﻿A new stream treefrog of the genus Hyloscirtus (Amphibia, Hylidae) from the Río Negro-Sopladora National Park, Ecuador

﻿Abstract Recent surveys in the Río Negro-Sopladora National Park revealed a striking new species of Hyloscirtus. The new species is easily diagnosed from all other congeners by its large body size (64.9 mm SVL in adult female); broad dermal fringes in fingers and toes; prepollex not projected into a prepollical spine and hidden under thenar tubercle; dorsum greyish-green, with paler-hued reticulum, yellow spots and black speckles; throat, venter, flanks and hidden surfaces of limbs golden-yellow with large black blotches and spots; fingers, toes and webbing yellow with black bars and spots; iris pale pink with black periphery. It is currently known only from its type locality, in the high montane forest on the southern slopes of the Cordillera Oriental of the Andes, southeastern Ecuador. The new species might be related to the H.larinopygion species group based on its morphology.

The Hyloscirtus larinopygion species group has been diagnosed by having a large body size (SVL>60 mm) and dark brown or grey dorsum with pale marks (Duellman et al. 2016;Ron et al. 2018). Two strongly supported clades are identified within this species group, showing latitudinal replacement among each other and sympatry in central and southern Ecuador (Almendáriz et al. 2014;Rivera-Correa et al. 2016;Rojas-Runjaic et al. 2018;Ron et al. 2018;Reyes-Puig et al. 2022). Fourteen species distributed across the Andes of southern Colombia and southern Ecuador are part of the northern clade of the H. larinopygion species group. The southern clade currently includes four species inhabiting the Andes of southern Ecuador and northern Peru (Ron et al. 2018;Reyes-Puig et al. 2022).
Recent expeditions to the Río Negro-Sopladora National Park, on the eastern slopes of the Andes of southeastern Ecuador, resulted in the discovery of a magnificent new species of Hyloscirtus. Herein, we describe this new species based on its distinctive morphology and colouration.

Materials and methods
Fieldwork was carried out between February and March 2020 at the Río Negro-Sopladora National Park, on the border between the provinces of Morona-Santiago and Azuay, southeastern Andes of Ecuador. Surveyed ecosystems included paramo grasslands and montane and foothill evergreen forests, between 1000 and 3400 m elevation on the River Paute basin. We used the complete species inventory field methodology (Angulo et al. 2006), with nocturnal surveys carried out between 19:00 and 23:00. Field coordinates were obtained using a Garmin Handheld Navigator GPS and are referenced to datum WGS84.
The specimen was euthanised with a 5% lidocaine solution, fixed in 10% formalin, and preserved in 70% ethanol, following recommendations by McDiarmid (1994) and Simmons and Muñoz Saba (2005). All procedures in this study comply with the guidelines for managing live amphibians and reptiles in field investigations (Beaupre et al. 2004). The study was carried out under scientific research authorisation N° 019-2018-IC-FAU-DNB/MAE and framework contract for access to genetic resources N° MAE-DNB-CM-2018-0106.
We reviewed diagnostic characters used for the taxonomy of the Hyloscirtus larinopygion species group based on data obtained from the direct study of specimens, photographs of preserved and live frogs with verified identification from Anfibios del Ecuador BioWeb database (Ron et al. 2019), CalPhotos (Berkeley Natural History Museums 2012) and MCZbase (Museum of Comparative Zoology 2022); and from the literature, including original descriptions (Duellman 1973;Duellman and Altig 1978;Duellman and Berger 1982;Ruiz-Carranza and Lynch 1982;Duellman and Hillis 1990;Ardila-Robayo et al. 1993;Duellman and Coloma 1993;Faivovich et al. 2005;Faivovich and De la Riva 2006;Mueses-Cisneros and Anganoy-Criollo 2008;Coloma et al. 2012;Rivera-Correa and Faivovich 2013;Almendáriz et al. 2014;Duellman et al. 2016;Rivera-Correa et al. 2016;Rojas-Runjaic et al. 2018;Ron et al. 2018;Yánez-Muñoz et al. 2021;Reyes-Puig et al. 2022 Format, definitions, and terminology used for the species description follow standards proposed by Duellman (1970) and Duellman and Hillis (1990). Webbing formulae follow the notation system proposed by Savage and Heyer (1967) and Myers and Duellman (1982). We use the definitions and terminology for the colouration patterns of body and limbs proposed by Savage (2002) and for eye colouration descriptions by Glaw and Vences (1997). Sex and maturity were determined by inspection of gonads through a dorsolateral incision. The following measurements were taken with digital callipers (0.01 mm accuracy, rounded to the nearest 0.1 mm) under a stereomicroscope by a single person: Snout-vent length (SVL), head length (HL), head width (HW), internarial distance (IND), interorbital distance (IOD), eye width (EW), eye-nostril distance (EN), eye diameter (ED), tympanum diameter (TD), tibial length (TL), foot length (FL), disc of Finger III width (Fin3DW). Colouration patterns in life and other relevant characteristics were obtained from field notes and photographs taken in the field.

