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Research Article
One hundred years of solitude: The rediscovery of Pristimantis ruidus (Anura, Strabomantidae) in the southern Andes, Ecuador and its phylogenetic relationships
expand article infoJuan C. Sánchez-Nivicela§|, Diana Székely#¤, Luis A. Salagaje M.«, Nicolás Astudillo-Abad«, Jaime Culebras«», Ernesto Arbeláez Ortiz«, Paul Székely¤#
‡ Fundación y Bioparque AMARU, Cuenca, Ecuador
§ Universidad Nacional de Colombia, Bogotá, Colombia
| Universidad San Francisco de Quito, Quito, Ecuador
¶ Instituto Nacional de Biodiversidad del Ecuador, Quito, Ecuador
# Ovidius University Constanţa, Constanța, Romania
¤ Universidad Técnica Particular de Loja, Loja, Ecuador
« Fundación Cóndor Andino, Quito, Ecuador
» Photo Wildlife Tours, Quito, Ecuador
Open Access

Abstract

We report the rediscovery, a century after its last record, of two specimens of Pristimantis ruidus from approximately 6 km northeast of its type locality, in a montane forest from the western Andes of southern Ecuador. This species, described by John D. Lynch in 1979, based on specimens collected in 1922 by George H. Tate, was previously known only from the type series, all specimens originating from a single locality (Molleturo, Provincia Azuay, Ecuador, 2317 m elev.). The rediscovery occurred in November 2022 in the Reserva de Conservación Quitahuaycu, Molleturo Parish, Azuay Province. The two specimens, both females, are characterised by rough skin with numerous rounded low warts and tubercles on their dorsum, a W-shaped occipital fold, tympanum and tympanic ring not externally visible and by the lack of cranial crests, characters that correspond with the description of the holotype. We evaluate the phylogenetic relationships, based on two mitochondrial genes (12S and 16S rRNA) and one nuclear gene (RAG-1). These analyses reveal Pristimantis ruidus as a member of the subgenus Huicundomantis, being most closely related to P. philipi, a species with a distribution restricted to the paramo of the same mountain region (Macizo del Cajas). Pristimantis ruidus co-exists with Pristimantis jimenezi, three additional species of Pristimantis and one Noblella with uncertain taxonomic status. This rediscovery underscores the urgency of implementing effective conservation and monitoring measures for the few remnants of the original ecosystems from western Ecuador, which face ongoing anthropogenic threats and harbour a diversity that remains insufficiently studied.

Resumen

Reportamos el redescubrimiento, un siglo después de su último registro, de dos especímenes de Pristimantis ruidus en aproximadamente 6 km al noreste de su localidad tipo, en un bosque montano en los Andes occidentales del sur de Ecuador. Esta especie, descrita por John D. Lynch en 1979, con base en especímenes recolectados en 1922 por George H. Tate, se conocía solo por la serie tipo con todos los especímenes originarios de la misma localidad (Molleturo, Provincia Azuay, Ecuador, 2317 m.). El redescubrimiento ocurrió en noviembre de 2022 en la Reserva de Conservación Quitahuaycu, parroquia Molleturo, provincia de Azuay. Los dos ejemplares son hembras y se caracterizan por presentar una piel rugosa con numerosas verrugas bajas y tubérculos redondeados en su textura dérmica dorsal, un pliegue occipital en forma de W, tímpano y anillo timpánico no visibles externamente y carecen de crestas craneales, caracteres que concuerdan con el holotipo. Evaluamos relaciones filogenéticas basadas en dos genes mitocondriales (12S y 16S rRNA) y un gen nuclear (RAG-1). Estos análisis revelan a Pristimantis ruidus como miembro del subgénero Huicundomantis, estando más estrechamente relacionado con P. philipi, especie de distribución restringida que habita el páramo de la misma región montañosa (Macizo del Cajas). Pristimantis ruidus coexiste con Pristimantis jimenezi, tres especies adicionales de Pristimantis y una Noblella con estatus taxonómico incierto. Este redescubrimiento subraya la urgencia de implementar planes efectivos de conservación y monitoreo para todos los remanentes de los ecosistemas del oeste de Ecuador, que enfrentan amenazas antropogénicas continuas y albergan una diversidad que aún no está suficientemente estudiada.

