A Phylogenomic Analysis of Turtles

YMPEV 5061 No. of Pages 8, Model 5G 8 November 2014

Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx 1 Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

4 5

3 A phylogenomic analysis of turtles

a,b,1 c,⇑,1 a d e 6 Q1 Nicholas G. Crawford , James F. Parham , Anna B. Sellas , Brant C. Faircloth , Travis C. Glenn , f a a,g a 7 Theodore J. Papenfuss , James B. Henderson , Madison H. Hansen , W. Brian Simison

8 a Center for Comparative Genomics, California Academy of Sciences, 55 Music Concourse Drive, San Francisco, CA 94118, USA 9 b Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104, USA 10 c John D. Cooper Archaeological and Paleontological Center, Department of Geological Sciences, California State University, Fullerton, CA 92834, USA 11 d Department of Biological Sciences and Museum of Natural Science, Louisiana State University, Baton Rouge, LA 70803, USA 12 e Department of Environmental Health Science, University of Georgia, Athens, GA 30602, USA 13 f Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720, USA 14 g Mathematical and Computational Biology Department, Harvey Mudd College, 301 Platt Boulevard, Claremont, CA 9171, USA

1516 17 article info abstract 3219 20 Article history: Molecular analyses of turtle relationships have overturned prevailing morphological hypotheses and 33 21 Received 11 July 2014 prompted the development of a new taxonomy. Here we provide the first genome-scale analysis of turtle 34 22 Revised 16 October 2014 phylogeny. We sequenced 2381 ultraconserved element (UCE) loci representing a total of 1,718,154 bp of 35 23 Accepted 28 October 2014 aligned sequence. Our sampling includes 32 turtle taxa representing all 14 recognized turtle families and 36 24 Available online xxxx an additional six outgroups. Maximum likelihood, Bayesian, and species tree methods produce a single 37 resolved phylogeny. This robust phylogeny shows that proposed phylogenetic names correspond to 38 25 Keywords: well-supported clades, and this topology is more consistent with the temporal appearance of clades 39 26 Turtles and paleobiogeography. Future studies of turtle phylogeny using fossil turtles should use this topology 40 27 Ultraconserved elements 28 Biogeography as a scaffold for their morphological phylogenetic analyses. 41 29 Phylogeography Ó 2014 Published by Elsevier Inc. 42 30 Systematics 43 31 44 45 46 1. Introduction snorkel-like proboscis. In the morphology-based hypothesis, 65 morphologically bizarre trionychians are nested high in one of 66 47 Q2 The evolutionary relationships of turtles (Testudines) are con- two fundamental branches of the turtle tree, the diverse clade 67 48 tentious. Until recently, the placement of turtles within Amniota Cryptodira (Fig. 1b). Molecular studies disagree with this place- 68 49 was uncertain (i.e., Hedges and Poling, 1999). Genome-scale and ment but are equivocal on the alternate position of trionychians. 69 50 whole genome analyses have confirmed the phylogenetic position Some studies remove trionychians from their highly nested 70 51 of turtles as the sister group to archosaurs (Crawford et al., 2012; position within Cryptodira (Fig. 1c) and place them as the sister 71 52 Field et al., 2014; Fong et al., 2012; Shaffer et al., 2013; Wang taxon of all other cryptodires. Other studies place Trionychia as 72 53 et al., 2013), rejecting a putative relationship between turtles and sister taxon to Pleurodira, the other branch of the turtle tree 73 54 lepidosaurs (Lyson et al., 2012). However, relationships among tur- (Fig. 1d), or as the sister taxon to both Cryptodira and Pleurodira 74 55 tles have not been studied using phylogenomic techniques. Similar (Fig. 1e). As molecular phylogenies changed the position of 75 56 to the placement of turtles relative to their amniote ancestors, Trionychia and other branches of the turtle tree of life, these 76 57 Q3 molecular studies within Testudines (Shaffer et al., 1997; Fujita changes prompted the simultaneous development of new nomen- 77 58 et al., 2003; Krenz et al., 2005; Parham et al., 2006a; Barley et al., clature, and phylogenetically defined clade names were created for 78 59 2010) have challenged prevailing phylogenetic hypotheses based several higher-level nodes (Joyce et al., 2004; Danilov and Parham, 79 60 on cladistic analyses of morphological data (e.g., Gaffney and 2006; Knauss et al., 2011; Joyce et al., 2013) in the turtle 80 61 Meylan, 1988; Gaffney et al., 1991). phylogeny. 81 62 One example of the discrepancies among previous phylogenetic Here, we use sequence data collected from thousands of ultra- 82 63 approaches involves the position of Trionychians (Fig. 1a), a group conserved elements (UCEs; sensu Faircloth et al., 2012) to infer a 83 64 of turtles that have lost their scales and developed a fleshy genome-scale phylogeny of turtles. We use this phylogeny to 84 assess and update the phylogenetic nomenclature and to compare 85 86 ⇑ Corresponding author. the evolutionary relationships of turtles to broad temporal and E-mail address: [email protected] (J.F. Parham). spatial patterns from the fossil record. 87 1 Co-equal authors.

