One hundred years of solitude: The rediscovery of Pristimantis ruidus (Anura, Strabomantidae) in the southern Andes, Ecuador and its phylogenetic relationships Author Sánchez-Nivicela, Juan C. 0000-0001-7971-1216 Universidad Nacional de Colombia, Facultad de Ciencias, Grupo de Investigación Evolución y Ecología de Fauna Neotropical, Bogotá, D. C., Colombia & Universidad San Francisco de Quito USFQ, Colegio de Ciencias Biológicas y Ambientales COCIBA, Laboratorio de Zoología Terrestre y Museo de Zoología, Campus Cumbayá, Quito 170901, Ecuador & Instituto Nacional de Biodiversidad del Ecuador, calle Rumipamba 341 y Av. de los Shyris, Casilla 17 - 07 - 8976, Quito, Ecuador & Fundación y Bioparque AMARU, Cuenca, Ecuador Author Székely, Diana 0000-0002-3786-3729 Museo de Zoología, Universidad Técnica Particular de Loja, San Cayetano Alto, calle París s / n, 110107, Loja, Ecuador & Laboratorio de Ecología Tropical y Servicios Ecosistémicos (EcoSs-Lab), Facultad de Ciencias Exactas y Naturales, Departamento de Ciencias Biológicas y Agropecuarias, Universidad Técnica Particular de Loja, San Cayetano Alto s / n, 110107, Loja, Ecuador Author Salagaje M., Luis A. 0000-0002-9545-6865 Research Center of the Department of Natural Sciences, Faculty of Natural and Agricultural Sciences, Ovidius University Constanţa, Al. Universității no. 1, 900470, Constanța, Romania Author Astudillo-Abad, Nicolás Research Center of the Department of Natural Sciences, Faculty of Natural and Agricultural Sciences, Ovidius University Constanţa, Al. Universității no. 1, 900470, Constanța, Romania Author Culebras, Jaime 0000-0003-2211-6605 Research Center of the Department of Natural Sciences, Faculty of Natural and Agricultural Sciences, Ovidius University Constanţa, Al. Universității no. 1, 900470, Constanța, Romania & Fundación Cóndor Andino, Quito, Ecuador Author Ortiz, Ernesto Arbeláez Fundación y Bioparque AMARU, Cuenca, Ecuador & Research Center of the Department of Natural Sciences, Faculty of Natural and Agricultural Sciences, Ovidius University Constanţa, Al. Universității no. 1, 900470, Constanța, Romania Author Székely, Paul 0000-0002-9252-1596 Museo de Zoología, Universidad Técnica Particular de Loja, San Cayetano Alto, calle París s / n, 110107, Loja, Ecuador & Laboratorio de Ecología Tropical y Servicios Ecosistémicos (EcoSs-Lab), Facultad de Ciencias Exactas y Naturales, Departamento de Ciencias Biológicas y Agropecuarias, Universidad Técnica Particular de Loja, San Cayetano Alto s / n, 110107, Loja, Ecuador text Zoosystematics and Evolution 2024 2024-08-13 100 3 1107 1120 journal article 10.3897/zse.100.119662 38565FEC-15BA-4832-8DC5-875963431C79 Pristimantis ruidus ( Lynch, 1979 ) Figs 2 , 3 , 4 , 5 , 6 Common English name: Molleturo Robber Frog Common Spanish name: Cutín de Molleturo Etymology. Latin, meaning rough, in reference to the skin texture ( Lynch 1979 ). Types and type locality. Holotype ( AMNH 17590 ), adult female obtained at Molleturo , Provincia Azuay , Ecuador , 2317 m elev., on 5–19 June 1922 by G. H. Tate . Paratypes ( AMNH 17588, 89, 17591–96, 17598–601, 17603), topoparatypes ( Lynch 1979 ). Newly-collected material. Two females : DHMECN 19106 (field series: JCS- 2081) and MUTPL 1613 (field series: JCS- 2084). The first individual was found amongst the remains of a felled tree ( 75 cm from the ground), along a stream edge ( - 2.73185 , - 79.34213 , elevation 2677 m ); the second individual, was on the leaf of a low shrub ( 40 cm from the ground), also by a stream edge ( - 2.73387 , - 79.34182 , elevation 2525 m ); both specimens were collected in the Reserva de Conservación Quitahuaycu, Molleturo Parish, Azuay Province , Ecuador , by Luis Salagaje, Ernesto Arbeláez, Jaime Culebras and Nicolás Astudillo, on 27 November 2022 (Fig. 1 ). Definition. A moderate-sized species of Pristimantis in the subgenus Huicundomantis as inferred from molecular phylogenetic relationships, characterised by the following combination of traits: (1) skin on head, dorsum, flanks and extremities shagreen with scattered tubercles, pustules and W-shaped scapular fold; venter coarsely areolate; discoidal fold absent; ventral texture