Centrolene zarza, Székely & Córdova-Díaz & Hualpa-Vega & Hualpa-Vega & Székely, 2023

Szekely, Paul, Cordova-Diaz, Maria, Hualpa-Vega, Daniel, Hualpa-Vega, Santiago & Szekely, Diana, 2023, A new glassfrog species of the genus Centrolene (Amphibia, Anura, Centrolenidae) from Cordillera del Condor, southern Ecuador, ZooKeys 1149, pp. 53-84 : 53

Centrolene zarza sp. nov.

Figs 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 Common English name: Zarza Glassfrog Common Spanish name: Rana de cristal del Zarza

Etymology.

The specific name zarza is a noun in apposition and refers to the species’ type locality: Refugio de Vida Silvestre El Zarza. This relatively small wildlife refuge conserves an impressive biodiversity with countless species of plants and birds, more than 50 species of amphibians and reptiles and several emblematic mammals, like the Amazonian tapir, jaguar, oncilla or the spectacled bear. It is surrounded by active mining concessions and thus fulfills an important role as a conservation island for the region, with an urgent need to expand connectivity between the reserve and neighboring conservation areas.

Type material.

Holotype. MUTPL 932 (field no. SC 425; Figs 2 , 3 , 5A ), an adult female from Ecuador, Zamora Chinchipe Province, Refugio de Vida Silvestre El Zarza, quebrada "Las Mariposas" (3.8341°S, 78.5458°W; datum WGS84), 1434 m a.s.l., collected by Joselyn Vinueza, Santiago Hualpa-Vega, María Córdova-Díaz, Daniel Hualpa-Vega, Angel Hualpa, Dalton Morocho, Luis León, and Ramiro Sarango on 10 October 2020.

Paratypes. (1 female, 5 males). MUTPL 933 (field no. SC 428; Fig. 6C, D ) an adult male from Refugio de Vida Silvestre El Zarza, quebrada "Las Mariposas" (3.8371°S, 78.5424°W), 1461 m a.s.l. collected by Luis León, Dalton Morocho, Ramiro Sarango, Santiago Hualpa-Vega, María Córdova-Díaz, Daniel Hualpa-Vega, Angel Hualpa and Joselyn Vinueza on 10 October 2020; MUTPL 1022 (field no. SC 435; Fig. 6E, F ), MUTPL 1023 (field no. SC 436; Figs 4 , 5B ), and MUTPL 1024 (field no. SC 437), adult males from Refugio de Vida Silvestre El Zarza, quebrada "Las Mariposas" (3.8376°S, 78.5421°W), 1469 m a.s.l., collected by Santiago Hualpa-Vega, María Córdova-Díaz, Daniel Hualpa-Vega, Camilo López, Dalton Morocho, Dalton Bustán and Luis León on 24 January 2021; MUTPL 1050 (field no. SC 443) adult male and MUTPL 1051 (field no. SC 444; Fig. 6A, B ) adult female from Refugio de Vida Silvestre El Zarza, quebrada "Las Mariposas" (3.8373°S, 78.5423°W), 1471 m a.s.l., collected by Santiago Hualpa-Vega, María Córdova-Díaz, Daniel Hualpa-Vega, Joselyn Vinueza, Luis León, Dalton Morocho and Álex Armijos on 13 March 2021.

Diagnosis.