Results
The specimen collected at Río Negro-Sopladora National Park has broad dermal fringes in fingers and toes, a large body size (64.9 mm in SVL) and lacks mental glands. Broad dermal fringes are a putative morphological synapomorphy of the genus Hyloscirtus (Faivovich et al. 2005;Rivera-Correa and Faivovich 2013), and the other two characteristics suggest that this specimen might be related to species included in the Hyloscirtus larinopygion species group (Faivovich et al. 2005;Rivera-Correa and Faivovich 2013;Duellman et al. 2016)-although some species of the group have mental glands, e.g., H. caucanus (Brunetti et al. 2015). The specimen from Río Negro-Sopladora National Park shows some phenetic characteristics like those present in species of the northern clade of the H. larinopygion group. Species of the northern clade are morphological distinct from species of the southern clade as follows (condition for species of the southern clade in parentheses): HW/HL < 1.1 (HW/HL ≥ 1.1); longer snouts, usually EN/ED > 0.75 (EN/ED < 0.65); dentigerous processes of vomer in contact or slightly separated and having numerous vomerine teeth (widely separated, with few vomerine teeth); forearms robust and slightly thicker than upper arm (forearms and arms hypertrophied, similar to species of the Hyloscirtus armatus species group); enlarged, broad, elliptical prepollex, hidden under thenar tubercle (protruding, curved prepollical spine); colouration on dorsum different from colouration on flanks, hidden surfaces of thighs and venter (coloration similar on dorsal, flanks and venter) (Fig. 1). The distinction between both clades of the H. larinopygion species group has been consistently identified in several studies (Almendáriz et al. 2014;Rivera-Correa et al. 2016;Rojas-Runjaic et al. 2018;Ron et al. 2018).
The specimen from Río Negro-Sopladora National Park shows a unique colouration pattern with pale coloured background and dark marks on dorsal, lateral, and ventral surfaces, while most species currently under the H. larinopygion group have dark-coloured backgrounds with dark or pale marks (except for H. sarampiona and some specimens of H. larinopygion and H. psarolaimus). While it is known from a single individual, we propose that the population of Hyloscirtus from the Río Negro-Sopladora National Park corresponds to an undescribed taxon, and we described it below. Diagnosis. Hyloscirtus tolkieni differs from other congeneric species by the following combination of characters: large body size (64.9 mm SVL in a single adult female); broad dermal fringes in fingers and toes; discs slightly expanded; head 7% wider than long; snout truncate in dorsal and lateral view; tympanic membrane and annulus evident, partially covered by supratympanic fold; dentigerous process of vomers slightly separated, with 9-13 vomerine teeth; forearm robust and slightly thicker than arm; discs slightly expanded; broad dermal fringes in fingers and toes; prepollex enlarged, hidden under thenar tubercle and not projected into a prepollical spine; subarticular tubercles on hands and feet rounded and poorly projected; calcar tubercle present; dorsum greyish-green, with paler-hued reticulum, yellow spots and black speckles; throat, venter, flanks and hidden surfaces of limbs yellow with large black blotches and spots; fingers, toes and webbing yellow with black bars and spots; iris pale pink with black periphery, sclera greyish-blue, and nictitating membrane yellow (Figs 2-4).
Hyloscirtus tolkieni is readily distinguishable from all other species of Hyloscirtus by its greyish-green dorsum with paler reticulations, yellow spots, and black speckles. Based on its colouration pattern, Hyloscirtus tolkieni (characteristics in parentheses) is easily differentiated from all other species of the northern clade of the Hyloscirtus larinopygion species group ( (present).
Hyloscirtus tolkieni differs from species of the H. armatus species group by the absence of clusters of keratinised spines on the prepollex and the proximal ventral surface of the humerus (present in H. armatus and H. charazani), non-expanded prepollex (expanded in H. armatus and H. charazani), robust but not hypertrophied forearms (hypertrophied in H. armatus and H. charazani), and absence of a skin fold in the proximoventral portion of upper arm (present in H. armatus, H. charazani, and H. chlorostea).
