Molecular Phylogenetics and Evolution 69 (2013) 929–939

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Molecular Phylogenetics and Evolution

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Molecular phylogenetics of the mud and musk turtle familyKinosternidae

1055-7903/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ympev.2013.06.011

⇑ Corresponding author. Fax: +1 765 983 1497.E-mail address: johni@earlham.edu (J.B. Iverson).

1 Present address: Department of Biology, University of Virginia, Charlottesville, VA22904, United States.

John B. Iverson a,⇑, Minh Le b,c,d, Colleen Ingram d,1

a Department of Biology, Earlham College, Richmond, IN 47374, United Statesb Faculty of Environmental Sciences, Hanoi University of Science, 334 Nguyen Trai Road, Hanoi, Viet Namc Centre for Natural Resources and Environmental Studies, Vietnam National University, 19 Le Thanh Tong Street, Hanoi, Viet Namd Department of Herpetology, American Museum of Natural History, Central Park West at 79th Street, NY 10024, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 November 2012Revised 15 May 2013Accepted 18 June 2013Available online 30 June 2013

Keywords:ClaudiusDNAEvolutionKinosternonStaurotypusSternotherus

The turtle family Kinosternidae comprises 25 living species of mud and musk turtles confined to the NewWorld. Previous attempts to reconstruct a phylogenetic history of the group have employed morpholog-ical, isozyme, and limited mitochondrial DNA sequence data, but have not been successful in producing awell-resolved phylogeny. With tissues from every recognized species and most subspecies, we sequencedthree mitochondrial (cyt b, 12S, 16S) and three nuclear markers (C-mos, RAG1, RAG2). Our analysesrevealed the existence of three well-resolved clades within the Kinosterninae (aged >22 mya), onlytwo of which have been named: Sternotherus and Kinosternon. We here describe the third clade as anew genus. The evolutionary relationships among most species were well resolved, although thosebelonging to the K. scorpioides species group will require more extensive geographic and genetic sam-pling. Divergence time estimates and ancestral area reconstructions permitted the development of thefirst rigorous hypothesis of the zoogeographic history of the group, including support for three separatedispersals into South America, at least two of which preceded the closure of the Panamanian portal.

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1. Introduction cause of a paucity of meristic characters and the existence of signif-

The turtle family Kinosternidae includes 25 recognized extantaquatic to semiaquatic species (38 taxa including subspecies) dis-tributed in the New World from Canada to Argentina (Iverson,1992a; TTWG, 2012). Although the greatest living diversity is inMesoamerica (Iverson, 1992a, 1992b), fossil taxa are most diversein the Eocene and Oligocene of Wyoming and South Dakota(Hutchison, 1991). Two subfamilies, the Staurotypinae (includingthe genera Staurotypus and Claudius) and the Kinosterninae(including Kinosternon and Sternotherus), have been generally rec-ognized as distinct, with some authors elevating them to the familylevel (Bickham and Carr, 1983; Vetter, 2005).

Referred to as ‘‘mud’’ or ‘‘musk’’ turtles, members of this familyare small, secretive, malodorous, and generally non-descript (Bo-nin et al., 2006; Schilde, 2001; Vetter, 2005). As a result of thiscombination of traits, recognition of the living species diversityin the family has been delayed compared to most other turtle fam-ilies. For example, ten species (40% of total) and 8 subspecies (of 13total) have been described since 1922 (TTWG, 2012), with one ortwo species described each decade since then. Furthermore, be-

icant (though drab) color variation within and among populations,species boundaries have often been difficult to establish (e.g., Bour-que, 2012c; Iverson, 2010; Lamb and Lovich, 1990; Serb et al.,2001). Nonetheless, undescribed taxonomic diversity is suspectedto exist (Webb, 1984; Iverson, unpublished).

The family Kinosternidae also exhibits a stunning diversity oflife history traits when compared to other turtle families. It rangesfrom north temperate to tropical habitats, and from rain forest tograsslands to desert (Bonin et al., 2006). It includes totally aquaticto semi-terrestrial species, with adult carapace lengths of 10–38 cm (Bonin et al., 2006), and female-dominated to male-domi-nated sexual size dimorphism (Ceballos et al., 2013). At least onespecies exhibits close to the maximum skeletal mass relative tobody mass among all vertebrates (Iverson, 1984). Some specieshave a greatly reduced plastron, whereas others have a plastronso extensive as to completely close the shell (Hutchison, 1991).The group includes members capable of submerged, fully aquaticrespiration (Belkin, 1968), and others capable of estivating under-ground for up to two years (Rose, 1980). Some species produce asingle clutch in the spring, others nest multiple times in the sum-mer, and others nest nearly year-round (Iverson, 2010), withclutches ranging from one or two relatively huge eggs to ten ormore relatively tiny eggs (Iverson, 1999). Embryonic developmentis direct in some species, whereas others exhibit early embryonicdiapause and/or late embryonic estivation, with incubation times

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from 56 to over 366 days (Ewert, 1991). Finally, sex determinationin the family ranges from genetic (with sex chromosomes) to tem-perature-dependent (Ewert et al., 2004). Unfortunately, under-standing the evolution of these diverse traits has been impededby the lack of a well-resolved phylogeny for the group.

