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

3x elongated and rounded outer metatarsal tubercle; subarticular tubercles prominent, round and rounded in section; plantar supernumerary tubercles rounded, smaller than subarticular tubercles; toes bearing prominent lateral fringes; webbing basal; discs on toes slightly Fig 20. Relative size of the fingers and toes in Pristimantis colodactylus (MUTPL 388, SVL 20.5 mm), P. muranunka (MUTPL 652, SVL 20.1 mm) and P. matildae sp. nov. (Holotype, MUTPL 731, SVL 20.2 mm). https://doi.org/10.1371/journal.pone.0238306.g020 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 35 / 59

expanded, truncated, about same size as those on fingers; toes with ventral pads well defined by circumferential grooves (Fig 18D); relative length of toes I < II < III < V < IV; Toe V much longer than Toe III (tip of Toe III barely reaching the penultimate subarticular tubercle on Toe IV, tip of Toe V extends to the distal edge of distal subarticular tubercle on Toe IV). Coloration of holotype. In life (Fig 16): dorsum, upper parts of flanks, dorsal surfaces of hindlimbs and arms grayish tan with dark markings; on the middle of the back a large dark X- shaped marking is visible; the head with dark brown interorbital bar, canthal and supratympa- nic stripes; lower parts of flanks, venter, throat and ventral surfaces of hindlimbs and arms pinkish gray; iris bronze with a dark median horizontal streak and fine black reticulations. In preservative (Fig 17): the coloration of dorsum and dorsal surfaces of hindlimbs and arms dark gray, with the dark X-shaped marking evident; venter brownish gray; throat and ventral surfaces of hindlimbs and arms light gray. Measurements of holotype (in mm): SVL 20.2; HW 6.7; HL 6.6; IOD 2.2; IND 1.8; EW 1.7; ED 2.2; EN 1.8; snout to eye distance 2.9; FL 8.8; TL 9.3; FoL 7.9; HaL 5.1; Finger I length 2.4. Body mass of holotype: 0.50 g. Variation. Morphometric variation is shown in Table 1. Except for the female MUTPL 394 (Fig 19B), all specimens had a light interorbital bar flanked by a dark border. This female had the uniquely yellowish tan broad middorsal band, bordered by dark brown dorsolateral stripes

that continue onto the snout and pinkish gray flanks, dorsal surfaces of hindlimbs and arms and venter. Female MUTPL 361 (Fig 19A) had a tan background coloration with dark brown reticulum and the female MUTPL 813 (Fig 19C) had a general grayish coloration with dark markings. Two individuals, MUTPL 813 and 362, additionally had yellowish white spots of different sizes on their dorsum and flanks (Fig 19C and 19D). Several individuals had a large dark X-shaped marking on their back which in case of the female MUTPL 361(Fig 19A), male MUTPL 362 (Fig 19D) and juvenile MUTPL 733 (Fig 19F) is more or less similar to an “hour- glass” shaped marking. The male MUTPL 360 (Fig 19E) had a general dark brown coloration. Advertisement call. Unknown. Distribution. Pristimantis matildae is known only from Abra de Zamora and the Cajanuma sector from the Parque Nacional Podocarpus, about 13 km to the south (Fig 6). We were not able to encounter this species in adjacent areas, with similar ecosystems. However, about 35 km south from the type locality, we found another bromeliad specialist from the P. colodacty- lus subgroup, a genetically and morphologically similar, undescribed species. Pristimantis matildae was encountered at an altitudinal range between 2800 and 3360 m a.s.l. in subpa´ramo (Fig 2B) and shrub pa´ramo ecosystems. Natural history. This is a common species and one of the most abundant species encoun- tered inside of bromeliads. All the specimens were encountered in terrestrial or arboreal bro-

meliads or in their close proximity (perching on the leaves), during the day or night. Similarly to P. colodactylus and P. muranunka, this species displayed the same defensive behavior of rapidly retreating to the water from the bromeliads when threatened. No calling males were heard. Conservation status. Pristimantis matildae is known from only two close localities, from a small area estimated to be less than 50 km2. Nonetheless, we recommend that this species to be categorized as Near Threatened following the IUCN criteria [47], due the fact that it is a rela- tively abundant species and its habitat currently does not face any major threats (because most of the population is situated within a national protected area—Parque Nacional Podocarpus). Remarks. Pristimantis matildae is part of the P. colodactylus subgroup and is closely related to P. colodactylus, P. muranunka and an undescribed species (Fig 7). The genetic distance between P. matildae and its closest relatives from the P. colodactylus subgroup ranges between 5.4–5.8% (P. colodactylus) and 6.6–7.5% (P. muranunka) (S3 Table). 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 36 / 59

Subgenus Huicundomantis Pa´ez and Ron, 2019 Four species of this subgenus were described from Abra de Zamora. Phylogeny For the Huicundomantis subgenus PartitionFinder under AICc (for ML) identified three parti- tion schemes as the best strategy (best model in parentheses): 12S and 16S (GTR+I+G), RAG-1 1st position (HKY+G), and RAG-1 2nd and 3rd positions (TIM+I+G). Under BIC (for BI) Parti- tionFinder identified three partitions: 12S and 16S (GTR+I+G), RAG-1 1st position (K80+G), and RAG-1 2nd and 3rd positions (K81UF+I). The phylogenetic trees constructed by Bayesian inference and Maximum likelihood showed mostly the same topology with differences in the position of some of the unresolved branches as P. atratus, P. totoroi, P jimenezi, and the P. teslai/P. UCS2 subgroup (Fig 21). In general, the branches had stronger support in the case of the Bayesian inference. Similarly to the results of Pa´ez & Ron [30], we recovered the Huicundomantis subgenus as monophyletic with strong support (bootstrap values = 98.7%; posterior probabilities = 1) in both Maximum likelihood and Bayesian analyses (Fig 21). However, in our analysis, the P. cryptomelas group had a strong support only in the case of the Bayesian inference, the ML analysis yielding moderate support. Overall, the phylogenetic tree of our analysis showed the same topology with the one constructed by Pa´ez & Ron [30]. The main differences were in the position of several unresolved branches such as P. jimenezi, P. phoxocephalus, P. totoroi and P.

