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

each processes bearing 3 to 9 teeth; (6) males with a large subgular vocal sac and vocal slits; nuptial pads absent; (7) Finger I shorter than Finger II; discs on fingers slightly expanded, rounded; circumferential grooves present; fingers lacking lateral fringes; (8) subarticular tuber- cles prominent, round; supernumerary palmar tubercles evident; palmar tubercle completely divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle oval, smaller than the inner palmar tubercle; (9) small, inconspicuous, ulnar tubercles present (trait more visible in life); (10) heel with 2 or 3 larger and several smaller tubercles (trait more visible in life); outer edge of tarsus bearing a row of small tubercles; inner tarsal tubercles coalesced into a long tarsal fold; (11) inner metatarsal tubercle broadly ovoid, about 1.5 to 2x rounded outer metatarsal tubercle; supernumerary plantar tubercles present; (12) Toe V slightly longer than Toe III; discs on toes slightly expanded, rounded, about same size as those on fingers; circum- ferential grooves present; toes lacking lateral fringes; webbing absent; (13) in life, dorsum tan to brown or grey, mottled with darker brown or bearing dark dorsolateral stripes; venter cream or pinkish white; no markings in axilla, groin or on concealed limb surfaces; iris whitish gray, with a reddish broad median horizontal streak, that seems to cover all the lower part of the iris, and with fine black reticulations; (14) SVL 17.9–26.8 mm in adult females (22.4 ± 2.35 SD, n = 16) and 16.1–19.5 mm in adult males (17.6 ± 1.28 SD, n = 11).

Variation. Morphometric variation is shown in Table 1. The females are more colorful and variable than the males (Fig 8). The female specimen MUTPL 312 (Fig 8A) had a coloration very similar with the holotype KU 120082 and paratype KU 120089 (Fig 13D in [9]), with light orange dorsolateral stripes and spots and pale dorsolateral folds that continue with stripes onto the snout. Many females have particular dark dorsolateral stripes on lighter background (orange, yellow or cream), as in the case of MUTPL 164 and 586 (Fig 8B and 8C). One female, MUTPL 590, had the head and snout distinctly yellowish beige, contrasting with the rest of the body which was dark brown (Fig 8D). Other females are dark brown with light upper lips and canthal stripes (MUTPL 153, Fig 8E), or completely pinkish beige, as MUTPL 150 (Fig 8F). Most of the encountered males are similar in color with MUTPL 495, with reddish brown mid- dorsal band and members, dark dorsolateral folds and lighter flanks (Fig 8G), or alternatively have a uniform grey coloration, with some darker stripes and black supratympanic stripes, as in MUTPL 496 (Fig 8H). One male, MUTPL 148 had a different coloration, with a brown mid- dorsal band, and yellowish dorsolateral stripes that continued onto the snout and yellowish flanks (Fig 8I). 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 17 / 59

Advertisement call. The advertisement call of one male from Abra de Zamora was recorded in 2017 and of two males from Reserva Madrigal del Podocarpus in 2018. 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 vidua has an advertisement Table 1. Body mass (in grams), measurements (in mm) and morphological proportions (in percentages) of adult females and males of the species from the Pristi- mantis orestes species group described from Abra de Zamora. Character Pristimantis vidua Pristimantis samaniegoi sp. nov. Pristimantis colodactylus Pristimantis matildae sp. nov. females (n = 16) males (n = 11) females (n = 4) males (n = 3) females (n = 2) males (n = 1) females (n = 4) males (n = 3) Body mass (BM) 0.97 ± 0.29 (0.54– 1.61) 0.49 ± 0.10 (0.35– 0.66) 0.63 ± 0.15 (0.44– 0.80) 0.34 ± 0.04 (0.31– 0.38) 0.61–0.62 0.31 0.84 ± 0.16 (0.66– 0.97) 0.55 ± 0.06 (0.50– 0.61) Snout-vent length (SVL) 22.4 ± 2.35 (17.9– 26.8) 17.6 ± 1.28 (16.1– 19.5) 19.3 ± 1.58 (17.1– 20.7) 16.0 ± 1.17 (14.7– 16.9) 20.2–20.5 16.9 22.5 ± 1.12 (20.8– 23.3) 20.4 ± 0.68 (19.9– 21.2) Head width (HW) 8.2 ± 0.85 (6.9– 10.1) 6.3 ± 0.54 (5.7– 7.2) 6.6 ± 0.37 (6.1– 7.0) 5.3 ± 0.47 (4.8– 5.7) 6.5–6.9 5.4 8.1 ± 0.27 (7.7– 8.3) 6.9 ± 0.35 (6.7– 7.3) Head length (HL) 7.4 ± 0.90 (5.5– 9.2) 5.6 ± 0.53 (4.6– 6.4) 5.9 ± 0.28 (5.6– 6.2) 4.7 ± 0.44 (4.2– 5.0) 5.9–6.0 5.3 7.2 ± 0.15 (7.0– 7.3) 6.5 ± 0.27 (6.2– 6.7) Interorbital distance (IOD) 2.6 ± 0.24 (2.2– 3.1) 2.1 ± 0.13 (1.9–

