**3. Results**

We obtained two maximum likelihood trees from IQ-TREE and two species trees from ASTRAL-III. A summarized genus-level phylogeny with divergence times is shown in Figure 1. The large IQ-TREE phylogeny (Figure S1) and small IQ-TREE phylogeny (Figure S2a) had identical topologies in terms of relationships between species. The large ASTRAL-III phylogeny (where 63 samples were coalesced into 38 species; Figure S2b) was nearly identical to the small ASTRAL-III phylogeny (Figure S2c) and the small IQ-TREE phylogeny (Figure S2a), with the exceptions of the additional unidentified *Allobates*

*sp.* sample and the rearrangemen<sup>t</sup> of several *Ameerega* species (Figure S2b, gray labels). Topologies for the small IQ-TREE and small ASTRAL-III analyses were also identical (Figure S2), pointing to an overall concordance between methods. In terms of species relationships, the large ASTRAL-III topology differed from the large IQ-TREE topology only in the rearrangemen<sup>t</sup> of the three *Ameerega* species mentioned above. Topologies and support values were extremely similar across trees. Each genus was always monophyletic with high support, as well as the three dendrobatid subfamilies. We found Hyloxalinae sister to Colostethinae rather than Dendrobatinae. In all trees, *Oophaga* was recovered as sister to *Dendrobates*, and *Minyobates* was recovered as sister to *Adelphobates* (Figure 1). Regarding other generic relationships, we found that the clade containing *Oophaga* and *Dendrobates* is sister to the clade containing *Adelphobates* and *Minyobates. Ranitomeya* and *Andinobates* are recovered as sister genera, with *Excidobates* sister to this clade. *Phyllobates* is recovered as the sister genus to all other dendrobatines. In Colostethinae, we recovered *Epipedobates* and *Silverstoneia* as sister genera, with this clade itself sister to the clade containing *Ameerega* and *Colostethus*.

**Figure 1.** Time-calibrated genus-level phylogeny of Dendrobatidae produced using BEAST. Node labels indicate divergence times (mya). This figure is reduced to one tip per genus from the species-level chronogram in Figure S3. Art by WXG.

Node age estimates and associated error bars (representing uncertainty in node age estimates) are summarized in Table S2 and visualized in a time-calibrated phylogeny in Figure S3. Uncertainty in node age generally increases with deeper time. Our analyses indicate the subfamilies Colostethinae

and Hyloxalinae diverged around 30 ± 10 mya. Dendrobatinae diverged from the common ancestor of Hyloxalinae and Colostethinae around 32 ± 10 mya. Dendrobatidae diverged from its sister family Aromobatidae 36 ± 10 mya.
