Three new species of frogs of the genus Pristimantis (Anura, Strabomantidae) with a redefinition of the P. lacrimosus species group

Abstract A new phylogeny for the Pristimantis lacrimosus species group is presented, its species content reviewed, and three new species described from the eastern slopes of the Ecuadorian Andes. Our phylogeny includes, for the first time, samples of P. aureolineatus, P. bromeliaceus, and P. lacrimosus. The morphology of hyperdistal subarticular tubercles is also assessed among 21 species of Pristimantis. The P. lacrimosus species group is composed of 36 species distributed in the Chocó, Guiana, and Amazon regions of tropical South America with a single species reaching Central America. Ancestral area reconstruction indicates that, despite its high diversity in the Amazon region, the P. lacrimosus group originated in the Pacific basin, Chocó region of Ecuador and Colombia. Pristimantis amaguanaesp. nov. is most closely related to P. bromeliaceus. It differs from P. bromeliaceus by being smaller, having transversal dark bands in the hindlimbs (absent or faint in P. bromeliaceus) and the absence of discoidal fold (present in P. bromeliaceus). Pristimantis nankintssp. nov. and P. romeroaesp. nov. are part of a clade of predominantly light-green frogs that includes P. acuminatus, P. enigmaticus, P. limoncochensis, and P. omeviridis. Pristimantis nankintssp. nov. and P. romeroaesp. nov. can be distinguished from all of them by the presence of a dark dorsolateral stripe that borders a light green band on a green background. Hyperdistal tubercles are present in all examined species of the P. lacrimosus species group and its sister clade. Species with hyperdistal tubercles are characterized by having relatively long terminal phalanges and narrow T-shaped expansion at the end of the terminal phalange. We discuss the phylogenetic distribution of these characters and their potential diagnostic significance.

preserved in 10% formalin and stored in 70% ethanol. Sex was determined by examining the presence of vocal slits and gonadal inspection. Measurements were taken with digital calipers (to nearest 0.01 mm) following the methodology described by Navarrete et al. (2016). Examined specimens belong to the herpetological collection at Museo de Zoología, Pontificia Universidad Católica del Ecuador, Quito (QCAZ), and are listed in Appendix 1.
Fingers and toes are numbered preaxially to postaxially from I to IV and I to V, respectively. Relative lengths of toes III and V were determined by adpressing them against toe IV; lengths of fingers I and II were compared by appressing them to each other. While describing the hands and feet of the new species, we noticed the presence of an additional distal subarticular tubercle. These tubercles were recently named by Ospina-Sarria and Duellman (2019) as "hyperdistal". We use the following terminology for the subarticular tubercles of Pristimantis, including hyperdistal tubercles ( Fig. 1): (1) "basal" for the tubercles at the base of fingers and toes, (2) "penultimate" occurs only on toe IV and refers to the tubercle underlying the distal articulation of the first phalange, (3) "distal" for the tubercles underlying the proximal articulation of the penultimate phalange, and (4) "hyperdistal" for the tubercle underlying the articulation of the last phalange of each finger and toe. Except for the exclusion of hyperdistal tubercles, this terminology has been used in most systematic accounts of Pristimantis at least since Lynch and Duellman (1997) seminal publication (e.g., Duellman and Lehr 2007;Urgiles et al. 2019;Valencia et al. 2019). We recommend its use, as amended by Ospina-Sarria and Duellman (2019), for future terminological consistency. Subarticular tubercles underly articulations between phalanges (Lynch and Duellman 1997). To understand the osteological anatomy of the hyperdistal subarticular tubercles, we obtained X-ray images of 21 species of Pristimantis including two of the new species and three additional members of the P. lacrimosus species group (P. acuminatus, P. enigmaticus, and P. limoncochensis). The other species represent assorted lineages within Pristimantis (based on the phylogeny of Jetz and Pyron 2018). Digital X-ray images were obtained with the Thermo Kevex X-ray Imaging System at the QCAZ museum. To explore the relationship between phalange size and the presence of hyperdistal tubercles, we compared the length of the ultimate and penultimate phalanges of finger III (Fig. 2). Pristimantis have T-shaped terminal phalanges for which we measured the terminal expansion width in finger III. We measured the left hand except when fingers were twisted as result of fixation problems. If both hands had fixation problems, we excluded the specimen. All measurements were made with software ImageJ 2 version 1.52q (Rasband 2020).

Phylogeny
We estimated the phylogenetic relationships of the new species based on DNA sequences of mitochondrial genes 12S rRNA (12S), 16S rRNA (16S), NADH dehydrogenase subunit 1 (ND1), their flanking tRNAs and the nuclear gene Recombination activating gene 1 (RAG-1). DNA was extracted from muscle or liver tissue preserved in 95% ethanol, using standard guanidine thiocyanate extraction protocols. We used a polymerase chain reaction (PCR) to amplify DNA fragments. The primers used for the 12S amplification were obtained from Goebel et al. (1999), for 16S from Heinicke et al. (2007), for ND-1 from Zhang et al. (2013), Wiens et al. (2005), Moen and Wiens (2009) and Brito et al. (2017) and for RAG-1 from Hedges et al. (2008). PCR amplification was performed under standard protocols and sequenced by the Macrogen Sequencing Team (Macrogen Inc., Seoul, Korea).