http://dx.doi.org/10.1016/j.ympev.2014.10.021 1055-7903/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Crawford, N.G., et al. A phylogenomic analysis of turtles. Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/ j.ympev.2014.10.021 YMPEV 5061 No. of Pages 8, Model 5G 8 November 2014

2 N.G. Crawford et al. / Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx

88 2. Materials and methods

89 2.1. Materials

90 We selected 32 turtle/ingroup operational taxonomic units 91 (OTUs) and six outgroups (three lepidosaurs [Sphenodon, two squa- 92 mates], two archosaurs [crocodilians and birds], and one mammal 93 [humans]; Table 1) for analysis. Our 32 ingroup OTUs represent all 94 of the major lineages of turtles where we deﬁne a major lineage as 95 an established, uncontroversial monophyletic group of extant lin- a 96 eages. Although the inferred relationships among these lineages 97 can vary (Fig. 1), all recent studies accept the monophyly of six Trionychia 98 major lineages: Pleurodira, Trionychia, Testudinoidea, Chelonioi- Pleurodira 99 dea, Chelydridae, and Kinosternoidea. In addition to sampling Other 100 these six lineages we also included samples from OTUs represent- Cryptodira 101 ing all 14 traditionally accepted families (Turtle Taxonomy b 102 Working Group, 2014). 103 With the exception of Pelodiscus sinensis and Chelonia mydas, Cryptodira 104 which have sequenced genomes, we sampled tissues for all 105 ingroup OTUs from vouchered specimens kept at the California Other 106 Academy of Sciences and the Museum of Vertebrate Zoology Pleurodira Trionychia Cryptodira 107 (Table 1). We used the phylogenetic nomenclature from Joyce 108 et al. (2004), except where otherwise noted. For the sake of sim- 109 plicity we refer to OTUs/specimens in the text and ﬁgures by their c 110 assigned genera according to a recent checklist (Turtle Taxonomy Cryptodira 111 Working Group, 2014). As parts of a species binomial can be unsta- 112 ble and/or controversial, Table 1 includes full species names that 113 can be compared to the aforementioned annotated checklist. We 114 also use this checklist for the counts of species given in the text. Other Cryptodira Pleurodira Trionychia 115 2.2. UCE methods

116 We extracted DNA from approximately 25 mg of tissue using d 117 Qiagen DNeasy Tissue kits following the manufacturer’s protocols, 118 and we ran all genomic DNA extractions on an agarose gel to assess 119 quality. We then sheared 1–2 lg of DNA to 400–600 bps in length 120 using a Diagenode BioruptorÒ Standard (UCD 200) with 6–8 cycles 121 of sonication (depending on DNA quality). We prepared sequencing Other Trionychia Pleurodira Cryptodira 122 libraries from DNA extracts using KAPA library prep kits (Kapa 123 Biosystems) following the library preparation protocols available 124 at . We attached sequence e 125 tags, designed by Faircloth (2014), to each library using individually 126 barcoded primers during the library amplification step. After 127 library amplification, we quantified 2 lL of each library using 128 fluorometry (Qubit, Life Technologies), and we prepared six pools 129 of eight libraries totaling 500 ng per pool (62.5 ng each library). Fig. 1. (a) An extant trionychian showing some of the bizarre diagnostic characters 130 We concentrated library pools using a Savant ISS110 SpeedVac for the group such as the lack of scales and a fleshy proboscis; (b) prevailing morphological hypothesis (Gaffney and Meylan, 1988; Gaffney et al., 1991; Gaffney, 131 Concentrator (Thermo Fisher) and rehydrated each library in 1996); (c) molecular hypothesis with trionychians and sister taxa to all other 132 3.4 lL of ddH2O. cryptodires (Shaffer et al., 1997 [mtDNA]; Fujita et al., (2003) [intron]; Krenz et al., 133 We enriched these pooled libraries using a synthesis of 2560 (2005) [intron]); (d, e) Topologies showing alternative roots for the crown group 134 RNA probes (Mycroarray, Inc.) targeting 2386 ultraconserved Testudines (Barley et al., 2010 [nuDNA]; Sterli, 2010 [morphology, mtDNA, intron]; Field et al., 2014 [miRNAs]). Photo credit: Dogania subplana from Indonesia taken by 135 elements (UCEs) and their flanking sequence (Faircloth et al., Peter Paul van Dijk. 136 2012). Detailed methods of library enrichment, post-enrichment 137 PCR and validation using relative qPCR may be found at . We generated sequences for each create a database of UCE loci recovered, and prepare FASTA files 150 139 enriched library using paired-end 150 base-pair sequencing on for sequence alignment. We generated alignments from this mono- 151 140 an Illumina HiSeq 2500 in ‘‘rapid-run’’ mode. After using scythe lithic FASTA file using MAFFT (version 7.130b) (Katoh, 2002; Katoh 152 141 (http://github.com/vsbuffalo/scythe) to remove adapter contami- and Standley, 2013), and we trimmed resulting alignments using 153 142 nation and sickle to quality-trim sequence reads (version 1.210) the trimming algorithm implemented by the seqcap_align2.py 154 143 (Joshi and Fass, 2011), we assembled reads into contigs using Vel- script within phyluce. From the trimmed alignments, we created 155 144 vet (version 1.2.10) (Zerbino and Birney, 2008). We used VelvetOp- two datasets: one where each locus contained all 36 taxa (100% 156 145 timiser.pl (version 2.2.5) to find the kmer value that produced the complete), and one where we allowed up to 25% missing taxa 157 146 most contigs. Because this method is computationally expensive, per locus (i.e., we required data from a minimum of 29 taxa per 158 147 we limited the search range to kmer values between 89 and 121. locus). We estimated the appropriate finite-sites substitution 159 148 We used phyluce (Faircloth et al., 2012) to identify those model for each locus in all datasets using CloudForest (Crawford 160 149 contigs that were UCE loci, remove putatively duplicate UCE loci, and Faircloth, 2014), and we prepared a concatenated dataset for 161

