Pterygoplichthys zuliaensis Weber 1991

© 2018 Academia Sinica, Taiwan Open Access The True Identity of the New World Iguanid Lizard Liolaemus chillanensis Müller and Hellmich 1932 (Iguania: Liolaemidae) and Description of a New Species in the Liolaemus elongatus Group Jaime Troncoso-Palacios1,*, Damien Esquerré2, Félix A. Urra3,4, Hugo A. Díaz5, Carlos CastroPastene6, and María Soledad Ruiz7 1Programa de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile 2Division of Ecology and Evolution, Research School of Biology, The Australian National University 0200, Canberra, Australia 3Programa de Anatomía y Biología del Desarrollo, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile and Fondap Geroscience Center for Brain Health and Metabolism, Independencia 1027, Santiago, Chile 4Programa de Farmacología Molecular y Clínica, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile 5Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Santiago, Chile 6Laboratorio de Vida Silvestre y Ecología, Facultad de Ciencias Veterinarias, Universidad de Concepción, Vicente Mendéz 595,Chillán, Chile 7Instituto de Bio y Geociencias del NOA (IBIGEO)CONICET. 9 de Julio 14, Rosario de Lerma, Salta, Argentina (Received 27 August 2017; Accepted 19 April 2018; Published 31 May 2018; Communicated by Benny K.K. Chan) Citation: Troncoso-Palacios J, Esquerré D, Urra FA, Díaz HA, Castro-Pastene C, Ruiz MS. The true identity of the new world iguanid lizard Liolaemus chillanensis Müller and Hellmich 1932 (Iguania: Liolaemidae) and description of a new species in the Liolaemus elongatus group. Zool Stud 57:22. doi:10.6620/ZS.2018.57-22. Jaime Troncoso-Palacios, Damien Esquerré, Félix A. Urra, Hugo A. Díaz, Carlos Castro-Pastene, and María Soledad Ruiz (2018) Liolaemus is a particularly species-rich radiation of New World iguanid lizards from southern South America. Thanks to intense systematic and taxonomic research, the knowledge on its specieslevel diversity has increased rapidly over the past several years. The L. elongatus species-complex has received considerable attention and a remarkable case is Liolaemus chillanensis, a name that has been used for two different species that are sympatric in Termas de Chillán, central Chile. Since the holotype is lost, we propose that the first step to identify the true L. chillanensis is through the analysis of the original description. Then we provide a morphological and molecular characterization of L. chillanensis based on topotypes and a description of the taxon previously confused with it. Key words: Chile, Cytochrome b, Liolaemus monticola, Phylogeny, Principal component analysis. *Correspondence: E-mail: [email protected] BACKGROUND Liolaemus Wiegmann, 1834 is the second most diverse genera of lizards, currently with 257 species (Abdala and Quinteros 2014) and new species described almost every year, especially in the Andean and Patagonian regions of Chile and Argentina (e.g. Avila et al. 2015; Esquerré et al. 2013; Troncoso-Palacios et al. 2015). Both morphological and molecular data support two main clades that are considered subgenera, roughly separated by the Andes: the Liolaemus (sensu stricto) subgenus or “Chilean” group and the Eulaemus subgenus or “Argentinean” group Zoological Studies 57: 22 (2018) doi:10.6620/ZS.2018.57-22 1 © 2018 Academia Sinica, Taiwan (Espinoza et al. 2004; Etheridge 1995; Laurent 1985; Schulte et al. 2000). There is a group of Andean and Patagonian viviparous and saxicolous lizards within the Liolaemus (sensu stricto) subgenus known as the L. elongatus clade, which is part of a complex of clades known as the L. elongatus-kriegi complex (Cei 1975 1979). Research on this complex has supported four main clades: punmahuida, petrophilus, kriegi and elongatus (Avila et al. 2004 2012; Morando et al. 2003). The L. elongatus clade comprises species almost exclusively confined to rocky environments along the eastern slope of the Andes and south of the Mendoza River basin (Argentina), extending to the volcanic hills of Patagonia in Chubut Province, Argentina, and the Araucanía Region in