Key Words

Amphibia, biodiversity, conservation, Huicundomantis, molecular genetics, morphology, new record, phylogeny, systematics, taxonomy

Palabras clave

Anfibios, biodiversidad, conservación, filogenia, genética molecular, Huicundomantis, morfología, nuevo registro, sistemática, taxonomía

Introduction

The direct-developing frogs of the genus Pristimantis Jiménez de la Espada, 1870 are the most species-rich group of all amphibians, with more than 600 formally recognised species (Frost 2023). The high diversity of the genus is also accompanied by exceptional levels of ecological specialisation, many species presenting small ranges of distribution, especially at higher elevations in the Andes (Ron et al. 2022). With respect to southern Ecuador, numerous studies of the Pristimantis have revealed an extraordinary diversity and endemism, but also massive threats (Lynch 1979; Lynch and Duellman 1997; Urgiles et al. 2014, 2017, 2019; Yánez-Muñoz et al. 2016, 2019; Sánchez-Nivicela et al. 2018, 2021; Székely et al. 2018, 2020, 2021; Páez and Ron 2019; Ortega-Andrade et al. 2021).

Pristimantis ruidus (Lynch, 1979) has eluded field researchers for over a hundred years. The 16 known specimens used in the description were collected by the naturalist George Henry Hamilton Tate (1894–1953) during his 1922 expedition for the American Museum of Natural History. The specimens were collected in south-western Ecuador, in Molleturo, Azuay Province, on the Pacific slopes of the Cordillera Occidental, at an elevation of 2317 m (Lynch 1979; Lynch and Duellman 1997).

In 2022, during an expedition to the Reserva de Conservación Quitahuaycu, led by Fundación y Bioparque AMARU, members of the exploration team collected two specimens of Pristimantis, which, after comparison with the original description and holotype photographs, were identified as P. ruidus. Based on the newly-acquired specimens, we present a re-description of this species, provide for the first time images of living specimens, as well as a genetic assessment and evaluation of phylogenetic relationships of P. ruidus.

Methods

Specimen collection and study site

Fieldwork was carried out at Reserva de Conservación Quitahuaycu (-2.74047, -79.33532 [WGS84]), Molleturo Parish, Azuay Province, Ecuador (Fig. 1). The study area is a montane forest, situated at an elevation between 2400 and 2900 m, approximately 6 km southwest from the type locality of P. ruidus. For the species habitat description, we follow the ecosystem classification system proposed by the Ministerio del Ambiente del Ecuador (MAE 2012).

Figure 1. 

Map of Ecuador showing the distribution of Pristimantis ruidus in Azuay Province, Ecuador. On the left, an enlarged view of the area. Source: Esri, Maxar, Earthstar geographics and the GIS User Community.

We used the complete species inventory methodology (Angulo et al. 2006), with diurnal and nocturnal samplings, visits being conducted in November 2022. The collected specimens were deposited at the División de Herpetología (DHMECN) of the Instituto Nacional de Biodiversidad (INABIO), Quito, Ecuador and Museo de Zoología, Universidad Técnica Particular de Loja (MUTPL), Loja, Ecuador. Specimens were manipulated in accordance with Beaupre et al. (2004), photographed alive and euthanised with a 5% Roxicain solution. Liver tissue samples were extracted and preserved in 90% ethanol, after which the specimens were fixed with 10% formalin and preserved in 70% ethanol, following the methods proposed by Heyer et al. (2001) and Simmons and Muñoz-Saba (2005). Research permits were issued by the Ecuadorian Ministry of Environment (019-2018-IC-FAU-DNB/MAE, MAE-DNB-CM-2018-0106 and MAATE-DBI-CM-2021-0181).

Morphological analysis

The collected specimens were identified, based on the original description (Lynch 1979) and compared with photographs of the holotype AMNH 17590 (AMNH = American Museum of Natural History). The definition follows the characters and diagnostic terms proposed by Duellman and Lehr (2009). The sex and maturity of the specimens were determined by inspecting the sexual organs through a dorsolateral incision and the external characteristics. Fingers and Toes are numbered pre-axially to postaxially from I to IV and I to V, respectively. The relative lengths of Toes III and V were determined by adpressing them against Toe IV and Fingers I and II were compared by adpressing them against each other.