Published phylogenetic hypotheses to date have been basedon morphology (Iverson, 1991), protein electromorph data (Iver-son, 1991; Seidel et al., 1986), and small segments of the mito-chondrial genome, using limited taxonomic sampling (Iverson,1998; Serb et al., 2001). None employed complete taxonomicsampling or nuclear markers, nor applied modern phylogeneticmethods. Hence, the phylogenetic structure for the family isnot well-resolved (e.g., see Iverson, 1998). To solve this defi-ciency, we sequenced 3 mitochondrial (cyt b, 12S, and 16S)and 3 nuclear (C-mos, RAG1, and RAG2) markers from represen-tatives of every recognized species and most subspecies in thisfamily (a total of 34 samples). Contemporary phylogenetic meth-ods were used to test previous hypotheses regarding the rela-tionships among the known kinosternid turtles and to directfuture research in clarifying cryptic diversity in the family. Inaddition, the recovered phylogeny was calibrated using knownfossils to permit a reconstruction of the zoogeographic historyof the family, particularly the timing of its multiple dispersalsinto South America.

2. Materials and methods

2.1. Sampling and laboratory methods

All 25 species of the family Kinosternidae were included in thestudy (Table S1). Outgroup polarity was provided by the sister fam-ily Dermatemydidae (Barley et al., 2010; Krenz et al., 2005; Nearet al., 2005). We sequenced three regions of the mitochondrial gen-ome, the complete cytochrome b (cyt b) sequence, and the partial12S and 16S rRNA genes (1958 total aligned bp). We also se-quenced three nuclear fragments of the C-mos, RAG1, and RAG2genes (2553 total aligned bp). Primers used for this study are listedin Table S2.

DNA was extracted from tissues and blood samples using theDNeasy kit (QIAGEN, Valencia, CA, USA) following manufacturer’sinstructions for animal tissues. We also extracted DNA from bonesamples using the procedures described in Le et al. (2007). PCR vol-ume consisted of 30 ll (9 ll of water, 2 ll of each primers, 15 ll ofHotStar Taq Master Mix (QIAGEN, Valencia, CA, USA), and 2 ll ofdiluted DNA). PCR conditions for the mitochondrial genes were:95 �C for 15 min to activate the Taq; with 42 cycles at 95 �C for30 s, 45 �C for 45 s, 72 �C for 60 s; and a final extension of 72 �Cfor 6 min. Nuclear DNA was amplified using the same PCR condi-tions, while the annealing temperatures were 52 �C for RAG1 andthe second fragment of RAG2, 56 �C for the first fragment ofRAG2, and 58 �C for C-mos.

PCR products were visualized using electrophoresis through a2% low melting-point agarose gel (NuSieve GTG, FMC Biopolymers)stained with ethidium bromide and/or Safe DNA (SYBR�). PCRproducts were cleaned using PerfectPrep� PCR Cleanup 96 plate(Eppendorf Scientific Inc., Hamburg, Germany) and cycle se-quenced using ABI prism big-dye terminator (Applied Biosystems,Foster City, CA, USA) according to manufacturer recommendation.Sequences were generated in both directions on an ABI 3130xl Ge-netic Analyzer (Applied Biosystems, Foster City, CA, USA).

2.2. Phylogenetic analyses

DNA sequences were edited, checked for ambiguities, andaligned using Geneious v5.4 (Drummond et al., 2011). For coding

regions, alignments were refined by eye to translated sequencesto confirm reading frame conservation and checked for prematurestop codons.

The loci were analyzed individually, mtDNA only (1958 bp),nuDNA only (2553 bp) and as a single concatenated dataset(4511 bp) under maximum likelihood (GARLI 2.0: Zwickl, 2006),and Bayesian inference (MrBayes v3.1.2). Coding regions were par-titioned into first, second, and third position. jModelTest 2.1.1(Darriba et al., 2012) was used to determine the appropriate modelof sequence evolution for each partition for all model-based anal-yses. The best models were GTR + G + I for the mtDNA regions16S rRNA, 12S rRNA, and the first and second positions of cyt b;GTR + G for the third position of cyt b; HKY + I for the first and sec-ond positions of C-mos; GTR for the third position of C-mos and firstand second positions of RAG1; GTR + I for the third position ofRAG1; HKY + G for the first and second positions of RAG2; andHKY for the third position of RAG2. For the ML analyses, four inde-pendent searches were performed on the concatenated mtDNA,nuDNA, and combined datasets with partitions modeled sepa-rately. All other parameters of GARLI were left at the default set-tings. The four independent searches were compared to confirmthat the heuristic searches were converging on the same likelihoodand topology, and the topology with the highest likelihood valuewas considered the best tree. 100 non-parametric ML bootstrapreplicates were examined to determine support for each node.

