A peer-reviewed open-access journal Zookeys 606: 77-97 (2016) RRS a! #ZooKeys http:/ /Zz00 keys -pen soft.net Launched to accelerate biodiversity research A new species of Pristimantis from southern Ecuador (Anura, Craugastoridae) 1,2,4 Paul Székely!”, Dan Cogalniceanu'?, Diana Székely!%, Nadia Pdez°*, Santiago R. Ron? | Ovidius University Constanta, Faculty of Natural and Agricultural Sciences, Al. Universitatii, nr. 1, corp B, 900470, Constanta, Romania 2 Universidad Técnica Particular de Loja, Departamento de Ciencias Naturales, San Cayetano Alto, calle Marcelino Champagnat s/n, Loja, Ecuador 3 Universidad Nacional de Loja, CI- TIAB, Ciudadela Universitaria, La Argelia, EC 110101, Loja, Ecuador 4 Laboratory of Fish and Amphibian Ethology, Behavioural Biology Unit, University of Liege, 22 Quai van Beneden, 4020, Liége, Belgium § Museo de Zoologia, Departamento de Ciencias Bioldégicas, Pontificia Universidad Catélica del Ecuador, Avenida 12 de Octubre 1076 y Roca, Apartado 17-01-2184, Quito, Ecuador Corresponding author: Dan Cogdalniceanu (dcogalniceanu@uniy-ovidius.ro) Academic editor: E Andreone | Received 9 May 2016 | Accepted 7 July 2016 | Published 21 July 2016 http://zoobank. org/C7ED1BFE-9310-4279-9021-56D94AAOIACT Citation: Székely P, Cogalniceanu D, Székely D, Paez N, Ron SR (2016) A new species of Pristimantis from southern Ecuador (Anura, Craugastoridae). ZooKeys 606: 77-97. doi: 10.3897/zookeys.606.9121 Abstract A new species of Pristimantis is described from Reserva Buenaventura, southern Ecuador, at elevations between 878 and 1082 m. A molecular phylogeny based on nuclear and mitochondrial genes shows that the new species is closely related to P phoxocephalus, P riveti, and P versicolor. The new species differs from them and other morphologically similar congeners in having a low W-shaped dermal ridge in the scapular region, a large conical tubercle on the upper eyelid and on the heel, a thin mid dorsal fold, and a longitudinal lateral fold starting behind the tympanic fold and extending along the anterior two thirds of the flank. The new species inhabits cloud forests in the Pacific slopes of the Andes. Copyright Paul Székely et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 78 Paul Székely et al. / ZooKeys 606: 77—97 (2016) Resumen Describimos una nueva especie de Pristimantis de la Reserva Buenaventura, al sur del Ecuador, entre eleva- ciones de 878 y 1082 m. Una filogenia molecular basada en genes nucleares y mitocondriales revela que la nueva especie esta cercanamente relacionada a P phoxocephalus, P. riveti y P. versicolor. La nueva especie difiere de ellas y otros congéneres morfoldgicamente similares por presentar un pliegue bajo en forma de “W” en la regién escapular, un tubérculo cénico sobre el parpado y en el talén, un delgado pliegue medi- odorsal y un pliegue lateral longitudinal que se inicia detras del pliegue timpanico y se extiende a lo largo de dos tercios del flanco. La nueva especie vive en bosques nublados de las estribaciones pacificas de los Andes. Keywords Anura, Craugastoridae, Pristimantis prometeii sp. n., Reserva Buenaventura Introduction The Neotropics have the highest amphibian species diversity in the world, housing almost half the number of known species (Bolafos et al. 2008). This high species diver- sity is almost entirely endemic, with 96% occurring only in the Neotropics (Bolafos et al. 2008). Our knowledge of Neotropical amphibians is mediocre at best (Duellman 1999), with nearly one-quarter of all known species described over the last decade and 150 to 200 new species described yearly (Catenazzi 2015). A large proportion of South American frogs belong to Terrarana, a clade of direct developing frogs (Hedges et al. 2008; Heinicke et al. 2009) or otherwise known as the superfamily Brachycephaloidea (Padial et al. 2014; Frost 2016). Their eggs are deposited in terrestrial habitats and the embryos develop directly into froglets, bypassing the