Zoosyst. Evol. 100 (3) 2024, 1107-1120 | DOI 10.3897/zse.100.119662

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One hundred years of solitude: The rediscovery of Pristimantis ruidus (Anura, Strabomantidae) in the southern Andes, Ecuador and its phylogenetic relationships

Juan C. Sanchez-Nivicela!:***, Diana Székely°>’, Luis A. Salagaje M.°, Nicolas Astudillo-Abad?, Jaime Culebras®’, Ernesto Arbeldez Ortiz*®, Paul Székely>®’

1 Universidad Nacional de Colombia, Facultad de Ciencias, Grupo de Investigacion Evolucion y Ecologia de Fauna Neotropical, Bogota, D.C., Colombia

2 Universidad San Francisco de Quito USFQ, Colegio de Ciencias Biolégicas y Ambientales COCIBA, Laboratorio de Zoologia Terrestre y Museo

de Zoologia, Campus Cumbayad, Quito 170901, Ecuador

Instituto Nacional de Biodiversidad del Ecuador, calle Rumipamba 341 y Ay. de los Shyris, Casilla 17-07-8976, Quito, Ecuador

Fundacion y Bioparque AMARU, Cuenca, Ecuador

Museo de Zoologia, Universidad Técnica Particular de Loja, San Cayetano Alto, calle Paris s/n, 110107, Loja, Ecuador

Dn oO fF W

Laboratorio de Ecologia Tropical y Servicios Ecosistémicos (EcoSs-Lab), Facultad de Ciencias Exactas y Naturales, Departamento de Ciencias Biologicas y Agropecuarias, Universidad Técnica Particular de Loja, San Cayetano Alto s/n, 110107, Loja, Ecuador 7 Research Center of the Department of Natural Sciences, Faculty of Natural and Agricultural Sciences, Ovidius University Constanta, Al. Universitdtii no.1, 900470, Constanta, Romania Fundacion Condor Andino, Quito, Ecuador 9 Photo Wildlife Tours, Quito, Ecuador

https://zoobank. org/38565FEC-15BA-4832-8DC5-8 7596343 1C79

Corresponding author: Juan C. Sanchez-Nivicela (juan.sancheznl 3@gmail.com)

Academic editor: Pedro Taucce # Received 27 January 2024 # Accepted 16 July 2024 Published 13 August 2024

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 orig- inating from a single locality (Molleturo, Provincia Azuay, Ecuador, 2317 m elev.). The rediscovery occurred in November 2022 in the Reserva de Conservacion 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 eval- uate the phylogenetic relationships, based on two mitochondrial genes (/2S and /6S rRNA) and one nuclear gene (RAG-/). 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 eco- systems 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 ultimo registro, de dos especimenes de Pristimantis ruidus en aproxima- damente 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 especimenes recolectados en 1922 por George H. Tate, se conocia solo por la serie tipo con todos los especimenes originarios de la misma localidad (Molleturo, Provincia Azuay, Ecuador, 2317 m.). El redescubri- miento ocurrid en noviembre de 2022 en la Reserva de Conservacion Quitahuaycu, parroquia Molleturo, provincia de Azuay. Los dos ejemplares son hembras y se caracterizan por presentar una piel rugosa con numerosas verrugas bayjas y tubérculos redondeados

Copyright Sanchez-Nivicela, J.C. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unre- stricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

1108 Sanchez-Nivicela, J.C. et al.: Rediscovery of Pristimantis ruidus in Ecuador en su textura dérmica dorsal, un pliegue occipital en forma de W, timpano y anillo timpanico no visibles externamente y carecen de crestas craneales, caracteres que concuerdan con el holotipo. Evaluamos relaciones filogenéticas basadas en dos genes mitocon- driales (J2S y 16S rRNA) y un gen nuclear (RAG-/). Estos analisis revelan a Pristimantis ruidus como miembro del subgénero Huicundomantis, estando mas estrechamente relacionado con P. philipi, especie de distribucion restringida que habita el paramo de la misma regidn montafiosa (Macizo del Cajas). Pristimantis ruidus coexiste con Pristimantis jimenezi, tres especies adicionales de Pristimantis y una Noblella con estatus taxonomico incierto. Este redescubrimiento subraya la urgencia de implementar planes efectivos de conservacion y monitoreo para todos los remanentes de los ecosistemas del oeste de Ecuador, que enfrentan amenazas

antropogénicas continuas y albergan una diversidad que aun no esta suficientemente estudiada.

