Eleutherodactylus viejas, Lynch & Rueda-Almonacid, 1999

Sepúlveda-Seguro, Ana M., Marín, Carlos M., Amézquita, Adolfo, García, Yuly A. & Daza, Juan M., 2022, Phylogeographic structure suggests environmental gradient speciation in a montane frog from the northern Andes of Colombia, Organisms Diversity & Evolution (New York, N. Y.) 22 (3), pp. 803-820 : 812-816

Eleutherodactylus viejas ( Lynch & Rueda-Almonacid, 1999:311; part).

Holotype MHUA-A 12022 , adult male ( Fig. 4 ) from Colombia, Department of Antioquia, municipality of Anorí, vereda El Retiro (6.9912, -75.1196; 1,669 m a.s.l., collected on September 30, 2019 by AMS, CMM and YAG.

Paratypes 31 specimens, all from Colombia, Department of Antioquia. From municipality of Anorí , vereda El Nevado (6.9783, -75.1111; 1,650 m a.s.l): MHUA-A 5555 (adult female) collected on 2007 by Laura Bravo ; from vereda San Benigno (6.9869, -75.1375; 1,600 m a.s.l.): MHUA-A 5562 (adult female) collected on 2007 by Laura Bravo ; from Vereda El Retiro , Porce III hydroelectric project protected area–EPM (6.9913, -75.1196; 1,689 m a.s.l.): MHUA-A 11731 , 11733 , 11734 , 11914 , 12019 , 12021 , 12027 (adult males), MHUA-A 11737 , 11913 , 12023 , 12024 (adult females) collected on 2019 by AMS, CMM, and YAG. From municipality of Guatape, vereda El Tronco (6.3028, -75.1434; 1,875 m a.s.l.): MHUA-A 8059 , 8088 (subadult females), MHUA-A 8099 (adult male) collected on 2013 by JMD, MHUA-A 8810 (juvenile) collected on 2015 by JMD, MHUA-A 11164 (adult male) collected on 2018 by Adriana Restrepo; from vereda Los Naranjos: MHUA-A 11385 (adult male) collected on 2018 by Diego Alzate- Estrada. From municipality of Amalfi, vereda Guayabito (6.8216, -75.0747; 1,906 m a.s.l.): MHUA-A 9428 (subadult female) collected on 2015 by JMD. From municipality of Granada, vereda Tafetanes (6.1267, -75.1088; 1,823 m a.s.l.): MHUA-A 10473 (adult male) collected on 2016 by

9744, g MHUA-A 11468, and h MHUA-A 9759; and P. factiosus , i MHUA-A 11736, j MHUA-A 11735, k MHUA-A 11732, and l MHUA-A 11736. Photos by Carlos M. Marin. Illustration by Ana M. Sepúlveda-Seguro

CMM; from vereda Los Medios (6.1325, -75.0966; 1,723 m a.s.l.): MHUA-A 10477, 10508 (juveniles), MHUA-A 10489, 10514 (adult males) collected on 2016 by CMM and MHUA-A 10814 (adult male) collected on 2016 by JMD.

Etymology The specific name “campesino ” (farmer in Spanish) is a noun used in apposition, in reference to people living in the rural areas. The name honors the people who live and work in the Colombian rural areas for their resilience and perseverance in the struggle towards achieving a peaceful and equitable country.

Diagnosis We assign the new species to the Pristimantis ridens species group based on our morphological and phylogenetic results (Fig. 1). Morphological description follows the terminology by Lynch and Duellman (1997). Description of the coloration patterns was based on field observations and photos of live specimens. Specimens were determined as adults by examination of secondary sexual characters (presence of vocal slits and vocal sacs) and sex was determined by the presence of ovaries or testes. Pristimantis campesino sp. nov. is characterized by the following combination of characters: (1) skin texture of the dorsum weakly tuberculate; venter areolate; dorsolateral folds presents; discoidal fold absent; (2) tympanic membrane and tympanic annulus visible, supratympanic fold usually prominent; diameter of tympanum 35–40% of eye diameter; (3) snout short and rounded in dorsal and lateral views; (4) one to three conical tubercles on the upper eyelid; without cranial crests; (5) vomerine odontophores arched and barely separated, that extend laterally from margins of the choanae; (6) in males vocal slits and small subgular vocal sac present; nuptial pads absent; (7) finger I shorter than II, with large rounded digital discs and defined pads; (8) fingers with narrow lateral fringes; (9) small ulnar tubercles present; (10) a short line of low tubercles on tarsus; inner tarsal fold absent; calcar tubercles present (11) inner metatarsal tubercle oval and prominent, four to five times bigger than external circular metatarsal tubercle; smalls supernumerary plantar tubercles present (12) toes with narrow lateral fringes; third toe shorter than fifth; a small webbing between toes IV and V reaching the middle-distal portion of the basal subarticular tubercles; (13) dorsum ranging from dark brown to bright yellow in males and uniform brown to almost black in females (some individuals present longitudinal stripes or a cream middorsal stripe); dark brown vertical bands on lips; oblique dark brown bars on the dorsal surface of the hind limbs; males with bright translucent yellow gular and ventral coloration that extends to the middle area of the forelimbs, with dark brown thighs; females with creamy orange belly, mottling, with thighs and forelimbs browning towards the distal region; eye with reddish-terracotta iris with thin dark brown reticulations and light blue sclera; (14) adult males small 14.4–19.0 mm (mean ± SD = 16.6 ± 1.42; n = 15) and females 20.2–30.3 mm (mean ± SD = 25.5 ± 3.2; n = 13).