tadpole stage. Currently this large group that contains more than 1000 species, consists of three families, Brachycephali- dae, Craugastoridae and Eleutherodactylidae. Most craugastorids belong to Pristimantis Jiménez de la Espada 1870), the most speciose genus among terrestrial vertebrates with 494 species (Duellman 1993; Hedges et al. 2008; Frost 2016). Members of this genus, commonly called rain frogs, robber frogs or dirt frogs, are largely restricted to moist, forested habitats in the Andes of Colombia, Ec- uador and Peru (Lynch and Duellman 1997; Frost 2016). The taxonomy of these species is challenging because of their high cryptic diversity, intraspecific variation, and the scant morphological characters available to diagnose species (Duellman and Lehr 2009). De- spite recent reviews (e.g. Hedges et al. 2008; Padial et al. 2014) the phylogenetic afhnities of most species are unknown and numerous new species are discovered and described each year. During the last decade, 125 new species of Pristimantis have been described, 30% of which occur in Ecuador (AmphibiaWeb 2016). Just in the past several months, nine new Pristimantis species were described from Ecuador (Hutter and Guayasamin 2015; Reyes- Puig et al. 2015; Arteaga et al. 2016; Brito et al. 2016; Navarrete et al. 2016) with prob- ably many more awaiting descriptions. Herein we describe a new species of Pristimantis from Reserva Buenaventura, El Oro province, southern Ecuador. A new species of Pristimantis from southern Ecuador (Anura, Craugastoridae) is) Materials and methods Specimen collection Field work was carried out between July and September in 2014 and March, April, and July to September in 2015 at several sites in Reserva Buenaventura. ‘The reserve is private and belongs to the Jocotoco Conservation Foundation. The protected area has an altitudinal range between 400 and 1200 ma.s.l. and occurs in a transition zone between Deciduous Costa Forest and Western Montane Forest (sensu Ron et al. 2016). We made intensive visual encounter surveys, auditory surveys and leaf litter searches during evenings (18h00—01h00) and also daytime searches in bromeliads. Collected specimens were photographed alive and euthanized using 20% benzocaine, fixed in 10% formalin, and stored in 70% ethanol. Tissue samples that were used for genetic analyses were preserved in 96% ethanol. Examined specimens (listed in the type-series and Appendix I) are housed in Museo de Zoologia de la Pontificia Universidad Catdli- ca del Ecuador (QCAZ). Morphology For the description of qualitative and quantitative morphological characters Du- ellman and Lehr (2009) was followed. Sex was determined by the presence of vo- cal slits and/or by gonadal inspection. Color data in life were based on field notes and digital photos. The capitalized colors and their corresponding color codes (in parentheses) used in the color in life descriptions follow Kohler (2012). Measure- ments were taken with a digital caliper and rounded to the nearest 0.1 mm. All well-preserved specimens were measured for the following morphometric variables: (1) snout-vent length (SVL), distance from tip snout to posterior margin of vent; (2) head width (HW), greatest width of head measured at level of jaw articulation; (3) head length (HL), distance from the tip of snout to posterior angle of jaw articula- tion; (4) interorbital distance (IOD), distance between the inner margins of the orbits; (5) internarial distance (IND), distance between the inner edges of the narial openings; (6) upper eyelid width (EW), the perpendicular distance to the outer edge of the eyelid; (7) eye diameter (ED), distance between anterior and posterior borders of eye; (8) eye-nostril distance (EN), distance from posterior margin of nostril to anterior margin of eye; (9) tympanum diameter (TD), horizontal distance between peripheral borders of tympanic annulus; (10) femur length (FL), length of femur from vent to knee; (11) tibia