Key Words

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

Palabras clave

Anfibios, biodiversidad, conservacion, filogenia, genética molecular, Huicundomantis, morfologia, nuevo registro, sistematica, taxonomia

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 re- vealed an extraordinary diversity and endemism, but also massive threats (Lynch 1979; Lynch and Duellman 1997; Urgiles et al. 2014, 2017, 2019; Yanez-Mufioz et al. 2016, 2019; Sanchez-Nivicela et al. 2018, 2021; Székely et al. 2018, 2020, 2021; Paez 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 Du- ellman 1997).

In 2022, during an expedition to the Reserva de Con- servaciOn Quitahuaycu, led by Fundacion 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 spec- imens, 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 rela- tionships of P. ruidus.

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Methods

Specimen collection and study site

Fieldwork was carried out at Reserva de Conservacion 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).

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 Division de Herpetologia (DHMECN) of the Instituto Nacional de Biodiversidad (INABIO), Quito, Ecuador and Museo de Zoologia, Uni- versidad Técnica Particular de Loja (MUTPL), Loja, Ec- uador. 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 Mufioz-Saba (2005). Re- search 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 orig- inal description (Lynch 1979) and compared with photo- graphs of the holotype AMNH 17590 (AMNH =American Museum of Natural History). The definition follows the

Zoosyst. Evol. 100 (3) 2024, 1107-1120

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COLOMBIA

O Pristimantis ruidus (Type locality)

[ | Pristimantis ruidus (Rediscovery) Provinces

ELEVATION Valu

e High : 6050

Low : 0

Kilometers 0 25 50 100 150 200

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.

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 HI 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 preci- sion caliper and rounded to the nearest 0.1 mm. The ab- breviations 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 dis- tance to the outer edge of the upper eyelid); IND = Internar- ial 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 Kohler (2012). The photographs were taken by Ernesto Arbelaez, Jaime Culebras and Juan Sanchez.

Molecular analysis

Genomic extraction, amplification and sequencing were done as described in Székely et al. (2020); the six new- ly-generated DNA sequences were deposited in GenBank (Appendix 1). We analysed the molecular data by using se- quences of two mitochondrial genes (/2S and /6S rRNA) and one nuclear gene (RAG-/) from 61 specimens (60 from Ecuador and one from Peru) of 35 species or candidate species available in GenBank, representing all the current- ly 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 (Gunther, 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. colodac- tylus (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 /2S, 16S and RAG-I 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

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method; Katoh and Standley (2013) in Geneious Prime; the /2S, 16S and RAG-/ aligned sequences were visually inspected to correct alignment errors in PhyDE (Miller 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 /2S, 877 bp for /6S and 612 bp for RAG-/); 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 Infor- mation Criterion (BIC) as a model of selection; Partition- Finder identified three partition schemes (best model in parentheses): /2S and /6S (GTR+I+G), RAG-/ 1* posi- tion (K80+G) and RAG-/ 2™ and 3" 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 sum- marised 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 likeli- hood 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 Partition- Finder 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) tn 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 boot- strap (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;

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Sanchez-Nivicela, J.C. et al.: Rediscovery of Pristimantis ruidus in Ecuador

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 tu- bercles); 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 H—IV; toes with lateral fringes, inter- nal toe pads smaller than those of outer fingers (Figs 2—5). 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 tu- bercles 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).

Phylogeny

The Bayesian Inference and the four Maximum Likeli- hood phylogenetic trees showed almost the same topol- ogy, 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 sup- port values), followed by the NNI tree (third run). Similar to Ortega et al. (2022), we recovered the subge- nus 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 & Vene- gas, 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 fur- ther 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 topolo- gy 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 re- vealed as the sister species of P. philipi and both together

Zoosyst. Evol. 100 (3) 2024, 1107-1120

form a basal group in Huicundomantis (Fig. 7). Uncor- rected genetic p-distances for the /6S gene between P. ru- idus and its sister species, P. philipi, range from 4.1% to 5.3% and between them and the other species of the sub- genus Huicundomantis from 4.6% to 11.4%.