Bayesian posterior probabilities were calculated using theMetropolis-coupled Markov chain Monte Carlo (MC3) sampling ap-proach in MrBayes v3.01 (Ronquist et al., 2012). Four independentsearches were performed for each dataset; each search consisted ofa cold chain and 6 heated chains. All searches started with randomtrees and uniform prior probabilities for all possible trees. For alldatasets, the original Markov chains were run for 4 � 107 genera-tions and trees were sampled every 10,000 generations. To deter-mine that stationarity had been reached, we compared both thefluctuating values of the likelihood from the four independentsearches using TRACER v1.4 (Rambaut and Drummond, 2007)and convergence rates of posterior split probabilities and branchlengths using AWTY (Nylander et al., 2008). The ‘‘burn-in’’ valuewas conservatively set at 1000; the first 1000 (10,000,000 genera-tions) trees were eliminated from the approximation of posteriorprobabilities. The trees retained from each run were combinedand a 50% majority rule consensus tree was produced. The ML treesfrom each independent GARLI and MrBayes search were compared.The likelihood scores from the ‘‘best tree’’ recovered using GARLIand the ML tree from each MrBayes search were optimized usingPAUP* for comparison of ML and topology testing. Uncorrected pdistances between each sample pair were calculated for the cyt bsequence data using PAUP* (Swofford, 2002).

2.3. Divergence time estimates

Divergence times were estimated using an uncorrelated log-normal relaxed clock model (Drummond et al., 2006) as imple-mented in the program BEAST v.1.7.5 (Drummond and Rambaut,2007), using the subroutine BEAUti v1.7.5 to set the analysisparameters. The model of evolution for partitions that fit GTR wereconservatively set to HKY. This was only after initial runs usingGTR had low ESS values for prior and posterior, with one of the rel-ative rates in each of the GTR modeled partition going to zero.Reducing the model to HKY fixed this issue without any changein results. Two calibration points were used. The minimum agefor the divergence between the Dermatemydidae and Kinosterni-dae was constrained to 74.8 mya, based on stem kinosternid fossilsfrom the lower third of the Kaiparowits Formation (Brinkman andRodriguez De La Rosa, 2006; Brinkman et al., 2013; Hutchisonet al., 2013; Knauss et al., 2011). Hutchison (1991) described the

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oldest known kinosternine fossils from the early Eocene (Lysitian),and suggested the divergence of the subfamilies Kinosterninae andStaurotypinae at between 56 and 65 mya. For our analysis we con-strained the minimum age of the Kinosterninae/Staurotypinae splitto 53 mya based on Koch and Morrill (2000). In each analysis, theMarkov chain was run for 100 million generations and sampledevery 10,000. Convergence to stationarity was checked using TRA-CER v. 1.5 (Rambaut and Drummond, 2007) for each search andcompared across all runs. The search results were summarizedusing TreeAnnotator v. 1.7.5 and visualized using Figtree v.1.3.1(http://tree.bio.ed.ac.uk/software/figtree/).

2.4. Biogeography

Ancestral distributions of all extant kinosternid turtles werereconstructed using both the Statistical Dispersal-Vicariance Anal-ysis (S-DIVA) and the Bayesian Binary method (BBM) implementedin RASP (Reconstruct Ancestral State in Phylogenies: Yu et al.,2011). We used a time-calibrated phylogeny that includedrepresentatives from all 25 species of Kinosternidae and a singleoutgroup (Dermatemys). We coded eleven geographical areas:A – South America, B – Central America (south of Mesoamerica,Honduras to Panama), C – Mesoamerica (Isthmus of Tehuantepecto NW Honduras), D – Atlantic Mexico (Tampico embayment,Tamaulipas to northern Veracruz), E – Mexican Plateau, F – Pacificcoastal Mexico (Sinaloa to Oaxaca), G – Northwest Mexico(Sonora), H – Southwest USA (Arizona), I – Central USA,J – Southeast USA, and K – Northeast USA. Although more fine scalecoding is possible, we find this simplistic matrix more appropriatefor the questions at hand, rather than making data overly complexand over-parameterized, exceeding their explanatory value.Because most sampled taxa occupied only one or two areas, andonly one occupied three (leucostomum), we set the maximum num-ber of areas for reconstruction in both S-DIVA and BBM at three.