We assign this species to Centrolene based on phylogenetic evidence (Fig. 1 ) and on the general morphological similarity to other members of the genus (presence of humeral spines in males, liver divided into lobes and the hepatic peritoneum lacking an iridophore layer, and green bones in life). Centrolene zarza has the following combination of characters: (1) dentigerous processes of vomers ovoid, in transverse row between the choanae, separated medially by distance slightly lower than the width of processes; each process bearing 3-5 teeth; (2) snout rounded in dorsal view, sloping in profile; nostrils slightly elevated, producing depression in the internarial area; canthus rostralis not evident in dorsal view, rounded in cross section; (3) tympanic annulus and tympanic membrane evident but with coloration similar to that of surrounding skin; tympanum large, its diameter ~ 46% of eye diameter; weak supratympanic fold present, slightly concealing the upper margin of the tympanum; (4) dorsal skin shagreen with elevated, and some enameled, warts corresponding to white spots; (5) ventral skin coarsely areolate; ventral surfaces of thighs below vent with a pair of large, round, flat tubercles, flat tubercles (subcloacal warts); cloacal region bordered ventrally by many enameled, white, warts; (6) half or more than half of the upper parietal peritoneum covered by iridophores (condition P3); iridophores absent on all visceral peritonea, including the pericardium (condition V0); (7) liver lobed, lacking iridophores (condition H0); (8) adult males with small projecting humeral spines, round vocal slits and large subgular vocal sac; (9) webbing absent between Fingers I and II, basal between II and III, moderate between outer fingers: III2+-2IV; (10) webbing between toes moderate: I1-- -2II1--2III1--2IV2-1+V; (11) outer edge of forearms and tarsus with row of enameled warts that often continue into the external edges of Finger IV and/or Toe V; fingers and toes with broad lateral fringes; (12) unpigmented Type I nuptial pads present in males; concealed prepollex; (13) Finger I shorter than Finger II; (14) diameter of eye ~ 2 × wider than disc on Finger III; (15) in life, dorsum light green with many white or whitish, elevated, spots and flecks of various sizes; bones green; (16) in preservative, dorsal surfaces greyish with white spots; (17) in life, iris white or yellowish white with thick and thin black reticulations; rounded points on the upper and lower side of the iris and no circumpupillary ring; (18) fingers and toes yellowish, usually lacking melanophores from the dorsal surfaces, except for Finger IV and Toes IV and V; (19) males call from the upper surfaces of leaves; advertisement call consisting of a high pitched, pulsed, single note, with every call/note featuring three clearly distinguishable pulses and a mean dominant frequency of 5309.8 Hz; courtship call composed by multi-noted, pulsed calls of usually five notes/call and a mean dominant frequency of 5127.4 Hz; (20) fighting behavior unknown; (21) egg clutches attached to the upper side of leaves; clutch size of 13-33 embryos (n = 2); probably without parental care; (22) tadpoles with elongated, oval-depressed body; sinistral spiracle; vent tube situated medially, caudal and with dextral opening; tail 2.4 × the length of the body; labial tooth row formula 0/2(1) in Gosner 26 but without tooth rows in Gosner 31; mostly pinkish coloration; (23) medium body size (sensu Guayasamin et al. 2020), SVL 25.5-27.0 mm in adult females (n = 2) and 23.2-26.2 mm in adult males (24.1 ± 1.21, n = 5).

Comparisons with similar species.

Due to its unique combination of characters, Centrolene zarza is easily distinguished from all other glassfrog species. The few congener species that generally resemble C. zarza , specifically that have green dorsum with white spots and/or flecks, are as follows: C. condor , has a very different general habitus with a more robust body, smaller eyes, less evident tympanum and dark bluish-black/brown flecks and punctuations along with the white flecks (vs. slender body, larger eyes, evident tympanic annulus and tympanic membrane and only white spots and/or flecks in C. zarza ); C. pipilata , has a dorsum with yellowish-white flecks and diffuse dark green/black marks, and a distinct prepollex (vs. only white spots and/or flecks and concealed prepollex); C. sanchezi , has a smaller body size and the presence of white warts in an area that extends from below the eye to the insertion of the arm (vs. absence of the white warts); specimens of the C. buckleyi species complex that have white spots have less evident markings, dentigerous processes of vomers without teeth, less evident tympanum, condition V1 of the visceral peritonea and also live at much higher elevations of 2050-3070 m (vs. dentigerous processes of vomers with teeth and condition V0 of the visceral peritonea).

