Phylogenetic Analysis
Maximum likelihood phylogenies produced from the All_M10, All_M20, All_M30, and All_M40 datasets all support three main clades: A. microunicolor , A. unicolor A, and A. unicolor B (Figure 2). However, low support values (< 70) for nodes in the All_M30 and All_M40 are more frequent when compared to All_M10 and All_M20 (see Discussion). Both All_M10 and All_M20 recovered a similar topology except for several sister relationships at the tips but with the majority of nodes having higher support values (> 70). The data matrix All_M20 was preferred over All_M10 for downstream analyses due to All_M20 having a higher taxon coverage than All_M10 while still maintaining well-supported relationships.
The Bayesian inference phylogeny produced from the All_M20 dataset was overall well-supported with posterior probabilities ≥ 0.95 and very closely resembled the maximum likelihood All_M20 phylogeny with the exception of a few sister relationships within A. unicolor B (Supplemental Figure S1). Specifically, the clade comprising MY2542, MY2541, MY2806, MY2807, ANTR74, and ANTR73 is sister to the rest ofA. unicolor B in the Bayesian, whereas the clade is sister to all other samples within A. unicolor B3 in the maximum likelihood phylogeny. The node denoting the split between this clade and its sister group has low support for both All_M20 RAxML and Bayesian phylogenies; however, the All_M10 RAxML tree, which has the same sister group relationship as the RAxML All_M20, has moderate support. Therefore, the All_M20 maximum likelihood phylogeny was preferred over the Bayesian tree for downstream analyses.
The quartet-based species tree estimated in ASTRAL-III (Supplemental Figure S2) comprised 868,916,301 induced quartet trees from the gene trees, which represented 46.19% of all quartets present in the species tree. The low normalized quartet score indicates a very high level of gene tree discordance. The species tree yielded a different topology from both the Bayesian and maximum likelihood trees (Figure 2; Supplemental Figures S1 & S2). Two of the main clades A. microunicolor and A. unicolor A remain monophyletic; however, there is significant uncertainty in the relationships between individuals within A. unicolor B, which reflects a high level of gene tree discordance (see Discussion).