Systematics of Pholidobolus lizards (Squamata, Gymnophthalmidae) from southern Ecuador, with descriptions of four new species

Abstract Four new species of Pholidobolus lizards are described from poorly explored areas in the Andes of southern Ecuador based on morphological and genetic evidence. Among other morphological characters, Pholidobolus sameksp. nov. and P. condorsp. nov. differ from their congeners in having green dorsolateral stripes on head. Males of P. condorsp. nov. differ from those of P. sameksp. nov. in having reddish flanks and venter. P. dolichoderessp. nov. is distinguished by having a long neck, with more scales between orbit and tympanum, whereas P. fascinatussp. nov. is distinguished by lacking enlarged medial scales on collar and a conspicuous vertebral stripe. In addition, the phylogenetic position of the new species is inferred using DNA sequences of mitochondrial and nuclear genes. The phylogeny supports strongly monophyly of each of the new species and renders P. macbrydei paraphyletic and split into six subclades. Available data suggest that the new species have restricted distribution ranges (< 100 km2 each), and it is proposed that their classification be as Data Deficient or Critically Endangered species. The results reveal unexpected levels of diversity within Pholidobolus in the Andes of southern Ecuador and highlight the importance of improving scientific collections and conservation efforts in this area.


Introduction
The uplift of the Andes mountains was one of the most influential geological events for the evolution and diversification of the South American biota during the Cenozoic. For example, it created many habitats and microclimates that became important centers of biodiversity and endemism (Pérez-Escobar et al. 2017). Therefore, the evolution of diverse Andean taxa is a complex research topic that has attracted the attention of many scientists (Castoe et al. 2004, Torres-Carvajal et al. 2015, Betancourt et al. 2018, Moravec et al. 2018, Lehr et al. 2019. With more than 250 species, Gymnophthalmidae is one of the most diverse lizard clades in the Neotropics. The uplift of the Andes had a strong influence on the radiation of gymnophthalmid lizards, resulting in high levels of diversity and endemism along the Tropical Andes Moravec et al. 2018).
The study of Pholidobolus and other gymnophthalmid taxa has been often hampered by the paucity of specimens in collections. For example, the recent description of P. paramuno reveals the importance of increased sampling effort in the Paramo ecosystem in the northern Andes of Colombia. Similarly, recent collections in poorly explored areas of the southern Andes of Ecuador yielded new specimens of Pholidobolus lizards, which we were unable to assign to any of the currently recognized species. Based on these specimens, here we combine evidence from morphology and DNA sequences to describe four new species of Pholidobolus and infer their phylogenetic affinities.

Genetic data
Total genomic DNA was digested and extracted from liver or muscle tissue using a guanidinium isothiocyanate extraction protocol. Tissue samples were first mixed with Proteinase K and a lysis buffer and digested overnight prior to extraction. DNA samples were quantified using a Nanodrop ND-1000 (NanoDrop Technologies, Inc.), resuspended and diluted to 25 ng/µl in ddH2O prior to amplification.
Using primers and amplification protocols from the literature (Pellegrino et al. 2001; Torres-Carvajal and Mafla-Endara 2013), we obtained 1,493 aligned nucleotides (nt) encompassing three mitochondrial genes, 12S (339 nt), 16S (533 nt), and ND4 (621 nt) from 16 individuals of the four new species herein described, as well as 21 individuals of Pholidobolus macbrydei. In addition, we obtained 411 nucleotides of the Dynein Axonemal Heavy Chain 3 (DNAH3) nuclear gene from 65 individuals of Anadia rhombifera, Macropholidus annectens, M. huancabambae, M. labiopunctatus, M. ruthveni, Pholidobolus affinis, P. dicrus, P. hillisi, P. macbrydei, P. montium, P. prefrontalis, P. ulisesi, P. vertebralis, and the four new species. DNAH3 was amplified using the primers DNAH3_f1 (GG-TAAAATGATAGAAGAYTACTG) and DNAH3_r6 (CTKGAGTTRGAHACAAT-KATGCCAT). The amplification protocol consisted of 1 cycle of initial denaturation for 5 min at 95 °C, 40 cycles of denaturation for 35s at 94 °C, annealing for 1 min at 72 °C, and extension for 1 min at 72 °C, as well as a final extension for 10 min at 72 °C (Townsend et al. 2008). Positive PCR products were visualized in agarose electrophoretic gels and treated with ExoSAP-IT to remove unincorporated primers and dNTPs. Cycle sequencing reactions were carried out by Macrogen Inc. GenBank accession numbers of sequences generated in this study are shown in Table 1. After incorporating GenBank sequences, our data matrix for phylogenetic analyses contained 74 taxa and 1904 characters.

Phylogenetic analyses
Data were assembled and aligned in Geneious v5.4.6. (Kearse et al. 2012) under default settings for MAFFT Multiple Alignment (Katoh and Toh 2010). ND4 and DNAH3 sequences were translated into amino acids for confirmation of alignment. The best-fit nucleotide substitution models and partitioning scheme were chosen simultaneously using PartitionFinder v2.1.1 (Lanfear et al. 2012) (ML) and Bayesian inference (BI) methods were used to obtain the optimal tree topology of the combined, partitioned dataset using the programs RAxML v.8.2.12 (Stamatakis 2014) and MrBayes v3.2.6 (Ronquist et al. 2012), respectively. The ML analysis was performed under the GTRGAMMA model for all partitions. Nodal support (BS) was assessed with the rapid bootstrapping algorithm under the MRE-based Boot-stopping criterion (252 replicates). For BI analysis, all parameters were unlinked between partitions (except topology and branch lengths), and rate variation (prset ratepr = variable) was invoked. Four independent runs, each with four MCMC chains, were set for ten million generations, sampling every 10,000 generations. All analyses were performed using the CIPRES platform (Miller et al. 2010). Results were analyzed in Tracer 1.6 (Rambaut and Drummond 2007) to assess convergence and effective sample sizes (ESS) for all parameters, based on which the first 10% of trees were removed from each run. The remaining trees were used to calculate posterior probabilities (PP) for each bipartition in a Maximum Clade Credibility Tree. The phylogenetic trees were visualized and edited using FigTree v1.4. 2 (Rambaut 2014). In order to address interspecific genetic differentiation, uncorrected genetic distances were calculated in MEGA 7 (Kumar et al. 2016) after removing ambiguous positions for each sequence pair (pairwise deletion option).