Some congener species have a similar habitus, but live in other countries: C. solitaria (one of the few species of Centrolene for which we lack molecular data) is endemic to the Andes of Colombia and has green spots along with the white flecks, iridophores covering parts of the gastrointestinal peritoneum and the males lack humeral spines (vs. only white spots and/or flecks, iridophores absent on all visceral peritonea and males with humeral spines); C. altitudinalis (Rivero, 1968) is endemic to Andes of Mérida State from Venezuela ( Barrio-Amorós et al. 2019), and has golden brown iris and lacks teeth on the dentigerous processes of vomers (vs. iris white or yellowish white with thick and thin black reticulations and dentigerous processes of vomers with teeth); C. daidalea ( Ruiz-Carranza and Lynch 1991c), is reported from Colombia and Venezuela, and has black spots along with the white ones, iridophores covering parts of the gastrointestinal peritoneum and the males lack humeral spines (vs. only white spots, iridophores absent on all visceral peritonea and males with humeral spines).

Somewhat similar species of the genus which live on the Pacific versant of the Ecuadorian Andes are C. ballux (Duellman & Burrowes, 1989), that has a minute body size, a distinct prepollex and lacks teeth on the dentigerous processes of vomers (vs. medium body size, concealed prepollex and dentigerous processes of vomers with teeth), and C. heloderma (Duellman, 1981) which has a unique pustular dorsum, condition V1 of the visceral peritonea and vomers lacking teeth (vs. shagreen dorsum with elevated warts, condition V0 of the visceral peritonea and vomers with teeth). Finally, species from other genera that superficially resemble C. zarza are Cochranella resplendens Lynch & Duellman, 1973, which has iridophores in pericardium and peritonea covering intestines and stomach and the males lack humeral spines (vs. iridophores absent on all visceral peritonea and males with humeral spines) and Nymphargus posadae (Ruiz-Carranza & Lynch, 1995a) that has almost indistinguishable tympanum, condition P2 and V1, and the males lack humeral spines (vs. evident tympanum, condition P3 and V0, and males with humeral spines).

Description of the holotype.

Adult female (MUTPL 932; Figs 2 , 3 , 5A ), medium sized, with many large and small yellowish white eggs. Head slightly narrower than the body, wider than long, head length 91% of head width, head width 34% of SVL, head length 31% of SVL; snout relatively short, snout to eye distance 13% of SVL, eye-nostril distance 24% of head length; snout rounded in dorsal view, sloping in profile; nostrils slightly elevated producing a shallow depression in the internarial area; canthus rostralis indistinct in dorsal view, rounded in cross section; loreal region slightly concave; lips non-flared; moderate sized eyes (sensu Guayasamin et al. 2020), eye diameter 11% of SVL, 34% of head length; eye-nostril distance 69% of eye diameter; eye diameter 93% of IOD; eyes directed anterolaterally at ~ 40° from midline, slightly visible from below; upper eyelid width 85% of IOD; tympanic annulus and tympanic membrane evident, but the membrane with coloration similar to that of surrounding skin; tympanum oriented slightly dorsolaterally; tympanum large (sensu Guayasamin et al. 2020), its diameter ~ 46% of eye diameter; weak supratympanic fold present, slightly concealing the upper margin of the tympanum; choanae large, ovoid, not concealed by palatal shelf of maxillary arch, closer to the distal margin of the dentigerous processes of vomers than to the margin of mouth; dentigerous processes of vomers ovoid, in transverse row between the choanae, separated medially by distance slightly lower than the width of processes; each process bearing three teeth; tongue just slightly longer as wide, not notched posteriorly, and only ~ 1/5 of posterior part not adherent to the floor of mouth.

Skin of dorsal surfaces shagreen with elevated, and some enameled, warts corresponding to white spots; throat smooth; ventral skin coarsely areolate; ventral surfaces of thighs below vent with a pair of large, round, flat tubercles (subcloacal warts); cloacal opening directed posteriorly at upper level of thighs, no distinct cloacal sheath; cloacal region bordered ventrally by many enameled (white) warts.

Upper arm thin, forearm somewhat robust; outer edge of forearms with row of enameled warts that continue into the external edges of Finger IV; hand length 34% of SVL; palmar tubercle large, elliptical; thenar tubercle large, ovoid; subarticular tubercles prominent, round and round in section; numerous round palmar supernumerary tubercles present, much smaller than subarticular tubercles; relative lengths of fingers I <II <IV <III; concealed prepollex; fingers with broad lateral fringes; webbing absent between Fingers I and II, basal between II and III, moderate between outer fingers: III2+-2IV (Fig. 5A ); bulla absent; discs on fingers expanded, truncate; disc pads nearly triangular.