Chile (Avila et al. 2015; Escobar-Huerta et al. 2015b; Minoli et al. 2013; Morando et al. 2003). Members of the L. elongatus clade are medium to large sized Liolaemus (maximum snout vent length = 107.8), long-tailed, with absent or reduced sexual dichromatism, viviparous, insectivorous, and almost exclusively saxicolous, with a high amount of midbody, ventral and dorsal scales (Abdala et al. 2010; Avila et al. 2015). Currently, this clade is comprised of nine species: L. antumalguen Avila et al. 2010, L. burmeisteri Avila et al. 2012, L. choique Abdala et al. 2010, L. crandalli Avila et al. 2015, L. elongatus Koslowsky 1896, L. janequeoae Troncoso-Palacios et al. 2016, L. lonquimayensis Escobar-Huerta et al. 2015b, L. shitan Abdala et al. 2010, L. smaug Abdala et al. 2010; and possibly L. carlosgarini Esquerré et al. 2013 (fide Esquerré et al. 2013) and L. cristiani Núñez et al. 1991 (fide Medina et al. 2014); but recently L. lonquimayensis and L. shitan were suggested as junior synonyms of L. elongatus in Avila et al. (2015) and TroncosoPalacios et al. (2016), respectively. One of the most confusing issues in the Liolaemus elongatus clade is the identity of the true L. chillanensis Müller & Hellmich 1932, a name that has been used indistinctly for two different species of Liolaemus that inhabit Termas de Chillán, Biobío Region, Chile. This species has been both included (Avila et al. 2010 2012 2015; Escobar-Huerta et al. 2015b; Medina et al. 2017; Torres-Pérez et al. 2009) and excluded from the L. elongatus clade (Lobo et al. 2010; TroncosoPalacios et al. 2015). One major problem is that the holotype of L. chillanensis is lost (Franzen and Glaw 2007). Here, we analyze the original description (Müller and Hellmich 1932), review several vouchers used by Torres-Pérez et al. (2009) and several specimens from different collections, and provide an identification for the species that we conclude is the true L. chillanensis. We use both a molecular (our data and data from GenBank) and a morphological characterization. We also provide a description for the taxon previously confused with L. chillanensis. MATERIALS AND METHODS Morphological data and analyses We collected specimens in the field by hand or noose. Specimens were dissected to extract a sample of liver/muscle for DNA extraction, and fixed in 99% ethanol. These and all examined specimens (n = 80) are indicated in Appendix 1. Morphological characters were examined according to Etheridge (1995), Lobo (2005) and Avila et al. (2010 2012 2015). Body measurements were taken using a digital Vernier calliper (0.02 mm precision). Body measurements are provided as mean ± standard deviation. The stomach and intestinal contents of the individuals were observed under a binocular microscope for a preliminary description of the species’ diet. Data for: L. choique and L. smaug were taken from Abdala et al. (2010); L. antumalguen were mainly taken from Avila et al. (2010), plus some specimens reviewed by us; L. burmeisteri were taken from Avila et al. (2012); L. crandalli were taken from Avila et al. (2015); L. lonquimayensis were taken from Escobar-Huerta et al. (2015b); and L. cristiani were taken from Núñez et al. (1991) and two specimens that we reviewed. As a visualization and exploratory analysis, we performed a Principal Component Analysis (PCA) on the morphometric and meristic variables with the R package FactoMineR (Lê et al. 2008). These analyses were performed in the R statistical environment version 3.2.3 (RDCT 2016). We used the residuals of a regression between the snoutvent length (SVL) and the following six variables: head length (distance between anterior edge of auditory meatus and tip of the snout), head width, head height, axilla-groin distance, arm length and foot length. Missing data from some measurements of some individuals were imputed using the imputePCA function from the MissMDA R package (Josse and Husson 2012). We performed a nonparametric KruskalWallis test on the meristic variables with a Dunn test for post hoc comparisons (Dunn 1964) of page 2 of 19Zoological Studies 