**Species Tree Analyses with ASTRAL-III** We used the sequence capture data set to estimate a species tree for Hyperoliidae using ASTRAL-III, which uses unrooted gene trees to estimate the species tree. This method employs a quartet-based approach that is consistent under the multi-species coalescent process, and therefore appropriate for resolving gene tree discordance resulting from incomplete lineage sorting. This approach also allows for missing taxa in alignments, which were present in our sequence capture data and are problematic for other coalescent-based summary methods. Using a single representative per lineage, we generated unrooted maximum likelihood (ML) gene trees with 200 bootstrap replicates for each locus using RAxML v8 (Stamatakis 2014) under the GTRCAT model. To automate these analyses for the set of 1,047 alignments we used a script available [here][1], which uses the following RAxML call (with automatic input and output flags not shown): raxmlHPC -f a -m GTRCAT -p 12345 -x 12345 -# 200 We concatenated the set of 1,047 best tree files to create the input for ASTRAL-III. This was accomplished with Unix: cd tree_folder/ cat *.tre > Gene_Trees_Input.tre For the first analysis we began by inferring a species tree from the set of gene trees using the following command: java -jar astral.5.6.3.jar -i Gene_Trees_Input.tre -o Species_Tree.tre We then used the -q option in ASTRAL to score the existing species tree to produce its quartet score, compute its branch lengths, and compute its ASTRAL branch support values: java -jar astral.5.6.3.jar -q Species_Tree.tre -i Gene_Trees_Input.tre -o Astral_Scores.tre The second analysis used 200 replicates of multi-locus bootstrapping, where each of the 200 RAxML bootstrap trees per locus were used to infer a species tree and a greedy consensus tree is created from the 200 species trees to calculate percent support across nodes. We used the following command to perform this analysis: java -Xmx10000M -jar astral.5.6.3.jar -i Gene_Trees_Input.tre -b BS_Map_System_v2.txt -r 200 -o BS_output.tre 2 > BS.log The output file *BS_output.tre* contains 200 boostrapped replicate trees, a greedy consensus tree of the 200 replicates, and the 'main' ASTRAL tree with bootstrap values drawn on. I copied the latter two trees to a new file called *BS_Trimmed.tre*, so that you don't need to scroll through 200 trees before getting to the actual results of interest. [1]: https://github.com/dportik/Phylo_Wrapper_Scripts/tree/master/RAxML