2.3) 2.4 ± 0.13 (2.2– 2.5) 1.9 ± 0.17 (1.7– 2.0) 2.3–2.4 1.8 2.5 ± 0.24 (2.3– 2.8) 2.2 ± 0.10 (2.1– 2.3) Internarial distance (IND) 2.0 ± 0.17 (1.7– 2.3) 1.7 ± 0.19 (1.4– 2.0) 1.8 ± 0.22 (1.5– 2.0) 1.5 ± 0.15 (1.4– 1.7) 1.6 1.4 1.9 ± 0.10 (1.8– 2.0) 1.9 ± 0.12 (1.8– 2.0) Upper eyelid width (EW) 1.6 ± 0.21 (1.3– 1.9) 1.4 ± 0.18 (1.2– 1.7) 1.6 ± 0.13 (1.4– 1.7) 1.3 ± 0.06 (1.3– 1.4) 1.4–1.5 1.3 1.9 ± 0.14 (1.7– 2.0) 1.7 ± 0.15 (1.6– 1.9) Eye diameter (ED) 2.4 ± 0.27 (2.0– 2.8) 2.0 ± 0.18 (1.8– 2.3) 2.1 ± 0.10 (2.0– 2.2) 1.7 ± 0.15 (1.6– 1.9) 2.1 1.9 2.3 ± 0.08 (2.2– 2.4) 2.2 ± 0.06 (2.1– 2.2) Eye-nostril distance (EN) 1.8 ± 0.26 (1.4– 2.3) 1.3 ± 0.13 (1.1– 1.5) 1.7 ± 0.08 (1.6– 1.8) 1.4 ± 0.06 (1.3– 1.4) 1.8–1.9 1.6 2.1 ± 0.05 (2.0– 2.1) 1.8 ± 0.10 (1.7– 1.9) Tympanum diameter (TD) 1.0 ± 0.19 (0.8– 1.4) 0.9 ± 0.14 (0.6– 1.1) - - - - - - Femur length (FL) 9.0 ± 0.55 (7.4– 10.0) 7.2 ± 0.59 (6.3– 8.0) 7.7 ± 0.42 (7.3– 8.3) 6.3 ± 0.44 (5.8– 6.60) 8.1–8.3 7.0 9.5 ± 0.66 (9.1– 10.5) 8.9 ± 0.36 (8.6– 9.30 Tibia length (TL) 9.5 ± 0.58 (8.4– 10.4) 7.5 ± 0.39 (6.7– 8.2) 8.3 ± 0.35 (7.9– 8.7) 6.7 ± 0.35 (6.4– 7.1) 8.6–9.0 7.6 10.5 ± 0.49 (10.2– 11.2) 9.4 ± 0.56 (8.9– 10.0) Foot length (FoL) 8.9 ± 0.85 (7.6– 10.2) 6.9 ± 0.55 (5.7– 7.5) 7.7 ± 0.45 (7.3– 8.3) 5.9 ± 0.25 (5.7– 6.2) 7.2–7.5 6.1 9.1 ± 0.50 (8.7– 9.8) 8.1 ± 0.38 (7.8– 8.5) Hand length (HaL) 5.6 ± 0.62 (4.4– 6.9) 4.2 ± 0.27 (3.7– 4.7) 4.7 ± 0.39 (4.2– 5.1) 3.6 ± 0.31 (3.3– 3.9) 4.3–4.4 3.7 5.7 ± 0.34 (5.3– 6.1) 5.2 ± 0.12 (5.1– 5.3)

call characterized by a call series composed by clicking calls repeated for long periods of time, similar to the call of P. tiktik (Fig 9). The calls are composed only by two short, single-pulsed notes (tonal sounds). 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 0.022 s (SD = 0.004), a mean inter-call interval of 0.196 s (SD = 0.028) and a mean call rate of 291.3 calls/min (SD = 36.817). The mean dominant frequency of the call was 3302.2 Hz (SD = 124.359), with a mean 90% bandwidth of 3109.6–3605.6 Hz. The fundamental frequency is not recognizable, but 2 to 4 harmonics are usually visible. Compared to the call of P. tiktik, which is single-noted, the call of P.vidua is composed of two notes: a first short one, and a second, slightly longer. Also, its call is very similar to the advertisement call of P. samaniegoi sp. nov. (the differences between the two calls are pre- sented further below, in the description of the P. samaniegoi sp. nov. call). Distribution. Pristimantis vidua presence is confirmed (with DNA samples) from Parque Nacional Podocarpus (Cajanuma entrance), Reserva Madrigal del Podocarpus, Abra de Zamora and Bosque Protector Washapamba, in Loja and Zamora Chinchipe provinces (Fig 6). The presence of this species in the Azuay province [9] is yet to be confirmed. We consider this unlikely, since we do not have any valid records of this species in any inventoried similar eco-

systems north of Saraguro (close to the interprovincial border). The species was encountered at an altitudinal range between 2750 and 3000 m a.s.l. in montane cloud forest and subpa´ramo ecosystems. Natural history. Lynch (1979) described the species based on 15 females, and hypothesized that maybe this species might be “all-female”, or at the least that P. vidua exhibits a “prepon- derance of females” (female-biased sex ratio). We found males although most of the Table 2. Quantitative description of the advertisement calls (mean ± SD, range and n) of the Pristimantis tiktik, P. vidua, P. samaniegoi sp. nov., P. balionotus, and P. versicolor. Pristimantis tiktik (11) Pristimantis vidua (3) P. samaniegoi sp. nov. (5) Pristimantis balionotus (3) Pristimantis versicolor (4) Notes per call 1 2 usually 2 1–8 (usually 1 or 2) 1–5 (usually 1) Call duration (s) 0.013 ± 0.004 (0.007–0.024) n = 330 0.022 ± 0.004 (0.013–0.029) n = 117 0.027 ± 0.008 (0.017–0.040) n = 116 0.069 ± 0.005 (0.061– 0.078) n = 31 0.084 ± 0.006 (0.070– 0.099) n = 109 Inter-call interval (s) 0.287 ± 0.029 (0.225–0.354) n = 319 0.196 ± 0.028 (0.157–0.259) n = 114 0.272 ± 0.027 (0.234–0.362) n = 112 6.075 ± 2.371 (3.499– 15.677) n = 67 1.804 ± 0.315 (1.364– 3.337) n = 136 Call rate (calls/ min) 200.6 ± 18.905 (173.6– 237.0) n = 11 291.3 ± 36.817 (249.0– 316.4) n = 3 185.9 ± 25.398 (160.8– 212.6) n = 5 10.0 ± 1.610 (8.6–11.8) n = 3 30.3 ± 3.138 (27.0–33.8) n = 4 Short notes duration (s) N/A 0.004 ± 0.001 (0.002–0.006) n = 97 0.004 ± 0.001 (0.002–0.006) n = 116 N/A N/A Long notes duration (s)