57: 22 (2018) © 2018 Academia Sinica, Taiwan the following: midbody scales, dorsal scales (counted between the occiput and the level of the anterior border of the hind limbs), ventral scales, supralabial scales, infralabial scales and fourth toe lamellae; using data from the following species: L. antumalguen, L. carlosgarini, L. chillanensis, L. curis, L. elongatus, L. janequeoae, L. scorialis, the candidate species L. sp. Chillán and L. sp. 6. We excluded species without a published dataset from the PCA and Kruskal-Wallis test, but we performed a diagnosis based on scale count and SVL ranges following the diagnosis previously published for the description of Liolaemus included in this work (Abdala et al. 2010; Avila et al. 2010 2012 2015; Escobar-Huerta et al. 2015b; Esquerré et al. 2013). Color pattern features were used as qualitative features of diagnosis for all species. Molecular laboratory procedures Genomic DNA was extracted using a saltextraction protocol (slightly modified from Miller et al. 1988). We amplified the mitochondrial gene cytochrome b (Cyt-b) with Polymerase Chain Reaction (PCR) using the IguaCytob_F2 and IguaCytob_R2 primers (Corl et al. 2010), with the following thermocycle: denaturation at 94°C for 5 minutes, then cycle 35 times at 94°C for 30 seconds, 50°C for 30 seconds, 72°C for 1.5 minutes, and then a final hold at 72°C for 5 minutes. Sequencing reactions were done using a Big Dye Kit (Applied Biosystems, Foster City, CA). Sequencing was run on an ABI 3130xl Genetic Analyzer. Sequences were edited on Geneious 9.0.4 (Biomatters, Auckland, New Zealand, 2015). Phylogenetic reconstruction Accession numbers of the Cyt-b mitochondrial loci sequences generated in this study and the sequences obtained from GenBank are indicated in Appendix 2. Sequences of the L. lonquimayensis type specimens (n = 4) were provided by G. Escobar-Huerta. The 164 nucleotide sequences involved in the analysis were aligned using MUSCLE (Edgar 2004). We used JModelTest v2.1.7 (Darriba et al. 2012; Guidon and Gascuel 2003) to select a substitution model (HKY + G), considering both BIC and AIC information criteria. We identified the non-redundant haplotypes of each terminal taxa using DnaSP v5.10 (Librado and Rozas 2009) and performed a Bayesian inference (BI) analyses with MrBayes v3.2.6 (Ronquist et al. 2012). Two independent analyses, each consisting of two groups of four chains that run independently, were run for 10 × 106 generations, sample frequency = 1,000. Priors were left by default. Phymaturus vociferator Pincheira-Donoso, 2004 was selected as out group because Phymaturus is the sister genus of Liolaemus (Schulte et al. 2000). The initial 25% of the samples were discarded as burn-in when calculating the convergence diagnostic, assessed by examining values of average standard deviation of the Potential Scale Reduction Factor (PSRF = 1.000 for all parameters; Gelmar and Rubin 1992) and the minimum and average Estimated Sample Size (ESS ˃ 5000 for all parameters). Then, to estimate speciation, we used Bayesian implementation of the Poisson Tree Processes (bPTP, Zhang et al. 2013) for species delimitation using the non-ultrametric consensus tree obtained through the BI analyses described above, in the online server (http://species.h-its.org/) run with 50000 MCMC generations (all other parameters by default). Additionally, we generated a maximum clade credibility tree (MCC) using TreeAnnotator v1.8 (Drummond et al. 2012), from the posterior distribution of an MCMC Bayesian phylogenetic inference with BEASTv1.81 (Drummond and Rambaut 2007), which was run for 100 × 106 generations. Relative divergence times (branch lengths) were calculated using the logNormal relaxed clock in BEAST. We then used this ultrametric tree to run the Generalized Mixed Yule Coalescent model (GMYC, Pons et al. 2006). The two sequences of L. aff. chillanensis from Avila et al. (2015) were not included in the GMYC analysis. RESULTS Identification of the true Liolaemus chillanensis Müller and Hellmich (1932) described L. monticola and included three subspecies, all