Phylogenetic Analysis
We estimated the phylogeny for several datasets (see Table 1 for dataset
descriptions) within a maximum likelihood framework implemented in RAxML
v8.2.4 (Stamatakis, 2014). We used the GTR+G model of sequence
evolution. For each analysis, 1000 bootstrap replicates were calculated
using the rapid bootstrapping option implemented in RAxML. The
phylogenies constructed from these datasets were viewed in Figtree
v1.4.1 with midpoint rooting. The phylogenies were then compared to look
for congruence among clades and bootstrap values, with the final tree
having the largest amount of taxon coverage and high support values used
for downstream analyses (All_M20 dataset with all 157 individuals and a
minimum of 20% locus occupancy; Figure 2).
Bayesian inference of the All_M20 data set was conducted in ExaBayes
v1.4.1 (Aberer et al., 2014). Two independent runs of
6x107 generations with four coupled chains each were
run simultaneously starting with a random starting tree. Standard
deviation of split frequencies was monitored (< 0.05), and the
first 25% were discarded as burn-in. An extended majority rule
consensus tree was generated using the program consense within ExaBayes
(Supplemental Figure S1; Aberer, Kobert, and Stamatakis 2014).
A species tree was generated from a set of gene trees using the software
ASTRAL-III (Supplemental Figure S2; Zhang et al. 2018). We used the
MAGNET v0.1.5 pipeline (Bagley, 2019) to estimate a gene tree for each
RAD locus within the All_M20 data set using RAxML v.8.2.4, with each
RAxML run implementing the GTR+G model. These gene trees were used as
input for ASTRAL-III. With uncertainty in the relationships amongA. unicolor B, a well-supported clade in both maximum likelihood
and Bayesian analyses but slightly differing sister relationships (see
Results), additional species trees were generated using A.
unicolor B -only matrices (UniB_M60 and UniB_M80; see Table 1) for aid
in elucidating relationships among A. unicolor B.