Molecular Phylogenetics and Evolution 83 (2015) 250–257

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev

A phylogenomic analysis of turtles
Nicholas G. Crawford a,b,1, James F. Parham c,⇑,1, Anna B. Sellas a, Brant C. Faircloth d, Travis C. Glenn e,
Theodore J. Papenfuss f, James B. Henderson a, Madison H. Hansen a,g, W. Brian Simison aaCenter for Comparative Genomics, California Academy of Sciences, 55 Music Concourse Drive, San Francisco, CA 94118, USA
Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104, USAcJohn D. Cooper Archaeological and Paleontological Center, Department of Geological Sciences, California State University, Fullerton, CA 92834, USAd
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USAe
Department of Environmental Health Science, University of Georgia, Athens, GA 30602, USAf
Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720, USAg
Mathematical and Computational Biology Department, Harvey Mudd College, 301 Platt Boulevard, Claremont, CA 9171, USAba r t i c l e

i n f oArticle history:
Received 11 July 2014
Revised 16 October 2014
Accepted 28 October 2014
Available online 4 November 2014
Keywords:
Turtles
Ultraconserved elements
Biogeography
Phylogeography
Systematics

a b s t r a c t
Molecular analyses of turtle relationships have overturned prevailing morphological hypotheses andprompted the development of a new taxonomy. Here we provide the ﬁrst genome-scale analysis of turtle
phylogeny. We sequenced 2381 ultraconserved element (UCE) loci representing a total of 1,718,154 bp ofaligned sequence. Our sampling includes 32 turtle taxa representing all 14 recognized turtle families andan additional six outgroups. Maximum likelihood, Bayesian, and species tree methods produce a single
resolved phylogeny. This robust phylogeny shows that proposed phylogenetic names correspond towell-supported clades, and this topology is more consistent with the temporal appearance of clades
and paleobiogeography. Future studies of turtle phylogeny using fossil turtles should use this topology
as a scaffold for their morphological phylogenetic analyses.
Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction
The evolutionary relationships of turtles (Testudines) are contentious. Until recently, the placement of turtles within Amniota
was uncertain (Hedges and Poling, 1999). Genome-scale and wholegenome analyses have conﬁrmed the phylogenetic position of turtles as the sister group to archosaurs (Crawford et al., 2012; Field
et al., 2014; Fong et al., 2012; Shaffer et al., 2013; Wang et al.,2013), rejecting a putative relationship between turtles and lepidosaurs (Lyson et al., 2012). However, relationships among turtles
have not been studied using phylogenomic techniques. Similar tothe placement of turtles relative to their amniote ancestors, molecular studies within Testudines (Shaffer et al., 1997; Fujita et al.,
2004; Krenz et al., 2005; Parham et al., 2006a; Barley et al.,2010) have challenged prevailing phylogenetic hypotheses basedon cladistic analyses of morphological data (e.g., Gaffney andMeylan, 1988; Gaffney et al., 1991).
One example of the discrepancies among previous phylogenetic
approaches involves the position of trionychians (Fig. 1a), a group⇑ Corresponding author.
1E-mail address: jparham@fullerton.edu (J.F. Parham).
Co-equal authors.

http://dx.doi.org/10.1016/j.ympev.2014.10.021
1055-7903/Ó 2014 Elsevier Inc. All rights reserved.

of turtles that have lost their scales and developed a ﬂeshy snorkellike proboscis. In the morphology-based hypothesis, the morphologically bizarre trionychians are nested high in one of two fundamental branches of the turtle tree, the diverse clade Cryptodira(Fig. 1b). Molecular studies disagree with this placement but areequivocal on the alternate position of trionychians. Some studies
remove trionychians from their highly nested position within
Cryptodira and place them as the sister taxon of all other cryptodires (Fig. 1c). Other studies place Trionychia as sister taxon to Pleurodira, the other branch of the turtle tree (Fig. 1d), or as the sistertaxon to both Cryptodira and Pleurodira (Fig. 1e). As molecularphylogenies changed the position of Trionychia and other branchesof the turtle tree of life, it prompted the simultaneous developmentof new nomenclature, and phylogenetically deﬁned clade names
were created for several higher-level nodes (Joyce et al., 2004;
Danilov and Parham, 2006; Knauss et al., 2011; Joyce et al., 2013)
in the turtle phylogeny.
Here, we use sequence data collected from thousands of ultraconserved elements (UCEs; sensu Faircloth et al., 2012) to infer a
genome-scale phylogeny of turtles. We use this phylogeny toassess and update the phylogenetic nomenclature and to compare
the evolutionary relationships of turtles to broad temporal andspatial patterns from the fossil record.

251

N.G. Crawford et al. / Molecular Phylogenetics and Evolution 83 (2015) 250–257

2. Materials and methods
2.1. Materials

2.2. UCE methods
We extracted DNA from approximately 25 mg of tissue using
Qiagen DNeasy Tissue kits following the manufacturer’s protocols,
and we ran all genomic DNA extractions on an agarose gel to assess
quality. We then sheared 1–2 lg of DNA to 400–600 bps in length
using a Diagenode BioruptorÒ Standard (UCD 200) with 6–8 cycles
of sonication (depending on DNA quality). We prepared sequencing libraries from DNA extracts using KAPA library prep kits (Kapa
Biosystems) following the library preparation protocols available
at <http://ultraconserved.org/#protocols>. We attached sequencetags, designed by Faircloth (2012), to each library using individually barcoded primers during the library ampliﬁcation step. Afterlibrary ampliﬁcation, we quantiﬁed 2 lL of each library using ﬂuorometry (Qubit, Life Technologies), and we prepared six pools ofeight libraries totaling 500 ng per pool (62.5 ng each library). We
concentrated library pools using a Savant ISS110 SpeedVac Concentrator (Thermo Fisher) and rehydrated each library in 3.4 lL
of ddH2O.
We enriched these pooled libraries using a synthesis of 2560
RNA probes (Mycroarray, Inc.) targeting 2386 ultraconserved elements (UCEs) and their ﬂanking sequence (Faircloth et al., 2012).
Detailed methods of library enrichment, post-enrichment PCRand validation using relative qPCR may be found at <http://ultraconserved.org/#protocols>. We generated sequences for eachenriched library using paired-end 150 base-pair sequencing onan Illumina HiSeq 2500 in ‘‘rapid-run’’ mode. After using scythe(http://github.com/vsbuffalo/scythe) to remove adapter contamination and sickle to quality-trim sequence reads (version 1.210)(Joshi and Fass, 2011), we assembled reads into contigs using Velvet (version 1.2.10) (Zerbino and Birney, 2008). We used VelvetOptimiser.pl (version 2.2.5) to ﬁnd the kmer value that produced themost contigs. Because this method is computationally expensive,
we limited the search range to kmer values between 89 and 121.

