Molecular Phylogenetics and Evolution 94 (2016) 410–423

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Molecular Phylogenetics and Evolution

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Molecular data reveal spatial and temporal patterns of diversification and a cryptic new species of lowland Stenocercus Duméril & Bibron, 1837 (Squamata: Tropiduridae) q ⇑ Mauro Teixeira Jr. a, , Ivan Prates b, Carolina Nisa a, Nathalia Suzan Camarão Silva-Martins c, Christine Strüssmann c, Miguel Trefaut Rodrigues a a Laboratório de Herpetologia, Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, 05508-090 São Paulo, SP, Brazil b Department of Biology, City College and Graduate Center, City University of New York, New York, NY 10031, USA c Departamento de Ciências Básicas e Produção Animal, Faculdade de Agronomia, Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso, 78060-900 Cuiabá, MT, Brazil article info abstract

Article history: Phylogenetic studies have uncovered biogeographic patterns and the associated diversification processes Received 11 April 2015 of Neotropical wet forest taxa, yet the extensive open and drier biomes have received much less Revised 28 July 2015 attention. In the Stenocercus lizard radiation, restricted sampling and phylogenetic information have Accepted 9 September 2015 limited inferences about the timing, spatial context, and environmental drivers of diversification in the Available online 30 September 2015 open and dry lowland settings of eastern and southern South America. Based on new DNA sequence data of previously unsampled species, we provide an updated historical biogeographic hypothesis of Keywords: Stenocercus. We infer phylogenetic relationships, estimate divergence times, and track ancestral distribu- Andes tions, asking whether cladogenetic events within the genus correlate to reported shifts in South American Climatic niche Historical biogeography landscapes during the past 30 million years, focusing in the open and drier areas. To examine correlations Iguania between genetic and ecological divergence, we extracted environmental data from occurrence records Stenocercini and estimated climatic envelopes occupied by lowland taxa. Our results suggest that Stenocercus began Taxonomy to diversify around the South American Midwest by the late Oligocene. We recovered two main lowland and two main Andean clades within the genus; within both Andean clades, most cladogenetic events date back to the Miocene, synchronously with the most intense phase of Andean uplift. In the western clade of lowland Stenocercus, species ranges and divergence times are consistent with major landscape shifts at the upper Guaporé and Paraguay River basins as a result of Andean orogeny, suggesting vicariant speci- ation. By contrast, in the ‘horned’ lowland clade, we find evidence that dispersal and ecological differen- tiation have shaped species divergences and current ranges in the Brazilian Cerrado, Caatinga, Pampas and Atlantic Forest, possibly under a vanishing refuge scenario. Lastly, our phylogenetic results indicate two divergent clades within the formerly recognized taxon S. sinesaccus, and further evaluation of mor- phological data corroborates the existence of a distinct, new species of Stenocercus, here described. The new taxon occurs in the Chapada dos Parecis massif in the Brazilian states of Mato Grosso and Rondônia. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction 2005, 2007). Unfortunately, this focus on wet environments has often resulted in exclusion of species occurring in open and drier Phylogenetic approaches aiming to uncover historical biogeog- biomes from major discussions about the origin of Neotropical bio- raphy patterns and the associated drivers of diversification in diversity (Redford et al., 1990; Sánchez-Azofeifa et al., 2005). As a Neotropical taxa have focused primarily on species from wet result, early views of a poor and homogeneous biotic composition forested environments (Carnaval et al., 2009; Costa, 2003; across the great dry South American diagonal composed of Chaco, Cracraft and Prum, 1988; Hall and Harvey, 2002; Ribas et al., Cerrado, Caatinga and Dry Forest biomes have prevailed (Marris, 2005; Vanzolini, 1988, 1994). More recently, biotic surveys across previously unsampled q This paper was edited by the Associate Editor J.A. Schulte. areas have provided new material for biodiversity studies in open ⇑ Corresponding author. and dry Neotropical settings, supporting biogeographic and E-mail address: [email protected] (M. Teixeira Jr.). http://dx.doi.org/10.1016/j.ympev.2015.09.010 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423 411 phylogenetic approaches (Pennington et al., 2004; Nogueira et al., 2. Materials and methods 2011; Werneck et al., 2012; Guedes et al., 2014). This renewed interest has led to unexpected discovery of biodiversity. A notice- 2.1. Molecular analyses able case is that of Stenocercus lizards, for which a number of spe- cies occurring in open settings has only recently been described 2.1.1. Sampling of molecular data and recorded (Nogueira and Rodrigues, 2006; Ribeiro et al., 2009; We generated DNA sequences for 15 specimens: two Stenocer- Torres-Carvajal, 2005). Currently, Stenocercus is one of the most cus caducus, one S. doellojuradoi, four S. quinarius, five specimens speciose South American lizard genera, with 66 valid taxa assigned to S. sinesaccus, two S. squarrosus, and one S. cf. tricristatus. (Torres-Carvajal, 2007a; Torres-Carvajal and Mafla-Endara, 2013; We combined our new sequences with GenBank data of several Venegas et al., 2013, 2014; Köhler and Lehr, 2015). The genus is Tropiduridae taxa, including species of Stenocercus, Microlophus, particularly diverse along the Andes, from Venezuela in the north Plica, Tropidurus, Uracentron, and Uranoscodon. Our final dataset to central Argentina in the south, reaching up to 4000 m in eleva- comprised 73 samples representing 62 species (Supplementary tion (Torres-Carvajal, 2007a). Yet, a number of species are also Table S1). found in the wet Amazonian lowlands, and in the drier, open set- We extracted total genomic DNA from liver or tail fragments tings of southern, central and northeastern Brazil (Nogueira and preserved in ethanol using standard protocols. Matching available Rodrigues, 2006; Torres-Carvajal, 2007a). datasets (Torres-Carvajal, 2007b; Torres-Carvajal et al., 2006), we Stenocercus has been addressed by a number of comprehensive generated sequences of a long mitochondrial fragment containing systematic revisions (Cadle, 1991; Fritts, 1974; Torres-Carvajal, the NADH dehydrogenase subunit 1 (ND1) and 2 (ND2) and eight 2007a). Recently, the implementation of molecular tools and transfer RNAs (tRNA-Ile, tRNA-Gln, tRNA-Met, tRNA-trp, tRNA- detailed morphological analyses have shed light onto phylogenetic Ala, tRNA-Asx, tRNA-Cys, tRNA-Tyr), following Torres-Carvajal relationships and geographic patterns of diversification within the et al. (2006). Sequences were edited and aligned using Geneious genus (Torres-Carvajal, 2000, 2007a, 2009; Torres-Carvajal et al., Pro 6 (Biomatters, Auckland). Models of nucleotide evolution and 2006). Importantly, these studies revealed that Andean species best-fit partition schemes were determined with Partition Finder compose two main clades, which likely originated in the Central v.1.1.1 (Lanfear et al., 2012), implementing PhyML for likelihood Andes and subsequently dispersed and diversified throughout the estimation (Guindon and Gascuel, 2003) and the Akaike informa- Andes. However, because some non-Andean species were tion criterion for model selection (Akaike, 1974). described only recently, the full diversity of lowland Stenocercus has not yet been addressed in biogeographic studies (e.g., Torres- 2.1.2. Inferring phylogenetic relationships Carvajal, 2007b; Torres-Carvajal et al., 2006). These gaps have lim- We performed phylogenetic inference under a Bayesian frame- ited our understanding of the temporal and spatial diversification work using MrBayes 3.2.1 (Ronquist et al., 2012), implementing of Stenocercus in the open and drier South American lowlands east three independent runs of four Markov chains of 20 million gener- to the Andes. These species occur in a range of contrasting environ- ations each, and sampling every 1000 steps. We partitioned ments, such as S. azureus (Müller, 1880) in the southern Atlantic protein-coding genes by codon position as indicated by Partition Forest and Pampas grasslands, S. quinarius Nogueira and Finder. Due to the short length of each of the eight tRNAs Rodrigues, 2006 and S. squarrosus Nogueira and Rodrigues, 2006 (70 base pairs), we treated them as a single partition (631 base in Atlantic dry forests, eastern Cerrado savannas and western pairs total). We assessed convergence and stationarity of model Caatinga scrublands, S. caducus (Cope, 1862) and S. sinesaccus parameters using Tracer 1.5, combined runs in LogCombiner 1.8 Torres-Carvajal, 2005 in western Cerrado savannahs, S. tricristatus (with 25% discarded as burn-in), and summarized a maximum (Duméril, 1851) in southeastern Cerrado savannahs (Torres-Carvajal, clade credibility tree in TreeAnnotator 1.8 (Drummond et al., 2007a), S. dumerilii (Steindachner, 1867) in eastern Amazonian 2012). We unlinked parameters of substitution rates and nucleo- forest, and S. fimbriatus Avila-Pires, 1995 and S. roseiventris tide frequencies between partitions. The resulting topologies were (Duméril and Bibron, 1837) in western Amazonian forest. visualized in FigTree 1.4 (available from http://tree.bio.ed.ac. To improve our understanding about diversification processes uk/software/figtree/). in open and dry Neotropical biomes, we provide an updated For descriptive purposes, we also estimated Tamura–Nei historical biogeographic scenario of Stenocercus, focusing on the corrected pairwise genetic distances (Tamura and Nei, 1993) for genus’ diversification to the east of the Andes. We generated new the mitochondrial DNA fragment of all populations assigned to DNA sequence data of previously unsampled Stenocercus species, Stenocercus sinesaccus and S. caducus, using the APE 3.1 package including S. dumerilii, S. quinarius, S. sinesaccus, S. squarrosus, and (Paradis et al., 2004) of the R 3.0.2 platform (R Core Team, 2015). S. cf. tricristatus recently collected in Brazil. By combining these data with existing molecular datasets, we inferred phylogenetic relationships, estimated divergence times, and tracked ancestral 2.1.3. Divergence time estimation distributions, to examine whether cladogenetic events within To estimate divergence times between Stenocercus species, as Stenocercus correlate with reported shifts in South American well as between the genus and related Tropiduridae taxa, we per- landscapes during the past 30 million years. We also used environ- formed simultaneous phylogenetic reconstruction and divergence mental data to estimate the climatic envelopes currently occupied time estimation using BEAST v.1.8 (Drummond et al., 2012). To cal- by lowland Stenocercus. We integrated niche and phylogenetic data ibrate the root of Tropiduridae, we set a normally-distributed prior to test for correlations between genetic and ecological divergence on the node corresponding to the most recent common ancestor of (Graham et al., 2004) and to examine the role of past climatic Stenocercus, Uranoscodon and Plica (mean = 49 Mya, standard devi- fluctuations, habitat change and local adaptation in shaping ation = 5.1 Mya) following Prates et al. (2015). We rooted our tro- current distribution patterns. Instead of focusing on the better pidurid tree by implementing a molecular clock strategy, which known Andean Stenocercus, we emphasize those species from the yields rooted trees in the absence of outgroups through the simul- dry and open lowland settings of South America. taneous estimation of tree topology and branch lengths Our new molecular information reveals a highly divergent clade (Felsenstein, 2004; Drummond and Rambaut, 2007). Molecular within previously recognized S. sinesaccus. As morphological data clock-based rooting has been found to be a robust and effective support that this is a distinct species from the closely related method (Huelsenbeck et al., 2002), especially useful in cases where S. caducus and S. sinesaccus, we formally describe the new taxon herein. an outgroup is not available or where deep divergence between the 412 M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423 group of interest and its sister leads to extensive substitution sat- initially performed analyses including 20 tropidurid outgroup taxa uration (e.g., Ribas et al., 2012; Cohen, 2013). We implemented an available in GenBank. However, presumably because outgroup uncorrelated lognormal relaxed clock strategy (Drummond et al., genera (e.g., Microlophus, Plica, Tropidurus) often contain species 2006) with a uniform prior distribution (interval = 0–1) to the having broad ranges in South America, and because most species mean rate of the molecular clock (ucld.mean parameter), while within those genera have not yet been sampled genetically, their implementing default settings for the parameters relative to sub- inclusion provided little or no information about ancestral ranges stitution rates, nucleotide frequencies, and the Yule tree prior. within Stenocercus. As a result, we also performed ancestral area We ran three independent chains of 100 million steps, sampling reconstructions including no outgroups (see Section 3.4.1). every 10,000 steps. After assessing stationarity and convergence of model parameters in Tracer, we discarded 25% as burn-in, com- 2.2.2. Environmental analyses bined the runs, and summarized results into a maximum clade To explore climatic space occupancy that underlies the distribu- credibility tree as described in Section 2.1.2. tion of analyzed Stenocercus species, we extracted environmental data from 19 bioclimatic variables (Hijmans et al., 2005) at each 2.2. Biogeographical analyses geographical record of the lowland Stenocercus species sampled in this study, using ArcGIS v. 10.0 (ESRI, 2011) at a 30-s resolution. 2.2.1. Ancestral area reconstruction We included S. prionotus and S. pectinatus, which are not included To perform ancestral area reconstructions, we combined the in our molecular analyses but are likely closely related to South American ecoregions of Olson et al. (2001) (proposed based S. caducus/S. sinesaccus and to S. doellojuradoi/S. azureus, respec- on fauna and flora distributions) into 13 geographic units. Due to tively, as suggested by morphological data (Torres-Carvajal, computational limits on the maximum number of areas 2007b). We implemented a principal component analysis (PCA) implemented in the analyses (see below), we also merged ecore- (Field, 2009) on the environmental variables upon a correlation gions sharing the same Stenocercus fauna when defining these geo- matrix, using SPSS v.20 (SPSS, 2011). Following best practices for graphic units. This strategy resulted in the following areas PCA (Field, 2009), we avoided both redundant (e.g. correla- (Supplementary Fig. S1): (1) the semi-arid Caatinga in northeast- tion > 0.9) and non-correlated variables (p > 0.05) by checking the ern Brazil (Caa), (2) Chaco shrublands and the Bolivian savannahs correlation matrix and their significance levels prior to analyses. of Beni (Cha), (3) the eastern Cerrado in Brazil (ECe), (4) the west- ern Cerrado in Brazil (WCe), (5) the Atlantic Forest and Pampas 2.3. Morphological analyses grasslands (SAF), (6) northern (NAn), (7) central (CAn), (8) and southern Andes (SAn), (9) western Amazonia, adjacent to the Because we found high genetic divergence between two clades Andean foothills (WAm), (10) eastern Amazonia and the Brazilian originally assigned to Stenocercus sinesaccus (see results in Sec- Babaçu forests of Maranhão (EAm), and (11) the southern tion 3.1), we evaluate the species’ current taxonomic status by car- Amazonia (SAm). In analyses including outgroup taxa, we also rying out an examination of morphological variation in this and in included (12) a broad northern Amazonian area which included closely related S. caducus. We refer to the lineage that comprises Tepui mountains, Llanos, and Guiana grasslands (NAm), and lastly specimens from the vicinities of S. sinesaccus’ type locality, and (13) the Galapagos islands (Gal). To define the distributions of which exhibit the species’ typical morphology, simply as S. sinesaccus; Stenocercus taxa, we obtained geographical data from our own we refer to the second, highly divergent lineage as an ‘‘unconfirmed collection records and from published references; a complete list candidate species” (UCS), following Padial et al. (2010). is provided in the online Supplementary References S1. A list of specimens examined for morphological comparisons is We performed ancestral area reconstruction under a presented in the Supplementary data 4. A detailed description of maximum likelihood framework using the BioGeoBEARS package the statistical procedures performed in our morphological (Biogeography with Bayesian and Likelihood Evolutionary Analysis in analyses, including tests of data normality and homoscedasticity, RScripts;(Matzke, 2013)inR(RCoreTeam,2015). BioGeoBEARS is presented in the Supplementary data 5 (Morphological estimates the geographic distribution of ancestral nodes on a methodology S1). time-calibrated phylogeny. It iteratively optimizes a likelihood function incorporating a range of free parameters that describe the rate or the relative weights of several biogeographic events, includ- 3. Results ing range expansion, local extinction, and cladogenesis through vicariance and sympatric speciation. Importantly, BioGeoBEARS 3.1. Molecular analyses allows for comparisons of model fitting across different parameter combinations, using the Akaike information criterion (AIC) and 3.1.1. Phylogenetic relationships of Stenocercus AIC weights (Burnham and Anderson, 2002; Wagenmakers and Phylogenetic analyses recovered two main clades within Farrell, 2004). Stenocercus: one composed solely of Andean species (A) and We compared six biogeographic models using BioGeoBEARS: another harboring both Andean and lowland taxa (B; Fig. 1). Clade (1) the dispersal-extinction-cladogenesis (DEC), originally imple- A includes 16 sampled taxa (Fig. 1). Most occur in the central mented in Lagrange (Ree et al., 2005); (2) DEC with founder events Andes, except for S. humeralis, S. imitator, and S. varius, which are (DECj); (3) DIVALIKE, a maximum likelihood implementation of restricted to the northern portion of the cordillera, and for the parsimony-based dispersal-vicariance analyses (DIVA) S. marmoratus, which is distributed throughout the central and (Ronquist, 1997); (4) DIVALIKE with founder events (DIVALIKEj); southern Andes and in the adjacent Chaco. (5) BayAreaLIKE, a maximum likelihood implementation of the The second main Stenocercus clade (B) is composed of lowland BayArea model, which incorporates no cladogenetic events (clades 1, 2) and Andean species. Clade 1 was maximally supported (Landis et al., 2013); and (6), BayAreaLIKE with founder events (PP = 1) and includes six lowland species exhibiting largely allopa- (BayAreaLIKEj). We allowed for a maximum of six areas per node, tric distributions across a diversity of habitats, from the Andean which corresponds to the highest observed number of areas foothills in Argentina to the northern Atlantic coast in Brazil. This occupied by a sampled taxon (Stenocercus caducus). clade was recovered as the sister of Stenocercus roseiventris from To conduct ancestral range inferences, we pruned our resulting western Amazonia and northwestern Argentina, although with dated phylogeny to include only one terminal per species. We low support. Within the clade 1, we recovered the ‘horned’ species M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423 413

