Fifty years after: A taxonomic revision of the amphibian species from the Ecuadorian biodiversity hotspot Abra de Zamora, with description of two new Pristimantis species

(Fig 29D) Common English name. Santiago Robber Frog Common Spanish name. Cutı´n del Suro Rancho Identification. This small sized species is easily distinguished from all the other Pristiman- tis species present in Abra de Zamora by its whitish, yellowish coloration with pale brown strips, yellowish orange iris, skin on dorsum with low ridges, and black and white (sometimes yellow or orange) markings in axilla, groin and concealed limb surfaces [9]. Fig 29. Other amphibian species present in Abra de Zamora. A. Rhinella margaritifera, from Parque Nacional Podocarpus (Bombuscaro entrance); B. Gastrotheca elicioi, from Loja city; C. Gastrotheca testudínea, from Abra de Zamora; D. Pristimantis atratus, from Abra de Zamora; E. Pristimantis galdi, from Reserva Tapichalaca; F. Pristimantis lymani, from Loja city. https://doi.org/10.1371/journal.pone.0238306.g029 PLOS ONE Taxonomic revision of the amphibian species from Abra de Zamora PLOS ONE | https://doi.org/10.1371/journal.pone.0238306 September 10, 2020 52 / 59

Remarks. This is an uncommon species in Abra de Zamora. Can be encountered on the eastern slope in evergreen upper montane forest (Fig 2C), between 2700 and 2500 m. Pristimantis galdi Jime´nez de la Espada, 1870 (Fig 29E) Common English name. Espada’s Robber Frog Common Spanish name. Cutı´n verde amazo´nico Identification. This small sized species is easily distinguished from all other Pristimantis species present in Abra de Zamora by the bright green dorsum, green iris, acuminate snout, flared lips, serrated cranial crests (especially in females), a conical tubercle on the upper eyelid and greatly expanded, truncate discs on Fingers II–IV [25]. Remarks. This is an uncommon species in Abra de Zamora. Can be encountered on the eastern slope in evergreen lower montane forest (Fig 2D), around 1900 m, near the El Tambo sector. Pristimantis lymani (Barbour and Noble, 1920) (Fig 29F) Common English name. Lyman’s Robber Frog Common Spanish name. Cutı´n de Lyman Identification. This species is easily distinguished from all the other Pristimantis species present in Abra de Zamora by its large size, prominent tympanum, smooth skin on the venter, reddish brown dorsum, canthal and postorbital dark stripes, chevrons on the back, and trans- verse bars on the limbs [25]. It is the only member of the Pristimantis conspicillatus species group, and the only terrestrial Pristimantis species from the area. Remarks. This is a common species in Abra de Zamora and the most common species in the nearby Loja city. Can be encountered on the western slope in evergreen upper montane

forest bellow 2600 m, and in the pasturelands neighboring the city. Additionally, 10 potential new species were encountered in Abra de Zamora: • from the western slope, in the vicinity of Loja city, Noblela aff. heyeri and two Pristimantis species (P. aff. cajamarcensis and P. aff. torresi); • from the eastern slope, heading to El Tambo, an undescribed species of salamander (Bolito- glossa sp.), Atelopus aff. halihelos, two undescribed species of glass frogs (Centrolene sp.), Noblela aff. heyeri, and three undescribed species of Pristimantis. Unfortunately, from the 29 species registered (belonging to six families, Plethodontidae, Bufonidae, Centrolenidae, Hemiphractidae, Strabomantidae and Telmatobiidae), one (A. aff. halihelos) was not encountered in the past ten years and three others (A. podocarpus, G. psy- chrophila, and T. cirrhacelis) were never seen again for over twenty years. Discussion Systematics Already in 1979, Lynch [9] observed the similarity between P. vidua and P. orestes and hypoth- esized a close relationship; this was later recognized as a distinct assemblage in 1997 by Lynch and Duellman [59], composed by three Ecuadorian species (P. orestes, P. simonbolivari and P. vidua). We now have the phylogenetic confirmation that indeed P. vidua is a member of the resurrected P. orestes group (sensu [29]), being closely related to the species of the P. orestes subgroup: P. andinognomus, P. orestes, P. cajanuma and an undescribed species (Fig 7). The situation was different for P. colodactylus, which, due to its particular morphological

