A new species of Alopoglossus lizard (Squamata, Gymnophthalmidae) from the tropical Andes, with a molecular phylogeny of the genus

Abstract We describe a new species of Alopoglossus from the Pacific slopes of the Andes in northern Ecuador based on morphological and molecular evidence. The new species differs most significantly from all other congeners in having a double longitudinal row of widened gular scales, lanceolate dorsal scales in transverse rows, 29–32 dorsal scales in a transverse row at midbody, and 4 longitudinal rows of ventrals at midbody. It is most similar in morphology to A. festae, the only species of Alopoglossus currently recognized in western Ecuador. We analyze the phylogenetic relationships among species of Alopoglossus based on the mitochondrial gene ND4. Cis-Andean [east of the Andes] and Trans-Andean [west of the Andes] species are nested in two separate clades, suggesting that the uplift of these mountains had an important effect in the diversification of Alopoglossus. In addition, we present an updated key to the species of Alopoglossus.


Introduction
The New World lizard clade Gymnophthalmidae Merrem 1820 includes 241 extant species assigned to 46 taxa traditionally ranked as genera (Uetz 2014). One of them is Alopoglossus, which differs from other gymnophthalmid genera except Ptychoglossus in having the dorsal surface of the tongue completely covered with anteromedially converging plicae rather than scale-like papillae (Harris 1994;Hoogmoed and Avila-Pires 1992). Alopoglossus differs from Ptychoglossus (character states for Ptychoglossus in parentheses) in having keeled scales on forelimbs (smooth forelimb scales), and rhomboid, laterally imbricating dorsal scales (parallel-sided dorsal scales; Harris 1994).
The close relationship between Alopoglossus and Ptychoglossus suggested by Harris (1994) based on morphological similarities has been corroborated by phylogenetic analyses of DNA sequence data; these genera are sister taxa and form the clade Alopoglossinae (Castoe et al. 2004). Moreover, this clade seems to be sister to all other gymnophthalmids (Pellegrino et al. 2001;Castoe et al. 2004;Trefaut et al. 2007) as first suggested by Harris (1994). Therefore, studying the phylogenetic systematics of Alopoglossinae is crucial to understand the evolution of gymnophthalmid lizards.
Alopoglossus includes six currently recognized species (A. angulatus, A. atriventris, A. buckleyi, A. copii, A. festae, and A. lehmanni) widely distributed across tropical South America (Köhler et al. 2012). Of these, only A. lehmanni (endemic to Colombia) does not occur in Ecuador; A. festae occurs west of the Andes, whereas the remaining species occur east of the Andes (Köhler et al. 2012;Torres-Carvajal et al. 2014;Torres-Carvajal 2001). In this paper we describe a new species of Alopoglossus from northwestern Ecuador and infer its phylogenetic affinities to other species in the genus as currently understood.

Morphological data
All type specimens of the new species described in this paper are listed in the type series below, and were deposited at the Museo de Zoología, Pontificia Universidad Católica del Ecuador, Quito (QCAZ). Specimens of other species of Alopoglossus examined in this study are listed in the Appendix. All measurements were made with digital calipers and recorded to the nearest 0.01 mm: head length (HL), head width (HW), shank length (ShL), axilla-groin distance (AGD), lateral neck scale size (ANS), snout-vent length (SVL), and tail length (TL). Each measurement was taken twice and averaged. Sex was determined by noting the presence of everted hemipenes or by dissection. We follow the terminology of Avila-Pires (1995) and Köhler et al. (2012) for measurements and squamation.