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Table 1 2.3. Trachemys whole genome sequencing, assembly, and UCE 190 Taxon IDs are either from the California Academy of Science (CAS), the Museum of identification 191 Vertebrate Zoology at Berkeley (MVZ), the LSU Museum of Natural Science (H), the University of Michigan Museum of Zoology (UMFS), or represent University of California Santa Cruz (UCSC) genome builds in which UCE loci were identified. Whole genome sequencing libraries were prepared using Illu- 192 mina’s Nextera library preparation kit, following manufacturer 193 Binomial Specimen ID or SRA accession protocols, with the following modifications: after library prepara- 194 genome build number tion, a 600–700 bp size selection was performed using the BluePip- 195 Alligator mississippiensis HCD-2620 Crawford et al. (2012) pin size selection system (Sage Science). Size selected products 196 Anolis carolinensis UCSC anoCar1 NA Crocodylus porosus UCSC croPor1 NA were amplified in a 7 cycle PCR using the KAPA Real Time Library 197 Pantherophis guttata H15909 NA Amplification kit (KAPA Biosystems) following manufacturer’s 198 Python molurus UCSC pytMol1 NA instructions. PCR products were cleaned using the standard 199 Sphenodon punctatus UMFS 10956 NA Ampure XP (Beckman Coulter Inc.) bead clean-up method with a 200 Agrionemys horsfieldii CAS 22863 SAMN02900523 201 Apalone ferox CAS 202549 SAMN02900524 0.8:1 bead to PCR product ratio. Libraries were validated by Carettochelys insculpta MVZ 238114 SAMN02900525 running 1 ll of product on an Agilent 2100 Bioanalyzer (Agilent 202 Chelydra serpentina MVZ 265668 SAMN02900526 Technologies) and quantified using a Qubit fluorometer (Life 203 Chrysemys picta H2662 (Crawford, 2012) Technologies). Final libraries were sequenced on a HiSeq2500 (Illu- 204 Cyclemys dentata CAS 243787 SAMN02900527 mina). Two lanes of 150 bp paired-end sequencing were run using 205 Deirochelys reticularia MVZ 204282 SAMN02900528 Dermatemys mawii MVZ 269552 SAMN02900529 the rapid run output mode, with each lane containing two libraries 206 Dermochelys coriacea MVZ 149844 SAMN02900530 pooled in equimolar amounts. These same libraries were also 207 Emys marmorata CAS 224202 SAMN02900531 sequenced on two runs of the CCG MiSeq (Illumina) sequencer 208 Erymnochelys madagascariensis MVZ 238759 SAMN02900532 with 600 cycle v3 kits and 300 bp paired-end sequencing mode. 209 Geoemyda spengleri MVZ 208234 SAMN02900533 210 Gopherus berlandieri MVZ 250594 SAMN02900534 After adapter trimming and quality filtering using Trimmomatic Graptemys pseudogeographica MVZ 250644 SAMN02900535 version 0.32 (Bolger et al., 2014), we assembled 12,731,817 Trache- 211 Kinosternon arizonense CAS 228101 SAMN02900536 mys scripta elegans contigs from 138,894,454 HiSeq and 53,677,903 212 Lepidochelys olivacea CAS180267 SAMN02900537 MiSeq reads with Soapdenovo2 (Luo et al., 2012) using its multi- 213 Lissemys punctata CAS 232082 SAMN02900538 kmer method. Kmer sizes ranged from 23 to 127. To identify those 214 Mesoclemmys nasuta MVZ 247578 SAMN02900539 Nilssonia formosa CAS 246283 SAMN02900540 contigs that contained UCE loci we used standalone BLAT version 215 Pelodiscus sinensis UCSC pelSin1 NA 35 (Kent, 2002) to match a 2560 UCE probe set (obtained from 216 Pelomedusa subrufa MVZ 236628 SAMN02900541 http://ultraconserved.org) to the assembled contigs. BLAT was 217 Pelusios castaneus CAS 219222 SAMN02900542 run with default parameters on contigs greater than 300 bp. Then 218 Platemys platycephala MVZ 247579 SAMN02900543 219 Platysternon megacephalum MVZ 230486 SAMN02900544 a custom PYTHON script was used to extract those contigs match- Podocnemis erythracephala MVZ 269553 SAMN02900545 ing the UCE probes with reported E-value scores of 1e-1 or lower. 220 Rhinoclemmys punctularia MVZ 247582 SAMN02900546 These 2926 contigs ranged in size from 301 bp to 33,369 bp. This 221 Staurotypus triporcatus MVZ 263984 SAMN02900547 final set of 2926 Trachemys scripta elegans contigs enriched for 222 Sternotherus minor CAS 221865 SAMN02900548 UCE sequences was used in all subsequent phylogenetic analyses. 223 Stigmochelys pardalis MVZ 241333 SAMN02900549 Terrapene ornata MVZ 230553 SAMN02900550 2.4. Data availability 224