Stenocercus orientalis Stenocercus latebrosus Stenocercus ornatissimus Stenocercus chrysopygus Stenocercus stigmosus Stenocercus melanopygus Stenocercus boettgeri Stenocercus cupreus A Stenocercus imitator Stenocercus eunetopsis Stenocercus empetrus Stenocercus varius Stenocercus humeralis Stenocercus quinarius Stenocercus marmoratus Stenocercus crassicaudatus Stenocercus torquatus Stenocercus quinarius PHV2204 Jalapão Stenocercus quinarius MZUSP 99475 Peruaçu Stenocercus quinarius MZUSP 99482 Peruaçu To outgroups Stenocercus quinarius MZUSP 99471 Peruaçu Stenocercus squarrosus LG 2033 Serra das Confusões Stenocercus squarrosus LG 2032 Serra das Confusões a Stenocercus dumerilii MZUSP 94471 Palmeirante Stenocercus dumerilii MZUSP 94470 Estreito 1 Stenocercus cf. tricristatus MZUSP 88874 Serra da Canastra Stenocercus azureus ZVC-R 5969 Bajada de Pena b Stenocercus doellojuradoi FML 9298 Finca Los Colorados Stenocercus doellojuradoi MTR 00325 Fuerte Esperanza Stenocercus roseiventris Stenocercus caducus MHNHP 6383 Parque Nacional San Luis de la Sierra Stenocercus caducus FSFL 1019 Corumbá B Stenocercus caducus LG 1297 Cáceres 2 Stenocercus sinesaccus LG 1578 Manso Stenocercus sinesaccus MZUSP 89549 Cocalinho Stenocercus UFMT 3110 Guapore Candidate Stenocercus UFMT 3076 Guapore new species Stenocercus UFMT 3111 Guapore Stenocercus apurimacus Stenocercus scapularis Stenocercus ochoai Stenocercus formosus Stenocercus limitaris Stenocercus ornatus Stenocercus percultus Stenocercus iridescens Stenocercus puyango Stenocercus rhodomelas Stenocercus angulifer Stenocercus chota Stenocercus sinesaccus > 95% posterior probability Stenocercus angel Stenocercus guentheri Stenocercus festae 0.1 Stenocercus cadlei

Fig. 1. Phylogram showing the phylogenetic relationship of Stenocercus species addressed in the study, recovered through a Bayesian analysis (MrBayes) on the concatenated dataset of mitochondrial genes. Scale bar represents estimated substitution per site. Black circles on nodes represent clades with posterior probability values higher than 95%. Posterior probability values for all nodes are presented in the complete tree provided in the Supplementary Material (Fig. S2).

(clade 1a) Stenocercus quinarius and S. squarrosus from Atlantic dry Corrected pairwise genetic distances between the candidate forests as sister taxa, closely related to the eastern Amazonian S. species and the closely related S. sinesaccus ranged between dumerilii. The most recent common ancestor of these three taxa 9.6% and 10.3%. Similarly, the candidate species and S. caducus is sister to S. cf. tricristatus (Fig. 1), which is restricted to the eastern exhibited pairwise genetic distances ranging from 9.6% to 11% Cerrado. Altogether, these four species share a common ancestor (Table 1). with the more southern S. doellojuradoi and S. azureus (clade 1b), a pair of sister species whose distributions span the Chaco and the southern Atlantic Forest including Pampas grasslands, Table 1 respectively. Corrected pairwise genetic distances (%) for mtDNA, among species in the clade 2, S. Stenocercus clade 2 (the western lowland clade, Fig. 1)is sinesaccus, S. caducus and the unconfirmed candidate species (UCS). composed of S. caducus and of two highly divergent clades 1234567 assigned to S. sinesaccus. We refer to such clades as S. sinesac- cus, in the western Cerrado, and as an ‘unconfirmed candidate 1 Stenocercus UCS – UFMT3076 species’, whose distribution includes both Cerrado and southern 2 Stenocercus UCS 0.6 – Amazonia. We recovered Stenocercus Clade 2 as sister to UFMT3110 another major Andean clade composed of 16 species, yet this 3 Stenocercus UCS 0.1 0.4 – relationship was poorly supported (PP = 0.54). Most species UFMT3111 4 S. sinesaccus MZUSP89549 9.6 10.0 9.8 – within this clade occur in the northern Andes, although 5 S. sinesaccus LG1578 10.1 9.8 10.3 2.6 – S. apurimacus, S. formosus, S. ochoai, and S. scapularis comprise 6 S. caducus FSFL1019 10.0 9.6 9.8 12.3 10.9 – a maximally supported clade restricted to the central portion 7 S. caducus LG1297 11.0 10.7 10.8 12.8 11.1 1.6 – of the cordillera (Fig. 1). 8 S. caducus MHNHP6383 10.2 9.8 10.0 12.3 11.3 0.6 1.6 414 M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423

3.1.2. Divergence times within Stenocercus morphological and morphometric variables, as described in detail Dating analyses suggest that the most recent common ancestor in the Supplementary data 6 (Morphological Results S1). (MRCA) of sampled Stenocercus dates back to 27.1 Mya (median value; 95% of the highest posterior density [HPD] = 20–34.4 Mya) 3.3. Taxonomic results (Supplementary Fig. S2). Diversification within Stenocercus clade A, which occurs mostly in the central Andes, dates back to approx- 3.3.1. A new species of Stenocercus imately the early Miocene, at 18 Mya (95% HPD = 13–23 Mya) In the face of deep, old divergences (see Section 3.1.2), and of a (Supplementary Fig. S2). On the other hand, the MRCA of the north- large number of significant differences between Stenocercus sine- ern Andean species within clade B dates back to 22 Mya (95% saccus and the proposed candidate species in both quantitative HPD = 16–28 Mya). Nearly all diversification events within these (see Section 3.2) and qualitative variables (see Section 3.3.1.1), clades date back to the Miocene, between 6.7 and 22 Mya (median and between them and S. caducus (Fig. 2), we recognize our candi- values). Exceptions are the younger divergence between S. festae date species of Stenocercus as a ‘confirmed candidate species’ and S. cadlei, which dates back to the Pliocene (5.2 Mya, 95% (Padial et al., 2010). We describe the new taxon in the following HPD = 3.3–7.4 Mya), and that between S. angel and S. guentheri, sections. dating back to the Pleistocene (2.4 Mya, 95% HPD = 1.4–3.6 Mya). (Supplementary Fig. S2). The MRCA of Stenocercus species in clade 1a dates back the late 3.3.1.1. Taxon description. Stenocercus albolineatus sp. nov. Miocene at 8.4 Mya (95% HPD = 5.5–11.7 Mya), while the MRCA of Fig. 3, Figs. S3, S4, S5A and S6 S. dumerilii, S. quinarius and S. squarrosus was found to be more recent, dating back to the transition between the Pliocene and Stenocercus caducus – Silva et al., 2010 Pleistocene, approximately 2.7 Mya (95% HPD = 1.8–3.9 Mya). The Stenocercus sinesaccus – Nogueira and Rodrigues, 2006 – part; latter two taxa diverged during the Pleistocene, around 1.5 Mya Torres-Carvajal, 2007a – part (95% HPD = 0.9–2.2 Mya). The MRCA of species in the clade 1b, Stenocercus sp. – Silva, 2007 – part S. azureus and S. doellojuradoi, dates back to the late Miocene, around 8.4 Mya (95% HPD = 4.4–10.13 Mya). Both clade 1a Holotype: MZUSP 97964, adult male; Brazil: Mato Grosso: and 1b shared a MRCA approximately 12.4 Mya (95% HPD = municipality of Vale de São Domingos, UHE Guaporé hydroelectric 8.7–16.7 Mya). In the second lowland clade of Stenocercus (clade dam (approximate coordinates 15°070S, 58°570W). Collected by the 2), S. caducus and S. sinesaccus and our candidate species share rescue team during lake filling in 2002. a MRCA approximately 11 million years old (95% HPD = Paratypes: Brazil: Mato Grosso: UHE Guaporé: MZUSP 97956– 7.6–15 Mya). The MRCA of S. sinesaccus and of the candidate 97963, MZUSP 97965, UFMT 3076–3114, UFMT 3510–3511, UFMT species dates back to the late Miocene (7.9 Mya, 95% HPD = 5.2– 6277–6280, UFMT 7268; same data as the holotype. 11 Mya), which is older than the divergences between several Referred specimens: Brazil: Mato Grosso: Araputanga: INPA recognized Stenocercus species (Supplementary Fig. S2). 15963–15964, INPA 15968–15969; Cotriguaçu: UFMT 8964; Indi- avai: INPA 1562, INPA 15967; PCH Ombreiras: UFMT 2871–2879, 3.2. Morphological analyses UFMT 9537; Juína: PCH Juína: UFMT 5745; Lambari d’Oeste: MZUSP 102986–102987; Quatro Marcos: INPA 15065; Rio Branco: We examined 87 individuals of Stenocercus caducus,53of INPA 1966; Sapezal: UFMT 7365–7366, UFMT 7450, UFMT 7462, S. sinesaccus, and 160 of our candidate species in morphological UFMT 7494–7495; Vale de São Domingos: UFMT 7401, UFMT analyses (Supplementary Examined Specimens S1). Comparisons 7404, UFMT 7406, Vila Bela da Santissima Trindade: Serra da between species revealed significant differences in several Borda: UFMT 2290. Rondônia: Espigão d’Oeste: MPEG 21937,