Taxonomy

Pristimantis ruidus (Lynch, 1979) Figs 2-6

Common English name: Molleturo Robber Frog Common Spanish name: Cutin 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, Ec- uador, 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, el- evation 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 spec- imens were collected in the Reserva de Conservacion Quitahuaycu, Molleturo Parish, Azuay Province, Ecua- dor, by Luis Salagaje, Ernesto Arbelaez, Jaime Culebras and Nicolas 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 ab- sent; (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 round- ed tubercles; tarsal tubercles rounded; inner tarsal fold present; (11) inner metatarsal tubercle oval, twice the size of round outer metatarsal tubercle; supernumerary plan- tar 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

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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 lack- ing a tympanic membrane and a tympanic annulus, head wider than long, short snout, cranial crest absent, thick supratympanic fold and by the presence of subacuminat- ed 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. phil- ipi) 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 posterodor- sally; canthus rostralis concave; cranial crest absent; one rounded interocular tubercle; upper eyelid with rounded tubercles (three and four each) and few lower: supra- tympanic fold thick and discontinuous; four large sub- acuminated postrictal tubercles; tympanic annulus and membrane absent; few small and subconical maxillary tu- bercles; choana barely visible in the palatal shelf, posteri- orly 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. Dor- sum skin shagreen with numerous low warts; dorsolateral and medial fold absent; flanks shagreen with low warts and scarcely subconical and low tubercles; venter areo- late 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 tu- bercles rounded; fingers with lateral fringes, without in- terdigital 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,

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barely expanded and elliptical in Toes I—V; relative length of Toes I< I< 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, pos- tocular 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 surfac- es, 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-Yel- low (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). Morphomet- ric variation is presented in Table 1.

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 Paez & Ron, 2019 and three additional Pristimantis species and one Noblella Barbour, 1930 species with uncertain taxonomic status.

Conservation status. Currently, the forests on the An- dean foothills in western Ecuador, which include the Mol-

Sanchez-Nivicela, J.C. et al.: Rediscovery of Pristimantis ruidus in Ecuador

leturo 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 Area de Vegetacion y Bosques Protectores (AVBP) Molletu- ro-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 En- vironmental Protection Policy (Politica Ambiental Nacion- al del Ecuador); this makes the entire area highly vulnera- ble to loss and destruction of natural vegetation cover. Various significant threats have been identified in the region, mostly linked to activities of anthropogenic or- igin, such as deforestation for the expansion of grazing areas and the extraction of wood for fire. The situation is ageravated by the presence of numerous mining conces- sions throughout the area, as well as the proliferation of il- legal 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 Conservacién Quitahuaycu comprises approximately 900 hectares (0.6% of the total area of Mol- leturo-Mollepungo AVBP), protecting ecosystems of high montane and montane evergreen forests of the western mountain of the Andean cordillera (0.14%) and the Paramo 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 Miguir, together with the Fundacion y Bioparque AMARU, aim- ing to preserve threatened species of amphibians which are present here, such as Ate/opus nanay Coloma, 2002 (CR), Pristimantis verrucolatus Paez & Ron, 2019 (CR), Pristi- mantis Jimenezi (CR), in addition to species with taxonom- ic uncertainty that are probably new species to science. Pristimantis ruidus has a very low detectability. De- Spite 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 individu- als. We suspect that it only inhabits densely-forested areas with ravines and humid areas, so that the species is restrict- ed to the best conserved parts of the ecosystem, Bosque siempreverde montano de Cordillera Occidental de Los An- des (MAE 2012), between elevations of 2317 and 2677 m. Pristimantis ruidus 1s categorised as “Critically Endan- gered (Possibly Extinct) D” according to a recent assess- ment, 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 ma- ture individuals)’ (IUCN SSC Amphibian Specialist Group 2023). We consider the Critically Endangered status to be

Table 1. Morphological measurements (in mm) of the two newly-collected Pristimantis ruidus specimens and for the holotype as provid- ed 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