of thighs areolate; (2) tympanic membrane and tympanic annulus absent; (3) snout rounded in dorsal and profile views; (4) upper eyelid with small rounded tubercles, 85.2 % of the IOD ; interocular space with rounded tubercle; cranial crests absent; (5) dentigerous processes of vomers rounded, with four and five teeth each, triangular in contour; (6) males lacking vocal sac and slits; (7) Finger I shorter than Finger II; disc expanded, elliptical; (8) fingers with narrow lateral fringes; (9) ulnar tubercles present, rounded, on the outer border of the forearm; (10) heel with several small rounded tubercles; tarsal tubercles rounded; inner tarsal fold present; (11) inner metatarsal tubercle oval, twice the size of round outer metatarsal tubercle; supernumerary plantar tubercles few in number; (12) toes with narrow lateral fringes; webbing absent; Toe V longer than Toe III; discs slightly shorter in size than those on outer fingers; (13) dorsum dark olive or brown with dark brown spots; dark W-shaped mark in scapular region, with orange spots in the higher portions; interorbital blotch, post ocular stripe and labial bars dark brown; venter dusty brown with white spots; (14) SVL females 20.1 and 30.0 mm ( type series: males = 25.8–31.1 mm , females = 37.1–39.8 mm ). Diagnosis. Pristimantis ruidus is distinguished from its congeners in the subgenus Huicundomantis by lacking a tympanic membrane and a tympanic annulus, head wider than long, short snout, cranial crest absent, thick supratympanic fold and by the presence of subacuminated postrictal tubercles. Pristimantis philipi is the only species lacking the middle ear and vocal apparatus and cranial crests ( Lynch and Duellman 1997 ). Pristimantis ruidus differs from P. philipi by the broadly expanded discs of the digits (discs barely expanded in P. philipi ), the evident scapular fold (lack of scapular fold in P. philipi ) and the colouration pattern composed by dark olive or brown with dark brown spots, dark W-shaped marks in the scapular region, with orange spots in the higher portions (two colour patterns in dorsum, black and pale yellow blotches or irregular flecks and blotches green, black, yellowish-green and grey in P. philipi ). Description of female DHMECN 19106. Head wider than long, short snout ( HL = 33 % of SVL ), rounded in dorsal and lateral profile, nostrils orientated posterodorsally; canthus rostralis concave; cranial crest absent; one rounded interocular tubercle; upper eyelid with rounded tubercles (three and four each) and few lower; supratympanic fold thick and discontinuous; four large subacuminated postrictal tubercles; tympanic annulus and membrane absent; few small and subconical maxillary tubercles; choana barely visible in the palatal shelf, posteriorly to level of choana and broadly separated, dentigerous process of vomers conspicuous, triangular, with four and five vomerine teeth each; tongue wider than long, 30 % adhering to the floor mouth; scapular fold W-shaped. Dorsum skin shagreen with numerous low warts; dorsolateral and medial fold absent; flanks shagreen with low warts and scarcely subconical and low tubercles; venter areolate with large and scattered warts; discoidal and pectoral fold absent; cloacal vent with rounded warts. Arms with low and oblique ridges and small subconical tubercles; subconical ulnar tubercles; U-shaped palmar tubercle, same length to thenar tubercle; thenar tubercle oval; low and few palmar supernumerary tubercles; subarticular tubercles rounded; fingers with lateral fringes, without interdigital membranes; Finger I shorter than Finger II; all digits with ventral pads, narrow and rounded in Finger I, barely expanded and elliptical in Finger II, expanded and elliptical in Fingers III and IV; discs in Fingers II and IV with circumferential grooves. Legs with low and oblique ridges, more noticeable in hind limbs and foot and small rounded tubercles; TL = 51 % of SVL ; heel and tarsus with subconical tubercles; inner tarsal fold low and extending along the tarsus; inner metatarsal tubercle oval, three times larger than external which is round; few low plantar supernumerary tubercles; subarticular tubercles