A new species of Liolaemus related to L. nigroviridis from the Andean highlands of Central Chile (Iguania, Liolaemidae).

The Liolaemus nigroviridis group is a clade of highland lizards endemic to Chile. These species are distributed from northern to central Chile, and currently there are no cases of sympatric distribution. This study describes a new species, Liolaemus uniformis sp. n., from this group, and provides a detailed morphological characterization and mitochondrial phylogeny using cytochrome-b. Liolaemus uniformis was found in sympatry with Liolaemus nigroviridis but noticeably differed in size, scalation, and markedly in the color pattern, without sexual dichromatism. This new species has probably been confused with Liolaemus monticola and Liolaemus bellii, both of which do not belong to the nigroviridis group. The taxonomic issues of this group that remain uncertain are also discussed.

Introduction

The group is a clade of highland lizards endemic to central and northern Chile, the species of which are allopatrically distributed (Pincheira-Donoso and Núñez 2005). Almost all species of this group have a complicated taxonomic history with several cases of synonymies (e.g. Núñez and Jaksic 1992, Pincheira-Donoso and Núñez 2007, Troncoso-Palacios 2013). In his book on lizards from northwest, northeast, and eastern Argentina, Cei (1993) proposed the group and included within it Donoso-Barros, 1961. Lobo (2001) performed the first cladistic analysis of this group and, based on morphological characteristics, included the following species: Hellmich, 1950, Hellmich, 1950, Núñez, Navarro & Loyola, 1991, and Müller & Hellmich, 1932. Later, Lobo (2005), updating the morphological phylogeny, added to the group Donoso-Barros, 1966 and Navarro & Núñez, 1993, but excluded .

Pincheira-Donoso and Núñez (2005), through phenetic analysis recovered all of the species listed by Lobo (2005) within the group and also reincorporated and . Furthermore, was listed with two subspecies, and Ortiz, 1975. These authors also incorporated Young-Downey & Moreno, 1991 and (Philippi, 1860), the latter of which was included as incertae sedis. Moreover, was considered a junior synonym of , as has also been proposed by other authors (Núñez and Jaksic 1992, Troncoso-Palacios 2013), while was regarded as a junior synonym of . In fact, was previously described as a subspecies of (Hellmich 1950) and later proposed to be a synonym of (Núñez and Jaksic 1992, Valencia et al. 1979).

Lobo et al. (2010) accepted all of the species listed by Pincheira-Donoso and Núñez (2005) as members of the group, except for which they placed into the group. Finally, Troncoso-Palacios (2013) indicated that is a junior synonym of , as previously suggested (Núñez and Jaksic 1992, Veloso et al. 1982), and recognized the seven species listed by Lobo et al. (2010) as members of the group – , , , , , , and .

Very few studies have used molecular data within this group. Schulte and Moreno-Roark (2010) constructed a mitochondrial phylogeny of 733 Iguanian lizards. The authors concluded that , and are sister taxa and that does not belong to the group. Cianferoni et al. (2013) performed a cytochrome-b (Cyt-b) phylogeographic study in populations and proposed that this species could contain at least two different species-level lineages.

In a field trip to the vicinity of Piuquenes (Valparaíso Region, Chile), we believe we found some populations probably previously assigned to Müller & Hellmich, 1932 by Núñez et al. (2010:57). Subsequent Cyt-b phylogenetic analysis PageBreakand morphological comparisons determined that this population represents a new species that belongs to the group. This new species occurred in sympatry with , constituting the first case of sympatry within this group of lizards.

The current study describes this new species and provides a full diagnosis in regards to other species of the group. Although the color pattern of this new species resembles and , the scalation is markedly different and the distribution is allopatric (> 240 km of separation). Moreover, various taxonomical aspects of the group that require attention are discussed.