3.0 A S. caducus B S. caducus S. sinesaccus S. sinesaccus Stenocercus (UCS) 3.0 Stenocercus (UCS)

2.0 2.0

1.0 1.0

0.0

0.0 PC2 (12.1%) PC2 (15.1%) -1.0

-1.0 -2.0

-3.0 -2.0

-4.0 -2.0 -1.0 0.0 1.0 2.0 -2.0 0.0 2.0 4.0 PC1 (47.3%) PC1 (52.7%)

Fig. 2. Ordination diagram of principal component analysis, upon meristic (A) and size-corrected morphometric (B) data of Stenocercus caducus, S. sinesaccus and the unconfirmed candidate species (UCS). M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423 415

Fig. 3. Live individual of Stenocercus albolineatus sp. nov. from Cotriguaçu, Mato Grosso, Brazil (A); lateral views of the heads of S. albolineatus sp. nov. (B), S. sinesaccus (C) and S. caducus (D).

MPEG 21487; Pimenta Bueno: CHUNB 18042–18049; Vilhena: Characterization: (1) SVL in males 70.2–85.5 mm (n = 26); (2) CHUNB 11473–11474. SVL in females 73.3–92.2 mm (n = 49); (3) vertebrals 32–45; (4) Etymology: The specific epithet albolineatus is derived from the paravertebrals 35–53; (5) scales around midbody 31–41; (6) gular Latin words albus (=white) and linea (=line) and atus (=that bears), scales 11–20; (7) transversal dorsals between crests 9–13; (8) as all members of the new species present a whitish line on the ventral scales 19–27; (9) caudal scales 111–134; (10) supraoculars anterior surface of the forelimbs, characteristic of this species. 3–6; (11) internasals 4–7; (12) subdigitals on Finger IV 13–19; (13) Diagnosis: Data from species in comparison are given in paren- subdigitals on Toe IV 21–30; (14) posthumeral mite pocket absent; theses. The new species differs from all other species of Stenocercus (15) postfemoral mite pocket absent; (16) parietal eye visible except S. caducus, S. dumerilii, S. prionotus, S. quinarius, S. sinesaccus, through interparietal cornea; (17) scales on occipitoparietal region S. squarrosus and S. tricristatus by having strongly keeled and large, keeled, juxtaposed; (18) row of enlarged supraoculars occu- mucronate body scales, laterally oriented nostrils, and lacking a pying most of supraocular region absent; (19) preauricular fringe postfemoral mite pocket. present; (20) neck folds absent; (21) lateral and dorsal nuchals It differs from Stenocercus caducus and S. prionotus by lacking a similar in size; (22) posterior gulars rhomboidal, projected posteri- posthumeral mite pocket (present). It can be distinguished from orly, strongly keeled and imbricate, not notched; (23) dorsal body S. dumerilii, S. tricristatus, S. quinarius and S. squarrosus by lacking scales larger than laterals; (24) vertebrals larger than adjacent par- triangularly enlarged horn-like post-supraciliaries (present). From avertebrals; (25) dorsolateral crest present; (26) ventrals keeled, S. caducus it can be further distinguished by the presence of com- imbricate; (27) scales on posterior surfaces of thighs keeled, imbri- plete dorsolateral crests (incomplete) and higher number of scales cate; (28) inguinal granular pocket absent; (29) inguinal groove along the white line on forelimb, 4–10 (1–5) (Supplementary absent; (30) tail not compressed laterally in adult males; (31) tail Fig. S5). From S. sinesaccus, the new species differs by having a length 68–71% of total length; (32) caudal autotomic segments higher number of vertebral scales, 32–45 (26–34), paravertebrals, absent; (33) caudals not spinose; (34) dark patch extensively cov- 36–53 (32–41), scales around body, 31–41 (27–36), transverse ering gular region of females absent; (35) dark patch extensively dorsals between dorsolateral crests, 11 in 75% of the individuals (9 covering gular region of adult males absent; (36) black patch on in 88% of the individuals), scales along the white line on forelimb, ventral surface of neck in adult males absent; (37) dark midventral 4–10 (1–5) (Supplementary Fig. S5) and ventrals, 19–27 (16–21). longitudinal mark such as faint line, conspicuous stripe, or Furthermore, S. albolineatus sp. nov. lacks a complex pattern of extensive patch in adult males absent; (38) dark patches on ventral markings over the shoulder (present in S. caducus)andawhitish surface of thighs in adult males absent; (39) white line over fore- scale on the anterior border of the ear (present in S. sinesaccus). limb; (40) scales along anterior border of ear homogeneously The new species is currently known to be sympatric with dark-colored. Stenocercus roseiventris. Besides overall body color and enlarged Coloration in preservative: In males, background coloration scales on tail, which produce a spiny shape in S. roseiventris, the ranges from dark to pale brown; dorsal color, limited at each side new species can be distinguished by lower number of vertebral by the dorsolateral keel, is always paler than flanks and limbs. Dor- scales 32–45 (50–59), paravertebrals, 36–53 (64–75), scales sum and flanks with blurred inconspicuous bars, those on dorsum around body, 31–41 (59–64), gulars, 11–20 (30–35), and oblique at each side forming a chevron pointing posteriorly. Post- transverse dorsals between dorsolateral crests, 9–13 (15–19), as pelvic chevrons darker and with sharper edges than pre-pelvic well as higher number of lamellae under the fourth toe, 21–30 ones. Tail banded with darker and pale brown; sharp-edged marks (17–20) and caudal scales, 111–136 (49–52). along tail are often observed. Forelimbs mostly homogeneous 416 M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423 brown, bearing a long whitish longitudinal line on arm; hindlimbs Dorsals phylloid, imbricate. Eleven transverse rows of dorsal scales usually with transverse bar on tight and shank (Supplementary at midbody, between dorsolateral crests (included). Thirty-nine Fig. S6A). Venter pale brown, with irregular scattered faded blurred vertebral scales from occiput to base of tail. Flanks with similar markings. but distinctively smaller scales. Twenty-three rows of ventral In females, background coloration ranges from dark to pale and scales between anterior level of forelimbs and anterior level of grayish brown; dorsal color, limited at each side by the dorsolat- hind limbs. Thirty-three scales around midbody. eral keel, is sometimes paler than flanks and limbs. Dorsum and Scales on preanal plate in irregular transverse rows, mite flanks mostly with blurred conspicuous bars, sometimes inconspic- pockets absent. Scales on both dorsal and ventral part of tail similar uous; those on dorsum oblique at each side forming a chevron to dorsals, phylloid, flat, keeled, anteriormost ones bearing a short pointing posteriorly, often with sharp edges. The series of sharp mucron, distal ones smaller. No distinct caudal verticils. edged chevrons start at the level of forelimbs and extends to the Limbs with phylloid, imbricate, strongly keeled and mucronate tail. Tail banded with darker and pale brown; sharp-edged marks scales, smaller than dorsals. The keels form distinct ridges on dor- along tail are often observed. Forelimbs mostly homogeneous sal, anterior and posterior aspect of limbs. Subdigital lamellae sin- brown, bearing a long whitish longitudinal line on arm; hindlimbs gle, tricarinate, 17 under fourth finger and 26 under fourth toe. usually with transverse bar on tight and shank (Supplementary Variation: all variables presented some variation (see Supplementary Fig. S6B). Venter ranging from cream to pale brown, usually with Tables S8 and S9). The transverse dorsal scales (TD), although irregular scattered faded blurred dark markings, throat and chin ranging from 9 to 13, 121 specimens out of the 160 examined with oblique series of blurred dark bars on each side. presented 11 TD (75%). The number of scales along the whitish line Description of the holotype: (Supplementary Figs. S3 and S4) over the forearm (LIN) ranged from 6 to 9 on 131, out of the 160 Adult male, 78.6 mm SVL, 184 mm TL. Head length 0.23 times (81%). Comparatively in Stenocercus sinesaccus, also although TD SVL, 1.4 times as long as wide, 1.7 times as wide as high. Snout ranges from 8 to 11, 47 out of 53 specimens had 9 TD (88%), and bluntly pointed in dorsal view, pointed in profile. Canthal ridge only two individuals presented 11 TD, and in LI, 46 out of well defined, continuous with supraciliaries. Neck slightly nar- the 53 specimens presented 2–4 scales along the white line on rower than head and body. Body roughly cylindrical. Limbs slen- arm (86%). In the morphometric variables, there was a strong der, forelimbs 0.4 times SVL, hindlimbs 0.7 times. Tibia 0.2 times variation regarding the sexes (Supplementary Table S9). SVL, and 0.3 times length of hindlimb. Tail roughly rounded in Distribution: Currently known from localities at the top and sur- cross-section from base to tip, 2.3 times SVL. rounding Chapada dos Parecis, from the western portion of Mato Rostral semicircular, largest width about three times median Grosso to eastern Rondônia states, Brazil (Fig. 4). This area encom- height, barely visible from above. Three postrostrals, wider than passes a large Mesozoic sandstone plateau, limited by the Guaporé long. Occipitals, parietals, interparietal, and postparietals large, River at its western side and by the Paraguay River at its eastern strongly keeled, juxtaposed. Supraorbital semicircle anteriorly dis- side (Supplementary Fig. S7). tinct and supraocular scales differentiated from surrounding Natural history: Stenocercus albolineatus sp. nov. seems to be scales. Canthal single, not reaching the postrostrals, bordered ante- more abundant during the rainy season, when adults are com- riorly by an elongate, keeled internasal. Supraciliaries seven, first monly found; during the dry season only juveniles were observed, four overlapping posteriorly, with similar size. The two last smal- suggesting a seasonal reproduction by the end of the wet and ler, sixth acuminate. Canthals and supraciliaries form a distinct beginning of the dry season (Silva et al., 2010). It is a generalist crest that ends in a strongly keeled post-supraciliary. Interparietal species in habitat occupancy, occurring over pasturelands, forest moderately enlarged, in the shape of an asymmetric pentagon, edge, and pristine forest; feeds on terrestrial arthropods, with parietal eye distinct. Posterior region of head bearing irregular, Coleoptera, Hymenoptera (Formicidae) and Isoptera as the most keeled shields; interparietal and parietals not differentiated. frequent (Silva et al., 2010). Nasal lateral, large, undivided; nostril in posterior part of nasal, directed laterally. Loreal region with three vertical rows of slightly 3.4. Biogeography keeled scales, first and third double, second triple. One horizontal row of mostly elongate lorilabials. One subocular, about four times 3.4.1. Historical biogeography of Stenocercus as long as high, with a longitudinal keel close to its upper margin. Of the six historical biogeographic models compared using Bio- Subocular separated from supralabials by lorilabials. Supralabials GeoBEARS, the Dispersal-extinction-cladogenesis model with four, narrow, anterior one smallest, fourth largest, below center founder events (DECj) demonstrated the best fit for the data as of eye. Temporal region with irregularly polygonal, strongly keeled supported by AIC weights (>0.94). Results suggest that the most scales; seven scales in an oblique row from lower posterior border probable (p = 0.28) distribution of the MRCA of all Stenocercus of orbit to above ear opening. Ear opening relatively large, verti- was a broad are encompassing the Chaco, central and southern cally oval, anteriormost part covered by a fringe. Tympanum Andes, and western and southern Amazonia simultaneously slightly recessed. (Fig. 5). Mental small, not distinctively larger than adjacent infralabials; The resulting model recovered broad ancestral ranges for both bordered posteriorly by two bulky, keeled scales and by two (one lowland clades of Stenocercus. The most likely distribution of the on each side) elongate and keeled infralabials. Infralabials five, MRCA of S. caducus, S. sinesaccus and S. albolineatus sp. nov. elongate, strongly keeled; first smallest, third one largest, below included the Chaco, central and southern Andes, and western Cer- anterior half of eye. One row of elongated sublabials, with a right rado (p = 0.83). The MRCA of the S. sinesaccus and S. albolineatus sp. median keel. Scales on chin anteriorly relatively small, elongate, nov. was likely distributed in the western Cerrado (p = 0.54) or in subimbricate, posteriorly grading into gulars. Gulars larger than both western Cerrado and southern Amazonia (p = 0.44). In turn, scales on chin and smaller than adjacent ventrals, strongly keeled we inferred a broad ancestral distribution for the horned lowland and mucronate, in irregular longitudinal rows; no gular or lateral clade of Stenocercus, including at least part of the Chaco, central folds. and southern Andes, and western and southern Amazonia Scales on nape similar to dorsals, anteriormost ones smaller. A (p = 0.75). The MRCA of S. quinarius, S. squarrosus, S. dumerilii and vertebral crest of enlarged, prominent and sharply keeled scales S. tricristatus likely occurred in the eastern Cerrado (p = 0.92); col- extends from nape to first third of tail. A dorsolateral crest of scales onization of eastern Amazonia and Caatinga seem to have favored extends from above ear opening to anteriormost part of tail. the divergences of S. dumerilii and S. squarrosus, respectively, from M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423 417