Comparisons with other species Pristimantis campesino sp. nov. shares with P. viejas the presence of lateral fringes, vocal slits, texture skin of venter areolate, finger I shorter than finger II and the color pattern of the concealed surfaces of thighs and shanks (having orange and/or red dots usually in a black background, Fig. 5 b-d, f–h) but differs from P. viejas by having a iris solid reddish-terracotta with thin dark brown and black reticulations ( Fig. 5a ) versus iris golden at the upper and lower portions with coarse black reticulations in P. viejas ( Fig. 5e ); in having shorter calls (0.027 ± 0.004 s in P. campesino sp. nov. and 0.053 ± 0.004 s in P. viejas ; Online Resource 2, Fig. S5 ); a wider frequency band (ranging from 2.328 to 3.503 kHz in P. campesino sp. nov., and 2.670 to 3.282 kHz in P. viejas ); and disjunct distribution in both altitudinal range (from 1500 to 2150 m a.s.l in P. campesino sp. nov. and 100 to nearly 1600 m a.s.l in P. viejas ; Fig. 2b–c ), and occupied climatic space ( Fig. 3b ). Additionally, the distinctiveness of P. campesino sp. nov. and P. viejas is further corroborated by uncorrected pairwise distances of 6.3% in 16S and 9.6% in coi (Fig. S1).

As Pristimantis campesino sp. nov. belongs to the Pristimantis ridens species group sensu Padial et al. (2014), we compared the new taxon with the species within this clade. Comparisons were conducted by a detailed revision of the original descriptions and, when possible, by direct examination of museum specimens. For a summary of diagnostic characters of the species belonging the Pristimantis ridens group see Online Resource 2, Table S1. Comparing the remaining species of the Pristimantis ridens group, Pristimantis campesino sp. nov. differs from P. crenunguis ( Lynch, 1976) and P. jorgevelosai (Lynch, 1994) , by lacking cranial crest; from P. actites ( Lynch, 1979) , P. adnus ( Crawford et al., 2010) , P. almendariz ( Brito & Pozo-Zamora, 2013) , P. bicolor ( Rueda-Almonacid & Lynch, 1983) , P. factiosus ( Lynch & Rueda-Almonacid, 1998) , P. ocellatus ( Lynch & Burrowes, 1990) , P. rosadoi ( Flores, 1988), P. sanguineus (Lynch, 1998) , and P. tanyrhynchus ( Lehr, 2007) by lacking inner tarsal folds ( P. factiosus is also readily distinguishable from P. campesino sp. nov. by lacking dark-brown vertical bands on lips; Fig. 5i ); from P. cerasinus ( Cope, 1875) , P. colomai ( Lynch & Duellman, 1997) , P. educatoris ( Ryan et al., 2010) , P. labiosus ( Lynch et al., 1994) , P. lanthanites ( Lynch, 1975) , P. museosus ( Ibáñez et al., 1994) , P. orpacobates ( Lynch et al., 1994) , and P. tenebrionis ( Lynch & Miyata, 1980) by having lateral fringes; from P. cremnobates ( Lynch & Duellman, 1980) , P. cruentus ( Peters, 1873) , P. erythropleura ( Boulenger, 1896) , P. ixalus ( Lynch, 2003) , P. laticlavius ( Lynch & Burrowes, 1990) , P. latidiscus ( Boulenger, 1898) , P. penelopus ( Lynch & Rueda-Almonacid, 1999) , P. rubicundus ( Jiménez de la Espada, 1875) , and P. thectopternus ( Lynch, 1975) by having vocal slits; from P. epacrus ( Lynch & Suárez-Mayorga, 2000) and P. w-nigrum ( Boettger, 1892) by lacking nuptial pads; and from P. ridens ( Cope, 1866) and P. caryophyllaceus ( Barbour, 1928) by having skin texture on dorsum weakly tuberculate (smoot in P. ridens and P. caryophyllaceus ).