Hyloscirtus tolkieni differs from species of the H. bogotensis species group, including H. albopunctulatus and H. phyllognathus that inhabit the eastern Andes of Ecuador, and from H. jahni, single member of its homonym group, by its larger body size with 64.9 mm in SVL (smaller in the H. bogotensis and H. jahni species groups with SVL<36 mm), greyish-green dorsum with paler reticulum, yellow spots and black speckles (green or brown dorsum with or without pale or dark spots and speckles and pale lines in the H. bogotensis and H. jahni species groups), ventral surfaces yellow with large black blotches and spots (venter cream or yellowish without dark marks in the H. bogotensis and H. jahni species groups).
Skin on dorsum shagreen, throat slightly granular, flanks and venter granular, posterior surfaces of limbs strongly granular; pectoral fold absent; cloacal opening directed posteroventrally at upper level of thighs; supracloacal flap present; two pairs of swollen, thick, vertical, pericloacal folds.
Colouration in life. Dorsal surfaces of head, body and limbs greyish-green, with thick paler-hued reticulum, yellow spots, and black speckles; head with a light greyishgreen medial line; throat, venter and flanks yellow (more intense on the throat and turning greyish towards posterior end of venter) with large black blotches and spots; hidden surfaces of limbs yellow with transversely distributed black oval dots; fingers, toes and webbing yellow with black bars and spots; iris pale pink with black periphery, sclera greyish-blue, and nictitating membrane yellow (Fig. 2) Colouration in preservative. Same colouration patterns as described for the colouration in life, but greyish-green dorsal areas turned darker grey, yellow on venter and flanks turned golden-grey to grey (Figs 3-4). Measurements of the holotype (in mm). SVL=64.9, HL=18.8, HW=20.2, IND=4.6, IOD=6.3, EW=4.9, EN=5.2, ED=6.9; TD=2.8, TL=31.2, FL=30.9, Fin3DW=3.1.
Etymology. The specific epithet tolkieni is in honour of the writer, poet, philologist, and academic John Ronald Reuel Tolkien (J.R.R. Tolkien, 1892Tolkien, -1973, creator of Middle-earth and author of fantasy works like "The Hobbit" and "The Lord of the Rings". The amazing colours of the new species evoke the magnificent creatures that seem to only exist in fantasy worlds. Distribution, natural history, and conservation status. Hyloscirtus tolkieni is only known from its type locality on the southeastern slopes of the Cordillera Oriental of the Andes of Ecuador, at 3190 m elevation, in the Río Negro-Sopladora National Park, province of Morona Santiago (Fig. 8). The ecosystem in the area is High Montane Forest of the Eastern Cordillera of the Southern Andes of Ecuador (MAE et al. 2013). The holotype was active at night at 20:30 amidst tree branches, c. 5 m above ground and 8 m from the nearest stream (Fig. 9). It was found in sympatry with an undescribed species of Pristimantis.
Very few herpetological surveys have been conducted in the region, with James A. Peters being one of the few herpetologists that visited the area (Peters 1973). Our surveys were carried out over 13 effective days, and we could not detect additional individuals of H. tolkieni, despite focalised searches. The type locality of H. tolkieni is officially protected as part of the Río Negro-Sopladora National Park, a protected area created in 2018 where little habitat loss has occurred. Large, forested areas remain unstudied in the national park, and the species may have a wider distribution beyond the immediate surrounding of its type locality. In the absence of sufficient information to evaluate the conservation status and extinction risk of H. tolkieni, we propose that it be classified under the Data Deficiency category until more data is obtained (IUCN 2012(IUCN , 2017Ortega-Andrade et al. 2021). Urgent research and monitoring actions should be established to study its life history and ecology, population size and trends, survey new sites where additional populations may exist and evaluate if threats are impacting its long-term conservation, such as invasive species, emerging diseases, or climate changes.
Key to the species of the northern clade of the Hyloscirtus larinopygion species group This key helps to identify adult female and male stream treefrogs of the northern clade of the H. larinopygion species group, using characters that can easily be observed in the field and lab (no dissections required). This key is probably not useful to identify juveniles and ontogenetic variation in many species of the group remains unknown. This key was expanded and corrected from the keys presented by Duellman and Hillis (1990) and Duellman and Coloma (1993). Colours in preservative are shown in parentheses.