N/A 0.009 ± 0.004 (0.003–0.017) n = 97 0.007 ± 0.001 (0.003–0.010) n = 116 N/A N/A Inter-note interval (s) N/A 0.009 ± 0.005 (0.002–0.017) n = 97 0.015 ± 0.007 (0.006–0.028) n = 115 0.215 ± 0.011 (0.196– 0.249) n = 39 0.208 ± 0.027 (0.168– 0.325) n = 94 Dominant frequency (Hz) 3190.8 ± 99.873 (3014.6– 3359.2) n = 330 3302.2 ± 124.359 (3100.8– 3531.5) n = 273 3140.3 ± 144.886 (2842.4– 3359.2) n = 294 2431.7 ± 59.371 (2325.6– 2584.0) n = 69 1753.6 ± 80.323 (1636.5– 2067.2) n = 139 Frequency 5% (Hz) 3059.5 ± 112.207 (2842.4– 3273.1) n = 330 3109.6 ± 93.588 (2842.4– 3273.1) n = 273 2994.4 ± 156.623 (2670.1– 3186.9) n = 294 2331.8 ± 39.947 (2239.5– 2411.7) n = 69 1542.3 ± 60.568 (1378.1– 1636.5) n = 138 Frequency 95% (Hz) 3390.5 ± 86.571 (3186.9– 3617.6) n = 330 3605.6 ± 170.730 (3273.1– 3876.0) n = 273 3496.2 ± 284.2 (3186.9– 4306.6) n = 294 2542.8 ± 45.772 (2497.9– 2670.1) n = 69 3269.2 ± 283.50 (2842.4– 4478.9) n = 138 The call of P. tiktik is presented with comparative purposes as it is very similar to the P. vidua and P. samaniegoi sp. nov. calls. In the case of P. balionotus and P. versicolor the call duration is presented for the one-noted calls and the Inter-note interval is for the multi-noted calls. The number of recorded individuals is given in brackets after species name. https://doi.org/10.1371/journal.pone.0238306.t002 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 19 / 59

encountered individuals proved to be females. We thus agree that the species has a female- biased sex ratio; alternatively, the detectability might be different between the sexes, as usually the females were found perched on leaves (at 20 cm to 1.5 m above the ground), while the males were encountered somewhat hidden in the vegetation or calling from inside the moss at Fig 9. Advertisement call of Pristimantis vidua (FUTPL-A 239). A. Oscillogram of a 10 calls section of the call series; B. Oscillogram of a single call; C. Spectrogram of a single call; D. Power spectrum of a single call. https://doi.org/10.1371/journal.pone.0238306.g009 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 20 / 59

the base of the shrubs. This is a common species. Calling males were encountered all year round but more frequently on rainy nights. Conservation status. Pristimantis vidua is currently categorized as Endangered [49] based on criteria B1 ab(iii). Even if this is a relatively abundant species at all localities, and its habitat does not face any major threats (it is encountered in one national protected area and two pri- vate and/or community protected areas), we suggest maintaining this category. This is due the fact that the species Extent of occurrence (EOO) and Area of occupancy (AOO) are estimated to be less than 200 km2, it is currently known only from 4 localities, and the existing popula- tions are severely fragmented. Remarks. Lynch [9] provides a detailed description of this species, including a brief description of the cranial osteology. Our diagnosis concurs with most of the morphological features described by the author (characters given in parentheses), but we also added descrip- tions of the characters that are more evident in life. Some of the differences that we observed are: skin on dorsum finely tuberculated (in contrast to the finely shagreened dorsum described by Lynch); dentigerous processes of vomers bearing 3 to 9 teeth (2 to 4 teeth); small, inconspic- uous, ulnar tubercles present (no ulnar tubercles or fold); heel with 2 or 3 larger and several smaller tubercles (no heel tubercles); inner tarsal tubercles coalesced into a long tarsal fold (tar- sus bearing ill-defined tubercle along inner edge). The diagnosis provided herein is based on

12 specimens from the original description (KU 120082–89, 120090–92, 141994), 3 adult females (MUTPL 295, 312, 805) and 6 adult males (MUTPL 296, 495–499, 501), collected from the type locality and 25 specimens collected from the other localities. Pristimantis vidua is part of the P. orestes subgroup, and is most closely related to an unde- scribed species from Parque Nacional Podocarpus (Lagunas del Compadre). However, its exact position in the subgroup remains non-resolved (Fig 7). The genetic distance between P. vidua and its closest relatives from the P. orestes subgroup ranges between 4.6–9.2% (S3 Table). Pristimantis samaniegoi sp. nov. Sze´kely, Eguiguren, Ordo´ñez-Delgado, Armijos-Ojeda, and Sze´kely urn:lsid:zoobank.org:act:0B9E2517-1BE9-4361-BB7D-D5B34120C248 (Figs 10–13) Common English name. Samaniego’s Rain Frog Common Spanish name. Cutı´n de Samaniego Etymology. The specific epithet is a noun in the genitive case and is a patronym for Dr. Gustavo Samaniego Rodrı´guez. The name is given as a tribute to his important contribution to the conservation of biodiversity in southern Ecuador. Through a generous donation of an important part of his estate, he contributed to the creation of the administrative and interpre- tation center in the Cajanuma sector of the Parque Nacional Podocarpus, which is one of the most important Ecuadorian sites of endemism and biodiversity. He was a university professor for over 40 years, and currently still maintains the "Reserva El Cristal" agro-ecological farm as an example of sustainable land management.

Holotype. MUTPL 357 (Figs 10–13A), field no. SC 170, an adult female from Ecuador, Loja Province, Cajanuma entrance to the Parque Nacional Podocarpus, on Los Miradores trail (4.1151˚ S, 79.1651˚ W; datum WGS84), 3258 m above sea level, collected by Paul Sze´kely and Diana Sze´kely on 28 June 2018. Paratypes (3 females, 3 males and 1 juvenile). Two specimens collected syntopically with the holotype: MUTPL 356 (SC 169) and MUTPL 358 (SC 171), both adult males (Fig 13A and 13D). Five specimens from Ecuador, Loja Province, Abra de Zamora: MUTPL 365 (SC 178), an adult female collected by Paul Sze´kely (3.9912˚ S, 79.1460˚ W; datum WGS84), 2817 m, on 19 July 2018; MUTPL 660 (SC 253), a juvenile (Fig 13F) collected by Stalin Go´mez, Alejandro 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 21 / 59

Ochoa, Michelle Angamarca and Paul Sze´kely (3.9836˚ S, 79.1307˚ W; datum WGS84), 2569 m, on 27 April 2019; MUTPL 674 (SC 993), an adult male (Fig 13E) collected by Paul Sze´kely, Stalin Go´mez, Alejandro Ochoa and Michelle Angamarca (3.9928˚ S, 79.1469˚ W; datum WGS84), 2865 m, on 17 May 2019; MUTPL 676 (SC 936), an adult female (Fig 13B) collected by Paul Sze´kely, Stalin Go´mez and Alejandro Ochoa (3.9869˚ S, 79.1443˚ W; datum WGS84), 2827 m, on 24 May 2019; MUTPL 777 (SC 1030) an adult female (Fig 13C) collected by Stalin Go´mez and Alejandro Ochoa (3.9965˚ S, 79.1469˚ W; datum WGS84), 2891 m, on 22 Septem- ber 2019. Diagnosis. We assign this species to Pristimantis based on phylogenetic evidence (Fig 7) and on the general morphological similarity to other members of the genus. Pristimantis sama- niegoi is a small species, distinguished by the following combination of traits: (1) skin on dor- sum finely tuberculated (in life the skin tuberculated texture is more evident); skin on venter coarsely areolate; thoracic and discoidal folds evident (trait more visible in preservative); dor- solateral folds absent; low middorsal fold present; (2) tympanic annulus and tympanic mem- brane absent; supratympanic fold present; (3) snout short, rounded in dorsal view and in profile; canthus rostralis weakly concave in dorsal view, rounded in profile; (4) upper eyelid bearing several small tubercles (trait more visible in life), about 67% IOD in females and 70% Fig 10. Holotype of Pristimantis samaniegoi sp. nov. (MUTPL 357, adult female), SVL 20.1 mm, in life. A. Lateral

view; B. Ventral view; C. Dorsal view. https://doi.org/10.1371/journal.pone.0238306.g010 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 22 / 59