Our matrix included 74 newly generated sequences of Pristimantis (Table 1). We also included congeneric sequences available on GenBank. To optimize taxon sampling, we blasted the 16S sequences of the new species to the GenBank database (blastn procedure). This search showed that the most similar sequences belong to species of the Pristimantis lacrimosus group. Therefore, we concluded that the new species are closely related to the P. lacrimosus species group clade B (sensu Rivera-Correa et al. 2016). We included all GenBank sequences of that clade as well as closely related species (based on Guayasamin et al. 2017) and representative samples of other species groups of Pristimantis. The Gen-Bank sequence of specimen QCAZ 19664 (EU130579) was assigned to P. acuminatus by Elmer et al. (2007) but is updated to P. omeviridis based on Ortega-Andrade et al. (2015). For the outgroup we included several species of the subgenus Hypodictyon.
The new sequences were assembled in Geneious 9.0 and then exported to Mesquite 3.61 (Maddison and Maddison 2019) where each genomic region was aligned using default parameters with Muscle 3.8 (Edgar 2004). Phylogenetic relationships were inferred for all genes concatenated using maximum likelihood (ML) as optimality cri- terion. The concatenated DNA matrix had 3974 bp and is available at http://zenodo. org under https://doi.org/10.5281/zenodo.4005737. Because different evolutionary processes have influenced each gene, we partitioned the data by gene and codon position to find the best model of evolution and partition scheme. To accomplish both tasks, we used the command MFP + MERGE (Chernomor et al. 2016;Kalyaanamoorthy et al. 2017) in software IQ-TREE multicore version 2.0 (Nguyen et al. 2015). To find the best phylogeny we run a search using IQ-TREE 2.0 under default settings. To assess branch support, we made 200 non-parametric bootstrap searches (-b 200 command) and 1000 replicates for the SH-like approximate likelihood ratio test (-alrt 1000 command; Guindon et al. 2010). We considered that branches with bootstrap values > 70 and SH-aLRT values > 80 had strong support. To calculate uncorrected p-distances of 16S we used MEGA 7.0 (Kumar et al. 2016).

Biogeographic origin of the P. lacrimosus species group
Most species of the Pristimantis lacrimosus group occur in the Amazon and Pacific basins. To determine the biogeographic origin of the group, we carried out ancestral state reconstruction. Regions were coded as a binary character (Pacific or Amazon) and the reconstruction employed maximum likelihood as optimality criterion. The

Phylogeny and biogeography
The phylogeny (Fig. 3)  We also found strong support (SH-aLRT = 94, bootstrap = 74) for a sister clade relationship between the P. lacrimosus group (as redefined below) and a clade composed of 11 species including P. appendiculatus, P. calcarulatus, P. cedros, P. jaguensis, P. pycnodermis, and P. orcesi. Within the P. lacrimosus species group, Pristimantis amaguanae sp. nov. is most closely related to P. bromeliaceus and an undescribed species from Bombuscaro, Podocarpus National Park, Ecuador. Branch lengths in the phylogeny and its morphological distinctiveness indicate that P. amaguanae sp. nov. is evolutionarily independent from P. bromeliaceus and the undescribed species from Bombuscaro. Uncorrected p-genetic distances (gene 16S) between P. amaguanae sp. nov. and its sister clade range between 13.0 and 13.8%. Pristimantis nankints sp. nov. is sister to an undescribed species from Sardinayacu, Sangay National Park, Ecuador. Their genetic distance is 2.7%. Both species are sister to P. romeroae sp. nov. from Napo Province. They are separated by distances of 6.9 (P. sp.) and 7.0% (P. nankints sp. nov.) These three species are sister to a clade composed by P. jorgevelosai, P. enigmaticus, P. limoncochensis, P. omeviridis, and P. tantanti.
The reconstruction of ancestral basin indicates that the group originated in the Pacific basin with a single colonization event to the Amazon basin (Fig. 4). There is one colonization event from the Amazon to Central America and the Pacific basin in P. moro and to the Río Magdalena basin (Colombia) in P. jorgevelosai.

Hyperdistal subarticular tubercles and phalange morphology
Most examined species of Pristimantis lack hyperdistal tubercles (Table 2). However, the five species of the P. lacrimosus group have hyperdistal tubercles as well as P. eriphus, P. katoptroides, and P. orcesi. As expected, the hyperdistal tubercle is located in the articulation of the two terminal phalanges (Fig. 1). Among the 21 species of Pristimantis, the five examined species of the P. lacrimosus group are characterized by having: (1) long terminal phalanges, and (2) narrow T-shaped expansions in the terminal phalanges. Species with shorter terminal phalanges and long T-shaped expansions lack distinct hyperdistal tubercles (e.g., P. crenunguis, P. condor; Fig. 2). We found large variation in width of the T-shaped expansion in the terminal phalange. In some species the expansion is almost as long as the phalange (P. crenunguis) while in others it represents less than 1/3 of its length (P. acuminatus).