Materials and methods

Morphological data and analyses

Specimens of all species currently considered within the group were examined. Morphological characteristics were examined according to Etheridge (1995) and Lobo (2005). Body measurements were taken with a digital Vernier caliper (0.02 mm precision) and given as the mean ± standard deviation (x ± SD). We applied a Kolmogorov-Smirnov test to verify data normality, a subsequent t-test or Mann-Whitney U test was used if data passed or failed the normality test, respectively, to compare scale count (midbody, dorsal and ventral) and size () of the new species against some related species (, , and ). Only significant results are presented. Scales were observed with different magnifying lenses. Scalation and measurements were recorded on the right side of the specimen. Dorsal scales were counted between the occiput and the anterior border of the hind limbs. Ventral scales were counted from the mental scale to the anterior margin of the cloacal opening. Stomach and intestinal contents were analyzed under a binocular stereoscope for one specimen of the new species. Data for the midbody scales of were taken from one revised specimen and six reported in Young-Downey and Moreno (1991). Classification was carried out considering species currently assigned to the group (Troncoso-Palacios 2013). is included in the comparison but the relationship of this species with the group is uncertain (see Discussion). The examined specimens are listed in Appendix I. Some mapping data were taken from existing literature or field observations without specimen collection: 1) from Manque (Mella 2005), El Arpa and El Roble (Cianferoni et al. 2013), Riecillo (Núñez et al. 2010), Campana (Hellmich 1050), Chepical and Juncal (field observations, 32°16'S - 70°30'W and 32°53'S - 70°07'W respectively); 2) from Los Molles (Núñez et al. 1991). Acronyms used are: , and .

snout vent length,

Museo Nacional de Historia Natural de Chile

Museo de Zoología de la Universidad de Concepción

Colección de Flora y Fauna, Profesor Patricio Sánchez Reyes of the Pontificia Universidad Católica de Chile

DNA purification, PCR amplification, and sequencing

Samples from liver and thigh muscle were obtained from ethanol-fixed lizards which were subject to a rehydration process according to Coura (2005). Samples were washed twice in distilled water for 5 min at 55 °C to remove the fixative and then rehydrated with 1x Tris/EDTA for 5 min at 55 °C and then 1M Tris pH 7.5, at 55 °C overnight. Right after, samples were digested with proteinase K (20 mg/ml) at 55 °C overnight. Genomic DNA isolation (mitochondrial and nuclear) was done with the Wizard® Genomic DNA Purification kit (Cat # A1120, Promega, USA) following manufacturer´s instructions. The mitochondrial gene Cyt-b was amplified from total DNA through two phase conventional PCR with the primers GLUDGL (5´-TGA CTT GAA RAA CCA YCG TTG-3´) and CB3 (5´-GGC AAA TAG GAA RTA TCA TTC-3´), reported in Torres-Pérez et al. (2009), to generate a 700bp amplicon. PCR reactions were performed with the SapphireAmp® Fast PCR Master Mix (Cat # RR350A, Takara Clontech, USA) using 100 ng of total genomic DNA as a template and following the instruction manual. Two-phase PCR cycling was as follows: Phase 1, initial 98 °C denaturation for 3 min, then 5 cycles of 98 °C denaturation for 30 s, 47 °C annealing for 45 s and 72 °C extension for 45 s. The Phase 2, next 40 cycles of 98 °C denaturation for 30 s, 58 °C annealing for 45 s and 72 °C extension for 45 s. A final 72 °C extension step for 5 min was added to finish the PCR. The 700 bp PCR amplicon was checked by DNA electrophoresis on a 1% agarose gel in 1x Tris-Acetate-EDTA (TAE) buffer. The amplicons were purified with the E.Z.N.A.® Cycle-Pure Kit (Cat # D6492-02, Omega Biotek, USA) and sent for capillary sequencing to Macrogen, Korea.

Phylogenetic reconstruction

The accession numbers of the Cyt-b mitochondrial loci sequences generated in this study and the sequences obtained from GenBank are indicated in Appendix II. Forty three nucleotide sequences involved in the analysis were aligned using MUSCLE (Edgar 2004). We used the JModelTest v2.1.7 (Darriba et al. 2012, Guidon and Gascuel 2003) to select an appropriate substitution model (HKY + G + I), with a BIC index. We performed a analyses with MrBayes v3.1.5 (Ronquist and Huelsenbeck 2003). Two independent analyses, each consisting of two groups of four chains that run independently, that were run for 15.0 × 106 generation and a at sample frequency = 1000. Priors were let by default. Pincheira-Donoso 2004, was selected as the outgroup. The 25% of samples were discarded as burnin when calculating the convergence diagnostic, assessed examining values of average standard deviation of the Potential Scale Reduction Factor (PSRF) for all parameters.