these with allopatric distributions: L. monticola monticola, L. monticola chillanensis and L. monticola villaricensis. In the same publication, they referenced an additional L. monticola ssp. sympatric with L. monticola chillanensis, but did not describe it. Currently, these three described taxa are accepted at the species level (Abdala and Quinteros 2014; Lobo et al. 2010). The identification of Liolaemus chillanensis is problematic because this species name has been used for two different species of Liolaemus that occur sympatrically in Termas de Chillán and page 3 of 19Zoological Studies 57: 22 (2018) © 2018 Academia Sinica, Taiwan share two features: a fragmented vertebral line and a black lateral band. However, one of these Liolaemus species has an olive or bluish dorsal and ventral color, lacks precloacal pores and does not belong to the L. elongatus clade. The other does not have an olive or bluish dorsal and ventral color, its males have precloacal pores and it belongs to the L. elongatus clade. Unfortunately, the holotype of L. chillanensis, collected in Termas de Chillán at 1700 m, is lost (Franzen and Glaw 2007) and was not illustrated in the original description by Müller and Hellmich (1932). However, the authors did provide a black and white dorsal picture of one male paratype. Müller and Hellmich (1932) indicated that the color pattern of the holotype’s dorsum is olive-brown (“Färbung der Oberfläche ein dunkles, leicht ins Oliv gehendes Rehbraun”, p. 184) and the ventral color is olive (“Gesamte Unterseite olivgrün”, p. 185). They do not describe the coloration of the male paratypes, but stated that the female paratypes have green or blue shades (“dunkel-bis hellblaugrün”, Müller and Hellmich 1932:186). Later, Hellmich (1950 1952) stated that this species has a dark “gray-blue” dorsal and ventral color. At this point it is clear that the true L. chillanensis is the lizard from Chillán with olive or bluish dorsal and ventral color. However, Müller and Hellmich (1932) point out that the holotype of L. chillanensis has four precloacal pores, although these are difficult to see (“schwer sichtbar”, p. 184), and according to Hellmich (1950) the precloacal pores in L. chillanensis are extraordinarily difficult to see (“auBerordentlich schwer sichtbar”, p. 147). Note that in the time of L. chillanensis description Müller and Hellmich (1932), the absence of precloacal pores in male Liolaemus species was unknown, being first reported by Cei and Scolaro (1982), so it is very likely that the mention of precloacal pores in the holotype of L. chillanensis was a mistake or that precloacal pores in L. chillanensis males are present at an extremely low frequency (we failed to find a topotype with them). Based on the original description (Müller and Hellmich 1932) and the posterior publications of one of the species authorities (Hellmich 1950 1952), we conclude that the true L. chillanensis is the lizard that occurs in Termas de Chillán (Chile) and that it can be differentiated by its olive or bluish dorsal and ventral coloration (Fig. 1; see more comparative features in Table 1). This has already been noted by other authors (DonosoBarros 1966; Pincheira-Donoso and Núñez 2005). In fact, a photograph of the first record of L. chillanensis from Argentina (Avila et al. 2013:228) clearly displays the olive coloration. In a recent phylogenetic work based on Cyt-b, Torres-Pérez et al. (2009) included both “monticola” taxa from Chillán: L. monticola chillanensis (currently L. chillanensis) and the taxon that they identified as the undescribed L. monticola ssp. However, we recently reviewed part of the vouchers used as L. chillanensis (MZUC 28249 and 28251, Fig. 2) and determined that these do not belong to L. chillanensis because they lack olive or bluish dorsal and ventral color and the male has precloacal pores. Thus, we conclude that these belong to an undescribed species. We also reviewed the vouchers MZUC 28257-59 and 28263, used by Torres-Pérez et al. (2009) as L. monticola ssp., and noted that they have olive or bluish dorsal