Morphological measurements were taken with a precision caliper and rounded to the nearest 0.1 mm. The abbreviations used follow the format of Duellman and Lehr (2009): SVL = Snout-vent length (distance from the tip of snout to the vent); TL = Tibia length (distance from the knee to the distal tibia end); FL = Foot length (distance from the proximal margin of inner metatarsal tubercle to tip of Toe IV); HL = Head length (distance from angle of the jaw to the tip of the snout); HW = Head width (distance between the angle of the jaws); IOD = Interorbital distance (between the orbits); EW = Width of upper eyelid (perpendicular distance to the outer edge of the upper eyelid); IND = Internarial distance (distance between the inner edges of the narial openings); EN = Eye-nostril distance (between the anterior corner of orbit and posterior margin of the narial opening); ED = Eye diameter (horizontal length of the orbit).

Colour patterns in life and other important external characteristics were based on field notes and photographs taken in the field and laboratory. The colour identification in life follows Köhler (2012). The photographs were taken by Ernesto Arbeláez, Jaime Culebras and Juan Sánchez.

Molecular analysis

Genomic extraction, amplification and sequencing were done as described in Székely et al. (2020); the six newly-generated DNA sequences were deposited in GenBank (Appendix 1). We analysed the molecular data by using sequences of two mitochondrial genes (12S and 16S rRNA) and one nuclear gene (RAG-1) from 61 specimens (60 from Ecuador and one from Peru) of 35 species or candidate species available in GenBank, representing all the currently confirmed species of the subgenus Huicundomantis of Pristimantis (sensu Ortega et al. (2022)). As outgroups, we used the species from Székely et al. (2021): Pristimantis unistrigatus (Günther, 1859), P. ceuthospilus (Duellman & Wild, 1993), P. imitatrix (Duellman, 1978), P. diadematus (Jiménez de la Espada, 1875), P. rhodoplichus (Duellman & Wild, 1993), P. melanogaster (Duellman & Pramuk, 1999), P. wiensi (Duellman & Wild, 1993), P. simonsii (Boulenger, 1900), P. orestes (Lynch, 1979), P. colodactylus (Lynch, 1979) and P. orcesi (Lynch, 1972), the trees being rooted with P. galdi Jiménez de la Espada, 1870. We conducted BLAST searches to identify similar 12S, 16S and RAG-1 sequences in GenBank and used FastTree v.2.1 (Price et al. 2010), implemented in the programme Geneious Prime (v.2023.1.2, Biomatters Ltd., Auckland, New Zealand) to build an exploratory tree with most of the available Ecuadorian Pristimantis sequences in order to determine the identity and relationships of our species.

The sequences were edited, assembled and aligned (MAFFT algorithm with the G-INS-i iterative refinement method; Katoh and Standley (2013) in Geneious Prime; the 12S, 16S and RAG-1 aligned sequences were visually inspected to correct alignment errors in PhyDE (Müller et al. 2010), concatenated into a single matrix and then used for the phylogenetic analyses. The analyses were based on a 2413-base pair (bp) dataset (924 bp for 12S, 877 bp for 16S and 612 bp for RAG-1); the matrix is available at https://doi.org/10.5281/zenodo.11099783.