162 subsequent analyses by grouping together loci having the same With the exception for Pelodiscus sinensis for which a published 225 163 substitution model into a partition. genome is available, all ingroup OTUs have specimen vouchers, and 226 164 We performed Bayesian analysis of the concatenated alignment all tissues and specimens are available to qualified researchers. The 227 165 data using two runs of MrBayes version 3.2.2 (r879) (Ronquist data we included for all outgroup taxa and P. sinensis are publicly 228 166 et al., 2012) for 500,000 iterations (4 chains; burn-in: 25%; thin- available at: 229 167 ning: 500). We assessed convergence of the runs using TRACER (Faircloth et al., 2012). Additional details concerning UCE sequence 230 168 (http://tree.bio.ed.ac.uk/software/tracer/). We performed maxi- capture methods and phylogenetic methods are described in 231 169 mum likelihood analyses of the concatenated data using RAxML Faircloth et al. (2012) and detailed protocols are available at 232 170 version 7.2.6 (Stamatakis, 2006) with the ‘‘GTRGAMMA’’ option . Sequenced reads are available in the 233 171 and 10,000 bootstrap replicates. We also performed gene-tree spe- short read archive (PRJNA254176) and alignments and trees at 234 172 cies-tree analysis by estimating gene trees for each UCE locus data dryad (doi:10.5061/dryad.t77q4). 235 173 incorporating 100 multi-locus bootstrap replicates, which we inte- 174 grated into STEAC and STAR species trees (Liu and Yu, 2010; Liu 175 et al., 2009). A posteriori bootstrapping analysis conducted with 3. Results 236 176 RAxML’s autoMRE tool indicated that trees converged after 50 177 replicates. We sequenced a total of 86 million read pairs with a mean of 237 178 We root our tree with the mammals following the approach of 3,083,947 per sample from 28 taxa (Table 1). We assembled a 238 179 recent analyses that confirm the archosaur affinities of Testudines mean of 5377.86 contigs per sample (95CI, min = 1919, max = 239 180 (Crawford et al., 2012; Fong et al., 2012; Field et al., 2014). Lyson 12,511) (Supp. S1). We also incorporated an average of 2939.9 240 181 et al. (2012) phylogenetically defined Ankylopoda for an alterna- UCEs drawn from eight taxa with published genomes. Combining 241 182 tive placement for turtles, the crown clade of turtles and lepido- the UCEs identified in published genomes with the contigs assem- 242 183 saurs, but the crown clade of turtles and archosaurs is an bled from the 28 UCE enriched genomic libraries and running the 243 184 unnamed amniote lineage. We fill this important nomenclatural matrix generation procedures produced: (1) a 100% complete 244 185 gap, and phylogenetically define the name ‘Archelosauria’ to refer matrix containing 233 alignments having a mean length of 245 186 to the clade that originated from the most recent common ancestor 820.26 bp (±48.58 CI) per alignment, totaling 191,121 bp of aligned 246 187 of Crocodylus niloticus Laurenti, 1768 and Testudo graeca Linnaeus, sequence and (2) a 75% complete matrix containing 2381 align- 247 188 1758. The name was chosen to evoke the two included lineages, ments having a mean length of 721.61 bp (±15.65 CI) per align- 248 189 archosaurs and chelonians (Testudines). ment, totaling 1,718,154 bp of aligned sequence. 249

