Copyedited by: AV MANUSCRIPT CATEGORY: Systematic Biology Syst. Biol. 69(3):502–520, 2020 © The Author(s) 2019. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: [email protected] DOI:10.1093/sysbio/syz062 Advance Access publication September 24, 2019 Interrogating Genomic-Scale Data for Squamata (Lizards, Snakes, and Amphisbaenians) Shows no Support for Key Traditional Morphological Relationships , FRANK T. BURBRINK1,FELIPE G. GRAZZIOTIN2,R.ALEXANDER PYRON3,DAVID CUNDALL4,STEVE DONNELLAN5 6,FRANCES IRISH7,J.SCOTT KEOGH8,FRED KRAUS9,ROBERT W. M URPHY10,BRICE NOONAN11,CHRISTOPHER J. RAXWORTHY1,SARA , ,∗ RUANE12,ALAN R. LEMMON13,EMILY MORIARTY LEMMON14 , AND HUSSAM ZAHER15 16 1Department of Herpetology, The American Museum of Natural History, 79th Street at Central Park West, New York, NY 10024, USA; 2Laboratório de Coleções Zoológicas, Instituto Butantan, Av. Vital Brasil, 1500—Butantã, São Paulo—SP 05503-900, Brazil; 3Department of Biological Sciences, The George Washington University, Washington, DC 20052, USA; 4Department of Biological Sciences, 1 W. Packer Avenue, Lehigh University, Bethlehem, PA 18015, USA; 5South Australian Museum, North Terrace, Adelaide SA 5000, Australia; 6School of Biological Sciences, University of Adelaide, SA 5005 Australia; 7Department of Biological Sciences, Moravian College, 1200 Main St, Bethlehem, PA 18018, US; 8Division of Ecology and Evolution, Research School of Downloaded from https://academic.oup.com/sysbio/article-abstract/69/3/502/5573126 by Rutgers University user on 21 May 2020 Biology, The Australian National University, Canberra, ACT 2601, Australia; 9Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109, USA; 10 Department of Natural History, Royal Ontario Museum, 100 Queens Park, Toronto, ON M5S 2C6, Canada; 11 Department of Biology, University of Mississippi, Oxford, MS 38677, USA; 12Department of Biological Sciences, 206 Boyden Hall, Rutgers University, 195 University Avenue, Newark, NJ 07102, USA; 13Department of Scientific Computing, Florida State University, Dirac Science Library, Tallahassee, FL 32306-4102, USA; 14 Department of Biological Science, Florida State University, 319 Stadium Drive, Tallahassee, FL 32306-4295, USA; 15Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil CEP 04263-000, Brazil; and 16 Centre de Recherche sur la Paléobiodiversité et les Paléoenvironnements (CR2P), UMR 7207 CNRS/MNHN/Sorbonne Université, Muséum national d’Histoire naturelle, 8 rue Buffon, CP 38, 75005 Paris, France ∗ Correspondence to be sent to: Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil CEP 04263-000, Brazil; E-mail: [email protected]. Received 15 January 2019; reviews returned 5 September 2019; accepted 10 September 2019 Associate Editor: Robert Thomson Abstract.—Genomics is narrowing uncertainty in the phylogenetic structure for many amniote groups. For one of the most diverse and species-rich groups, the squamate reptiles (lizards, snakes, and amphisbaenians), an inverse correlation between the number of taxa and loci sampled still persists across all publications using DNA sequence data and reaching a consensus on the relationships among them has been highly problematic. In this study, we use high-throughput sequence data from 289 samples covering 75 families of squamates to address phylogenetic affinities, estimate divergence times, and characterize residual topological uncertainty in the presence of genome-scale data. Importantly, we address genomic support for the traditional taxonomic groupings Scleroglossa and Macrostomata using novel machine-learning techniques. We interrogate genes using various metrics inherent to these loci, including parsimony-informative sites (PIS), phylogenetic informativeness, length, gaps, number of substitutions, and site concordance to understand why certain loci fail to find previously well- supported molecular clades and how they fail to support species-tree estimates. We show that both incomplete lineage sorting and poor gene-tree estimation (due to a few undesirable gene properties, such as an insufficient number of PIS), may account for most gene and species-tree discordance. We find overwhelming signal for Toxicofera, and also show that none of the loci included in this study supports Scleroglossa or Macrostomata. We comment on the origins and diversification of Squamata throughout the Mesozoic and underscore remaining uncertainties that persist in both deeper parts of the tree (e.g., relationships between Dibamia, Gekkota, and remaining squamates; among the three toxicoferan clades Iguania, Serpentes, and Anguiformes) and within specific clades (e.g., affinities among gekkotan, pleurodont iguanians, and colubroid families). [Neural