aOtpt herod
ira

Trionychiary

PleurodiraC

We selected 32 turtle/ingroup operational taxonomic units(OTUs) and six outgroups (three lepidosaurs [Sphenodon and twosquamates], two archosaurs [a crocodilian and a bird], and one
mammal [human]; Table 1) for analysis. Our 32 ingroup OTUs represent all of the ‘major lineages’ of turtles; we deﬁne a ‘major lineage’ as an established, uncontroversial, monophyletic group of
extant species. Although the inferred relationships among theselineages can vary (Fig. 1), all recent studies accept the monophylyof six major lineages: Pleurodira, Trionychia, Testudinoidea, Chelonioidea, Chelydridae, and Kinosternoidea. In addition to samplingthese six lineages we also included samples from OTUs representing all 14 traditionally accepted families (Turtle Taxonomy
Working Group, 2014).
With the exception of Pelodiscus sinensis and Chelonia mydas,
which have sequenced genomes, we sampled tissues for all
ingroup OTUs from vouchered specimens kept at the California
Academy of Sciences and the Museum of Vertebrate Zoology(Table 1). We used the phylogenetic nomenclature from Joyce
et al. (2004), except where otherwise noted. For the sake of simplicity we refer to OTUs/specimens in the text and ﬁgures by
their assigned genera according to a recent checklist (Turtle
Taxonomy Working Group, 2014). As parts of a species binomial
can be unstable and/or controversial, Table 1 includes full speciesnames that can be compared to the aforementioned annotatedchecklist. We also use this checklist for the counts of species
given in the text.

b
Cryptodira

Pleurodira

Trionychia

c

Other
Cryptodira

Other
Cryptodira

Cryptodira

Pleurodira

Trionychia

Pleurodira

Other
CryptodiradTrionychiaeFig. 1. (a) An extant trionychian showing some of the bizarre diagnostic charactersfor the group such as the lack of scales and a ﬂeshy proboscis; (b) prevailingmorphological hypothesis (Gaffney and Meylan, 1988; Gaffney et al., 1991; Gaffney,1996); (c) molecular hypothesis with trionychians as the sister taxon to all othercryptodires (Shaffer et al., 1997 [mtDNA]; Fujita et al., (2004) [intron]; Krenz et al.,(2005) [intron]); (d, e) Topologies showing alternative roots for the crown group
Testudines (Barley et al., 2010 [nuDNA]; Sterli, 2010 [morphology, mtDNA, intron];Field et al., 2014 [miRNAs]). Photo credit: Dogania subplana from Indonesia taken byPeter Paul van Dijk.

We used phyluce (Faircloth et al., 2012) to identify those contigs that were UCE loci, remove putatively duplicate UCE loci, create a database of UCE loci recovered, and prepare FASTA ﬁles forsequence alignment. We generated alignments from this monolithic FASTA ﬁle using MAFFT (version 7.130b) (Katoh, 2002;
Katoh and Standley, 2013), and we trimmed resulting alignments
using the trimming algorithm implemented by the seqcap_align2.py script within phyluce. From the trimmed alignments,
we created two datasets: one where each locus contained all 36
taxa (100% complete), and one where we allowed up to 25% missing taxa per locus (i.e., we required data from a minimum of 29
taxa per locus). We estimated the appropriate ﬁnite-sites substitu-

252

N.G. Crawford et al. / Molecular Phylogenetics and Evolution 83 (2015) 250–257

Table 1
Newly sequenced samples are from museum specimens housed at either the
California Academy of Science (CAS), the Museum of Vertebrate Zoology at Berkeley(MVZ). For each of the newly sequenced samples, a Sequence Read Archive (http://
www.ncbi.nlm.nih.gov/sra) number follows the museum specimen number. For
samples from previously published papers the reference and genome builds in which
UCE loci were identiﬁed (see Section 2.4).
Binomial

Specimen/Genome

Reference

Outgroup OTUs:
Anolis carolinensis
Crocodylus porosus
Gallus gallus
Homo sapiens
Python molurus
Sphenodon punctatus

anoCar1
croPor1
galGal3hg19
pytMol1
UMFS 10956

Alföldi et al. (2011)
St. John et al. (2012)
Cons ICGS (2004)
Lander et al. (2001)
Castoe et al. (2013)
Crawford et al. (2012)

Ingroup OTUs:
Agrionemys horsﬁeldii
Apalone ferox
Carettochelys insculpta
Chelydra serpentina
Chelonia mydas
Chrysemys picta
Cyclemys dentata
Deirochelys reticularia
Dermatemys mawii
Dermochelys coriacea
Emys marmorata
Erymnochelys madagascariensis
Geoemyda spengleri
Gopherus berlandieri
Graptemys pseudogeographica
Kinosternon arizonense
Lepidochelys olivacea
Lissemys punctata
Mesoclemmys nasuta
Nilssonia formosa
Pelodiscus sinensis
Pelomedusa subrufa
Pelusios castaneus
Platemys platycephala
Platysternon megacephalum
Podocnemis erythracephalaRhinoclemmys punctularia
Staurotypus triporcatus
Sternotherus minor
Stigmochelys pardalis
Terrapene ornata
Trachemys scripta

CAS 228637
CAS 202549
MVZ 238114
MVZ 265668
cheMyd1
H2662
CAS 243787
MVZ 204282
MVZ 269552
MVZ 149844
CAS 224202
MVZ 238759
MVZ 208234
MVZ 250594
MVZ 250644
CAS 228101
CAS180267
CAS 232082
MVZ 247578
CAS 246283
pelSin1
MVZ 236628
CAS 219222
MVZ 247579
MVZ 230486
MVZ 269553
MVZ 247582
MVZ 263984
CAS 221865
MVZ 241333
MVZ 230553
CAS 252979

SAMN02900523
SAMN02900524
SAMN02900525
SAMN02900526
Wang et al. (2013)
Crawford et al. (2012)
SAMN02900527
SAMN02900528
SAMN02900529
SAMN02900530
SAMN02900531
SAMN02900532
SAMN02900533
SAMN02900534
SAMN02900535
SAMN02900536
SAMN02900537
SAMN02900538
SAMN02900539
SAMN02900540
Wang et al. (2013)
SAMN02900541
SAMN02900542
SAMN02900543
SAMN02900544
SAMN02900545
SAMN02900546
SAMN02900547
SAMN02900548
SAMN02900549
SAMN02900550
SAMN03102956