DNA sequence 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 lysis buffer and digested overnight prior to extraction. DNA samples were quantified using a Nanodrop® ND-1000 (NanoDrop Technologies, Inc), re-suspended and diluted to 25 ng/ul in ddH2O prior to amplification.
We amplified 596 nucleotides (nt) of the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) from one individual each of Alopoglossus angulatus, A. atriventris, A. buckleyi, A. copii, A. festae, and the new species described herein. ND4 was amplified using the primers ND4F and ND4R (Pellegrino et al. 2001). Additionally, we used sequences of A. angulatus (erroneously identified as A. copii in Pellegrino et al. 2001), Bachia flavescens, Ecpleopus gaudichaudii, Heterodactylus imbricatus, Ptychoglossus brevifrontalis, Rhachisaurus brachylepis and Riama unicolor from GenBank. Gene regions of taxa included in phylogenetic analyses along with their GenBank accession numbers and locality data are shown in Table 1. Amplification of genomic DNA consisted of an initial cycle at 96 °C for 3 min, followed by 40 cycles of a denaturation at 95 °C for 30 s, annealing at 52 °C for 1 min, and extension at 72 °C for 1 min, as well as a final extension at 72 °C for 10 min.

Phylogenetic analyses
Editing, assembly, and alignment of sequences were performed with Geneious ProTM 5.5 (Drummond et al. 2010). Phylogenetic relationships were assessed under a Bayesian approach in MrBayes 3.2.0 (Ronquist and Huelsenbeck 2003). The data matrix was partitioned by codon. The model of character evolution for each partition was obtained in JModeltest (Posada 2008) under the Bayesian information criterion. Four independent analyses were performed to reduce the chance of converging on a local optimum. Each analysis consisted of ten million generations and four Markov chains with default heating values. Trees were sampled every 1,000 generations resulting in 10,000 saved trees per analysis. Stationarity was confirmed by plotting the log-likelihood scores per generation in the program Tracer 1.2 (Rambaut et al. 2013). Additionally, the standard deviation of the partition frequencies and the potential scale reduction factor (Gelman and Rubin 1992) were used as convergence diagnostics for the posterior probabilities of bipartitions and branch lengths, respectively. Adequacy of mixing was assessed by examining the acceptance rates for the parameters in MrBayes and the effective sample sizes (ESS) in Tracer. After analyzing convergence and mixing, 1,000 trees were discarded as "burn-in" from each run. We then confirmed that the four analyses reached stationarity at a similar likelihood score and that the topologies were similar, and used the resultant 36,000 trees to calculate posterior probabilities (PP) for each bipartition on a 50% majority rule consensus tree. Interspecific sequence divergence was assessed with uncorrected distances, which were obtained in PAUP* (Swofford 2003).

