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Supplementary Appendix S1
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Methods for estimating new time-calibrated phylogenies
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i) Emydid turtles
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To estimate a time-calibrated phylogeny for emydid turtles, we used the data set
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of six combined nuclear genes (ETS, GAPD, NGFB, ODC, R35, Vimentin) from Wiens
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et al. (2010). Although not all genes have data for all species, these combined data
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nevertheless yield a generally well-supported phylogeny, with seemingly little impact of
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missing data (Wiens et al. 2010; Wiens & Morrill 2011). We did not include the
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mitochondrial genes, as they appear to seriously distort branch lengths in some emydid
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groups (Wiens et al. 2010).
The phylogeny was estimated using the Bayesian uncorrelated lognormal
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approach (Drummond et al. 2006) in BEAST version 1.5.4 (Drummond & Rambaut
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2007). We used substitution models with parameters estimated separately for each gene
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but with a tree model and clock model shared among genes. We used the following
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general substitution models for each gene, as estimated by Wiens et al. (2010): HKY+
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gamma (GAPD, NGFB, ODC, R35), and GTR + gamma (ETS, Vim). We used
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estimated base frequencies and used four-rate categories for gamma. We used a relaxed,
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uncorrelated, clock model with an estimated clock rate and randomly generated trees with
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a Yule speciation tree prior.
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We used two fossil calibration points. First, the oldest described emydid species
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is Chrysemys antiqua (Hutchinson 1996), from the Chadronian (minimum age 33.9
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million years ago, based on the Paleobiology Database). Joyce & Bell (2004) showed
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that characters used to assign this species to Chrysemys are plesiomorphic for Emydidae
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or Deirocheylinae. We therefore used this fossil to merely constrain the minimum age of
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the emydid crown group (the age of the split between Emydinae and Deirocheylinae).
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For this clade, we used a lognormal distribution with an offset of 33.9 Myr, a mean of 5.0
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Myr, and a standard deviation of 1.0. The mean and standard deviation were selected to
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reflect that emydids are at least as old as this fossil, are most likely similar in age to this
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fossil, but might be considerably older. The 95% highest probability density limits were
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34.49–49.61 Mya.
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The second calibration point involves the minimum age of the split between Emys
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and its sister group (Emydoidea). According to Lenk et al. (1999), the earliest appearance
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of Emys in the fossil record is 12 Myr ago. We used this fossil to constrain the minimum
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age of this split. We used a lognormal distribution with an offset of 12.0 Myr, a mean of
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5.0 Myr, and a standard deviation of 1.0. The mean and standard deviation were selected
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to reflect that the clade is at least as old as this fossil, but might also be considerably
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older. The 95% highest probability density limits were 12.59–27.71 Mya.
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We performed two replicate analyses of 20 million generations each, sampling
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every 1,000 generations. These analyses yielded very similar likelihood values, identical
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majority-rule topologies, and mean estimated dates for all clades within ~1 Myr of each
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other. We therefore combined the results of these two analyses using LogCombiner in
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the BEAST package. Using Tracer (also in the BEAST package), we identified the first 2
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million generations as burnin. We then combined results from the subsequent
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generations of each analysis. The combined results had an effective sample size (ESS) >
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1,000 for all estimated dates and other parameters. The estimated tree was based on the
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tree with the highest clade credibility and ages were estimated based on mean ages across
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the postburnin trees.
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The estimated phylogeny (including posterior probabilities of clades) is shown in
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Fig. S1, and mean estimated divergence dates are shown in Fig. S2. In general, the
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estimated tree is strongly supported and similar to other recent estimates (e.g. Wiens et
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al. 2010).
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ii) New World ranid frogs
Wiens et al. (2009) estimated an extensive time-calibrated phylogeny for ranid
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frogs, but this phylogeny lacked many New World species. However, a relatively
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comprehensive phylogeny of New World species was provided by Pyron & Wiens
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(2011). We therefore took the data from Pyron & Wiens (2011) and estimated a time-
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calibrated phylogeny for the New World ranid species.
