Data Analyses
Aligned nucleotide sequences were edited using with Geneious Prime® v. 2021.0.0 (Biomatters, www.geneious.com). To find the phylogenetic position of our study, we obtained 82 additional sequences from Genbank which were added to our dataset (see Table S1). To alignments of the gene dataset, MAFFT v. 7 (https://mafft.cbrc.jp/alignment/server/) was used in a final 617 bp alignment and Hemiculter beleekeri was chosen as outgroup.
We used the PartitionFinder v.2 (Lanfear et al., 2016) to best-fit partitioning scheme.
The phylogenetic tree from maximum likelihood (ML) and Bayesian Inference (BI) was inferred by MrBayes v.3.2 (Huelsenbeck & Ronquist, 2001). Analysis was performed with Markov Chain Monte Carlo (MCMC) sampling for six million generations and parameters were saved every 100 iterations, which produced 6001 trees during the analysis. Finally, 10% of trees were discarded as burn-in, and the remaining trees were used to reconstruct the 50% majority-rule consensus tree.
The performance of each run and evaluate convergence explored using Tracer v.1.7 (Rambaut et al., 2009).
DnaSP v.5 was used to determine molecular diversity indices that contains the number of haplotypes (H), haplotype diversity (Hd), number of polymorphic sites (s) and nucleotide diversity (π). These diversity indices were estimated for the regional clades based on cytb.
A median-joining (MJ) network was constructed using NETWORK v.10.2 (Bandelt et al., 1999) to find the possible origins of introductions for each detected specimens in Iran.