70°W 60°W 50°W S. albolineatus sp. nov. S. pectinatus 1 S. azureus S. prionotus S. caducus S. quinarius S. doellojuradoi S. sinesaccus S. dumerilii S. squarrosus

10°S 10°S S. cf. tricristatus Biomes Rain Forests Dry Savannas Temperate Grasslands Dry Forests Caatinga Flooded Savannas

50°W 40°W

2 0°

Pa ci fic O

20°S c 20°S e a n 10°S

0165 330 660

km 70°W 60°W 50°W 20°S 20°S

2 1 30°S 30°S

an ce O tic n a tl A

km 0275 550 1,100 40°S 40°S

70°W 60°W 50°W 40°W 30°W

Fig. 4. Map showing the known distribution of Stenocercus albolineatus sp. nov., S. caducus, S. prionotus and S. sinesaccus (A), and S. azureus, S. doellojuradoi, S. dumerilii, S. pectinatus, S. quinarius, S. squarrosus and S. cf. tricristatus (B). Markers with a black central dot represent examined specimens; white central circles indicate molecular samples. Bold-outlined markers represent type localities, when available. their eastern Cerrado sister lineages. The common ancestor of of environmental data (Supplementary Table S10). The resulting S. doellojuradoi and S. azureus probably occurred in the Chaco correlation matrix included variables having correlation coeffi- (p = 0.61); subsequent expansion into the southern Atlantic Forest cients ranging from 0.3 to 0.9, most of which significant; commu- and Pampas seems to correlate to the divergence between these nalities were above 0.7. two species. Our analyses suggest that the most likely ancestral For clade 1, three components were extracted in a PCA of distribution of the six aforementioned Brazilian Stenocercus species environmental data (eigenvalues > 1), with 40.6% of the observed corresponds to the Chaco area. Lastly, we found that the most variance explained by PC1 and 35.5% by PC2 (Supplementary likely ancestral range for both Andean clades was the central Andes Table S10; Fig. 6A). PC1 was positively correlated with precipita- (p > 0.84), from which different lineages dispersed into the north- tion on the warmest and driest quarter, annual mean temperature ern and southern Andes, Chaco, and western Amazonia. and temperature of the wettest quarter, and negatively correlated with minimum temperature of the coldest month. PC2 was posi- 3.4.2. Environmental envelopes tively correlated with annual precipitation and precipitation of After evaluating communalities, multicolinearity and uncorre- the driest month and negatively correlated with precipitation of lated variables, we retained seven bioclimatic variables for a PCA the wettest month and mean diurnal temperature range 418 M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423

Alpine Mountains & hills Shields Rivers, lakes & wetlands Marine incursion

Stenocercus festae Stenocercus cadlei Stenocercus guentheri Stenocercus angel Stenocercus chota Stenocercus angulifer Stenocercus rhodomelas Stenocercus puyango Stenocercus iridescens Stenocercus percultus Stenocercus ornatus Stenocercus limitaris Stenocercus scapularis Stenocercus apurimacus Stenocercus ochoai Stenocercus formosus Stenocercus albolineatus sp. nov. Stenocercus sinesaccus Stenocercus caducus Stenocercus quinarius Stenocercus squarrosus Stenocercus dumerilli Stenocercus tricristatus Stenocercus doellojuradoi Stenocercus azureus Stenocercus roseiventris Stenocercus crassicaudatus Stenocercus torquatus Stenocercus marmoratus Caa SAF Stenocercus humeralis CAn SAm Stenocercus varius Stenocercus eunetopsis Cha SAn Stenocercus empetrus EAm WAm Stenocercus imitator Stenocercus boettgeri ECe WCe Stenocercus cupreus NAn Stenocercus stigmosus CAnSAnCha Stenocercus melanopygus Stenocercus orientalis CAnSAnWCeCha Stenocercus latebrosus CAnSAnSAmWAmCha Stenocercus ornatissimus Stenocercus chrysopygus NAnWAm SAmWCe Eocene Oligocene Miocene Plio Quat

30.0 20.0 10.0 0,0 mya +8 5 km 5 +4 4

0 3 T (°C) [δ O] -4 18 2 2 CAn 1 4 1 - Garzione et al 2008 1 2 - Insel et al 2012 -8 3a NAn 3 - Hoorn et al 2010 (a,b: different locations) 3b 4 - Gregory-Wodzicki 2000 0 5 - Zachos et al 2001

Fig. 5. Biogeographical history of Stenocercus; top panel: landscape changes (adapted from Cook et al., 2012; Hoorn et al., 2010; Lundberg et al., 1998); middle panel: ancestral area reconstruction (Caa = Caatinga; CAn = Central Andes; Cha = Chaco; EAm = Eastern Amazonia; ECe = Eastern Cerrado; NAn = Northern Andes; SAF = South Atlantic Forest; SAm = Southern Amazonia; SAn = Southern Andes; WAm = Western Amazonia; WCe = Western Cerrado); lower panel: temperature changes (adapted from Zachos et al., 2001) and Andean uplift (adapted from Garzione et al., 2008; Gregory-Wodzicki, 2000; Hoorn et al., 2010; Insel et al., 2012).

(Supplementary Table S10). Each species occupied distinct regions and wetter areas, and S. pectinatus ranging from warmer and drier of the environmental space: Stenocercus dumerilii occurs in warmer to cooler and wetter areas (Fig. 6A). and wetter areas, S. squarrosus, S. quinarius and S. doellojuradoi in For the Stenocercus clade 2 PCA, three components were warmer and drier areas, S. azureus and S. cf. tricristatus in cooler extracted, with 46.5% of the observed variance explained by PC1 M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423 419

3.0 A S. azureus B S. albolineatus sp. nov. S. doellojuradoi S. caducus S. dumerilii S. prionotus S. pectinatus 2.0 S. sinesaccus S. quinarius S. squarrosus S. cf. tricristatus 2.0 1.0

0.0 1.0

-1.0 PC2 (35.5%) PC2 (25.30%)

0.0 -2.0

-3.0 -1.0

-4.0 -1.0 0.0 1.0 2.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 PC1 (40.6%) PC1 (46.5%)