tion model for each locus in all datasets using CloudForest(Crawford and Faircloth, 2014), and we prepared a concatenateddataset for subsequent analyses by grouping together loci having
the same substitution model into a partition.
We performed Bayesian analysis of the concatenated alignment
data using two runs of MrBayes version 3.2.2 (r879) (Ronquist
et al., 2012) for 500,000 iterations (4 chains; burn-in: 25%; thinning: 500). We assessed convergence of the runs using TRACER(http://tree.bio.ed.ac.uk/software/tracer/). We performed maximum likelihood analyses of the concatenated data using RAxML
version 7.2.6 (Stamatakis, 2006) with the ‘‘GTRGAMMA’’ option
and 10,000 bootstrap replicates. We also performed gene-tree species-tree analysis by estimating gene trees for each UCE locus
incorporating 100 multi-locus bootstrap replicates, which we integrated into STEAC and STAR species trees (Liu and Yu, 2010; Liuet al., 2009). A posteriori bootstrapping analysis conducted with
RAxML’s autoMRE tool indicated that trees converged after 50
replicates.
We root our tree with the mammals following the approach ofrecent analyses that conﬁrm the archosaur afﬁnities of Testudines(Crawford et al., 2012; Fong et al., 2012; Field et al., 2014).
Lyson et al. (2012) phylogenetically deﬁned Ankylopoda for analternative placement for turtles, the crown clade of turtles andlepidosaurs, but the crown clade of turtles and archosaurs is an
unnamed amniote lineage. We ﬁll this important nomenclaturalgap, and phylogenetically deﬁne the name ‘Archelosauria’ to referto the clade that originated from the most recent common ancestorof Crocodylus niloticus Laurenti, 1768 and Testudo graeca Linnaeus,
1758. The name was chosen to evoke the two included lineages,archosaurs and chelonians (Testudines).
2.3. Trachemys whole genome sequencing, assembly, and UCE
identiﬁcation
Whole genome sequencing libraries were prepared using Illumina’s Nextera library preparation kit, following manufacturer
protocols, with the following modiﬁcations: after library preparation, a 600–700 bp size selection was performed using the BluePippin size selection system (Sage Science). Size selected products
were ampliﬁed in a 7 cycle PCR using the KAPA Real Time Library
Ampliﬁcation kit (KAPA Biosystems) following manufacturer’s
instructions. PCR products were cleaned using the standard
Ampure XP (Beckman Coulter Inc.) bead clean-up method with a
0.8:1 bead to PCR product ratio. Libraries were validated by running 1 ll of product on an Agilent 2100 Bioanalyzer (Agilent Technologies) and quantiﬁed using a Qubit ﬂuorometer (LifeTechnologies). Final libraries were sequenced on a HiSeq2500 (Illumina). Two lanes of 150 bp paired-end sequencing were run using
the rapid run output mode, with each lane containing two libraries
pooled in equimolar amounts. These same libraries were alsosequenced on two runs of an Illumina MiSeq with 600 cycle v3 kits
and 300 bp paired-end sequencing mode.
After adapter trimming and quality ﬁltering using Trimmomatic
version 0.32 (Bolger et al., 2014), we assembled 12,731,817 Trachemys scripta elegans contigs from 138,894,454 HiSeq and 53,677,903
MiSeq reads with Soapdenovo2 (Luo et al., 2012) using its multikmer method. Kmer sizes ranged from 23 to 127. To identify thosecontigs that contained UCE loci we used standalone BLAT version
35 (Kent, 2002) to match a 2560 UCE probe set (obtained fromhttp://ultraconserved.org) to the assembled contigs. BLAT was
run with default parameters on contigs greater than 300 bp. Thena custom PYTHON script was used to extract those contigs matching the UCE probes with reported E-value scores of 1e-1 or lower.
These 2926 contigs ranged in size from 301 bp to 33,369 bp. Thisﬁnal set of 2926 Trachemys scripta elegans contigs enriched for
UCE sequences was used in all subsequent phylogenetic analyses.
2.4. Data availability
With the exception for Pelodiscus sinensis for which a publishedgenome is available, all ingroup OTUs have specimen vouchers, andall tissues and specimens are available to qualiﬁed researchers. The
data we included for all outgroup taxa and P. sinensis are publicly
available at: <https://github.com/faircloth-lab/uce-probe-sets>(Faircloth et al., 2012). Additional details concerning UCE sequence
capture methods and phylogenetic methods are described in
Faircloth et al. (2012) and detailed protocols are available at<http://ultraconserved.org>. Sequenced reads are available in the
short read archive (PRJNA254176) and alignments and trees atdata dryad (doi:10.5061/dryad.t77q4).
3. Results
We sequenced a total of 86 million read pairs with a mean of
3,083,947 per sample from 28 taxa (Table 1). We assembled amean of 5377.86 contigs per sample (95CI, min = 1919,
max = 12,511) (Supp. S1). We also incorporated an average of

N.G. Crawford et al. / Molecular Phylogenetics and Evolution 83 (2015) 250–257

2939.9 UCEs drawn from eight taxa with published genomes.
Combining the UCEs identiﬁed in published genomes with the contigs assembled from the 28 UCE enriched genomic libraries andrunning the matrix generation procedures produced: (1) a 100%
complete matrix containing 233 alignments having a mean lengthof 820.26 bp (±48.58 CI) per alignment, totaling 191,121 bp ofaligned sequence and (2) a 75% complete matrix containing 2381
alignments having a mean length of 721.61 bp (±15.65 CI) peralignment, totaling 1,718,154 bp of aligned sequence.
We recover a phylogeny of Testudines that is identical across
Bayesian, maximum likelihood approaches and where every nodeis fully resolved (e.g., 100% bootstrap support or posterior probabilities of 1.0). The topologies of the STEAC and STAR species trees
contain a few inconsistencies when compared to the ML andBayesian trees. These include the position of Trachemys scripta
within Emydidae and the position of Chelonioidea within Durocryptodira. This is likely caused by both incomplete lineage sorting,and poorly resolved gene trees due to the small amount of DNA
sequence used to infer individual trees (mean = 820.26 bp)(McCormack et al., 2013). Alternately the high support of thesegroups in the ML and Bayesian trees may be from systematic biases
in inferring trees from concatenated datasets (Mossel and Vigoda,
2005; Kubatko and Degnan, 2007).
Our analysis of the UCE data recovers a monophyletic Pleurodira and Cryptodira. Within Pleurodira, we recover the traditionalfamilies based on inclusion of two representatives, each, of
Chelidae, Pelomedusidae, and Podocnemididae. We also recover amonophyletic Pelomedusoides (Pelomedusidae + Podocnemididae),a long-recognized group. Within Cryptodira, Trionychia is the sistergroup to all of the other cryptodire lineages (Durocryptodira).
Within durocryptodires, Testudinoidea is the sister group of a clade
including all of the other lineages. Platysternon megacephalum isresolved as the sister taxon to Emydidae as in Parham et al.
(2006a). The phylogeny places emydid OTUs consistent with theirsubfamilial designations, with the two emydine taxa (Emys andTerrapene) forming a monophyletic group. On the deirochelyine
side, Deirochelys is the sister taxon of a clade that includes Chrysemys, Trachemys, and Graptemys. This result differs from that of
Spinks et al. (2009), which placed Chrysemys outside of a clade that
includes Deirochelys and Graptemys. We also recovered theTestuguria clade (Geoemydidae + Testudinidae), and within eachof those included clades the topology of the tree matches previous
phylogenetic analyses (Spinks et al., 2004; Parham et al., 2006b). A
monophyletic Kinosternoidea is the sister taxon of Chelydridae,
thereby afﬁrming the Chelydroidea clade codiﬁed by Knauss
et al. (2011). The chelonioids are the sister taxon of the chelydroids, which together form the recently named Americhelydia(Joyce et al., 2013). The ability for UCEs to reconstruct relatively
recent divergences (e.g., within the traditional families) was
previously demonstrated by Smith et al. (2014) and is supported
here.