Hindlimbs long, slender; femur length 55% of SVL; tibia length 57% of SVL; foot length 48% of SVL; outer edge of tarsus with row of enameled warts that often continue into the external edges of Toe V; inner edge of tarsus bearing a long fold; inner metatarsal tubercle large, elliptical; outer metatarsal tubercle indistinct; subarticular tubercles rounded and flat; plantar supernumerary tubercles inconspicuous; relative length of toes I <II <III <V <IV; toes with broad lateral fringes; webbing between toes moderate: I1---2II1--2III1--2IV2-1+V (Fig. 5A ); discs on toes expanded, truncate, lacking papillae; disc pads nearly triangular.

Coloration of holotype.

In life (Fig. 2 ): dorsum light green, head and dorsal surfaces of arms and of hindlimbs darker green, with many white or whitish, elevated, spots and flecks of various sizes. The green vertebral column, sacrum, ileum, and urostyle visible through the skin (Fig. 2B ). Flanks white or transparent (Fig. 2A ); venter with more than half of the upper parietal peritoneum covered by white iridophores; lower parietal peritoneum transparent with part of the large intestine, urinary bladder, and some large yellowish white eggs visible (Fig. 2C ); ventral vein red (Fig. 2C ). Fingers and toes yellowish (more evident ventrally; Fig. 2C ) lacking melanophores from the dorsal surfaces, except for Fingers III and IV and Toes IV and V. Iris yellowish white with thick and thin black reticulations and no circumpupillary ring. Bones green.

In preservative (Fig. 3 ): dorsal surfaces greyish with white spots; vertebral column, sacrum, ileum and urostyle no longer visible through the skin (Fig. 3A ). Throat yellowish, upper parietal peritoneum on the venter white, lower part transparent (Fig. 3B ). Melanophores absent from hands and feet, except few present on dorsal surfaces of Finger III and many on Finger IV and Toes IV and V.

Measurements of holotype

(in mm). SVL 25.5; HW 8.7; HL 7.9; IOD 2.9; IND 2.1; EW 2.5; ED 2.7; EN 1.9; snout to eye distance 3.4; TD 1.4; FL 14.1; TL 14.5; FoL 12.2; HaL 8.6; 3DW 1.6.

Body mass of holotype.

1.05 g.

Variation.

Morphometric variation is shown in Table 1 . The females are larger, with slender bodies and longer limbs (Figs 3 , 6A, B ). The males have more robust bodies, slightly thicker forearms and have humeral spines (Figs 4 , 6C-F ). One male (MUTPL 1022, Fig. 6E ) had a slightly little lighter dorsal coloration, but overall, no significant variation in dorsal coloration, iris coloration or pattern of the spots or flecks was observed between the encountered individuals. The green bones of limbs and vertebral column were visible dorsally, through the skin, in all specimens.

Eggs and tadpoles.

Two egg clutches in stage Gosner 19 (Fig. 7 ) were collected from the type locality (3.8379°S, 78.5418°W; 1460 m a.s.l.) on 9 June 2021 (MUTPL-T22). Both egg clutches were attached to the upper side of a leaf at ~ 3 m above the stream. The clutches contained 13 and 33 embryos; no adults were observed guarding the eggs or in the near proximity. The tadpoles hatched in the laboratory after 5 days and survived for more than 6 months, until 2 January 2022. They developed well in the beginning, but halted their development at Gosner stage 31 (ca. 20 November), and unfortunately started to die in January 2022 without completing their metamorphosis. It is possible that the tadpoles died due to inadequate rearing conditions, or their death was produced by chytridiomycosis (see the discussion section).