Specimens and morphological data
We examined 98 specimens of Pholidobolus macbrydei (Appendix I) and 41 of the new species described herein (see corresponding type series). All specimens are deposited in the herpetological collection at Museo de Zoología, Pontificia Universidad Católica del Ecuador, Quito (QCAZ). The following measurements were taken with a digital caliper (to the nearest 0.1 mm): AGD axilla-groin distance; HL head length; HW head width; ShL shank length; SVL and snout-vent length.
Tail length (TL) was measured with a ruler. Sex was determined by dissection or by noting the presence of everted hemipenes. We followed the terminology of Montanucci (1973) and Kizirian (1996) (Peters 1964, Montanucci 1973. Hemipenes were prepared following the procedures described by Manzani and Abe (1988), as modified by Pesantes (1994) and Zaher (1999). Organs were everted after immersion in a potassium hydroxide solution, the retractor muscles were manually separated, and the everted organs filled with blue-stained petroleum jelly. Hemipenes were then immersed in an alcoholic solution of Alizarin Red for 24 hours in order to stain eventual calcified structures (e.g., spines or spicules), in an adaptation proposed by Nunes et al. (2012) on the procedures described by Uzzell (1973) and Harvey and Embert (2008). The terminology of hemipenial structures follows previous literature (Dowling and Savage 1960;Hurtado-Gómez et al. 2018;Nunes et al. 2012;Sánchez-Pacheco et al. 2017;Savage 1997;Venegas et al. 2016).

Systematics
The taxonomic conclusions of this study are based on the observation of morphological features and color pattern, as well as inferred phylogenetic relationships. We consider this information as species delimitation criteria following a general lineage or unified species concept (de Queiroz 1998;2007).
The new species share with all known species of Pholidobolus the presence of a ventrolateral fold between fore and hind limbs and the absence of a single transparent palpebral disc (Montanucci 1973).

Phylogenetic relationships and genetic distances
Tree topologies under ML and BI approaches were generally similar; here we describe the maximum clade credibility tree (Fig. 1). Our hypothesis supports the monophyly of Pholidobolus (BS= 60, PP = 0.99) and is congruent with previous molecular phylogenies in that P. ulisesi and P. hillisi form a clade (BS = 62, PP = 0.92) sister to all other congeners (Torres-Carvajal et al. 2015, 2016Hurtado-Gómez et al. 2018). Following branching order, the strongly supported species pair P. affinis, P. montium is sister to all remaining species, which form a clade where (P. prefrontalis (P. paramuno (P. dicrus, P. vertebralis))) is sister to a subclade containing the new species described in this paper and a paraphyletic P. macbrydei. Hereafter we refer to the latter subclade as the "P. macbrydei" species complex.

Morphological comparisons among species
Two components with eigenvalues > 1.0 were extracted from the PCA (Table 5). These components accounted for 50.7% of the total variation. The highest loadings corresponded to supratympanic temporals (NTS) and number of scales along margin of upper jaw (SUJ) for PC I, and number of scales around the tail (SAT) and number of scales around the body (SAB) for PC II (Table 5). In general, there is wide overlap in morphological space among species of the "P. macbrydei" complex ( Fig. 2).