Bayesian inference

Results

The genetic tree constructed from (Fig. 1) placed the newly identified species as a sister taxon of (posterior probability pp = 1). However, no data are available for most of the species in the group as sample collection is hampered by the high altitudes where these species inhabit. Therefore, the discovered topology should be considered preliminary (see Discussion). is nested with strong support (pp = 1) in the group, the sister clade of the group. is not closely related to the new or , and is nested in a node with polytomy.

Figure 1.

Bayesian inference of phylogeny tree using Cyt-b showing phylogenetic relationships of sp. n. (red) and related species (HKY+G+I model). and , probably confused with the new species, are also in red. Posterior probability is indicated at each node. Scale shows the number of substitutions per site. Number between parentheses indicates the number of sequences for the collapsed nodes.

mitochondrial DNA

sp. n.

http://zoobank.org/B412BEF2-C337-4472-A4CE-9AFD73876B07

Fig. 2A, B

Figure 2.

sp. n. A, B Holotype, male C, D Paratype, female E Paratype, male F Paratype, juvenile (unknown sex). All from the type locality.

(in part?),

?,

Holotype.

SSUC Re 674. Adult male. Collected in the west shore of the Chepical Lagoon (32°15'S – 70°30'W), approximately 30 km NE Alicahue, San Felipe de Aconcagua Province, Valparaíso Region, Chile. Collectors: J. Troncoso-Palacios and E. Alfaro. December, 2012.

Paratypes

(Fig. 2C, D, E, F). SSUC Re 675, male. SSUC Re 676–78, three females. SSUC Re 679, juvenile. The same data as the holotype.

Etymology.

The species name “” (Latin) refers to the lack of dorsal pattern and uniform color found for both males and females.

Diagnosis.

is larger than (Mann–Whitney U = 0.5, P < 0.01, Table 1). has sexual dichromatism, a feature absent in . Males of have a black vertebral line and black spots on the paravertebral fields (Fig. 3A), whereas has no dorsal pattern. Additionally, the southern distributional limit of in Agua Verde, Antofagasta Region, Chile (Ortiz 1975), is more than 750 km north of the type locality recorded for .

Table 1.

Scalation and morphological characteristics for the species of the group. Juvenile specimens examined are excluded. M = males; F = females. (*) Taken from Navarro and Núñez (1993). (**) Examined specimen plus Young-Downey and Moreno (1991) data. (***) Taken from Young-Downey and Moreno (1991). (****) Counted only for one specimen.

	Liolaemus constanzae (M = 14, F = 13)	Liolaemus isabelae (M = 4)	Liolaemus juanortizi (M = 1)	Liolaemus lorenzmuelleri (M = 3, F = 5)	Liolaemus maldonadae (M = 3)	Liolaemus melanopleurus (M = 2)	Liolaemus nigroviridis (M = 9, F = 4)	Liolaemus uniformis sp. n. (M = 2, F = 3)
Midbody scales	54–64	54–60	54–59**	50–62	58–64	42–56	55–64	58–62
Dorsal scales	56–67	56–67	52	44–55	48–50	40–51	45–53	56–63
Vental scales	86–96	86–97	88	86–96	83–91	91****	85–97	91–102
Nasal-rostral contact	92.6%	25%	100%	100%	100%	100%	100%	100%
Sexual dichromatism	Present	Present*	Absent***	Absent	?	?	Present	Absent
Vertebral line (males)	Present	Present/ absent	Present/ absent	Present	Absent/ inconspicuous	Absent	Absent/ inconspicuous	Absent
Maximum SVL (mm)	75,3	82,8	94.4***	88.8	85.6	70.6	73,8	89.1

Figure 3.