Phylogenetic relationships were inferred using both Bayesian Inference (BI) and Maximum Likelihood (ML). We used PartitionFinder v. 2.1.1 (Lanfear et al. 2017) to select the best partition scheme with the Bayesian Information Criterion (BIC) as a model of selection; PartitionFinder identified three partition schemes (best model in parentheses): 12S and 16S (GTR+I+G), RAG-1 1st position (K80+G) and RAG-1 2nd and 3rd position (F81+I+G). The BI analysis was implemented in MrBayes v.3.2.6 (Ronquist et al. 2012), the Markov Chain Monte Carlo runs being performed twice, independently, for 40 million generations, with trees sampled every 1,000 generations; convergence of the runs was assessed from the average split frequency of standard deviations (p < 0.001) and by checking the potential scale reduction factors (PSRF ~ 1.0) for all model parameters; consensus trees were summarised after discarding the initial 25% as burn-in. The ML analyses were conducted in IQ-TREE 2 (Minh et al. 2020). We performed four different runs, in order to test the tree’s topologies: one with IQ-TREE’s default settings, in which the programme determined the best-fit model for our alignment (TIM2+F+I+R4 under BIC), with 100,000 bootstrap replicates for the SH-like approximate likelihood ratio branch test (SH-aLRT; Guindon et al. (2010)) and with 100,000 ultrafast bootstrap replicates (UFBoot; Hoang et al. (2018)) to assess the branch support; a second run with the three partition schemes identified by PartitionFinder and with the same number of SH-aLRT and UFBoot replicates (100,000); a third run with the default settings, but with the -bnni option (optimised, hill-climbing nearest neighbour interchange, NNI, search) in order to reduce the risk of overestimating the branch supports of the UFBoot, with 10,000 replicates for both SH-aLRT and UFBoot; and finally, a fourth run with the default settings, but the branch support evaluated with 500 standard non-parametric bootstrap (Boot; Felsenstein (1985)) searches. The phylograms were visualised and edited with FigTree (Rambaut 2014) and the uncorrected genetic p-distances were calculated for 16S with MEGA6 (Tamura et al. 2013).

Results

Morphological identification of Pristimantis ruidus

Using the diagnosis and description from Lynch (1979), the two specimens (DHMECN 19106 and MUTPL 1613) were recognised as P. ruidus, based on the following characters: dorsal skin warty, W-shaped ridge on occiput; some larger warts on flanks; no dorsolateral folds; venter coarsely areolate without discoidal folds; head wider than body, wider than long; tympanum, tympanic membrane and annulus absent; two large postrictal tubercles; snout rounded in dorsal and lateral view; cranial crest absent; vomerine odontophores triangular in contour; canthus rostralis rounded; loreal region concave, sloping to non-flared lips; upper eyelid warty (not bearing enlarged tubercles); supratympanic fold prominent, extending from corner of eye to base of arm; Finger I shorter than Finger II, digits bearing broad discs on dilated pads; fingers with lateral fringes; non-conical tubercles on heel; inner tarsal fold present; two metatarsal tubercles, inner oval, twice the size of round outer; supernumerary plantar tubercles only at bases of Toes II–IV; toes with lateral fringes, internal toe pads smaller than those of outer fingers (Figs 25).

Figure 2. 

Pristimantis ruidus in life. Female DHMECN 19106: A. Dorso-lateral view; B. Dorsal view; C. Ventral view; SVL = 30.0 mm. Female MUTPL 1613; D. Dorso-lateral view; E. Dorsal view; SVL = 20.1 mm. Photographs: Jaime Culebras, Juan Sánchez.

Figure 3. 

Pristimantis ruidus, adult female DHMECN 19106 in life: A. Lateral view of head; B. Dorsal view of head; C. Dorsal view of hand; D. Dorsal view of foot. Scale bar: 4 mm. Photographs: Juan Sánchez.

Figure 4. 

Pristimantis ruidus, adult female DHMECN 19106 in preservative: A. Dorsal view; B. Ventral view; C. Lateral view. Scale bar: 4 mm. Photographs: Juan Sánchez.

Figure 5. 

Pristimantis ruidus, adult female DHMECN 19106, details in preservative: A. Lateral view of head; B. Dorsal view of head; C. Ventral view of hand; D. Ventral view of foot. Scale bar: 4 mm. Photographs: Juan Sánchez.

Additionally, the photographs of holotype (AMNH 17590, Fig. 6) show that P. ruidus has a little tubercle in the interorbital space; digital discs are elliptical; ulnar tubercles are present, low, and rounded, present on the outer border of the forearm; basal web between toes is absent, the membrane appearance corresponds to dilated lateral fringes (all these characteristics being present also in the specimens DHMECN 19106 and MUTPL 1613).

Figure 6. 

Holotype of Pristimantis ruidus AMNH 17590: A. Dorsal view; B. Ventral view; C. Ventral view of hand; D. Ventral view of foot; E. Lateral view of head F. Dorsal view of head; SVL = 38.4 mm. Scale bar: 10 mm (Above the black line); 4 mm (Below the black line). Photographs: Lauren Vonnahme.