Comparative hemipenial morphology
Hemipenes of holotypes of the four new species described herein are approximately 4-5 mm and 5−7 subcaudal scales long. The organs are fully everted in specimens of P. fascinatus, P. condor, and P. samek and partially everted in P. dolichoderes; the hemipenes of the holotype of P. fascinatus and P. condor are fully expanded, whereas the organs of P. dolichoderes and P. samek are partially expanded (Fig. 3). All hemipenes have two small lobes detached from the hemipenial body when the organ is fully everted. The hemipenis of P. condor presents a distinctive capitular groove originating at the median hemipenial body and extending toward the lobes. The lobes of P. fascinatus, P. condor, and P. samek present folds on their tips, which are not visible in P. dolichoderes due to the partial eversion. The hemipenial body is   Table 5 for character loadings on each component. cylindrical in P. dolichoderes and P. condor, whereas in P. samek and P. fascinatus the body is conical, with the basis distinctly thinner than the rest of the body. The sulcus spermaticus is broad in P. fascinatus, P. dolichoderes, and P. samek, narrower in P. condor; in P. fascinatus and P. condor, the sulcus spermaticus is deeper than in P. dolichoderes and P. samek. The sulcus originates medially at the base of the organ and extends in a straight line throughout the body towards the lobes in all species. However, unlike P. dolichoderes and P. condor, the sulcus originates between thick lips in P. samek and P. fascinatus. In all species, the sulcus spermaticus bifurcates at the lobular crotch, with each branch extending along the medial face of each lobe. The sides and borders of the sulcate and asulcate faces are ornamented with a series of roughly equidistant and chevron-shaped flounces, with the chevron vertices aligned medially on each side and directed proximally. All flounces bear calcified comb-like series of spicules, distinctively stained in red with Alizarin. The number of flounces extending along the hemipenial body varies slightly among species: 21 in P. condor and P. samek and 22 in P. dolichoderes and P. fascinatus. The base of the asulcate face bears three medial flounces in P. condor, P. dolichoderes, and P. samek, and four in P. fascinatus. All species have a conspicuous unevenness forming a bulge along the margins of the asulcate face. Description of holotype. Adult male (QCAZ 14955) (Figs 4, 5); SVL 46.7 mm; TL 80.9 mm; dorsal and lateral head scales juxtaposed, finely wrinkled; rostral hexagonal, 2.06 times as wide as high; frontonasal irregularly quadrangular, wider than long, laterally in contact with nasal, loreal and first superciliary, slightly bigger than frontal; prefrontal scales absent; frontal longer than wide, in contact with one supraocular on the left side, and two on the right side; frontoparietals pentagonal, longer than wide, slightly wider posteriorly, each in contact laterally with supraocular II; interparietal roughly heptagonal; parietals slightly bigger than interparietal, hexagonal, and positioned anterolaterally to interparietal, each in contact anteriorly with supraocular II (and supraocular III on right side) and dorsalmost postocular; postparietals three, medial scale smaller than laterals; seven supralabials, fourth one longest and below center of eye; six infralabials, fourth one shortest and below center of eye; temporals enlarged, irregularly hexagonal, juxtaposed, smooth; two large supratemporal scales, smooth; nasal slightly divided, irregularly pentagonal, longer than high, in contact with rostral anteriorly, first and second supralabials ventrally, frontonasal dorsally, loreal posterodorsally and frenocular posteroventrally; nostril on ventral aspect of nasal, directed lateroposteriorly; loreal rectangular, wider dorsally; frenocular higher than long, in contact with nasal, separating loreal from supralabials; two supraoculars on left side, three on right side (posteriormost much smaller), with the first one being the largest; four elongate superciliaries, first one enlarged, in contact with loreal; palpebral disc divided into four enlarged, pigmented scales; suboculars three (on the left side the medial subocular is fragmented), elongated and homogeneous in size; two postoculars, the dorsalmost wider than the other; ear opening vertically oval, without denticulate margins; tympanum recessed into a shallow auditory meatus; mental semicircular, wider than long; postmental pentagonal, slightly wider than long, followed posteriorly by three pairs of genials, the anterior two in contact medially and the posterior one separated by postgenials; all genials in contact with infralabials; gulars imbricate, smooth, posteriorly widened in two longitudinal rows; posterior row of gulars (collar) with six scales, the medial two widened.
Nuchal scales similar in size to dorsals, except for the anteriormost that are widened; scales on sides of neck small and granular; dorsal scales hexagonal, elongate, imbricate, arranged in transverse rows; scales on dorsal surface of neck striated, becoming progressively keeled from forelimbs to tail; number of dorsal scales between occipital and posterior margin of hindlimbs 27; dorsal scale rows in a transverse line at midbody 26; one longitudinal row of smooth, enlarged ventrolateral scales on each side; dorsals separated from ventrals by two rows of small scales at level of 13 th row of ventrals; lateral body fold between fore and hindlimbs present; ventrals smooth, wider than long, arranged in 20 transverse rows between collar fold and preanals; six ventral scales in a transverse row at midbody; subcaudals smooth; axillary region with granular scales; scales on dorsal surface of forelimb striated, imbricate; scales on ventral surface of forelimb granular; two thick, smooth thenar scales; supradigitals (left/right) 3/3 on finger I, 6/7 on II, 8/8 on III, 9/9 on IV, 6/6 on V; supradigitals 3/4 on toe I, 6/6 on II, 10/9 on III, 11/12 on IV, 7/7 on V; subdigital lamellae of fingers I and II single, paired on III (except the four distalmost), paired at base on IV, on finger V all single; subdigital lamellae 5/5 on finger I, 11/12 on II, 15/16 on III, 17/16 on IV, 9/10 on V; subdigital lamellae on toes I and II single, on toe III, IV and V all paired, except for the three distalmost subdigitals; subdigital lamellae 6/6 on toe I, 11/10 on II, 16/15 on III, 21/21 on IV, 14/14 on V; groin region with small, imbricate scales; scales on dorsal surface of hindlimbs smooth and imbricate; scales on ventral surface of hindlimbs smooth; scales on posterior surface of hindlimbs granular; femoral pores absent; preanal pores absent; cloacal plate paired, bordered by four scales anteriorly, of which the two medialmost are enlarged.
Color of holotype in life. Dorsal background from head to base of tail grayish brown, with a golden light brown vertebral stripe extending from occiput to tail; bright green dorsolateral stripes on head; cream white longitudinal stripe extending from first supralabial to shoulder; sides of neck, flanks and limbs dark brown; reddish brown narrow stripe extending from tympanum to arm insertion; ventrolateral region of body grayish brown; throat cream; chest, belly and base of tail cream orange (Figs 4C, 6B).