Pristimantis lacrimosus species group
Content. We present a new definition of the group based in our phylogeny and recent taxonomic reviews. We define the P. lacrimosus species group to include all species descendant of the most recent common ancestor of P. eremitus and P. lacrimosus (Fig. 3). Our definition is based on our phylogeny and the data presented by Arteaga et al. (2013)  Reconstructions were based on maximum likelihood inference. Pie charts at nodes represent conditional probabilities for the Pacific (black) and Amazon (green) basins. The asterisk indicates significant support for the Pacific basin as ancestral area for the most recent common ancestor of the P. lacrimosus species group. White circles indicate equivocal state, striped circles indicate distribution outside the Pacific or Amazonian basins (coded as missing data). Shepack et al. (2016), Gonzalez-Duran et al. (2017), and Ospina-Sarria and Duellman (2019). We exclude P. apiculatus from this group based on its close resemblance to P. calcarulatus (Lynch and Duellman 1997), a species that is not closely related to the P. lacrimosus species group (e.g., Pyron 2014). We exclude from the group P. eugeniae based on the phylogeny by Carrión (2020) which shows that it belongs to the sister clade of the P. lacrimosus species group. Ospina-Sarria and Duellman (2019) indicated that Table 2. Phalange morphometry and hyperdistal tubercle condition in several species of Pristimantis. Species of the Pristimantis lacrimosus species group are shown in bold. Species are ordered according to the relative length of the terminal phalanges (from low to high, terminal/penultimate). The five lowest T-width/terminal values are shown with italics. Note that species of the P. lacrimosus species group are characterized by having longer terminal phalanges, narrower T-expansions at the end of the terminal phalange, and hyperdistal tubercles. Hyperdistal tubercles were coded as "present" when they were similar in size to other hyperdistal tubercles in the same finger or toe. sneiderni is most similar to P. schultei and P. deyi, two members of the P. lacrimosus species group. However, they also stated a resemblance to P. boulengeri, a species belonging to the P. boulengeri species group. Given this inconsistency we refrain from assigning P. sneiderni to the P. lacrimosus group until genetic evidence is available. We suspect P. sneiderni is not a member of the P. lacrimosus species group because it inhabits paramo, a habitat type on which the P. lacrimosus species group is absent. Most species of the P. lacrimosus species group inhabit foothill Andean forest or lowland tropical rain forest. The group contains 36 described species: P. acuminatus (Shreve 1935), P. amaguanae sp. nov. (herein), P. aureolineatus (Guayasamin et al. 2006), P. bromeliaceus (Lynch 1979), P. calima Ospina-Sarria and Duellman 2019, P. crucifer (Boulenger 1899), P. deyi Lehr et al. 2013, P. ecuadoriensis Guayasamin et al. 2017, P. enigmaticus (Ortega-Andrade et al. 2015, P. eremitus (Lynch 1980), P. galdi (Jiménez de la Espada 1870), P. jorgevelosai (Lynch 1994), P. lacrimosus (Jiménez de la Espada 1875), P. latericius Brito 2014, P. limoncochensis Ortega-Andrade et al. 2015, P. mendax (Duellman 1978), P. mindo Arteaga et al. 2013, P. moro (Savage 1965), P. nankints sp. nov. (herein), P. nyctophylax (Lynch 1976), P. olivaceus (Köhler et al. 1998), P. omeviridis Ortega-Andrade et al. 2015, P. ornatissimus (Despax 1911), P. padiali Moravec et al. 2010, P. pardalinus, (Lehr et al. 2006), P. petersi (Lynch and Duellman 1980), P. pluvialis Shepack et al. 2016, P. royi (Morales 2007), P. pseudoacuminatus (Shreve 1935), P. romeroae sp. nov. (herein), P. schultei (Duellman 1990), P. subsigillatus (Boulenger 1902), P. tantanti (Lehr et al. 2007), P. tayrona (Lynch and Ruíz-Carranza 1985), P. waoranii (McCracken et al. 2007), and P. zimmermanae (Heyer and Hardy 1991 Diagnosis. A species of Pristimantis characterized by the following combination of characters: (1) skin on dorsum shagreen with conical tubercles, skin on venter areolate with light green warts on the chest; discoidal fold absent; dorsolateral folds absent (Fig. 5); (2) tympanic membrane and tympanic annulus present, its dorsoposterior border converges with supratympanic fold; (3) snout acuminate in dorsal, protruding in lateral profile, with rostral papilla; (4) upper eyelid with several small conical tubercles; cranial crests absent; (5) dentigerous processes of vomers absent; (6) male having vocal slits, nuptial pads absent; (7) finger I shorter than finger II; discs of digits expanded, rounded (Fig. 6); (8) fingers with lateral fringes; hyperdistal subarticular tubercles present; (9) ulnar tubercles present, low and rounded; (10) heel bearing conical tubercles varying from prominent to inconspicuous; inner tarsal fold absent; (11) inner metatarsal tubercle ovoid, elevated, five times the size of round outer metatarsal tubercle; supernumerary plantar tubercles present; (12) toes with narrow lateral fringes; basal toe webbing absent; toe V much longer than toe III (disc on toe III reaches the proximal border of the penultimate subarticular tubercle on toe IV, disc on toe V reaches the distal subarticular tubercle on toe IV); hyperdistal subarticular tubercles present; toe discs as large as those on fingers (Fig. 6); (13) in life, dorsal surfaces of body and limbs olive green or olive brown with black markings; canthal stripe and supratympanic fold black; lips cream with black bars; flanks cream with one broad oblique black bar; chest light green with greenish cream warts; belly yellowish white; iris bronze to reddish copper with black reticulations (Fig. 5); (14) SVL in adult female 20.4 mm (n = 1), in adult male 16.3 mm (n = 1).