Chilean species of the group (with the exception of ), ordered from north to south. A , male from vicinity of San Pedro (picture by JTP) B , male from Atacama (picture by JTP) C , male from Montandón (picture by JTP) D , unknown sex specimen from road to Negro Francisco (picture by F. de Grotee) E , unknown sex specimen from Embalse La Laguna (picture by A. Labra) F , male from vicinity of Alcohuaz (picture by JTP).

differs from (Fig. 3C), because in the latter the nasal and the rostral scales are in contact only in 25% of specimens, whereas in , these scales are always in contact. Males of have black ventral coloration, a yellow dorsal color with a black vertebral line, black bars in the paravertebral fields, and a black lateral band, or some males have a completely black dorsal color; all traits that are absent in . Additionally, the southern distributional limit of in Salar de Pedernales, Atacama Region, Chile (Pincheira-Donoso and Núñez 2005) is more than 650 km north of the type locality recorded for .

resembles (Fig. 3E) and (Fig. 3D), species suggested as conspecific (Pincheira-Donoso and Núñez 2005). However, the dorsal scales in and are noticeably larger than those of , and have a distinct “ovoid” shape. has more dorsal scales (60.0 ± 2.9) than (48.4 ± 4.2) (t = -5.4, P < 0.01). On the other hand, while only one specimen of was examined, this one has 52 dorsal scales, which is below of the range for (Table 1). has more midbody scales (60.4 ± 1.7) than (54.9 ± 4.5) (t = 2.6, P < 0.05) and (56.7 ± 2.1) (t = 3.2, P < 0.05). has a dark vertebral line and dark transversal lines running from the paravertebral fields to the flanks, whereas has no dorsal pattern. The dorsal pattern of is very similar to , but some specimens have a black ventral coloration, a black lateral band, and the lack of a dark vertebral line, whereas has no PageBreakblack ventral color or black lateral band. Additionally, the southern distributional limit of (Embalse La Laguna, Coquimbo Region, Chile) is more than 240 km north of the type locality recorded for ; and the southern distributional limit of in Quebrada Contrabando, Atacama Region, Chile (MNHNCL collection catalog, unpublished) is more than 520 km north of the type locality recorded for .

differs from (a species with only three known specimens from an undetermined location, Fig. 3B) in that the latter has a blue-gray dorsal coloration (Philippi 1860) and a black lateral band running from the axilla to the midbody, features absent in . Although the type locality of is undetermined, the syntypes were collected by Philippi in his journey through the Atacama Desert, between the vicinities of Copiapó (27°23'S) and San Pedro de Atacama (22°54'S), more than 530 km north of the type locality recorded for .

differs from (Fig. 3F), because males of the latter have a yellowish or reddish dorsal color with black transverse dorsal and ventral bars and black lateral band, whereas has no dorsal pattern or black transPageBreakverse ventral bars. Dorsal scales in are noticeably larger than found in , and they have an “ovoid” shape. Dorsal and ventral scale counts in do not overlap with the same scale counts in (Table 1). Finally, the southern distributional limit of in Los Molles (Núñez et al. 1991) is more than 150 km north of the type locality of .

is found in sympatry with (Fig. 4), but is larger than (Mann–Whitney U = 8.0, P < 0.05, Table 1). also has more dorsal scales (60.0 ± 2.9) than (49.4 ± 2.7) (t = 7.4, P < 0.01). has strongly mucronated dorsal scales, whereas has no mucrons (Fig. 5). has sexual dichromatism, absent in . Males of have a bluish or yellowish green dorsal color PageBreakPageBreakwith black reticulation, and females have a brown dorsal color with a black lateral band, black vertebral line, and black paravertebral spots. In contrast, has a brown dorsal color without any pattern.

Figure 4.

Variation in . A Male from Farellones (picture by H. Díaz) B Male from Carpa Mountain (picture by JTP) C Male from Provincia Mountain (picture by JTP) D Female from Juncal (picture by JTP).

Figure 5.

Dorsal scales, 8 mm width of view. A Male of sp. n. B .