Color of holotype in preservative. Dorsal background uniformly grayish brown, with a golden-gray vertebral stripe extending from occiput to tail; vertebral stripe wider anteriorly, becoming slightly slender at most posterior part of body; dorsal and lateral surfaces of head brown (rostral, frontonasal, frontal, frontoparietals, and supraoculars); bluish white longitudinal stripe extending from first supralabial to shoulder and fading on flanks; ventrolateral aspect of neck dark brown with a dorsolateral light brown stripe extending posteriorly along flanks to hindlimbs; forelimbs with scattered ocelli (black with white center); flanks grayish brown with two dorsolateral stripes on each side, the dorsal one dark brown and the most ventral one brown diffuse with dark brown spots; tail brown dorsally; ventral surface of head gray, chest and venter dark gray, ventral surface of tail slightly gray, with scattered dark brown marks.
Distribution and natural history. Pholidobolus samek inhabits cloud forests in Cordillera del Cóndor, southeastern Ecuador at elevations between 2324−2844 m (Fig. 7). The new species is known only from Zamora-Chinchipe province, on the
Etymology. The specific epithet samek means green in the Shuar language, in allusion to the green dorsolateral head stripes distinguishing the new species from other congeners. The type locality of Pholidobolus samek lies within territory of Shuar indigenous people, who inhabit the Amazonian rainforest in Ecuador and Peru.
Remarks. Pholidobolus samek sp. nov. is very similar morphologically and genetically to P. condor sp. nov. These species can be easily distinguished from each other by coloration in adult males, although we recognize that our sample size is small (N = 7 and 4, respectively) and includes only one adult male per species. However, further evidence supports recognition of P. samek and P. condor as different species. First, they are reciprocally monophyletic and they are not sister taxa, with P. samek being sister to "P. macbrydei" Clade A (Fig. 1), which is very different in color patterns from either P. samek or P. condor (V. Parra and O. Torres-Carvajal, personal observation). Second, unlike the 12S gene (the less variable gene in this study), genetic distances between P. samek and P. condor for 16S and ND4 are not the lowest (Tables 2 and 4, respectively) within Pholidobolus. For example, the 16S distance between P. samek and P. condor (3%) is the same as the distance between the well-recognized species P. paramuno and P. affinis. In addition, genetic exchange among P. samek, P. condor and Clade A is very unlikely as they are isolated from each other on mountaintops above 2000 m (Fig. 7). Diagnosis. Pholidobolus condor is unique among its congeners, except P. samek sp. nov., in having green dorsolateral stripes on the head. However, adult males of P. condor differ from those of P. samek sp. nov. in having lighter dorsolateral head stripes, and reddish flanks and venter. In addition, P. ulisesi, P. dicrus, P. hillisi, and P. vertebralis differ from P. condor (character states of P. condor in parentheses) in having a conspicuous light vertebral stripe (light vertebral stripe absent). Pholidobolus affinis, P. prefrontalis, P. dicrus, P. hillisi, and P. vertebralis further differ from P. condor in having prefrontal scales (prefrontal scales absent). Additionally, P. condor sp. nov. has fewer dorsal scales (26−30) than P. affinis (45−55), P. montium (35−50), P. prefrontalis (37−46), P. macbrydei (31−43), and P. dolichoderes sp. nov. (35−40). Pholidobolus condor can be further distinguished from P. fascinatus sp. nov. by having widened medial scales on collar, and from P. dolichoderes sp. nov. by having fewer temporals (7-9 and 4-5, respectively), fewer ventrals (18-20 and 25-27), and fewer gulars (14-16 and 22-23).
Description of holotype. Adult male (QCAZ 15844) (Figs 9, 10); SVL 42.7 mm; TL 74.8 mm; dorsal and lateral head scales juxtaposed, finely wrinkled; rostral hexagonal, 1.67 times as wide as high; frontonasal quadrangular, slightly bigger than frontal, laterally in contact with nasal, loreal and first superciliary; prefrontal scales absent; frontal pentagonal, longer than wide, wider anteriorly, in contact with first superciliary and supraocular; frontoparietals hexagonal, longer than wide, slightly wider in the middle, each in contact laterally with supraocular II; interparietal octagonal, with a short medial suture posteriorly, lateral borders nearly parallel to each other; parietals larger than interparietal, hexagonal and positioned anterolaterally to interparietal, each in contact laterally with supraocular II and dorsalmost postocular; postparietals three, medial scale smaller than lateral ones; eight supralabials, fourth one longest and below center of eye; six infralabials, fourth one below center of eye; temporals enlarged, irregularly hexagonal, smooth; two A B C large and smooth supratemporals; nasal shield slightly divided above nostril, irregularly pentagonal, longer than high, in contact with rostral anteriorly, first and second supralabials ventrally, frontonasal dorsally, loreal posterodorsally and frenocular posteroventrally; nostril on ventral aspect of nasal, directed laterally; loreal quadrangular, slightly wider dorsally, not in contact with supralabials; frenocular higher than long, in contact with nasal; nasal separating loreal from supralabials; two supraoculars, anteriormost one the widest; four elongate superciliaries, anteriormost enlarged, in contact with loreal; palpebral disc divided into five pigmented scales; four suboculars, anteriormost three elongated and homogeneous in size, posteriormost widest; two postoculars, the dorsalmost wider than the other; ear opening vertically oval, without denticulate margins; tympanum recessed into a shallow auditory meatus; mental wider than long; postmental pentagonal, slightly wider than long, followed posteriorly by three pairs of genials, the anterior two pairs in contact medially and the third pair separated by postgenials; all genials in contact with infralabials; gulars imbricate, smooth, widened in two longitudinal rows; posterior row of gulars (collar) with nine scales, the medial three slightly widened.