DHMECN 19106 S005 19:5 b.s> 10.0 MUTPL 1613 20:1, S1Q2= ‘iG 6.7 ihe AMNH 17590 (Holotype) 38.4 16.3 - 11.4

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SVL TL FL HL HW 10.9

13.4

10D EW IND EN ED Sex State 3.0 2.6 2: 2.6 333 Female adult 2.0 1.8 LZ 1.6 255 Female adult 2.9 30 - S32 4.5 Female adult

Zoosyst. Evol. 100 (3) 2024, 1107-1120 LilS

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 Sanchez.

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 Sanchez.

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1114 Sanchez-Nivicela, J.C. et al.: Rediscovery of Pristimantis ruidus in Ecuador

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 Sanchez.

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 Sanchez.

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Zoosyst. Evol. 100 (3) 2024, 1107-1120 1115

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.

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Sanchez-Nivicela, J.C. et al.: Rediscovery of Pristimantis ruidus in Ecuador

e | KU217863 Pristimantis philipi (Azuay, nearby PN Cajas)

QCAZ37537 Pristimantis philipi (Azuay, PN Cajas) eo DHMECN19106 Pristimantis ruidus (Azuay, Quitahuaycu)

©@! MUTPL1613 Pristimantis ruidus (Azuay, Quitahuaycu) e QCA245720 Pristimantis sp. (Pastaza, Ankaku) ©! QCAZ45945 Pristimantis sp. (Pastaza, Ankaku) QCAZ58855 Pristimantis sp. (Morona Santiago, PN Sangay)

e@ | QCAZ45744 Pristimantis mallii (Pastaza, Ankaku) ®! QCAZ45770 Pristimantis mallii (Pastaza, Ankaku)

GGU807 Pristimantis miktos (Peru)

QCAZ55445 Pristimantis miktos (Orellana, PN Yasuni) e; QCAZ59643 Pristimantis tamia (Pastaza, PN Llanganates) e QCAZ59630 Pristimantis tamia (Pastaza, PN Llanganates)

QCAZ41710 Pristimantis nangaritza (Zamora Chinchipe, Nangaritza) QCA2Z46738 Pristimantis gagliardoi (Cafar, Mazar) QCAZ42575 Pristimantis gagliardoi (Morona Santiago, PN Sangay) QCAZ54857 Pristimantis muscosus (Zamora Chinchipe, Tapichalaca)

KU218052 Pristimantis spinosus (Morona Santiago, Plan de Milagro) e MUTPL168 Pristimantis cryptomelas (Loja, Washapamba) ®_ MUTPL135 Pristimantis cryptomelas (Loja, Abra de Zamora) e, QCAZ47397 Pristimantis torresi (Loja, Celica)

MUTPL996 Pristimantis torresi (Loja, Guachanama) e; MUTPL191 Pristimantis lojanus (Loja, Cristal) ®! MUTPL178 Pristimantis lojanus (Loja, Loja)

QCAZ58463 Pristimantis phoxocephalus (Cotopaxi, Pilal6)

e KU218025 Pristimantis totoroi (Chimborazo, Pallatanga) QCAZ58425 Pristimantis totoroi (Cotopaxi, Pilald)

e QCAZ47213 Pristimantis multicolor (Loja, PN Yacuri)

e; MUTPL810 ©! MUTPL812

®! QCAZ47214 Pristimantis multicolor (Loja, PN Yacuri) e; QCAZ45580 Pristimantis atratus (Loja, Cajanuma) ©! QCAZ45645 Pristimantis atratus (Loja, PN Podocarpus)

Pristimantis percultus (Loja, PN Podocarpus) Pristimantis percultus (Loja, PN Podocarpus)

e MUTPL524 Pristimantis chomskyi (Loja, Cerro Toledo)

QCAZ45666 Pristimantis chomskyi (Zamora Chinchipe, Tapichalaca)

MUTPL359 Pristimantis andinogigas (Loja, PN Podocarpus)