Two species distributed in the Cordillera Central which can be potentially confused with P. campesino sp. nov. are P. actinolaimus ( Lynch & Rueda-Almonacid, 1998) and P. suetus ( Lynch & Rueda-Almonacid, 1998) both species described from Selva de Florencia, Department of Caldas (the same type locality from P. factiosus ). However, P. campesino sp. nov. differs from P. actinolaimus by having vocal slits and texture skin of the dorsum weakly tuberculate (shagreen in P. actinolaimus ) and from P. suetus by lacking nuptial pads and skin texture of the dorsum weakly tuberculate (finely shagreen in P. suetus ).

Measurements of the holotype (in millimeters) SVL: 17.4; HL: 6.1; HW: 6.9; ED: 2.6; UEW: 1.9; EN: 2.1; SL: 2.7; IND: 1.8; IOD: 2.2; TD: 1.0; FLL: 4.2; HAL: 4.9; THL: 9.8; TL: 10.6; FL: 8.4; Fin4DW: 0.5 and Toe4DW: 0.5.

Description of the holotype A male, 17.4 mm SVL. Texture of skin on dorsum and flanks weakly tuberculate; thin and broken dorsolateral folds; venter areolate; discoidal fold absents. Head barely wider than long; rounded snout in dorsal and lateral views, relatively short (snout length 15.3% of SVL); protuberant and sideways-facing nostrils; canthus rostralis distinct; loreal region weakly concave; small subgular vocal sac. Flattened interorbital area, narrower than upper eyelid width; one to three conical tubercles on upper eyelid; cranial crests absent; eye diameter greater than eye-nostril distance. Supratympanic fold present; tympanic membrane and tympanic annulus distinct and rounded; diameter of tympanum 40% of eye diameter.

Forearm slender, 24.9% of SVL; ulnar tubercles present; hand length 28.2% of SLV; finger I shorter than II; fingers with defined pads and broadly expanded elliptical digital discs; narrow lateral fringes on fingers; smalls supernumerary palmar tubercles; nuptial pads absent. Hind limbs relative proportional to body; tibia length 60.7% of SVL; thigh length 56.3% of SVL; tarsal fold absent; tubercles on tarsus, lows; calcar tubercles. Foot length 79.4% of TL; lateral fringes on toes; basal webbing between toes IV and V; inner metatarsal oval, about 4 × times bigger than external metatarsal tubercle; low rounded supernumerary plantar tubercles; defined pads and broadly expanded elliptical digital discs on toes; toe III shorter than toe V; tip of toe III reaching the middle of penultimate subarticular tubercle of toe IV; tip of toe V reaching the distal border of the distal subarticular tubercle of toe IV.

Coloration of holotype in life Dorsum dark brown and ochre with small and darker spots and a dark ochre patch covering the eyelids and the front of the snout. Translucent yellow belly with dark brown spots in the medial and posterior area; throat region with few small dark brown spots near the lips. Rostral dark-brown edge that lightens towards the area of the lips, with dark-brown vertical bands on lips. Supratympanic dark brown membrane in the upper area, which becomes lighter with the tympanum towards the lower area. Extremities dark brown with multiple dark spots with no defined pattern. Concealed surfaces the of thighs and shanks with orange blotches on a black background. Axillary and groin region with dark brown spots on a yellow background. Reddish-terracotta iris with thin dark brown reticulations and light blue sclera ( Fig. 5a ).

Coloration of the holotype in ethanol Dorsum pale brown with light brown blotches and granules dark brown. Flanks dark brown. Loreal region cream; snout dark brown. Lower lip cream with some scattered gray dots. Throat and anterior portion of chest cream. Belly with light cream granules. Dorsal surface of upper arm cream with dark brown blotches; anterior surface cream, posterior surface with dark brown blotches; ventrally cream. Dorsal, anterior, and posterior surfaces of forearm cream lacking distinctive marks. Concealed surface of forearm cream. Palmar surfaces brown. Dorsal surface of hand with pale brown blotches on fingers. Dorsal surface of thigh light cream with dark brown blotches. Ventral surface of thigh and concealed surfaces of shank and foot cream. Plantar surfaces dark brown (Online Resource 2, Fig. S6).