rounded; toes with lateral fringes, without interdigital membranes; all digits with ventral pads and circumferential grooves, barely expanded and elliptical in Toes I – V ; relative length of Toes I <II <III < V <IV (Figs 2 – 5 ). In preservative, dorsum brown with dark brown blotches, interocular bar dark brown, scapular fold dark brown with yellowish-cream blotches in the higher portions. Flanks greyish-cream with white spots in tubercles tip or warts, postocular stripe black, labial bars dark brown with thin white lines or blotches. Venter yellowish-cream with white spots in warts. Extremities yellowish-cream in concealed surfaces, dorsally brown with diagonal dark brown bars with white spots in tubercles tip, ventrally yellowish-cream with some ash shading and white spots in tip tubercles or warts (Fig. 4 ). In life, dorsally Olive Clay Colour (85), with Verona Brown (37) blotches including scapular fold, and diagonal bars in extremities; snout Olive Sulphur Yellow (90), tip of snout and upper eyelids Lime Green (105); tip of tubercles in scapular fold Brunt Orange (10). In lateral view, flanks Olive Clay Colour (85) with Russet (44) botches in the upper middle portion, change to Medium Greenish-Yellow (88) and ashen Ferruginous (35) in the lower middle portion; russet labial bars, canthal blotch and postocular stripe; Smoky White (261) blotches and lines in labials. Ventrally, ashen Cream Colour (12) with Smoky White (261) spots. Interior surfaces of digits, Spectrum Yellow (79) with Verona Brown (37) splashes. Iris Light Chrome Orange (76) with black reticulations and sclera (Figs 2 , 3 ). Variation. In the case of the MUTPL 1613 specimen, the Olive Clay Colour (85) of the dorsum and other olive or green colour tones are replaced with Salmon Colour (83) (Fig. 2 ). The two female specimens ( MUTPL 1613 and DHMECN 19106) are smaller in size ( SVL 20.1 and 30.0 mm, respectively) than the females of the type series ( 37.1–39.8 mm ), reported by Lynch (1979) . Morphometric variation is presented in Table 1 . Table 1. Morphological measurements (in mm) of the two newly-collected Pristimantis ruidus specimens and for the holotype as provided by Lynch (1979) . Abbreviations: SVL = Snout-vent length, TL = Tibia length, FL = Foot length, HL = Head length, HW = Head width, IOD = Interorbital distance, EW = Width of upper eyelid, IND = Internarial distance, EN = Eye-nostril distance, ED = Eye diameter.

Museum Code	SVL	TL	FL	HL	HW	IOD	EW	IND	EN	ED	Sex	State
DHMECN 19106	30.0	15.5	15.3	10.0	10.9	3.0	2.6	2.5	2.6	3.3	Female	adult
MUTPL 1613	20.1	10.2	9.6	6.7	7.5	2.0	1.8	1.7	1.6	2.3	Female	adult
AMNH 17590 (Holotype)	38.4	16.3	--	11.4	13.4	2.9	3.6	--	3.2	4.5	Female	adult

Distribution and natural history. Pristimantis ruidus was known only from its type locality Molleturo ( Lynch 1979 ). The new record is approximately 6 km from the town of Molleturo, from the Western Andean montane evergreen forest ( MAE 2012 ), on the same western slope of the Macizo del Cajas (Fig. 1 ). Both individuals were found during the night, with full moon lighting (21: 58 h – 00: 09 h), close to the ground (< 70 cm ), in forested areas with abundant shrubby and herbaceous vegetation, soil covered by leaf litter and close to streams (Fig. 8 ). Pristimantis ruidus was observed in sympatry with P. jimenezi Páez & Ron, 2019 and three additional Pristimantis species and one Noblella Barbour, 1930 species with uncertain taxonomic status. Figure 8. Above: Panoramic view of Reserva de Conservación Quitahuaycu (Amaru Foundation). Below: Montane evergreen forest habitat of Pristimantis ruidus . Photographs: Ernesto Arbeláez. Conservation status. Currently, the forests on the Andean foothills in western Ecuador , which include the Molleturo Forest, retain only 30 % of their original extent, this being one of the most degraded and fragmented ecosystems in Ecuador ( Sierra et al. 2021 ). Molleturo is part of the Área de Vegetación y Bosques Protectores ( AVBP ) Molleturo-Mollepungo, a form of community reserve that covers over 140,000 hectares; however, currently it does not have a management plan and is not included in the National Environmental