The tadpoles of C. zarza (Fig. 8 ) belong to Type IV tadpole of Orton (1953), and the exotroph, lotic, and burrower ecomorphological guild of McDiarmid and Altig (1999). The following description is based on tadpoles at Gosner stages 26 and 31 (from the MUTPL-T22 series). For the tadpoles in Gosner stage 31 the total length was 26.9-34.7 mm (31.9 ± 2.67, n = 6) and the body length was 8.2-10.4 mm (9.5 ± 0.73, n = 6), body length being ~ 30% of total length.

Body elongated, oval-depressed, wider than high; snout rounded in dorsal view and sloped and rounded in lateral view. Eyes located on dorsal surface of head and C-shaped (at least until Gosner stage 31). Nostrils positioned dorsally, protruding, with very small narial apertures oriented anteriorly. Spiracle short, single, sinistral, located at the posterolateral region of the body; spiracular opening slightly below body axis, oriented posteriorly and upwards (dorsoposterior orientation). Vent tube situated medially, short, attached to the ventral fin (caudal), with a dextral opening directed postero-ventrally. Tail long, ~ 2.4 × the length of the body, with subacute tip. Dorsal fin originating at ca. mid-length of tail; myotomes of tail musculature weakly visible in the first half of tail length.

Oral disc large (oral disc width ~ 65% of body width), not emarginated, located near tip of snout, directed anteroventrally, protruding ventrally but not laterally (Fig. 8 ), beyond body. Marginal papillae uniserial, large, distributed around oral disc (~ 43-47 papillae); large part of the anterior (upper) margin of labium lacking papillae and instead having an involuted fold with smooth surface, but with a row of submarginal flattened papillae-like ridge in the proximity of the upper jaw sheath (Fig. 9 ). Upper jaw sheath broadly arched, slender (with less than half depth of upper jaw cartilages keratinized) and with serrated edge; lower jaw sheath slightly U-shaped, slender (with less than half depth of lower jaw cartilages keratinized) and with serrated edge (Fig. 9 ). Labial tooth row formula (LTRF) 0/2(1), P-1 with medial gap, and with a row of submarginal flattened papillae-like ridge (composed by seven or eight papillae) on the posterior (lower) labium in Gosner stage 26 (Fig. 9A ). However, in Gosner stage 31, all the tooth rows were lost and only the tooth (dermal) ridges were visible beside the submarginal ridge of papillae (Fig. 9B ). It is not clear if the loss of the tooth rows is a natural process throughout the metamorphosis or it is caused by disease or other factors (see the discussion section).

General coloration varied in the different developmental stages (Fig. 8 ). Hatchlings (sensu McDiarmid and Altig 1999) in Gosner stage 24 were slightly pinkish but with a special green coloration of the abdomen (Fig. 8A ). The tadpoles had a more evident pinkish coloration in Gosner stage 25 (Fig. 8B ) and became almost red by the Gosner stage 26 (Fig. 8C ). However, after a couple of months, by the Gosner stage 31, they had lost the reddish coloration and had the body just slightly pink and the tail almost brown (Fig. 8D ). We do not know if this discoloration was a natural developmental process or the tadpoles were actually suffering from a disease or had other problems.

Vocalizations.

On 3 September 2022 we recorded the calls of several males from the type locality (Refugio de Vida Silvestre El Zarza, quebrada "Las Mariposas", Suppl. material 2). The males were calling (advertisement calls sensu Wells 2007) from above, at several meters high, in the vegetation bordering a small stream. For the description of these calls we used two recordings: FUTPL-A 263 and FUTPL-A 264 (the detailed information of each of the separate recordings is presented in the Suppl. material 2). Because the males were calling out of reach, up in the trees, we were not able to distinguish and pinpoint the calling males and so the recordings contain the calls of several males. Thus, the call description is based on the calls of probably three of the closest calling males. For this reason, we were not able to measure some of the temporal parameters, like the inter-call interval and call rate. We used for the analysis only the calls clearly distinguishable in each of the recordings, which were not overlapped by other calls. The advertisement call of C. zarza is characterized by a high pitched, pulsed, single note, with every call/note featuring 3 clearly distinguishable pulses (Fig. 10A-C ). The calls had a duration of 0.242-0.318 s (0.268 ± 0.02, n = 17), with a pulse duration of 0.072-0.159 s (0.089 ± 0.02, n = 51), and an average pulse rate of 11.8 pulses/s. The mean dominant frequency of the calls was 5309.8 Hz, with a mean 90% bandwidth of 5041.3-5487.2 Hz (Suppl. material 2). The fundamental frequency is not recognizable, and no harmonics are visible.