3. Results

3.1. Phylogenetic analyses

The final data matrix contained 4511 aligned characters, andamong the six markers, cyt b and 16S were the most phylogeneti-cally informative (Table 1). Phylogenies estimated for each locuswere variable in the amount of resolution; mitochondrial genetrees were resolved with support while the nuclear gene treesshowed variable and poor resolution distributed across the tree(not shown). Phylogenies based on the combined nuclear data alsodemonstrated minimal resolution (e.g., Fig. S1), whereas thosebased on the combined mitochondrial sequences were almost fullyresolved (e.g., Fig. S2) and nearly identical to those based on thecombined nuclear and mitochondrial data set. While these datasetsvaried in the amount of phylogenetic information, comparisons donot reveal any major conflict; areas that were not completely con-gruent among trees were restricted to nodes involving shortbranches and/or weak bootstrap support, and therefore we focusedon the results from the total concatenated dataset.

Table 1Variation among markers used in our phylogenetic analysis of kinosternid turtles.

Marker Total # characters # Variable # Informative

cyt b 1085 471 35512S 363 61 2916S 510 141 108C-mos 522 61 39RAG1 872 56 23RAG2 1159 90 35

The four different analyses (ML and BI, codon and non-codon-based) recovered almost identical topologies, differing only in nodesupport values (Fig. 1). The two previously recognized subfamilies,Staurotypinae and Kinosterninae (TTWG, 2012), were recovered asmonophyletic in all analyses (ML bootstrap [BP] = 100%, BI poster-ior probability [PP] = 100%; only codon-based support values re-ported in the text, but see Fig. 1).

Within the Kinosterninae, three major clades were resolvedwith high support (ML BP = 89–97%, BI PP = 100%), although therelationship among the three clades was not well resolved (MLBP < 50%, BI PP = 51%). A monophyletic genus Sternotherus wasstrongly supported (ML BP = 96%, BI PP = 100%); however, thegenus Kinosternon as previously recognized was recovered consis-tently as paraphyletic with respect to Sternotherus. Most formermembers of Kinosternon (sensu lato) belong to the clade includingK. scorpioides (type species of the genus Kinosternon; Iverson,1992a), with high support (ML BP = 89%, BI PP = 100%. However,the leucostomum group (also including acutum, angustipons, crea-seri, dunni, and herrerai) was recovered as monophyletic with re-spect to Sternotherus and the restricted genus Kinosternon withstrong support (ML = 97%, BI = 100%). Mean uncorrected p dis-tances for cyt b among species within each clade (0.058–0.076)were substantially lower than average distances between clades(0.10–0.12: Table 2). No unique genus name has ever been pro-posed for any member of the leucostomum group (Fritz and Havas,2007).

Within Sternotherus, odoratus was weakly supported as sister tothe carinatus complex (including minor and depressus; see also Tin-kle, 1958; ML = 56%, BI = 82%), suggesting that the divergence ofodoratus and the carinatus group was nearly coincident in time.The mtDNA only dataset strongly supported S. odoratus as sisterto S. carinatus (ML = 96%, BI = 100%; Fig. S2), while the nuDNA onlyanalysis did not recover odoratus within the Sternotherus clade.This may be explained by the limited amount of nuDNA signaland the lack of RAG1 sequence for S. odoratus.

Within the unnamed clade including leucostomum, two radia-tions were well supported. The ‘‘northern’’ group (ML BP = 100%,BI PP = 100%) included the three parapatric taxa found on theAtlantic versant of Mexico, Guatemala, and Belize, whereas the‘‘southern’’ group (ML = 73%, BI = 94%) included the two disjunct,southern, small-plastroned species angustipons (Nicaragua toPanama) and dunni (Colombia), and the wide-ranging leucosto-mum (Veracruz to Peru; Iverson, 1992a), which is sympatric withthose two species as well as the southern members of the‘‘northern’’ clade. The remaining kinosternine species belong tothe restricted, well-supported (see above), monophyletic genusKinosternon, with a distribution across the USA and Mexico(where its diversity is greatest), but also into Central and SouthAmerica (Iverson, 1992a).

Within Kinosternon (sensu stricto) those taxa found in the cen-tral and eastern USA represent a well-separated monophyleticclade divergent from the other included taxa (ML = 88%,BI = 100%). The latter includes the previously recognized ‘‘scorpio-ides group’’ (as defined by Berry et al. (1997) and Iverson (1991)but excluding acutum, creaseri, chimalhuaca, and alamosae), whichis resolved as monophyletic (ML = 98% BI = 100%). However, therelationships among the members of this derived group are not re-solved with high support values (Fig. 1). On the contrary, the rela-tionships among nearly all of the other members of the restrictedgenus Kinosternon were consistently resolved.

3.2. Divergence estimates and biogeography

Program BEAST estimated the divergence of the three extantkinosternine clades at 22–25 mya (Fig. 2; Table S4), but most ofthe diversification of extant kinosternine species took place

Fig. 1. GARLI partitioned maximum likelihood tree for the Kinosternidae based on concatenated sequences of three nuclear and three mitochondrial genes. Numbers at nodesare bootstrap percentage support values (above: right, partitioned by codon position, and left, not so partitioned) and Bayesian posterior probability (below: right, partitionedby codon position, and left, not so partitioned). Asterisk indicates 100% value. Nodes lacking support values have 100% support for all four partitions. Abbreviations are PL(plastron), GSD (genetic sex determination), and TSD (temperature-dependent sex determination).