4. Discussion
4.1. The phylogeny of turtles based on UCEs
4.1.1. Pleurodira, Trionychia, and Durocryptodira
The UCE phylogeny supports the monophyly of Cryptodira, with
Trionychia as the sister taxon to all other cryptodires (Figs. 2, 3a, c).
The clade including non-trionychian cryptodires was phylogenetically deﬁned as ‘Durocryptodira’ by Danilov and Parham (2006).
The topology from ultraconserved elements and other molecularstudies (Shaffer et al., 1997; Krenz et al., 2005; Barley et al.,2010) support the monophyly and recognition of Durocryptodira,
which contrasts with the morphological hypothesis (Figs. 1, 3b).

253

A monophyletic Durocryptodira is consistent with the temporalappearance of lineages in the fossil record. Pan-trionychians (trionychians and their stem) are the most ancient cryptodire lineage.
Therefore, the molecular phylogenetic placement of Trionychia as
the sister taxon to all other cryptodires (Durocryptodira) is mostconsistent with their antiquity (Fig. 3a, b). In contrast, the morphological hypothesis requires signiﬁcant missing time for several ofthe major lineages of turtles (Fig. 3b). These ‘ghost lineages’(Norell, 1992) are hard to accept given the relatively rich fossil
record of turtles. On the other hand, even though the phylogenetic
position of many fossils at the base of Cryptodira is poorly constrained, there are several candidate taxa that could ﬁll the ghost
lineage on the stem of Durocryptodira (Fig. 3a).
4.1.2. Americhelydia
The UCE phylogeny supports the division of durocryptodiresinto the diverse and long recognized Testudinoidea (183 species)
and the recently named Americhelydia (38 species, Joyce et al.,2013). Americhelydia is comprised of three major lineages thatshare a common ancestor in the Cretaceous of North America.
Two of these lineages, the chelydroids and kinosternoids, are still
North American endemics. The third lineage is the chelonioids,
extant marine turtles, which have a cosmopolitan, oceanic distribution. Whereas the exclusive monophyly of extant marine turtles
relative to extant, non-marine lineages is not controversial, therelationships of many fossil marine turtles are confounded bypotential polyphyly (multiple origins of marine turtles) and parallel evolution (Joyce, 2007; Joyce et al., 2013). Given this confusion
it is unclear whether the chelonioid lineage diverged from other
Americhelydians in the Early or Late Cretaceous, but in either casetheir oldest fossils and presumed origins are in the Americas(Zangerl, 1953; Hirayama, 1998; Joyce, 2007). Therefore, just as
the UCE phylogeny ﬁts with the temporal appearance of clades in
the fossil record, it also coincides well with biogeography by uniting some American durocryptodires into a monophyletic group.
4.1.3. Testudinoidea
Testudinoidea (183 species, more than half of turtle diversity)
has deep fossil roots in Asia where it maintains a high diversity
today. Within testudinoids, the sister taxon relationship between
terrestrial tortoises (testudinids) and the geoemydids was
phylogenetically deﬁned as Testuguria by Joyce et al. (2004). At
that time the phylogenetic position of the big-headed turtle (Platysternon) was not well established. Early molecular phylogenies
placed it outside of Testudinoidea (Shaffer et al., 1997) or as thesister taxon to Testuguria (Krenz et al., 2005). Parham et al.
(2006a) placed Platysternon as the sister group of the Emydidae
based on an analysis of complete mitochondrial genomes, and thisresult has been conﬁrmed by more comprehensive nuclear data
sets (Barley et al., 2010; this study). The Platysternon – Emydidae
node is the only node uniting two or more of the traditional families of turtles that does not have a name. We ﬁll this importantnomenclatural gap, and phylogenetically deﬁne the name ‘Emysternia’ to refer to the clade that originated from the most recent
common ancestor of Platysternon megacephalum Gray, 1831 andEmys orbicularis (Linnaeus, 1758). The name was chosen to evoke
the two included lineages Emydidae and Platysternon.
4.2. Global paleobiogeography of turtles based on the UCE phylogeny
Combining the UCE phylogeny with the known fossil record of
turtles allows us to reconstruct some global biogeographic patterns(Fig. 3c). Intercontinental dispersal of turtles is common, usually
involving a limited number of species. For our discussion we focus
primarily on the broad patterns of vicariance and dispersal events
that generated signiﬁcant turtle diversity (i.e., speak to geographic

254

N.G. Crawford et al. / Molecular Phylogenetics and Evolution 83 (2015) 250–257
Homo
Sphenodon

Amniota

Lepidosauria

AnolisPythonGallusSquamata

Archosauria

Crocodylus

Chelidae

Platemys
Pelusios

Pelomedusidae

Erymnochelys
Podocnemis

Pelomedusoides

Podocnemididae

Carettochelys insculpta
Lissemys
ApaloneTrionychidae
Nilssonia
Pelodiscus

Trionychia

Archelosauria

Pelomedusa

Pleurodira

SauriaMesoclemmys

Testudinidae

Agrionemys

Lepidochelys

Cheloniidae

Chelonioidea

Dermatemys mawii
Staurotypus
Sternotherus Kinosternidae
Kinosternon

Kinosternoidea

Chelydroidea

Chelydra / Chelydridae

Americhelydia

Dermochelys coriacea
Chelonia

Cryptodira

Gopherus
Stigmochelys

Testuguria

Rhinoclemmys
Cyclemys
Geoemydidae
Geoemyda

Durocryptodira

Terrapene
Emys

Testudinoidea

Emydidae

Graptemys
Trachemys

Emysternia

Deirochelys
Chrysemys

Testudines

Platysternon megacephalum

0.02

Fig. 2. Phylogenetic hypothesis based on RAxML analysis of UCE data showing phylogenetically deﬁned crown clades of turtles (Testudines). All clades were supported bylikelihood 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 ofsubstitutions per site.