and ventral color and the males lack precloacal pores (Fig. 3). To confirm the identification of these two taxa in Torres-Pérez et al. (2009), we sequenced Cyt-b for the two species from our own samples, the undescribed Liolaemus sp. and L. chillanensis. In our Cyt-b phylogeny, our sample of L. chillanensis is nested with the L. monticola ssp. from Torres-Pérez et al. (2009) and our undescribed species is nested with L. monticola chillanensis from Torres-Pérez et al. (2009), so the obvious conclusion is that TorresPérez et al. (2009) used the name “L. chillanensis” for the undescribed species from Termas de Chillán (= L. sp. Chillán) and used “L. monticola ssp.” for L. chillanensis. Phylogenetic relationships Our Bayesian inference found that neither Liolaemus chillanensis nor the undescribed taxon from “Termas de Chillán” (L. sp. Chillán) are closely related to L. monticola (50% consensus tree) (Fig. 4); this was also found by Torres-Pérez et al. (2009). The inference also concluded that L. chillanensis is not part of the L. elongatus clade, but is instead sister to L. cristiani - a species that also features olive dorsal color and lacks precloacal pores - with strong support. We propose that the name “L. chillanensis clade” refer to this group in the future. Liolaemus sp. Chillán is recovered as part of the L. elongatus clade, with strong support. This clade is also comprised of L. antumalguen, L. burmeisteri, L. carlosgarini, L. choique, L. curis, L. leopardinus, L. elongatus, L. scorialis, L. shitan, L. smaug, two candidate species (Liolaemus sp. 6 and Liolaemus sp. 7) proposed by Morando et al. (2003) and Liolaemus sp. from Lircay, this last page 4 of 19Zoological Studies 57: 22 (2018) © 2018 Academia Sinica, Taiwan referred as L. carlosgarini by Escobar-Huerta et al. (2015a). Liolaemus sp. 7 and L. antumalguen were not found to be reciprocally monophyletic (see below). The same occurs with L. shitan and L. elongatus, which raises doubts about the validity of the first (see Avila et al. 2015). Liolaemus sp. Chillán is closely related to L. antumalguen, L. sp. 6 and L. sp. 7 with strong support. On the other hand, L. aff. chillanensis by Avila et al. (2015), listed by Medina et al. (2017) as L. sp. 1, is closely related to L. scorialis, a species with type locality in Laja, 5 km NW from where the samples of L. aff. chillanensis were collected (Fig. 4). The species delimitation through GMYC shows the presence of several species in the data set (likelihood ratio = 12.08; P = 0.002). Liolaemus chillanensis was recovered as a full species, while eleven lineages were recognized in the L. elongatus clade (Fig. 5), L. sp. Chillán among them. In the MCC tree, L. sp. Chillán is sister to the lineage comprised of L. antumalguen + L. sp. 7, with L. sp. 6 basal to this clade. The GMYC model did not discriminate several species and candidate species as independent lineages: 1) L. antumalguen + L. sp. 7, therefore considered here as conspecifics, in fact Medina et al. (2017) were also unable to differentiate these two taxa at the genetic level, even with a larger data set. 2) L. elongatus + L. shitan + L. lonquimayensis, previously thought to be junior synonyms of L. Fig. 1. Variation in the true Liolaemus chillanensis. (A-B) Male from Termas de Chillán (SSUC Re 708). (C) Male from near Aguas Calientes (SSUC Re 710). (D) Male from Termas de Chillán (SSUC Re 707). (E-F) Female from Termas de Chillán (SSUC Re 709). (A) (C) (E) (B) (D) (F) page 5 of 19Zoological Studies 57: 22 (2018) © 2018 Academia Sinica, Taiwan Table 1. Characteristics of Liolaemus antonietae sp. nov. and the species of the L. elongatus clade that occur near it, plus L. chillanensis. Juvenile specimens examined are excluded. M = males; F = females L. antonietae sp. nov. (M = 6, F = 2) L. antumalguen L. burmeisteri L. carlosgarini (M = 6, F = 11) L. chillanensis (M = 9, F = 4) L. elongatus (M = 3, F = 3) L. scorialis (M = 8, F = 3) L. smaug Max SVL (mm) 77.6 107.8 85.2 68.8 85.8 73.7 69.9 71.3 Midbody scales range 86-98 72-82 70-81 80-95 82-92 76-88 76-90 73-80 Ventral scales 