Table 2Summary of uncorrected p distances within and among proposed genera of kinosternid turtles based on the cyt b sequence data. Mean distance of samples between generaappears below diagonal; mean distance between samples within genera appears along diagonal. Ranges appear in parentheses. Full compilation of distances is in Table S3.

Claudius Staurotypus Cryptochelys Sternotherus Kinosternon

Staurotypus 9.0 (8.7–9.3) 1.4Cryptochelys 16.0 (15.1–17.3) 17.8 (17.0–18.3) 7.6 (1.2–10.6)Sternotherus 16.3 (16.1–16.5) 16.8 (15.6–17.6) 11.3 (10.0–13.2) 5.8 (0.0–8.3)Kinosternon 17.1 (15.7–18.2) 16.7 (14.9–17.7) 12.1 (9.0–14.3) 10.3 (8.7–12.4) 6.8 (2.0–9.8)

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between 5 and 10 mya. At the species level, only the divergence oftriporcatus from salvinii, acutum from creaseri and depressus fromminor were aged at less than 4 mya. The topology of the BEAST treeat the species level was identical to that in the ML and BI trees, ex-cept for the placement of S. odoratus.

Bayesian (BBM) and event based (S-DIVA) method reconstruc-tion of ancestral areas based on extant taxa produced conflictingresults for the basal nodes. The BBM method in RASP (Fig. 3) sup-ported a Mesoamerican origin (Node 69, marginal probabil-ity = 99%) for the family Kinosternidae during the late Cretaceous.In this reconstruction, the two subfamilies diverged within Meso-america (Node 68, marginal probability = 97%) during the Paleo-cene, with the Staurotypinae confined to Mesoamerica (Nodes 66and 67, marginal probability >99%). Mesoamerica is hypothesizedas the ancestral area for the extant Kinosterninae (Node 65, 84%)followed by the divergence of the Mesoamerican clade (the leuco-stomum group; Node 42, 65%) from the ancestor of the remainingKinosternon plus Sternotherus which is hypothesized to have dis-

persed into the Southeastern USA (Node 64, marginal probabil-ity = 87%). S-DIVA (Fig. S3) reconstructed the ancestral areas forthese nodes with much larger distributions, with support distrib-uted across a number of combinations including large areas ofthe current distribution of the Kinosternidae. This is not unex-pected due to the nature of the different methods; S-DIVA maxi-mizes vicariance and minimizes dispersal/extinction leading to apreference for larger ancestral areas (Yu et al., 2011). At almostevery node, BBM showed strong support for a much smaller geo-graphic area, typically one to at most three areas, while S-DIVA in-creased the number of areas from the tips to the base of the tree,with the basal node reconstructed as potentially including nearlythe entire geographic range the Kinosternidae. Predicted ancestralareas for the BBM analyses were not impacted by the maximumnumber of areas chosen (i.e., three, six, or twelve; latter notshown), whereas ancestral areas were strongly influenced by thenumber of areas for S-DIVA, increasing to the maximal setting(whether three, six or twelve; latter not shown).

Fig. 2. Relaxed clock model of divergence times for kinosternid turtles from program BEAST (see Methods). Shaded bars across nodes are 95% confidence intervals for age ofnode. Nodes are numbered for text reference (Section 4.3). Numerical estimates of ages of nodes are in Table S4. Calibration nodes are indicated by circled C.P + Q = Pliocene + Quaternary.

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4. Discussion

4.1. Taxonomic implications

Our analysis confirmed the monophyly of the two previouslyrecognized clades within the family Kinosternidae, one includingthe genera Staurotypus and Claudius, and the other includingSternotherus and Kinosternon. The fossil record dates the diver-gence of these two clades at P54 mya (see Section 2.3). Given(1) that the other North American turtle subfamilies date fromonly 34 mya (emydids) to 52 mya (geoemydids; Spinks and Shaf-fer, 2009), (2) that other speciose cryptodiran turtle families datefrom ca. 52 to 70 mya (Testudinidae and Geoemydidae), 60–90 mya (Emydidae), and 100–129 mya (Trionychidae) (Lourençoet al., 2012; Spinks and Shaffer, 2009; Wang et al., 2012), and(3) that the two subfamilies Staurotypinae and Kinosterninaeare unambiguously distinct morphologically (Hutchison, 1991)and in their sex-determining mechanisms (Fig. 1; Ewert et al.,2004), we follow Bickham and Carr (1983; among others) in rec-ognizing these two clades as separate families (Staurotypidaeand Kinosternidae; Table 3).

