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

We made intensive visual encounter surveys and auditory surveys both during the day and during the night (12h00–02h00). Due to the heterogeneity of the surveyed ecosystems and sampling methods, we could not calculate the relative abundance or the survey effort. However, we use the following terms in ranking the relative abundance (adapted from [23, 24]): common: when the species presence was detected (seen or heard), in the proper habitat, in large or moderate numbers, on 50– 100% of the sampling days/nights; uncommon: when the species presence was detected in moderate or small numbers, on 25–50% of the sampling days/nights; rare: when the species presence was detected in small numbers on less than 25% of the sampling days/nights. Collected specimens were photographed alive, after which they were euthanized using 20% benzocaine, fixed in 10% formalin, and stored in 70% ethanol. Tissue samples for genetic anal- yses were preserved in 96% ethanol. Examined specimens (listed in the type-series and S1 Appendix) are housed in Museo de Zoologı´a, Universidad Te´cnica Particular de Loja, Loja, Ecuador (MUTPL), Museo de Zoologı´a de la Universidad del Azuay, Cuenca, Ecuador (MZUA), Museo de Zoologı´a, Pontificia Universidad Cato´lica del Ecuador, Quito, Ecuador (QCAZ) and Museo de Zoologı´a, Universidad Tecnolo´gica Indoame´rica, Quito, Ecuador (MZUTI). Morphology The description of qualitative and quantitative morphological characters, as well as the format of the description follows Duellman and Lehr [25]. Sex was determined by the presence of

vocal slits and/or by gonadal inspection. Color data in life were based on field notes and digital photos. The specimens were weighted (body mass: BM) before euthanasia using a My Weigh Triton T3 portable scale with 0.01 g precision. Measurements were taken under a stereo micro- scope, with a Vernier caliper, and rounded to the nearest 0.1 mm. Specimens were measured for the following morphometric variables: (1) snout-vent length (SVL), distance from the tip of snout to posterior margin of vent; (2) head width (HW), widest portion of the head, mea- sured at level of jaw articulation; (3) head length (HL), distance from the tip of snout to poste- rior angle of jaw articulation; (4) interorbital distance (IOD), distance between the inner margins of the orbits; (5) internarial distance (IND), distance between the inner edges of the narial openings; (6) upper eyelid width (EW), the perpendicular distance to the outer edge of the eyelid; (7) eye diameter (ED), distance between anterior and posterior borders of eye; (8) eye-nostril distance (EN), distance from posterior margin of nostril to anterior margin of eye; (9) tympanum diameter (TD), horizontal distance between peripheral borders of tympanic annulus; (10) femur length (FL), length of femur from vent to knee; (11) tibia length (TL), length of flexed leg from knee to heel; (12) foot length (FoL), distance from proximal margin of inner metatarsal tubercle to tip of Toe IV; (13) hand length (HaL), distance from proximal edge of palmar tubercle to the tip of Finger III. Measurements are given as mean ± SD.

DNA extraction, amplification and sequencing DNA extraction was performed directly from 96% ethanol-preserved liver tissue, using the Extract-N-Amp™ Tissue PCR Kit (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), fol- lowed by PCR reactions under the manufacturer’s protocol. Additionally, in the case of hard to identify and potentially endangered groups (especially Gastrotheca spp.), species identity was confirmed by non-destructive technique, using buccal swabs for DNA extraction [26]. Individuals were afterwards released at capture site. The 12S and 16S rRNA mitochondrial genes, and the nuclear RAG-1 gene were amplified using the primers and annealing tempera- tures given in S1 Table. Success of PCR amplification was tested by gel electrophoresis, using a 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 5 / 59