Fig. 6. Ordination diagram of a principal component analysis upon environmental data from each known record of species within clade 1, Stenocercus azureus, S. doellojuradoi, S. dumerilii, S. prionotus, S. quinarius, S. squarrosus and S. cf. tricristatus (A), and clade 2, S. albolineatus sp. nov., S. caducus, S. pectinatus and S. sinesaccus (B). and 25.3% by PC2 (Supplementary Table S10; Fig. 6B). PC1 was pos- viously explored. Lastly, differences in resulting tree topologies are itively correlated with annual precipitation, minimum tempera- expected to have affected the outcome of ancestral area analyses. ture of the coldest month, precipitation of the wettest month, Previous works (Torres-Carvajal et al., 2006; Torres-Carvajal, annual mean temperature and precipitation of the driest month, 2007b), also found two major clades within Stenocercus composed negatively correlated with mean diurnal temperature range. In mostly of Andean species, while the species from the dry lowlands turn, PC2 was positively correlated with maximum temperature were all grouped in a single clade. Those results presented of the warmest month and annual mean temperature, and Stenocercus caducus as sister to S. prionotus,andbothtoS. sinesaccus. negatively correlated with precipitation of the driest month This group was inferred to be sister to a group comprising S. dumerilii (Supplementary Table S10). Species in clade 2 presented a broad and S. tricristatus. Although low supported, this clade had overlap across the environmental space, with S. caducus and S. roseiventris as sister, forming a clade sister to S. doellojuradoi, S. prionotus occupying a broad range along both PCs, and with S. azureus and S. pectinatus. Our results however present those S. sinesaccus and S. albolineatus sp. nov. presenting an intermediate species in two distinct clades (clades 1 and 2; Fig. 1; see following position along PC1 and a broad occurrence along PC2 in both sections). Nonetheless, basal splits in our recovered phylogeny positive and negative directions (Fig. 6B). are poorly supported, and thus a different history could be recovered with the inclusion of more genetic markers and taxa. We found that a biogeographic model incorporating founder 4. Discussion event speciation (DECj) had the best fit to our data. Founder event describes a particular case of allopatric speciation in which a small 4.1. Historical biogeography and the timing of Stenocercus set of individuals, or a single individual, perform a long-distance diversification dispersal and colonization, presenting a random subsample of the genetic pool of the source population, prompting it to subse- Our analyses found that the most recent common ancestor quent differentiation (Mayr, 1954; MacDonald, 2003; Lomolino (MRCA) of Stenocercus dates back to the late Oligocene, and that et al., 2006; Cox and Moore, 2010; Matzke, 2014). The parameter it exhibited a broad distribution simultaneously including at least ‘j’ has been shown to be highly predictive in island systems (see part of the Chaco, central and southern Andes, and western and Matzke, 2014), in which species often show high dispersal capabil- southern Amazonia (Fig. 5; Supplementary Fig. S7). These findings ities (e.g., birds or flight invertebrates), or are helped by wind and are in partial agreement with a previous ancestral area analysis of ocean currents; nonetheless it has not been broadly addressed in this group; Torres-Carvajal (2007b) found that the MRCA of Steno- intracontinental terrestrial species. Such a model is thus problem- cercus occupied the eastern cordillera of the central Andes, an area atic as the major promoter of speciation in continental, terrestrial, that is included within our resulting ancestral range. Our finding of sedentary species, as no analogous mechanism to ocean currents is a broader ancestral area for Stenocercus may be related to a number known to affect such species. Dispersal capabilities in Stenocercus of factors. First, we defined a larger number of areas, which are not is unknown for most species, but for some forms in the clade 1 they fully equivalent to those implemented previously. This is especially seem to be strictly sedentary, moving only a few meters per day true for taxa occurring in the lowlands to the east of the Andes, (Teixeira Jr., 2010). Nevertheless, a scenario of colonization and such as S. caducus, S. tricristatus, S. doellojuradoi, S. azureus and S. divergence fits some Stenocercus species pairs. For instance, we pectinatus, previously assigned to a broad ‘Atlantic lowlands’ area found that the ancestor of S. quinarius (an eastern Cerrado species) (Torres-Carvajal, 2007b). Moreover, we implemented an additional and S. squarrosus (a Caatinga species) was most likely distributed in set of ancestral area reconstruction algorithms, obtaining a higher the eastern Cerrado, suggesting that colonization of the Caatinga fit to the data for the dispersal-extinction-cladogenesis model with subsequent interruption of gene flow has favored divergence (DEC) as compared to the dispersal-vicariance analysis (DIVA) pre- between S. quinarius and S. squarrosus (Fig. 5). Similarly, we found 420 M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423 that the ancestor of S. doellojuradoi (a Chaco species) and S. azureus colonization between distinct habitats, as described in the vanish- (a Pampas species) was most likely distributed in the Chaco, indi- ing refugia model (VRM) (Damasceno et al., 2014; Vanzolini and cating that range expansion to a new area likely triggered cladoge- Williams, 1981). This model proposes a situation in which gradual nesis in this case as well. A role for founder event speciation in the environmental change allows for populations to progressively diversification of such terrestrial organisms deserves to be exam- adapt locally, ultimately triggering divergence (Damasceno et al., ined in additional continental groups. 2014; Vanzolini and Williams, 1981). Speciation under the VRM predicts distinct ecologies between closely related parapatric taxa, 4.1.1. The clade 1 – the ‘‘horned” Stenocercus and related taxa which contrasts sharply with the common pattern under Our biogeographic analyses recovered the most likely ancestral phylogenetic niche conservatism, in which a species pair is range of Stenocercus lowland clade 1 to the Chaco biome, from ecologically similar (Crisp and Cook, 2012; Losos, 2008). In which lineages have reached the eastern Cerrado and the area the face of geographic and environmental segregation, and of comprising the southern Atlantic Forest and Pampas grasslands temporal congruence between species divergences and periods of (Fig. 5). Subsequently, both the arid Caatinga and eastern Amazonia extensive climatic fluctuations and habitat fragmentation in were likely colonized from eastern Cerrado ancestors (Fig. 5). South America (Fig. 5), the VRM may be a candidate mechanistic This inferred role of dispersal in the diversification of lizards model for the initial divergence between some lowland Stenocercus occurring in the dry diagonal contrasts with the traditional view pairs. that establishment of vicariant barriers has been the major promoter of population divergence and subsequent speciation in continental South America (Moritz et al., 2000). Allopatric 4.1.2. The clade 2 – the western lowland Stenocercus distributions of closely related Neotropical taxa have been widely Contrasting with the horned clade, our results indicate that the attributed, for instance, to the formation of rivers, mountain three species with available molecular data in the Stenocercus low- chains, and geologic faults (Nascimento et al., 2013; Ribas et al., land clade 2, which presumably includes also S. prionotus (see 2012; Ribeiro et al., 2013; Smith et al., 2014). Even in the absence results of Torres-Carvajal, 2007b), may have evolved allopatrically of evident barriers, clusters of co-distributed taxa have been within the western Cerrado and adjacent Amazonia. Marked mor- interpreted as a result of vicariant speciation in the Cerrado and phological differentiation and deep genetic divergences pose the Caatinga (Guedes et al., 2014; Nogueira et al., 2011). While broadly question of how these ecologically similar species, which show distributed taxa across the entire dry diagonal are likely exposed to broad overlap in environmental space occupancy (Fig. 6), have dif- a complex set of geographical features that could promote ferentiated. Niche conservatism may lead populations to diverge differentiation (Werneck et al., 2012), it has been challenging to when isolated by intervening unsuitable environmental condi- identify landscape features that correlate with the deep genetic tions, as in the case of populations occurring in high elevations breaks observed within the core regions of the Caatinga and separated by lowlands (Wiens and Graham, 2005). It has been Cerrado (Domingos et al., 2014; Gamble et al., 2012; Prado et al., shown that closely related species occurring in parapatry and shar- 2012; Santos et al., 2014). As a result, it is still largely unclear what ing similar ecological requirements may have diverged in allopatry are the mechanistic factors involved with species divergences followed by secondary contact (Graham et al., 2004; Wiens and within the South American dry diagonal. Graham, 2005). In the Stenocercus clade 2, contact areas between Our analyses of environmental space occupancy in the species ranges broadly coincide with the limits of the upper Stenocercus clade 1 indicate that, following colonization of a Guaporé and Paraguay depressions (see definition in Ross, 1985). new area, ecological differentiation may have favored genetic The Guaporé depression currently separates the Chapada dos divergence and speciation even in the absence of pronounced Parecis plateau in western Brazil from the pre-Andean highlands geographic discontinuities. An examination of climatic envelopes to the west, where S. caducus is found; the Paraguay depression reveals nearly complete environmental segregation among most separates this plateau from the Brazilian shield to the east, where species in this clade (Figs. 4 and 6), suggesting that they are S. sinesaccus and some populations of S. caducus are found; and the subjected to contrasting regimes of temperature and water avail- area between (and north to) both depressions is occupied by ability. In the Cerrado, environmental heterogeneity correlates to S. albolineatus sp. nov. (Supplementary Fig. S6). spatial turnover of anuran assemblages, suggesting physiological Divergence times between these three species are consistent and life history constraints to species’ distributions (Valdujo with the timing of reported shifts in this region as a result of et al., 2013). Strong natural selection favoring locally-adapted Andean orogeny. Lowlands between the Andean foothills and the phenotypes may promote genetic divergence between populations Brazilian shield underwent large scale landscape transformations even in the presence of substantial gene flow, a process referred between 11.8 and 8 Mya, with shifts in the course and contacts to as ecological speciation (Nosil, 2012). Our data on the environ- of major rivers, marine incursions coming from both north and mental spaces occupied by Stenocercus clade 1 support the largely south, and the establishment and vanishing of large lacustrine or unexplored idea that prominent environmental gradients and marine systems (Cook et al., 2012; Hernández et al., 2005; ecological differentiation have shaped biodiversity patterns in Hoorn, 2006; Lovejoy et al., 2006; Lundberg et al., 1998; Räsänen open and dry Neotropical settings. et al., 1995). Formation of large water bodies seems to have However, dispersal and colonization alone are unlikely to have affected the composition of ecological communities at the Para- produced the observed distributional patterns in this clade. guay and Guaporé depressions (Cook et al., 2012; Lundberg et al., Although typically found in forested habitats (with the exception 1998), with connections between basins promoting faunal of Stenocercus. cf. tricristatus), species in the horned clade often exchange (Lundberg et al., 1998; Montoya-Burgos, 2003; Ribeiro show broad habitat tolerance, being found in lower abundances et al., 2013). Our results are consistent with the hypothesis that at adjacent open areas (Avila-Pires, 1995; Nogueira and the formation of these water systems may have isolated the ances- Rodrigues, 2006; Teixeira Jr., 2010). These habitats have undergone tor of Stenocercus. caducus from its eastern sister, while the later a turbulent mixing at this portion of the dry diagonal in the last establishment or shifting courses of the Paraguay River could have few million years (Ab’Saber, 2000), during climatic fluctuations of led to the divergence of S. sinesaccus and S. albolineatus sp. nov.,in high amplitude (Zachos et al., 2001). This local fragmentation the absence of ecological differentiation. Geomorphological change and disappearance of habitats coupled with the exhibited in this region has also been associated with species divergences in environmental tolerances may have facilitated the dispersal and other lizard groups (Werneck et al., 2012). M. Teixeira Jr. et al. / Molecular Phylogenetics and Evolution 94 (2016) 410–423 421

4.1.3. The Andean Stenocercus Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP The diversification and history of colonization in both Andean 2003/10335-8 and 2011/50146-6), Conselho Nacional de Desen- clades of Stenocercus (clades A and B) have been comprehensively volvimento Científico e Tecnológico (CNPq), and by the Dimensions addressed in the literature (Torres-Carvajal, 2007b), yet no infor- of Biodiversity Program [FAPESP (BIOTA, 2013/50297-0), NSF (DOB mation about the timing of diversification was previously avail- 1343578), and NASA]. CS acknowledges funding from CNPq able. Our results indicate that these Andean clades are as old as research fellowship (process # 309541/2012-3). the cordillera itself; the highest areas corresponding to the present-day Andes reached up to 2000 m above the sea level by Appendix A. Supplementary material the onset of Andean Stenocercus diversification, at around 27 Mya (Garzione et al., 2008; Insel et al., 2012). On the other hand, while Supplementary data associated with this article can be found, in the MRCA of Stenocercus likely occupied part of the developing cor- the online version, at http://dx.doi.org/10.1016/j.ympev.2015.09. dillera in the late Oligocene, our dating analysis indicates that most 010. extant Andean lineages originated more recently, between the early and the late Miocene (Fig. 5). During this time, major oroge- netic processes took place on both the central and northern Andes, References resulting in pronounced uplift of the cordillera (Benjamin et al., Ab’Saber, A.N., 2000. Spaces occupied by the expansion of dry climates in South 1987; Garzione et al., 2008; Garzione et al., 2006, 2007; Ghosh America during the quaternary ice ages. Rev. Inst. Geol. 21, 71–78. et al., 2006; Gregory-Wodzicki, 2000). In other animal groups, lin- Akaike, H., 1974. New look at statistical-model identification. IEEE Trans. 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Table S1: GenBank accession numbers for newly generated sequences

LG=Laboratory number for lizards; FSFL=Felipe Sá Fortes Leite, UFMT=Universidade Federal do Mato

Grosso; MZUSP=Museu de Zoologia da Universidade de São Paulo; PHV; Paula Hanna Valdujo.

Species Voucher Locality State Country GenBank Stenocercus caducus LG1297 Cáceres MT Brazil KT827772 Stenocercus caducus FSFL1019 Corumbá MT Brazil KT827771 Stenocercus albolineatus sp. nov. UFMT3076 UHE Guaporé MT Brazil KT827768 Stenocercus albolineatus sp. nov. UFMT3111 UHE Guaporé MT Brazil KT827770 Stenocercus albolineatus sp. nov. UFMT3110 UHE Guaporé MT Brazil KT827769 Stenocercus sinesaccus MZUSP89549 Cocalinho MT Brazil KT827782 Stenocercus sinesaccus LG1578 UHE Manso MT Brazil KT827781 Stenocercus doellojuradoi LG1094 Fuerte Esperanza Chaco Argentina KT827776 Stenocercus quinarius MZUSP99471 Peruaçu valley MG Brazil KT827775 Stenocercus quinarius MZUSP99482 Peruaçu valley MG Brazil KT827774 Stenocercus quinarius MZUSP99475 Peruaçu valley MG Brazil KT827779 Stenocercus quinarius PHV2204 EE Serra Geral do Tocantins BA Brazil KT827780 Stenocercus squarrosus LG2032 Serra das Confusões PI Brazil KT827783 Stenocercus squarrosus LG2033 Serra das Confusões PI Brazil KT827784 Stenocercus dumerilli MZUSP94470 Estreito TO Brazil KT827777 Stenocercus dumerilli MZUSP94471 Palmeirante TO Brazil KT827778 Stenocercus cf. tricristatus MZUSP88874 Serra da Canastra LG1308 MG Brazil KT827773

Table S2. Results for Kolmogorov-Smirnov (K-S) normality test and Levene’s (F) homoscedasticity test, for each meristic variable, along with their respective probability values (p), between typical Stenocercus sinesaccus and the unconfirmed candidate species (UCS), and the UCS and S. caducus.

Abbreviations of each variable are explained in the text; values in bold are the significant ones.