Phylogeny

The Bayesian Inference and the four Maximum Likelihood phylogenetic trees showed almost the same topology, with only minor differences in the position of some of the unresolved branches; the BI tree presented fewer unresolved branches and relatively higher branch support (Fig. 7). In the case of the four ML trees, values of the branch support differed slightly between the runs; the non-parametric bootstrap run (fourth run) proved to be the most conservative (having overall the smallest support values), followed by the NNI tree (third run).

Figure 7. 

Bayesian phylogenetic tree of the Huicundomantis subgenus of Pristimantis. The analysis is based on 2413 base-pairs of concatenated mitochondrial DNA from 12S and 16S and nuclear DNA RAG-1 gene fragments. Outgroup is not shown. The catalogue number, species name, province and short locality name of the vouchers (in the case of Ecuadorian specimens) are shown next to each terminal (associated data are listed in Appendix 1). Abbreviations: SH-aLRT = SH-like approximate likelihood ratio branch test, UFBoot = ultrafast bootstrap, Boot = standard non-parametric bootstrap, PP = Bayesian posterior probabilities.

Similar to Ortega et al. (2022), we recovered the subgenus Huicundomantis of Pristimantis as monophyletic, with strong support (SH-aLRT = 99.8–99.9%; UFBoot = 100%; Boot = 97%; posterior probabilities (PP) = 1). However, in our tree (Fig. 7), the basal terminals are those represented by P. philipi (Lynch & Duellman, 1995) and P. ruidus and not the samples including P. miktos Ortego-Andrade & Venegas, 2014 (Ortega et al. 2022), although with strong support only in the BI and the SH-like approximate likelihood ratio branch test (SH-aLRT = 80.4–85.5%; UFBoot = 81–93%; Boot = 69%; PP = 0.98); this difference is most likely a consequence of the different gene sampling scheme and further analyses (with matrices that contain longer sequences and more genes) are needed to clarify the relationships of the species in this subgenus. However, our tree has a topology very similar to the one presented in Székely et al. (2021).

The initial exploratory FastTree, as well as the BI and ML phylogenetic trees, recovered P. ruidus as a species in the subgenus Huicundomantis. Pristimantis ruidus is revealed as the sister species of P. philipi and both together form a basal group in Huicundomantis (Fig. 7). Uncorrected genetic p-distances for the 16S gene between P. ruidus and its sister species, P. philipi, range from 4.1% to 5.3% and between them and the other species of the subgenus Huicundomantis from 4.6% to 11.4%.

Taxonomy

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 25).

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 km2, the Area of Occupancy (AOO) is less than 10 km2, 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.

Discussion

The rediscovery of P. ruidus after one hundred years shows that there still exist montane forests that preserve conditions suitable for the survival of rare and threatened species. Despite several unsuccessful efforts to record the species in the type locality, leading to an evaluation of possibly extinct (IUCN 2023), we found that a population still persists in the area, emphasising the importance of conserving this habitat, one of the last remnants of West Andean Montane Forest in southern Ecuador (Sierra et al. 2021). For any effective conservation measures, efforts should focus on the protection and monitoring of montane forests and paramos in the region, along with the collection of basic ecological data, as well as intensive searches for additional populations.

In the original description of this species, Lynch (1979) reports that the specimens were found under rocks. This might be because, in the past, most searches for amphibians were done during the day, due to complicated logistics of night-time fieldwork. In several species of Pristimantis whose activity is nocturnal, they take refuge on the ground, under rocks or logs during the day (Arroyo et al. 2008). Based on our observations, P. ruidus similarly displays nocturnal activity. Additionally, both specimens of P. ruidus were found on shrubs or vegetation at the edge of streams, a microhabitat preference which we have also noticed in other Pristimantis species that lack an external tympanum, such as P. philipi and P. cryophilius (personal observation).

The phylogenetic closeness between P. ruidus and P. philipi and their basal position within the entire clade of the subgenus Huicundomantis (Páez and Ron 2019) implies an allopatric speciation process associated with the Macizo del Cajas. This region is isolated due to its high mountain ecosystems (Barnett 1999) and houses endemic species of amphibians, such as Atelopus nanay, Atelopus onorei Coloma, Lötters, Duellman & Miranda-Leiva, 2007, P. erythros Sánchez-Nivicela, Celi-Piedra, Posse-Sarmiento, Urgilés, Yánez-Muñoz & Cisneros-Heredia, 2018, P. philipi and P. ruidus (Lynch 1979; Lynch and Duellman 1997; Coloma 2002; Coloma et al. 2007; Sánchez-Nivicela et al. 2018).

