bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185025; this version posted July 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. An integrative multi-approach workflow resolves species limits in the southernmost members of the Liolaemus kingii group (Squamata: Liolaemini) Kevin I. Sanchez´ a,∗, Luciano J. Avilaa, Jack W. Sites Jr.b,1, Mariana Morandoa aInstituto Patag´onicopara el Estudio de los Ecosistemas Continentales, Consejo Nacional de Investigaciones Cient´ıficasy T´ecnicas(IPEEC-CONICET), Boulevard Almirante Brown 2915, Puerto Madryn, CT U9120ACD, Argentina bDepartment of Biology and M.L. Bean Life Science Museum, Brigham Young University (BYU), Provo, UT 84602, USA Abstract Recent conceptual and methodological advances have enabled an increasing number of studies to address the problem of species delimitation in a comprehensive manner. This is of particular interest in cases of species whose divergence times are recent, where the con- clusions obtained from a single source of evidence can lead to the incorrect delimitation of entities or assignment of individuals to species. The southernmost species of the Liolaemus kingii group (namely L. baguali, L. escarchadosi, L. sarmientoi, L. tari and the candidate species L. sp. A) show widely overlapping distributions as well as recent mitochondrial divergences, thus phylogenetic relationships and species boundaries are ambiguous. Here we use a comprehensive approach to assess species limits and corroborate their status as independent lineages through the use of four sources of molecular and morphological in- formation (mitochondrial cytochrome-b, nuclear sequences collected by ddRADseq, and linear, meristic and landmark-based morphometrics). We found concordance among the different datasets, but signs of admixture were detected between some of the species. Our results indicate that the L. kingii group can serve as a model system in studies of diversi- fication accompanied by hybridization in nature. We emphasize the importance of using multiple lines of evidence in order to solve evolutionary stories, and minimizing potential erroneous results that may arise when relying on a single source of information. ∗Corresponding author Email address: [email protected] (Kevin I. Sanchez)´ 1Current address: Department of Biology, Austin Peay State University, Clarksville, Tennessee 37044, USA Preprint submitted to Molecular Phylogenetics and Evolution July 2, 2020 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185025; this version posted July 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Keywords: Integrative taxonomy, Patagonia, Liolaemus kingii group, ddRADseq, introgression 1 1. Introduction 2 Delimiting species objectively is critical in a wide range of biological research in- 3 cluding the fields of evolution, biodiversity assessment and conservation. This may be a 4 challenging task in recently derived populations due to the influence of the limited time 5 since their split, which may heavily impact patterns of genetic differentiation (e.g. Nieto- 6 Montes de Oca et al., 2017; Villamil et al., 2019). This is due to insufficient time for fixed 7 differences to accumulate, which may compromise a consistent diagnosis (Struck et al., 8 2018), while admixture between closely related species can result in individuals with phe- 9 notypically and genetically intermediate states, generating controversial inferences with 10 different data types (e.g. Sistrom et al., 2013; Olave et al., 2017; Singhal et al., 2018). On 11 the other hand, there are documented cases of species with ancient divergence times that do 12 not differ sufficiently in their morphological attributes, for example by niche conservatism 13 (Wiens and Graham, 2005; Jezkova and Wiens, 2018), making difficult the assignment of 14 individuals to putative species based solely on this class of characters. Processes such as 15 past hybridization and incomplete lineage sorting of ancestral polymorphisms (ILS) can 16 also mask the signatures of true phylogenetic divergence, and hence complicate the precise 17 delimitation of species boundaries, especially, when relying on a single source of evidence 18 (Maddison, 1997; Knowles and Carstens, 2007; Degnan and Rosenberg, 2009). Both of 19 these processes have a high prevalence in recently diverged populations/species (Knowles 20 and Carstens, 2007). But more recently, species delimitation research (SDL) has been 21 characterised by a pluralistic approach in which independent sources of information (e.g. 22 genetics, morphology, ecology) are used in order to objectively test species boundaries 23 (Dayrat, 2005; Padial et al., 2010; Edwards and Knowles, 2014). Moreover, the increas- 24 ing availability of genomic datasets comprised of hundreds/thousands of independent loci, 25 and coalescent-based approaches for species delimitation, have opened the door for the 26 detection of independent lineages with increased levels of statistical rigor and objectivity 27 (e.g. Eaton and Ree, 2013; Leache´ et al., 2014; Leache´ and Oaks, 2017; Esquerre´ et al., 28 2019; Villamil et al., 2019; Dufresnes et al., 2020). 