Our analysis also resolved three relatively old (22–25 my), dis-tinctly monophyletic clades within the restricted Kinosternidae(Fig. 2). The age of these clades exceeds the estimated ages of mostof the recognized genera in the other primarily North Americanradiation (the Emydidae) for which data are available. For example,Martin et al., 2013 provided the following estimates: Actinemys andEmys (ca. 7.5 mya), Emydoidea (ca. 12.5 mya), Glyptemys (20 mya),and Terrapene and Clemmys (ca. 21 mya); and Spinks and Shaffer(2009) dated the genera Trachemys and Graptemys at only ca.15 mya. Furthermore, mean cyt b uncorrected p distances betweenmembers of these three kinosternid clades range from 10.3% to12.3% (Table 1), generally exceeding distances found among spe-cies in the same genus (reviewed by Vargas-Ramirez et al., 2010).The three clades are also differentiated by at least their carinationand life style patterns (Fig. 1).

These data argue that the three kinosternid clades merit recog-nition as genera, but only two of the clades have previously been sonamed (Sternotherus and Kinosternon). Because members of thethird radiation have been known since at least 1831, but the dis-tinction of that clade has been unrecognized and undiagnosed untilnow, we here describe this ‘‘hidden’’, early, tropical radiation of theturtle subfamily Kinosterninae as a distinct genus (Table 3).

Family Kinosternidae Agassiz, 1857Tribe Kinosternini Hutchison, 1991Cryptochelys gen. nov.

Synonymy: Kinosternon Duméril and Bibron (in Duméril andDuméril, 1851: 17 (in part) (and nearly all subsequent authors).

Etymology: From the Greek, kruptos (cryptic, hidden) and chelus(tortoise, turtle). The genus is feminine, requiring a feminine suffixfor adjectival species names.

Type species: Kinosternon leucostomum Duméril and Bibron (inDuméril and Duméril 1851) [= Cryptochelys leucostoma].

Content: Cryptochelys acuta (Gray 1831), C. angustipons (Legler,1965), C. creaseri (Hartweg, 1934), C. dunni (Schmidt, 1947), C.herrerai (Stejneger, 1925), and C. leucostoma (Duméril and Bibronin Duméril and Duméril, 1851).

Diagnosis: Kinosternid (sensu stricto) turtles lacking an entopl-astron (present in Baltemys and Xenochelys), with reduced carina-tion (basically unicarinate; usually tricarinate in Baltemys,Xenochelys, Sternotherus, and Kinosternon, though nearly acarinatein some in the latter genus), a reduced neural series (typically fivebones, all posteriorly symmetric; six in C. creaseri) not in contactwith the nuchal bone (usually six with neural contact in otherkinosternids; Iverson, 1988b), the presence of clasping organs onthe posterior crus and thigh (except absent in C. acuta and C. crea-seri; also present in Sternotherus, but absent in many Kinosternon),the anterior end of the anterior musk duct groove reaching only tothe anterior half of the third peripheral (unknown for dunni; reach-ing to the second peripheral in Sternotherus and most Kinosternon,

Fig. 3. Graphical output from RASP (Reconstruct Ancestral State in Phylogenetics; see Section 2.4 for details). (A) Reconstruction of ancestral distributions at each node basedon the Bayesian Binary Method (BBM). (B) Color key to predicted ancestral ranges: A – South America, B – Central America (south of Mesoamerica, Honduras to Panama),C – Mesoamerica (defined as Isthmus of Tehuantepec to NW Honduras), D – Atlantic Mexico (Tampico embayment, Tamaulipas to northern Veracruz), E – Mexican Plateau,F – Pacific coastal Mexico (Sinaloa to Oaxaca), G – Northwest Mexico (Sonora), H – Southwest USA (Arizona), I – Central USA, J – Southeast USA, and K – Northeast USA. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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and to the first peripheral in Baltemys and Xenochelys), a gular scuteof intermediate width (much narrower in Sternotherus and usuallybroader in Kinosternon; see Appendix in Iverson, 1991), and dis-tinctive mitochondrial DNA.

Phylogenetic definition: All members of the Kinosternini moreclosely related to Cryptochelys leucostoma than to Sternotherus odo-ratus or Kinosternon flavescens.

Distribution: Atlantic versant of Mexico, Central, and extremenorthwestern South America, and Pacific versant of South Americafrom Panama to northern Peru.

Fossil history: Langebartel (1953) reported post-Pleistocene re-mains on the Yucatan that may represent C. creaseri, and Cadena

et al. (2007) described kinosternid fragments from the latePleistocene of Colombia that likely represent C. leucostoma (seeSection 4.3).

4.2. Phylogenetics

The relationships among the species of Cryptochelys have previ-ously been obscure, primarily because of the unavailability of tis-sues from rare taxa (especially angustipons, dunni, and creaseri).Iverson (1988a) first proposed that acuta and creaseri were sistertaxa, based on their parapatry and similar morphology and ecol-ogy. On morphological grounds, Legler (1965) suggested that

Table 3Explicit taxonomic changes recommended in this study.