QCAZ45178 Pristimantis jimenezi (Azuay, San Antonio) QCAZ46978 Pristimantis jimenezi (Azuay, Molleturo)

e@, QCAZ53728 Pristimantis lutzae (Azuay, PN Cajas) ®L QCAZ32785 Pristimantis lutzae (Azuay, Yanuncay-Irquis)

QCAZ45129 Pristimantis sp. (El Oro, Chillacocha) MUTPL223 Pristimantis gloria (Loja, Urdaneta)

She QCAZ16448 Pristimantis gloria (Azuay, Gualaceo-Macas) KU218035 Pristimantis gloria (Azuay, Gualaceo-Limon)

e,; MUTPL180 Pristimantis balionotus (Loja, Madrigal)

©! MUTPL392 Pristimantis balionotus (Loja, Abra de Zamora)

ML - Maximum Likelihood

BI - Bayesian inference QCAz46982

SH-aLRT 2 80 | UFBoot 2 95 | Boot > 75

| PP > 0.95

QCAZ26642 Pristimantis sp. (Azuay, San Antonio)

e QCAZ58042 Pristimantis hampatusami (El Oro, Buenaventura) e

QCAZ58044 Pristimantis hampatusami (El Oro, Buenaventura)

QCAZ32790 Pristimantis sp. (Azuay, Yanuncay-Irquis)

Pristimantis verrucolatus (Azuay, Yumate)

QCAZ46993 Pristimantis verrucolatus (Azuay, Cochapamba) ef QCAZ40582 Pristimantis tinguichaca (Morona Santiago, PN Sangay) ®. QCAZ31945 Pristimantis tinguichaca (Morona Santiago, PN Sangay) QCAZ53999 Pristimantis sp. (Zamora Chinchipe, Romerillos) e MUTPL494 Pristimantis versicolor (Loja, Madrigal)

®L KU218096 Pristimantis versicolor (Loja, Abra de Zamora)

e, QCA2Z42488 Pristimantis atillo (Morona Santiago, PN Sangay)

®! QCAZ42498 Pristimantis atillo (Morona Santiago, PN Sangay)

0.03 le

QCAZ46213 Pristimantis teslai (Tungurahua, Llanganatillo) QCAZ45029 Pristimantis sp. (Morona Santiago, PN Sangay)

Figure 7. Bayesian phylogenetic tree of the Huicundomantis subgenus of Pristimantis. The analysis is based on 2413 base-pairs of concatenated mitochondrial DNA from /2S and /6S and nuclear DNA RAG-/ gene fragments. Outgroup is not shown. The cata- logue 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.

accurate, as the Extent of Occurrence (EOO) is less than 20 km?, the Area of Occupancy (AOO) is less than 10 km/?, 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 Di- agnosis and Description sections from Lynch (1979), as well as characteristics that could have been lost due to the

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preservation of the original specimens and which are easily recognisable by examining the living specimens and recent- ly 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

Zoosyst. Evol. 100 (3) 2024, 1107-1120

Figure 8. Above: Panoramic view of Reserva de Conservacion Quitahuaycu (Amaru Foundation). Below: Montane evergreen for- est habitat of Pristimantis ruidus. Photographs: Ernesto Arbelaez.

specimens and the holotype photographs in concordance with Diagnosis (Lynch 1979) but differs from the Descrip- tion in the same publication and is misidentified as relative- ly prominent. In Lynch and Duellman (1997), the authors mentioned that Toe V was much longer than Toe III; how- ever, when examining the holotype photographs, we can ob- serve 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 IT) (Fig. 6). According to Lynch (1979), AMNH 17597 (SVL = 29.0 mm) is not a mature fe- male, having moderate convolutions and small eggs. How- ever, 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. De- spite 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 protec- tion 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

Lely,

might be because, in the past, most searches for amphibi- ans 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 (Paez 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 Ate/opus nanay, Atelopus onorei Coloma, Lotters, Duellman & Miran- da-Leiva, 2007, P. erythros Sanchez-Nivicela, Celi-Pie- dra, Posse-Sarmiento, Urgilés, Yanez-Mufioz & Cisner- os-Heredia, 2018, P. philipi and P. ruidus (Lynch 1979; Lynch and Duellman 1997; Coloma 2002; Coloma et al. 2007; Sanchez-Nivicela et al. 2018).