IOD in males; cranial crests absent; (5) dentigerous processes of vomers prominent, oblique, triangular or oval in outline, separated medially by distance almost equal with the width of processes; each processes bearing 4 to 6 teeth; (6) males with large subgular vocal sac and vocal slits; nuptial pads absent; (7) Finger I shorter than Finger II; discs on fingers just slightly expanded, rounded; circumferential grooves present; fingers lacking lateral fringes; (8) subarti- cular tubercles prominent, round and subconical in section; supernumerary palmar tubercles rounded, large, just slightly smaller than subarticular tubercles; palmar tubercle completely divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle oval, smaller than the inner palmar tubercle; (9) ulnar tubercles absent or very small, inconspicuous (trait more visible in life); (10) heel with several small tubercles (trait more visible in life); outer edge of tarsus with a row of small tubercles (trait more visible in life); inner tarsal tubercles coalesced into a long tarsal fold; (11) inner metatarsal tubercle broadly ovoid, about 3x elongated and subconical outer metatarsal tubercle; supernumerary plantar tubercles ovoid, large, just slightly smaller than subarticular tubercles; (12) Toe V slightly longer than Toe III; discs on toes just slightly expanded, rounded, about same size as those on fingers; circumferential grooves pres- ent; toes lacking lateral fringes; webbing basal; (13) evident sexual dimorphism: in life, the

females with dorsum, flanks, dorsal surfaces of hindlimbs and arms dark brown with various white, irregular spots and dark gray venter; males dorsum light brown, reddish brown or red, without white spots and much lighter venter, pinkish white or light gray; no markings in axilla, groin or on concealed limb surfaces; iris whitish gray with a reddish bronze, broad, median Fig 11. Holotype of Pristimantis samaniegoi sp. nov. (MUTPL 357, adult female) in preservative. A. Dorsal view; B. Ventral view; C. Lateral view. https://doi.org/10.1371/journal.pone.0238306.g011 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 23 / 59

Fig 12. Holotype of Pristimantis samaniegoi sp. nov. (MUTPL 357, adult female) in preservative. A. Dorsal view of head; B. Profile view of head; C. Palmar view of hand: D. Plantar view of foot. https://doi.org/10.1371/journal.pone.0238306.g012 Fig 13. Color variation of Pristimantis samaniegoi sp. nov. in life. A. amplexus: female, holotype (MUTPL 357) and male, paratype (MUTPL 356); B–C females, paratypes: B. MUTPL 676, C. MUTPL 777; D–E males, paratypes: D. MUTPL 358, E. MUTPL 674; F. juvenile, paratype: MUTPL 660. Abra de Zamora (B, C, E, F), Parque Nacional Podocarpus—Cajanuma (A, D). https://doi.org/10.1371/journal.pone.0238306.g013 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 24 / 59

horizontal streak and with fine black reticulations; (14) SVL 17.1–20.7 mm in adult females (19.3 ± 1.58 SD, n = 4) and 14.7–16.9 mm in adult males (16.0 ± 1.17 SD, n = 3). Comparisons with similar species. Pristimantis samaniegoi is morphologically similar to its closest relatives, the species from the P. orestes group, but it can be easily distinguished from all the resembling species. From the 14 species that are currently included in this group, only three species lack tympanic annulus and tympanic membrane: P. samaniegoi, P. matildae sp. nov. and P. colodactylus. However, P. colodactylus and P. matildae sp. nov. belong to the P. colodactylus subgroup of species (Fig 7), specialized to live inside bromeliads, that have a very distinct habitus, with longer snouts/heads, longer legs, head as wide or wider than the body and usually very short fingers and toes (Table 1). Additionally, P. samaniegoi can be easily dis- tinguished from P. matildae sp. nov. and P. colodactylus as follows (characters of P. samaniegoi in parenthesis): discoidal fold absent (thoracic and discoidal folds evident), supratympanic fold absent (present), upper eyelid bearing one prominent tubercle (upper eyelid bearing only several small tubercles), dentigerous processes of vomers concealed in buccal mucosa (denti- gerous processes of vomers prominent), Toe V much longer than Toe III (Toe V slightly lon- ger than Toe III), and males lacking vocal sac and slits (males with large subgular vocal sac and vocal slits). Pristimantis samaniegoi can be easily distinguished from other closely related species,

which are somewhat similar, as follows (characters of P. samaniegoi in parenthesis): P. quinta- nai, P. saturninoi, P. simonbolivari, P. bambu and P. mazar have spots—black, white or yellow —in axilla, groin, concealed limb surfaces, or on venter (spots absent) and P. tiktik has tuber- culated dorsum (finely tuberculated) and the venter, axillae and groins of the females is white with black reticulum (venter dark gray, without reticulum). From the sympatric species, P. andinognomus is significantly smaller, with females on average 16 mm, males 13 mm (larger, with females on average 19 mm, males 16 mm), has inguinal spots (no inguinal spots), and Toe V is much longer than Toe III (Toe V is slightly longer than Toe III), P. cajanuma has inguinal spots (spots absent) and P. vidua is larger, with females on average 22 mm, males 17 mm (smaller, with females on average 19 mm, males 16 mm) and has dorsolateral folds (dorso- lateral folds absent). And finally, none of the other species have the typical dark coloration with white spots of the females of P. samaniegoi. Description of the holotype. Adult female (MUTPL 357; Figs 10–13A), head narrower than body, wider than long, head length 92% of head width, head width 33% of SVL; head length 30% of SVL; snout short (snout to eye distance 13% of SVL), rounded in dorsal view and in profile (Fig 12A and 12B); canthus rostralis weakly concave in dorsal view, rounded in profile; loreal region flat; eye diameter notably greater than eye-nostril distance; nostrils not