length (TL), length of flexed leg from knee to heel; (12) foot length (FoL), distance from proximal margin of inner metatarsal tubercle to tip of Toe IV; (13) hand length (HaL), distance from proximal edge of palmar tubercle to the tip of Finger III. 80 Paul Székely et al. / ZooKeys 606: 77-97 (2016) DNA extraction amplification and sequencing DNA was extracted from muscle or liver tissue preserved in 96% ethanol or tissue storage buffer, using standard phenol—chloroform extraction protocols (Sambrook et al. 1989). We used a polymerase chain reaction (PCR) to amplify DNA fragments for the mitochondrial gene 16S rRNA (16S) and the nuclear gene RAG-1, using primers listed in Goebel et al. (1999), Moen and Wiens (2009) and Wiens et al. (2005). PCR amplification was performed under standard protocols and sequenced by the Mac- rogen Sequencing Team (Macrogen Inc., Seoul, Korea). The newly generated DNA sequences are available on GenBank (Table 1). We also included 12S, 16S and RAG-1 sequences from GenBank. To optimize taxon sampling within Pristimantis we carried out a preliminary phylogenetic analysis including all available sequences from Gen- Bank. These analyses showed that the new species was closely related to P phoxocepha- lus. Therefore, P phoxocephalus and closely related species (based on Padial et al. 2014) are included as well as representative species of all major clades within Pristimantis. As outgroup we included sequences of Diasporus, Eleutherodactylus, Holoaden, Hypodacty- lus, Ischnocnema, Lynchius, and Strabomantis. The combined DNA matrix had up to 2914 bp. Preliminary sequence alignment was done with MAFFT 7.2 software with the L-INS-i algorithm (Katoh and Standley 2013). The matrix was partitioned to allow independent inferences of models of evolu- tion by gene and by codon position in coding genes. We used software PartitionFinder v. 1.1.1 (Lanfear et al. 2012) to simultaneously estimate both the best-fit model for each partition and the best partition strategy for our data. We defined five a priori par- titions (12S, 16S, first, second and third codon position of RAG1). The best partition strategy was selected using the Akaike information criterion (AIC). Phylogenetic analysis and genetic distances Phylogenetic trees were obtained using maximum likelihood searches with software GARLI 2.0 (Zwickl 2006). We made two independent searches with 10 replicates each. The first search started with random trees and the second with stepwise addition trees. We increased the setting “genthreshfortopoterm” until all 10 searches resulted in similar likelihood values, indicating an efficient search. The final setting of “genthresh- fortopoterm” was 100,000. Other settings were set to default values. Node support was assessed with 200 pseudoreplicate non-parametric bootstraps (npb; Felsenstein 1985), starting from random trees configured with the same settings of the full search, but with one replicate per run. Uncorrected p-genetic distances were estimated with software Mesquite 2.75 excluding ambiguous sites and gaps (Maddison and Maddison 2011). A new species of Pristimantis from southern Ecuador (Anura, Craugastoridae) 81 Table |. Voucher and GenBank accession numbers for specimens used in the phylogenetic analysis. Voucher number 12S USNM314179 Eleutherodactylus caribe | EF493385 - - USNM327822__| Elewtherodactylus pantoni | EF493616 |= : MZUSP131872 Holoaden luederwaldti EU186710 - KU178258 | Hypodactylus brunneus_| GQ345248 | = : Chere ied LeBos99 | 2 Ischnocnema hoehnei EF493359 : USNM318165 | ___schnocnema holti__| EU186722 | = : KU181408 Lynchius nebulanastes EU186704 - KU212327 | _ Oreobates saxatilis | EU186708 | = KU217786 Pristimantis acerus EF493678 EF493678 AJC0573 JN991420 JN991485 KU215460 Pristimantis altamazonicus | EF493670 EF493441 EF493670 KU177637 ee EF493524 EF493524 appendiculatus KU291638 Pristimantis bipunctatus | EF493702 EF493430 EF493702 KU291702 | Pristimantis