Our comprehensive description of the morphological characters of P. ruidus, along with the additional informa- tion 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 Fundacion Condor Andino for being a part of the field exploration team. Additionally, we extend our thanks to the friends and associates from Fundacion 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 “Descripcion de nuevas especies de anfibios y reptiles del sur de Ecuador a partir de las colecciones del Museo de Zoologia, UTPL” (PROY_INV_CCBIO_2020 2794), Rainforest Trust and Naturaleza y Cultura Internacional through the “Investigacion cientifica, para la proteccién y monitoreo de especies de anfibios en el Area 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 Fabian Rodas from Naturaleza y Cultura Internacional were essential to promote conservation and biodiversity research of Reserva de Conservacion Quitahuaycu.

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Urgiles VL, Székely P, Székely D, Christodoulides N, Sanchez-Nivicela JC, Savage AE (2019) Genetic delimitation of Pristimantis orestes (Lynch, 1979) and P. saturninoi Brito et al., 2017 and description of two new terrestrial frogs from the Pristimantis orestes species group (Anura, Strabomantidae). ZooKeys 864: 111-146. https://doi. org/10.3897/zookeys.864.35102

Yanez-Mufioz MH, Sanchez-Nivicela JC, Reyes-Puig C (2016) Tres nuevas especies de ranas terrestres Pristimantis (Anura, Craugasto- ridae) de la Provincia de El Oro, Ecuador. ACI Avances en Ciencias e Ingenierias 8(1): 5—25. https://doi.org/10.18272/aci.v8114.455

Yanez-Mufioz MH, Veintimilla-Yanez D, Batallas D, Cisneros-Heredia DF (2019) A new giant Pristimantis (Anura, Craugastoridae) from the paramos of the Podocarpus National Park, southern Ecuador. ZooKeys 852: 137-156. https://doi.org/10.3897/zookeys.852.24557

Table Al. 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

Pristimantis andinogigas Pristimantis atillo

Pristimantis atillo Pristimantis atratus Pristimantis atratus Pristimantis balionotus Pristimantis balionotus Pristimantis ceuthospilus Pristimantis Chomsky! Pristimantis chomsky! Pristimantis colodactylus Pristimantis cryptomelas Pristimantis cryptomelas Pristimantis diadematus Pristimantis gagliardoi

Pristimantis gagliardoi Pristimantis galdi Pristimantis gloria

Pristimantis gloria Pristimantis gloria Pristimantis hampatusami Pristimantis hampatusami

Voucher number MUTPL359 QCAZ42488

QCAZ42498 QCAZ45580 QCAZ45645 MUTPL180 MUTPL392 KU212216 MUTPL524 QCAZ45666 MUTPL388 MUTPL135 MUTPL168 KU221999 QCAZ42575

QCAZ46738 QCAZ32368 KU218035

MUTPL223 QCAZ16448 QCAZ58042 QCAZ58044

GenBank accession no.

12S MT 764339

MT778069 MT778071 EF493520 MZ678943

MT778072 MT778073 MT778075 EU186668

EU186670 EF493348

MT778079

16S MT 756022 MK881440

MK881444 MK881471 MK881473 MT 756023 MT 756025 EF493520 MZ678934 MK881476 MT 762200 MT 756026 MT 756028 EU186668 MK881456

MK881480 EU186670 EF493348

MT 756032 MK881402 MK881504 KX525478

Locality RAGI MT810305 Ecuador: Loja, Parque Nacional Podocarpus, Cajanuma MK881340 — Ecuador: Morona Santiago, Parque Nacional Sangay, Lagunas de Atillo

MK881344 Ecuador: Morona Santiago, Parque Nacional Sangay MK881364 Ecuador: Loja, Cajanuma MK881366 Ecuador: Loja, Parque Nacional Podocarpus, Cajanuma MT810306 Ecuador: Loja, Reserva Madrigal del Podocarpus MT810308 Ecuador: Loja, Abra de Zamora