Molecular data show that is not closely related to (Fig. 1). Moreover, is smaller (maximum SVL = 65.6 mm) than (max. SVL = 89.1 mm) (t = 3.9, P < 0.01) according to our samples, and although Pincheira-Donoso and Núñez (2005) recorded a max. SVL = 67.3 mm for , the difference between both species is marked. Moreover, exhibit a characteristic black lateral band between the axilla and midbody (diffuse in females), and males have white dots dispersed on the dorsum and a series of small black spots on the dorsum (Fig. 6). All these traits are absent in . The upper altitudinal limit of distributions is 2000 m a.s.l. (Espinoza et al. 2004, Fuentes and Ipinza 1979), whereas has a lower altitudinal distribution limit of 2820 m a.s.l.

Figure 6.

Variation in species probably confused with sp. n. A from Salto de Apoquindo (picture by JTP) B from La Cruz Mountain (picture by J. Abarca-Díaz) C from Provincia Mountain (picture by JTP) D from La Parva (JR Martini) E from Lagunillas (picture by JTP) F from San Ramón Mountain (picture by JTP).

Molecular data show that is not closely related to (Fig. 1). Moreover, is smaller (maximum SVL = 80.8 mm) than (max. SVL = 89.1 mm) (t = 2.7, P < 0.05). has more midbody scales (60.4 ± 1.7) than (52.9 ± 2.6) (t = 6.1, P < 0.01); more dorsal scales (60.0 ±2.9) than (43.3 ±3.1) (t = 10.2, P < 0.01); and more ventral scales (96.2 ±4.8) than (89.7 ±4.6) (Mann–Whitney U = 10.5, P < 0.05). Dorsal scales in are strongly keeled and mucronated, whereas there are no mucrons in . PageBreakMoreover, exhibit a characteristic series of black dorsal “W” o “V” shaped spots (Fig. 6), whereas has no dorsal pattern.

Description of the holotype.

Adult male. SVL = 84.7 mm. Horizontal diameter of the eye: 4.3 mm. Subocular length: 4.5 mm. Length of the fourth supralabial: 4.1 mm. Head length (from the posterior border of the auditory meatus to the tip of the snout): 22.1 mm. Head height (distance between the two ear openings): 10.4 mm. Head width (at the level of ear openings): 15.8 mm. Neck width: 12.4 mm. Interorbital distance: 6.3 mm. Ear-eye distance: 7.5 mm. Internarine distance: 3.8 mm. Ear width: 2.5 mm. Ear height: 3.5 mm. Axillary-groin distance: 34.9 mm. Body width: 24.7 mm. Forelimb length: 25.7 mm. Hindlimb length: 46.1 mm. Length of the right hand: 10.4 mm. Length of the right foot: 22.4 mm. Tail length (not autotomized): 132.4 mm, with relation tail length/SVL = 1.56. Pentagonal rostral scale, wider (4.2 mm) than high (1.4 mm).

Two postrostrals. Four internasals. Heptagonal interparietal, with a central, small, and whitish central spot marking the position of the parietal eye. Interparietal smaller than the parietals, surrounded by seven scales. Seven scales between the interparietal and rostral. Thirteen scales between the occiput and the rostral. Orbital semicircle incomplete on the right side and complete on the left (formed by thirteen scales). Three supraoculars on the left side and four on the right. Six superciliary scales. Frontal area divided into three scales (1 posterior and 2 anterior). Preocular separated from the lorilabials by one loreal scale. Two scales between nasal and canthal. Nasal in contact with the rostral, surrounded by six scales. One row of lorilabials between the supralabials and subocular. Four lorilabials in contact with the subocular. Six supralabials, the fourth is curved upward without contacting the subocular. Four infralabials scales. Pentagonal mental scale, in contact with four scales. Four pairs of post-mental shields, the second is separated by two scales. Temporal scales smooth or slightly keeled, imbricated. Six temporal scales between the level of superciliary scales and the rictal level. Four scales on the anterior edge of the ear, which do not cover the auditory meatus. Poorly differPageBreakentiated auricular scale, pentagonal and located at the upper part of the meatus. Thirty gulars between the auditory meatus. Lateral neck fold is “Y” shaped. Ventrolateral fold running from the neck to the groin. Dorsolateral fold slightly developed, running from the ear to the base of the tail. Midbody scales: 60. Dorsal scales are lanceolated, imbricated, keeled (without mucrons), with few interstitial granules. Dorsal smaller than the ventrals. Dorsal scales: 58. Ventrals scales are polymorphic (rounded, rhomboidal, pentagonal or hexagonal) smooth, imbricated, without interstitial granules. Ventrals: 91. Three precloacal pores. Supra-femoral scales lanceolate, imbricated, smooth or keeled. Infra-femoral scales lanceolate or rounded, smooth and imbricated. Supra-antebrachials scales are rounded or lanceolated, imbricated and smooth or keeled. Infra-antebrachials are rounded, imbricated and smooth. Dorsal scales of tail are pentagonal or rhomboidal, imbricated and keeled. Ventral tail scales are rounded or rhomboidal, smooth and imbricated. Lamellae of the fingers: I: 9, II: 13, III: 20, IV: 20 and V: 13. Lamellae of the toes: I: 11, II: 15, III: 21, VI: 27 and V: 17.