tinguichaca. Additionally, with the inclusion of the newly generated P. cryptomelas sequences, this species position, relative to P. spinosus, became weakly supported (BI) or only moderately supported (ML) (Fig 21). Our analysis confirmed the membership of P. percultus, P. andinogigas, and P. balionotus to the P. phoxocephalus species group. In the case of P. andinogigas the pairwise genetic distance from its closest congener, P. chomskyi, it was only 2.3% (S3 Table) thus a careful revision of these species statuses is needed. Pristimantis cryptomelas species group One species of this species group was described from Abra de Zamora. Pristimantis cryptomelas (Lynch, 1979) (Fig 22) Eleutherodactylus cryptomelas Lynch, 1979 Eleutherodactylus (Eleutherodactylus) cryptomelas: Lynch, 1996; Lynch and Duellman, 1997 Pristimantis cryptomelas: Heinicke, Duellman, and Hedges, 2007 Pristimantis (Pristimantis) cryptomelas: Hedges, Duellman, and Heinicke, 2008 Pristimantis (Huicundomantis) cryptomelas: Pa´ez and Ron, 2019 Common English name. Cryptic Robber Frog Common Spanish name. Cutı´n crı´ptico Etymology. The specific name is derived from the Greek kryptos, meaning “hidden” and the Greek melas, meaning “black” and refers to the black areas in the axilla and groin and on the hidden surfaces of the limbs [25]. Holotype. KU 141992, an immature female, taken 15 km E Loja, Provincia Zamora- Chinchipe, Ecuador, 2710 m, May 1971 by William E. Duellman. Paratypes. KU 141993, taken syntopically with holotype; KU 120095–96, 8–9 km N San

Lucas, Provincia Loja, Ecuador, 3000 m; USNM 198480–82, Sapote, Provincia Morona-Santi- ago, Ecuador, 2470 m; USNM 198483, 2 km W Sapote, Provincia Morona-Santiago, Ecuador, 2560 m. 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 37 / 59

Diagnosis. Pristimantis cryptomelas is a medium sized species distinguished by the follow- ing combination of traits: (1) skin on dorsum shagreened; skin on venter areolate; discoidal fold weak; dorsolateral folds absent; low middorsal fold present; (2) tympanic membrane absent but tympanic annulus evident, oval, its length about 30% of the length of eye; Fig 21. Maximum likelihood phylogram of the Huicundomantis subgenus of Pristimantis based on 2463 base pairs of concatenated DNA from 12S, 16S, and RAG-1 gene fragments. Bootstrap values (%) and Bayesian posterior probabilities (decimal) are shown except when they are below 50 (bootstrap) or 0.5 (posterior probability); asterisks indicate support values of 100% or 1 (posterior probabilities). Outgroup is not shown; the tree was routed with Pristimantis galdi. With green are marked the newly generated sequences for the present study. The collection number, species name, province and short locality name of the samples are shown next to each terminal; all samples are from Ecuador (associated data listed in S2 Table). Abbreviations: CCS = confirmed candidate species, UCS = unconfirmed candidate species [30]. https://doi.org/10.1371/journal.pone.0238306.g021 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 38 / 59

supratympanic fold present; (3) snout subacuminate in dorsal view, rounded in profile; can- thus rostralis straight in dorsal view, rounded in profile; (4) upper eyelid bearing 1 to 3 larger tubercles (trait more visible in life), about 84% IOD; cranial crests absent but prominent) (or > < -shaped scapular folds are present (trait more visible in life); (5) dentigerous processes of vomers prominent, oblique, ovoid, separated medially by distance equal or lower than the width of processes; each processes bearing 3 to 5 teeth; (6) males lacking vocal sac and slits; nuptial pads absent; (7) Finger I shorter than Finger II; discs on fingers broadly expanded, truncated; circumferential grooves present; fingers bearing narrow lateral fringes; (8) subarti- cular tubercles prominent; supernumerary palmar tubercles evident; palmar tubercle partially (sometimes completely) divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle elliptical, smaller than the palmar tubercle but larger than either palmar tubercles (inner or outer); (9) prominent ulnar tubercles present (trait more visible in life); (10) heel with one large, conical and several smaller tubercles; outer edge of tarsus bearing a row of enlarged spine like tubercles (trait more visible in life); inner tarsal tubercles coalesced into a short tarsal fold; (11) inner metatarsal tubercle elongated, not compressed, about 4 to 6x ovoid, subconical (in profile), outer metatarsal tubercle; supernumerary plantar tubercles pres-

ent; (12) Toe V much longer than Toe III; discs on toes broadly expanded, truncated, slightly smaller than those of fingers; circumferential grooves present; toes bearing narrow lateral Fig 22. Color variation of Pristimantis cryptomelas in life. A. female: MUTPL 168; B. male: MUTPL 470; C. subadult: MUTPL 135; D. juvenile: MUTPL 493. Abra de Zamora (B, C), Bosque Protector Washapamba (A), Parque Nacional Podocarpus—Cajanuma (D). Most common coloration of this species is presented in A. https://doi.org/10.1371/journal.pone.0238306.g022 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 39 / 59

fringes; webbing basal; (13) in life, dorsum gray to brown with sparse darker markings or chev- rons; venter white to cream with brown reticulations; hindlimbs usually with dark transverse bars; axilla, groin, anterior and posterior surfaces of thighs, and concealed surfaces of shanks black bordered by white, yellow or orange blotches; iris dark bronze with a reddish median horizontal streak; (14) SVL 28.9–45.7 mm in adult females (35.3 ± 9.06 SD, n = 3) and 25.6– 31.2 mm in adult males (n = 2). Variation. Morphometric variation is shown in Table 3. The females are significantly larger than the males, with one female (MUTPL 168, Fig 22A) having 45.7 mm SVL. All encountered specimens had the groin (but frequently also the axilla, anterior and posterior surfaces of thighs, and concealed surfaces of shanks) black, with the bordering blotches (or stripes) vary- ing from reddish orange (Fig 22A and 22C), yellow (Fig 22B) to whitish yellow (Fig 22D). Manny encountered individuals had the coloration of the female MUTPL 168 (Fig 22A), with brown or light brown dorsum and whitish, light markings. A few individuals had some white, branch-like white markings on their flanks and dorsum like in the case of the male MUTPL 470 (Fig 22B), and several individuals had the head and snout distinctly colored (white or beige) compared to the rest of the brownish body (MUTPL 135, Fig 22C). The juveniles had a more rugged general appearance and it seems that they lack the intense yellow or orange color- ation of the inguinal and axial blotches (MUTPL 493, Fig 22D). Advertisement call. Unknown.