bromeliaceus | EF493351 | = ——|_—_—CEFA493351 KU217857 Pristimantis condor EF493701 EF493443 EF493701 KU177733 |__Pristimantis crucifer | EU186718 |___—- Ss |_~—_—EU186736 QCAZ48309 KX525474 KX525470 - KU179090 Pristimantis dissimulatus | EF493522 - EF493522 KU217998 | Pristimantis duellmani_|___- __|__EF493438__| NRPS0055 JN991445 JQ025182 JN991509 NRPS0009 Pristimantis gaigei JN991449 JQ025186 JN991513 KU218002 |_Pristimantis glandulosus_| EF493676 | = SEF 493676 KU218227 Pristimantis leoni EF493684 EF493433 EF493684 MTD45080____|_Pristimantis cf. mendax_| EU186659 |__—- ~—|_——CEU186659 aici INS AJC1860 Pristimantis moro JN991454 JQ025191 JN991520 NRPS0048 JN991522 KU222023 Pristimantis ockendeni EF493519 EF493434 EF493519 KU218021 |__Pristimantis orcesi___|_EF493679 | __—- Ss |_—_—CEF493679 KU218025 Pristimantis phoxocephalus | EF493349 - EF493349 AJC0594 JN991528 cA 08 i QCAZ58042 Pristimantis prometeii KX525476 KX525471 - QCAZ58043 82 Paul Székely et al. / ZooKeys 606: 77-97 (2016) Voucher number 12S QCAZ58044 KX525478 KX525472 : KU218028 Pristimantis pycnodermis | EF493680 | ——- ~—s—s«s~Ss«EF 493680 KU218035 Pristimantis riveti EF493348 - EF493348 KU291635 Pristimantis sagittulus EF493705 EF493439 EF493705 NRPS0085 Pristimantis savagei JN991467 JQ025205 JN991536 KU212220 EF493681 | | EF493681 KU218052 EF493673 | | EBFA93673 KU291659 Pristimantis stictogaster | EF493704 EF493445 EF493704 KU218147 Pristimantis subsigillatus | EF493525 - EF493525 NRPS0067 Pristimantis aff. taeniatus | JN991429 JQ025171 JN991493 NRPS0001 JN991430 JQ025172 JN991494 USNM336098 Pristimantis urichi EF493699 EF493426 EF493699 KU218096 Pristimantis versicolor EF493389 EF493431 EF493389 KU218116 Pristimantis walkeri EF493518 EF493428 EF493518 ROM43978 Eu186678 | - | EU186678 NRPS0060 JN991479 jn JN991549 CVULA7073 Strabomantis biporcatus | GQ345249 - SIUC7062 Strabomantis bufoniformis | DQ283165 - - Results Phylogeny The best partitioning scheme consisted of three partitions with their models of evolu- tion in parenthesis: 12S and 16S (GTR + 1+ G), RAG 1* and 2™ position (HKY + G), and RAG 3" position (IrNef + G). The phylogeny shows that the new species is most closely related to P versicolor, P riveti, PR phoxocephalus, and P. spinosus (Fig. 1). This strongly supported clade is distributed in the Andes of northern Peru and central and southern Ecuador. Uncorrected p-genetic distances for the gene 16S between the new species and its closest relative, P versicolor, range from 0.074 to 0.103. Distances with P phoxocephalus, P riveti, and P. spinosus range from 0.075 and 0.130. These large genetic distances and its morphological distinctiveness, clearly demonstrate P prometeii sp. n. is in fact undescribed. We describe it below. Taxonomy Pristimantis prometeii sp. n. http://zoobank.org/EFAA799F-ODE2-4EE2-BDD3-3F22A5B648AB Common names. English: Prometeo Rain Frog. Spanish: Cutin Prometeo Holotype (Figs 2-4). QCAZ 58044 (field no. SC-PUCE 47291), an adult female from Ecuador, Provincia El Oro, canton Pifias, Reserva Buenaventura, on the reserve’s A new species of Pristimantis from southern Ecuador (Anura, Craugastoridae) 83 NRPS0055 Pristimantis erythropleura Vereda La Esperanza CO T7 > KU217830 P. actites Pilalo EC 100 —— KU217998 P. duelimani 27 km E Maldonado EC USNM336098 P. urichi Arima Valley TT MHNSM9267 P. peruvianus PE 100 KU291659 P. stictogaster Oxapampa PE lL KU291635 P. sagittulus Oxapampa PE NRPS0085 P. savagei Villavicencio-Restrepo CO 100 ee P. nervicus Parque Nacional Chingaza CO NRPS0009 P. gaigei La Guayana CO KU291638 P. bipunctatus Oxapampa PE 100 — KU217857 P. condor 4.6 km N Gualaquiza EC 100 AJC0573 P. achatinus Cana PA - KU218227 P. leoni Tulcan EC KU217877 QCAZ48309 P. curtipes Mojanda EC KU215460 P. altamazonicus Cuzco Amazénico PE AJC0594 P. pirrensis Cana PA KU218116 P. walkeri 5 km W La Florida EC KU222023 P. ockendeni 1.5 km N Teniente Lépez PE NRPSOO60 P. zophus Reserva Alto de San Miguel