origin of ‘major lineages’ and clades that have been recognized as
families, especially in North America). We assign each lineage toa continent based on the their area of origin as shown by the fossil
record (stem taxa). For the timing of events we use the simple
appearance of lineages in the fossil record used to construct divergence-dating priors by Joyce et al. (2013). For the divergences discussed below, the fossil record of turtles is complete enough that
there is no discrepancy between prior and posterior estimates(Joyce et al., 2013) and so molecular divergence dating of the
UCE phylogeny would be superﬂuous.
The earliest fossils of stem testudinoids, stem trionychians, andstem cryptodires are from Eurasia (Danilov and Parham, 2006,
2008; Joyce et al., 2013; Pérez-García et al., 2014). Mapping thesedata onto the UCE phylogeny demonstrates that cryptodires have a
Jurassic (>145 Ma) Eurasian origin (Fig. 3c). The emergence of cryptodires in Eurasia is complemented by the concurrent origin of

pan-pleurodires in the Southern Hemisphere (Gondwana; Joyce
et al., 2013). Given the distribution of the clades and the timingof their origin, the geography of the cryptodire-pleurodire split
can be plausibly linked to the breakup of the supercontinent Pangaea (Scotese, 2001; Rogers and Santosh, 2003; Smith et al., 2004).
In this way turtles demonstrate a pattern common to other terrestrial vertebrates (e.g., placental vs. marsupial mammals).
Despite their Jurassic (>145 Ma) origin, cryptodires did not
dominate the northern continents for almost 100 million years(until the Cenozoic). Instead, stem turtles (especially the extinct
clade Paracryptodira) were diverse and abundant in North America
throughout the Cretaceous (145–66 Ma) and into the Cenozoic(<66 Ma; Lyson and Joyce, 2009; Lyson et al., 2011). In the Late Cretaceous (100–66 Ma), cryptodires (trionychians and durocryptodires) began to appear in North America, invading through highlatitude dispersal routes (Hirayama et al., 2000; Parham and

255

ea

a

ch

ny

Tr
io

te
r
os

ia

no

id

de

a

oi
in
Ki
n

Te
s

tu
d

ni

oi

id
lo

dr

he

ly
C

b

C

he

Tr
io

a

de

ae

ny
Te chiastudinC
he oi dloe
C ni o ahe i dealyKi drid
no
a
st e
er
no
id
ea

N.G. Crawford et al. / Molecular Phylogenetics and Evolution 83 (2015) 250–257~70 MaMacrobaenids
Sinemydids
Xinjiangchelyids~120 Ma

id

ys

te

ae

rn

ni
er
st

Pa

n-

Pl

at

yd
Em

st Ste
ud m
in
oi
ds
te S
st te
ud m
in
oi
ds
Te
st
ug
ur
ia

Emysternia

te

ia
ch
ny

?

?

TestudinoideaTr
ioJurassic
Pangaea breakup

ra
di
ro
eu
Pl

GONDWANA

Americhelydia

on

da

e

em
at
m
er
no

Pa

C

he

Ki

n-

dr
ly

oi
ni
lo
C

he

D

id

de

a

NORTH
AMERICAaecPaleogene
Dispersal

ysCretaceous
Dispersal

Cryptodira

Durocryptodira

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. Somefossil 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) prevailingmorphological hypothesis requiring multiple, long ghost lineages; (c) UCE phylogeny mapped against intercontinental paleobiogeography. Two dispersal events ofdurocryptodires 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.

Hutchison, 2003; Brinkman and Tarduno, 2005; Vandermark et al.,2009). The UCE phylogeny conﬁrms that one of the North American
durocryptodire lineages (Americhelydia) underwent a modest
radiation, accounting for three of the six ‘major lineages’ of extant
turtles (38 extant species, Fig. 3c). The relatively short branches
among the Americhelyidian lineages suggest this radiation was
rapid.
The Paleogene experienced periods of extremely warm climate(e.g., the Late Paleocene Thermal Maximum and the Early Eocene
Climatic Optimum) that are responsible for the dispersal of manyorganisms into North America through high latitude dispersal
routes (Zachos et al., 2001), including a wave of testudinoids(Estes and Hutchison, 1980; Holroyd et al., 2001; Eberle et al.,2010; Hutchison, 2013). Three of these testudinoid lineages persist in North America into modern times. Two are modest radiations of testugurians (four species of Gopherus Testudinidae; ninespecies of Rhinoclemmys, Geoemydidae). Previous studies suggested that these genera are sister taxa to all of the Old World
members of their respective clades (Parham et al., 2006b;
Spinks et al., 2004). We sequenced Gopherus, Rhinoclemmys, andrepresentative divergent members of geoemydids and testudinids; the UCE data conﬁrm the basal position of these North
American genera. This pattern links the overall diversiﬁcation at
the base of these clades with their intercontinental dispersal,
which can logically be attributed to periods of warm climate.
Similar to the Americhelydia, short branches within the testudinoids also suggest a rapid adaptive radiation that coincides withhigh latitude intercontinental dispersal events. This patternsuggests that global climate change has a major impact on thediversity and distribution of turtles.
The end of the Paleogene (45–23 Ma) coincides with global
environmental changes, with the climate becoming signiﬁcantly
cooler and drier (Zachos et al., 2001), i.e., much less favorable toturtles. Many turtle lineages that inhabited the Western Interior,including the last stem cryptodires in North America, go extinct
at this time (Hutchison, 1982, 1992, 1998). One testudinoid lineage
took advantage of the subtropical southeastern portions of the continent (Parmley et al., 2006), to radiate into the diverse clade Emydidae (53 species, Figs. 2, 3c). The recent description of a fossil
taxon on the stem of Platysternon megacephalum from the Eocene