Our comprehensive description of the morphological characters of P. ruidus, along with the additional information regarding its phylogenetic position, distribution and natural history, enhances our understanding of a species that has eluded researchers for decades. This will enable future taxonomic and systematic research of this group of frogs and assist in conservation efforts for a threatened fauna.

Acknowledgements

We express our gratitude to the colleagues and partners of the Fundación Cóndor Andino for being a part of the field exploration team. Additionally, we extend our thanks to the friends and associates from Fundación y Bioparque AMARU for their invaluable assistance during research field trips and collaboration for the development of this research. Likewise, we are indebted to the Instituto de Biodiversidad del Ecuador (INABIO) for accepting the valuable specimen in their collections. The phylogenetic work was partially funded by Universidad Técnica Particular de Loja, through the project “Descripción de nuevas especies de anfibios y reptiles del sur de Ecuador a partir de las colecciones del Museo de Zoología, UTPL” (PROY_INV_CCBIO_2020_2794), Rainforest Trust and Naturaleza y Cultura Internacional through the “Investigación científica, para la protección y monitoreo de especies de anfibios en el Área Clave de Biodiversidad Abra de Zamora” project (PROY_INV_BA_2022_3502 PY3502). We are kindly grateful to David A. Kizirian, Lauren Vonnahme and Jesse Delia of the American Museum of Natural History for the holotype photographs. The financial support and advice of Byron Swift from ReWild and Fabián Rodas from Naturaleza y Cultura Internacional were essential to promote conservation and biodiversity research of Reserva de Conservación Quitahuaycu.

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Appendix 1

Table A1.

Voucher number, GenBank accession number and locality for the Pristimantis (Huicundomantis) specimens used in the phylogenetic analysis. Bold letters mark the sequences generated by the present study.