CO NRPS0067 P-. aff. taeniatus Alto de San Miguel CO NRPS0001 P. aff. taeniatus Reserva Alto de San Miguel CO KU218052 P. spinosus 10.6 km W Plan de Milagro EC KU218025 P. phoxocephalus 70km W Riobamba via Pallatanga EC KU218035 P. riveti 8.1 kmW Morona Santiago border EC KU218096 P. versicolor Zamora Chinchipe 1.7 km E Loja border EC QCAZ58042 P. prometeii Buenaventura EC 100 | QCAZ58040 P. prometeii Buenaventura EC {0 Q CAZ58043 P. prometeii Buenaventura EC CAZ58044 P. prometeii Buenaventura EC KU217786 P. acerus 6.8 km E Papallacta EC KU218015 P. inusitatus 31 km N Jondachi EC KU218002 P. glandulosus 2.7 km W Cuyuja EC KU218021 P. orcesi Bosque de Pasochoa EC KU218028 P. pycnodermis Gualaceo Limén EC KU177637 P. appendiculatus Quebradas Zapadores EC KU179090 P. dissimulatus Quebrada Zapadores EC KU177658 P. calcarulatus Tandapi EC KU177733 P. crucifer Tandapi EC KU218147 P. subsigillatus 11.2 km W Luz Maria EC KU177812 P. nyctophylax Tandapi EC AJC1753 P. moro Chilibre PA \_______ AJC 1860 P. moro Pinogana PA ROM43978 P. zeuctotylus Mount Wokomung GU KU212220 P. schultei Chachapoyas 5 km N Levan PE — KU291702 P. bromeliaceus 2.9 km N 5.5 km E Oxapampa PE 100 — _ MTD45080 P. cf. mendax Aquimarca PE 0.09 Figure |. Maximum likelihood phylogram depicting phylogenetic relationships of Pristimantis prometeii sp. n. Bootstrap support values are shown under each branch. 84 Paul Székely et al. / ZooKeys 606: 77-97 (2016) rg , aa a le = cs a = = a Figure 2. Holotype of Pristimantis prometeii sp. n. in life, QCAZ 58044, adult female, SVL 37.6 mm: A lateral view B dorsal view C ventral view. Sendero del Perico de Orcés (3.6470°S, 79.7565°W; datum WGS84), 878 m above sea level, collected by Dan Cogalniceanu and Paul Székely on 14 September 2014. Paratopotypes. QCAZ 58045 (field no. SC-PUCE 47292), an adult female and QCAZ 58042 (field no. SC-PUCE 47289), an adult male (Fig. 5C, D) collected with the holotype; QCAZ 62540 (field no. SC-PUCE 51624), an adult female (Fig. 5A, B) A new species of Pristimantis from southern Ecuador (Anura, Craugastoridae) 85 Figure 3. Holotype of Pristimantis prometeii sp. n. (QCAZ 58044, adult female) in preservative: A dorsal view B ventral view C head, lateral view D head, dorsal view. and QCAZ 62541 (field no. SC-PUCE 51625), an adult male, same data as the holo- type but collected by Dan Cogalniceanu on 13 September 2015. Paratypes. QCAZ 58056 (field no. SC-PUCE 47353), an adult male and QCAZ 58058 (field no. SC-PUCE 47355), an adult female from Ecuador, Provincia El Oro, canton Pifas, Reserva Buenaventura, close to Finca Ramirez (3.6311°S, 79.7618°W), 1082 m above sea level, collected by Dan Cogalniceanu on 7 September 2014; QCAZ 62547 (field no. SC-PUCE 51631), an adult female and QCAZ 62548 (field no. SC- PUCE 51632), an adult male from Ecuador, Provincia El Oro, canton Pifas, Reserva 86 Paul Székely et al. / ZooKeys 606: 77-97 (2016) Figure 4. Hand and feet of the holotype of Pristimantis prometeii sp. n. in life, QCAZ 58044, adult female: A palmar view of hand B plantar view of foot. Buenaventura, Quebrada Oscura (3.6652°S, 79.7417°W), 948 m above sea level, col- lected by Dan Cogalniceanu on 15 September 2015. Additional specimens. Juveniles, QCAZ 58040 (field no. SC-PUCE 47287) (Fig. 5E, F) and QCAZ 58043 (field no. SC-PUCE 47290) with the same collecting data as the holotype. Diagnosis. This species is placed in the genus Pristimantis based on the general morphological similarity to other members of the genus (e.g. characteristic T-shaped terminal phalanges, toes without membranes, and Toe V longer than Toe III) and based on phylogenetic evidence (Fig. 1). Pristimantis prometeii is a medium-sized spe- cies distinguished by the following combination of traits: (1) skin on dorsum shagreen with numerous small tubercles; a low W-shaped ridge in the scapular region, usually with four larger warts on it; skin on venter areolate; discoidal fold weak; thoracic fold present; dorsolateral