256

N.G. Crawford et al. / Molecular Phylogenetics and Evolution 83 (2015) 250–257

of North America (Hutchison, 2013) raises the possibility that themore inclusive Emysternia may also have an American origin.
Depending on the resolution of that possibility, the UCE topology
indicates that two dispersal events into North America led to theorigin of 36 or 43% (5 or 6 of 14) of the recognized families (Fig. 3c).
4.3. Consilience in the turtle tree of life: a scaffold for paleontologicalstudies
Because it is more consilient with temporal (stratigraphic,
Fig. 3a, b) and spatial (biogeographic, Fig. 3c) patterns, we argue
that the molecular phylogenetic topology is more plausible thanthe morphological topology. In this way, the genetic data fromthe modern turtle fauna provide an important window into theevolutionary history of turtles. However signiﬁcant, this window
is limited by extinction, and the living species represent only afraction of past turtle diversity. Fortunately, by virtue of theiraquatic tendencies, past abundance, and bony shell, turtles are
one of the most common vertebrate fossils since the Late Jurassic(earlier fossils exist, but are rare). This rich fossil record of turtles
provides crucial insights into their geographic origin (see Sections
4.1.2 and 4.2), temporal appearance (Joyce et al., 2013), and morphological evolution (Miyashita, 2013; Rabi et al., 2013, 2014).
Consequently, any discussion of these patterns arising from molecular phylogenetic studies must consider fossil data. But it is alsoessential that paleontological studies take advantage of insightsfrom molecular phylogenetics. In particular, paleontologists working on the systematics of lineages that include extant members
must address and reconcile phylogenetic hypotheses based on
DNA evidence (Parham et al., 2012).
Paleontological studies continue to generate phylogenies thatunite Trionychia with the americhelyidian lineage Kinosternoidea(e.g., Bardet et al., 2013; Tong and Meylan, 2013). The conclusionsof these studies are compromised because of the strong molecularsignal rejecting that topology. Even studies that are not focused ontrionychians or their putative close relatives suffer from the incorrect polarization of characters resulting from demonstrably incorrect topologies. The reasons that some paleontological studies do
not incorporate information from molecular phylogenetics areusually not stated (but see Sterli, 2010). Explanations likely include
a distrust of molecular data and/or the logistical hurdle associatedwith synthesizing these disparate data types. For the latter, combined analyses are understandably difﬁcult because of non-overlapping taxa and unfamiliarity with analyzing molecular data sets.
The solution is to use a ‘‘molecular scaffold’’ (Springer et al.,2001), i.e., a backbone constraint tree for well-supported nodes
involving extant lineages. Molecular scaffolds are useful because
they do not require a statistical analysis of molecular data by paleontologists, just a determination of which nodes should be constrained. A molecular scaffold prevents incorrect morphologicalnodes, such as a highly nested Trionychia, from appearing in thetree, while allowing all fossil taxa to be placed anywhere in the
topology. Danilov and Parham (2006) were the ﬁrst to use thistechnique for turtles, and other workers have since adopted thismethod (e.g., Lyson and Joyce, 2010; Rabi et al., 2013; Rabi et al.,2014). We strongly recommend that all future phylogenetic studies
of fossil turtles that include extant lineages use molecular scaffolds
so that the resultant patterns and discussions can be more conﬁdently interpreted.
Acknowledgments
We thank Jens Vindum (California Academy of Sciences) and
Carol Spencer (Museum of Vertebrate Zoology) for facilitating the
procurement of tissue samples. We thank the California Academyof Sciences’ Center for Comparative Genomics and its staff for theirassistance with lab work. We also thank Dongbo Hu and the Tishkoff lab who helped provide computational resources. This work
used the Vincent J. Coates Genomics Sequencing Laboratory at
UC Berkeley, supported by NIH S10 Instrumentation Grants
S10RR029668 and S10RR027303. This work used the Extreme
Science and Engineering Discovery Environment (XSEDE), which
is supported by National Science Foundation grant number
OCI-1053575. NSF DEB-1242260 to BCF supported computational
portions of this work.

References
Alföldi, J. et al., 2011. The genome of the green anole lizard and a comparative
analysis with birds and mammals. Nature 477, 587–591.
Barley, A.J., Spinks, P.Q., Thomson, R.C., Shaffer, H.B., 2010. Fourteen nuclear genes
provide phylogenetic resolution for difﬁcult nodes in the turtle tree of life. Mol.
Phylog. Evol. 55, 1189–1194.
Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a ﬂexible trimmer for
illumina sequence data. Bioinformatics 5, 2114–2120.
Brinkman, D.B., Tarduno, J.A., 2005. A Late Cretaceous (Turonian-Coniacian) highlatitude turtle assemblage from the Canadian Arctic. Can. J. Earth Sci. 42, 2073–
2080.
Cons, I.C.G.S., 2004. Sequence and comparative analysis of the chicken genome
provide unique perspectives on vertebrate evolution. Nature 432, 695–716.
Crawford, N.G., Faircloth, B.C., 2014. CloudForest: Code to calculate species trees
from large genomic datasets. doi:10.5281/zenodo.12259.
Crawford, N.G., Faircloth, B.C., McCormack, J.E., Brumﬁeld, D., Winker, K., Glenn, T.C.,
2012. More than 1000 ultraconserved elements provide evidence that turtles
are the sister group of archosaurs. Biol. Lett. 8, 783–786.
Danilov, I.G., Parham, J.F., 2006. A redescription of ‘Plesiochelys’ tatsuensis, a turtle
from the Late Jurassic of China, and its bearing on the antiquity of the crownclade Cryptodira. J. Vert. Paleo. 26, 573–580.
Danilov, I.G., Parham, J.F., 2008. A reassessment of some poorly known turtles from
the Middle Jurassic of China with comments on the antiquity of extant turtles. J.
Vert. Paleo. 28, 306–318.
Eberle, J.J., Fricke, H.C., Humphrey, J.D., Hackett, L., Newbrey, M.G., Hutchison, J.H.,
2010. Seasonal variability in Arctic temperatures during early Eocene time.
Earth Planet. Sci. Lett. 296, 481–486.
Estes, R., Hutchison, J.H., 1980. Eocene lower vertebrates from Ellesmere Island,
Canadian Arctic Archipelago. Palaeog. Palaeoc. Palaeoe. 30, 325–347.
Faircloth, B.C., 2012. Phyluce: phylogenetic estimation from ultraconserved
elements. doi:10.6079/J9PHYL.
Faircloth, B.C., McCormack, J.E., Crawford, N.G., Harvey, M.G., Brumﬁeld, R.T., Glenn,
T.C., 2012. Ultraconserved elements anchor thousands of genetic markers
spanning multiple evolutionary timescales. Syst. Biol. 61, 717–726.
Field, D.J., Gauthier, J.A., King, B.L., Pisani, D., Lyson, T.R., Peterson, K.J., 2014. Toward
consilience in reptile phylogeny: miRNAs support an archosaur, not lepidosaur,
afﬁnity for turtles. Evol. Dev.. http://dx.doi.org/10.1111/ede.12081.
Fong, J.J., Brown, J.M., Fujita, M.K., Boussau, B., 2012. A phylogenomic approach tothe vertebrate phylogeny supports a turtle-archosaur afﬁnity and a possible
paraphyletic Lissamphibia. PLoS One 7, e48990.
Fujita, M.K., Engstrom, T.N., Starkey, D.E., Shaffer, H.B., 2004. Turtle phylogeny:
insights from a novel nuclear intron. Mol. Phylogenet. Evol. 31, 1031–1040.
Gaffney, E.S., 1996. The postcranial morphology of Meiolania platyceps and a reviewof the Meiolaniidae. Bull. Am. Mus. Nat. Hist. 229, 1–165.
Gaffney, E.S., Meylan, P.A., 1988. A phylogeny of turtles. In the phylogeny andclassiﬁcation of the tetrapods. In: Benton, M.J. (Ed.), Amphibians, Reptiles, Birds.
Systematics Association Special Volume, vol. 35. Clarendon Press, Oxford, pp.
157–219.
Gaffney, E.S., Meylan, P.A., Wyss, A., 1991. A computer assisted analysis of therelationships of the higher categories of turtles. Cladistics 7, 313–335.
Gray, J.E., 1831. A specimen of a tortoise regarded as the type of a new genus in thefamily Emydidae. Proc. Zool. Soc. Lond. 183, 106–107.
Hedges, S., Poling, L., 1999. A molecular phylogeny of reptiles. Science 283, 998–
1001.
Hirayama, R., 1998. Oldest known sea turtle. Nature 392, 705–708.
Hirayama, R., Brinkman, D.B., Danilov, I.G., 2000. Distribution and biogeography ofnonmarine Cretaceous turtles. Russ. J. Herpetol. 7, 181–198.
Holroyd, P.A., Hutchison, J.H., Strait, S.G., 2001. Changes in turtle diversity andabundance through the earliest Eocene Willwood Formation: preliminary
results: University of Michigan Papers in Paleontology 33, pp. 97–108.
Hutchison, J.H., 1982. Turtle, crocodilian, and champsosaur diversity changes in the
Cenozoic of the north-central region of western United States. Palaeog. Palaeoc.
Palaeoe. 37, 149–164.
Hutchison, J.H., 1992. Western North American reptile and amphibian record acrossthe Eocene/Oligocene boundary and its climatic implications. In: Prothero, D.R.,
Berggren, W.A. (Eds.), Eocene-Oligocene Climate and Biotic Evolution. Princeton
University Press, Princeton, NJ, pp. 451–467.
Hutchison, J.H., 1998. Turtles across the Paleocene/Eocene Epoch boundary in westcentral North America. In: Aubry, M.-P., Lucas, S.G., Berggren, W.A. (Eds.), Late
Paleocene-Early Eocene Climatic and Biotic Events in the Marine and Terrestrial
Records. Columbia University Press, New York, pp. 401–408.