Species Voucher number GenBank accession no. Locality
12S 16S RAG1
Pristimantis andinogigas MUTPL359 MT764339 MT756022 MT810305 Ecuador: Loja, Parque Nacional Podocarpus, Cajanuma
Pristimantis atillo QCAZ42488 - MK881440 MK881340 Ecuador: Morona Santiago, Parque Nacional Sangay, Lagunas de Atillo
Pristimantis atillo QCAZ42498 - MK881444 MK881344 Ecuador: Morona Santiago, Parque Nacional Sangay
Pristimantis atratus QCAZ45580 - MK881471 MK881364 Ecuador: Loja, Cajanuma
Pristimantis atratus QCAZ45645 - MK881473 MK881366 Ecuador: Loja, Parque Nacional Podocarpus, Cajanuma
Pristimantis balionotus MUTPL180 MT778069 MT756023 MT810306 Ecuador: Loja, Reserva Madrigal del Podocarpus
Pristimantis balionotus MUTPL392 MT778071 MT756025 MT810308 Ecuador: Loja, Abra de Zamora
Pristimantis ceuthospilus KU212216 EF493520 EF493520 - Peru: Cajamarca, Chota, 12 km W Llama
Pristimantis chomskyi MUTPL524 MZ678943 MZ678934 MZ700220 Ecuador: Loja, Parque Nacional Podocarpus, Cerro Toledo
Pristimantis chomskyi QCAZ45666 - MK881476 MK881369 Ecuador: Zamora Chinchipe, Reserva Tapichalaca
Pristimantis colodactylus MUTPL388 MT778072 MT762200 MT810310 Loja, Abra de Zamora
Pristimantis cryptomelas MUTPL135 MT778073 MT756026 MT810311 Ecuador: Loja, Abra de Zamora
Pristimantis cryptomelas MUTPL168 MT778075 MT756028 MT810313 Ecuador: Loja, Bosque Protector Washapamba
Pristimantis diadematus KU221999 EU186668 EU186668 - Peru: Loreto, Teniente Lopez
Pristimantis gagliardoi QCAZ42575 - MK881456 MK881355 Ecuador: Morona Santiago, Parque Nacional Sangay, Ranger Station, Tinguichaca river
Pristimantis gagliardoi QCAZ46738 - MK881480 MK881372 Ecuador: Cañar, Reserva Mazar
Pristimantis galdi QCAZ32368 EU186670 EU186670 EU186746 Ecuador: Zamora Chinchipe, El Pangui
Pristimantis gloria KU218035 EF493348 EF493348 - Ecuador: Azuay, 8.1 km W Morona Santiago border, Gualaceo-Limón road
Pristimantis gloria MUTPL223 MT778079 MT756032 MT810317 Ecuador: Loja, 21 km E Urdaneta
Pristimantis gloria QCAZ16448 - MK881402 MK881316 Ecuador: Azuay, Gualaceo-Macas road
Pristimantis hampatusami QCAZ58042 - MK881504 MK881387 Ecuador: El Oro, Reserva Buenaventura
Pristimantis hampatusami QCAZ58044 - KX525478 KX525472 Ecuador: El Oro, Reserva Buenaventura
Pristimantis imitatrix KU215476 EF493824 EF493667 - Peru: Madre de Dios, Cuzco Amazonico, 15 km E Puerto Maldonado
Pristimantis jimenezi QCAZ45178 - MK881468 MK881362 Ecuador: Azuay, San Antonio, Parque Nacional Cajas border
Pristimantis jimenezi QCAZ46978 - MK881482 MK881374 Ecuador: Azuay, Molleturo, Zadracay river
Pristimantis lojanus MUTPL178 MZ678945 MZ678936 MZ700222 Ecuador: Loja, Loja, Quebrada San Simon
Pristimantis lojanus MUTPL191 MZ678946 MZ678937 MZ700223 Ecuador: Loja, Cristal
Pristimantis lutzae QCAZ32785 - MK881421 MK881326 Ecuador: Azuay, Bosque Protector Yanuncay-Irquis, Páramo de Quimsacocha
Pristimantis lutzae QCAZ53728 - MK881495 - Ecuador: Azuay, Parque Nacional Cajas, El Capo, Laguna Toreadora
Pristimantis mallii QCAZ45744 MZ330729 MZ241492 - Ecuador: Pastaza, Reserva Comunitaria Ankaku
Pristimantis melanogaster MHNSM56846 EF493826 EF493664 - Peru: Amazonas, N. Slobe Abra Barro Negro, 28 km SSW Leimebambe
Pristimantis mallii QCAZ45770 MZ330730 MZ241496 MZ332932 Ecuador: Pastaza, Reserva Comunitaria Ankaku
Pristimantis miktos GGU807 - KP064151 - Peru: Loreto, Nanay, Lote 123
Pristimantis miktos QCAZ55445 - MZ241510 MK391383 Ecuador: Orellana, Parque Nacional Yasuní, Tambococha
Pristimantis multicolor QCAZ47213 - MK881488 - Ecuador: Loja, Parque Nacional Yacuri, Laguna Negra
Pristimantis multicolor QCAZ47214 - MK881489 - Ecuador: Loja, Parque Nacional Yacuri, Laguna Negra
Pristimantis muscosus QCAZ54857 - MK881501 MK881386 Ecuador: Zamora Chinchipe, Reserva Tapichalaca