118-131 105-118 99-110 112-124 113-127 119-129 115-131 119-131 Dorsal scales 71-78 70-78 76-85 68-82 75-88 67-73 72-81 69-83 Dorsal pattern Fragmented vertebral line, dark paravertebral spots and lateral dark bands Variable, from patternless to two dorsolateral series of black ocelli sometimes fused longitudinally Light brown speckled with white spots, flanked by band of dark brown between axilla and groin, with few white spots Marked or inconspicuous dark occipital band and lateral dark bands Fragmented vertebral line, dark paravertebral spots and lateral dark bands Vertebral and lateral bands Marked dark occipital band and lateral dark bands Dark occipital band with white dots in males, and lateral dark bands Ventral melanism Absent Present Absent Absent Absent Absent Absent Absent Head color Light brown Variable, from completely black to light-tan or ochre Ochre Light brown Brown Dark brown Brown/Light brown Gray to brown Body color Light brown Light gray to ochre Light brown/kaki Yellowish brown or light brown Olive-brown/ bluish brown Ochre to almost black Brown/Gray Yellowish brown (males) or brown (females) Tail rings Marked/Weak Absent Weak Marked/Weak Absent/Weak Marked/Weak Marked Absent Precloacal pores in males 3-5 3-4 0-5 0-3 0 4-5 3-4 3-4 Behavior Arboreal-saxicolous Saxicolous Saxicolous Saxicolous Saxicolous Saxicolous Saxicolous Arenicolous Fig. 2. Some specimens previously misidentified as L. chillanensis in Torres-Pérez et al. (2009): (A-B) MZUC 28251 and (C-D) MZUC 28249. Here identified as L. sp. Chillán. (A) (C) (B) (D) page 6 of 19Zoological Studies 57: 22 (2018) © 2018 Academia Sinica, Taiwan Fig. 3. Some misidentified specimens of Liolaemus chillanensis. Part of the vouchers used as “Liolaemus monticola ssp.” in TorresPérez et al. (2009) from Termas de Chillán: (A-B) MZUC 28257, (C-D) MZUC 28259. Part of the specimens of “L. cristiani” from Termas de Chillán deposited in the MZUC collection (E-F). elongatus in Avila et al. (2015) and TroncosoPalacios et al. (2016) and therefore considered here as part of the L. elongatus variation. 3) L. carlosgarini was not found to be monophyletic and instead forms part of two not closely related clades: L. smaug + L. choique + L. carlosgarini and L. carlosgarini + L. scorialis. Our sample of L. carlosgarini consists of the type specimens and several topotypes, among which it is not possible to differentiate two morphotypes and should all be assigned to L. carlosgarini according to the features provided by Esquerré et al. (2013). These results suggest hybridization or introgression in L. carlosgarini, as have been recorded for other Liolaemus (Morando et al. 2004 2007). 4) L. choique was not found to be an independent lineage (forming part of the L. smaug + L. choique + L. carlosgarini clade); therefore, its taxonomic status should be evaluated in the future. Besides, our bPTP species delimitation analysis recognized thirteen different lineages in the L. elongatus clade, among them L. sp. Chillán as the fourth with the highest posterior probability (Fig. 6). Results only differ from GMYC in that part of L. carlosgarini was recovered as a full species, sister to L. scorialis; and part of L. aff. chillanensis was also recovered as a full species. Morphologic analyses results The first three Principal Components (PCs) cumulatively account for 76.86% of the total variation (Table 2). PC1 is mainly explained by variation in head length, head height and head width (Fig. 7). PC2 mostly represents variation in arm length, foot length and AGD (Fig. 7). PC3 (A) (C) (E) (B) (D) (F) page 7 of 19Zoological Studies 57: 22 (2018) © 2018 Academia Sinica, Taiwan mostly represents variation in AGD, head width and foot length (Fig. 7). The PCA plots (Fig. 7) have 95% confidence ellipses around the centroid of the species and illustrate the morphometric differences between the species included in the analysis. The PC1 vs PC2 graphic shows a partial overlap between Liolaemus sp. Chillán, L. chillanensis, L. carlosgarini, L. scorialis and L. elongatus, but there is no overlap between Liolaemus