folds absent but with a longitudinal lateral fold from behind the tympanic fold along the 2/3 of the flank length; low mid dorsal fold with rows of small tubercles, especially on the head; (2) tympanic membrane and tympanic annulus prominent, its length about 40% of the length of eye; supratympanic fold obscuring 87 A new species of Pristimantis from southern Ecuador (Anura, Craugastoridae) peace Treaty Seeks oe reas ater perry ah ran porary cee female paratopotype, QCAZ 62540, ; male paratopotype, QCAZ 58042, SVL 24.9 mm QCAZ 58040, SVL 10.4 mm in life wz Sp. Nn. in istimantis promete f Pr A dorsolateral view B ventral v lation oO Figure 5. Color var SVL 32.6 mm 1eWw E dorsolateral view F ventral juvenile, 1ew; iew D ventral v C dorsolateral v view. i in dorsal inate subacum b upper and posterodorsal edges of tympanum; (3) snout short Ing (4) upper eyelid bd is angular in profile; canthus rostral ightly protrud sl view, rounded bd bearing one larger conical tubercle and numerous small tubercles, about 90% IOD igerous processes of (5) dent b) 1 crests absent cranla b in males females and 85% IOD In ith a subgular vocal sac males w (6) b lar with 3 to 4 teeth , triangu vomers prominent discs on fingers broadly expanded, inger II; inges I shorter than Fi inger (8) fingers bearing broad lateral fr sua 1ts and vocal sl ell into ; (9) ulnar tubercles coalesced 2) ical ipt 88 Paul Székely et al. / ZooKeys 606: 77-97 (2016) low ulnar fold; (10) heel bearing one larger, conical tubercle and several smaller tu- bercles; outer edge of tarsus with row of small, conical tubercles; inner edge of tarsus bearing a low fold; (11) inner metatarsal tubercle broadly ovoid, about 5x ovoid outer metatarsal tubercle; supernumerary plantar tubercles present; (12) toes bearing broad lateral fringes; webbing absent; Toe V much longer than Toe II]; discs elliptical, about same size as those on fingers; (13) in life, dorsum of various shades of brown, with or without white spots, blotches, or dark brown bars or reticulum; flanks cream, yellow, or green; venter cream with dark flecks and blotches; yellow blotches on the groin, anterior, and posterior surfaces of thighs; iris bronze with fine black reticulations and a median, horizontal read streak; (14) SVL 20.4—24.9 mm in adult males (22.4 + 1.86 SD, 2 = 4) and 29.9—37.6 mm in adult females (32.7 + 2.91 SD, 2 = 5). Comparisons with other species. Comparisons are based on molecular evidence to compare Pristimantis prometeii with close relatives and on morphologically similar species present in southern Ecuador and Northern Peru. The phylogenetically closest species are Pristimantis versicolor, P. phoxocephalus and P. riveti (Fig. 1). From these three P. phoxo- cephalus (Lynch 1979) is the most similar. However, it is easily distinguished from P. pro- meteii by a fleshy vertical keel on the snout. Furthermore, P. phoxocephalus lacks tubercles on the upper eyelid, heel and tarsus, and the low W-shaped dermal ridge in the scapular region. Pristimantis riveti (Despax 1911) differs from P. prometeii in having a heel without prominent tubercles (one prominent tubercle in P. prometeii), smaller finger and toe discs, and W-shaped dermal ridge in the scapular region absent. In P. versicolor (Lynch 1979), males lack vocal slits and vocal sacs (both present in P. prometeii), the tarsus lacks distinct tubercles (tubercles present in P. prometeii), lateral fringes are absent in toes (present in P. prometeii), and the dorsum lacks the low W-shaped dermal ridge in the scapular region. Additionally, all these three Pristimantis species inhabit upper humid montane forest and subparamo, habitats at higher elevations than those of P. prometeii: 1800-3100 m, in P. phoxocephalus (Lynch and Duellman 1997), 2.620-3.600 m in P. riveti (Coloma et al. 2004) and 2500-3100 m in P. versicolor (Frenkel et al. 2013). The related Pristimantis spinosus (Lynch 1979) is also easily distinguished by