Color of the holotype in life.

The specimen is notable for its lack of pattern and uniform color. The head is brown and darker than the body. There are several white dots dispersed over the head and cheeks. The dorsum is coppery brown and has a few white-spotted scales that did not form a pattern. The subocular is brown and crossed by three white, vertical lines. The dorsal surface of the tail is light brown and without a pattern. The limbs are a dorsal-brown, similar to the dorsal surface, with white dots PageBreakPageBreakdispersed on the forelimbs and white transversal lines on the hindlimbs. The flanks are whitish with abundant dark brown scales. Ventrally, the hands, feet, thighs, vent, and tail are yellowish. The belly is whitish with dark dispersed spots and a dark ventral stripe. The throat is whitish with a dark thick reticulation. The precloacal pores are orange.

Variation in the type series.

Males are larger and more corpulent than females. In two males: SVL: 84.7–89.1 mm. Axilla-groin distance: 34.9–37.8 mm. Head length: 21.9–22.1 mm. Head width: 15.8–16.3 mm. Head height: 10.4–11.2 mm. Leg length: 45.4–46.1 mm. Arm length: 25.0–25.8 mm. Tail length: 132.4 mm in one specimen, with relation tail length/SVL = 1.56 (autotomized in the other). In three females: SVL: 67.7–73.1 mm. Axilla-groin distance: 33.1–35.7 mm. Head length: 17.8–20.0 mm. Head width: 11.8–13.3 mm. Head height: 7.5–8.3 mm. Leg length: 32.0–34.8 mm. Arm length: 19.2–21.3 mm. Tail length: 98.1 mm in one specimen, with relation tail length/SVL = 1.45 (autotomized in other).

The variation of the scalation in is as follows. Midbody scales: 58–62 (60.4 ±1.7). Dorsal scales: 56–63 (60.0 ±2.9). Ventral scales 91–102 (96.2 ±4.8). Fourth finger lamellae: 17–20 (19.0 ±1.4). Fourth toe lamellae: 25–27 (26.4 ±0.9). Supralabial scales: 6. Infralabial scales: 4–5 (4.4 ±0.6). Interparietal scale pentagonal, hexagonal or heptagonal, bordered by 5–7 scales (6.0 ±0.7). Nasal and rostral always in contact. Precloacal pores in males: 3. Precloacal pores are absent in females.

In general, all specimens have the pattern and color described for the holotype, with slight variations in shade. The male paratype has a dark brown throat. Two females have inconspicuous dark rings and an inconspicuous vertebral stripe on the dorsal surface of the tail. Also, two females have an olive hue on the snout. One female has a very inconspicuous series of dark crossbars on the paravertebral fields, which, while difficult to count, approximated eight. The juvenile has a similar pattern and color as the holotype, but it has an inconspicuous and fragmented dark vertebral line and inconspicuous dark spots on the paravertebral fields.

Distribution and natural history.

