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

López-Hervas, Karem1 ; Santos, Juan C.2; Ron, Santiago R.3; Betancourth-Cundar, Mileidy4; Cannatella, David C.5; Tarvin, Rebecca6

Published Dec 26, 2023; Updated Mar 27, 2024 on Dryad. https://doi.org/10.5061/dryad.4xgxd25h3

Data files

Dec 26, 2023 version files 1.43 MB

Feb 29, 2024 version files 1.43 MB

Mar 27, 2024 version files 1.43 MB

Abstract

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.

README: Supplementary material from: Deep Divergences Among Inconspicuously Colored Clades of Epipedobates Poison Frogs

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,†

Facultad de Ciencias Biológicas y Ambientales, Universidad Central del Ecuador, Quito, Ecuador
Museo de Zoología, Escuela de Biología, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador, Quito, Ecuador
Department of Biological Sciences, St. John's University, Jamaica, NY 11439, USA
Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá, Colombia
Department of Integrative Biology and Biodiversity Center, University of Texas, Austin, TX 78712, USA
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

Methods

Works referencing this dataset

Citation

Subject keywords

Funding

Society of Systematic Biologists
North Carolina Herpetological Society
Society for the Study of Reptiles and Amphibians
Chicago Herpetological Society
Texas Herpetological Society
The University of Texas at Austin, EEB Program
National Science Foundation, Graduate Research Fellowship Program
National Science Foundation, Graduate Research Opportunities Worldwide
National Geographic Society: #9468-14, Young Explorer Grant
National Science Foundation: 1556967, DEB
Secretaría Nacional de Educación Superior, Ciencia, Tecnología e Innovación del Ecuador , Arca de Noé initiative
Pontificia Universidad Católica del Ecuador, Dirección General Académica
National Science Foundation: 2319711, IOS