Biological Journal of the Linnean Society, 2009, 96, 635–650. With 4 figures Systematics and evolutionary relationships of the mountain lizard Liolaemus monticola (Liolaemini): how morphological and molecular evidence contributes to reveal hidden species diversity FERNANDO TORRES-PÉREZ1,2*, MARCO A. MÉNDEZ2,3, EDGAR BENAVIDES4, RODRIGO A. MORENO5,6, MADELEINE LAMBOROT6, R. EDUARDO PALMA2,7 and JUAN CARLOS ORTIZ8 1Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA 2Center for Advanced Studies in Ecology and Biodiversity, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile 3Laboratorio de Genómica Evolutiva (INTA), Universidad de Chile, Macul 5540, Casilla 138-11, Santiago, Chile 4Department of Integrative Biology and M.L. Bean Life Science Museum, Brigham Young University, Provo, UT 84602, USA 5Instituto Milenio de Ecología y Biodiversidad (IEB), Casilla 653, Santiago, Chile 6Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile 7Departamento de Ecología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, 657613, Chile 8Departamento de Zoología, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Casilla 160-C, Concepción, Chile Received 14 March 2008; accepted for publication 20 June 2008 The delimitation of species is a major issue in systematic biology and has been a re-emerging discipline in the last decade. A number of studies have shown that the use of multiple data sets is critical for the identification of cryptic species, particularly in groups with complex evolutionary histories. Liolaemus monticola is a montane lizard species distributed in central Chile (32°–42°S), with four described subspecies in a latitudinal gradient from north to south: L. m. monticola, L. m. chillanensis, L. monticola ssp. and L. m. villaricensis. In order to test the systematic status and phylogenetic relationships of the taxa included in the L. monticola group, we analysed morphological (morphometric and meristic) and molecular (allozyme and mitochondrial DNA) data sets. The results of the morphological analyses showed that meristic variables correctly assigned individuals with higher accuracy than did morphometric characters. The results of the analyses of allozyme data revealed eight diagnostic loci that are evidence for significant differences among the four L. monticola subspecies. Phylogenetic analyses with mitochondrial DNA data, including additional species, showed that the L. monticola group is polyphyletic. We postulate that the four current subspecies represent independent evolutionary lineages and must be raised to the specific level as L. monticola, L. chillanensis and L. villaricensis. The taxonomic status of the unnamed L. monticola ssp. remains unresolved, although we provide a preliminary proposal. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 635–650. ADDITIONAL KEYWORDS: allozymes – Andean range – biometric data – Chile – cytochrome b gene – meristic data – molecular phylogeny – polytypic species – species delimitation. *Corresponding author. Current address: Biology Department, University of New Mexico, MSC03-2020, Albuquerque, NM 87131, USA. E-mail: [email protected] © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 635–650 635 636 F. TORRES-PÉREZ ET AL. INTRODUCTION tional patterns. The nominal subspecies, Liolaemus monticola monticola, has been described in the San The delimitation of species boundaries is a major Francisco valley in the Santiago de Chile mountains goal in solving systematic uncertainties using pre- (32°22′S, 70°25′W) at 1700 m (Fig. 1). It has also viously proposed systematic groups as hypotheses to been reported in coastal and transversal mountain be tested (Sites & Marshall, 2003, 2004). The choice ranges (33°S) between 600 and 1800 m in central of a single method for the evaluation of species Chile (Lamborot & Alvarez-Sarret, 1993; Lamborot boundaries may be problematic, especially in taxo- & Eaton, 1997). Two additional subspecies have nomic groups with complex evolutionary histories. been described from the same region (‘Cordillera de Even the use of a variety of methods with different Chillán’, 36°52′S), albeit at a different elevation: statistical power may lead to conflicting answers Liolaemus monticola chillanensis inhabits environ- when elucidating the same systematic issue (Hey ments at c. 1700 m in Termas de Chillán (Fig. 1), et al., 2003; Marshall et al., 2006). These issues are whereas the unnamed Liolaemus monticola ssp. enforced when cryptic species are suspected within a occupies habitats above 2300 m (on ‘Lavas del taxonomic group, and the comparison of different Volcán Chillán’, VIII Region, Chile). However, L. m. types