Distribution. Pristimantis cryptomelas presence is confirmed (with DNA samples) from Reserva Tapichalaca, Parque Nacional Podocarpus (Cajanuma entrance), Abra de Zamora, Huacapamba (5 km north to the city of Loja) and Bosque Protector Washapamba, in Loja and Zamora Chinchipe provinces (Fig 23). The species has been reported from Morona Santiago province [9] and Peru [25]; however, due to the large distance between the sites, we recom- mend genetic analyses to confirm that these populations in fact belong to P. cryptomelas. The species was encountered at an altitudinal range between 2480 and 2960 m a.s.l. in montane cloud forest, evergreen upper montane forest (Fig 2C) and subpa´ramo ecosystems. Natural history. This is a common species, even in habitats with anthropic disturbance. All specimens were encountered during the night, perching on the vegetation (usually at 30 cm–2 m above the ground), sometimes in the vicinity of streams. No calling males were heard. Conservation status. Pristimantis cryptomelas is currently categorized as Near Threatened following the IUCN criteria [51]. We consider this category as adequate due the fact that it is a relatively abundant species and some of its populations are inside protected areas (Parque Nacional Podocarpus, Bosque Protector Washapamba), but also because it is currently known from only 5 localities, the existing populations are severely fragmented and there is a continu- ing decline in the extent and quality of its habitat due to cattle grazing and deforestation.

Remarks. Lynch [9] provides a detailed description of this species. Our diagnosis concurs with most of the morphological features described by the author, and we add descriptions of the characters that are more evident in life. Some of the differences that we observed are: tym- panum oval (in contrast to the round tympanum described by Lynch); discoidal fold weak (discoidal folds prominent); upper eyelid bearing 1 to 3 larger tubercles (upper eyelids tuber- culate, no tubercle greatly enlarged). The diagnosis provided herein is based on 8 specimens from the original description (KU 141992, 141993, 120095, 120096, 198480–83), one adult male (MUTPL 470) and two subadults (MUTPL 135, 471) collected from the type locality and 10 specimens collected from the other localities. Pristimantis phoxocephalus species group Three species of this species group were described from Abra de Zamora. 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 40 / 59

Pristimantis percultus (Lynch, 1979) (Fig 24) Eleutherodactylus percultus Lynch, 1979 Eleutherodactylus (Eleutherodactylus) percultus: Lynch, 1996; Lynch and Duellman, 1997 Pristimantis percultus: Heinicke, Duellman, and Hedges, 2007 Table 3. Body mass (in grams), measurements (in mm) and morphological proportions (in percentages) of adult females and males of the species from the Subge- nus Huicundomantis described from Abra de Zamora. Character Pristimantis cryptomelas Pristimantis percultus Pristimantis balionotus Pristimantis versicolor females (n = 3) males (n = 2) females� (n = 3) males� (n = 1) females� (n = 2) males (n = 6) females (n = 3) males (n = 4) Body mass (BM) 3.39 ± 2.69 (1.68– 6.49) 1.19–1.84 2.08–2.52 - 2.48 1.12 ± 0.21 (0.88– 1.44) 2.13 ± 0.69 (1.62– 2.92) 1.05 ± 0.13 (0.96– 1.24) Snout-vent length (SVL) 35.3 ± 9.06 (28.9– 45.7) 25.6–31.2 33.6 ± 4.15 (30.1– 38.2) 29.8 28.4–32.4 24.7 ± 0.83 (23.6– 25.8) 30.8 ± 1.40 (29.4– 32.2) 24.0 ± 1.08 (22.9– 25.4) Head width (HW) 12.5 ± 3.38 (10.5– 16.4) 9.6–11.6 13.1 ± 1.71 (11.7– 15.0) 12.1 11.3–11.5 9.0 ± 0.47 (8.4– 9.8) 11.5 ± 1.26 (10.3– 12.8) 8.8 ± 0.39 (8.4– 9.2) Head length (HL) 11.6 ± 3.15 (9.7– 15.2) 7.6–9.5 11.9 ± 1.30 (10.6– 13.2) 11.0 10.4–10.7 8.7 ± 0.50 (7.9– 9.3) 10.3 ± 0.95 (9.4– 11.3) 8.0 ± 0.84 (7.2– 8.9) Interorbital distance (IOD) 4.0 ± 1.00 (3.2– 5.1) 2.8–3.4 4.0 ± 0.30 (3.7– 4.3) 3.9 2.8–4.0 3.0 ± 0.49 (2.6– 3.9) 3.3 ± 0.27 (3.1– 3.6) 2.5 ± 0.19 (2.4– 2.8) Internarial distance (IND) 2.7 ± 0.46 (2.4– 3.2) 2.1–2.5 2.4–2.6 - 2.4 2.0 ± 0.09 (1.9– 2.1) 2.4 ± 0.17 (2.2–