S. sinesaccus Stenocercus S. sinesaccus Stenocercus (UCS) S. caducus typical x UCS UCS x S. caducus N K-S p N K-S p N K-S p F p F p

VS 53 1.030 0.239 160 1.371 0.047 87 1.240 0.092 13.440 0.000 5.519 0.020

PS 53 0.861 0.449 160 1.523 0.019 87 1.280 0.076 1.233 0.268 1.339 0.248

SAB 53 1.080 0.194 160 1.963 0.001 87 0.866 0.442 4.287 0.040 0.415 0.520

GS 53 2.120 0.000 160 2.043 0.000 87 1.582 0.013 7.571 0.006 0.276 0.600

TD 53 3.651 0.000 160 5.617 0.000 86 2.078 0.000 15.840 0.000 25.425 0.000

SO 53 2.646 0.000 160 4.361 0.000 87 2.556 0.000 0.065 0.799 2.577 0.110

IN 53 1.734 0.005 160 3.622 0.000 86 2.803 0.000 0.137 0.711 5.056 0.025

F4 48 1.138 0.150 160 1.976 0.001 87 1.463 0.028 0.001 0.972 0.946 0.332

T4 48 0.927 0.357 160 2.093 0.000 87 0.944 0.336 0.310 0.578 6.316 0.013

LIN 53 1.529 0.019 159 1.694 0.006 86 2.049 0.000 9.791 0.002 32.638 0.000

NS 53 1.618 0.011 160 2.064 0.000 87 1.353 0.051 4.804 0.029 2.318 0.129

VE 49 1.451 0.030 158 1.877 0.002 86 1.071 0.201 8.099 0.005 1.841 0.176

CS 43 0.773 0.773 136 1.019 0.250 73 0.732 0.658 0.236 0.628 8.467 0.004

Table S3. Results for Kolmogorov-Smirnov (K-S) normality test, Levene’s (F) homoscedasticity test, and Student’s (t) test for equality of means, for each morphometric variable across sexes, in the typical Stenocercus sinesaccus, the unconfirmed candidate new species (UCS), and S. caducus, with their respective probability values (p). Abbreviations of each variable are explained in the text; values in bold are the significant ones. Normality Comparisons Homoscedasticity females males ♀ x ♂

N K-S p N K-S p F p t p

HH 9 0.360 0.999 14 0.468 0.981 6.094 0.022 4.084 0.002

HW 9 0.364 0.999 14 0.674 0.754 0.605 0.445 8.464 0.000

HL 9 0.379 0.999 14 0.640 0.807 3.331 0.082 6.418 0.000

SVL 9 0.448 0.988 14 0.622 0.834 2.873 0.105 7.552 0.000

DBM 9 0.530 0.942 14 0.488 0.971 4.689 0.042 5.132 0.000

TL 8 0.563 0.909 14 0.776 0.584 0.000 0.989 2.602 0.017

FoL 8 0.822 0.509 14 0.555 0.918 4.251 0.052 -0.776 0.446

SL 8 0.576 0.894 14 0.526 0.945 5.349 0.031 -0.240 0.816

FeL 8 0.445 0.989 14 0.523 0.947 4.120 0.055 -0.322 0.751

Stenocercus sinesaccus Stenocercus HaL 9 0.465 0.982 14 0.785 0.569 0.630 0.436 1.814 0.084

RL 9 0.703 0.707 14 0.456 0.985 0.156 0.697 1.040 0.310

HuL 9 0.457 0.985 14 0.750 0.628 0.086 0.772 2.136 0.045

HH 49 0.551 0.921 26 0.532 0.940 0.017 0.897 4.635 0.000

HW 49 0.557 0.916 26 0.594 0.873 0.241 0.625 6.477 0.000

HL 49 0.540 0.932 26 0.690 0.728 0.273 0.603 6.324 0.000

) SVL 49 0.629 0.823 26 0.966 0.309 0.826 0.366 7.291 0.000

UCS DBM 49 0.838 0.484 26 0.554 0.919 0.000 0.984 6.819 0.000 ( TL 40 0.594 0.872 23 0.840 0.481 21.308 0.000 -2.354 0.022

FoL 49 0.537 0.935 26 0.698 0.714 1.328 0.253 2.490 0.015

SL 49 0.864 0.444 26 0.572 0.899 0.059 0.809 4.469 0.000

Stenocercus Stenocercus FeL 49 0.762 0.607 26 0.427 0.993 0.419 0.520 4.210 0.000

HaL 49 0.629 0.823 26 0.668 0.763 0.748 0.390 2.908 0.005

RL 49 0.906 0.384 26 0.583 0.886 1.280 0.262 3.838 0.000

HuL 49 0.764 0.603 26 0.997 0.997 0.330 0.567 3.627 0.001

HH 12 0.534 0.938 29 0.780 0.577 0.016 0.900 1.823 0.076 HW 12 0.668 0.764 29 1.173 0.128 0.067 0.798 2.837 0.007 HL 12 0.375 0.999 29 0.616 0.842 2.591 0.116 4.176 0.000 SVL 12 0.421 0.994 29 0.977 0.295 1.618 0.211 4.618 0.000 DBM 12 0.744 0.638 29 0.824 0.506 0.014 0.907 5.301 0.000 TL 8 0.583 0.886 25 0.665 0.768 103.252 0.000 -2.197 0.050 FoL 12 0.484 0.973 29 0.675 0.753 0.015 0.903 1.168 0.250 SL 12 0.646 0.798 29 0.853 0.461 0.030 0.863 2.734 0.009 FeL 12 0.465 0.982 29 0.457 0.985 3.353 0.075 1.087 0.284 Stenocercus caducus Stenocercus HaL 12 0.504 0.961 29 0.694 0.720 0.542 0.466 2.468 0.018 RL 12 0.361 0.999 29 0.937 0.344 0.745 0.393 1.839 0.074 HuL 11 0.605 0.858 29 0.842 0.478 3.274 0.078 -0.720 0.476

Table S4. Results for Levene’s (F) homoscedasticity test, for each morphometric variable between typical Stenocercus sinesaccus and the unconfirmed candidate new species (UCS), and the UCS and S. caducus, with their respective probability values (p). Abbreviations of each variable are explained in the text; values in bold are the significant ones.

N S. sinesaccus Stenocercus

typical x UCS UCS x S. caducus S. sinesaccus S. typical UCS caducus F p F p

HH 9 49 12 3.499 0.067 0.544 0.464

HW 9 49 12 0.071 0.791 0.011 0.918

HL 9 49 12 0.672 0.416 3.845 0.055

SVL 9 49 12 0.565 0.456 0.662 0.419

DBM 9 49 12 0.644 0.426 0.075 0.785

TL 8 40 8 0.085 0.772 0.793 0.378

FoL 8 49 12 1.125 0.293 0.100 0.753

Females SL 8 49 12 1.295 0.260 0.009 0.924

FeL 8 49 12 0.702 0.406 11.399 0.001

HaL 9 49 12 0.022 0.882 0.004 0.948

RL 9 49 12 0.036 0.851 0.942 0.336

HuL 9 49 11 2.027 0.160 0.894 0.348

HH 14 26 29 1.671 0.204 1.750 0.192

HW 14 26 29 0.599 0.444 0.001 0.981

HL 14 26 29 3.623 0.065 0.144 0.706

SVL 14 26 29 0.507 0.481 2.214 0.143

DBM 14 26 29 4.393 0.043 0.306 0.583

TL 14 23 25 0.027 0.871 0.849 0.362

FoL 14 26 29 0.082 0.776 2.084 0.155 Males SL 14 26 29 0.858 0.360 0.025 0.876

FeL 14 26 29 0.009 0.925 0.476 0.493

HaL 14 26 29 0.122 0.729 0.026 0.873

RL 14 26 29 0.365 0.549 0.971 0.329

HuL 14 26 29 0.517 0.476 0.006 0.938

Table S5. Comparison results between typical Stenocercus sinesaccus and the unconfirmed candidate new species (UCS), and the UCS and S. caducus, under Mann-Whitney (U) test, on each meristic variable. Abbreviations of each variable are explained in the text; values in bold are the significant ones.

N S. sinesaccus Stenocercus

S. sinesaccus S. typical x UCS UCS x S. caducus typical UCS caducus U p U p VS 53 160 87 58.000 0.000 6571.000 0.464

PS 53 160 87 557.000 0.000 13591.000 0.000

SAB 53 160 87 296.000 0.000 12293.000 0.000

GS 53 160 87 271.500 0.000 11168.500 0.000

TD 53 160 86 672.000 0.000 10643.000 0.000

SO 53 160 87 3077.000 0.001 6800.500 0.736

IN 53 160 86 5296.500 0.003 8004.000 0.022

F4 48 160 87 2.238 0.000 11709.000 0.000

T4 48 160 87 2756.000 0.003 9915.000 0.000

LIN 53 159 86 48.000 0.000 93.000 0.000

NS 53 160 87 1065.000 0.000 11352.000 0.000

VE 49 158 86 321.500 0.000 11951.500 0.000

CS 43 136 73 3824.000 0.002 9392.000 0.000

Table S6 Comparison results between typical Stenocercus sinesaccus and the unconfirmed candidate new species (UCS), and the UCS and S. caducus, under Student’s (t) test, on each meristic variable, and its corresponding probability (p). Abbreviations of each variable are explained in the text; values in bold are the significant ones.

N S. sinesaccus Stenocercus

S. sinesaccus S. typical x UCS UCS x S. caducus typical UCS caducus t p t p

HH 11 55 17 -0.532 0.597 -1.135 0.261 HW 11 55 17 0.549 0.585 0.133 0.894 HL 11 55 17 0.023 0.982 -0.219 0.827 SVL 11 55 17 0.718 0.476 -1.264 0.211

DBM 11 55 17 1.253 0.216 -0.161 0.873 TL 11 45 13 0.032 0.974 -3.065 0.004 FoL 11 55 17 5.374 0.000 -2.462 0.017

Females SL 11 55 17 5.499 0.000 -2.368 0.021 FeL 11 55 17 7.432 0.000 -0.546 0.595 HaL 11 55 17 3.472 0.001 -1.477 0.145 RL 11 55 17 4.015 0.000 0.565 0.574 HuL 11 55 16 4.971 0.000 3.805 0.000 HH 15 36 39 3.431 0.001 -2.748 0.008 HW 15 36 39 5.505 0.000 -2.156 0.036 HL 15 36 39 3.386 0.002 -0.943 0.350 SVL 15 36 39 6.360 0.000 -1.677 0.099 DBM 15 36 39 4.207 0.000 -0.040 0.969

TL 15 33 35 1.352 0.185 -4.973 0.000 FoL 14 36 39 Males 7.058 0.000 -3.954 0.000 SL 14 36 39 7.147 0.000 -2.391 0.020 FeL 14 36 39 7.938 0.000 -2.474 0.017 HaL 15 36 39 4.323 0.000 -1.338 0.187 RL 15 36 39 2.204 0.034 -0.371 0.712 HuL 15 36 39 5.587 0.000 2.507 0.015

Table S7. Loadings for each variable on extracted principal components, upon meristic and size-corrected morphometric data for Stenocercus sinesaccus, S. caducus and the yet unconfirmed candidate species. Abbreviations of each variable are explained in the text. Bold values indicate those variables with higher loadings in each component.

Components Variables 1 2 3 PS 0.898 -0.113 0.110 SAB 0.886 0.109 0.060 VE 0.843 -0.005 -0.118 GS 0.823 0.235 0.167 TD 0.793 0.197 0.192

NS 0.710 -0.007 0.175

data F4 0.713 -0.245 -0.261 VS 0.640 0.606 0.032 LIN -0.062 0.873 -0.155

Meristic IN 0.009 -0.225 0.825 T4 0.569 -0.25 -0.504 CS 0.564 -0.627 0.064 Total 5.701 1.946 1.174 % Variance 43.852 14.971 9.028 HaL 0.902 -0.185 – HuL 0.880 -0.135 – RL 0.854 -0.196 – FeL 0.793 -0.306 –

SL 0.780 -0.343 –

data FoL 0.773 -0.349 – DBM 0.684 0.277 – HH 0.670 0.291 – TL 0.654 0.125 – HW 0.607 0.276 –

Morphometric SVL 0.490 0.612 – HL 0.470 0.636 – Total 6.326 1.455 – % Variance 52.718 12.123 –

Table S8. Meristic variables for Stenocercus albolineatus sp. nov., S. sinesaccus and S. caducus. Abbreviations of each variable are explained in the text.

S. albolineatus S. S. sp. nov. sinesaccus caducus N mean (range) N mean (range) N mean (range) VS 160 36.71 (32–45) 53 30.00 (26–34) 87 36.44 (32–41) PS 160 41.09 (35–53) 53 36.13 (32–41) 87 49.31 (42–56) SAB 160 36.12 (31–41) 53 30.9 (27–36) 87 40.03 (34–47) GS 160 15.41 (11–20) 53 12.43 (11–14) 87 16.98 (14–20) TD 160 10.69 (9–13) 53 9.11 (8–11) 86 11.73 (9–15) SO 160 4.76 (3–6) 53 4.45 (4–5) 87 4.75 (4–6) IN 160 5.48 (4–7) 53 5.84 (4–7) 86 5.77 (5–7) F4 160 16.28 (13–19) 48 15.29 (13–18) 87 18.21 (15–22) T4 160 25.01 (21–30) 48 24.14 (21–28) 87 26.48 (22–32) LIN 159 7.35 (4–10) 53 2.79 (1–5) 86 3.00 (1–5) NS 160 11.88 (9–16) 53 9.86 (8–12) 87 13.87 (11–22) VE 158 22.62 (19–27) 49 19.14 (16–21) 86 25.51 (21–30) CS 136 122.44 (111–136) 43 125.18 (114–134) 73 135.93 (115–149)

Table S9. Morphometric variables for Stenocercus albolineatus sp. nov., S. sinesaccus and S. caducus. Abbreviations of each variable are explained in the text.