Former name Proposed name

Staurotypinae StaurotypidaeKinosternon (leucostomum group) CryptochelysK. leucostomum C. leucostomaK. acutum C. acutaK. subrubrum steindachneri K. steindachneriK. scorpioides abaxillare K. abaxillare

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angustipons and dunni were sister taxa, and using combined mor-phology and preliminary mtDNA sequence data, Iverson (1998)first noted the close relationship of leucostoma and dunni, and ofacuta and herrerai. Our analysis is the first to clarify the relation-ships among the included taxa and the monophyly of this newgenus. However, more thorough geographic sampling is neededfor the wide-ranging species leucostoma, since preliminary mor-phological data indicate the existence of undescribed variation(Berry, 1978; Iverson, unpublished).

Within the genus Sternotherus the most primitive species hasbeen hypothesized to be S. carinatus (Zug, 1966) or S. odoratus(Iverson, 1998; Tinkle, 1958). Our ML and BI analyses supportedthe latter, though with relatively low support indices (Fig. 1), andour BEAST analysis placed odoratus as sister to carinatus (Fig. 2).These data suggest that the divergence of odoratus, carinatus, andminor (including depressus) may have been nearly simultaneous.S. depressus was consistently (ML BP = 100%, BI PP = 100%) foundto be sister to S. m. peltifer, from which it has long been assumedto have been derived (e.g., Iverson, 1977; Tinkle, 1958; Walkeret al., 1998b). The controversy concerning the recognition ofdepressus as a species or subspecies (Walker et al., 1998b) is stillan open question.

The restricted genus Kinosternon comprises two well-supportedclades, the ‘‘subrubrum group’’ (previously identified by Iverson(1998); including, baurii, flavescens, and subrubrum [includingsteindachneri], for which the name Thyrosternum Agassiz, 1857could be applied as a subgenus), and the remaining species. Withinthe former, the paraphyly of K. subrubrum (as previously recog-nized; e.g., Walker et al., 1998a) was fully supported in all analyses.This suggests that steindachneri may represent a distinct species(see also Bourque, 2012a). Our analysis also supports the conclu-sion that K. durangoense and K. arizonense, once considered subspe-cies of K. flavescens because of obvious morphological similarity(Iverson, 1979a, 1979b, 1989b), are independent radiations andhence, separate species (Serb et al., 2001).

With two exceptions, the relationships among the remainingmembers of the genus Kinosternon (sensu stricto) are not well re-solved. First, the ‘‘scorpioides group’’ (as defined by Berry et al.,1997, but excluding K. alamosae and K. chimalhuaca; Fig. 1 Node24) is supported with high confidence (ML BP = 98%; BIPP = 100%), although the four currently recognized (parapatric)subspecies of K. scorpioides were recovered as paraphyletic with re-spect to K. integrum and K. oaxacae in each analysis, with generallyweak support (e.g., Fig. 1). This suggests that K. scorpioides likelyrepresents a multispecies complex (see also Iverson, 2010), butmuch more complete geographic sampling will be necessary toclarify species boundaries in this clade. Future work should alsoreconsider the validity of the South American taxa currently syn-onymized under K. s. scorpioides (TTWG, 2012): carajascensis daCunha, 1970 in central Brazil and (especially) the disjunct serieiFreiberg, 1936 in Paraguay, Argentina, and Bolivia.

Second, samples of K. integrum from Colima, Puebla, and Oaxacawere resolved (together) as monophyletic with complete supportin all analyses. Webb (1984) suggested that integrum is polytypic,but confirmation of that will require much more thorough

geographic sampling, including comparisons with Pleistocene fos-sils from the Mexican Plateau (Cruz et al., 2009; Mooser, 1980).

Iverson (1981, 1998) hypothesized that the morphologicallysimilar, precisely parapatric K. sonoriense and K. hirtipes were sistertaxa. That relationship was resolved with reasonably high supportin this study (ML BP = 75%; BI PP = 100%), although additional sam-pling across both of these wide-ranging taxa is needed, particularlygiven the subspecific variation in morphology that has been de-scribed in both (Iverson, 1981).

The resolution of K. alamosae and K. chimalhuaca outside the K.scorpioides group is an enigma. All previous authors examining thegroup have concurred with their inclusion therein, and their closerelationship with K. integrum (Berry and Legler, 1980; Berry et al.,1997; Iverson, 1991, 1998), albeit on morphological grounds (e.g.,each share the assumed synapomorphy of the lack of clasping or-gans; Iverson, 1991). Since its description (Berry and Legler,1980), K. alamosae has been assumed to be most closely relatedto K. integrum, despite its general external similarity to K. arizon-ense (Iverson, 1989a).