2.5) 1.9 ± 0.08 (1.8– 2.0) Upper eyelid width (EW) 3.3 ± 0.78 (2.7– 4.2) 2.3–2.9 3.0 ± 0.21 (2.8– 3.2) 3.4 2.6–3.1 2.5 ± 0.09 (2.4– 2.6) 2.8 ± 0.10 (2.7– 2.9) 2.5 ± 0.06 (2.4– 2.5) Eye diameter (ED) 4.0 ± 1.19 (3.3– 5.4) 3.0–3.6 3.8 ± 0.55 (3.4– 4.4) 3.5 3.3 2.9 ± 0.14 (2.8– 3.1) 3.5 ± 0.15 (3.3– 3.6) 2.9 ± 0.06 (2.8– 2.9) Eye-nostril distance (EN) 3.6 ± 1.29 (2.5– 5.0) 2.4–3.5 3.6 ± 0.58 (3.3– 4.3) 3.4 3.6–4.0 2.7 ± 0.15 (2.5– 2.9) 3.1 ± 0.15 (3.0– 3.3) 2.6 ± 0.22 (2.3– 2.8) Tympanum diameter (TD) 1.1 ± 0.21 (0.9– 1.3) 0.9–1.1 1.8 ± 0.20 (1.6– 2.0) 1.5 1.7–1.8 1.4 ± 0.11 (1.3– 1.6) 1.6 ± 0.15 (1.4– 1.7) 1.3 ± 0.08 (1.2– 1.4) Femur length (FL) 16.1 ± 4.16 (13.6– 20.9) 12.3–14.7 15.2–17.3 - 13.3 10.6 ± 0.30 (10.2– 10.8) 14.5 ± 1.05 (13.5– 15.6) 11.0 ± 0.44 (10.4– 11.4) Tibia length (TL) 18.3 ± 3.92 (15.5– 22.8) 14.4–15.2 18.6 ± 2.33 (16.7– 21.2) 15.5 12.8–14.3 11.5 ± 0.24 (11.2– 11.9) 16.4 ± 0.72 (15.8– 17.2) 12.6 ± 0.72 (11.8– 13.5) Foot length (FoL) 17.4 ± 4.77 (14.1– 22.9) 12.4–15.3 16.0–16.6 - 13.1 9.8 ± 0.30 (9.3– 10.2) 14.6 ± 1.23 (13.7– 16.0) 11.4 ± 0.81 (10.4– 12.3) Hand length (HaL) 11.2 ± 3.71 (8.5– 15.4) 8.2–9.3 9.6–10.8 - 7.7 6.4 ± 0.33 (6.0– 6.9) 9.8 ± 1.15 (8.9– 11.1) 7.3 ± 0.43 (6.9– 7.9) HW/SVL 33.8–36.3 37.2–37.5 38.7–39.3 40.6 34.9–40.5 34.1–38.1 35.0–39.8 36.2–37.0 HL/SVL 31.2–33.6 29.7–30.4 34.6–36.8 36.9 32.1–37.7 31.7–36.8 32.0–35.1 31.1–35.0 HL/HW 92.4–92.7 79.2–81.9 88.0–95.2 90.9 92.0–93.0 88.8–98.9 88.3–91.3 85.9–96.7

EN/HL 25.8–32.9 31.6–36.8 27.5–32.6 30.9 33.6–38.5 30.1–34.2 29.2–31.9 31.5–34.2 ED/HL 33.7–35.5 37.9–39.5 29.2–33.3 31.8 30.8–31.7 31.1–36.7 31.9–35.1 32.6–38.9 EW/IOD 82.4–86.1 82.1–85.3 72.5–75.7 87.2 65.0–110.7 66.7–96.2 80.6–87.5 89.3–104.2 EN/ED 73.5–97.0 80.0–97.2 94.3–97.7 97.1 109.1–121.2 87.1–103.6 88.6–91.7 82.1–96.6 TD/ED 22.2–39.4 30.0–30.6 45.5–51.4 42.9 51.5–54.5 44.8–51.6 42.4–47.2 42.9–48.3 FL/SVL 43.9–47.1 47.1–48.0 50.5–53.1 - 41.0 41.9–43.9 45.9–48.4 44.3–47.2 TL/SVL 49.9–53.6 48.7–56.3 54.9–55.5 52.0 44.1–45.1 44.6–49.8 52.4–53.7 50.2–53.7 FoL/SVL 48.7–50.1 48.4–49.0 50.9–53.2 - 40.4 38.4–42.7 45.6–49.7 45.4–48.4 HaL/SVL 29.4–33.7 29.8–32.0 31.9–33.1 - 23.8 24.1–26.7 30.3–34.5 30.0–31.1 Values are given as mean ± SD (range). Female body mass includes eggs. �—indicates that some measurements were included from the original descriptions [9]. https://doi.org/10.1371/journal.pone.0238306.t003 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 41 / 59

Pristimantis (Huicundomantis) percultus: Pa´ez and Ron, 2019 Common English name. Zamora Robber Frog Common Spanish name. Cutı´n de pa´ramo bajo Etymology. Latin, meaning highly adorned, in reference to the orange lip stripes. Fig 23. Distribution of the species of the Huicundomantis subgenus described from Abra de Zamora. Records are based on specimens (and DNA sequences) deposited at the Museo de Zoologı´a, Universidad Te´cnica Particular de Loja (MUTPL). 1. Bosque Protector Washapamba, 2. Ramos Urcu, 3. Huacapamba, 4. Reserva Madrigal del Podocarpus, 5. Parque Nacional Podocarpus (Cajanuma sector), 6. Reserva Tapichalaca. https://doi.org/10.1371/journal.pone.0238306.g023 Fig 24. Color variation of Pristimantis percultus in life. A–B females: A. MUTPL 810, B. MUTPL 812; C. subadult: MUTPL 811. All specimens are from Parque Nacional Podocarpus—Cajanuma. https://doi.org/10.1371/journal.pone.0238306.g024 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 42 / 59

Holotype. KU 166058, an adult male, found in Abra de Zamora, Provincia Zamora- Chinchipe, Ecuador, 2850 m, on 8 March 1975 by Dana K. Duellman. Paratype. KU 166057, an adult female topotype, obtained on 7 March 1975. Diagnosis. Pristimantis percultus is a medium sized species distinguished by the following combination of traits: (1) skin on dorsum and flanks pustulated, covered with large flat warts or pustules (in life the pustulated skin texture is more evident); skin on venter coarsely areo- late; discoidal fold weak; dorsolateral folds absent; (2) tympanic annulus evident and tympanic membrane differentiated, its length about 47% of the length of eye; supratympanic fold pres- ent; (3) snout subacuminate in dorsal view, rounded in profile, with pointed tip; canthus ros- tralis weakly concave in dorsal view, rounded in profile; (4) upper eyelid bearing one larger and several small tubercles (trait more visible in life), about 74% IOD in females and 87% IOD in males; barely visible, low cranial crests present; (5) dentigerous processes of vomers promi- nent, oblique, triangular, separated medially by distance lower than the width of processes; each processes bearing 4 to 8 teeth; (6) males lacking vocal sac and vocal slits; (7) Finger I shorter than Finger II; discs on fingers broadly expanded, rounded; circumferential grooves present; fingers bearing narrow lateral fringes; (8) subarticular tubercles prominent, round, non-conical; supernumerary palmar tubercles present; palmar tubercle bifid, partially divided