This species is currently only known from the type locality in the surroundings of the Chepical Lagoon, approximately 30 km NE of Alicahue, in the San Felipe de Aconcagua Province, Valparaíso Region, Chile (Fig. 7). Specimens were collected on the west shore of the Chepical Lagoon (32°15'S – 70°30'W, 3050 m a.s.l.). This new species was found inhabiting rocky areas with little shrubby vegetation composed mainly of high-Andean forbs, such as and sp. (Fig. 8). This lizard was found in abundance and was observed to have saxicolous habits. It was active between 9:00 h and 18:00 h and took refuge under rocks. Moreover, this species was found in syntopy with Núñez, Veloso, Espejo, Veloso, Cortés & Araya 2010. Specimens were also observed at lower altitudes (32°16'S - 70°30'W, 2820 m a.s.l.) in similar environments, altitudes at which this species was found in sympatry with a few specimens of .

Figure 7.

Distributional map for sp. n. along with geographically proximate species of the group. Red star: sp. n., Chepical Lagoon, type locality. Green circles: (1 = Manque, 2 = El Arpa, 3 = Juncal, 4 = Riecillo, 5 = La Campana, 6 = El Roble, without number = near Chepical Lagoon). Pink squares: (1 = near Alcohuaz, 2 = Los Molles). Yellow triangles: (1 = Baños del Toro, 2 = Embalse La Laguna).

Figure 8.

View of the type locality of sp. n., a high Andean environment.

One of the collected specimens had a yellow flower inside of its mouth. An analysis of intestinal contents showed that is omnivorous; plant and remains were found. A large quantity of nematodes from an unidentified species was PageBreakfound in the intestines. While the reproductive mode is yet unknown, at the time of sampling (December) no evidence of embryos was found but one female had several small oocytes. Comparisons with the reproductive modes of other species in the group would not be helpful as there is little available data. It is known that is viviparous (Donoso-Barros 1966) and is oviparous (Cortés et al. 1995). Pincheira-Donoso and Núñez (2005) reported that and are viviparous, but the source of this information is unclear (see Lobo et al. 2010:4) since the reproductive mode was not mentioned in the original descriptions (Navarro and Núñez 1993, Núñez et al. 1991).

Discussion

Almost no molecular data are currently available for the group, probably due to the great difficulties of obtaining samples since all of these species inhabit high altitude mountainous areas (Pincheira-Donoso and Núñez 2005), with only (Ortiz 1975) and (Espinoza et al. 2004) recorded below 2000 m a.s.l. (1400 m a.s.l. and 500 m a.s.l., respectively). Moreover, most specimens from the MNHNCL and MZUC collections (the two major herpetological collections in Chile) are fixed with formaldehyde, making DNA extraction and amplification challenging (Lin et al. 2009). In PageBreakregards to previous works, Torres-Pérez et al. (2009) performed three phylogenetic analysis (Bayesian inference, ML and maximum parsimony) and found that is the basalmost species of a clade also composed of + + and that this clade is closely related to + clade. Our results are very similar with the and clades as sister groups, but we did not want to include “” from GenBank (Torres-Pérez et al. 2009) because the true identity of this species was only recently clarified (Troncoso-Palacios and Garín 2013) and although a specimen voucher is indicated (CUCH-3143), no locality data is provided. Since we have not seen this specimen we are not sure if it belongs to the true or to . We also did not include “” from GenBank (Torres-Pérez et al. 2009) because the specimen voucher (MZUC-30556) was collected in Laja Lagoon, Chile (according to MZUC Book catalog, unpublished) out of the known range for (Troncoso-Palacios and Marambio-Alfaro 2011), so it could be misidentified. In a recently mitochondrial ML phylogeny performed for a region spanning ND1-COI, Troncoso-Palacios et al. (2015b) found that the + clade is the sister group of the clade ( + + ). This is also very similar to our result, but since there are not Cyt-b data for , it could not be included in the present analysis.

