III. Phylogenetic Analyses
Consensus sequences were created by assembling the forward and reverse sequences using Geneious Pro v.5.3.6 (Biomatters, Auckland, New Zealand). Multiple sequence alignments were performed using Clustal X (Larkin et al., 2007). Haplotype sequences of Bufo species obtained in this study were deposited in GenBank (Table S2 ). Genetic distances within and between species were calculated with MEGA v5.2 (Tamura et al., 2007).
Phylogenetic analyses were performed on the seven individual gene datasets (cytb , ND2 , Rho-3 , Sox9-2 ,CCNB2-3 , DBI-2 , and UCH-2 ), and a concatenated dataset (all genes except cytb ) (Table S1 ). Cytbwas not included in the combined dataset to enable comparison with Zhan and Fu (2011). A selection of 1–4 outgroups (B. bufo , B. stejnegeri , B. japonicus [two individuals]) was used for the individual analyses (Table S2 ). For the individual gene datasets, both maximum likelihood (ML) and Bayesian inference (BI) analyses were performed. The ML analyses were conducted using the combined ML search and rapid bootstrap in RAxML v.8.0.2 (Stamatakis, 2014). A GTR+G model of sequence evolution was used for the ML tree search with 1000 bootstrap replicates. BI analyses were performed using MrBayes v3.2 (Ronquist et al., 2012) by running four chains for 2 million generations, sampling every 1000th generation. The best fit model of evolution was estimated based on the Bayesian Information Criterion (BIC) in jModeltest v.1.0 (Table S5 ) (Guindon & Gascuel, 2003; Posada, 2008). All analyses were conducted using the CIPRES Science Gateway (Miller, Pfeiffer & Schwartz, 2010). Trees were viewed and edited using FigTree v.1.4.0 (Rambaut, 2012).
For the concatenated dataset, POFAD (Joly & Bruneau, 2006) was used to construct a multilocus phylogeny. The POFAD algorithm combines genetic distance matrices generated from allelic data of individual loci into a single genetic-distance matrix (Joly & Bruneau, 2006). The analysis included 79 individuals (68 from Zhan and Fu [2011] and 11 from this study). The uncorrected pairwise distances of each marker were generated in MEGA v.5.2 (Tamura et al., 2007), then POFAD was used to obtain a standardized combined-locus distance matrix. Subsequently, a neighbor joining (NJ) phylogenetic tree was reconstructed based on the combined genetic-distance matrix using MEGA v.5.2 (Tamura et al., 2007).