QCAZ
Comparison with other species. In this section, coloration refers to live individuals unless otherwise noticed. The coloration of Pristimantis amaguanae resembles that of P. bromeliaceus and P. petersi (Fig. 5). Pristimantis amaguanae can be easily recognized by the presence of transversal dark bands in the hindlimbs (absent or faint in P. bromeliaceus and P. petersi) and the absence of discoidal fold (present in P. bromeliaceus and P. petersi). Pristimantis amaguanae further differs from P. bromeliaceus by its smaller adult size (female SVL = 20.4 mm, males SVL = 16.3 mm vs. P. bromeliaceus female SVL = 23.0-28.5 mm, male SVL = 16.7-22.8 mm; Lynch and Duellman 1980). Another small, green Pristimantis from the Amazon basin is P. paululus. The new species differs by having more tuberculate dorsal skin, discoidal folds absent (folds prominent in P. paululus, Lynch 1974), and scattered enlarged light green warts in the venter (small white points in P. paululus ;Lynch 1974). Pristimantis pseudoacuminatus differs by having a truncate snout in profile (acuminate in P. amaguanae, Fig. 7) and by having a lighter and more uniform coloration in preservative.
Skin on dorsum shagreen with scattered tubercles; dorsolateral folds absent; skin on lower flanks and belly areolate with scattered tubercles; skin on throat and chest smooth; discoidal fold absent; skin in upper cloacal region shagreen, wrinkled ventrally, with several tubercles below the cloacal sheath. Forearms slender; conical ulnar tubercles present along outer edge of forearm; all digits bearing pads and discs, broadly expanded and rounded but those of fingers II-IV clearly larger than that on thumb; fingers bearing narrow lateral fringes; relative lengths of fingers I < II < IV < III; subarticular tubercles single, well defined, round in ventral and lateral view; hyperdistal subarticular tubercles present in all fingers; several supernumerary tubercles at base of fingers present, distinct; palmar tubercle bifid, approximately 1.5 size of ovoid thenar tubercle (Fig. 6).
Hindlimbs slender; upper surfaces of hindlimbs smooth; posterior surfaces of thighs smooth, ventral surfaces of thighs slightly areolate; heel bearing low conical tubercles; inner surface of tarsus bearing small, low tubercles; toes with lateral fringes; webbing between toes absent; discs on toes expanded, elliptical, as large as those on fingers; all toes having pads surrounded by circumferential grooves; relative lengths of toes: I < II < III < V < IV; subarticular tubercles rounded, simple; hyperdistal subarticular tubercles present; plantar surface with supernumerary tubercles; inner metatarsal tubercle prominent, ovoid approximately five times of rounded outer metatarsal tubercle (Fig. 6).
Color of holotype in preservative. (Fig. 7) Background color pale brown with a dark brown interorbital bar and chevron marks in the scapular and sacrum region; a white mark extending from the posterior border of the upper eyelid to the scapular region; canthal and supratympanic stripe black, extending as a post-axial stripe on lower flanks; a dark brown Y-shaped mark at the tip of snout; dark brown transversal bars on dorsal surfaces of the limbs (three on the forearm, four to five on the thigh, five on the shank, and four on the foot); anal triangle dark brown; flanks and hidden surfaces of thighs pale brown; venter cream with white tubercles; scattered brown flecks on the neck, chest, and lips; ventral surfaces of hindlimbs and forelimbs creamy white with a brown suffusion.
Color of holotype in life. (Fig. 5) Dorsal surfaces of body and limbs olive green with black markings; canthal stripe and supratympanic fold black; flanks cream with one broad oblique bar; chest light green with small white spots; belly yellowish white; ventral surfaces of forelimbs and shanks faint green wash; ventral surfaces of thighs pale brown; iris bronze with black reticulations.
Color in life (based on digital photographs; Fig. 5). Background coloration is olive brown with faint green dorsolaterally. Marks on dorsum and flanks are similar to the holotype, except for the interorbital bar that is interconnected with the chevron mark in the scapular region. Iris is reddish copper.
Distribution, natural history, and conservation status. This species is only known from the type locality in Provincia de Pastaza, Ecuador at 430 m above sea level (Fig. 8). Natural region is Amazonian Tropical Rainforest (as defined by Ron et al. 2019). The forest is characterized by a high canopy (up to 30 m) with emergent trees that can reach 40 m. Annual precipitation is above 3000 mm and seasonality is low. The amplectant pair was on a leaf 0.4 m above the ground in primary forest near a stream at night.
We recommend assigning Pristimantis amaguanae to the Endangered Red List category according to the B2ab(iii) criteria (based on IUCN 2017 guidelines) because it is known from a single locality, its Area of Occupancy is less than 500 km 2 and its only known locality is at a distance of 1.5 km from deforested areas (based on Google Earth satellite images dating from 2017). A road was built in the area ca. five years ago. Road building is the main predictor of forest destruction in the Ecuadorian Amazon (Sierra 2013).
Etymology. The specific name amaguanae is a noun in the genitive case and is a patronym for Tránsito Amaguaña, a leading female figure of the indigenous movement in Ecuador. In 1930 she helped to form the first indigenous organization in Ecuador and during all her life she fought for equality and justice for Ecuadorian poor people.  Lynch (1974), Lynch (1979), and Lynch and Duellman (1990). Suggested common name. English: Nankints Rain Frog. Spanish: Cutín de Nankints.