29 The lizard genus Liolaemus contains 271 species (Uetz, 2020) distributed in two main 30 clades or subgenera: Liolaemus sensu stricto (the chilean group) and Eulaemus (the argen- 31 tinean group; Laurent, 1983, 1985). The members of this genus are distributed throughout 32 the Andes and adjacent lowlands of South America, from Peru´ to Tierra del Fuego in ◦ 0 ◦ 0 33 Argentina (Cei, 1986), having one of the largest latitudinal (14 ± 30 - 52 ± 30 S), ele- 34 vational (from sea level to at least 5176 m.a.s.l.), and climatic distributions among lizards 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185025; this version posted July 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 35 worldwide (Cei, 1986). This may explain the great diversity of biological characteristics 36 observed in these reptiles, including body size (Pincheira-Donoso et al., 2015), karyotype 37 (Morando, 2004; Aiassa et al., 2005), reproductive modes (viviparity/oviparity/parthenogenetic; 38 Abdala et al., 2016), and dietary habits (insectivores/herbivores/omnivores; Cei, 1986, 39 1993; Espinoza et al., 2004). For this reason, this genus is increasingly being employed 40 as a study model in numerous research areas such as evolution of herbivory, parity modes, 41 community structure, comparative anatomy, physiology, behavioral ecology, and conser- 42 vation (e.g. Espinoza et al., 2004; Tulli et al., 2009; Ibarguengoyt¨ ´ıa et al., 2010; Kacoliris 43 et al., 2010; Corbalan´ et al., 2011; Kacoliris et al., 2011; Labra, 2011; Tulli et al., 2011). 44 However, due to the species-richness and geographic range of the genus, taxonomy and 45 systematics remain the most intensely studied focus (see Avila et al., in press, for a recent 46 review). 47 Horizontal gene flow has been documented between non-sister species (e.g. L. gracilis 48 × L. bibronii) (Olave et al., 2011, 2018), and many other instances of hybridization have 49 been hypothesized with various levels of evidence for several groups within this genus. For 50 example, Avila et al. (2006) have suggested the presence of hybridization between mem- 51 bers of two deeply diverged species complexes (L. boulengeri and L. rothi complexes) 52 based on extensive mitochondrial paraphyly, and more recently supported by morpholog- 53 ical and nuclear sequence data analysed by recently developed coalescent-based methods 54 to detect interspecific gene flow (Olave et al., 2018). For a detailed review of hybridization 55 cases in Liolaemus see Morando et al. (2020). 56 The Patagonian lizards of the Liolaemus kingii group are included in the L. lineomac- 57 ulatus section of the subgenus Eulaemus (Cei and Scolaro, 1982; Breitman et al., 2011, 58 2013), and currently includes 13 described species distributed in two main clades: the L. 59 kingii clade (11 species) and the L. somuncurae clade (2 species) (Breitman et al., 2011, 60 2013; Olave et al., 2014). In addition, numerous candidate species have been identified 61 based on various approaches, revealing that this group is characterized by high levels of 62 genetic and morphological variation within and between populations, and phenotypic in- 63 tergradation between adjacent populations (Breitman et al., 2013, 2015). The geographi- 64 cal distribution of this complex ranges mainly from southern R´ıo Negro to southern Santa ◦ 0 ◦ 0 65 Cruz provinces in Argentina (41 ± 30 - 52 ± 30 S). Within the L. kingii clade, a widely 66 distributed group of southern lineages, including L. baguali, L. escarchadosi, L sarmientoi, 67 L. tari and the candidate species L. sp A identified by Breitman et al. (2015), is distributed 68 across a heterogeneous area, ranging from sea level to high-altitude areas in the Andean 69 cordillera, and characterised by recent divergence times (Late Pliocene to Late Pleistocene: 70 ≈ 3 Mya to 0.75 Mya) (Breitman et al., 2011, 2013, 2015; Esquerre´ et al., 2018). These 71 species were resolved as a well supported clade in a previous study attempting to infer 72 relationships within the L. lineomaculatus section (Breitman et al.,