Supplementary material from: Deep divergences among inconspicuously colored clades of Epipedobates poison frogs

Poison frogs (Dendrobatidae) are famous for their aposematic species, having a combination of diverse color patterns and defensive skin toxins, yet most species in this family are inconspicuously colored and considered non-aposematic. Epipedobates is among the youngest genus-level clades of Dendrobatidae that includes both aposematic and inconspicuous species. Using Sanger-sequenced mitochondrial and nuclear markers, we demonstrate deep genetic divergences among inconspicuous species of Epipedobates but relatively shallow genetic divergences among conspicuous species. Our phylogenetic analysis includes broad geographic sampling of the inconspicuous lineages typically identified as E. boulengeri and E. espinosai, which reveals two putative new species, one in west-central Colombia (E. sp. 1) and the other in north-central Ecuador (E. aff. espinosai). We conclude that E. darwinwallacei is a junior subjective synonym of E. espinosai. We also clarify the geographic distributions of inconspicuous Epipedobates species including the widespread E. boulengeri. We provide a qualitative assessment of the phenotypic diversity in each nominal species, with a focus on the color and pattern of inconspicuous species. We conclude that Epipedobates contains eight known valid species, six of which are inconspicuous. A relaxed molecular clock analysis suggests that the most recent common ancestor of Epipedobates is ~11.1 million years old, which nearly doubles previous estimates. Last, genetic information points to a center of species diversity in the Chocó at the southwestern border of Colombia with Ecuador. A Spanish translation of this text is available in the supplementary materials.

Methods
Please see López-Hervas et al. (2024) for the full methods. In this study, we evaluate and clarify the phylogeny of the dendrobatid poison frog genus Epipedobates. Although Epipedobates is considered one of the youngest clades in the Dendrobatidae family, we find tremendous phenotypic variation within and among species, making them an interesting group for taxonomists and evolutionary biologists. We aimed to comprehensively assess the phylogenetic relationships among species within this clade, which until now has remained incomplete despite several in-depth studies. Briefly, we sequenced mtDNA and nDNA and estimated phylogenetic trees using Bayesian and Maximum Likelihood approaches (see Data S1, Fig. S1, Fig. S2, Table S2). We estimated and reviewed haplotypes based on each sequenced gene (Table S3). We then used alignments of mtDNA to estimate putative species limits in 1) Assemble Species by Automatic Partitioning (ASAP) and 2) Generalized Mixed Yule Coalescent (GYMC) (see Data S2 and Fig. S3). We reviewed published specimen data to estimate species ranges and differences in body sizes among putative species (see Data S3). Finally, we reviewed images of all examined specimens (Fig. S4-S27) as well as accompanying metadata (Table S1) to qualitatively compare color among species.