features, was included in a different group. In the original description [9], Lynch hypothesized PLOS ONE Taxonomic revision of the amphibian species from Abra de Zamora PLOS ONE | https://doi.org/10.1371/journal.pone.0238306 September 10, 2020 53 / 59

that P. colodactylus may be related to P. proserpens and P. celator, and in 1980 Lynch and Duellman [59] assigned it to the P. celator assemblage of the P. unistrigatus species group. The membership to the P. unistrigatus species group was maintained by Duellman and Lehr [25] and Hedges et al. [41]. With the description of P. muranunka, Brito et al. [29] recognized the morphological similarities of this species with P. colodactylus (and also with P. proserpens, P. paquishae, and P. tinajillas). In 2019, Urgilles et al. [16] recognized P. colodactylus (together with P. vidua and P. tinajillas) as a potential member of the P. orestes group. We provide genetic evidence that P. colodactylus is a member of the P. orestes group, which, together with P. muranunka, P. matildae and an undescribed species, forms a subgroup of closely related, morphologically distinct, bromeliad specialist species (Fig 7). In the original description of the species Lynch [9] observed several similarities between P. balionotus and P. percultus and “very tentatively” associated them with the balionotus-riveti- ruidus series. In 1980, Lynch and Duellman [59] assigned both species to the P. unistrigatus species group, although to two different assemblages: P. balionotus to the P. unistrigatus assem- bly and P. percultus to the P. glandulosus assembly. Hedges et al. [41] assigned both species to the P. unistrigatus species group. In 2019, Pa´ez and Ron [30] assigned P. balionotus and P. per- cultus, based on their similar morphology, to the P. phoxocephalus species group of the newly

tected, being inside Parque Nacional Podocarpus, the amphibians of this area face several threats. Among the main threats we can mention are the loss and degradation of habitats, due to cattle farming, and the introduction of exotic species (Rainbow Trout, Oncorhynchus mykiss and Bullfrog, Lithobates catesbeianus), species that have already been proven to have a direct negative effect on Andean amphibian populations [61]. On the other hand, forest fires are also a potential threat, since they occur frequently in nearby sectors intended for agriculture or livestock. As for the infectious diseases that are affecting the Ecuadorian amphibian populations, we have limited information from the study area. The presence of chytrid fungus (Batrachochy- trium dendrobatidis) is documented from the Andes of central and northern Ecuador [62, 63]. A recent survey conducted by researchers from University of Central Florida, yielded negative PLOS ONE Taxonomic revision of the amphibian species from Abra de Zamora PLOS ONE | https://doi.org/10.1371/journal.pone.0238306 September 10, 2020 54 / 59

results for samples collected from Abra de Zamora, (Urgiles et al., pers. com.). However, it is possible that the decline of several species in Abra de Zamora could be caused by the chytrid fungus [62]. To assure an adequate protection of the areas that currently are not included in Parque Nacional Podocarpus, we are working on a proposal, in collaboration with the local govern- ments and Ecuadorian Ministry of Environment. Additionally, by the end of 2020, the Action Plan for the conservation of the amphibians of Abra de Zamora will be available, generated by the EcoSs Lab group of Universidad Te´cnica Particular de Loja and supported by funding from the Critical Ecosystem Partnership Fund (CEPF). Supporting information S1 Appendix. Additional specimens examined. (DOCX) S1 Table. Primers used for PCR amplification of 12S rRNA, 16S rRNA, and RAG1 nDNA. (DOCX) S2 Table. Voucher, GenBank accession numbers and locality for the specimens used in the phylogenetic analysis. (DOCX) S3 Table. Uncorrected pairwise distances (%), for the mitochondrial gene 16S fragment, for Lynchius, Pristimantis orestes group and Huicundomantis subgenus of Pristimantis. (XLSX) S4 Table. Information regarding the call recordings and the bioacoustic measurements for each of the recorded males. Values are given as average ± SD (range) and n = sample size. (XLSX) S5 Table. Amphibian species recorded in Abra de Zamora. (XLSX) Acknowledgments We want to thank Enrique Armijos Cano as well all the other rangers of Podocarpus National