Results
The taxonomic conclusions of this study are based on the observation of morphological features and color patterns, as well as inferred phylogenetic relationships. We consider this information as species delimitation criteria following the general species concept (de Queiroz 1998(de Queiroz , 2007.  Diagnosis. Alopoglossus viridiceps can be distinguished from all other known congeners except A. festae by having a double longitudinal row of widened gular scales and lanceolate dorsal scales in transverse rows. From A. festae (character states in parentheses, taken from Köhler et al. 2012), the new species differs in having 29-32 dorsal scales in a transverse row at midbody (16-24, mean = 19.14 ± 2.25), four ventral scales in a transverse row at midbody (six), and a distinct longitudinal light stripe from mouth commissure to shoulder (Fig. 3). Scale counts and measurements of A. festae and A. viridiceps are presented in Table 2.
Rostral hexagonal, 2.08 times as wide as high, visible from above, in broad contact with frontonasal. Frontonasal irregularly pentagonal, wider than long, laterally in contact with nasal. Prefrontals irregularly pentagonal, nearly as wide as long, with medial suture; laterally in contact with nasal, loreal, and first and second supraocular. Frontal irregularly hexagonal, nearly twice as long as wide, slightly wider anteriorly; at each side in contact with supraoculars II-III. Frontoparietals irregularly pentagonal, longer than wide, with a wide medial suture; each in contact with supraoculars III-IV. Interparietal pentagonal, lateral borders parallel to each other. A pair of irregularly hexagonal parietals, approximately as wide and as long as interparietal. Interparietal and parietals forming slightly undulating posterior head margin. Occipitals absent. Four supraoculars, first one smallest and second one largest. Four elongate superciliaries, first one widest, followed by a postsuperciliary scale, which is also in contact with supraocular IV and anterior supratemporal. Nasal divided, irregularly pentagonal, longer than wide, in contact with rostral anteriorly, first and second supralabials ventrally, frontonasal and prefrontals dorsally, loreal posterodorsally, and frenocular posteroventrally. Nostril in lower part of nasal, directed lateroposteriorly. Loreal small, quadrangular. Frenocular in contact with nasal, separating loreal from supralabials. Three suboculars, the one below eye very elongated (nearly three times the size of adjacent suboculars). Posterior subocular continuous with three postoculars. Semitransparent disc in lower eyelid with vertical sections delimiting six large scales on right side and five scales on left side. Five supralabials, third one longest  and below center of eye. Two postsupralabials. Temporals small, irregularly polygonal, juxtaposed, keeled. Two large supratemporal scales, posterior one keeled. Ear opening vertically oval, without denticulate margins. Tympanum recessed into a short auditory meatus. All dorsal and lateral head scales juxtaposed. Interparietal and parietals with lateral ridges, other dorsal head scales smooth. Mental trapezoidal, anterior margin nearly  forming a semicircle. Postmental irregularly heptagonal, wider than long. Four infralabials, third one longest and below center of eye. One postinfralabial. Three pairs of chin shields, first two in contact medially and with infralabials; third one in contact medially but separated from infralabials. Third pair of chin shields separated from gulars by two transverse rows of scales. Anterior row composed laterally by two scales (one on each side) similar in size to the scales on the posterior row, and medially by two enlarged scales (not in contact medially) similar in size to the enlarged gulars. Gulars imbricate, smooth, in four longitudinal rows, the medial double row formed by five pairs of distinctly widened scales. Posterior row (collar) with five scales, the medial three distinctly widened (Fig. 2).
Scales on nape similar to dorsals, except that anterior ones are shorter. Scales on sides of neck small, keeled and mostly granular. Dorsals and scales on flanks lanceolate, strongly keeled and mucronate, imbricate, in transverse rows; number of scales along a middorsal line from nape to base of tail 30; transversal dorsal count 31. Ventrals smooth, imbricate, with round posterior margin; 18 in a longitudinal count; four in a transverse count. Scales on flanks similar to dorsals. One femoral pore on each side, in preanal position, separated from each other by four ventral scales. Scales on tail keeled, slightly mucronate, imbricate; in transverse and longitudinal rows; dorsal keels sharp, forming four distinct longitudinal ridges. Scales on limbs mostly rhomboidal, imbricate, sharply keeled, and mucronate; smooth on ventral aspect of hind limbs, small and keeled or tuberculate on ventral aspect of upper arms and posterior aspect of thighs. Subdigital lamellae of fingers and toes single, transversely enlarged and smooth; 20 under fourth toe.
Color in life of holotype (Fig. 1). Dorsal background uniformly dark brown with a wide light brown vertebral stripe extending from occiput onto tail; vertebral stripe wider anteriorly; dorsal surface of head bright metallic green medially (rostral, frontonasal, prefrontals, frontal and frontoparietals) and dark brown laterally (supraoculars and supratemporals), with a lateral bright green stripe on each side extending posteriorly from the border between the loreal and the first supraocular, over the superciliaries, to the lateral border of the parietal; lateral aspect of neck with a longitudinal yellowish-green stripe extending posteriorly from mouth commissure, over ventral margin of tympanum, to shoulder; most scales between lateral neck stripe and gular region reddish brown forming a short irregular stripe between last infralabial and shoulder; ventral surface of head light green, brighter laterally; gular and pectoral regions same tone as chin shields but lighter; ventral aspect of body orange with scattered light green or light blue small marks; ventral aspect of tail with dark brown marks that form transverse bars on the posterior half.
Variation. Intraspecific variation in scale counts and measurements in Alopoglossus viridiceps sp. n. is presented in Table 2. Color in preservative of holotype is similar to its color in life, except that the bright green tones of the head and orange tones of the venter have faded away.
Color in life of juvenile paratypes QCAZ10671, QCAZ11854-55 is similar to that of the holotype except that these juveniles have a reddish-brown longitudinal stripe extending from the dorsal border of the tympanum to the shoulder and fading away on the flanks (Fig. 3). The orange ventral coloration of male juvenile QCAZ11854 does not extend onto tail as in the holotype; female juvenile QCAZ10671 and juvenile QCAZ11855 (undetermined sex) have a light yellowish green background color on the venter, similar to that on gular region and chin (Fig. 3).
Distribution and ecology. Alopoglossus viridiceps sp. n. inhabits cloud forests on the Pacific slopes of the Andes in northwestern Ecuador (Fig. 4). It occurs at elevations of 1246-1911 m in the province of Pichincha. Most type specimens were collected at   Table 1. Santa Lucía Cloud Forest Reserve, which extends between 1400-2560 m and has an area of 756 ha; annual precipitation ranges from 1500 to 2800 mm, and average annual temperature is recorded at 16 °C (Rivas-Martínez and Navarro 1995). Specimens of A. viridiceps sp. n. were found active between 9h30-11h30 on leaf litter in primary forest, or on the border of sugar cane plantations. Other species of small ground lizards collected in the same area include the sphaerodactylid gecko Lepidoblepharis conolepis, the gymnophthalmids Cercosaura vertebralis and Echinosaura brachycephala, as well as an undescribed species of the gymnophthalmid genus Riama. Etymology. The specific epithet viridiceps is an adjective derived from the Latin words "viridis" and "ceps", which mean "green" and "head", respectively. It refers to the distinctive bright green coloration of the dorsal and ventral aspects of the head of Alopoglossus viridiceps sp. n.
Phylogenetic relationships. Of the 596 nucleotide characters included in our analysis 290 were constant, 70 parsimony uninformative, and 236 were parsimony informative.
Uncorrected genetic distances for ND4 are presented in Table 3. Distance values between Ptychoglossus brevifrontalis and species of Alopoglossus ranged between 0.197-0.225. The genetic distance between A. viridiceps sp. n. and its sister species A. festae (0.124) is slightly lower than all other interspecific distance values within Alopoglossus (0.148-0.185). A. angulatus, the only species for which we had two samples, had an intraspecific distance value of 0.06.

