*2.1. Laboratory Work*

We followed protocols of extraction, amplification, and sequencing of DNA previously used for terrestrial-breeding frogs [1,20,22]. For the focal taxa (the three species members of the new genus), we extracted DNA from tissue samples obtained from six specimens collected in the field (two specimens per species). We also obtained DNA sequences from seven specimens in five other species of *Bryophryne*, and two specimens representing two species in other genera (*Noblella* and *Psychrophrynella*), and the remaining sequences are legacy data from GenBank.

We extracted DNA from liver tissue preserved in 70% ethanol by using a commercial extraction kit (IBI Scientific, Dubuque, IA, USA). We used selected primers (Table 2) to amplify DNA from each gene using the polymerase chain reaction (PCR) [22,32]. We obtained sequence data by running purified PCR products in an ABI 3730 Sequence Analyzer (Applied Biosystems), except sequences of *B. mancoinca* and *B. phuyuhampatu*, which we shipped to MCLAB (San Francisco, CA) for sequencing. We deposited all new sequences in GenBank (Table 1). We provide updated names of 86 terminals included in the analysis for 314 GenBank sequences.


**Table 2.** Primers used in this study.

## *2.2. Molecular Phylogenetic Analyses*

We inferred the phylogenetic relationships among taxa through analysis of concatenated DNA sequences of the five gene fragments (16S, 12S, COI, RAG1, Tyr). We used *Niceforonia dolops* to root the tree. We aligned sequences with Geneious R6, v. 6.1.8 (Biomatters 2013), using the built-in Geneious Aligner program. We then used PartitionFinder, v. 1.1.1 [36] to select the best partitioning scheme and substitution model for each gene using the Bayesian information criterion (BIC). The best partitioning scheme included the following six subsets (best fitting substitution models are in parentheses): partition subset 1 includes 12S and 16S sequences (GTR + I + G), partition 2 is the first codon position of COI (SYM + G), partition 3 is the second codon position of COI (F81), partition 4 is the third codon position of COI (HKY + G), partition 5 includes the first and second codon positions of RAG together with the first and second codon positions of Tyr (HKY + I + G), and partition 6 includes the third codon position of RAG together with the third codon position of Tyr (K80 + G).

We used MrBayes, v. 3.2.0 [37] to infer a molecular phylogeny for the 106 terminals and 2632 bp concatenated partitioned dataset (16S, 12S, COI, RAG1, Tyr). We performed an MCMC Bayesian analysis that included two simultaneous runs of 10 million generations, sampled once every 1000 generations. Each run had one "cold" chain and three heated chains, and the burn-in was set to discard 25% samples from the cold chain. Upon completion of the MCMC Bayesian analysis, the average standard deviation of split frequencies was 0.003916. We used Tracer version 1.5 [38] to examine the effective sample sizes (ESS), to verify convergence, and to verify that the runs reached stationarity. The observed effective sample sizes were satisfactory for all parameters (ESS > 200). Lastly, we used FigTree v. 1.4.2 [39] to visualize the majority-rule consensus tree and assess node support (based on posterior probability values).

Our research was approved by the Institutional Animal Care and Use Committee of Florida International University (18-009). The Dirección General Forestal y de Fauna Silvestre, Ministerio de Agricultura y Riego issued the permit authorizing this research (collecting permits #292-2014-MINAGRI-DGFFS-DGEFFS, SERNANP-Machu Picchu 054-2012-SERNANP-JEF, Contrato de Acceso Marco a Recursos Genéticos, No 359-2013-MINAGRI-DGFFS-DGEFFS).

The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) and the associated information can be viewed through any standard web browser at http://zoobank.org/urn:lsid:zoobank.org:pub:0B8FFBEE-96AA-46E1-BA6F-541DC9FA73BF.