N.G. Crawford et al. / Molecular Phylogenetics and Evolution 83 (2015) 250–257
Hutchison, J.H., 2013. New turtles from the Paleogene of North America. In:
Brinkman, D.B., Holroyd, P.A., Gardner, J.D. (Eds.), Morphology and Evolution of
Turtles. Springer, pp. 477–497.
Joshi, N.A., Fass, J.N., 2011. Sickle: A sliding-window, adaptive, quality-based
trimming tool for FastQ ﬁles. Software.
Joyce, W.G., 2007. Phylogenetic relationships of Mesozoic turtles. Bull. PeabodyMus. Nat. Hist. 48, 3–102.
Joyce, W.G., Parham, J.F., Gauthier, J., 2004. Developing a protocol for the conversionof rank-based taxon names to phylogenetically deﬁned clade names, as
exempliﬁed by turtles. J. Paleo. 78, 989–1013.
Joyce, W.G., Parham, J.F., Lyson, T.R., Warnock, R.C.M., Donoghue, P.C.J., 2013. A
divergence dating analysis of turtles using fossil calibrations: an example ofbest practices. J. Paleo. 87, 612–634.
Katoh, K., 2002. MAFFT: a novel method for rapid multiple sequence alignment
based on fast Fourier transform. Nucl. Acids Res. 30, 3059–3066.
Katoh, K., Standley, D.M., 2013. MAFFT Multiple sequence alignment software
version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–
780.
Kent, W.J., 2002. BLAT-the BLAST-like alignment tool. Genome Res. 12 (4), 656–664.
Knauss, G.E., Joyce, W.G., Lyson, T.R., Pearson, D., 2011. A new kinosternoid from theLate Cretaceous Hell Creek Formation of North Dakota and Montana and theorigin of the Dermatemys mawii lineage. Paläont. Zeit., 1–18.
Krenz, J.G., Naylor, G.J.P., Shaffer, H.B., Janzen, F.J., 2005. Molecular phylogenetics
and evolution of turtles. Mol. Phylogenet. Evol. 37, 178–191.
Kubatko, L.S., Degnan, J.H., 2007. Inconsistency of phylogenetic estimates fromconcatenated data under coalescence. Syst. Biol. 56, 17–24.
Lander, E.S., Linton, L.M., Birren, B., et al., 2001. Initial sequencing and analysis of thehuman genome. Nature 409, 860–921.
Laurenti, J.N., 1768. Specimen medicum, exhibens synopsin reptilium emendatam
cum experimentis circa venena et antidota reptilium austracorum, quodauthoritate et consensu. Vienna, Joan. Thomae, 217pp.
Linnaeus, C., 1758. Systema Naturae, Volume 1 (tenth edition). Laurentius Salvius,
Holmia, 824pp.
Liu, L., Yu, L., 2010. Phybase: an R package for species tree analysis. Bioinformatics
26 (7), 962–963.
Liu, L., Yu, L., Pearl, D., Edwards, S., 2009. Estimating species phylogenies using
coalescence times among sequences. Syst. Biol. 58, 468.
Luo et al., 2012. SOAPdenovo2: an empirically improved memory-efﬁcient shortread de novo assembler. GigaScience 2012 (1), 18.
Lyson, T.R., Joyce, W.G., 2009. A revision of Plesiobaena (Testudines: Baenidae) andan assessment of baenid ecology across the K/T boundary. J. Paleo. 83, 833–853.
Lyson, T.R., Joyce, W.G., 2010. A neglected lineage of North American turtles ﬁlls amajor gap in the fossil record. Palaeontology 53, 241–248.
Lyson, T.R., Joyce, W.G., Knauss, G.E., Pearson, D.A., 2011. Boremys (Testudines,
Baenidae) from the latest Cretaceous and early Paleocene of North Dakota: An
11-million-year range extension and an additional K/T survivor. J. Vertebr.
Paleontol. 31, 729–737.
Lyson, T.R., Sperling, E.A., Heimberg, A.M., Gauthier, J.A., King, B.L., Peterson, K.J.,
2012. MicroRNAs support a turtle + lizard clade. Biol. Lett. 8, 104–107.
McCormack, J.E., Harvey, M.G., Faircloth, B.C., Crawford, N.G., Glenn, T.C., Brumﬁeld,
R.T., 2013. A phylogeny of birds based on over 1500 loci collected by target
enrichment and high-throughput sequencing. PLoS ONE 8 (1), e54848.
Miyashita, T., 2013. Geometric and developmental perspectives on the evolution of
the skull and internal carotid circulation in turtles. In: Brinkman, D.B., Holroyd,P.A., Gardner, J.D. (Eds.), Morphology and Evolution of Turtles, Vertebrate.
Paleobiology and Paleoanthropology. Springer Verlag, Dorsrecht, pp. 71–101.
Mossel, E., Vigoda, E., 2005. Phylogenetic MCMC algorithms are misleading on
mixtures of trees. Science 309, 2207–2209.
Norell, M.A., 1992. Taxic Origin and Temporal Diversity: The Effect of Phylogeny
Extinction and Phylogeny. Columbia University Press, New York, pp. 89–118.
Bardet, N., Jalil, N.-E., de Lapparent de Broin, F., Germain, D., Lambert, O., Amaghzaz,M., 2013. A giant chelonioid turtle from the late cretaceous of morocco with asuction feeding apparatus unique among tetrapods. PLoS ONE 8 (7), e63586.
http://dx.doi.org/10.1371/journal.pone.0063586.
Parham, J.F., Hutchison, J.H., 2003. A new eucryptodiran turtle from the Late
Cretaceous of North America (Dinosaur Provincial Park, Alberta, Canada). J. Vert.
Paleo. 23, 783–798.
Parham, J.F., Feldman, C.R., Boore, J.L., 2006a. The complete mitochondrial genomeof the enigmatic bigheaded turtle (Platysternon): description of unusual
genomic features and the reconciliation of phylogenetic hypotheses based on
mitochondrial and nuclear DNA. BMC Evol. Biol. 6 (11), 1–11.
Parham, J.F., Macey, J.R., Papenfuss, T.J., Feldman, C.R., Türkozan, O., Polymeni, R.,
Boore, J.L., 2006b. The phylogeny of Mediterranean tortoises and their close
relatives based on complete mitochondrial genome sequences from museum
specimens. Mol. Phylog. Evol. 38, 50–64.