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250 We recover a phylogeny of Testudines that is identical across position of many fossils at the base of Cryptodira is poorly con- 313 251 Bayesian, maximum likelihood approaches and where every node strained, there are several candidate taxa that could fill the ghost 314 252 is fully resolved (e.g., 100% bootstrap support or posterior probabil- lineage on the stem of Durocryptodira (Fig. 3a). 315 253 ities of 1.0). The topologies of the STEAC and STAR species trees 254 contain a few inconsistencies when compared to the ML and 4.1.2. Americhelydia 316 255 Bayesian trees. These include the position of Trachemys scripta The UCE phylogeny supports the division of durocryptodires 317 256 within Emydidae and the position of Chelonioidea within Duro- into the diverse and long recognized Testudinoidea (183 species) 318 257 cryptodira. This is likely caused by both incomplete lineage sorting, and the recently named Americhelydia (38 species, Joyce et al., 319 258 and poorly resolved gene trees due to the small amount of DNA 2013). Americhelydia is comprised of three of the six major lin- 320 259 sequence used to infer individual trees (mean = 820.26 bp) eages that presumably share a common ancestor in the Cretaceous 321 260 (McCormack et al., 2013). Alternately the high support of these of North America. Two of these lineages, the chelydroids and kino- 322 261 groups in the ML and Bayesian trees may be from systematic biases sternoids, are still North American endemics. The third lineage is 323 262 in inferring trees from concatenated datasets (Mossel and Vigoda, the chelonioids, extant marine turtles, which have a cosmopolitan, 324 263 2005; Kubatko and Degnan, 2007). oceanic distribution. Whereas the exclusive monophyly of extant 325 264 Our analysis of the UCE data recovers a monophyletic Pleurod- marine turtles relative to extant, non-marine lineages is not con- 326 265 ira and Cryptodira. Within Pleurodira, we recover the traditional troversial, the relationships of many fossil marine turtles are con- 327 266 families based on inclusion of two representatives, each, of Cheli- founded by potential polyphyly (multiple origins of marine turtles) 328 267 dae, Pelomedusidae, and Podocnemidae. We also recover a mono- and parallel evolution (Joyce, 2007; Joyce et al., 2013). Given this 329 268 phyletic Pelomedusoides (Pelomedusidae + Podocnemidae), a confusion it is unclear whether the chelonioid lineage diverged 330 269 long-recognized group. Within Cryptodira, Trionychia is the sister from other Americhelydians in the Early or Late Cretaceous, but 331 270 group to all of the other cryptodire lineages (Durocryptodira). in either case their oldest fossils and presumed origins are in the 332 271 Within durocryptodires, Testudinoidea is the sister group of a clade Americas (Zangerl, 1953; Hirayama, 1998; Joyce, 2007). Therefore, 333 272 including all of the other lineages. Platysternon megacephalum is just as the UCE phylogeny fits with the temporal appearance of 334 273 resolved as the sister taxon to Emydidae as in Parham et al. clades in the fossil record, it also coincides well with biogeography 335 274 (2006a). The phylogeny places emydid OTUs in a matter consistent by uniting American durocryptodires into a monophyletic group. 336 275 with their subfamilial designations, with the two emydine taxa 276 (Emys and Terrapene) forming a monophyletic group. On the 4.1.3. Testudinoidea 337 277 deirochelyine side, Deirochelys is the sister taxon of a clade that Testudinoidea (183 species, more than half of turtle diversity) 338 278 includes Chrysemys, Trachemys, and Graptemys. This result differs has deep fossil roots in Asia where it maintains a high diversity 339 279 from that of Spinks et al. (2009), which placed Chrysemys outside today. Within testudinoids, the sister taxon relationship between 340 280 of a clade that includes Deirochelys and Graptemys. We also recov- terrestrial tortoises (testudinids) and the geoemydids and was 341 281 ered the Testuguria clade (Geoemydidae + Testudinidae), and phylogenetically defined as Testuguria by Joyce et al. (2004).At 342 282 within