the specimen KU 202427. The subadult MUTPL 811 (Fig 24C) had a peculiar coloration with dorsum light brown and flanks multicolored with yellow, green, brown and pinkish gray blotches. Also, this specimen was the only one that had dark interorbital bars. In all encoun- tered specimens the dorsum and especially the flanks pustulated texture it was very evident. Most of the pustules had the yellowish orange color similar with those of the holotype. Addi- tionally, all three specimens presented some scar-like wrinkles on their flanks (more evident in the case of MUTPL 812, Fig 24b). Advertisement call. Unknown. Distribution. Despite intensive searches carried out since 2016 we did not find any individ- uals in the type locality, Abra de Zamora. Pristimantis percultus was reported from Parque Nacional Podocarpus (Cajanuma sector) in 2010 [52], in a grass pa´ramo ecosystem [44], from about 12 km south to Abra de Zamora (Fig 23). We also encountered there in 2019. The spe- cies was recorded at an altitudinal range between 2850 and 3380 m a.s.l. in subpa´ramo and grass pa´ramo ecosystems. 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 43 / 59

Natural history. This is a rare or hard to detect species. Lynch [9] described the species with only two specimens and only four more specimens were collected afterwards, in 1984 by W.E. Duellman, all from the type locality. Aguirre Mendoza et al. [52] found only seven indi- viduals through 21 sampling days, from December 2009 to April 2010, in Parque Nacional Podocarpus (Cajanuma sector). We encountered two females and a subadult, all during one night of our ongoing fieldwork carried out in this area since 2018. All specimens were encoun- tered during the night, perching on the vegetation (on leaves of Neurolepis sp., Valeriana sp. or of bromeliads) at 10 to 40 cm above the ground. No calling males were heard. Conservation status. Pristimantis percultus is currently categorized as Endangered based on criteria B1ab(iii) [53]. We suggest maintaining this category because, its Extent of occur- rence (EOO) and Area of occupancy (AOO) are estimated to be less than 50 km2, it is known from only two locations and it is possible that there is a decline in the number of locations or subpopulations. Remarks. Lynch [9] provides a detailed description of this species. Our diagnosis concurs with all the morphological features described by the author, to which we add descriptions of the characters that are more evident in life. The diagnosis provided herein is based on two specimens from the original description (KU 166057, 166058), two adult females (MUTPL 810, 812) and one subadult (MUTPL 811) collected from about 12 km south to the type locality.

Pristimantis percultus is part of the P. phoxocephalus group, and is closely related to P. chomskyi, P. andinogigas, P. atratus and P. multicolor. However, its exact kinship to these spe- cies remains non-resolved (Fig 21). The genetic distance between P. percultus and its closest relatives ranges between 3.3–3.7% (P. chomskyi) and 4% (P. multicolor) (S3 Table). Pristimantis balionotus (Lynch, 1979) (Fig 25) Eleutherodactylus balionotus Lynch, 1979 Eleutherodactylus (Eleutherodactylus) balionotus: Lynch and Duellman, 1997 Pristimantis balionotus: Heinicke, Duellman, and Hedges, 2007 Pristimantis (Pristimantis) balionotus: Hedges, Duellman, and Heinicke, 2008 Pristimantis (Huicundomantis) balionotus: Pa´ez and Ron, 2019 Common English name. Speckled Rain Frog (This is a newly proposed name as it is more appropriate than the previous one, “Crest Robber Frog”). Common Spanish name. Cutı´n de lomo manchado Etymology. Greek, meaning speckled back, in reference to the predominant color pattern. Holotype. KU 142136, an adult female from 13.5 km E Loja, at the crest of the Cordillera (Abra de Zamora) between Provincia Loja and Provincia Zamora-Chinchipe, Ecuador, 2800 m, obtained on 22 July 1971 by William E. Duellman. Paratypes. KU 142135, KU 142137–44, collected syntopically with the holotype. Diagnosis. Pristimantis balionotus is a medium sized species distinguished by the following combination of traits: (1) skin on dorsum tuberculated (in life the skin tuberculated texture is more evident); skin on venter coarsely areolate; discoidal fold weak; dorsolateral folds absent;

(2) tympanic annulus evident and tympanic membrane differentiated, its length about 49% of the length of eye; supratympanic fold present; (3) snout subacuminate in dorsal view, rounded in profile; canthus rostralis weakly concave in dorsal view, rounded in profile; (4) upper eyelid bearing several small tubercles, similar in size and shape with the ones from the dorsum, about 88% IOD in females and 84% IOD in males; cranial crests absent; (5) dentigerous processes of vomers prominent, oblique, ovoid, separated medially by distance equal or lower than the width of processes; each processes bearing 2 to 4 teeth; (6) males with a subgular vocal sac and vocal slits; nuptial pads absent; (7) Finger I shorter than Finger II; discs on fingers broadly 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 44 / 59

expanded, rounded; circumferential grooves present; fingers bearing narrow lateral fringes; (8) subarticular tubercles prominent; supernumerary palmar tubercles present; palmar tubercle partially divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle elliptical, smaller than the inner palmar tubercle; (9) small, inconspicuous, ulnar tubercles present (trait more visible in life); (10) heel with several small, rounded tubercles; outer edge of tarsus with a row of small tubercles; inner tarsal tubercles present, sometimes coalesced into a tarsal fold; (11) inner metatarsal tubercle broadly ovoid, about 4x ovoid, subconical (in profile), outer metatarsal tubercle; supernumerary plantar tubercles present; (12) Toe V slightly longer than Toe III; discs on toes broadly expanded, rounded, about same size as those on fingers; circum- ferential grooves present; toes bearing narrow lateral fringes; webbing absent; (13) in life, dor- sum gray or brown, with black flecks; venter light gray without markings; no markings in axilla, groin or on concealed limb surfaces; iris bronze with a reddish median horizontal streak and fine black reticulations; (14) SVL 28.4–32.4 mm in adult females (n = 2; [9]) and 23.6–25.8 mm in adult males (24.7 ± 0.83 SD, n = 6). Variation. Morphometric variation is shown in Table 3. Most of the encountered individu- als had a generally gray coloration, as was the case of the female MUTPL 392 (Fig 25A) and male MUTPL 677 (Fig 25B). The male MUTPL 677 (Fig 25B) had the same coloration as the