Pristimantis nangaritza QCAZ41710 - MK881436 MK881336 Ecuador: Zamora Chinchipe, Alto Nangaritza PF, Las Orquídeas, Tepuy forest
Pristimantis orcesi KU218021 EF493679 EF493679 - Ecuador: Pichincha, Bosque de Pasochoa
Pristimantis orestes MUTPL242 MT778087 MK604538 MK602185 Ecuador: Loja, 11 km NE Urdaneta
Pristimantis percultus MUTPL810 MT778088 MT756034 MT810325 Ecuador: Loja, Parque Nacional Podocarpus, Cajanuma
Pristimantis percultus MUTPL812 MT778089 MT756035 MT810326 Ecuador: Loja, Parque Nacional Podocarpus, Cajanuma
Pristimantis philipi KU217863 EF493672 EF493672 - Ecuador: Azuay, 4 km W Laguna Toreadora, nearby Parque Nacional Cajas
Pristimantis philipi QCAZ37537 - MK881426 MK881331 Ecuador: Azuay, Parque Nacional Cajas
Pristimantis phoxocephalus QCAZ58463 - MK881507 MK881390 Ecuador: Cotopaxi, Pilaló surroundings
Pristimantis rhodoplichus KU219788 EF493674 EF493674 - Peru: Piura, El Tambo
Pristimantis ruidus DHMECN19106 PP725379 PP723736 PP731015 Ecuador: Azuay, Quitahuaycu reserve
Pristimantis ruidus MUTPL1613 PP725380 PP723737 PP731016 Ecuador: Azuay, Quitahuaycu reserve
Pristimantis simonsii KU212350 EU186665 EU186665 - Peru: Cajamarca, S slope Abra Quilsh, 28 km NNW Cajamarca
Pristimantis spinosus KU218052 EF493673 EF493673 - Ecuador: Morona Santiago, 10.6 km W Plan de Milagro
Pristimantis tamia QCAZ59630 MZ330735 MZ241526 MZ332958 Ecuador: Pastaza, Parque Nacional Llanganates, Comunidad Zarentza
Pristimantis tamia QCAZ59643 MZ330736 MZ241528 MZ332960 Ecuador: Pastaza, Parque Nacional Llanganates, Comunidad Zarentza
Pristimantis teslai QCAZ46213 - MK881478 - Ecuador: Tungurahua, Llanganatillo, Parque Nacional Llanganates border
Pristimantis tinguichaca QCAZ31945 - MK881418 MK881323 Ecuador: Morona Santiago, Parque Nacional Sangay, San Vicente
Pristimantis tinguichaca QCAZ40582 - MK881433 MK881334 Ecuador: Morona Santiago, Parque Nacional Sangay, Lagunas de Atillo
Pristimantis torresi MUTPL996 MZ678947 MZ678938 MZ700224 Ecuador: Loja, Guachanamá, El Apretadero
Pristimantis torresi QCAZ47397 - MK881492 MK881380 Ecuador: Loja, Celica-Alamor road
Pristimantis totoroi KU218025 EF493349 EF493349 - Ecuador: Chimborazo, 70 km W Riobamba via Pallatanga
Pristimantis totoroi QCAZ58425 - MK881505 MK881388 Ecuador: Cotopaxi, Pilaló surroundings
Pristimantis unistrigatus KU218057 EF493387 EF493387 EF493444 Ecuador: Imbabura, 35 km E Pquela
Pristimantis verrucolatus QCAZ46982 - MK881483 MK881375 Ecuador: Azuay, Yumate, Shoupshe
Pristimantis verrucolatus QCAZ46993 - MK881485 MK881377 Ecuador: Azuay, Cochapamba
Pristimantis versicolor KU218096 EF493389 EF493389 EF493431 Ecuador: Loja, Abra de Zamora
Pristimantis versicolor MUTPL494 MT778095 MT756038 MT810332 Ecuador: Loja, Reserva Madrigal del Podocarpus
Pristimantis wiensi KU219796 EF493377 EF493668 - Peru: Piura, 12.7 km E Canchaque
Pristimantis sp. QCAZ26642 - MK881409 - Ecuador: Azuay, San Antonio de Chaucha
Pristimantis sp. QCAZ32790 - MK881423 MK881328 Ecuador: Azuay, Bosque Protector Yanuncay-Irquis, Páramo de Quimsacocha
Pristimantis sp. QCAZ45029 - MK881461 - Ecuador: Morona Santiago, Parque Nacional Sangay, Etén, Rio Culebrillas
Pristimantis sp. QCAZ45129 - MK881462 MK881358 Ecuador: El Oro, Chillacocha
Pristimantis sp. QCAZ45720 - MZ241490 MZ332927 Ecuador: Pastaza, Reserva Comunitaria Ankaku
Pristimantis sp. QCAZ45945 - MZ241502 MZ332938 Ecuador: Pastaza, Reserva Comunitaria Ankaku
Pristimantis sp. QCAZ53999 - MK881496 - Ecuador: Zamora Chinchipe, Yacuambi, Romerillos
Pristimantis sp. QCAZ58855 - MZ241514 MZ332949 Ecuador: Morona Santiago, Parque Nacional Sangay, Sardinayacu
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