Nuchal scales slightly smaller than dorsals, except for the anteriormost that are widened; scales on sides of neck small and granular; dorsal scales elongate, imbricate, arranged in transverse rows; scales on dorsal surface of neck striated, becoming progressively keeled from forelimbs to tail; dorsal scales between occipital and posterior margin of hindlimbs 27; dorsal scale rows in a transverse line at midbody 27; one longitudinal row of smooth, enlarged ventrolateral scales on each side; dorsals separated from ventrals by two rows of small scales at the level of 13 th row of ventrals; lateral body fold between fore and hindlimbs present; ventrals smooth, wider than long, arranged in 20 transverse rows between collar fold and preanals; six ventral scales in a transverse row at midbody; subcaudals smooth; axillary region with granular scales; scales on dorsal surface of forelimb striated, imbricate; scales on ventral surface of forelimb granular; two thick, smooth thenar scales; supradigitals (left/right) 3/3 on finger I, 6/6 on II, 8/8 on III, 9/9 on IV, 6/6 on V; supradigitals 3/3 on toe I, 6/6 on II, 9/9 on III, 12/12 on IV, 7/7 on V; subdigital lamellae of finger I, II, III, and V single, on finger IV few scales in the middle paired; subdigital lamellae 6/6 on finger I, 11/11 on II, 15/15 on III, 17/16 on IV, 10/10 on V; subdigital lamellae on toes I and II single, on toes III, IV and V paired, except for two or three distalmost subdigitals; subdigital lamellae 7/6 on toe I, 12/12 on II, 15/16 on III, 22/22 on IV, 12/12 on V; groin region with small, juxtaposed scales; scales on dorsal surface of hindlimbs striated and imbricate; scales on ventral surface of hindlimbs smooth; scales on posterior surface of hindlimbs granular; femoral pores absent; preanal pores absent; cloacal plate paired, bordered by four scales anteriorly, of which the two medialmost are enlarged.
Color of holotype in life. Dorsal background from head to base of tail dark brown, with a golden brown vertebral stripe extending from occiput to tail; greenish cream dorsolateral stripes on head, becoming light brown on posterior part of body; white longitudinal stripe extending from first supralabial to shoulder; sides of neck, flanks and limbs dark brown; chocolate brown narrow stripe extending from tympanum to arm insertion; ventrolateral region of body grayish brown; throat reddish cream; chest, belly, base of tail and lateral region of tail bright orange, with brown marks in some scales; ventral surface of hind limbs with orange diffuse marks (Fig. 9B,C).
Color of holotype in preservative. Dorsal background uniformly dark brown with a grayish brown middorsal stripe extending from occiput onto tail; dorsolateral stripe distinct, pale gray, extending from snout to near base of tail; head brown dorsally (rostral, frontonasal, frontal, frontoparietals and supraoculars) and dark brown laterally; white longitudinal stripe extending from first supralabial to forelimb; lateral aspect of neck dark brown with a dorsolateral light brown stripe extending posteriorly along flanks to hindlimbs; flanks grayish brown; tail dark brown dorsally and bronze laterally; ventral surface of head gray, with dirty cream genials and scattered black marks; chest, belly and ventral surface of tail light gray with light red spots; ventral surface of limbs dark gray (Fig. 9A).
Variations. Measurements and scale counts of Pholidobolus condor are presented in Table 6. Supraoculars three on left side in specimen QCAZ 16789; supralabials six in QCAZ 16789 and 16790, and seven in QCAZ 16788; two quadrangular frontonasals in QCAZ 16788; transverse rows of ventral scales between collar fold and preanals 18 in QCAZ 16788 and 19 in QCAZ 16790. Hatchlings with eight (QCAZ 16788-89) or six (QCAZ 16790) posterior gular (collar) scales. Unlike the adult male, hatchlings lack reddish color on tail.
Distribution and natural history. Pholidobolus condor occurs in Cordillera del Cóndor in southeastern Ecuador at elevations between 1994-2226 m. The new species is known from El Quimi Biological Reserve in Morona Santiago province (Fig. 7). The holotype was found active at 21h14 at the base of a bromeliad on a sandstone plateau of shrub vegetation (Fig. 11).
Several eggs were found within a bromeliad, suggesting that females of P. condor lay their eggs in communal nests. Four eggs that were found on the ground at the base of bromeliads and under a trunk were incubated in sphagnum and perlite in captivity for approximately three months. On average, hatchlings weighted 0.4 g and were 23.7 mm in SVL.
Conservation status. Pholidobolus condor is only known from Cordillera del Cóndor in southeastern Ecuador. This area is currently threatened by mining activities (Ron et al. 2018;Valencia et al. 2017;Van Teijlingen 2016). Habitat destruction and fragmentation is evident at a distance of ~11 km from the collection sites (Mazabanda et al. 2018). Because of the small known distribution and habitat disturbance, we suggest assigning P. condor to the Critically Endangered category under criteria B1a, b(iii); C1; D, according to IUCN (2012) guidelines.
Etymology. The specific epithet condor refers to Cordillera del Cóndor, where the new species was discovered. The Cordillera del Cóndor is an eastern outlier of the main Andean chain, where a significant number of species have been discovered in the last decade (Brito et al. 2017;Huamantupa-Chuquimaco and Neill 2018;Ron et al. 2018;Torres-Carvajal et al. 2009;Valencia et al. 2017).