protuberant; lips not flared; cranial crests absent; upper eyelid bearing several small tubercles (two slightly larger than the others), width of upper eyelid 64% of IOD; tympanic annulus and tympanic membrane absent (Fig 12B); evident supratympanic fold present; one larger, rounded postrictal tubercle present; choanae large, oval, not concealed by palatal shelf of max- illary arch; dentigerous processes of vomers prominent, slightly larger than the choanae, obli- que, situated posterior and median to choanae, triangular in outline, separated medially by distance almost equal to the width of processes, each processes bearing 4 to 5 teeth; tongue 1.5x as long as wide, slightly notched posteriorly, posterior half not adherent to floor of mouth. Skin on dorsum finely tuberculated; thin, low middorsal fold starting at tip of snout and ending at cloaca; dorsolateral folds absent; skin on throat, chest, belly, and ventral surfaces of thighs coarsely areolate; thoracic and discoidal folds evident (trait more visible in preservative; Figs 10B and 11B); ornamentation in cloacal region absent. Ulnar tubercles absent; outer palmar tubercle prominent, completely divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle oval, smaller than the inner palmar 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 25 / 59

tubercle; subarticular tubercles prominent, round and subconical in section; supernumerary palmar tubercles rounded, large, just slightly smaller than subarticular tubercles; fingers lack- ing lateral fringes; relative length of fingers I < II < IV < III; discs on fingers just slightly expanded, rounded; all fingers bearing pads well defined by circumferential grooves (Fig 12C). Hindlimbs short; tibia length 40% of SVL; foot length 38% of SVL; heel with several small tubercles; outer edge of tarsus with a row of small tubercles (trait more visible in life); inner edge of tarsus bearing a long fold; inner metatarsal tubercle broadly ovoid, about 3x elon- gated and subconical outer metatarsal tubercle; subarticular tubercles prominent, ovoid and subconical in section; plantar supernumerary tubercles ovoid, large, just slightly smaller than subarticular tubercles; toes lacking lateral fringes; webbing basal; discs on toes just slightly expanded, rounded, about same size as those on fingers; toes with ventral pads well defined by circumferential grooves (Fig 12D); relative length of toes I < II < III < V < IV; Toe V slightly longer than Toe III (tip of Toe III barely reaching the penultimate subarticular tuber- cle on Toe IV, tip of Toe V not reaching the proximal edge of distal subarticular tubercle on Toe IV). Coloration of holotype. In life (Fig 10): dorsum, flanks, dorsal surfaces of hindlimbs and arms dark brown with various yellowish white, irregular spots; venter and throat gray, without

spots; iris whitish gray with a dark bronze, broad, median horizontal streak and with fine black reticulations. In preservative (Fig 11): the coloration of dorsum, flanks and dorsal surfaces of hindlimbs became dark gray and the spots light gray; dorsal surfaces of arms, venter and throat became light gray. Measurements of holotype (in mm): SVL 20.1; HW 6.6; HL 6.1; IOD 2.5; IND 2.0; EW 1.6; ED 2.2; EN 1.8; snout to eye distance 2.6; FL 7.7; TL 8.1; FoL 7.6; HaL 4.8; Finger I length 2.2. Body mass of holotype: 0.80 g. Variation. Morphometric variation is shown in Table 1. This species displays an evident sexual dimorphism. The females are larger, and are dark brown with various white, irregular spots and have a dark gray venter (Fig 13A, 13B and 13C). One female (MUTPL 676, Fig 13B) had a whitish orange low middorsal fold. The same individual had very distinctive white lines on the center of the venter and throat, ventral surfaces of hindlimbs and arms. The males have lighter colors of the dorsum that varies from uniform light brown (MUTPL 674, Fig 13E), to reddish brown dorsum and light green and gray flanks (MUTPL 358, Fig 13D). The male MUTPL 356 (Fig 13A) had a bright red coloration of the dorsum and dorsal surfaces of hin- dlimbs and arms and gray flanks. The venter coloration of the males varied from pinkish white to light gray. None of the males had the characteristic white spots of the females. In all speci- mens, the supratympanic fold was black. Advertisement call. The advertisement call of one male from Abra de Zamora was

recorded in in 2017 and of four males from Cajanuma in 2018. 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 samaniegoi has an advertisement call characterized by a call series composed by clicking calls repeated for long periods of time, simi- lar to the calls of P. tiktik and P. vidua (Fig 14). The calls are composed by two short, single- pulsed notes (tonal sounds). The male MUTPL 356 (FUTPL-A-241) had the calls composed by only one note but it is possible that this is actually a recording artifact. 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 0.027 s (SD = 0.008), a mean inter-call interval of 0.272 s (SD = 0.027) and a mean call rate of 185.9 calls/min (SD = 25.398). The mean dominant fre- quency of the call was 3140.3 Hz (SD = 144.886), with a mean 90% bandwidth of 2994.4– 3496.2 Hz. The fundamental frequency is not recognizable but 3 to 4 harmonics are visible. 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 26 / 59

The advertisement call of P. samaniegoi is most similar to that of P. tiktik, with similar inter-call intervals, call rates, dominant frequency and 90% bandwidth frequency (Table 2). However, the main difference is that P. samaniegoi has double-noted calls as opposed to the single-noted calls of P. tiktik (Fig 14). The call of P. samaniegoi is also similar to that of P. Fig 14. Advertisement call of Pristimantis samaniegoi sp. nov. (FUTPL-A 242). A. Oscillogram of a 6 calls section of the call series; B. Oscillogram of a single call; C. Spectrogram of a single call; D. Power spectrum of a single call. https://doi.org/10.1371/journal.pone.0238306.g014 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 27 / 59

vidua, but has a longer call duration, longer inter-call interval, a lower call rate, longer inter- note interval and, most importantly, lower dominant and 90% bandwidth frequency (Table 2). Distribution. Pristimantis samaniegoi is known only from the Cajanuma entrance to the Parque Nacional Podocarpus and about 13 km in the north, in Abra de Zamora (Fig 6). We did not encounter this species in any similar habitats located in the vicinity of the type locality, despite fieldwork being carried out regularly since 2016. The species was encountered at an altitudinal range between 2560 and 3300 m a.s.l., in evergreen upper montane forest (Fig 2C), subpa´ramo (Fig 2B) and shrub pa´ramo ecosystems. Natural history. This is a common species. All the specimens were encountered during the night, close to the ground (usually at 5–20 cm above the ground), at the base of the grassy vege- tation, on the moss bed under the shrubs, or on shrub leaves. Individuals are hard to find, as they are well hidden at the base of the shrubs or inside the moss bed or grassy vegetation. How- ever, the male’s characteristic advertisement call can be commonly heard. Calling males were encountered year round, more frequently on rainy nights. All the females were encountered in the immediate proximity of the calling males. Conservation status. Pristimantis samaniegoi is known only from two close localities, from an estimated area of less than 50 km2. Nonetheless, we recommend that this species be catego- rized as Near Threatened following the IUCN criteria [47], due the fact that it is a relatively