The call of paratype MUTPL 933 (FUTPL-A 261 and FUTPL-A 262) was recorded on 14 October 2020, on the first night that the specimen arrived in the laboratory. The animal was encountered in the type locality on a leaf at a height of 1.5 m near the stream while vocalizing. In laboratory, a recorder was left running all night, in order to record the call. The male was left in the same plastic bag in which it arrived from the field and had in its proximity, in a different bag, a female (the holotype). The male vocalized almost all night, but the call had a different structure from the typical advertisement call heard and recorded in the field (Fig. 10D-F ). We identified this call as a courtship call (sensu Wells 2007), being composed not only by the single-noted calls, but mainly by multi-noted calls (usually of five notes/call, but up to six notes/call). The notes had the same structure as the single-noted calls, with the typical three pulses. Unfortunately, the pulses are not sufficiently visible in oscillograms or spectrograms in order to allow accurate measurements (Fig. 10D ), probably due to the special conditions from the laboratory (echo from the walls, animal in plastic bag, etc.). The multi-noted calls had a duration of 0.581 to 1.905 s (1.300 ± 0.45, n = 18), depending on the number of notes/calls. The individual notes had a duration similar to the ones from the single-noted calls of the advertisement calls, just slightly longer: 0.244-0.378 s (0.304 ± 0.03, n = 80). The inter-call interval varied from 40.2 to 596.9 s (255.5 ± 171.23, n = 18) and the call rate was ~ 0.23 calls/min, or rather, ~ 14 calls/hour (Suppl. material 2). The frequencies were slightly lower than the ones from the advertisement calls, with the mean dominant frequency of 5127.4 Hz, and a mean 90% bandwidth of 4914.7-5411.2 Hz (Suppl. material 2). The fundamental frequency was not recognizable, but 2 to 3 harmonics were visible, although these could be artificially produced by the echo from the walls in the laboratory (Fig. 10E ).

It seems that in this species the males emit multi-note courtship calls when they detect the nearby presence of females and are used to interact with them, as in our case, where the male was probably aware of the female’s presence. Similar behavior was observed in other glassfrog species ( Greer and Wells 1980; Hutter et al. 2013) and it is well documented in various anuran species (see Wells 2007 for a detailed discussion). No multi-note calls were heard or recorded in the field but we did not witness any female-male interactions.

Distribution.

Centrolene zarza is currently known only from Refugio de Vida Silvestre El Zarza, Zamora Chinchipe province, southern Ecuador (Fig. 11 ). The specimens were encountered at an altitudinal range between 1434 and 1480 m a.s.l. in an evergreen lower montane forest ecosystem.

Natural history.

This is a (locally) common species in the sense that the species presence was detected (seen or heard), in the proper habitat, in large or moderate numbers, on more than 50% of the sampling days/nights ( Székely et al. 2020). All specimens were encountered during the night, on the upper surfaces of leaves of the vegetation bordering two small streams (Fig. 12 ). Calling males were heard during January, March, June, October, and November, but intense activity (many males calling from lower heights, 50 cm to 3 m above the streams) was recorded between October and March. The female paratype MUTPL 1051 was encountered nearby the calling male MUTPL 1050 at ~ 4 m high over the stream. However, no multi-note courtship calls were heard, nor was any direct interaction observed between these individuals. Observed syntopic glassfrog species were mainly Espadarana audax (Lynch & Duellman, 1973), in large numbers, but also Nymphargus posadae , Chimerella mariaelenae , and Rulyrana mcdiarmidi (Cisneros-Heredia, Venegas, Rada & Schulte, 2008).

Conservation status.