the presence of cranial crests (absent in P. prometeii), males lacking vocal slits and vocal sacs, and the coloration of groins and concealed surfaces of hind limbs which are black with white spots. Among the few morphologically similar congeners from southern Ecuador, Pristi- mantis sternothylax (Duellman and Wild 1993) can be distinguished by lacking promi- nent tubercles on the upper eyelid, having smooth ulnar surfaces, and heel and tarsus lacking tubercles and folds. Pristimantis buenaventura (Arteaga et al. 2016) is some- what similar but it is significantly smaller, and lacks prominent tubercles on the upper eyelid and heel. It also differs by having orange-red spots on the groins. Similar spe- cies in northern Peru include Pristimantis rhodoplichus (Duellman and Wild 1993), P. wiensi (Duellman and Wild 1993), and P. petrobardus (Duellman 1991). Pristimantis rhodoplichus and P. petrobardus differ by lacking prominent tubercles on the upper eye- lid. The lack of tympanic membrane readily distinguishes P. wiensi from P. prometeii. Both species also differ in dorsal coloration: green dorsum with scattered bronze and dark blotches in P. wiensi vs. brown dorsum in P. prometeii. A new species of Pristimantis from southern Ecuador (Anura, Craugastoridae) 89 Description of the holotype. Adult female (Fig. 3) with head slightly narrower than body, wider than long, head length 89% of head width, head width 36% of SVL; head length 32% of SVL; snout short (snout to eye distance 14% of SVL), subacumi- nate in dorsal view, rounded, slightly protruding in profile; canthus rostralis angular; loreal region flat; eye diameter notably greater than eye-nostril distance; nostrils slight- ly protuberant laterally; lips not flared; cranial crests absent; upper eyelid bearing one larger conical tubercle and numerous small tubercles, width of upper eyelid 94% of IOD; tympanic annulus prominent, round, its upper and posterodorsal part obscured by rounded supratympanic fold; tympanic membrane differentiated, visible; diameter of tympanum 41% of the length of eye; one larger and several low postrictal tuber- cles situated posteroventrally to tympanic annulus; choanae big, oval, not concealed by palatal shelf of maxillary; vomerine odontophores prominent, triangular, about 3x size of choana, separated medially by distance lower than width of odontophore; each otontophore has 3 to 4 teeth; tongue longer than wide, bilobate, posterior half not adherent to floor of mouth. Skin on dorsum shagreen with numerous small tubercles; a low W-shaped dermal ridge is present in the scapular region, with 4 larger warts defining its corners (this trait is more visible in life, Fig. 2); thin, low mid dorsal fold starting at tip of snout and ending at cloaca, with rows of small tubercles, especially on the head (trait more visible in life, Fig. 2); dorsolateral folds absent; longitudinal lateral fold from behind the tym- panic fold along the 2/3 of the flank length (trait more visible in life, Fig. 2); skin on throat, chest, belly, and ventral surfaces of thighs areolate; discoidal fold weak; thoracic fold present (trait more visible in life, Fig. 2); ornamentation in cloacal region absent. Ulnar tubercles present, coalescing into low ulnar fold; outer palmar tubercle par- tially divided distally; thenar tubercle ovoid; subarticular tubercles prominent, round; supernumerary palmar tubercles rounded, smaller than subarticular tubercles; fingers bearing broad lateral fringes; Finger I shorter than Finger II; discs on fingers broadly expanded, elliptical; all fingers bearing pads well defined by circumferential grooves (Fig. 4). Hind limbs moderately robust; tibia length 46.5% of SVL; foot length 40.7% of SVL; heel bearing one larger, conical tubercle and several smaller tubercles; outer edge of tarsus with row of small, conical tubercles; inner edge of tarsus bearing a low fold; inner metatarsal tubercle broadly ovoid, about 5x ovoid outer