abundant species and its habitat currently does not face any major threats (most of the popula- tion is situated within a nationally protected area—Parque Nacional Podocarpus). Remarks. Pristimantis samaniegoi is part of the P. simonbolivari subgroup, but its kinship to the other members of the subgroup remains non-resolved (Fig 7). In our phylogram P. samaniegoi is most closely related to P. quintanai and an undescribed species, but with only weak BI support. The genetic distance between P. samaniegoi and its closest relatives from the P. simonbolivari subgroup ranges between 4.8–6.0% (P. tiktik) and 6.7–7.3% (P. bambu) (S3 Table). Pristimantis colodactylus (Lynch, 1979) (Fig 15) Eleutherodactylus colodactylus Lynch, 1979 Eleutherodactylus (Eleutherodactylus) colodactylus: Lynch, 1996; Lynch and Duellman, 1997 Pristimantis colodactylus: Heinicke, Duellman, and Hedges, 2007 Pristimantis (Pristimantis) colodactylus: Hedges, Duellman, and Heinicke, 2008 Common English name. Stubby Fingered Rain Frog (This is a newly proposed name as it is more appropriate. The previous one “Piura Robber Frog” makes no sense, as the species is known only from its type locality, Abra de Zamora, Ecuador). Common Spanish name. Cutı´n de dedos cortos Etymology. The specific name is derived from the Greek kolos, meaning “stubby” and the Greek daktylo, meaning “toes”, referring to the short fingers and toes of this species [25]. Holotype. KU 142151, an adult female from 13.5 km E Loja, at the crest (Abra de Zamora)

on the frontier between Loja and Zamora-Chinchipe provinces, Ecuador, 2800 m, obtained on 22 July 1971 by William E. Duellman. Paratypes. KU 142152–59 topotypes; KU 142160–61, 14 km E Loja, Provincia Zamora- Chinchipe, 2770 m; KU 142162–64, 15 km E Loja, Provincia Zamora-Chinchipe, 2710 m. Diagnosis. Pristimantis colodactylus is a small species distinguished by the following combi- nation of traits: (1) skin on dorsum areolate, almost tuberculate (in life the skin areolate texture is more evident); skin on venter areolate; discoidal fold and dorsolateral folds absent; (2) tym- panic membrane and tympanic annulus absent; supratympanic fold absent; (3) snout rounded in dorsal view and in profile; canthus rostralis straight in dorsal view, rounded in profile; (4) upper eyelid bearing one prominent tubercle and several small tubercles, about 62% IOD in 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 28 / 59

females and 72% IOD in males; cranial crests absent; (5) dentigerous processes of vomers con- cealed in buccal mucosa; each processes bearing 2 to 6 teeth; (6) males lacking vocal sac and slits; (7) fingers very short and stocky; Finger I shorter than Finger II; discs on fingers slightly expanded, rounded; circumferential grooves present; fingers bearing lateral fringes; (8) subar- ticular tubercles prominent; supernumerary palmar tubercles numerous, continuing onto fin- gers; palmar tubercle completely divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle oval, the same size or slightly smaller than the inner palmar tubercle; (9) ulnar tubercles absent; (10) heel bearing one moderate-sized conical tubercle and several small tubercles (trait more visible in life); inner and outer edges of tarsus lacking tubercles or folds; (11) inner metatarsal tubercle broadly ovoid, about 4 to 5x rounded outer metatarsal tubercle; supernumerary plantar tubercles numerous, continuing onto toes; (12) toes very short and stocky; Toe V much longer than Toe III; discs on toes slightly expanded, rounded, about same size as those on fingers; circumferential grooves present; toes bearing prominent lateral fringes; webbing basal; (13) in life, dorsum various shades of brown with or without pale dor- solateral stripes, interorbital bars, or dark X-shaped markings on the back; venter cream with Fig 15. Color variation of Pristimantis colodactylus in life. A–B females: A. MUTPL 311, B. MUTPL 388; C. male:

MUTPL 675; D–E juveniles: D. MZUA 2491, E. MUTPL 732. All specimens are from Abra de Zamora. https://doi.org/10.1371/journal.pone.0238306.g015 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 29 / 59

dark flecks; no markings in axilla, groin or on concealed limb surfaces; iris light bronze with a reddish median horizontal streak and fine black reticulations; (14) SVL 16.5–25.8 mm in adult females [9] and 14.0–20.7 mm in adult males [9]. Variation. Morphometric variation is shown in Table 1. The five encountered specimens had variable coloration (Fig 15). Both female MUTPL 311 (Fig 15A) and juvenile MUTPL 732 (Fig 15E) had a dark X-shaped marking on their back (equivalent to what Lynch described as “hour-glass” [9]), but the female had a general grayish coloration with light and dark brown spots and stripes, while the juvenile had a general reddish brown coloration. Both female MUTPL 388 (Fig 15B) and male MUTPL 675 (Fig 15C) had a dark brown middorsal band, but the female had darker flanks and members than the male. The coloration of the male MUTPL 675 is very similar to that of the holotype KU 142151, where the dark brown middorsal band is bordered by light whitish dorsolateral stripes that continue onto the snout (Fig 15C). The col- oration of juvenile MZUA 2491 (Fig 15D) is very similar with that of the male specimen KU 165219 (Fig 3D in [9] and Fig 139 in [25]), with dark brown dorsum and whitish yellow inter- orbital bar and snout. Advertisement call. Unknown. Distribution. For now, P. colodactylus presence is confirmed (with DNA samples) from only a small area in its type locality, Abra de Zamora (Fig 6). The majority of specimens were encountered outside of Parque Nacional Podocarpus borders. We did not encounter this spe-

