DOI: 10.5061/dryad.4xgxd25h3
title: Supplementary material from: Deep divergences among inconspicuously colored clades of Epipedobates poison frogs

description: 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.
description: 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 <em>Epipedobates</em>. Although
 <em>Epipedobates </em>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.
description: # Supplementary material from: Deep Divergences Among Inconspicuously
 Colored Clades of Epipedobates Poison Frogs
 [https://doi.org/10.5061/dryad.4xgxd25h3](https://doi.org/10.5061/dryad.4xgxd25h3) We examined genetic and phenotypic data from 116 *Epipedobates* frogs. The frogs were collected from 29 locations spanning southwest Colombia to southwest Ecuador, covering the entire range of *E. boulengeri* and including type localities of several species. Mean pairwise p-distances and haplotype networking were calculated, and species limits were estimated using ASAP and GMYC methods. Phylogenetic relationships were determined using Maximum Likelihood and Bayesian Inference methods. The study was carried out under permits from Colombian and Ecuadorian authorities and voucher specimens were deposited in Museo de Zoología de Pontificia Universidad Católica del Ecuador and Museo ANDES at Universidad de los Andes, Bogotá, Colombia, with specific codes for specimens at the Museo de Historia Natural C.J. Marinkelle. ## Author(s) Karem López-Hervas1,2,†, Juan C. Santos3, Santiago R. Ron2, Mileidy Betancourth-Cundar4, David C. Cannatella5,*, and Rebecca D. Tarvin * 5,6,† 1. Facultad de Ciencias Biológicas y Ambientales, Universidad Central del Ecuador, Quito, Ecuador 2. Museo de Zoología, Escuela de Biología, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador, Quito, Ecuador 3. Department of Biological Sciences, St. John's University, Jamaica, NY 11439, USA 4. Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá, Colombia 5. Department of Integrative Biology and Biodiversity Center, University of Texas, Austin, TX 78712, USA 6. Museum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, Berkeley, CA 94720, USA *Co-corresponding authors †Current Address: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Biology, Plön, Germany 24306 ## Description of the data and file structure ### Data (Raw data and scripts for analyses) ``` |-- Data S1. Raw data (alignments) and scripts for constructing trees and haplotype networks, including the resulting tree files in newick format. |-- IQTree_analysis |-- Data S1A: Epipedobates-6Genes-120-FINAL.nex – Alignment for IQTREE |-- Data S1B: run_Epipedobates_6Genes.sh – script for IQTREE |-- Data S1C: Epipedobates-6Genes-120-FINAL-Partitions-Invar-Omitted.nex – partition file for IQTREE |-- Data S1D: IQTREE2-MaximumLikelihood-Analysis8-For-FigS1.tre – Output tree file from IQTREE |-- MrBayes_All_Tips |-- Data S1E: Epipedobates-6Genes-120-FINAL-Analysis4.nex– Alignment for MrBayes |-- Data S1F: Bayesblock_Epipedobates.txt – Code to run MrBayes |-- Data S1G: MrBayes-120-FINAL-Analysis4-MCCT-For-Fig2.tre – Output tree file from MrBayes |-- MrBayes_TimeTree-18Tips |-- Data S1H: Epipedobates-Pruned_IGR-TT10.nex – Code and alignment to run MrBayes pruned timetree (18 tips) |-- Data S1I: MrBayes-Chronogram-18Tips-TT10-For-FigS2.tre – Output tree file from MrBayes pruned timetree analysis |-- Data S1J: Epipedobates_12S_haplotypes.fasta – Haplotypes for 12S-16S |-- R-Scripts-Haplotype_Network_Analyses |-- data |-- Data S1K: Epipedobates_ControlRegion_haplotypes.fasta – Haplotypes for CR |-- Data S1L: Epipedobates_cytb_haplotypes.fasta – Haplotypes for CYTB |-- Data S1M: Epipedobates_BMP2_haplotypes.fasta – Phased haplotypes for BMP2 |-- Data S1N: Epipedobates_H3_haplotypes.fasta – Phased haplotypes for H3 |-- Data S1O: Epipedobates_KV13_haplotypes.fasta – Phased haplotypes for KV1.3 |-- Data S1P: Epipedobates_Haplotype_Networks.Rmd – R script to analyze haplotypes |-- Data S2. Quantitative species delimitation analyses |-- ASAP |-- Data S2A: Epipedobates_mtDNA-fewremoved_only_ASAP.fasta – input alignment for ASAP |-- Data S2B: Epipedobates_mtDNA-fewremoved_only_ASAP.fasta.spart– ASAP logfile |-- Data S2C: Epipedobates_mtDNA-fewremoved_only_ASAP.fasta.Partition_1.txt – ASAP partition results for N = 2 species groups |-- GYMC |-- Mr-Bayes-GYMC |-- Data S2D: infile.nex – Infile for MrBayes mtDNA-only chronogram tree |-- Data S2E: GMYC4_Epipedobates-12S16S_CYTB_CR_IGR.log.txt – logfile for MrBayes |-- Data S2F: run_Epipedobates-CIPRES-IGR-for-GMYC4.nex – MrBayes script |-- Analysis-R |-- Data S2G: GMYC_Epipedobates.Rmd – R script to analyze GMYC results |-- Data S2H: my_1000_GMYC4_Trees.nexus – 1000 trees from MrBayes |-- Data S2I: GMYC4-Epipedobates-12S16S-CYTB-CR-IGR.con.tre – consensus mtDNA tree |-- Data S2J: Supplementary-Data-Y-Summary-GMYC-Results.txt – summary of R script results |-- Data S3. Locality data for each species used in constructing Figure 1 and specimen data and R script used to compare SVL among species and with elevation. |-- Data S3A: Epiped_KLH_NewModel_20181026.voucher.txt – Locality data for each species used in constructing Figure 1 |-- Data S3B: specimen-data-for-SVL_2023-02-05.csv – Specimen SVL, sex, and elevation data |-- Data S3C: specimen_data_2023-06-19.R– R script to analyze SVL, sex, and elevation ``` ### Supplemental Text, Tables, and Figures * Supplemental Text. A Spanish translation of the main text * Table S1. Information about specimens and GenBank accession numbers used in this study. * Table S2. PCR primers and conditions used to amplify CYTB, 12S-16S, BMP2, CR, and H3. * Table S3. Summary of haplotype data * Figure S1. Complete maximum likelihood phylogeny. * Figure S2. Top: Distribution of p-values from GMYC analyses of 1000 trees from the posterior distribution of MrBayes chronogram. The vertical blue line indicates p = 0.05. Bottom: Frequency of the number of inferred species based on GMYC analyses of 1000 trees from the posterior distribution. * Figure S3. Bayesian chronogram using a subset of tips that represent the major clades in Figures 1 and 2. The following are image plates of frogs examined as part of this study. Noted are the species name, collection localities, and museum numbers of the specimens. * Figure S4. Epipedobates narinensis, Biotopo: ANDES-A 3704–ANDES-A 3711 * Figure S5. Epipedobates sp. 1, Anchicayá: ANDES-A 371–ANDES-A 3720 * Figure S6. Epipedobates sp. 1, Ladrilleros: ANDES-A 2458–ANDES-A 2465 * Figure S7. Epipedobates sp. 1, La Barra and Pianguita: ANDES-A 2455, ANDES-A 3690–ANDES-A 3694 * Figure S8. Epipedobates boulengeri s. s., Isla Gorgona: ANDES-A 3695–ANDES-A 3701 * Figure S9. Epipedobates boulengeri s. s., Maragrícola: ANDES-A 2468–ANDES-A 2475 * Figure S10. Epipedobates aff. espinosai, Bilsa: QCAZ:A:53634–QCAZ:A:53642 * Figure S11. Epipedobates aff. espinosai, Cube: QCAZ:A:58233–QCAZ:A:58240 * Figure S12. Epipedobates aff. espinosai, Lita: QCAZ:A:58221–QCAZ:A:58228 * Figure S13. Epipedobates aff. espinosai, La Nutria and Pachijal: ANDES-A 2476, QCAZ:A:58335–QCAZ:A:58336 * Figure S14. Epipedobates espinosai, La Perla: QCAZ:A:53626–QCAZ:A:53633 * Figure S15. Epipedobates espinosai, Mindo: QCAZ:A:58278–QCAZ:A:58285 * Figure S16. Epipedobates espinosai, Otongachi: QCAZ:A:58259–QCAZ:A:58266 * Figure S17. Epipedobates espinosai, Río Palenque: QCAZ:A:58270–QCAZ:A:58277 * Figure S18. Epipedobates espinosai, Valle Hermoso: QCAZ:A:58245–QCAZ:A:58254 * Figure S19. Epipedobates machalilla, 5 de Agosto: QCAZ:A:53643–QCAZ:A:53650, QCAZ:A:58231–QCAZ:A:58232 * Figure S20. Epipedobates machalilla, Jouneche: QCAZ:A:53667–QCAZ:A:53674 * Figure S21. Epipedobates machalilla, Río Ayampe: QCAZ:A:58291–QCAZ:A:58298 * Figure S22. Epipedobates tricolor, Chazo Juan: QCAZ:A:53659–QCAZ:A:53666, QCAZ:A:58307 * Figure S23. Epipedobates tricolor, Guanujo: QCAZ:A:53651–QCAZ:A:53658, QCAZ:A:58308 * Figure S24. Epipedobates tricolor, San José de Tambo: QCAZ:A:58299–QCAZ:A:58306 * Figure S25. Epipedobates anthonyi, Moromoro: QCAZ:A:53683–QCAZ:A:53690, QCAZ:A:58290 * Figure S26. Epipedobates anthonyi, Pasaje: QCAZ:A:53675–QCAZ:A:53682 * Figure S27. Epipedobates anthonyi, Uzchurummi: QCAZ:A:53691–QCAZ:A:53698