast bootstrap UFB) and posterior probabilities (PP), respectively. The resulting tree was visualised and edited in FigTree (http://tree.bio.ed.ac.uk/software/figtree). The uncorrected pairwise distances (P–distance) were calculated in MEGA 7.1, with pairwise deletion of missing data and gaps.