sp. Chillán and L. chillanensis in the PC1 vs PC3 graphic. Although Liolaemus sp. Chillán partially overlaps with L. carlosgarini in the PC1 vs PC3 graphic, the ellipses have different orientation. Liolaemus sp. Chillán, L. scorialis and L. elongatus partially overlap in all PC analyses, but they are not sister species in our phylogenetic reconstruction. According to the mDNA phylogeny, Liolaemus sp. Chillán, L. antumalguen and L. sp. 6 are closely related, but show no overlap in the PC1 vs PC2 graphic; in the PC1 vs PC3 graphic only L. antumalguen and L. sp. 6 show partial overlap. Besides, Liolaemus sp. Chillán and L. janequeoae show no overlap in any of the PCs graphics. A Kruskal-Wallis test on the meristic variables revealed significant differences in three of the six variables analysed: scales around midbody, dorsal scales and ventral scales (Table 3). Liolaemus sp. Chillán differs from L. antumalguen (closely Fig. 4. Phylogenetic relationships using Bayesian inference (50% consensus tree) based on Cyt-b (HKY + G). In color: Liolaemus sp. Chillán (red, our data from Shangrila, Termas de Chillán and GenBank data from Termas de Chillán), L. chillanensis (blue, our data from Termas de Chillán and data from GenBank) and L. elongatus from Chubut, type locality (green). Red circle on the node denotes posterior probabilities ˃ 0.95. Scale shows the number of changes per site. Numbers between parentheses indicate the number of sequences of the collapsed nodes. page 8 of 19Zoological Studies 57: 22 (2018) © 2018 Academia Sinica, Taiwan Fig. 5. Maximum Clade Credibility ultrametric tree (MCC) with species delimitation found through the Generalized Mixed Yule Coalescent model (GMYC). Lineages of the L. elongatus clade and L. chillanensis are in color. Red circle on the node denotes posterior probabilities ˃ 0.95. Scale shows the number of changes per site. Numbers between parentheses indicate the number of sequences of the collapsed nodes. Table 2. Eigenvalues, the percentage of the total variance and the cumulative percentage of variance for PC1-3, and the correlation of each variable (residuals of a regression with the SVL) PCA Eigenvalues PC1 PC2 PC3 Eigenvalue per Component 2.41 1.25 0.94 % of var. 40.29 20.86 15.69 Cum. % of var. 40.29 61.16 76.86 Eigenvectors PC1 PC2 PC3 Head Length (HL) 0.885 -0.220 -0.081 Head Height (HH) 0.753 0.209 0.373 Head Width (HW) 0.790 -0.133 0.153 Axilla-Groin Distance (AGD) -0.579 0.373 0.393 Foot Length (FL) 0.225 0.559 -0.750 Arm Length (AL) 0.277 0.831 0.237 page 9 of 19Zoological Studies 57: 22 (2018) © 2018 Academia Sinica, Taiwan Fig. 9. Distributional map for Liolaemus antonietae sp. nov. along with geographically proximate species of the L. elongatus clade. Stars: Liolaemus antonietae sp. nov (red = Termas de Chillán, type locality; white = Shangrila). Green pentagon: L. smaug (1 = near Las Leñas, 2 = between Las Loicas and Peteroa Volcano, 3 = near Las Loicas). Lilac cross: L. carlosgarini (1 = Maule Lagoon, 2 = Lircay). Black diamond: L. choique (Paso el Choique). Gray square: L. antumalguen (1= Domuyo volcano, 2= Tromen Volcano). Pink haxagon: L. burmeisteri (Caepe Malal). White asterisk: L. crandalli (Auca Mahuida Volcano). Green triangle: L. scorialis (1 = Laja Lagoon, 2 = La Mula Lagoon). Orange circle: L. elongatus (formerly L. lonquimayensis, Lonquimay Volcano). Brown circle: L. elongatus used for morphology and DNA analyses (Llaima volcano). Yellow circles: L. elongatus used for DNA analyses (1 = Pampa de Lonco Luan, 2 = Primeros Pinos, 3 = Portal La Atravesada, 4 = Laguna Blanca, 5 = near Ingeniero Jacobacci, 6 = San Carlos de Bariloche, 7 = Ojo de Agua, 8 = El Maiten, 9 = Esquel, 10 = Tecka, 11 = Gobernador Costa and 12 = Los Manantiales). Blue circles: L. shitan (1 = Estancia Piedras Blancas, type locality, 2 = near San Antonio del Cuy). Pink inverted triangle: L. janequeoae (Tolhuaca). Blue ellipse: L. sp. 6 (Copahue Volcano). N page 16 of 19Zoological Studies 57: 22 (2018) © 2018 Academia Sinica, Taiwan of L. aff. chillanensis as the same species (Fig. 6). This is congruent with