**5. Conclusions**

In this work, the phylogenetic relationships among seven of the eight described species of Central American *Atelopus*, plus eight South American congeneric species, were inferred based on mitochondrial DNA sequence data. The phylogenetic analyses revealed a ladderized topology showing the Central American species as a monophyletic group, and placing the species geographically furthest from South America more nested in the tree. We detected two previously unrecognized candidate species, including an undescribed species from Costa Rica involved in a taxonomic confusion that requires further study. We showed that species in eastern Panama, also show geographic variation in their color patterns, and clarify the species identity of individuals from some of these populations. Biogeographic models supported either one or two colonization events from South America, indicating that *Atelopus* reached Central America prior to 4 million years ago (Ma), a timing slightly older than the traditional date estimated for the closure of the Isthmus. Furthermore, this study underscores the invaluable role of museum collections in documenting biodiversity, and the relevance of genetic analyses for guiding conservation efforts.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/12/9/360/s1; Resumen (Abstract in Spanish); Figure S1: One of the 97 most-parsimonious trees (selected arbitrarily) inferred from DNA sequences of two mitochondrial genes (COI and cy<sup>t</sup> *b*) for samples of *Atelopus* of Central and South America; Figure S2: Maximum likelihood phylogenetic tree inferred from DNA sequences of two mitochondrial genes (COI and cy<sup>t</sup> *b*) from species of *Atelopus* of Central and South America, including the extended non-*Atelopus* bufonid outgroup; Figure S3: Timetree of *Atelopus* based on a relaxed-clock MCMC Bayesian analysis of DNA sequences of two mitochondrial genes, COI and cy<sup>t</sup> *b*, calibrated using the estimated substitution rate for bufonid frogs of Macey et al. [53], as corrected by Crawford [54], and including the extended non-*Atelopus* bufonid outgroup; Table S1: Specimens of *Atelopus* and other bufonid frogs used for the present study, including museum voucher, locality, geographic coordinates, elevation and GenBank accession numbers; Table S2: Best-fit partitioning scheme for the two protein-coding mitochondrial genes, cytochrome *b* (cyt *b*) and cytochrome oxidase subunit I (COI) and models of nucleotide substitution for each partition, based on the Bayesian information criterion (BIC) as implemented in PartitionFinder 2 [45]; Table S3: Comparison among biogeographic models evaluated with the software BioGeoBEARS; Table S4: Estimated crown ages (in millions of years ago, Ma) and results of the ancestral area estimates under the Dispersal-Extinction-Cladogenesis (DEC) model, assuming the MCMC Bayesian consensus tree obtained using the software BEAST 2 and concatenating the two mitochondrial genes.

**Author Contributions:** J.P.R., C.A.J., E.D.L., A.J.C. and R.I. designed the study; C.A.J., E.D.L. and R.I. collected and procured tissues; C.A.J. and R.I. examined specimens; C.A.J. conducted laboratory work to extract and sequence DNA; J.P.R., C.J.A. and A.J.C. performed the bioinformatic analyses on DNA sequences; J.P.R., C.A.J. and A.J.C. performed data curation; J.P.R., A.J.C. and R.I. prepared the manuscript with the contributions and edits from all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by Jegg Ettling, Field Research for Conservation (FRC) Program, Saint Louis Zoological Park. A.J.C. was supported by Research Program INV-2017-51-1432 from the School of Sciences, Universidad de los Andes. R.I. was funded by the Panama Amphibian Rescue and Conservation project, Sistema Nacional de Investigación (Secretaría Nacional de Ciencia, Tecnología e Innovación–SENACYT) and Minera Panamá (First Quantum Minerals Ltd.), during the preparation of the manuscript.

**Acknowledgments:** We thank the following persons and museums for providing access to tissue samples and/or specimens: David B. Wake and Tami Mott (Museum of Vertebrate Zoology, University of California at Berkeley), David C. Cannatella, Travis LaDuc, and Santiago Ron (Texas Natural History Collection, Texas Memorial Museum, University of Texas at Austin), Luis A. Coloma (Museo de Zoología, Pontificia Universidad Católica del Ecuador), William E. Duellman and Juan M. Guayasamin (Natural History Museum and Biodiversity Research Center, University of Kansas), Federico Bolaños (Museo de Zoología, Universidad de Costa Rica), and Yiselle Cano (Museo de Historia Natural C. J. Marinkelle, Universidad de los Andes, Bogotá). Thanks to Kevin Zippel, Anthony P. Wisniewski, Edgardo J. Griffith, Vicky Poole, Peter B. Johantgen, Allan Gillogly, Fidel E. Jaramillo, José Luis Atehortúa, Carlos A. Navas, Shyh-Hwang Chen, Victoriano González, and Karla Aparicio for donating specimens or tissues. Field assistance in Panama was provided by Frank A. Solís, James Coronado, and G. Urrieta. Alessandro Catenazzi and Juan C. Chaparro helped with permits in Peru. This work was conducted under the following permits: Instituto Nacional de Recursos Naturales Renovables de Panamá (scientific permits No. 27-92, No. 27-93, No. 46-94, No. 58-95, No. 03-97, No. 10-98, No. 20-2000, No. 39-2000 and No. 41-2000), Autoridad Nacional del Ambiente de Panamá (scientific permits No. SC/A 013-2001, No. SE/A-083-2001, No. SE/A-104-2001, No. SE/A-2-03, No. SE/A-78-03 and No. SE/A-07902, scientific import permits No. SIM/A 007-02, No. SIM/A 011-02, No. SIM/A-14-02, No. SIM/A-14-03 and No. SIM/A-16-03, scientific re-exportation permit No. SEX/A 059-02 and No. SER/A-1-04), and the U.S. Fish and Wildlife Service (declaration for export). We owe thanks to Eldredge Bermingham for opening the doors of his molecular genetics laboratory to us, to Lee A. Weigt for preliminary molecular work of initial tissues, and to Nimiadina Herrera and Maribel González for support and guidance in the laboratory. Thanks also to the members of the @CrawLab, especially Paola Montoya and Luis A. Rueda, for their feedback on this project. We acknowledge David Cannatella, David M. Dennis, Sandra V. Flechas, Michael and Patricia Fogden, Marcos A. Guerra, and Guido Sterkendries for photographic images of frogs in the graphical abstract and Figure 4. J.P.R. thanks Viviana Romero-Alarcón for constructive discussions about phylogenetic analyses and Martha Ramírez-Pinilla for support.

**Conflicts of Interest:** The authors declare no conflict of interest.