Description of holotype. Adult male (QCAZ 16353) (Figs 12, 13); SVL 41.1 mm; TL 96.3 mm; dorsal and lateral head scales imbricated, smooth; rostral hexagonal, 1.75 times as wide as high; frontonasal heptagonal, slightly wider than long, laterally in contact with nasal, similar in size to frontal; prefrontals present, in wide contact medially, and in contact with loreal and first superciliary laterally; frontal hexagonal, longer than wide, wider anteriorly, in contact with first and second supraoculars; frontoparietals hexagonal, longer than wide, slightly wider posteriorly, each in contact with second and third supraoculars, parietals and interparietal; interparietal heptagonal, lateral borders nearly parallel to each other; parietals wider than interparietal, heptagonal, and positioned anterolaterally to interparietal, each in contact with third supraocular and dorsalmost postocular; postparietals three, medial scale smaller than lateral ones; seven supralabials, fourth one the longest and below center of eye; five infralabials, fourth one below center of eye; temporals small, irregularly, smooth; supratemporal scales not well differentiated, smooth; nasal shield divided above the nostril, longer than high, in contact with rostral anteriorly, first and second supralabials ventrally, frontonasal dorsally, loreal posteriorly; loreal pentagonal, slightly wider dorsally, in contact with second and third supralabials; frenocular longer than high, in contact with loreal; three supraoculars, with the first one being the widest; four elongate superciliaries, anteriormost one enlarged, in contact with loreal; palpebral disc oval, pigmented, divided into four scales; four suboculars, two elongated and similar in size, the anteriormost and posteriormost larger than the others; three postoculars, dorsalmost wider than the others; ear opening vertically oval, without denticulate margins; tympanum recessed into a shallow auditory meatus; mental wider than long; postmental pentagonal, slightly wider than long, followed posteriorly by three pairs of genials, the anterior two pairs in contact medially and the third pair separated by postgenials; all genials in contact with infralabials; gulars imbricate, smooth, widened in two longitudinal rows; gular fold complete, posterior row of gulars (collar) with six scales, the medial two distinctly widened.
Nuchal scales slightly smaller than dorsals, except for the anteriormost that are widened; scales on sides of neck small and granular; dorsal scales elongate, juxtaposed, arranged in transverse rows; scales on dorsal surface of neck striated, becoming slightly keeled from forelimbs to tail; dorsal scales between occipital and posterior margin of hindlimbs 35; dorsal scale rows in a transverse line at midbody 32; one longitudinal row of smooth, enlarged ventrolateral scales on each side; dorsals separated from ven-trals by three rows of granular scales at level of 13 th row of ventrals; lateral body fold between fore and hindlimbs poorly defined; ventrals smooth, arranged in 26 transverse rows between collar fold and preanals; six ventral scales in a transverse row at midbody; subcaudals smooth; axillary region with granular scales; scales on dorsal surface of forelimb smooth, imbricate; scales on ventral surface of forelimb granular; two thick, smooth thenar scales; supradigitals (left/right) 3/0 on finger I, 7/7 on II, 9/8 on III, 10/10 on IV, 5/5 on V; supradigitals 4/4 on toe I, 7/7 on II, 11/11 on III, 12/11 on IV, 9/8 on V; subdigital lamellae of fingers I and II mostly single, III and IV paired proximally, on finger V all single; subdigital lamellae 5 on left finger I (right finger missing), 10/10 on II, 14/14 on III, 14/14 on IV, 9/9 on V; subdigital lamellae on toe I single, on toe II paired at the middle, on toe III and IV paired along proximal half, and on toe V paired proximally; subdigital lamellae 5/5 on toe I, 10/10 on II, 14/14 on III, 18/19 on IV, 11/11 on V; groin region with small, imbricate scales; scales on dorsal surface of hindlimbs striated and imbricate; scales on ventral surface of hindlimbs smooth; scales on posterior surface of hindlimbs granular; femoral pores present, three on left leg and five on right leg; preanal pores absent; cloacal plate paired, bordered by four scales anteriorly, of which the two medialmost are enlarged.
Color of holotype in life. Dorsal background of head dark brown; diffuse pale brown vertebral stripe that becomes grayish brown towards tail; creamy white dorsolateral stripes on head extending posteriorly and fading away at midbody; white longitudinal stripe extending from first supralabial to shoulder; sides of neck brown; flanks grayish brown with diffuse dark brown marks; limbs brown; ventrolateral region of body grayish brown; throat and chest cream; belly grayish cream; base of tail gray with dark little spots (Figs 12, 14B).
Color of holotype in preservative. Dorsal background uniformly brown with a diffuse light brown vertebral stripe extending from occiput onto tail, but fading at posterior end of body; dorsal and ventral surface of head brown; flanks light brown, with scattered dark brown spots; head and neck with two distinct white longitudinal stripes, the ventral one extending from first supralabial to forelimb, and the dorsal one from canthus rostralis to scapular region, posterior to which if fades into a light brown stripe; lateral aspect of neck dark brown; tail grayish brown; gular, chest and venter regions pale gray; ventral surface of tail and limbs gray.
Adult females differ from holotype in having a grayish brown vertebral stripe, fading away posteriorly, and grayish brown flanks (Fig. 14). Juvenile QCAZ 16350 differs from holotype in having grayish brown flanks, without scattered dark brown spots; juvenile QCAZ 16351 is unique in having white spots on flanks and over forelimbs.
Distribution and natural history. Pholidobolus dolichoderes is known to occur between 2506−2675 m in San Felipe de Oña, southwestern Azuay province (Fig. 7). This area is composed of many different landscapes including small valleys, desert areas and wet paramo. Most specimens were found active at day (10h26-15h30), mostly on the ground or near spiny ground bromeliads known as achupallas (Puya sp.).
Conservation status. Pholidobolus dolichoderes is only known from unprotected localities around Oña. The population size of this species is unknown, but our sampling suggests low abundances. Because of the small known distribution and lack of additional data, we suggest assigning P. dolichoderes to the Data Deficient category according to IUCN (2012) guidelines.