257

Parham, J.F., Donoghue, P.C.J., Bell, C.J., Calway, T.D., Head, J.J., Holroyd, P.A., Inoue,
J.G., Irmis, R.B., Joyce, W.G., Ksepka, D.T., Patané, J.S.L., Smith, N.D., Tarver, J.E.,
Van Tuinen, M., Yang, Z., Angielczyk, K.D., Greenwood, J., Hipsley, C.A., Jacobs, L.,
Makovicky, P.J., Müller, J., Smith, K.T., Theodor, J.M., Warnock, R.C.M., Benton,
M.J., 2012. Best practices for justifying fossil calibrations. Syst. Biol. 61, 346–
359.
Parmley, D., Hutchison, J.H., Parham, J.F., 2006. A diverse turtle fauna from the
Eocene of Georgia (USA) including the oldest records of aquatic testudinoids insoutheastern North America. J. Herpetol. 40, 343–350.
Pérez-García, A., Gasulla, J.M., Ortega, F., 2014. A new turtle species of Brodiechelys
from the Early Cretaceous of Spain: systematic and palaeobiogeographic
implications. Acta Palaeont. Polon. 59, 333–342.
Rabi, M., Zhou, C.-F., Wings, O., Ge, S., Joyce, W.G., 2013. A new xinjiangchelyid
turtle from the middle Jurassic of Xinjiang, China and the evolution of thebasipterygoid process in Mesozoic turtles. BMC Evol. Biol. 2013 (13), 203.
Rabi, M., Sukhanov, V.B., Egorova, V.N., Danilov, I., Joyce, W.G., 2014. Osteology,
relationships, and ecology of Annemys (Testudines, Eucryptodira) from the Late
Jurassic of Shar Teg, Mongolia, and phylogenetic deﬁnitions for
Xinjiangchelyidae, Sinemydidae, and Macrobaenidae. J. Vert. Paleo. 34, 327–
352.
Rogers, J.J.W., Santosh, M., 2003. Supercontinents in earth history. Gondwana Res. 6,
357–368.
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget,
B., Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: efﬁcient Bayesian
phylogenetic inference and model choice across a large model space. Syst. Biol.
61, 539–542.
Scotese, C.R., 2001. Atlas of Earth History. Department of Geology, University ofTexas, Arlington, TX.
Shaffer, H.B., Meylan, P., McKnight, M.L., 1997. Tests of turtle phylogeny: molecular,
morphological, and paleontological approaches. Syst. Biol. 46, 235–268.
Shaffer, H.B. et al., 2013. The western painted turtle genome, a model for theevolution of extreme physiological adaptations in a slowly evolving lineage.
Genome Biol. 14, R28, <http://dx.doi.org/10.1186/gb-2013-14-3-r28>.
Smith, A.G., Smith, D.G., Funnell, B.M., 2004. Atlas of Cenozoic and Mesozoic
Coastlines. Cambridge Univ. Press, Cambridge, UK.
Smith, B.T., Harvey, M.G., Faircloth, B.C., Glenn, T.C., Brumﬁeld, R.T., 2014. Target
capture and massively parallel sequencing of ultraconserved elements forcomparative studies at shallow evolutionary time scales. Syst. Biol. 63, 83–95.
Spinks, P.Q., Shaffer, H.B., Iverson, J.B., McCord, W.P., 2004. Phylogenetic hypotheses
for the turtle family Geoemydidae. Mol. Phylog. Evol. 32, 164–182.
Spinks, P.Q., Thomson, R.C., Lovely, G.A., Shaffer, H.B., 2009. Assessing what isneeded to resolve a molecular phylogeny: simulations and empirical data fromemydid turtles. BMC Evol. Biol. 9, 56.
Springer, M.S., Teeling, E.C., Madsen, O., Stanhope, M.J., de Jong, W.W., 2001.
Integrated fossil and molecular data reconstruct bat echolocation. Proc. Natl.
Acad. Sci. USA 98, 6241–6246.
St. John, A. et al., 2012. Sequencing three crocodilian genomes to illuminate theevolution of archosaurs and amniotes. Gen. Biol. 13, 415.
Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic
analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–
2690.
Sterli, J., 2010. Phylogenetic relationships among extinct and extant turtles: theposition of Pleurodira and the effects of the fossils on rooting crown-group
turtles. Contrib. Zool. 79, 93–106.
Tong, H., Meylan, P.A., 2013. Morphology and relationships of brachyopsemys
tingitana gen. et sp. nov. from the Early Paleocene of Morocco and Recognitionof the New Eucryptodiran Turtle Family: Sandownidae. In: Brinkman, D.B.,
Holroyd, P.A., Gardner, J.D. (Eds.), Morphology and Evolution of Turtles,
Vertebrate. Paleobiology and Paleoanthropology. Springer Verlag, Dorsrecht,
pp. 71–101.
Turtle Taxonomy Working Group, 2014. Turtles of the world, 7th edition: annotatedchecklist of taxonomy, synonymy, distribution with maps, and conservation
status. Chelonian Res. Monogr. 5, 329–479.
Vandermark, D., Tarduno, J.A., Brinkman, D.B., Cottrell, R.D., Mason, S., 2009. New
Late Cretaceous macrobaenid turtle with Asian afﬁnities from the High
Canadian Arctic. Geology 37, 183–186.
Wang, Z. et al., 2013. The draft genomes of soft-shell turtle and green sea turtle
yield insights into the development and evolution of the turtle-speciﬁc bodyplan. Nat. Genet. 45, 701–706.
Zachos, J., Pagani, M., Sloan, L., Thomas, E., Billups, K., 2001. Trends, rhythms,
andaberrations in global climate 65 Ma to present. Science 292, 686–693.
Zangerl, R., 1953. The vertebrate fauna of the Selma Formation of Alabama. Part IV. The
turtles of the family Toxochelyidae. Fieldiana: Geology. Memoirs 633, 135–277.
Zerbino, D.R., Birney, E., 2008. Velvet: algorithms for de novo short read assembly
using de Bruijn graphs. Genome Res. 18, 821–829.