of marker has been of great utility in herpe- chillanensis and L. monticola ssp. have recently tozoan studies (Godinho et al., 2005; Camargo, De been found in syntopy, showing no evidence of Sa & Heyer, 2006; Sanders, Malhotra & Thorpe, hybridization or genetic introgression (Torres-Pérez 2006; Pinho, Harris & Ferrand, 2007). Mitochondrial et al., 2003). The latter subspecies has remained DNA (mtDNA) has been proven to be highly useful unnamed from its description for more than 70 in systematic studies (Wiens & Penkrot, 2002), and years. The southernmost subspecies of the group, has been widely used in species delimitation of ver- Liolaemus monticola villaricensis, inhabits the tebrate groups. Isozyme electrophoresis has tradi- lava fields of the Villarrica Volcano (41°S) at tionally been used as a tool to examine divergence 1400 m. at the specific level when morphological inference is Liolaemus monticola shows a remarkable charac- inconclusive (Buth & Murphy, 1999). However, tra- teristic within Liolaemus. There is extensive varia- ditional morphology-based methods are still widely tion in the chromosomal number from north to south. used in systematic studies, providing several advan- The nominal subspecies (L. m. monticola) exhibits tages when molecular methods cannot be applied high chromosomal polymorphism (2n = 34–44), sepa- (Hillis & Wiens, 2000). An integrative approach to rated by chromosomal races apparently maintained the delineation of species using multiple complemen- by riverine barriers (Lamborot, 1991, 1998b), whereas tary methods is expected to produce robust results the southern L. m. chillanensis and L. m. villaricensis of a specific systematic study (Dayrat, 2005; Sanders show 2n = 32. Males of L. m. villaricensis and L. et al., 2006). monticola ssp. do not have anal pores, a trait consid- The lizard genus Liolaemus is widely distributed in ered to be rare in the genus Liolaemus (Donoso- southern South America, ranging from arid Patago- Barros, 1966; Videla & Cei, 1998). Remarkably, anal nian to high-altitude Andean environments, including pores are present in males of L. m. monticola and L. valley and coastal ranges. The adaptive radiation of m. chillanensis. Phylogenetic analyses involving some Liolaemus has produced interesting patterns of of the taxa of the L. monticola group have been developmental (Lobo & Espinoza, 1999, 2004), genetic performed using allozymes (Young-Downey, 1998) (Morando et al., 2004; Avila, Morando & Sites, 2006) and morphological characters (Lobo, 2001, 2005). and morphological (Harmon et al., 2003; Schulte The morphological approach showed first monophyly et al., 2004) variation, as well as contrasting rates of (Lobo, 2001) and then paraphyly (Lobo, 2005) of the molecular evolution (Schulte et al., 2000). Some of group. A complete systematic study involving all four these recently described phenomena have produced, cited subspecies is essential to elucidate the status of as an indirect outcome, revised species taxonomy. In each current subspecies in the L. monticola group. In this article, we examine an interesting example of addition, we aim to determine the evolutionary rela- Liolaemus diversification and its implications for tionships among taxa in this group using molecular species classification and evolutionary relationships sequence data, and to test the monophyly/paraphyly in the genus. of L. monticola as proposed by previous morphological Liolaemus monticola (Müller & Hellmich, 1932) is phylogenetic analyses. an endemic lizard species distributed along the We therefore test the following hypotheses: (1) Andes between latitudes 32° and 41°S and at 600 all current subspecies of the L. monticola group to 2300 m (Donoso-Barros, 1966; Lamborot et al., represent different taxa at the species level; and 1981). Currently, it is composed of four subspecies (2) L. monticola constitutes a paraphyletic group. (morphotypes) based on morphological and distribu- Species boundaries were studied using nuclear © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 635–650 SYSTEMATICS AND PHYLOGENY OF L. MONTICOLA GROUP 637 Figure 1. Map of the sampling localities and currently known geographical distributions of the Liolaemus monticola group. Numbers correspond to the localities sampled for the four L. monticola subspecies: open circles, L. m. monticola; open squares, L. m. chillanensis; filled squares, L. monticola ssp.; filled triangle, L. m. villaricensis. See Appendix S1 for specific sampled localities. molecular markers (allozymes) and by performing MATERIAL AND METHODS multivariate analyses for the assessment of mor- MORPHOMETRIC