each of those included clades the topology of the tree that time the phylogenetic position of the big-headed turtle (Platy- 343 283 matches previous phylogenetic analyses (Spinks et al., 2004; sternon) was not well established. Early molecular phylogenies 344 284 Parham et al., 2006b). A monophyletic Kinosternoidea is the sister placed it outside of Testudinoidea (Shaffer et al., 1997) or as the 345 285 taxon of Chelydridae, thereby affirming the Chelydroidea clade sister taxon to Testuguria (Krenz et al., 2005). Parham et al. 346 286 codified by Knauss et al. (2011). The chelonioids are the sister (2006a) placed Platysternon as the sister group of the Emydidae 347 287 taxon of the chelydroids, which together form the recently named based on an analysis of complete mitochondrial genomes, and this 348 288 Americhelydia (Joyce et al., 2013). The ability for UCEs to recon- result has been confirmed by more comprehensive nuclear data 349 289 struct relatively recent divergences (e.g., within the traditional sets (Barley et al., 2010; this study). The Platysternon – Emydidae 350 290 families) was previously demonstrated by Smith et al. (2014) and node is the only node uniting two or more of the traditional 351 291 is supported here. families of turtles that does not have a name. We fill this important 352 nomenclatural gap, and phylogenetically define the name 353 292 4. Discussion ‘Emysternia’ to refer to the clade that originated from the most 354 recent common ancestor of Platysternon megacephalum Gray, 355 293 4.1. The phylogeny of turtles based on UCEs 1831 and Emys orbicularis (Linnaeus, 1758). The name was chosen 356 to evoke the two included lineages Emydidae and Platysternon. 357 294 4.1.1. Pleurodira, Trionychia, and Durocryptodira 295 The UCE phylogeny supports the monophyly of Cryptodira, with 4.2. Global paleobiogeography of turtles based on the UCE phylogeny 358 296 Trionychia as the sister taxon to all other cryptodires (Figs. 2, 3a, c). 297 The clade including non-trionychian cryptodires was phylogeneti- Combining the UCE phylogeny with the known fossil record of 359 298 cally defined as ‘Durocryptodira’ by Danilov and Parham (2006). turtles allows us to reconstruct some global biogeographic patterns 360 299 The topology from ultraconserved elements and other molecular (Fig. 3c). Intercontinental dispersal of turtles is common, usually 361 300 studies (Shaffer et al., 1997; Krenz et al., 2005; Barley et al., involving a limited number of species. For our discussion we focus 362 301 2010) support the monophyly and recognition of Durocryptodira, primarily on the broad patterns of vicariance and dispersal events 363 302 which contrasts with the morphological hypothesis (Figs. 1, 3b). that generated significant turtle diversity (i.e., speak to geographic 364 303 A monophyletic Durocryptodira is consistent with the temporal origin of ‘major lineages’ and clades that have been recognized as 365 304 appearance of lineages in the fossil record. Pan-Trionychians (tri- families, especially in North America). We assign each lineage to 366 305 onychians and their stem) are the most ancient cryptodire lineage. a continent based on the their area of origin as shown by the fossil 367 306 Therefore, the molecular phylogenetic placement of Trionychia as record (stem taxa). For the timing of events we use the simple 368 307 the sister taxon to all other cryptodires (Durocryptodira) is most appearance of lineages in the fossil record used to construct 369 308 consistent with their antiquity (Fig. 3a, b). In contrast, the morpho- divergence-dating priors by Joyce et al. (2013). For the divergences 370 309 logical hypothesis requires significant missing time for several of discussed below, the fossil record of turtles is complete enough 371 310 the major lineages of turtles (Fig. 3b). These ‘ghost lineages’ that there is no discrepancy between prior and posterior estimates 372 311 Q4 (Norrell, 1992) are hard to accept given the relatively rich fossil (Joyce et al., 2013) and so molecular divergence dating of the UCE 373 312 record of turtles. On the other hand, even though the phylogenetic phylogeny would be superfluous. 374