Description of holotype. Adult male (QCAZ 15120) (Figs 15, 16); SVL 52.5 mm; TL 37.6 mm; dorsal and lateral head scales juxtaposed, finely wrinkled; rostral hexagonal, 2.27 times as wide as high; frontonasal hexagonal, wider than long, in contact with nasal laterally, slightly larger than frontal; prefrontal scales irregularly pentagonal; frontal heptagonal, longer than wide, slightly wider anteriorly, in contact with prefrontals and frontonasal anteriorly, two supraoculars laterally, and frontoparietals posteriorly; frontoparietals pentagonal, longer than wide, slightly wider posteriorly, each in contact laterally with supraocular II; interparietal heptagonal, lateral borders nearly parallel to each other; parietals hexagonal, each in contact laterally with supraocular II and dorsalmost postocular; postparietals four, with medial scales less than half the size of lateral ones; eight supralabials, fourth one the longest and below center of eye; eight infralabials, third and fourth one below center of eye; temporals enlarged, irregularly hexagonal, juxtaposed, smooth; two large, smooth supratemporal scales; nasal divided, irregularly pentagonal, longer than high, in contact with rostral anteriorly, first and second supralabials ventrally, frontonasal dorsally, loreal posterodorsally and frenocular posteroventrally; nostril in center of nasal, directed lateroposteriorly; loreal rectangular, wider ventrally; frenocular longer than high, higher anteriorly, in contact with nasal, separating loreal from supralabials; two supraoculars, homogeneous in size; four superciliaries, anteriormost enlarged and in contact with loreal; palpebral disc divided into five pigmented scales; suboculars elongated, four on right side and three on left side; two postoculars, dorsalmost wider than the other; ear opening vertically oval, without denticulate margins; tympanum recessed into a shallow auditory meatus; mental semicircular, longer than wide; postmental pentagonal, slightly longer than wide, followed posteriorly by three pairs of genials, the anterior two in contact medially and the posterior one separated by postgenials; all genials in contact with infralabials; gulars imbricate, smooth, widened in two longitudinal rows; posterior row of gulars (collar) with 11 scales that are similar in size.
Nuchal scales similar in size to dorsals, except for the anteriormost that are widened; scales on sides of neck small and slightly granular; dorsal scales hexagonal, elongate, imbricate, arranged in transverse rows; scales on dorsal surface of neck smooth, becoming progressively striated from forelimbs to tail; dorsal scales between occipital and posterior margin of hindlimbs 33; dorsal scale rows in a transverse line at midbody 25; dorsals separated from ventrals by one row of small scales at level of 13 th row of ventrals; lateral body fold between fore and hindlimbs present; ventrals smooth, wider than long, arranged in 25 transverse rows between collar fold and preanals; six ventral scales in a transverse row at midbody; subcaudals smooth; axillary region with granular scales; scales on dorsal surface of forelimb striated, imbricate; scales on ventral surface of forelimb granular; two thick, smooth thenar scales; supradigitals (left/right) 3/3 on finger I, 7/6 on II, 8/8 on III, 10/10 on IV, 5/5 on V; supradigitals 3/3 on toe I, 6/6 on II, 8/9 on III, 11/11 on IV, 8/8 on V; subdigital lamellae of finger I single, on finger II all paired, except by the three distalmost, on finger III (proximal half ) paired, on finger IV slightly paired at the middle, on finger V all single in right finger and three paired in left finger; subdigital lamellae 5/5 on finger I, 9/9 on II, 13/13 on III, 14/15 on IV, 9/9 on V; subdigital lamellae on toes I and II paired proximally and single distally, on toes III, IV and V paired, except for the three to five distalmost subdigitals; subdigital lamellae 5/5 on toe I, 10/10 on II, 14/13 on III, 18/18 on IV, 11/12 on V; groin region with small, imbricate scales; scales on dorsal surface of hindlimbs smooth and imbricate; scales on ventral surface of hindlimbs smooth; scales on posterior surface of hindlimbs granular; femoral pores absent; preanal pores absent; cloacal plate paired, bordered anteriorly by two enlarged scales.
Color in life of the holotype. Dorsal background from head to base of tail brown, with a diffuse chocolate-brown middorsal stripe that fades away towards tail; light brown dorsolateral stripes on head extending posteriorly and fading away at midbody; white longitudinal stripe extending from third supralabial to shoulder; sides of neck, flanks, and limbs brown; reddish brown narrow stripe extending from tympanum to arm insertion; ventrolateral region of body grayish brown; throat and chest gray; belly background gray with conspicuous orange marks; tail orange anteriorly and laterally (Figs 15, 17A). Color in preservative of the holotype. Dorsal background uniformly brown with a cream brown vertebral stripe extending from head onto tail; vertebral stripe slender anteriorly, becoming slightly wider posteriorly; head light brown with black dots dorsally (rostral, frontonasal, frontal, frontoparietals and supraoculars) and brown laterally; cream longitudinal stripe extending from third supralabial to shoulder; ventrolateral aspect of neck brown; forelimbs with scattered black dots; flanks brown; tail brown dorsally; ventral surface of head light gray, chest and venter dark gray, ventral surface of tail slightly brown, with scattered dark brown marks.