- Peru: Cajamarca, Chota, 12 km W Llama MZ700220 Ecuador: Loja, Parque Nacional Podocarpus, Cerro Toledo MK881369 Ecuador: Zamora Chinchipe, Reserva Tapichalaca MT810310 Loja, Abra de Zamora MT810311 Ecuador: Loja, Abra de Zamora MT810313 Ecuador: Loja, Bosque Protector Washapamba

- Peru: Loreto, Teniente Lopez MK881355 — Ecuador: Morona Santiago, Parque Nacional Sangay,

Ranger Station, Tinguichaca river

MK881372 Ecuador: Canar, Reserva Mazar EU186746 Ecuador: Zamora Chinchipe, El Pangui

- Ecuador: Azuay, 8.1 km W Morona Santiago border,

Gualaceo-Limon road

MT810317 Ecuador: Loja, 21 km E Urdaneta MK881316 Ecuador: Azuay, Gualaceo-Macas road MK881387 Ecuador: El Oro, Reserva Buenaventura KX525472 Ecuador: El Oro, Reserva Buenaventura

zse.pensoft.net

1120

Species Pristimantis imitatrix Pristimantis Jimenezi

Pristimantis Jimenezi Pristimantis lojanus Pristimantis lojanus Pristimantis lutzae

Pristimantis lutzae

Pristimantis mallii

Pristimantis melanogaster MHNSM56846

Pristimantis mallii Pristimantis miktos Pristimantis miktos Pristimantis multicolor Pristimantis multicolor Pristimantis Muscosus Pristimantis nangaritza

Pristimantis orcesi Pristimantis orestes Pristimantis percultus Pristimantis percultus Pristimantis philipi

Pristimantis philipi Pristimantis phoxocephalus Pristimantis rhodoplichus Pristimantis ruidus Pristimantis ruidus Pristimantis simonsil

Pristimantis spinosus Pristimantis tamia

Pristimantis tamia Pristimantis teslal Pristimantis tinguichaca Pristimantis tinguichaca

Pristimantis torres! Pristimantis torres! Pristimantis totoroi Pristimantis totoroi Pristimantis unistrigatus Pristimantis verrucolatus Pristimantis verrucolatus Pristimantis versicolor Pristimantis versicolor Pristimantis wiensi Pristimantis sp. Pristimantis sp.

Pristimantis sp.

Pristimantis sp. Pristimantis sp. Pristimantis sp. Pristimantis sp. Pristimantis sp.

zse.pensoft.net

Voucher number

KU215476 QCAZ45178

QCAZ46978 MUTPL178 MUTPL191

QCAZ32785

QCAZ53728

QCAZ45744

QCAZ45770 GGU807 QCAZ55445 QCAZ47213 QCAZ47214 QCAZ54857 QCAZ41710

KU218021 MUTPL242 MUTPL810 MUTPL812 KU217863

QCAZ37537 QCAZ58463

KU219788

DHMECN19106

MUTPL1613 KU212350

KU218052 QCAZ59630

QCAZ59643 QCAZ46213 QCAZ31945 QCAZ40582

MUTPL996 QCAZ47397 KU218025 QCAZ58425 KU218057 QCAZ46982 QCAZ46993 KU218096 MUTPL494 KU219796 QCAZ26642 QCAZ32790

QCAZ45029

QCAZ45129 QCAZ45720 QCAZ45945 QCAZ53999 QCAZ58855

GenBank accession no.

