**4. Discussion**

In recent decades, field research conducted in the Andes–Amazon region has uncovered dozens of new species of terrestrial-breeding frogs [12,16,40,45–47], and most of these species have small geographic distributions. Prior to this study, the only species of *Noblella* previously known to occur in lowland rainforest was *N. myrmecoides* and it was assumed that it had a broad geographic distribution across western Amazonia [48]. Our findings indicate that populations of *Noblella* from lowland and montane forest in southern Peru previously ascribed to *N. myrmecoides* represent a new species. Furthermore, our phylogenetic analyses indicate that *N. myrmecoides* belongs to a different clade than the clade including *N. losamigos* sp. n., other species of *Noblella* from southern Peru, and species of *Psychrophrynella*.

*Noblella losamigos* sp. n. is the only species in the genus *Noblella* that inhabits both lowland and montane rainforest, covering an elevational range of 1250 m, and the only species in the "southern clade" of *Noblella* (likely to represent *Noblella* sensu stricto) that inhabits the lowland Amazon rainforest. Previous research has shown that terrestrial breeding frogs distributed at high-elevations tend to have larger body size and different body shape than species found at lower elevations [49–53]. Our data supported this prediction (Figure 11). It has been unclear as to whether ectotherms follow Bergmann's rule, in which individuals of a species tend to be larger in colder environments and smaller in warmer environments [54,55], though our data sugges<sup>t</sup> that *N. losamigos* sp. n. appears to follow Bergmann's rule, as body size increases with elevation. Our data also sugges<sup>t</sup> that body shape of *N. losamigos* sp. n. varies across elevation, with lowland populations occupying a smaller morphological space than highland populations.

Regardless of the variation in body size and shape exhibited by *N. losamigos* sp. n., all members of the clade containing *Noblella* and *Psychrophrynella* are miniaturized. It is thought that miniaturization may allow species to access microhabitats and food sources that are not available to larger taxa, and may be particularly advantageous to species inhabiting the leaf litter of wet, tropical rainforests [56–59]. However, a smaller body size might lead to higher exposure to water loss resulting from higher surface area-to-volume ratio [59,60]. A further inquiry into the potential effect of ecological factors correlated with elevation (e.g., resource availability, competition) will develop our understanding of the ecomorphology of *Noblella* and *Psychrophrynella*.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/11/9/145/s1, Table S1: Genbank accession numbers for the taxa sampled in this study.

**Author Contributions:** Conceptualization, R.S.-C., R.v.M. and A.C.; methodology, R.S.-C., R.v.M. and A.C.; software, R.v.M. and D.L.R.; validation, R.S.-C., R.v.M. and A.C.; formal analysis, R.S.-C. and R.v.M.; investigation, R.S.-C., R.v.M., A.C., C.W., E.L.T. and D.L.R.; resources, R.v.M., A.C., E.L.T. and D.L.R.; data curation, R.S.-C., R.v.M., A.C. and C.W.; writing—original draft preparation, R.S.-C. and R.v.M.; writing—review and editing, R.S.-C., R.v.M., A.C., C.W., E.L.T. and D.L.R.; visualization, R.S.-C., R.v.M. and C.W.; supervision, R.v.M. and D.L.R.; project administration, R.S.-C. and R.v.M.; funding acquisition, R.v.M., A.C. and D.L.R.

**Funding:** R.S.-C. thanks UNSAINVESTIGA for providing support ("PP-0120-2016" Internship). R.v.M. thanks the National Science Foundation (Postdoctoral Research Fellowship DBI-1103087), the American Philosophical Society, the National Geographic Society Committee for Research and Exploration (Grant # 9191-12), and the Amazon Conservation Association. A.C. was funded with grants from the Mohamed bin Zayed Species Conservation Fund, the Disney Worldwide Conservation Fund, the Foundation Matthey-Dupraz, the Andrew Sabin Family Foundation, and the Amazon Conservation Association. Research was supported in part by a fellowship from the David and Lucile Packard Foundation to D.L.R.

**Acknowledgments:** We thank Cesar Aguilar (MUSM) and Greg Schneider (UMMZ) for supporting our access to specimens in the MUSM and UMMZ museum collections, respectively. We thank the Amazon Conservation Association and the staffs at Los Amigos Biological Stations and Los Amigos Conservation Concession for facilitating our work at their field stations. We thank Michelle Lynch and Erin Westeen for their help in generating μCT images included in this paper. We thank the Servicio Nacional Forestal y de Fauna Silvestre, Peru (SERFOR) for providing collecting permits (R.D.G. N◦ 120-2012-AG-DGFFS-DGEFFS, N◦ 064-2013-AG-DGFFS-DGEFFS, N◦ 0146-2013-AG-DGFFS-DGEFFS, N◦ 292-2014-AG-DGFFS-DGEFFS, N◦ 029-2016-SERFOR-DGGSPFFS, N◦ 405-2016-SERFOR-DGGSPFFS, and Contrato de Acceso Marco a Recursos Genéticos N◦ 359-2013-MINAGRI-DGFFS-DGEFFS). R.S.-C. thanks Julieta Cabrera for her help and patience in the coordination and use of research and training funds, and Amaranta Canazas for her support in advancing this manuscript. A.C. also thanks Perú Verde for permission to work at their biological station in San Pedro, and within their protected area (Área de Conservación Privada Bosque Nublado).

**Conflicts of Interest:** The authors declare no conflict of interest. The funding organizations that provided support for this work had no role in the design of the study, data collection, analyses, or interpretation of data, writing of the manuscript, or the decision to publish the results.