Discussion
The phylogenetic tree presented in this paper (Fig. 5) supports strongly the monophyly of Alopoglossus and its sister taxon relationship with Ptychoglossus, as suggested by previous authors based on morphological evidence (Harris 1994). The basal split between cis-Andean and trans-Andean species of Alopoglossus suggests that the uplift of the Andes represented an important event that allowed allopatric speciation in Alopoglossus, whether resulting from dispersal or vicariance.
Alopoglossus viridiceps sp. n. can be distinguished readily from its sister species A. festae based on morphological and color characters (see Diagnosis). A. festae occurs as close as 30 km W from the known distribution of A. viridiceps sp. n. below 1000 m (Fig. 4), suggesting that these species originated by allopatric or parapatric speciation. Although we did not attempt to examine variation within A. festae, we found differences among some populations of this species that suggest it might represent a species complex as currently circumscribed. A more detailed systematic study of A. festae, as well as all species of Alopoglossus east of the Andes in Ecuador with extensive geographic sampling is underway.
Alopoglossus viridiceps sp. n. is one of two new species of lizards that have been discovered recently in the same area. The other one is an undescribed species of the gymnophthalmid genus Riama. These discoveries indicate that the herpetofauna of the cloud forests in this region is more diverse in species numbers than previously thought. We recommend increasing field surveys in this region as it includes several protected areas (e.g., Santa Lucía Cloud Forest Reserve, Mindo-Nambillo Protected forest, Maquipucuna Reserve, El Cedral Ecolodge) that provide an opportunity to find species of lizards that might not occur elsewhere.