cies in the Cajanuma sector of the Parque Nacional Podocarpus (about 13 km to the south), which has a similar species composition to Abra de Zamora. The presence of this species in Azuay and Morona Santiago provinces [9] is yet to be confirmed. We consider this highly unlikely, and that it is more plausible that these are different, currently undescribed species. Also, it is almost certain that the animals from Peru [9, 25] are different, undescribed species, taking into consideration the great distance from the type locality. Even Lynch noted some morphological differences between the specimens from the type locality and the ones from central Ecuador and Peru (see Table 1 in [9]). The species was encountered at an altitudinal range between 2800 and 2820 m a.s.l. in a subpa´ramo ecosystem (Fig 2B). Natural history. This species shares the same microhabitat (bromeliads) with P. matildae sp. nov., which dominates in term of abundance. It is a rare species. Between 2016 and 2020, in Abra de Zamora, we encountered only five specimens of P. colodactylus. This is a bromeliad specialist that most likely spends its entire life inside or nearby these plants. The short fingers of the species of the P. colodactylus subgroup (Fig 7) are probably adaptations for the life inside bromeliads, and possibly help to increase the adhesivity of the hands and feet [29]. Four speci- mens were found inside bromeliads (both terrestrial and arboreal) during the day or night. One specimen (MUTPL 675) was found outside, on the leaf of a bromeliad (probably forag-

ing), and when it detected our presence rapidly retreated backwards to the water inside the plant. No calling males were heard. Conservation status. Pristimantis colodactylus is currently categorized as Least Concern [50]. However, this assessment was done by considering the records from the provinces of Azuay and Morona Santiago and from Peru. Based on the available (revised) information we consider P. colodactylus to be Critically Endangered following the B1ab(iii,iv)+2ab(iii,iv) IUCN criteria [47] because: (1) its Extent of occurrence (EOO) and Area of occupancy (AOO) are estimated to be less than 10 km2; (2) it is known from only one location; and (3) its habitat could be severely affected in the near future by deforestation, as it is situated mostly outside a protected area and in the vicinity of a densely populated area. Remarks. Lynch [9] provides a detailed description of this species, including a brief description of the cranial osteology. Our diagnosis concurs with the morphological features described by the author (one small difference would be the coloration of the iris that we found 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 30 / 59

to be light bronze and not reddish bronze), and we add descriptions of the characters that are more evident in life. The diagnosis provided herein is based on 14 specimens from the original description (KU 142151–64), two adult females (MUTPL 311, 388) one adult male (MUTPL 675) and two juveniles (MUTPL 732, MZUA 2491) collected from the type locality. Pristimantis colodactylus is part of the P. colodactylus subgroup, and as such it is closely related to P. matildae sp. nov., P. muranunka and an additional undescribed species from Reserva Tapichalaca (Fig 7). The genetic distance between P. colodactylus and its closest rela- tives from the subgroup ranges between 5.4–7.5% (S3 Table). Pristimantis matildae sp. nov. Sze´kely, Eguiguren, Ordo´ñez-Delgado, Armijos-Ojeda, and Sze´kely urn:lsid:zoobank.org:act:E2E120CF-C257-4A95-9052-879CA1387C89 (Figs 16–19) Common English name. Matilde’s Rain Frog Common Spanish name. Cutı´n de Matilde Etymology. The specific epithet is a noun in the genitive case and is a patronym for Matilde Hidalgo Navarro (1889–1974). She was the first woman to obtain a medical degree in the country, and the first woman in Ecuador (and Latin America) to exercise the right to vote in a Fig 16. Holotype of Pristimantis matildae sp. nov. (MUTPL 731, adult male), SVL 20.2 mm, in life. A. Lateral view; B. Ventral view; C. Dorsal view. https://doi.org/10.1371/journal.pone.0238306.g016 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 31 / 59

Fig 18. Holotype of Pristimantis matildae sp. nov. (MUTPL 731, adult male) in preservative. A. Dorsal view of head; B. Profile view of head; C. Palmar view of hand: D. Plantar view of foot. https://doi.org/10.1371/journal.pone.0238306.g018 Fig 17. Holotype of Pristimantis matildae sp. nov. (MUTPL 731, adult male) in preservative. A. Dorsal view; B. Ventral view; C. Lateral view. https://doi.org/10.1371/journal.pone.0238306.g017 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 32 / 59

national election. A tireless fighter for women’s rights, she laid important foundations for the development of women in academia and science, nationally and in South America. In Ecua- dor, there are currently many women involved in biological sciences and with this species, in addition to being a tribute to Matilde, we honor their tireless work for the conservation of bio- logical diversity. Holotype. MUTPL 731 (Figs 16–18), field no. SC 958, an adult male from Ecuador, Loja Province, Abra de Zamora (3.9910˚ S, 79.1458˚ W; datum WGS84), 2815 m above sea level, collected by Paul Sze´kely, Stalin Go´mez and Alejandro Ochoa on 6 August 2019. Paratypes (4 females, 2 males and 1 juvenile). Three specimens collected in the type locality: MUTPL 733 (SC 960), a juvenile (Fig 19F) collected syntopically with the holotype; MUTPL 394 (SC 152), an adult female (Fig 19B) collected by Diana Sze´kely, Veronica Urgiles, Anna Savage and Paul Sze´kely (3.9910˚ S, 79.1455˚ W; datum WGS84), 2815 m, on 31 May 2018; MUTPL 366 (SC 179), an adult female collected by Paul Sze´kely (3.9912˚ S, 79.1460˚ W; datum WGS84), 2817 m, on 19 July 2018; four specimens from Ecuador, Loja Province, Caja- numa sector, Parque Nacional Podocarpus: MUTPL 361 (SC 174) an adult female (Fig 19A) and MUTPL 360 (SC 173) and MUTPL 362 (SC 175) adult males (Fig 19D and 19E) collected by Diana Sze´kely and Paul Sze´kely (4.1141˚ S, 79.1627˚ W; datum WGS84), 3361 m, on 29 June 2018; MUTPL 813 (SC 1113), an adult female (Fig 19C) collected by Diana Sze´kely and