Pristimantis nankints
Diagnosis. A species of Pristimantis characterized by the following combination of characters: (1) skin on dorsum shagreen, skin on venter areolate with scattered warts, smooth on throat; discoidal fold absent; dorsolateral folds absent; (2) tympanic membrane and tympanic annulus present, upper edge of tympanic annulus covered by supratympanic fold; (3) snout short, protruding in lateral profile, acuminate in dorsal view, with rostral papilla; (4) upper eyelid without conical tubercles; cranial crests absent; (5) dentigerous processes of vomers present, prominent, oblique; (6) male having vocal slits, nuptial pads absent; (7) finger I shorter than finger II; discs of digits moderately expanded, rounded; (8) fingers bearing narrow lateral fringes; hyperdistal subarticular tubercles present; (9) ulnar and tarsal tubercles present, those on the tarsus are flattened and low, nearly inconspicuous; (10) heel without conical tubercles; inner tarsal fold present; (11) inner metatarsal tubercle prominent, elliptical, approximately 3× as large as rounded, conical outer metatarsal tubercle; outer metatarsal tubercle small, rounded; supernumerary plantar tubercles inconspicuous (Fig. 10); (12) toes bearing narrow lateral fringes; toe webbing absent except for basal webbing between toes III and IV; toe V much longer than toe III (disc on toe III extends to the distal edge of the medial subarticular tubercle on toe IV, disc on toe V extends beyond the proximal edge of the distal subarticular tubercle on toe IV); hyperdistal subarticular tubercles present; toe discs smaller than those on fingers (Fig.  10); (13) in life, dorsum lime green to olive green with black to brown marks on dorsum; flanks green with black to brown longitudinal stripes on upper edge; chest and belly greenish cream, throat yellowish green; ventral surface of thighs lime green to pinkish green. Iris bronze with a broad dark brown horizontal band and black reticulations; (14) SVL in adult females 29.4 mm ± 2.2 (27.8-30.9, n = 2), SVL in one adult male 19.6 mm.
Comparisons with other species (Fig. 12). In this section, coloration refers to live individuals unless otherwise noticed. Pristimantis nankints and P. romeroae have similar coloration in preservative. Pristimantis nankints differs by having small warts in the ven- ter (numerous large warts in P. romeroae) and by the size and shape of discs on fingers: expanded and truncate in P. romeroae vs. moderately expanded and rounded in P. nankints. Its green dorsal coloration resembles that of P. acuminatus, P. enigmaticus, P. limoncochensis, P. omeviridis, P. pseudoacuminatus, and P. tantanti. It differs from all of them by having a dark stripe bordering the upper edge of the flanks (dark stripe absent or if present, it is an oblique-lateral stripe starting behind the eye and ending near the ventral edge of the flank at midbody). It further differs from P. acuminatus, P. limoncochensis, and P. tantanti by having a prominent tympanum (tympanum absent in the three species; Duellman and Lehr 2009). Pristimantis enigmaticus and P. omeviridis differ by having a smaller tympanum (12-13% of head length [Ortega-Andrade et al. 2015] vs. 20-25% in P. nankints). Pristimantis pseudoacuminatus differs by having sparse tubercles and warts on the dorsum (tubercles and warts absent in P. nankints; Shreve 1935).
Description of the holotype. Adult female (QCAZ 71457; Figs 9-11). Measurements (in mm): SVL 30.9; tibia length 13.2; foot length 14.9; head length 11.4; head width 10.6; eye diameter 3.0; tympanum diameter 2.3; interorbital distance 3.8; upper eyelid width 2.8; internarial distance 2.3; eye-nostril distance 3.7; tympanum-eye distance#1.7. Body slender; head slightly wider than long, wider than body; snout short, protruding in lateral profile, acuminate in dorsal view, with rostral papilla; canthus rostralis distinct, slightly curved in dorsal view; loreal region slightly concave; interorbital space flat, no cranial crests; upper eyelid ca. 75% of interorbital distance; lacking tubercles, no interocular fold. Tympanic membrane and annulus present, rounded in shape, with supratympanic fold covering upper edge; horizontal diameter of tympanum ca. 75% of eye diameter, separated from eye by a distance of a complete tympanum length; choanae large, rounded, not concealed by palatal shelf of maxillary arc; dentigerous processes of vomers present, prominent, oblique, bearing 5 teeth, tongue cordiform. Skin on dorsum shagreen; dorsolateral folds absent; skin on belly and posterior half of chest areolate with scattered warts; skin on anterior half of chest and throat smooth; discoidal fold absent; skin in upper cloacal region shagreen. Forearms slender with a row of four conical ulnar tubercles in outer edge of forearm; fingers large and slender, all with round discs; fingers bearing narrow lateral fringes; relative lengths of fingers I < II < IV < III; subarticular tubercles single, well defined, round in ventral view; hy-  perdistal subarticular tubercles present in all fingers; supernumerary palmar tubercles present, distinct; palmar tubercle bifid, 2× size of ovoid thenar tubercle (Fig. 10).
Hindlimbs slender; tibia length ca. 50% of SVL; upper surfaces of hindlimbs smooth; foot length ca. 46 % of SVL, posterior surfaces of thighs smooth, ventral surfaces of thighs slightly areolate; knee and heel lacking tubercles; inner surface of tarsus lacking tubercles; toes bearing narrow lateral fringes; webbing between III and IV toes present at the base; discs on toes broadly expanded, rounded, relatively smaller than fingers; all toes having pads surrounded by circumferential grooves; relative lengths of toes: I < II < III < V < IV; subarticular tubercles rounded; hyperdistal subarticular tubercles present in all toes; plantar surface with inconspicuous supernumerary tubercles; inner metatarsal tubercle prominent, elliptical, approximately three times as large as rounded, conical outer metatarsal tubercle (Fig. 10).
Color of holotype in preservative. (Fig. 11) Background color pale gray with dark brown interorbital bar; light canthal and supratympanic black stripe continued by a long dorsolateral stripe bordering the upper flank; dorsal brown marks scattered on all dorsum except for two longitudinal bands adjacent to the dark dorsolateral stripe; color of venter, chest, and ventral surfaces of the limbs pale cream.