network; gene interrogation; lizards; snakes; genomics; phylogeny.] Well-supported phylogenies inferred using both thor- few of intermediate range with ∼50 loci and 161 taxa ough taxon-sampling and genome-scale sequence data (Wiens et al., 2012; Reeder et al., 2015). Furthermore, are paramount for understanding phylogenetic struc- phylogenetic thinking about such groups often reflects ture and settling debates about higher-level taxonomy. historical morphological hypotheses that are weakly Phylogenomic analyses can provide reliable trees for congruent or incongruent with recent phylogenomic downstream use in comparative biology (Garland et al. estimates (Conrad 2008; Gauthier et al., 2012; Losos et al., 2005; Wortley et al., 2005; Heath et al., 2008; Ruane 2012). et al., 2015; Burbrink et al., 2019) and help unravel The order Squamata comprises almost 10,800 extant evolutionary complexity (Philippe et al., 2011), such lizards, snakes, and amphisbaenians (Uetz et al. 2018) as deep-time phylogenetic reticulation (Burbrink and showing continuous diversification since the Jurassic, Gehara, 2018). In recent years, well-resolved phylogenies with many groups surviving the Cretaceous/Tertiary of birds and mammals used both large numbers of mass extinction (Evans 2003; Evans and Jones 2010; genes and taxa (Prum et al., 2015; Liu et al., 2017). Jones et al., 2013). Extant squamates occur in nearly Unfortunately, among amniotes, squamates have fallen all habitats globally and show huge variation in body behind and all comparative and taxonomic studies still size, body shape, limb types (including repeated com- rely on phylogenetic structure estimated from either plete limb loss), oviparous and viviparous reproduct- a handful of genes and a large number of species ive modes, complex venoms, and extremely varied (Pyron et al., 2013), small number of lineages with diets that include plants, invertebrates and verteb- phylogenomic data (Streicher and Wiens 2017), and a rates (Vitt and Pianka 2005; Vitt and Caldwell 2009; 502 [07:45 3/4/2020 Sysbio-OP-SYSB190063.tex] Page: 502 502–520 Copyedited by: AV MANUSCRIPT CATEGORY: Systematic Biology 2020 BURBRINK ET AL.—GENOMIC RELATIONSHIPS OF SQUAMATES 503 Colston et al., 2010; Pyron and Burbrink, 2014; Zaher have found quantitatively similar hidden morphological et al., 2014; Fry 2015). Squamates are important research support for Toxicofera as well as for Scleroglossa (Reeder organisms in the fields of behavior, ecology, and macro- et al., 2015), which suggests that most traits supporting evolution, for which major studies on their speciation, a basal Iguania/Scleroglossa split are the result of con- biogeography, and latitudinal richness gradients have vergent ecological adaptations. Moreover, Simões et al. contributed to the basic understanding of how diversity (2018) recently rejected this basal split into Scleroglossa accumulates across the earth (O’Connor and Shine 2004; using a new morphological data matrix containing Ricklefs et al., 2007; Pyron and Burbrink 2012; Pyron, characters that support the molecular phylogeny (at least 2014; Burbrink et al., 2015; Esquerré and Scott Keogh 2016; in part). This suggests that the traditional coding of some Esquerré et al., 2017). of these morphological traits may have been in error or Attempts to understand relationships among squam- reflected homoplasy. ates over the last 250 years reflect the input from Many morphological studies recovered a single origin hundreds of researchers spanning morphological, small- for large-gaped snakes, referred to as Macrostomata Downloaded from https://academic.oup.com/sysbio/article-abstract/69/3/502/5573126 by Rutgers University user on 21 May 2020 scale molecular, and now phylogenomic data sets, (Cundall et al., 1993; Rieppel et al., 2003; Conrad 2008; marked by several key milestones (Oppel 1811; Camp Wilson et al., 2010; Gauthier et al., 2012; Zaher and 1923; Underwood 1967; Estes et al., 1988; Townsend Scanferla 2012; Simões et al., 2018). This group has been et al., 2004; Vidal and Hedges 2005, 2009; Conrad 2008; defined by a large number of skull features, all involving Wiens et al., 2010, 2012; Gauthier et al., 2012; Pyron the dentigerous upper and lower jaws, palatal, and et al., 2013, 2014; Reeder et al., 2015; Streicher and Wiens suspensorium bones, which contribute to an increase of 2016). Although research from both morphological gape size (Rieppel 1988; Cundall and Irish 2008). How- and molecular studies have converged toward sim- ever, Macrostomata was found to be paraphyletic based ilar content of most family-level groups, relationships on fossil taxa and morphological data alone (Lee and among these groups and, more