holotype KU 142136 (Fig 3B in [9]). Male MUTPL 487 (Fig 25C) was reddish brown, the juve- nile MUTPL 489 (Fig 25D) had a reddish brown back and gray flanks, juvenile MUTPL 490 (Fig 25E) had a brown broad middorsal band and gray flanks and juvenile MUTPL 491 (Fig 25F) had a dark brown broad middorsal band and beige flanks. All the encountered individu- als had the characteristic black flecks but some had only a few (MUTPL 490, 491), others had many flecks especially on the center of their backs (MUTPL 677, 487, 489), and in some cases all their dorsum and dorsal surfaces of hindlimbs and arms were covered with flecks (MUTPL 392). Advertisement call. The advertisement call of one male from Reserva Madrigal del Podo- carpus was recorded in 2018 and of two males from Abra de Zamora were recorded in 2019. Descriptive statistics of the acoustic variables are provided in Table 2 (the detailed information of each of the separate recordings is presented in the S4 Table). Pristimantis balionotus has an advertisement call characterized by a call series composed by whistles repeated over a period of time, somewhat similar to the calls of P. jimenezi and P. phoxocephalus (Fig 26). The calls Fig 25. Color variation of Pristimantis balionotus in life. A. female: MUTPL 392; B–C males: B. MUTPL 677, C. MUTPL 487; D–F juveniles: D. MUTPL 489, E. MUTPL 490, F. MUTPL 491. Abra de Zamora (A, B), Reserva Madrigal del Podocarpus (C, D, E, F). Most common coloration of this species is presented in B. https://doi.org/10.1371/journal.pone.0238306.g025 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 45 / 59

are composed usually by one or two notes (frequency modulated tonal sounds), but sometimes (probably in case of social interactions, triggered by the nearby presence of a female or a com- petitive male) the males can emit calls with up to 8 notes (FUTPL-A 230, Fig 26E and 26f). The calls are characterized by a mean duration of 0.069 s (SD = 0.005), a mean inter-call interval of 6.075 s (SD = 2.371) and a mean call rate of 10.0 calls/min (SD = 1.610). In the case of the dou- ble-noted calls the call duration was on average 0.355 s (SD = 0.110) and had a mean note rate Fig 26. Advertisement call of Pristimantis balionotus. A–D MUTPL 677, FUTPL-A 232: A. Oscillogram of a 6 calls section of the call series, with single and double-noted calls; B. Oscillogram of a single-noted call; C. Spectrogram of a single-noted call; D. Power spectrum of a single-noted call; E–F FUTPL-A 230: E. Oscillogram of a 6 notes section of a 8-noted call; F. Spectrogram of a 6 notes section of a 8-noted call. https://doi.org/10.1371/journal.pone.0238306.g026 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 46 / 59

of 3.56 notes/s (SD = 0.126) (S4 Table). The mean dominant frequency of the call was 2431.7 Hz (SD = 59.371), with a mean 90% bandwidth of 2331.8–2542.8 Hz (Table 2). The fundamen- tal frequency is not recognizable, but usually 4 harmonics are visible. Distribution. Pristimantis balionotus is known only from Abra de Zamora and about 9 km to the south, in Reserva Madrigal del Podocarpus, Loja province (Fig 23). We encountered most of the specimens, from Abra de Zamora, outside of Parque Nacional Podocarpus. The species was encountered at an altitudinal range between 2770 and 2960 m a.s.l., in subpa´ramo ecosystems (Fig 2B). Natural history. This is an uncommon species. Lynch [9] described the species based on 10 specimens, and only one more was collected afterwards, in 1984 by W.E. Duellman. Pristiman- tis balionotus is a bromeliad specialist, all the individuals being found inside or on the leaves of terrestrial bromeliads. The call of this species was recorded infrequently in the months of Feb- ruary, May, July, August, September, November and December, only on nights with high humidity (rain or dense fog). All calling males were found on bromeliads leaves. Conservation status. Pristimantis balionotus is currently categorized as Endangered [54] based on criteria B1 ab(iii). We suggest maintaining this category because its Extent of occur- rence (EOO) and Area of occupancy (AOO) are estimated to be less than 30 km2, it is known from only two locations, with part of its populations found outside protected areas which

could be severely affected in the near future by habitat destruction. Remarks. Lynch [9] provides a detailed description of this species. Our diagnosis concurs with all the morphological features described by the author, to which we add descriptions of the characters that are more evident in life. The diagnosis provided herein is based on 10 speci- mens from the original description (KU 142135–44), one adult female (MUTPL 392) and 4 adult males (MUTPL 292, 297, 391, 677) collected from the type locality, and 5 specimens col- lected from Reserva Madrigal del Podocarpus. Pristimantis balionotus is part of the P. phoxocephalus group, but its kinship to the other members of the group remains non-resolved (Fig 21). In our phylogram P. balionotus is closely related to P. hampatusami and an undescribed species (UCS3) but with only moderate BI sup- port. The genetic distance between P. balionotus and its relatives from the P. phoxocephalus group ranges between 2.3–4.4% (P. gloria), 2.5–3.5% (P. lutzae), 3.7–3.9% (P. verrucolatus), 3.6–4.3% (P. tinguichaca), 3.8–5.8% (P. hampatusami) and up to 6.5–8.0% (P. torresi) (S3 Table). Pristimantis versicolor (Lynch, 1979) (Fig 27) Eleutherodactylus versicolor Lynch, 1979 Eleutherodactylus (Eleutherodactylus) versicolor: Lynch, 1996; Lynch and Duellman, 1997 Pristimantis versicolor: Heinicke, Duellman, and Hedges, 2007 Pristimantis (Pristimantis) versicolor: Hedges, Duellman, and Heinicke, 2008 Pristimantis (Huicundomantis) versicolor: Pa´ez and Ron, 2019 Common English name. Variegated Rain Frog (This is a newly proposed name as it is

more appropriate than the previous one, “Loja Robber Frog”). Common Spanish name. Cutı´n versicolor Etymology. The specific name is a Latin adjective meaning of different colors or variegated [25]. Holotype. KU 119858, an adult female [25] obtained 13.5 km E. Loja, 2800 m, a locality just east of the crest on the mountain range dividing Loja and Zamora-Chinchipe provinces, Ecua- dor, on 10 June 1968 by Robert W. Henderson and J. D. Lynch. Paratypes. KU 119859–71 and KU 119911–44, topotypes collected on 10 June and 13 June 1968 by R. W. Henderson and J. D. Lynch. 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 47 / 59

