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).