We recognize that one limitation to our work is that it is based in a phylogenetic analysis of only one mtDNA gene and that a wider phylogenetic DNA analysis (including nuclear genes) should be conducted in the future. This is also true for most of the 21 species of (sensu stricto) described in the last five years, which have been classified through different methodologies in regards to DNA comparisons. For example, three species (, and ) include data from two mtDNA genes and shared data in GenBank (Aguilar et al. 2013). As our work, five species (, , , and ) have been described with only Cyt-b data, and one species has been described with two mtDNA genes (). However, DNA data from all these have not been shared in GenBank or other online databases (Avila et al. 2010, 2012, 2015, Escobar-Huerta et al. 2015, Esquerré et al. 2014, Martínez et al. 2011) which does not allow the replication of the provided phylogenies or genetic distances. Two described species (Quinteros 2012, Troncoso-Palacios et al. 2015a), and , are supported in regards to DNA features by previously published phylogenetic works. Nine species (, , , , , , , and ) have been described without the support of molecular data (Abdala et al. 2010, Esquerré et al. 2013, Marambio-Alfaro and Troncoso-Palacios 2014, Ocampo et al. 2012, Quinteros 2012, Quinteros et al. 2014, Troncoso-Palacios et al. 2015a). Finally, one species, , was described (Abdala et al. 2010) despite that no molecular differentiation was previously noted (Morando et al. 2003). No description in the last five year had included nuclear genes or more than two mtDNA genes and in most cases when DNA phylogeny is provided no data are shared in GenBank or other online databases. It is evident that researchers should put emphasis on trying to improve this situation in the future.

Although is strongly supported as a sister species of (pp = 1), a comprehensive phylogenetic study with more species of this group is needed. For example, was not placed within the group in a mitochondrial phylogenetic study that included one specimen (Schulte and Moreno-Roark 2010), despite that this species has been determined to be a member of this group in cladistic (Lobo 2005) and phenetic studies (Pincheira-Donoso and Núñez 2005) based on morphology. We included this species in our comparisons but for the time being, this should not be considered part of the group. Although the morphological cladistic analysis (Lobo 2005) found five apomorphies for the group (range of scale organs on postrostral scales, fourth supralabial - subocular not in contact, range of lamellae on the fourth finger, intraspecific female pattern and the relationship between the subocular length and the eye diameter), this study does not include all species currently accepted as part of the group and does not indicate the specific variation ranges of variation for these features in this group. On the other hand, the phenetic analysis of Pincheira-Donoso and Núñez (2005) does not provide supporting data for the features that were included in the matrix, so it cannot be replicated (see Lobo et al. 2010).

resembles and in that the three species share a similar background dorsal coloration. Although no molecular data exists to compare with these two species, we propose that the marked differences in scalation and the strongly allopatric distribution (> 240 km of separation), which is quiet considerable for lizards, support classifying as a new taxon. has probably been misidentified as by Núñez et al. (2010), who noted as the only lizard species to inhabit in syntopy with (no specimen collection indicated). However, the present study found residing at over 2900 m a.s.l, whereas the upper altitude limit for is 2000 m a.s.l. (Espinoza et al. 2004, Fuentes and Ipinza 1979). Therefore, the present data indicates that the only lizards occurring in syntopy with are and . Moreover, and shows deep morphological and molecular differences. has probably also been confused with (formerly ) by Mella (2005), who found presence of the latter species in the highlands of Putaendo (no specimen collection indicated). However, a field expedition to the highlands of Putaendo by the authors of the present study found no specimens of , and no additional records of in this zone are known. Taking into account these details, in addition to both species having a similar background dorsal color, we think that might have been confused with .

Several aspects of the group remain uncertain. For example, possibly contains at least two species, the nominal species from the Andean highlands and populations from Coastal highlands, formerly (Cianferoni et al. 2013). might be a junior synonym of (Pincheira-Donoso and Núñez 2005), and although both are certainly very similar, it is difficult to carry out a study on this matter because the type series of is lost (Valladares 2011) and there are very few samples of this species (Pincheira-Donoso and Núñez 2005). On the other hand, remains a problematic species PageBreakin terms of identification as the type locality is imprecise and no additional specimens have been found in more than 100 years (Troncoso-Palacios 2012).

The present work contributes to the existing taxonomical knowledge, but the group of lizards remains poorly studied, and new samples are required to better investigate its challenging taxonomy.