Diagnosis. Pristimantis versicolor is a medium sized species, distinguished by the following combination of traits: (1) skin on dorsum shagreen with fine tubercles; skin on venter areolate; discoidal fold visible, thoracic fold prominent; dorsolateral folds absent; (2) tympanic mem- brane and tympanic annulus prominent, its length about 45% of the length of eye; supratympa- nic fold present; (3) snout subacuminate in dorsal view, rounded in profile; canthus rostralis angular, straight or slightly concave in dorsal view, angular in profile; (4) upper eyelid usually bearing several larger tubercles (trait more visible in life), about 85% IOD in females and 97% IOD in males; cranial crests absent; (5) dentigerous processes of vomers prominent, oblique, ovoid, separated medially by distance equal to twice than the width of processes; each pro- cesses bearing 3 to 4 teeth; (6) males lacking vocal sac and slits; nuptial pads present; (7) Finger I shorter than Finger II; discs on fingers broadly expanded, elliptical; circumferential grooves present; fingers bearing weak lateral fringes or none; (8) subarticular tubercles prominent, round; supernumerary palmar tubercles evident; palmar tubercle partially divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle oval, equal in size with the inner palmar tubercle; (9) ulnar tubercles low, diffuse; (10) heel with one large and several smaller tubercles (trait more visible in life); outer edge of tarsus bearing a row of small, conical tubercles; inner

tarsal tubercles coalesced into a short tarsal fold; (11) inner metatarsal tubercle ovoid, about 3 to 4x round, conical (in profile), outer metatarsal tubercle; supernumerary plantar tubercles present, conical; (12) Toe V much longer than Toe III; discs on toes broadly expanded, ellipti- cal, slightly smaller than those of fingers; circumferential grooves present; toes lacking lateral fringes; webbing absent; (13) in life, dorsum brown or green, with dark markings (chevrons, interorbital bar, canthal and supratympanic stripes); flanks with vertical bars or spotted; limbs usually with transverse bars; venter cream with black or dark brown reticulations; no markings in axilla, groin or on concealed limb surfaces; iris whitish bronze with a broad reddish median horizontal streak, that almost covers all the lower part of the iris and fine black reticulations; (14) SVL 29.4–32.2 mm in adult females (30.8 ± 1.40 SD, n = 3) and 22.9–25.4 mm in adult males (24.0 ± 1.08 SD, n = 4). Variation. Morphometric variation is shown in Table 3. The “versicolor” (variegated) name is very fitting because this species displays an incredible color variation. Many individu- als have a general brown coloration with green blotches and whitish markings like female MUTPL 390 (Fig 27A) or a general green coloration (MUTPL 313, Fig 27F). In some Fig 27. Color variation of Pristimantis versicolor in life. A. female: MUTPL 390; B–C males: B. MUTPL 112, C. MUTPL 740; D–E subadults: D. MUTPL 497, E. MUTPL 293; F. juvenile: MUTPL 313. Abra de Zamora (A, B, D, E,

F), Reserva Tapichalaca (C). Most common coloration of this species is presented in A and F. https://doi.org/10.1371/journal.pone.0238306.g027 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 48 / 59

individuals the dorsum is reddish brown, with some pale green markings (MUTPL 112, Fig 27B), or have brown dorsum with orange or almost red flanks (MUTPL 112, Fig 27B). The specimen MUTPL 497 (Fig 27D) had a very special coloration with a brown broad middorsal band, bordered by dark brown dorsolateral strips, green upper part of the flanks and of the dorsal surfaces of limbs and orange with black reticulations lower part of the flanks. Many individuals have several white spots of various sizes (like MUTPL 390) but in some cases, the entire body is covered with white spots, like MUTPL 293 (Fig 27E). Almost all encountered individuals had on their flanks larger dark bars or spots. The subadults and juveniles have a coarser texture of their dorsum, flanks and of the dorsal surfaces of limbs (Fig 27D, 27E and 27F). Advertisement call. The advertisement call of this species has been overlooked for a long time, probably because it resembles more a sound produced by insects. The advertisement calls of four males from Abra de Zamora were recorded in 2016 and 2018. Descriptive statistics of the acoustic variables are provided in Table 2 (the detailed information of each of the sepa- rate recordings is presented in the S4 Table). Pristimantis versicolor has a peculiar advertise- ment call, characterized by a call series composed by low squeak/chirp-like calls repeated for long periods of time. (Fig 28). The calls are composed usually by one note (pulsed sound) but sometimes (probably in case of social interactions, where there is a female or a competitive

male nearby) the males can emit calls with up to 5 notes (MUTPL 112, FUTPL-A 234, Fig 28E and 28f). The barely detectable, low advertisement call might be related to the lack of vocal sac and slits. Because the males can call continuously for long periods of time, the call series duration is unknown. The calls are characterized by a mean duration of 0.084 s (SD = 0.006), a mean inter-call interval of 1.804 s (SD = 0.315) and a mean call rate of 30.3 calls/min (SD = 3.138). In the case of the double-noted calls, the call duration was on average 0.403 s (SD = 0.013), for the 5-noted calls was on average 1.211 s (SD = 0.068) and had a mean note rate of 3.48 notes/s (SD = 0.259) (S4 Table). The calls had 6 to 14 pulses/note, with a mean pulse rate of 114.58 pulses/s (SD = 21.640). The mean dominant frequency of the call was 1753.6 Hz (SD = 80.323), with a mean 90% bandwidth of 1542.3–3269.2 Hz (Table 2). The fun- damental frequency is not recognizable, but sometimes 3 to 5 harmonics are visible. Distribution. Pristimantis versicolor presence is confirmed (with DNA samples) from Reserva Cerro Plateado, Reserva Tapichalaca, Parque Nacional Podocarpus (Lagunas del Com- padre), Reserva Madrigal del Podocarpus, Abra de Zamora and Ramos Urcu (at about 5 km west to San Lucas), in Loja and Zamora Chinchipe provinces (Fig 23). Very probable the ani- mals from Peru [25] are in fact a different, currently undescribed species, taking into consider- ation the great distance from the type locality. The species was encountered at an altitudinal