Centrolene zarza is known, for now, from only two small streams inside the wildlife refuge, in an estimated area of less than 7 km2. Although this is a locally common species, which lives inside a nationally protected area, we recommend that C. zarza be categorized as Critically Endangered following the B1ab(i,ii,iii)+2ab(i,ii,iii) IUCN criteria ( IUCN 2001) because: (1) its Extent of occurrence (EOO) and Area of occupancy (AOO) are estimated to be less than 7 km2; (2) it is known from only one locality; and (3) its habitats could be affected in the near future by mining activities (both legal or illegal), as the wildlife refuge is surrounded by active mining concessions.

Figures

Figure 1. Maximum likelihood phylogram of Centrolene. The analysis is based on 2457 base pairs of concatenated mitochondrial DNA from 12 S and 16 S, and nuclear DNA from POMC gene fragments. Outgroup is not shown; the tree was routed with Allophryne ruthveni. In red, the new species and in green a sequence newly generated by the present study. The catalog number, species name, country, and in the case of Ecuadorian species province and short locality names, are shown next to each terminal (associated data are listed in Appendix 1).

Figure 2. Holotype of Centrolene zarza sp. nov. (MUTPL 932, adult female), SVL 25.5 mm, in life A lateral view B dorsolateral view C ventral view.

Figure 3. Holotype of Centrolene zarza sp. nov. (MUTPL 932, adult female) in preservative A dorsal view B ventral view C lateral view.

Figure 4. Paratype of Centrolene zarza sp. nov. (MUTPL 1023, adult male), SVL 23.2 mm, in preservative A dorsal view B ventral view C lateral view.

Figure 5. Palmar view of hand and plantar view of foot of A holotype of Centrolene zarza sp. nov. (MUTPL 932, adult female) and B paratype (MUTPL 1023, adult male) in preservative.

Figure 6. Morphological variation of Centrolene zarza sp. nov. in live specimens A, B female, paratype (MUTPL 1051) C, D male, paratype (MUTPL 933) E, F male, paratype (MUTPL 1022).

Figure 7. Egg-clutches of Centrolene zarza sp. nov. collected from the type locality (MUTPL-T 22) A the egg-clutches attached to a leaf (10 June 2021) B hatchlings (sensu McDiarmid and Altig 1999) in stage Gosner 20 (10 June 2021) C hatchlings in stage Gosner 22 (13 June 2021).

Figure 8. Hatchlings (sensu McDiarmid and Altig 1999) and tadpoles of Centrolene zarza sp. nov. (MUTPL-T 22) A Gosner stage 24 (26 June 2021) B Gosner stage 25 (5 August 2021) C Gosner stage 26 (23 August 2021) D Gosner stage 31 (20 November 2021).

Figure 9. Oral apparatus of the tadpoles of Centrolene zarza sp. nov. (MUTPL-T 22) A Gosner stage 26 B Gosner stage 31.

Figure 10. Vocalizations in Centrolene zarza sp. nov. Visual representation of the advertisement call (FUTPL-A 263; A-C) and courtship call (paratype MUTPL 933, FUTPL-A 261; D-F) A oscilogram of a single-noted call with the 3 pulses B spectrogram of a single-noted call C power spectrum of a single-noted call D oscilogram of a multi-noted call with 6 notes E spectrogram of a multi-noted call F power spectrum of a multi-noted call.

Figure 11. Distribution of Centrolene zarza sp. nov. Records are based on specimens deposited at the Museo de Zoologia, Universidad Tecnica Particular de Loja (MUTPL), Museo de Historia Natural Gustavo Orces, Escuela Politecnica Nacional (MEPN) and Museo de Zoologia, Pontificia Universidad Catolica del Ecuador (QCAZ). 1. Destacamento Militar Condor Mirador, the type locality of Centrolene condor. 2. Alto Machinaza, with the two collecting sites, Loma Tigres Alto (yellow dot) and Loma Tigres Bajo (green dot). 3. Rio Machinaza - Sector Colibri. 4. Paquisha Alto.

Figure 12. Habitat of Centrolene zarza sp. nov. in the type locality. Refugio de Vida Silvestre El Zarza, quebrada " Las Mariposas ".