Variations. Measurements and scale counts of Pholidobolus fascinatus are presented in Table 6. Supralabials 9/9 (left/right) in specimens QCAZ 15118 and 15122, and supraoculars 3/3 in QCAZ 15118; loreal scale absent in QCAZ 15118; prefrontals absent in QCAZ 15122 and 15173; little intrusive scales between postparietal and frontoparietals in QCAZ 15118, 15121 and 15122; frontonasal quadrangular in QCAZ 15122; frontal nonagonal and pentagonal in QCAZ 15118 and 15173, respectively; interparietal hexagonal in QCAZ 15122; parietal pentagonal in QCAZ 15170. Four posterior cloacal scales in QCAZ 15118. Males are slightly smaller (SVL 47.6 mm, N = 2) than female (maximum SVL 48.2 mm, N = 2). Adult male QCAZ 15122 differs from holotype in having sides of tail and chest dark brown without gray spots. Adult female QCAZ 15118 differs from holotype in having a light gray chest, a dark gray ventral surface of tail, dark brown sides of tail, and in lacking orange or red brown color on sides of neck (Fig. 17).
Distribution and natural history. Pholidobolus fascinatus inhabits wet paramo in the western slopes of the Andes of southern Ecuador (Fig. 7). The new species is known only from El Oro province, at 3348−3382 m. All specimens were found active at 14h00-17h00 mostly under stones.
We found 41 eggs (17 as fragmented eggshells) in a communal nest next to male QCAZ 15120. We incubated the 24 unhatched eggs in soil and perlite in captivity. They were 11.9-13.2 mm long, 5.5-9.2 mm wide, and weighted 0.5 g on average. Hatchlings (N = 20) weighted 0.4 g and were 26.2 mm in SVL on average. Conservation status. Pholidobolus fascinatus is only known from localities around Lake Chillacocha. The population size for this species is unknown, but our sampling suggests average abundances. Because of the small known distribution and lack of additional data, we suggest assigning P. fascinatus to the Data Deficient category, according to IUCN (2012) guidelines.
Etymology. The species epithet fascinatus is a Latin word meaning enchanted, in allusion to Lake Chillacocha, also known as the Enchanted Lake. According to local belief, this lake is enchanted and has healing powers.

Discussion
The systematics of Pholidobolus and its sister taxon Macropholidus have been controversial partly because morphological evidence has been misinterpreted. Nonetheless, the recent use of molecular phylogenies has reshaped the systematics and taxonomy of this clade (Torres-Carvajal and Mafla-Endara 2013; Torres-Carvajal et al. 2015). In addition, recent collections in poorly explored areas along the Andes of Colombia, Ecuador, and Peru have led to the discovery and description of new species (Hurtado-Gómez et al. 2018;Torres-Carvajal et al. 2014;Venegas et al. 2016). In this paper we use morphological and molecular evidence to describe four new species of Pholidobolus, all except P. dolichoderes sp. nov. from remote highlands, based mostly on recent collections in southern Ecuador. Unexpectedly, allocating the new species described herein within the phylogenetic tree of Pholidobolus rendered P. macbrydei paraphyletic, suggesting that populations currently assigned to this taxon represent multiple species, some of which (e.g., Clades A and F) match the evolutionary significant units identified by Mafla-Endara (2011). Nonetheless, we refrain from describing any of these putative species (Clades A-F) in this paper as we believe that further sampling and analysis are necessary. According to our PCA results, three of the four new species are morphologically different from other "Pholidobolus macbrydei" (Fig. 2). Components I and II in the PCA, however, explain less than 50% of the variation within the "P. macbrydei" clade (Table 5). Thus, it is necessary to study additional morphological characters and increase sample size to better elucidate morphological differences. Mafla-Endara (2011) also suggested hybridization between P. macbrydei from Cañar province and P. prefrontalis based on both the relatively great variation in morphology within the Cañar populations, and their morphological similarity to P. prefrontalis. Nevertheless, our nuclear phylogenetic tree does not suggest hybridization between P. macbrydei and P. prefrontalis (Appendix II).
Current evidence prevents us from assigning the name P. macbrydei to any of the recovered clades. However, we suspect that P. macbrydei belongs or is more closely related to Clades C, D, and E for two reasons (Fig. 1). First, adult males in these clades match closely the description of P. macbrydei (Montanucci 1973). Second, Clades C, D, and E lie nearby the type locality of P. macbrydei. It is noteworthy that Clade B also lies near the type locality of P. macbrydei (Fig. 7), although males in Clade B lack the red lateral stripes characteristic of P. macbrydei. DNA samples from the type locality should help clarify the taxonomy of this group.
The Cordillera del Cóndor is a sub-Andean mountain chain geologically similar to the Tepuis of the Guiana region. It is composed of marine and continental sediments (Neill 2005). This area is presently threatened by mining activities, despite discovery of a significant number of new species in the last ten years (Brito et al. 2017;Huamantupa-Chuquimaco and Neill 2018;Mashburn et al. 2020;Ron et al. 2018;Torres-Carvajal et al. 2009;Valencia et al. 2017) suggesting that Cordillera del Cóndor is a diversity hotspot. Our discovery of P. samek and P. condor further supports this idea. Therefore, we strongly advise authorities to improve conservation efforts for Cordillera del Cóndor.
The discovery of four new species and a paraphyletic P. macbrydei reveals high levels of unexpected diversity within Pholidobolus from southern Ecuador. This study supports the idea that Andean herpetofauna in this region is more diverse in species numbers than previously thought (Sánchez-Pacheco et al. 2012), especially for poorly explored areas like Cordillera del Cóndor. Collections in this area are usually scarce due to complex logistics. However, we recommend more intensive sampling efforts. Future studies should include larger samples and other types of evidence (e.g., genomic data, environmental variables) that might prove useful for species delimitation within Pholidobolus and other vertebrate taxa.