metatarsal tubercle; subarticular tubercles prominent, round; plantar supernumerary tubercles rounded, smaller than subarticular tubercles; toes bearing broad lateral fringes; webbing absent; discs on toes elliptical, about same size as those on fingers; toes with ventral pads well defined by circumferential grooves; relative length of toes I 3500 le ara ae Figure 7. Distribution of Pristimantis prometeii sp. n. in Ecuador. Occurrence records are marked with red dots. The Reserva Buenaventura was created in 1999 for the protection of two endemic species of birds, and despite its rather small size (about 2400 ha) is an important area for conservation in Southwestern Ecuador. Actually, the reserve is one of the most di- verse sites in El Oro province hosting more than 60 species of amphibians and reptiles and 320 bird species (MECN-INB-GADPEO 2015). As for the amphibians, until 2015 there were known 24 species, 22 anurans (from seven families) and 2 caecilians from the reserve (MECN-INB—GADPEO 2015, Yanez-Mufioz et al. 2013, authors’ personal observations). The Craugastoridae family is represented by nine species in the reserve, Barycholos pulcher and eight Pristimanis species, with several more undescribed 94 Paul Székely et al. / ZooKeys 606: 77-97 (2016) ones (authors’ personal observations). The description of this new Pristimantis species highlights both the still poor knowledge of amphibians in southern Ecuador and the importance for conservation of even small protected areas, like Reserva Buenaventura, in a constantly degrading environment. Acknowledgments Dan Cogalniceanu and Paul Székely received funding from Secretaria de Educacion Su- perior, Ciencia, Tecnologia e Innovacién, Republic of Ecuador (SENESCYT) through the Prometeo Project. Laboratory work was funded by a grant from SENESCYT (Arca de Noé Initiative; Santiago R. Ron principal investigator). The authors gratefully ac- knowledge the support of SYNTHESYS grants: SE-TAF-4807 to Paul Székely and GB- TAF-4710 to Dan Cogalniceanu. The SYNTHESYS Project is financed by the Euro- pean Community—Research Infrastructure Action under the FP7 “Capacities” Specific Programme. D. B. Provete and an anonymous reviewer provided helpful comments to the manuscript. The authors are also grateful to Jocotoco Foundation for provid- ing access to Reserva Buenaventura and to the staff working at the Reserve, especially to Marco Galvez, for his support during our stay. Collecting permits were granted by Ministerio de Ambiente del Ecuador (Permit no. 005-14 IC-FAU-DNB/MA). References AmphibiaWeb (2016) AmphibiaWeb: information on amphibian biology and conservation. University of California Berkeley. http://amphibiaweb.org [accessed 27 June 2016] Arteaga A, Pyron RA, Pefafiel N, Romero-Barreto P, Culebras J, Bustamante L, Yanez-Mufoz MH, Guayasamin JM (2016) Comparative phylogeography reveals cryptic diversity and repeated patterns of cladogenesis for amphibians and reptiles in Northwestern Ecuador. 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Pristimantis riveti Ecuador—Azuay: Parque Nacional El Cajas (QCAZ 7386); El Oro: Chillacocha, 8 km desde Chilla (QCAZ 45157); Chimborazo: via Pallatanga (QCAZ 20979); Loja: Amaluza—El Salado de Jimbura (QCAZ 30775); Tungurahua: Recinto—Caserio, mar- gen del Parque Nacional LLanganates (QCAZ 46107); Zamora Chinchipe: Reserva Tapichalaca (QCAZ 45677). Pristimantis spinosus Ecuador—Zamora Chinchipe: Villa Nueva (QCAZ 54002). Pristimantis sternothylax Ecuador—Loja: 5-10 km de Loja (QCAZ 30600, QCAZ 30602, QCAZ 30604). Pristimantis versicolor Ecuador—Loja: Reserva Ecoldgica El Madrigal, Unidad Educativa Amauta (QCAZ 50733); Loja: Shucos (QCAZ 54330); Loja: Via Yangana-Valladolid (QCAZ 36237). Pristimantis walkeri Ecuador—Azuay: Recinto La Lopez (QCAZ 53616); Carchi: Cabeceras del Rio Baboso al NE de Lita (QCAZ 49514); El Oro: Reserva Ecolégica Buenaventura, Rio Moro- moro (QCAZ 17126); Loja: Cangonama (QCAZ 49438).