Paul Sze´kely (4.1054˚ S, 79.1616˚ W; datum WGS84), 3347 m, on 23 November 2019. Diagnosis. We assign this species to Pristimantis based on phylogenetic evidence (Fig 7) and on the general morphological similarity to other members of the genus. Pristimantis matil- dae is a small species distinguished by the following combination of traits: (1) skin on dorsum finely tuberculated (trait more visible in life); skin on venter areolate; discoidal fold and dorso- lateral folds absent; low middorsal fold present; (2) tympanic annulus and tympanic mem- brane absent; supratympanic fold absent; (3) snout moderately long, subacuminate in dorsal view and subacuminate and slightly protruding in profile; canthus rostralis weakly concave in dorsal view, angular in profile; (4) upper eyelid bearing one prominent tubercle and several small tubercles, about 77% IOD in females and 79% IOD in males; cranial crests absent; (5) dentigerous processes of vomers concealed in buccal mucosa; each processes bearing 2 to 5 Fig 19. Color variation of Pristimantis matildae sp. nov. in life. A–C females, paratypes: A. MUTPL 361, B. MUTPL 394, C. MUTPL 813; D–E males, paratypes: D. MUTPL 362, E. MUTPL 360; F. juvenile, paratype: MUTPL 733. Abra de Zamora (B, F), Parque Nacional Podocarpus—Cajanuma (A, C, D, E). https://doi.org/10.1371/journal.pone.0238306.g019 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 33 / 59

teeth; (6) males lacking vocal sac and slits; nuptial pads absent; (7) Finger I shorter than Finger II; discs on fingers slightly expanded, truncated; circumferential grooves present; fingers bear- ing lateral fringes; (8) subarticular tubercles prominent, round and rounded in section; super- numerary palmar tubercles prominent, rounded; palmar tubercle completely divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle ovoid, larger than the inner pal- mar tubercle; (9) ulnar tubercles absent; (10) heel with several small tubercles (trait more visi- ble in life); outer edge of tarsus with a row of small, inconspicuous, tubercles (trait more visible in life); inner edge of tarsus bearing a long fold; (11) inner metatarsal tubercle broadly ovoid, about 3x elongated and rounded outer metatarsal tubercle; supernumerary plantar tubercles prominent, rounded; (12) Toe V much longer than Toe III; discs on toes slightly expanded, truncated, about same size as those on fingers; circumferential grooves present; toes bearing prominent lateral fringes; webbing basal; (13) in life, dorsum various shades of brown or gray with or without middorsal bands, interorbital bars, or dark X-shaped markings on the back; venter light gray or pinkish gray; no markings in axilla, groin or on concealed limb surfaces; iris bronze or reddish bronze with a dark median horizontal streak and fine black reticulations; (14) SVL 20.8–23.3 mm in adult females (22.5 ± 1.12 SD, n = 4) and 19.9–21.2 mm in adult males (20.4 ± 0.68 SD, n = 3).

Comparisons with similar species. Pristimantis matildae is morphologically similar to its closest relatives, the species from the P. orestes group, and especially to the species from the P. colodatylus subgroup (Fig 7), P. colodactylus and P. muranunka. The difference between P. matildae and the species from the other subgroups were presented in the description of P. samaniegoi. Pristimantis matildae is easily distinguished from P. muranunka by the lack of tympanic annulus and tympanic membrane (tympanic annulus and tympanic membrane evi- dent in P. muranunka). Pristimantis matildae best resembles P. colodactylus, but several mor- phological features can be used to distinguish between these two species. The most important feature is the relative size of the fingers and toes (Fig 20). From the three species currently included in this subgroup, P. colodactylus has the shortest fingers and toes (Hand length (length of Finger III) about 22% of SVL; Foot length (length Toe IV) about 36% of SVL), fol- lowed by P. muranunka (Hand length about 23% of SVL; Foot length about 38% of SVL), and finally, P. matildae, has the longest fingers and toes (Hand length about 25% of SVL; Foot length about 40% of SVL) (Fig 20, Table 1). Additionally, P. matildae can be distinguished from P. colodactylus as follows (characters of P. matildae in parenthesis): skin on dorsum and flanks areolate (finely tuberculate), low middorsal fold absent (present), heel with one moder- ate-sized conical tubercle and several small tubercles (without a larger tubercle, only with sev-

eral small ones), ornamentation in cloacal region absent (cloacal region bordered ventrally by two large, oval, tubercles), thenar tubercle the same size or slightly smaller than the inner pal- mar tubercle (thenar tubercle larger than the inner palmar tubercle), inner edge of tarsus lack- ing tubercles (inner edge of tarsus bearing a long fold) and inner metatarsal tubercle about 4 to 5x outer metatarsal tubercle (inner metatarsal tubercle about 3x outer metatarsal tubercle). Also, the iris coloration in P. matildae is more reddish than in the case of P. colodactylus. Description of the holotype. Adult male (MUTPL 731; Figs 16–18), head wider than body, just slightly wider than long, head length 99% of head width, head width 33% of SVL; head length 32% of SVL; snout moderately long (snout to eye distance 14% of SVL), subacuminate in dorsal view and subacuminate and slightly protruding in profile (Fig 18A and 18B); canthus rostralis weakly concave in dorsal view, angular in profile; loreal region slightly concave; eye diameter notably greater than eye-nostril distance; nostrils slightly protuberant, oriented pos- teriorly; lips not flared; cranial crests absent; upper eyelid bearing one prominent tubercle and several small tubercles, width of upper eyelid 77% of IOD; tympanic annulus and tympanic membrane absent (Fig 18B); supratympanic fold absent; one large, rounded postrictal tubercle 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 34 / 59

present; choanae large, oval, not concealed by palatal shelf of maxillary arch; dentigerous pro- cesses of vomers concealed in buccal mucosa but each processes bearing two teeth; tongue 0.5x as long as wide, slightly notched posteriorly, posterior half not adherent to floor of mouth; vocal sac, vocal slits and nuptial pads absent. Skin on dorsum and flanks finely tuberculated (trait more visible in life, Fig 16A and 16C); thin, low middorsal fold starting at tip of snout and ending at cloaca; dorsolateral folds absent; skin on chest, belly, and ventral surfaces of thighs areolate; thoracic and discoidal folds absent (Figs 16B and 17B); cloacal region bordered ventrally by two large, oval, tubercles. Ulnar tubercles absent; outer palmar tubercle prominent, completely divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle ovoid, larger than the inner palmar tubercle; subarticular tubercles prominent, round and rounded in section; supernumerary pal- mar tubercles rounded, smaller than subarticular tubercles; fingers bearing lateral fringes; rela- tive length of fingers I < II < IV < III; discs on fingers slightly expanded, truncated; all fingers bearing pads well defined by circumferential grooves (Fig 18C). Hindlimbs long; tibia length 46% of SVL; foot length 39% of SVL; heel with several small tubercles; outer edge of tarsus with a row of small, inconspicuous, tubercles (trait more visible in life); inner edge of tarsus bearing a long fold; inner metatarsal tubercle broadly ovoid, about