Park and the rangers of Reserva Cerro Plateado for their support during our fieldwork. We are grateful for the help and support offered by Ministerio del Ambiente y Agua de Ecuador, Zonal Loja and Zonal Zamora Chinchipe. Special thanks to our former and current students to their valuable help during fieldwork: Santiago Padilla Bustos, Gabriela Mora Castillo, Daniela Sa´nchez Ludeña, Marek Castel Tapia, Stalin Go´mez Eras, Alejandro Ochoa Carrillo, Michelle Angamarca Guartan, Santiago Hualpa Vega, Daniel Hualpa Vega, Maria Cordova Diaz, and Guillermo Enriquez Prado. In addition, we thank Naturaleza y Cultura Internacio- nal, especially Felipe Serrano, and Marı´a F. Ta´pia, Marı´a F. Burneo, Vı´ctor Romero for their support in the implementation of the project. We are grateful to the Tapia Castro family, and especially to Hugo, the administrator of Reserva Madrigal del Podocarpus, for their dedication to the conservation of this amazing place and for granting us access and helping with the field- work. We also want to thank Jose´ Napoleo´n Carrio´n Gordillo for his generous help during our fieldwork in La Siberia hacienda. Research permit was issued by Ministerio del Ambiente del Ecuador (MAE-DNB-CM-2015-0016, granted to Universidad Te´cnica Particular de Loja). PLOS ONE Taxonomic revision of the amphibian species from Abra de Zamora PLOS ONE | https://doi.org/10.1371/journal.pone.0238306 September 10, 2020 55 / 59

Author Contributions Conceptualization: Paul Sze´kely, Diana Sze´kely. Data curation: Paul Sze´kely, Juan Sebastia´n Eguiguren, Diego Armijos-Ojeda. Formal analysis: Paul Sze´kely, Juan Sebastia´n Eguiguren, Leonardo Ordo´ñez-Delgado, Diego Armijos-Ojeda. Funding acquisition: Paul Sze´kely, Diego Armijos-Ojeda, Diana Sze´kely. Investigation: Paul Sze´kely, Juan Sebastia´n Eguiguren, Leonardo Ordo´ñez-Delgado, Diego Armijos-Ojeda, Diana Sze´kely. Methodology: Paul Sze´kely, Juan Sebastia´n Eguiguren, Leonardo Ordo´ñez-Delgado, Diana Sze´kely. Project administration: Paul Sze´kely, Diego Armijos-Ojeda, Diana Sze´kely. Resources: Paul Sze´kely, Diego Armijos-Ojeda, Diana Sze´kely. Supervision: Paul Sze´kely. Validation: Paul Sze´kely. Visualization: Paul Sze´kely, Leonardo Ordo´ñez-Delgado, Diego Armijos-Ojeda, Diana Sze´kely. Writing – original draft: Paul Sze´kely. Writing – review & editing: Paul Sze´kely, Juan Sebastia´n Eguiguren, Leonardo Ordo´ñez-Del- gado, Diego Armijos-Ojeda, Diana Sze´kely. References 1. Ulloa Ulloa C, Acevedo-Rodrı´guez P, Beck S, Belgrano MJ, Bernal R, Berry PE, et al. An integrated assessment of the vascular plant species of the Americas. Science. 2017; 358: 1614–1617. https://doi. org/10.1126/science.aao0398 PMID: 29269477 2. Ron SR, Merino-Viteri A, Ortiz DA. Anfibios del Ecuador. Version 2019.0. https://bioweb.bio/faunaweb/ amphibiaweb, accessed on 19 May 2020. Museo de Zoologı´a, Pontificia Universidad Cato´ lica del Ecua- dor. 2019. 3. Torres-Carvajal O, Pazmiño-Otamendi G, Salazar-Valenzuela D. Reptiles del Ecuador. Version

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