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We first reduced the matrix of Pyron & Wiens (2011) to include only the 48
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sampled New World species. These species do not form a monophyletic group, but
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belong to two closely related clades (which can therefore serve as outgroups to each
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other). The data set of Pyron & Wiens (2011) includes 12 nuclear and mitochondrial
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genes. We deleted 4 nuclear genes that lacked sequences for any of these taxa (CXCR4,
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NCX1, POMC, SCL8A3), which left 3 mitochondrial genes (12S, 16S, cytb) and 5
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nuclear genes (H3A, RAG1, Rhod, SIA, Tyrosinase). However, not all genes were
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sampled for all species (although most taxa were sampled for 12S and 16S), and all
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nuclear genes were present in only a subset of species.
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The phylogeny was estimated using the Bayesian uncorrelated lognormal
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approach (Drummond et al. 2006) implemented in BEAST version 1.5.4 (Drummond &
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Rambaut 2007). We used separate substitution models for each gene and partition, but
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with a tree model and clock model shared among genes. Following Pyron & Wiens
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(2011), we used the GTR+I+gamma for all genes and partitions, with separate partitions
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for stems and loops in 12S, separate stem and loop partitions in 16S, and separate
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partitions for genes and codons in the protein-coding genes. We used estimated base
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frequencies and used four-rate categories for gamma. We used a relaxed, uncorrelated,
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clock model with an estimated clock rate and randomly generated trees with a Yule
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speciation tree prior. Wiens et al. (2009) estimated common ancestor of clade of North
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America species at 34.14 Myr, based on analyses using BEAST. We used this date as a
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secondary calibration point, with normal distribution and standard deviation of 1 (95%
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prior distribution: 32.5--35.8 Myr). Analyses were run for 20 million generations,
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sampling every 1000 generations
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Initial analyses yielded trees with higher-level relationships that were somewhat
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inconsistent with those of Pyron & Wiens (2011) and Wiens et al. (2009). We therefore
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performed analyses with the following clades constrained to be monophyletic: (1) boylii
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group, (2) all New World Rana excluding the boylii group, (3) the clade of the catesbiana
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group + R. sylvatica, (4) palmipes group + tarahumarae group+ leopard frog clade
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(Nenirana, Scurillirana, Stertirana), (5) palmipes group + tarahumarae groups, and (6)
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the leopard frog clade.
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Given these constraints, we then performed two analyses of 20 million
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generations each. Using Tracer (the BEAST package), we identified the first 2 million
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generations of each analysis as burnin, and excluded these results. The two analyses
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yielded similar trees and clade ages. We then combined the estimated post-burnin trees
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and parameters using LogCombiner in the BEAST package. Using Tracer in the BEAST
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package, we found the combined results yielded an effective sample size >200 on all ages
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and parameters. The estimated tree was based on the tree with the highest clade
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credibility and ages were estimated based on mean ages across the postburnin trees.
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The final estimated tree was similar to that from Wiens et al. (2009), Pyron &
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Wiens (2011), and earlier studies, and was generally strongly supported. The estimated
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tree, including posterior probabilities of clades and mean estimated divergence times is
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shown in Fig. S3.
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Additional references (not in main text).
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Drummond, A.J. & Rambaut, A. (2007). BEAST: Bayesian evolutionary analysis by
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sampling trees. BMC Evol. Biol., 7, 214.
Drummond, A.J., Ho, S.Y.W., Phillips, M.J., & Rambaut, A. (2006). Relaxed
phylogenetics and dating with confidence. PLoS Biology, 4, e88.
Hutchinson, J.H. (1996). Testudines. Pages 337–353 in D. R. Protrhero and R. J. Emry,
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eds. The terrestrial Eocene-Oligocene transition in North America. Cambridge
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University Press, Cambridge.
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Joyce, W.G. & Bell, C.J. (2004). A review of comparative morphology of extant
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testudinoid turtles (Reptilia: Testudines). Asiatic Herpetological Research, 10,
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53–109.
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Lenk, P., Fritz, U., Joger, U. & Winks, M. (1999). Mitochondrial phylogeography of the
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European pond turtle, Emys orbicularis (Linnaeus 1758). Mol. Ecol., 8, 1911–
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1922.
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Wiens, J.J. & Morrill, M.C. (2011). Missing data in phylogenetic analysis: reconciling
results from simulations and empirical data. Syst. Biol., 60, 719–731.