Females Males N Mean (range) N Mean (range)

HH 49 11.21 (8.98–13.64) 26 10.23 (8.93–12.16) HW 49 13.85 (12.08–15.86) 26 12.6 (11.05–14.35)

HL 49 18.41 (16.53–19.99) 26 16.94 (15.29–19.52) SVL 49 82.36 (73.34–92.24) 26 74.63 (70.17–85.5)

sp. nov. DBM 49 41.07 (33.21–47.84) 26 35.60 (27.67–40.69) TL 40 185.92 (161.00–215.00) 23 177.21 (159.00–211.00) FoL 49 28.76 (22.35–32.37) 26 27.65 (25.52–31.96) SL 49 17.6 (15.66–20.77) 26 16.32 (13.73–19.81) FeL 49 17.56 (15.32–20.03) 26 16.56 (14.43–18.96) S. albolineatus S. albolineatus HaL 49 13.7 (11.6–16.52) 26 12.87 (9.22–15.09) RL 49 9.66 (7.82–11.85) 26 8.89 (7.16–11.02) HuL 49 11.89 (9.64–13.6) 26 11.11 (9.23–13.23) HH 9 11.34 (9.24–13.87) 14 9.36 (8.22–10.93) HW 9 13.39 (11.65–14.95) 14 11.29 (10.53–12.18) HL 9 18.06 (15.77–20.06) 14 15.9 (15–17.36) SVL 9 79.27 (65.96–89.83) 14 66.89 (62.66–73.69)

DBM 9 38.28 (28.59–48.02) 14 31.58 (28.5–35.38) TL 8 182.00 (157.00–200.00) 14 171.57 (140.00–185.00) FoL 8 24.59 (22.10–26.83) 14 23.91 (20.36–26.73) SL 8 15.03 (12.36–16.85) 14 13.80 (11.86–15.14)

S. sinesaccus FeL 8 14.86 (13.93–15.93) 14 13.75 (11.91–15.9) HaL 9 12.03 (9.7–13.94) 14 11.24 (10.15–14.63) RL 9 8.44 (7.63–9.21) 14 8.25 (6.64–9.69) HuL 9 9.99 (8.48–11.96) 14 9.31 (7.06–10.62) HH 12 11.46 (9.40–12.9) 29 11 (9.03–13.39) HW 12 13.81 (11.82–15.34) 29 13.09 (11.24–14.46) HL 12 18.43 (17.47–19.54) 29 17.27 (14.74–19.48) SVL 12 83.77 (78.35–91.55) 29 77.03 (65.14–87.14) DBM 12 40.76 (34.86–48.02) 29 35.73 (30.17–39.77) TL 8 201.25 (181.00–229.00) 25 197.84 (158–222) FoL 12 30.20 (27.95–35.3) 29 29.57 (26.13–34.05)

S. caducus SL 12 18.38 (16.89–20.76) 29 17.25 (14.15–20.44) FeL 12 17.76 (14.67–20.93) 29 17.40 (14.03–19.44) HaL 12 14.24 (12.63–16.5) 29 13.30 (9.10–15.03) RL 12 9.46 (8.15–11.57) 29 9.03 (6.77–10.74) HuL 11 10.91 (9.60–11.97) 29 10.51 (7.66–13.20)

Table S10. Loadings for each of the seven retained bio-climatic variables used in the principal

components analyses over the environmental data, extracted from records of species of

Stenocercus belonging to Clade 1 and Clade 2. Bold values indicate higher loadings in each

component.

Clade 1- Components Clade 2- Components Variables 1 2 3 1 2 3 BIO01 Ann. Mean Temp. 0.939 -0.101 0.203 0.709 0.685 0.004 BIO02 Mean Diurnal Temp. Range -0.567 -0.723 0.225 -0.547 0.201 0.715 BIO05 Max. Temp. Warm. Month 0.479 -0.469 0.613 0.119 0.859 -0.124 BIO06 Min. Temp. Cold. Month -0.846 -0.343 0.201 0.851 0.316 -0.090 BIO07 Temp Ann. Range 0.729 -0.495 0.357 – – – BIO08 Mean Temp. Wet. Quarter 0.745 0.611 0.076 – – – BIO12 Ann. Precip. -0.272 0.886 0.338 0.858 -0.360 0.312 BIO13 Precip. Wet. Month 0.594 -0.699 -0.139 0.773 -0.151 0.558 BIO14 Precip. Driest Month -0.256 0.901 0.323 0.616 -0.521 -0.356 BIO15 Precip. Season -0.477 0.002 0.468 – – – BIO17 Precip. Driest Quarter 0.714 0.577 -0.032 – – – BIO18 Precip. Warm. Quarter 0.939 -0.101 0.203 – – – BIO19 Precip. Coldest Quarter -0.567 -0.723 0.225 – – – Total 4.465 3.905 1.099 3.253 1.771 1.070 Percentage of Variance 40.592 35.503 9.989 46.478 25.300 15.291

Supplementary data 2: Supplementary Figures

Figure S1. Area codification used in the ancestral area reconstruction analyses. Caa=Caatinga;

CAn=Central Andes; Cha=Chaco; EAm=Eastern Amazonia; ECe=Eastern Cerrado;

NAm=Northern Amazonia; NAn=Northern Andes; SAF=South Atlantic Forest; SAm=Southern

Amazonia; SAn=Southern Andes; WAm=Western Amazonia; WCe=Western Cerrado.

Figure S2. Dated phylogeny recovered through a Bayesian analysis on the concatenated dataset of NADH dehydrogenase subunit 1 and 2, and eight transfer RNAs, including all available samples of Stenocercus and the outgroups. Bars represent 95% of the highest posterior density.

Figure S3. Holotype of Stenocercus albolineatus sp. nov. (A) in dorsal and (B) ventral views.

SVL = 78.6 mm.

Figure S4. Head of the holotype of Stenocercus albolineatus sp. nov. in (A) lateral, (B) dorsal and (C) ventral views. Scale bar represents 10 mm.

Figure S5. Coloration on forearm of Stenocercus albolineatus sp. nov. (A), S. sinesaccus (B) and S. caducus (C).

Figure S6. Dorsal coloration variation in Stenocercus albolineatus sp. nov. in (A) males and

(B) females.

Figure S7. Close view of Chapada dos Parecis plateau, and the surrounding upper Paraguay and

Guaporé depressions, showing the distribution of Stenocercus albolineatus sp. nov., S. caducus and S. sinesaccus.

Supplementary data 3: References

Supplementary References S1

Source for the geographical records of Stenocercus species and outgroups

The records of Stenocercus used in geographical analyses were based on our own collection records and the literature data (Alvares, 2011; Avila-Pires, 1995; Carreira and

Baletta, 2004; Carreira et al., 2005; Cei, 1993; Dirksen and De la Riva, 1999; Duellman, 2005;

Freitas et al., 2011; Fritts, 1974; GBIF, 2013; Macedo et al., 2008a; Morais et al., 2010;

Nogueira, 2006; Nogueira and Rodrigues, 2006; Pansonato et al., 2008; Ribeiro et al., 2009;

Santo et al., 2011; Silva Jr et al., 2009; SpeciesLink, 2013; Torres-Carvajal, 2005, 2007; Torres-

Carvajal et al., 2013; Torres-Carvajal et al., 2006; Uetanabaro et al., 2007; Valdujo et al., 2009;

Vaz-Silva et al., 2007; Venegas et al., 2014).

The records of Microlophus, Plica, Tropidurus, Uracentron and Uranoscodon used in the geographical analyses were based on our own collection records and the literature data (Abreu et al., 2002; Alvarez et al., 1994; Avila-Pires et al., 2010; Avila and Kawashita-Ribeiro, 2011;

Barrio-Amoros and Brewer-Carias, 2008; Bernarde et al., 2011; Bernardo et al., 2012; Borges-

Leite et al., Morges; Borges-Nojosa and Caramaschi, 2003; Borges-Nojosa and Cascon, 2005;

Carvalho et al., 2005; Cei, 1982, 1986, 1993; Cunha, 1981; Dantas de Sales et al., 2009; de

Freitas et al., 2011; Dirksen and De la Riva, 1999; Donoso-Barros, 1968; Duellman, 2005;

Duellman and Mendelson, 1995; Etheridge, 1968; Fausto Filho, 1988; Fitzgerald et al., 1999;

Freire, 1996; Frost et al., 1998; Garda et al., 2013; GBIF, 2013; Greene, 1977; Hoogmoed,

1973; Hoogmoed and Lescure, 1975; Juncá, 2005; Kacoliris et al., 2006; Kizirian et al., 2004;

Lima, 2008; MacCulloch et al., 2007; MacCulloch and Reynolds, 2012; Macedo et al., 2008b;

Mendes-Pinto and Souza, 2011; Molina et al., 2004; Moraes, 2008; Morato et al., 2011;

Nogueira, 2006; Oliveira et al., 2014; Passoni et al., 2008; Pelegrin, 2007; Pellegrin and

Leynaud, 2006; Perez et al., 2011; Prudente et al., 2013; Reynolds and MacCulloch, 2012;

Ribeiro et al., 2008; Rocha et al., 2004; Rodrigues, 1987, 1996; Santana et al., 2008; Santos et al., 2008; Silva-Jr et al., 2008; Silva, 2008; SpeciesLink, 2013; Test et al., 1966; Torres- Carvajal et al., 2013; Valdujo et al., 2009; Valladares-Faundez, 2011; Vanzolini, 1986; Vogt et al., 2007; Waldez et al., 2013; Ziegler et al., 2002)

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Supplementary data 4: Examined material

Examined specimens S1

Stenocercus albolineatus sp. nov. (n=160): BRAZIL: Mato Grosso: unknown locality:

UFMT 6007; Araputanga: INPA 15963–15964, INPA 15968–15969, PCH Ombreiras:

UFMT 2758–2759, UFMT 2871–2879, UFMT 9537; Cáceres: UFMT 10216-10219;

Cotriguaçu: UFMT 8964, UFMT 9072; Indiavai: INPA1562, INPA 15967; Jauru: UFMT

9537, UFMT 9554; Juína: PCH Juína: UFMT 5745; Lambari d’Oeste: MZUSP 102986–

102987; Pontes e Lacerda: UFMT 10439, UFMT 10485, UFMT 10487, UFMT 10491,

UFMT 10690, UFMT 10696–10697, UFMT 11056-11057, UFMT 11070, UFMT 11072,

UFMT 11500, UFMT 11502; Quatro Marcos: INPA 15065; Reserva do Cabaçal: UFMT

937; Rio Branco: INPA 15966; Sapezal: UFMT 7365–7367, UFMT 7372–7375, UFMT

7445, UFMT 7450, UFMT 7456–7457, UFMT 7462, UFMT 7470–7471, UFMT 7475,

UFMT 7463, UFMT 7484–7485, UFMT 7494–7495, UFMT 7499, UFMT 7500–7504;

Sapezal: UHE Cachoeirão: MZUSP 96004–96008; Vale de São Domingos: UFMT 7401–

7409, UHE Guaporé: MZUSP 97956–97965, UFMT 887, UFMT 2632, UFMT 3076–3114,

UFMT 3510–3511, UFMT 6277–6280, UFMT 7268, UFMT 7494 (type series); Vila Bela

da Santissima Trindade: Serra da Borda: UFMT 2290. Rondônia: Espigão d’Oeste: MPEG

21937, MPEG 21487; Pimenta Bueno: CHUNB 18042–18049; Vilhena: CHUNB 11473–

11474.

Stenocercus caducus (n=87): BRAZIL: Mato Grosso do Sul: Bodoquena: MZUSP 93413,

MZUSP 94242–94248, MZUSP 94588–94597, MZUSP 94672–94675; Bonito: MTR

10312; Corumbá: UFMG 1250–1255, UFMT 1271, UFMT 1278, UFMT 1288–1289,

UFMT 1295, UFMT 1306, UFMT 1317, UFMT 1320, UFMT 6223–6224; Serra do

Urucum: MZUSP 7638. Mato Grosso: Porto Estrela: Estação Ecologica Serra das Araras:

UFMT 218, UFMT 2633, MPEG 14294, MTR 15672, MZUSP 94249–94251, MZUSP

94676–94677; Cáceres: JC 1519, MZUSP 83116–83143, MZUSP 89548; Porto Estrela:

UFMT 8303.Vila Bela da Santissima Trindade: MZUSP 82813–82816. BOLIVIA: Santa

Cruz: Santa Cruz de la Sierra: MPEG 19824. Stenocercus dumerilii (n=4): Brazil: Maranhão: Reserva Biológica Gurupi: MTR 23319.