Color of holotype in life. (Fig. 9) Dorsal surfaces dark green with black spots and limbs yellowish green; canthal stripe and supratympanic fold black, continued by black dorsolateral stripe bordering the upper flank, the black stripe gradually fades in the upper flank and limits dorsally with a parallel light green band; chest and belly greenish cream, throat yellowish cream; ventral surfaces of forelimbs yellowish green and shanks faint green; ventral surfaces of thighs pinkish green; iris bronze with a broad dark brown horizontal band and black reticulations.
Variation. (Fig. 11) In this section, coloration refers to preserved individuals. In the type series, the adult male has an SVL = 19.6 mm and the adult female 27.8 mm; (Table 3). The male has vocals slits and lacks nuptial pads. Dorsal coloration in preservative is light gray (e.g., QCAZ 71458) to yellowish cream (e.g., QCAZ 69137) with black dorsolateral bars and a black canthal stripe. Both paratypes have a lighter dorsolateral band bordered by the dark dorsolateral stripe and delimited medially by a faint black stripe (QCAZ 71458) or a row of black marks (QCAZ 69137). Both paratypes lack an interorbital bar but QCAZ 69137 has two dark marks instead. In QCAZ 69137 the flanks are yellowish cream; in QCAZ 71458 the flanks are gradually darker towards the black dorsolateral stripe. The tips of the fingers have the same color as the rest of the finger in QCAZ 69137 while in QCAZ 71458 they are darker.
Color in life (based on digital photographs of an adult male QCAZ 69137) (Fig. 9): dorsal surfaces are lime green; black-reddish canthal stripe is continued by black supratympanic fold and black dorsolateral stripe bordering the upper flank, the black stripe limits dorsally with a parallel light lime green band limited medially by two rows of black round marks; flanks, ventral and dorsal surfaces of limbs are green; belly and chest are greenish cream, throat greenish yellow; fingers, and toes greenish yellow; iris bronze.
Distribution, natural history, and conservation status. Pristimantis nankints has been recorded at one locality in the eastern Andean slopes of Ecuador, Provincia Morona Santiago, Cordillera del Cutucú, 1364-1413 m above sea level (Fig. 8). Natural Region is Andean Eastern Montane Forest (according to Ron et al. 2019 classification) which is characterized by evergreen trees covered by mosses and abundant epiphytic plants. Specimens were found at night along crystalline creek surrounded by secondary forest. The holotype was perching on a leaf 2 m above the ground, next to the stream; after capture, while in the plastic bag, the female deposited 22 eggs. The adult male was collected on secondary forest, on a terrestrial bromeliad. The female paratype was perching on a leaf 30 cm above the ground.
Pristimantis nankints distribution area is a mosaic of forest and deforested areas (based on Ministerio de Ambiente del Ecuador 2013). Its occurrence in secondary forest, near artificial open areas, indicates at least some level of resilience to anthropogenic habitat change. Nevertheless, there is not enough information to assess its risk of extinction. Therefore, we recommend its assignment to the Data Deficient Red List Category (DD) (based on IUCN 2017 guidelines).
Etymology. The species name is a noun in apposition that refers to Nankints, a small hamlet in Cordillera del Cóndor, Ecuador, that used to be inhabited by Shuar native Americans. Its dwellers were violently evicted and Nankints was destroyed in 2016 to establish a mining camp. Large scale mining projects generate widespread deforestation and pollution in the Andes. The species name is a tribute to Ecuadorian people who have resisted mining activities in defense of the environment. Nankints is a shuar word that means spear.

Diagnosis.
A species of Pristimantis characterized by the following combination of characters: (1) skin on dorsum shagreen, skin on venter areolate with scattered warts; discoidal fold absent; dorsolateral folds absent; (2) tympanic membrane and tympanic annulus present, upper edge of tympanic annulus covered by supratympanic fold; (3) snout short, truncate in dorsal view, slightly protruding in lateral profile, with small rostral papilla; (4) upper eyelid with several small tubercles; cranial crests absent; (5) dentigerous processes of vomers present, prominent, moderately oblique; (6) male having vocal slits, nuptial pads present on finger I; (7) finger I slightly shorter than finger II; discs of digits expanded, truncate; (8) fingers with lateral fringes; hyperdistal subarticular tubercles present; (9) ulnar tubercles absent, tarsal tubercles present, subconical, conspicuous; (10) heel with one, nearly inconspicuous, small subconical tubercle or without tubercles; inner tarsal fold absent; (11) inner metatarsal tubercle prominent, elliptical, approximately three times as large as rounded, conical outer metatarsal tubercle; supernumerary plantar tubercles present (Fig. 1); (12) toes with lateral fringes; toe webbing absent; toe V much longer than toe III (disc on toe III extends to the distal edge of the medial subarticular tubercle on toe IV, disc on toe V extends beyond the proximal edge Comparisons with other species. In this section, coloration refers to preserved individuals (Fig. 13). Pristimantis romeroae resembles P. nankints in coloration. Pristimantis romeroae differs by having numerous large warts on the venter (small warts in P. nankints) and by the size and shape of discs on fingers: expanded and truncate in P. romeroae vs. moderately expanded and rounded in P. nankints. Its predominately pale creamy orange dorsal coloration resembles that of preserved P. acuminatus, P. enigmaticus, P. limoncochensis, P. omeviridis, P. pseudoacuminatus, and P. tantanti. It differs from all of them by having a dark stripe bordering the upper edge of the flanks (dark stripe absent or if present, it is an oblique-lateral stripe starting behind the eye and ending near the ventral edge of the flank at midbody). It also differs from P. acuminatus, P. limoncochensis, and P. tantanti by having a conspicuous tympanum (absent in the three species). Pristimantis romeroae can be further distinguished from P. enigmaticus and P. omeviridis by having a larger tympanum (21-27% of head length in P. romeroae vs. 12-13% in both species; Ortega-Andrade et al. 2015). Pristimantis pseudoacuminatus differs by having sparse tubercles and warts on the dorsum (absent in P. romeroae; Shreve 1935).