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Testudines

Pleurodira Cryptodira Squamata Americhelydia Python Durocryptodira Chelydroidea

Pelomedusoides Trionychia Anolis Testudinoidea Testuguria Kinosternoidea Chelonioidea Podocnemidae Emysternia Trionychidae Pelomedusidae Kinosternidae Archosauria Testudinidae Chelidae Lissemys Geoemydidae Emydidae Cheloniidae Carettochelys insculpta Erymnochelys Gallus Podocnemis Pelodiscus Apalone Nilssonia / Chelydridae Pelusios Pelomedusa Staurotypus Stigmochelys Dermatemys mawii Kinosternon Dermochelys coriacea Agrionemys Sternotherus Platemys Gopherus Mesoclemmys Rhinoclemmys Lepidochelys Geoemyda Cyclemys Chelonia Chelydra Graptemys Deirochelys Chrysemys Trachemys Terrapene Terrapene Emys Platysternon megacephalum Crocodylus Sphenodon

Homo Lepidosauria Archelosauria Sauria 0.02

Amniota

Fig. 2. Phylogenetic hypothesis based on RAxML analysis of UCE data showing phylogenetically deﬁned crown clades of turtles (Testudines). All clades were supported by likelihood bootstrap percentages of 100 except for the position of Chelydra in the STAR species tree, which has a bootstrap support of 68. The scale bar is in units of substitutions per site.

375 The earliest fossils of stem testudinoids, stem trionychians, and The Paleogene experienced periods of extremely warm climate 404 376 stem cryptodires are from Eurasia (Danilov and Parham, 2006, (e.g., the Late Paleocene Thermal Maximum and the Early Eocene 405 377 2008; Joyce et al., 2013; Pérez-García et al., 2014). Mapping these Climatic Optimum) that are responsible for the dispersal of many 406 378 data onto the UCE phylogeny demonstrates that cryptodires have a organisms into North America through high latitude dispersal 407 379 Jurassic (>145 Ma) Eurasian origin (Fig. 3c). The emergence of cryp- routes (Zachos et al., 2001), including a wave of testudinoids 408 380 todires in Eurasia is complemented by the concurrent origin of (Estes and Hutchison, 1980; Holroyd et al., 2001; Eberle et al., 409 381 pan-pleurodires in the Southern Hemisphere (Gondwana; Joyce 2010; Hutchison, 2013). Three of these testudinoids lineage persist 410 382 et al., 2013). Given the distribution of the clades and the timing in North America into modern times. Two are modest radiations of 411 383 of their origin, the geography of the cryptodire-pleurodire split testugurians, (four species of Gopherus Testudinidae; nine species 412 384 can be plausibly linked to the breakup of the supercontinent Pan- of Rhinoclemmys, Geoemydidae). Previous studies suggested that 413 385 gaea (Scotese, 2001; Rogers and Santosh, 2003; Smith et al., 2004). these genera are sister taxa to all of the Old World members of 414 386 In this way turtles demonstrate a pattern common to other terres- their respective clades (Parham et al., 2006; Spinks et al., 2004). 415 387 trial vertebrates (e.g., placental vs. marsupial mammals). We sequenced Gopherus, Rhinoclemmys, and representative diver- 416 388 Despite their Jurassic (>145 Ma) origin, cryptodires did not gent members of geoemydids and testudinids; the UCE data con- 417 389 dominate the northern continents for almost 100 million years firm the basal position of these North American genera. This 418 390 (until the Cenozoic). Instead, stem turtles (especially the extinct pattern links the overall diversification at the base of these clades 419 391 clade Paracryptodira) were diverse and abundant in North America with their intercontinental dispersal, which can logically be attrib- 420 392 throughout the Cretaceous (145–66 Ma) and into the Cenozoic uted to periods of warm climate. Similar to the Americhelydia, 421 393 (<66 Ma; Lyson and Joyce, 2009; Lyson et al., 2011). In the Late short branches within the testudinoids also suggest a rapid adap- 422 394 Cretaceous (100–66 Ma), cryptodires (trionychians and durocryp- tive radiation that coincides with high latitude intercontinental 423 395 todires) began to appear in North America, invading through high dispersal events. This pattern suggests that global climate change 424 396 latitude dispersal routes (Hirayama et al., 2000; Parham and has a major impact on the diversity and distribution of turtles. 425 397 Hutchison, 2003; Brinkman and Tarduno, 2005; Vandermark The end of the Paleogene (45–23 Ma) coincides with global 426 398 et al., 2009). The UCE phylogeny confirms that one of the North environmental changes, with the climate becoming significantly 427 399 American durocryptodire lineages (Americhelydia) underwent a cooler and drier (Zachos et al., 2001), i.e., much less favorable to 428 400 modest radiation, accounting for three of the six ‘major lineages’ turtles. Many turtle lineages that inhabited the Western Interior, 429 401 of extant turtles (38 extant species, Fig. 3c). The relatively short including the last stem cryptodires in North America, go extinct 430 402 branches among the Americhelyidian lineages suggest this radia- at this time (Hutchison, 1982, 1992, 1998). One testudinoids line- 431 403 tion was rapid. age took advantage of the subtropical southeastern portions of the 432