Pará: Peixe Boi: MTR 33909; Tocantins: Estreito: MZUSP 94470–94471.

Stenocercus fimbriatus (n=1): Brazil: Amazonas: Tabatinga: INPA 1214.

Stenocercus quinarius (n=22): Brazil: Bahia: Correntina: MTR 27065–27066, MTR 27141;

Formosa do Rio Preto: Estação Ecológica Serra Geral do Tocantins: PHV 2204. Minas

Gerais: Parque Nacional Cavernas do Peruaçu: MTR 33262, MZUSP 99471–99475,

MZUSP 99478–99482, MZUSP 99485, MZUSP 99487–99488, MZUSP 99496, MZUSP

99498–99500.

Stenocercus roseiventris (n=5): Brazil: Rondônia: Espigão D’Oeste: MPEG 21494, MPEG

21934–21935; Vilhena: UFMT 6269.

Stenocercus sinesaccus (n=53): BRAZIL: Goiás: Aporé: CHUNB 38937, CHUNB 48192–

48194; Floresta Nacional de Silvania: ZUFG 130; Mineiros: CHUNB 32754; Montes

Claros de Goiás: UFMG 1201; PCH Mosquitão: MZUSP 98273–98274, MZUSP 98302,

MZUSP 98306–98310; Santa Rita do Araguaia: CHUNB 32755. Mato Grosso: unknown

locality: UFMT 11433; Barra do Garças: MPEG 29629–29630; Chapada dos Guimarães:

UFMT 6862, UHE Manso: MZUSP 92009, MZUSP 92012, MZUSP 92030, MZUSP

92040, UFMT 401–406, UFMT 1115–1119, UFMT 2459, UFMT 2460; Cocalinho:

MZUSP 89549; Guiratinga: MZUSP 98531–98534, MZUSP 98547–98550, MZUSP

98574; Primavera D’Oeste: UFMT 7748; Tesouro: MZUSP 99262–99266;.

Stenocercus squarrosus (n=19): Brazil: Ceará: Santana do Cariri: URCA 9, URCA 6824;

Piauí: Caldeirão Grande do Piauí: URCA 5906; Serra das Confusões: MZUSP 94057–

94067; Serra da Capivara: MTR 23543, MTR 25209, MTR, 25259, MTR 26299.

Stenocercus cf. tricristatus (n=2): Brazil: Minas Gerais: Serra da Canastra: MZUSP 94456,

MZUSP 88874. Supplementary data 5: Material and Methods

Morphological methodology S1

S2.3. Morphological analyses

We found a deep divergence (9.6%–10.3%), between the western and eastern populations, originally assigned to Stenocercus sinesaccus, and between the western populations and S. caducus (9.6–11%) (see section 3.1.1.), a range that lies within the known range between pairs oi sister species in Stenocercus (2.4–19.6%).

In order to assess the taxonomic status of the divergent lineage we performed an extensive examination of morphological variation across the entire distribution of Stenocercus sinesaccus; features were examined to check whether the morphological variation accompanied the genetic divergence. If robust morphological evidence matches the genetic data indicating a differentiation between the candidate species and S. sinesaccus, we will consider it a “confirmed candidate species” (Padial et al., 2010), describing it

We analyzed morphological data of all specimens available from Stenocercus sinesaccus, both the typical and the candidate populations, and S. caducus. Morphological characterizations, diagnostic features and comparisons are adapted from Torres-Carvajal (2005, 2007) and

Nogueira and Rodrigues (2006).

S2.3.1. Data

We quantified thirteen meristic and twelve morphometric variables on the left side of each specimen. Meristics were: vertebral scales along a mid-dorsal longitudinal row, from the post-occipital region to the level of the posterior edge of hindlimb insertion (VS); paravertebral scales along a longitudinal row adjacent to VS (PS); scales along a transversal row between dorsolateral crests, including those at its widest point (TD); scales around mid-body (SAB); gular scales in a transverse row, at the level of the ventral edge of the ear opening, starting on the first enlarged scale (GS); internasal scales in a transverse row between the nasal scales, right after the postrostrals (IN); number of supraocular scales along a transverse row, at the point of widest supraocular area (SO); ventral scales along a longitudinal row between the posterior edge of forelimb insertion and the anterior edge of hindlimb insertion (VE); number of scales under the fourth finger (F4); number of scales under the fourth toe (T4); scales along a caudal longitudinal row (CS); number of nuchal scales, along the first dorsal row after, and in contact with, the posterior enlarged head scales, comprised between the enlarged post supraciliaries

(NS); and number of transverse scale rows along the white line over the forearm (LIN).

Measurements were taken only in mature adult individuals. We considered matures all individuals with SVL larger than those presented by the smallest males with convoluted epididymis and females with vitellogenic follicles end/or corpora lutea. The following measurements were taken to nearest 0.01 mm with a digital caliper: snout-vent length, from the border of the cloaca to the tip of the snout (SVL); length between limbs, between the posterior margin of the forelimb to the anterior margin of the hindlimb (DBM); tail length, from the posterior margin of the cloaca to the tip of the tail (specimens with intact tails only) (TL); head height, at the highest point in a longitudinal axis (HH); head width, at its widest point (HW); head length, from the anterior margin of the tympanic aperture to the tip of the snout (HL); hand length, from the tip of the fourth finger to the hand insertion (HaL); radius length, from the elbow to the hand insertion (RL); humerus length, from the elbow to the arm insertion in the body (HuL); foot length, from the tip of the fourth toe to the leg insertion (FoL); shank length, from the knee to the foot insertion (SL); and femur length, from the knee to the leg insertion in the body (FeL). All specimens were also checked for the presence/absence of mite pockets.

Examined specimens are housed in the herpetological collection of the Museu de

Zoologia da Universidade de São Paulo (MZUSP), Universidade Federal de Mato Grosso

(UFMT), Coleção Herpetológica da Universidade de Brasília (CHUNB), Museu Paraense

Emílio Goeldi (MPEG), Instituto de Pesquisas da Amazônia (INPA), Universidade Federal de

Goiás (UFG), Universidade Regional do Cariri (URCA), Universidade Federal de Minas Gerais

(UFMG), and in the Laboratório de Herpetologia at the Universidade de São Paulo (JC = field numbers of José Cassimiro, MTR = field numbers of Miguel Trefaut Rodrigues). A complete list of specimens is presented in the online Supplementary Examined Specimens S1.

S2.3.2. Statistical treatment

To verify the assumption of normality of morphological variables in the comparisons between species, candidate species and sexes, we used the Kolmogorov-Smirnov test (K-S)

(Zar, 2014). We tested for homogeneity of variances across groups by using the Levene’s test

(F) (Levene, 1960). When normality and homoscedasticity were simultaneously met, we compared variable means using the parametric Student’s test (t); when normality but not homoscedasticity was met, we used the corrected Student’s test; (Zar, 2014). When normality and homoscedasticity were not met, we compared median values using the non-parametric

Mann-Whitney’s test (U) (Mann and Whitney, 1947). We performed all statistical analyses using SPSS.

To explore overall patterns among individuals of Stenocercus caducus and both S. sinesaccus and Stenocercus candidate species in a morphological multivariate space, we performed a principal component analysis (PCA) upon a correlation matrix of morphometric and meristic data separately, removing individuals exhibiting missing values. We avoided redundant and non-correlated variables as described in section 2.2.2. To remove the effect of body size, we performed a PCA on all morphometric variables for each species/candidate species separately for each sex. All variables showed high and positive loadings in PC1, which we considered as “size”. We then performed a regression of each variable upon the scores of

PC1, recorded unstandardized residuals, and used them in the PCA (Reist, 1985, 1986).

References

Levene, H., 1960. Robust tests for equality of variances. in: Olkin, I., Hotelling, H. (Eds.), Contributions to Probability and

Statistics: Essays in Honor of Harold Hotelling. Stanford University Press, Stanford, California, pp. 278–292.

Mann, H.B., Whitney, D.R., 1947. On a Test of Whether one of Two Random Variables is Stochastically Larger than the Other.

Ann. Math. Stat. 18, 50–60.

Nogueira, C., Rodrigues, M.T., 2006. The genus Stenocercus (Squamata: Tropiduridae) in extra–Amazonian Brazil, with the

description of two new species. S. Am. J. Herpetol. 1, 149–166.

Padial, J.M., Miralles, A., De la Riva, I., Vences, M., 2010. The integrative future of taxonomy. Front. Zool. 7, 1–14.

Reist, J.D., 1985. An Empirical–Evaluation of Several Univariate Methods That Adjust for Size Variation in Morphometric Data.

Can. J. Zool.–Rev. Can. Zool. 63, 1429–1439. Reist, J.D., 1986. An Empirical–Evaluation of Coefficients Used in Residual and Allometric Adjustment of Size Covariation. Can.

J. Zool.–Rev. Can. Zool. 64, 1363–1368.

Torres–Carvajal, O., 2005. A new species of Stenocercus (Squamata, Iguania) from central–western Brazil with a key to Brazilian

Stenocercus. Phyllomedusa 4, 123–132.

Torres–Carvajal, O., 2007. Phylogeny and biogeography of a large radiation of Andean lizards (Iguania, Stenocercus). Zool. Scr. 36,

311–326.

Zar, J.H., 2014. Biostatistical Analysis. Pearson Education Limited, United States of America.

Supplementary data 6: Results

Morphological Results S1

S1.1. Normality and homoscedasticity

We found departure from normality in Stenocercus sinesaccus meristic variables GS,

TD, SO, IN, LIN, NS and VE. For the candidate species, we found departure from normality in all variables except CS. For S. caducus, we found departure from normality in TD, SO, IN, F4 and LIN (Supplementary Table S2). We did not reject normality of any morphometric variable

(Supplementary Table S3).

We rejected homogeneity of variances between sexes of Stenocercus sinesaccus OTU 1 in morphometric variables HH, DBM and SL (Supplementary Table S3). For both the OTU 2 and S. caducus, we found no homogeneity of variances across sexes in TL. We found no homogeneity of variances between S. sinesaccus and the candidate species in meristic variables

VS, SAB, GS, TD, LIN, NS and VE, and between the candidate species and S. caducus in VS,

TD, IN, T4, LIN, and CS (Supplementary Table S2). We rejected homogeneity of variances between females of S. sinesaccus and the candidate species in all morphometric variables; between candidate species and S. caducus in FeL; and between males of S. sinesaccus and the candidate species in DBM (Supplementary Table S4).

S1.2. Sexual dimorphism

Regarding the morphometric data, we found significant differences between sexes of

Stenocercus sinesaccus in HH, HW, HL, SVL, DBM and TL. For the candidate species, we found significant differences in all variables. Lastly, we found significant differences between sexes of S. caducus in HW, HL, SVL, DBM, TL, SL and HaL (Supplementary Table S3). In all cases, females were larger than males.

S1.3. Comparisons between species

We found significant differences in all meristic variables between Stenocercus sinesaccus and the candidate species. We also found differences between candidate species and S. caducus in all meristic variables except SO (Supplementary Table S5). For females, we found significant differences in morphometric variables FoL, SL, FeL, HaL, RL and UL between S. sinesaccus and the candidate species, and in TL, FoL, SL and UL between candidate species and S. caducus. We also found significant differences between males of S. sinesaccus and the candidate species in all morphometric variables except TL, and in HH, HW, TL, FoL, SL, FeL and UL between candidate species and S. caducus (Supplementary Table S6).

After evaluating communalities, multicolinearity and presence of uncorrelated variables we removed SO from the PCA with meristic data (Supplementary Table S7). The resulting correlation matrix included variables having correlation coefficients ranging from 0.3 to 0.9, most of which were significant; most communalities were above 0.6. The PCA presented a well-structured distribution of individuals across multivariate space, with each species/candidates occupying a distinct region (Fig. 2A). Three components were extracted

(eigenvalues > 1), with 47.3% of the observed variance explained by PC1 and 15.1% by PC2.

Variables with the highest scores on PC1 were PS, SAB, VE, GS, TD, NS, F4 and VS; and LIN,

VS and CS on PC2 (Supplementary Table S7). By contrast, a PCA using morphometric data suggests broad overlap of each species/candidates across multivariate space. Two components were extracted, explaining 52.7 % and 12.1% of the observed variance, respectively. Variables loading on PC1 were HaL, UL, RL, FeL, SL, FoL, DBM, HH, TL and HW; and SVL and HL on PC2 (Supplementary Table S7) (Fig. 2B).