Medina et al. (2017), since they list LJAMM-CNP 14027 and 14029 as L. sp. 1 and include “12 taxa… one being described (L. sp. 1, Esquerré, personal communication)” in their L. elongatus clade analysis (p. 239). In fact, Troncoso-Palacios, Díaz, Esquerré and Urra, described L. scorialis (Troncoso-Palacios et al. 2015) from the Laja surroundings. Medina et al. (2017) found low Cyt-b distance between the taxon that they refer as “L. chillanensis” (here described as L. antonietae) and the lineage composed of L. antumalguen + L. sp. 7, but stated that both lineages were recover as full species in the species delimitation analysis. Moreover, Medina et al. (2017) shows in the “Supporting information Table 2” that L. antumalguen + L. sp. 7 do not share Cyt-b haplotypes with “L. chillanensis”. Congruent with this, our species delimitation analyses (GMYC and bPTP) found L. antonietae (referred as L. chillanensis in Medina et al. 2017) as a lineage at the species level, monophyletic in regards to the most closely related lineage (L. antumalguen + L. sp. 7). Additionally, the PCA shows that L. antonietae and L. antumalguen do not share the same morphological space; some meristic variables show statistical differentiation and striking differences in coloration were found. All these support the recognition of this taxon as an independently evolving lineage but the low Cyt-b distance suggests that L. antonietae is a young species. Acknowledgements: This work and the new species name have been registered with ZooBank under urn:lsid:zoobank.org:pub:A4F4AEED-1AB048E7-88DD-C2711FBACED2. We thank P. Zavala (Pontificia Universidad de Católica de Chile) for allowing us to review and deposit material into the collection under his care. We thank the following colleagues and institutions for allowing us to review specimens: H. Núñez (Museo Nacional de Historia Natural), J.N. Artigas (Museo de Zoología de la Universidad de Concepción) and M. Lamborot (Colección del Laboratorio de Citología de la Universidad de Chile); M. Morando, F. Breitman and C. Aguilar for providing use useful information on PCR procedures and primers; I. G. Brennan for extensive help in the lab; F. Ferri, D. Vieites, F. Torres-Pérez, L.J. Avila and J.S. Keogh for their comments on the early version of the manuscript; F. Ferri, C.S. Abdala and A. Laspiur for sending literature; G. Feuerhake for providing photographs. J. Troncoso-Palacios thanks M. Penna for his support and L. Rodríguez for her support in the field. D. Esquerré was supported by a Becas Chile-Conicyt Scholarship. F.A. Urra is supported by FONDECYT postdoctoral fellowship #3170813. The map (Fig. 9) was made with ArcGis 10.5.1 free trial. We thank the Servicio Agrícola y Ganadero (SAG) for collecting permits (N°4468). Authors’ contributions: JTP conceived and designed the study, drafted the manuscript (except the background section), collected specimens and morphological data, perform morphological and phylogenetic analyses and made some figures. DE drafted the background section, perform DNA laboratory procedures and perform morphological and phylogenetic analyses. FAU and HAD collected specimens and made some figures. CCP collected specimens. MSR collected specimens and morphological data. All authors contributed with the corrections. Competing interests: JTP, DE, FAU, HAD, CCP and MSR declare that they have no conflict of interest. Availability of data and materials: Sequences generated in the study have been deposited in GenBank (see Appendix 2). Consent for publication: The authors give their consent to publish this paper. Ethics approval consent to participate: Not applicable. 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Museum codes are as follow: MNHNCL (Museo Nacional de Historia Natural, Chile), MRC (Museo Regional de Concepción), MZUC (Museo de Zoología of Universidad de Concepción) and SSUC (Colección de Flora y Fauna Patricio Sánchez Reyes, Pontificia Universidad Católica de Chile). (download) Appendix 2. Specimens used for phylogenetic analysis. (download) page 19 of 19Zoological Studies 57: 22 (2018)