Skin on dorsum and flanks shagreen; dorsolateral folds absent; skin on belly and posterior half of chest areolate with scattered warts; skin on throat and anterior half of chest smooth; discoidal fold absent; skin in upper cloacal region smooth. Forearms slender with three ill-defined, low ulnar tubercles in distal, medial and proximal outer edge of forearm; fingers large and slender, all fingers with pads surrounded by circumferential grooves, truncate discs; bearing narrow lateral fringes; relative lengths of fingers I < II < IV < III; subarticular tubercles single, round in ventral and lateral view; hyperdistal subarticular tubercles present; bearing few, inconspicuous, low supernumerary tubercles, palmar tubercle bifid, twice the size of elliptical thenar tubercle (Fig. 1).
Hindlimbs slender; tibia length ca. 50% of SVL; upper surfaces of hindlimbs smooth; foot length ca. 45 % of SVL, posterior surfaces of thighs shagreen, ventral surfaces of thighs smooth; knee and heel lacking tubercles; inner surface of tarsus lacking tubercles; toes bearing narrow lateral fringes; webbing between toes absent; discs on toes broadly expanded, truncate, the same size than fingers; all toes having pads surrounded by circumferential grooves; relative lengths of toes: I < II < III < V < IV; subarticular tubercles rounded, simple; hyperdistal subarticular tubercles present; plantar surface with numerous indistinct supernumerary tubercles; inner metatarsal tubercle prominent, elliptical, approximately 3 times the size of rounded, conical outer metatarsal tubercle (Fig. 1).
Color of holotype in preservative. (Fig. 13) Background color pale creamy orange with faint interorbital line; long, thick, dark brown dorsolateral bars; dorsum with paler blotches clustered in two parallel stripes at the scapular region approximately half the length of the dorsolateral bars; face with dark brown canthal and supratympanic stripes, supratympanic stripe suffused with the dark brown dorsolateral bar; flanks the same back ground color with minute dark spots (visible under magnification) densely distributed; dorsal surfaces of limbs yellowish cream brighter than dorsum with scattered minute dark brown spots visible under magnification; ventral surface of body yellowish cream; plantar and palmar surfaces dirty cream.
Color of holotype in life. Unknown but presumably green, similar to its most closely relatives (e.g., P. nankints, P. enigmaticus) which have a similar clear coloration in preservative (Figs 9 and 12).
Variation. (Fig. 13) In this section, coloration refers to preserved individuals unless otherwise noted. In the type series, the adult male has an SVL = 23.8 mm, lower than the adult female SVL (range 31.1-33.8 mm; Table 3). Males have vocals slits and nuptial pads on finger I. Dorsal coloration is creamy tan (e.g., QCAZ 41121) with a black canthal stripe followed by black dorsolateral stripes. Marks on dorsum vary from scattered dark brown spots (e.g., QCAZ 41103) to two longitudinal brown strips starting behind the head and converging medially in the sacral region (QCAZ 41122) with or without a fine interorbital bar. Flanks are cream; venter and ventral surfaces of limbs vary from creamy white (e.g., QCAZ 41122) to yellowish cream (e.g., QCAZ 41103). The belly has scattered white warts (e.g., QCAZ 41103, 41121).
Color in life: unknown but presumably green (see description of the holotype). Distribution, natural history, and conservation status. Pristimantis romeroae is known from one locality at the eastern Andean slopes of Ecuador, Provincia de Napo, on the SSE slope of the Sumaco volcano, 1602 m above sea level (Fig. 8). Natural Region is Andean Eastern Montane Forest (according to Ron et al. 2019 classification) which is characterized by evergreen trees covered by mosses and abundant epiphytic plants. Except for QCAZ 41128, all specimens were found on a spiny bromeliad 6 cm from the ground by the day. QCAZ 41128 was found also by day on a bromeliad of a recently fallen tree.
In 2008, one year before the specimens were collected, the type locality was at a distance of < 1 km from agricultural deforested areas (based on Ministerio de Ambiente del Ecuador 2013) suggesting at least some level of tolerance to habitat degradation. Available information is insufficient to determine the risk of extinction of this species known from a single locality. Lack of records may partly be a consequence of its association with bromeliads which generally grow at heights unreachable during herpetological searches. We suggest to assigning P. romeroae to the Data Deficient Red List Category (DD) (based on IUCN 2017 guidelines).
Etymology. The species name is a noun in the genitive case and is a patronym for Giovanna Romero, an Ecuadorian botanist and SRR's wife. For almost two decades, she has supported SRR's research in countless ways and this is a long-overdue tribute.