Protection Policy (Política Ambiental Nacional del Ecuador ); this makes the entire area highly vulnerable to loss and destruction of natural vegetation cover. Various significant threats have been identified in the region, mostly linked to activities of anthropogenic origin, such as deforestation for the expansion of grazing areas and the extraction of wood for fire. The situation is aggravated by the presence of numerous mining concessions throughout the area, as well as the proliferation of illegal mining. The Molleturo Parish has one of the highest rates of poverty and inequality in the country ( Molina et al. 2015 ), making the area even more susceptible to rapid destruction and consequent loss of environmental quality. The Reserva de Conservación Quitahuaycu comprises approximately 900 hectares (0.6 % of the total area of Molleturo-Mollepungo AVBP ), protecting ecosystems of high montane and montane evergreen forests of the western mountain of the Andean cordillera (0.14 %) and the Páramo grassland (0.02 %) ( MAE 2012 ). This Reserve is the result of conservation efforts made by the community coalition of Arquillo, San José de Huigra, San Antonio and Migüir, together with the Fundación y Bioparque AMARU , aiming to preserve threatened species of amphibians which are present here, such as Atelopus nanay Coloma, 2002 (CR), Pristimantis verrucolatus Páez & Ron, 2019 (CR), Pristimantis jimenezi (CR) , in addition to species with taxonomic uncertainty that are probably new species to science. Pristimantis ruidus has a very low detectability. Despite intense search efforts carried out by different research groups in the area, its presence was missed over the last century and we were only able to encounter two individuals. We suspect that it only inhabits densely-forested areas with ravines and humid areas, so that the species is restricted to the best conserved parts of the ecosystem, Bosque siempreverde montano de Cordillera Occidental de Los Andes ( MAE 2012 ), between elevations of 2317 and 2677 m . Pristimantis ruidus is categorised as “ Critically Endangered (Possibly Extinct) D ” according to a recent assessment, because “ given survey efforts and lack of records since 1922, it was inferred that it is either extinct or if there is still an extant population, that it is very small (<50 mature individuals) ” ( IUCN SSC Amphibian Specialist Group 2023 ). We consider the Critically Endangered status to be accurate, as the Extent of Occurrence ( EOO ) is less than 20 km 2 , the Area of Occupancy ( AOO ) is less than 10 km 2 , probably only one viable population and a low number of individuals. However, the complement “ Possibly Extinct ” should be removed from the IUCN assessment. Remarks. We identified contradictions between the Diagnosis and Description sections from Lynch (1979) , as well as characteristics that could have been lost due to the preservation of the original specimens and which are easily recognisable by examining the living specimens and recently preserved specimens. In the case of the condition of digits without rounded pads, we recognised them as elliptical. The interorbital space originally described as flat, but without mention of the presence of the one evident rounded tubercle, as it is noticeable in the photographs of the holotype and the fig. 18 in Lynch (1979) . The discoidal fold is absent in our specimens and the holotype photographs in concordance with Diagnosis ( Lynch 1979 ) but differs from the Description in the same publication and is misidentified as relatively prominent. In Lynch and Duellman (1997) , the authors mentioned that Toe V was much longer than Toe III; however, when examining the holotype photographs, we can observe that, in reality, the size relationship between Toe V and Toe III is similar to that of our specimens (Toe V extends slightly beyond subarticular tubercle II and Toe III does not exceed subarticular tubercle II ) (Fig. 6 ). According to Lynch (1979) , AMNH 17597 ( SVL = 29.0 mm) is not a mature female, having moderate convolutions and small eggs. However, our specimen DHMECN 19106 ( SVL = 30.0 mm) is an adult female because it has developed ovaries.