Morphological variation Adult males of Pristimantis campesino sp. nov. are smaller than females (Online Resource 2, Fig. S3b ) and exhibit variation in dorsal coloration ranging from dark brown (MHUA-A 08810) to bright yellow (MHUA-A 10814). Dorsal coloration in females is variable, with individuals having a uniform brown (MHUA-A 11737) to almost black (MHUA-A 12024) dorsal surface and others having either a broad dark brown dorsal stripe (MHUA- A 08819) or a narrow cream middorsal stripe (MHUA-A 08088; Online Resource 2, Fig. S7). Variations in morphometric measurements of the type series of P. campesino sp. nov. is shown in Online Resource 2, Table S2.

Bioacoustics We recorded and analyzed 94 calls of eight individuals: four from the lowland and four from the highland lineage. Sound files were obtained using a digital TASCAM DR-40 recorder and the builtin microphone. Spontaneously calling males were recorded as near as 1 m of distance from the recorder. We analyzed the sound files using the Raven pro software version 1.5 (Center for Conservation Bioacoustics, 2014) using a Hanning sampling window, window size of 512 points, sampling rate of 44.1 kHz, 16-bit of precision, and overlap of 50%. Following Köhler et al. (2017), we measured five temporal and spectral variables: call duration in seconds and low frequency, high frequency, peak frequency, and delta frequency in KHz. Average values were estimated for each individual, our biological, and statistical unit of analysis. We compared the advertisement call variables which showed the highest inter-lineage variation by implementing the non-parametric Wilcoxon test after check for normality.

The advertisement call of Pristimantis campesino sp. nov. consists of a single chirp-like (i.e., pulsed) note lasting about 0.027 ± 0.004 s (mean ± SD, n = 53 calls from four individuals), with at least four visible harmonics but no appreciable frequency modulation. The calls are uttered in bouts with no apparent temporal structure. The note oscillogram (Online Resource 2, Fig. S5a ) further reveals asymmetric amplitude modulation, with a slow rise and fast decrease of amplitude, which peaks at about 80–90% of the note duration. The frequency band of the fundamental harmonic ranges from 2.328 ± 0.056 to 3.503 ± 0.246 kHz with frequency peaking at about 2.649 ± 0.175 kHz. The advertisement call of P. campesino sp. nov. is readily distinguished from the call of P. viejas by the presence of pulses (absent in P. viejas ), the absence of frequency modulation (present in P. viejas ), the shorter note duration (0.027 s in P. campesino sp. nov. and 0.053 s in P. viejas ; Wilcoxon test = 0, p <0.005), and the lower peak frequency (2.649 kHz in P. campesino sp. nov. and 2.916 kHz in P. viejas ; Wilcoxon = 256.5, p <0.005; Online Resource 2, Fig. S5 ). A list of the number of analyzed calls per specimen and a comparison of the advertisement call parameters between P. viejas and P. campesino sp. nov. is shown in Online Resource 2, Table S3.

Distribution and natural history Pristimantis campesino sp. nov. inhabits open and secondary habitats in the premontane forests of the Department of Antioquia, Colombia, on the eastern versant of the northern Cordillera Central between 1,500 and 2150 m a.s.l. approximately ( Fig. 2c ). Populations of this species are locally abundant and individuals are commonly found perched on branches and leaves of shrubs and trees at 0.5 to 2.0 m from the ground (Online Resource 2, Fig. S8). We observed and collected acoustically active individuals between 17:30 and 19:00 h. Relevant biological aspects concerning the reproductive activity period (whether continuous or not) and population dynamics remains unknown. At present P. campesino sp. nov. is known to occur in the protected areas of the hydroelectric projects Porce III in the municipality of Anorí and Calderas in the municipality of Granada, Department of Antioquia. Distribution of P. campesino sp. nov. in these protected areas can favor its conservation status.

Figures

Fig.4 Holotype of Pristimantis campesino sp. nov. adult male MHUA- A 12022 in life. SVL 17.40 mm. Photo by Carlos M. Marín

Fig. 5 Variation in the coloration of the iris (photographs) and concealed surfaces of thighs (illustrations) of Pristimantis campesino sp. nov., a MHUA-A 11913, b MHUA-A 11734, c MHUA-A 8059, and d MHUA-A 8089; P. viejas, e MHUA-A 11261, f MHUA-A

Fig. 2 a Geographic distribution of genetic sampling including the phylogenetic neighborhood of Pristimantis viejas. Color in symbols match lineages depicted in Fig. 1. b Altitudinal distribution across the latitudinal gradient of the two recovered lineages within the nominal species P. viejas. Red dots denote records from lowland lineage and

Fig. 3 Between lineages differences in the climatic space occupied by two lineages within Pristimantis viejas, as inferred from DAPC. a Posterior probability of assignment of encountered individuals to each lineage (bars) based on the climatic variables, compared to the actual lineage identity. b Distribution of individuals along the single