6 N.G. Crawford et al. / Molecular Phylogenetics and Evolution xxx (2014) xxx–xxx

abTrionychiaTestudinoideaChelonioideaChelydridaeKinosternoidea ChelydridaeChelonioideaTestudinoideaKinosternoideaTrionychia

~70 Ma

Macrobaenids Sinemeydids, Xinjiangchelyids, ~120 Ma

Cretaceous Paleogene c Dispersal Dispersal

NORTH Dermatemys

AMERICA Pan- Kinosternidae Platysternon Chelydridae Emydidae Chelonioidea Pan- ? Americhelydia ? Emysternia

Stem Testuguria Stem testudinoids Pleurodira testudinoids Testudinoidea Trionychia

Jurassic Pangaea breakup Durocryptodira GONDWANA Cryptodira EURASIA

Fig. 3. Phylogenetic hypotheses for the major lineages of turtles mapped against the ﬁrst appearance of clades in the fossil record and geography. (a) Based on UCE data. Some fossil taxa of basal cryptodires that cannot be attributed to any of the major lineages (i.e. potential stem durocryptodires; Rabi et al., 2013, 2014) are shown; (b) prevailing morphological hypothesis requiring multiple, long ghost lineages; (c) UCE phylogeny mapped against the intercontinental paleobiogeography. Two dispersal events of durocryptodires into North America are shown. The geographic origin of Pan-Platysternon is uncertain so the Paleogene radiation may have included the common ancestor of Emysternia.

433 continent (Parmley et al., 2006), to radiate into the diverse clade phological evolution (Miyashita, 2013; Rabi et al., 2013, 2014). 456 434 Emydidae (53 species, Figs. 2, 3c). The recent description of a fossil Consequently, any discussion of these patterns arising from molec- 457 435 taxon on the stem of Platysternon megacephalum from the Eocene ular phylogenetic studies must consider fossil data. But it is also 458 436 of North America (Hutchison, 2013) raises possibility that the more essential that paleontological studies take advantage of insights 459 437 inclusive Emysternia may also have an American origin. Depending from molecular phylogenetics. In particular, paleontologists work- 460 438 on the resolution of that possibility, the UCE topology indicates ing on the systematics of lineages that include extant members 461 439 that two dispersal events into North America led to the origin of must address and reconcile phylogenetic hypotheses based on 462 440 36–43% (5 or 6 of 14) of the recognized families (Fig. 3c). DNA evidence (Parham et al., 2012). 463 Paleontological studies continue to generate phylogenies that 464 441 4.3. Consilience in the turtle tree of life: a scaffold for paleontological unite Trionychia with the americhelyidian lineage Kinosternoidea 465 442 studies (e.g., Bardet et al., 2013; Tong and Meylan, 2013). The conclusions 466 of these studies are compromised because of the strong molecular 467 443 Because it is more consilient with temporal (stratigraphic, signal rejecting that topology. Even studies that are not focused on 468 444 Fig. 3a, b) and spatial (biogeographic, Fig. 3c) patterns, we argue trionychians or their putative close relatives suffer from the 469 445 that the molecular phylogenetic topology is more plausible than incorrect polarization of characters resulting from demonstrably 470 446 the morphological topology. In this way, the genetic data from incorrect topologies. The reasons that some paleontological studies 471 447 the modern turtle fauna provide an important window into the do not incorporate information from molecular phylogenetics are 472 448 evolutionary history of turtles. However signiﬁcant, this window usually not stated (but see Sterli, 2010). Explanations likely include 473 449 is limited by extinction, and the living species represent only a a distrust of molecular data and/or the logistical hurdle associated 474 450 fraction of past turtle diversity. Fortunately, by virtue of their with synthesizing these disparate data types. For the latter, 475 451 aquatic tendencies, past abundance, and bony shell, turtles are combined analyses are understandably difﬁcult because of non- 476 452 one of the most common vertebrate fossils since the Late Jurassic overlapping taxa and unfamiliarity with analyzing molecular data 477 453 (earlier fossils exist, but are rare). This rich fossil record of turtles sets. 478 454 provides crucial insights into their geographic origin (see Sections The solution is to use a ‘‘molecular scaffold’’ (Springer et al., 479 455 4.1.2 and 4.2), temporal appearance (Joyce et al., 2013), and mor- 2001), i.e., a backbone constraint tree for well-supported nodes 480

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Please cite this article in press as: Crawford, N.G., et al. A phylogenomic analysis of turtles. Mol. Phylogenet. Evol. (2014), http://dx.doi.org/10.1016/ j.ympev.2014.10.021