12S EF493824

MZ678945 MZ678946

MZ330729 EF493826

MZ330730

EF493679 MT778087 MT778088 MT778089 EF493672

EF493674

16S EF493667

MK881468

MK881482 MZ678936 MZ678937 MK881421

MK881495

MZ241492 EF493664

MZ241496 KP064151 MZ241510 MK881488 MK881489 MK881501 MK881436

EF493679 MK604538 MT756034 MT756035 EF493672

MK881426 MK881507

EF493674

RAG1

MK881362

MK881374 MZ700222 MZ700223 MK881326

MZ332932 MK391383

MK881386 MK881336

MK602185 MT810325 MT810326

MK881331 MK881390

PP725379 PP723736 PP731015 PP725380 PP723737 PP731016

EU186665

EF493673 MZ330735

MZ330736

MZ678947 EF493349 EF493387 EF493389

MT778095 EF493377

EU186665

EF493673 MZ241526

MZ241528 MK881478 MK881418 MK881433

MZ678938 MK881492 EF493349 MK881505 EF493387 MK881483 MK881485 EF493389 MT756038 EF493668 MK881409 MK881423

MK881461

MK881462 MZ241490 MZ241502 MK881496 MZ241514

MZ332958 MZ332960

MK881323 MK881334

MZ700224 MK881380

MK881388 EF493444 MK881375 MK881377 EF493431 MT810332

MK881328

MK881358 MZ332927 MZ332938

MZ332949

Sanchez-Nivicela, J.C. et al.: Rediscovery of Pristimantis ruidus in Ecuador

Locality

Peru: Madre de Dios, Cuzco Amazonico, 15 km E Puerto Maldonado Ecuador: Azuay, San Antonio, Parque Nacional Cajas border Ecuador: Azuay, Molleturo, Zadracay river Ecuador: Loja, Loja, Quebrada San Simon Ecuador: Loja, Cristal Ecuador: Azuay, Bosque Protector Yanuncay-Irquis, Paramo de Quimsacocha Ecuador: Azuay, Parque Nacional Cajas, El Capo, Laguna Toreadora Ecuador: Pastaza, Reserva Comunitaria Ankaku Peru: Amazonas, N. Slobe Abra Barro Negro, 28 km SSW Leimebambe Ecuador: Pastaza, Reserva Comunitaria Ankaku Peru: Loreto, Nanay, Lote 123

Ecuador: Orellana, Parque Nacional Yasuni, Tambococha

Ecuador: Loja, Parque Nacional Yacuri, Laguna Negra

Ecuador: Zamora Chinchipe, Reserva Tapichalaca

Ecuador: Zamora Chinchipe, Alto Nangaritza PF, Las Orquideas, Tepuy forest

Ecuador: Pichincha, Bosque de Pasochoa Ecuador: Loja, 11 km NE Urdaneta Ecuador: Loja, Parque Nacional Podocarpus, Cajanuma Ecuador: Loja, Parque Nacional Podocarpus, Cajanuma Ecuador: Azuay, 4 km W Laguna Toreadora, nearby Parque Nacional Cajas Ecuador: Azuay, Parque Nacional Cajas Ecuador: Cotopaxi, Pilald surroundings

Peru: Piura, El Tambo Ecuador: Azuay, Quitahuaycu reserve Ecuador: Azuay, Quitahuaycu reserve Peru: Cajamarca, S slope Abra Quilsh, 28 km NNW Cajamarca

Ecuador: Morona Santiago, 10.6 km W Plan de Milagro

Ecuador: Pastaza, Parque Nacional Llanganates, Comunidad Zarentza

Ecuador: Tungurahua, Llanganatillo, Parque Nacional Llanganates border Ecuador: Morona Santiago, Parque Nacional Sangay, San Vicente Ecuador: Morona Santiago, Parque Nacional Sangay, Lagunas de Atillo

Ecuador: Loja, Guachanama, El Apretadero Ecuador: Loja, Celica-Alamor road Ecuador: Chimborazo, 70 km W Riobamba via Pallatanga Ecuador: Cotopaxi, Pilald surroundings Ecuador: Imbabura, 35 km E Pquela Ecuador: Azuay, Yumate, Shoupshe Ecuador: Azuay, Cochapamba Ecuador: Loja, Abra de Zamora Ecuador: Loja, Reserva Madrigal del Podocarpus Peru: Piura, 12.7 km E Canchaque Ecuador: Azuay, San Antonio de Chaucha

Ecuador: Azuay, Bosque Protector Yanuncay-Irquis, Paramo de Quimsacocha

Ecuador: Morona Santiago, Parque Nacional Sangay, Etén, Rio Culebrillas Ecuador: El Oro, Chillacocha Ecuador: Pastaza, Reserva Comunitaria Ankaku Ecuador: Pastaza, Reserva Comunitaria Ankaku Ecuador: Zamora Chinchipe, Yacuambi, Romerillos Ecuador: Morona Santiago, Parque Nacional Sangay,

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