Subarticular tubercles and phalange morphology in Pristimantis
Subarticular tubercles are round dermal protuberances, below the articulations of phalanges, on the underside of the hands and feet of anurans. According to the most recent comprehensive reviews of morphological characters of Pristimantis (Lynch and Duellman 1997;Duellman and Lehr 2007), strabomantid frogs have: (1) one subarticular tubercle on fingers I, II and toes I, II, (2) two subarticular tubercles on fingers III, IV and toes III, IV, and (3) three subarticular tubercles on toe IV. While describing the morphology of the new species, we noticed the presence of an additional subarticular tubercle underlying the articulation of the last phalange on each finger and toe (arrows in Fig. 1). Those tubercles are similar in size to other subarticular tubercles. While preparing this publication, we became aware of a recent publication by Ospina-Sarria and Duellman (2019) on which they report the same tubercles and named them "hyperdistal tubercles". Prior to Ospina-Sarria and Duellman (2019), the only mention we could find of hyperdistal tubercles in Strabomantid frogs was made by Lynch (1999) who stated "in many species there is a tubercle poorly defined that corresponds to the articulation of the terminal phalange (which supports the disk) and the penultimate phalange. This is a subarticular tubercle but because it is not as developed as the others, conventionally we do not take it into account". Our observations indicate that hyperdistal tubercles can be as large or larger than other subarticular tubercles (e.g., Fig. 1). Variation between presence and absence of hyperdistal tubercles appear to be continuous. In some species, like P. phoxocephalus, there is an inconspicuous tubercle at the base of the disk. In others, like P. katoptroides, the tubercle is larger than that of P. phoxocephalus but smaller than the hyperdistal tubercles observed in the P. lacrimosus species group. Future studies on the evolution and functional significance of hyperdistal tubercles would require methodologies to code it as a continuous character.
The available information on the phylogenetic distribution of hyperdistal tubercles and the morphology of the terminal phalanges suggest that they have phylogenetic signal and diagnostic value. Hyperdistal tubercles are present in all examined species of the P. lacrimosus species group (ten species: Table 2, Ospina-Sarria and Duellman 2019). They are also present in P. orcesi and P. eugeniae, two members of the sister clade of the P. lacrimosus species group (Table 2, Ospina-Sarria and Duellman 2019). In contrast, they were absent in the distantly related P. crenunguis, P. achatinus, P. condor, and P. lanthanites (Table 2). The available evidence suggests that the hyperdistal tubercles may be shared by the P. lacrimosus species group and its sister clade. Hyperdistal tubercles were also found in three species belonging to the closely related P. boulengeri and P. leptolophus species groups: P. angustilineatus, P. boulengeri, and P. leptolophus (Ospina-Sarria and Duellman 2019). They are also present in P. eriphus, P. katoptroides, and P. orpacobates, species that are not closely related to the P. lacrimosus species group ( Table 2). The distribution of hyperdistal tubercles among Pristimantis suggests several independent origins. An exhaustive analysis of the distribution of hyperdistal tubercles on a phylogenetic context is necessary to understand its evolution.
Interestingly, our results suggest that the presence of hyperdistal tubercles is linked to two osteological characters of the terminal phalanges (Table 2). First, species with hyperdistal tubercles tend to have long terminal phalanges (relative to the penultimate phalange; Fig. 2, Table 2). Second, they tend to have narrow T-shaped expansions at the end of the terminal phalange. Our results on 21 species of Pristimantis (Table 2) suggest future venues of research to discover morphological synapomorphies for Pristimantis clades, a task that has been elusive until now.
The correlation between the presence of hyperdistal tubercles and the morphology of the terminal phalanges has not been tested. We suspect that in species with short ultimate phalanges, like P. crenunguis (Fig. 2), the disk pad overlaps the phalange articulation and, therefore, the hyperdistal tubercle. This overlap may explain the absence of hyperdistal tubercles in species with short terminal phalanges (Fig. 2). In species with long terminal phalanges, like P. romeroae, the disk pad does not overlap with the articulation and the subarticular tubercle is separate from the disk pad and, therefore, is distinct.
The Pristimantis lacrimosus species group originated in the Chocoan forests Our reconstruction of ancestral basin indicates that the P. lacrimosus species group originated in the Chocoan forests of the Pacific basin of Ecuador and Colombia. This result was unexpected because, by far, most species of the group occur in the Amazon basin (26 described species out of 36). Our reconstruction suggests that the Amazon basin was colonized on a single event. The paucity of colonization events across the Andes demonstrates the pivotal role of the Andean barrier in the diversification of Pristimantis. There is a single colonization event from the Amazon Basin to the Chocoan forests with P. moro. Because P. moro is also distributed in Central America, we suspect that its presence in the Chocoan forest is a result of a colonization from central America instead of colonization across the Andes. The Amazon basin was also the origin of P. jorgevelosai, a species distributed in the Magdalena river basin and embedded in an otherwise Amazonian clade (Fig. 4).
Our biogeographic reconstruction suggests that the P. lacrimosus species group had higher diversification rates in the Amazon basin relative to the Chocó region. There are two clades inhabiting the Chocoan region. One of them is older than the Amazonian clade and yet it only has four species. The second clade is sister to the Amazonian clade. Although both clades have the same age, the Chocoan clade has only four species compared to 19 species in the Amazonian clade. These differences in diversification rates are inconsistent with the time-for-speciation hypothesis, which predicts the highest richness in first colonized regions (Stephens and Wiens 2003). Additional studies are needed to determine if the higher diversification rate in the Amazon region, relative to the Chocó, observed in the P. lacrimosus group is a generality among Pristimantis inhabiting both regions.