To estimate the phylogenetic relationships among the 23 darter
species and two outgroups (Perca flavescens and Sander vitreus),
we compiled sequence data available on GenBank (see electronic
supplementary material, table S1) for three loci. The
loci included the mitochondrial cytochrome b (Cytb), nuclear
S7 ribosomal protein intron 1 (S7) and nuclear recombination
activating protein 1 (RAG1) exon 3. We first ran a partition
homogeneity test implemented in PAUP 4.0b10 to test for topological
incongruence among the loci [15]. Using each locus as a
single partition across 100 replicates that each had 100 replicates
of random taxon addition, we did not identify any significant
incongruence among the loci ( p ¼ 0.20). Therefore, we performed
a concatenated analysis of all three loci with sequence
alignments and models of molecular evolution taken from
Near et al. [10]. Using BEAST v. 1.7.5 [15], we ran a Bayesian
analysis on the dataset partitioned by gene for 10 million generations
with sampling every 5000 generations. We used TRACER
v. 1.5 [16] to estimate the effective sample size (ESS) of
parameters in the MCMC. ESS values more than 200 were
considered adequate.