range between 2440 and 3380 m a.s.l. in montane cloud forest, evergreen upper montane forest (Fig 2C), subpa´ramo (Fig 2B) and shrub pa´ramo ecosystems. Natural history. This is one of the most common species from the higher part of Abra de Zamora (together with P. andinognomus). All specimens were encountered during the night, perching on the vegetation (usually at 30 cm–1.5 m above the ground) or in bromeliads. Call- ing males were encountered year round, but more frequently on rainy nights. Conservation status. Pristimantis versicolor is currently categorized as Least Concern [55] following the IUCN criteria. We suggest maintaining this category due the fact that it is an abundant species (in all encountered localities), some of its populations are inside protected areas (Reserva Cerro Plateado, Reserva Tapichalaca, Parque Nacional Podocarpus and Reserva Madrigal del Podocarpus), and no major threats are predictable. Remarks. Lynch [9] provides a detailed description of this species. Our diagnosis concurs with almost all of the morphological features described by the author (the only difference was the presence of a large and several small tubercles on the heel in contrast with the lack of 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 49 / 59

tubercles described by Lynch); we added descriptions of the characters that are more evident in life. The diagnosis provided herein is based on 48 specimens from the original description (KU 119858–71, 119911–44), one adult female (MUTPL 390), three adult males (MUTPL 112, 294, 389) and two subadults (MUTPL 313, 497) collected from the type locality, and 8 speci- mens collected from the other localities. Fig 28. Advertisement call of Pristimantis versicolor (MUTPL 112, FUTPL-A 234). A. Oscillogram of a 9 calls section of the call series, with single, double and triple-noted calls; B. Oscillogram of a pulsed, single-noted call; C. Spectrogram of a pulsed, single-noted call; D. Power spectrum of a pulsed, single-noted call; E. Oscillogram of a 5-noted call; F. Spectrogram of a 5-noted call. https://doi.org/10.1371/journal.pone.0238306.g028 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 50 / 59

The populations from Reserva Cerro Plateado and Reserva Tapichalaca need a careful revi- sion because the genetic distance between specimens collected in these reserves and the ones from the type locality and nearby areas (northern populations) is around 2.0%. Family Telmatobiidae Fitzinger, 1843 Genus Telmatobius Wiegmann, 1834 Telmatobius cirrhacelis Trueb, 1979 Common English name. Loja Water Frog Common Spanish name. Uco de Loja Etymology. The specific name is derived from the Greek kirrhos, meaning “orange-colored, yellow, tawny”, and kelis, meaning “spot”, with reference to the bright orange spots that char- acterize this species. Holotype. KU 165991, an adult female from 13 km E Loja (Abra de Zamora), 2850 m, Pro- vincia Loja, Ecuador (latitude 03˚59’S, longitude 79˚08’W), taken on 8 March 1975 by William E. Duellman. Paratypes. KU 165989 (male skeleton) and KU 165990 (subadult female) from 15 km E Loja, 2700 m, Provincia Zamora-Chinchipe, Ecuador (latitude 03˚59’S, longitude 79˚07’W). Remarks. Despite numerous searching efforts carried out in the last 40 years [56] the spe- cies was not re-encountered. The last sighting of Telmatobius cirrhacelis dates from 1984, from Parque Nacional Podocarpus (Lagunas del Compadre), and the species is considered probably extinct. Other amphibian species present in Abra de Zamora Besides the 11 species (13, with the two new species presented herein) described from Abra de Zamora, 16 other amphibian species were recorded (S5 Table). Family Bufonidae Rhinella margaritifera (Laurenti, 1768) (Fig 29A)

Common English name. South American Common Toad Common Spanish name. Sapo comu´n sudamericano Identification. It is easily distinguished from all other species present in Abra de Zamora by the presence of developed canthal, antorbital, and supra-orbital crests (larger in females), small, triangular parotoid glands, and granulose skin on dorsum with many small tubercles [57]. Remarks. This is an uncommon species in Abra de Zamora. Can be encountered on the eastern slope, in evergreen lower montane forest (Fig 2D), bellow 2000 m, near the El Tambo sector. Its precise identity has yet to be confirmed, as it is part of a species complex. Family Hemiphractidae Gastrotheca elicioi Carvajal-Endara, Coloma, Morales-Mite, Guayasamin, Sze´kely, and Duellman, 2019 (Fig 29B) Common English name. Elicio’s Marsupial Frog Common Spanish name. Rana marsupial de Elicio Identification. For now, it is the only Gastrotheca species identified on the western slope of Abra de Zamora. Despite our intensive searches, we did not find G. lojana, the other species reported from the area. Based on morphological characters, G. elicioi can be difficult to distin- guish from G. lojana, the main differences being the color pattern (characters of G. lojana in parenthesis): distinct dark canthal stripes present (absent), groin and anterior and posterior surfaces of the thighs slightly mottled (heavily mottled), and the dark bars on the limbs, when 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 51 / 59

present, are shorter, thinner and less defined than in G. lojana [58]. However, the unique advertisement call of G. elicioi (the long, pulsed notes are produced after the short ones, whereas the other Southern Ecuadorian Gastrotheca species tend to produce the long, pulsed notes before the short ones) make it very easy to detect and identify. Remarks. This is a common species in Abra de Zamora and nearby Loja city. Its distribu- tion is restricted to the western slope, close to the city, in evergreen upper montane forest bellow 2600 m. The individuals are usually difficult to encounter, as they are very well camou- flaged in the vegetation; however, the males’ characteristic call can be heard frequently, espe- cially on rainy days. Gastrotheca testudinea (Jime´nez de la Espada, 1870) (Fig 29C) Common English name. Espada’s Marsupial Frog Common Spanish name. Rana marsupial de Jimenez de la Espada Identification. For now, it is the only Gastrotheca species recorded on the eastern slope of Abra de Zamora. It is easily distinguished from all the other species present in Abra de Zamora by its large size, robust body, wide head, flat head top, and characteristic advertisement call [8]. Remarks. This is an uncommon species in Abra de Zamora. It can be encountered on the eastern slope in evergreen lower montane forest (Fig 2D) bellow 2200 m, near the El Tambo sector. Individuals are difficult to find, especially in dense vegetation, but the males’ character- istic call can be heard frequently, especially on rainy days. Family Strabomantidae Pristimantis atratus (Lynch, 1979)