The identification of K. chimalhuaca as sister to K. hirtipes withvery high support (87–98%) in all analyses is even more surprising.The range of K. chimalhuaca is completely within that of K. integrum(Berry et al., 1997), with which it is parapatric, and the nearestpopulation of K. hirtipes lies at least 100 km to the northeast, acrossat least two mountain ranges (with only integrum inhabiting thevalley between them). Genetic sampling across the range of inte-grum may help explain the puzzling resolution of alamosae andchimalhuaca in our analyses.

Although it is clear that species (or genus) boundaries shouldnot be based on such variable measures as uncorrected p distances,those values can still be useful as relative measures of taxonomicdivergence. For example, published values of p distances for thecyt b gene between species of Asian pond turtles, Palearctic tor-toises, and Asian and North American softshells range from about2.8% to 13.9% (review in Vargas-Ramirez et al., 2010).

With only a few exceptions, p distances among kinosternid spe-cies fall in the middle of this range or higher (Table S3), and min-imum distances between samples of the three proposed generawere approximately 9% and averaged 10–12% (Table 1). Interest-ingly, we found K. subrubrum steindachneri to be less divergentfrom the broadly sympatric K. baurii (4.2%) than it was from pur-portedly conspecific K. s. hippocrepis (6.8%); however, we did notsequence the more geographically proximate K. s. subrubrum. Nev-ertheless, these data do support Bourque’s (2012a) recommenda-tion that steindachneri be elevated to species status (Table 3). Inaddition, K. scorpioides abaxillare, isolated in the Central Valley ofChiapas, is >4% divergent from all other samples except the dis-tinctly different K. oaxacae (3.7%), including the supposedly con-specific K. s. cruentatum, K. s. albogulare, and K. s. scorpioides.Given its morphological distinction (Berry, 1978), its allopatry withother Kinosternon, and its genetic divergence, we recommend theelevation of abaxillare to species status. Unexpectedly, we founda distance of only 1.2% between the parapatric sister taxa C. creaseriand C. acuta, but because of their morphometric distinction (Iver-son, 1988a), we continue to support their retention as separatespecies.

4.3. Historical biogeography

Our analysis confirmed the reciprocal monophyly of the previ-ously recognized Staurotypinae and Kinosterninae (here recog-nized as separate families). The former has a meager fossil recordin Central America (Cadena et al., 2012), but is currently distrib-uted only in Mesoamerica, where it may initially have been iso-lated in sympatry with Cryptochelys (see below). When onlyconsidering extant taxa, the BBM analyses reconstructed a clean

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history for the group, originating in Mesoamerica, then expandingits range into North America and South America (Fig. 3). Given theevidence provided by the fossil record, the early biogeographic his-tory for this group must be more complex and may be more closelyaligned with the prediction of S-DIVA (Fig. S3). For example, aqua-tic fossil kinosternids (Baltemys and Xenochelys) are hypothesizedto have occurred all over North America in the late Paleocene, sinceEocene kinosternine fossils are known from at least 23 sites fromFlorida to Arkansas to Texas to New Mexico to Wyoming to north-ern Canada (33–55 mya; Bourque, 2012c; Eberle and Greenwood,2012; Hutchison, 1991). Indeed, maximum known Cenozoic turtlediversity in at least western North America was in the Wasatchianof the early Eocene (50–55 mya; Hutchison, 1982; Zachos et al.,2001). Those early kinosternids (with an entoplastron and a smallnon-hinged plastron) likely diversified across the mid-continentduring the warm Eocene (Zachos et al., 2001) as noted above. How-ever, during the cooling period in the late Eocene and early Oligo-cene, the group likely survived only in the more tropical regions inMeso- and Central America (and Florida; see Bourque, 2012c), ashypothesized for other reptiles and amphibians (Savage, 2002; Leand McCord 2008). The well-documented decline in aquatic turtlediversity in at least western North America in the early Oligocene,between 33 and 28 mya (Corsini et al., 2011; Hutchison, 1992,2005), was presumably due to these cooling and drying conditions(Zachos et al., 2001). Hence, the hypothesized ancestral area for therestricted family Kinosternidae in Mesoamerica (Fig. 3) is actually arelict distribution compared to its former wide distribution andhigh diversity in the Paleocene and Eocene.

The Oligocene Mesoamerican relict kinosternids apparently re-tained the tricarinate shell and small plastron (but lost the entopl-astron) of Baltemys and Xenochelys, and were presumably aquatic.But when the climate warmed in the mid to late Oligocene (ca.28 mya), the group apparently dispersed both northward andsouthward. The common ancestor of Sternotherus and the re-stricted Kinosternon (node 10; Fig. 2) is hypothesized to have dis-persed north and east along the Gulf Coast, where one subclade,primarily aquatic, with a reduced plastron, diverged as the genusSternotherus. The second subclade (Kinosternon sensu stricto), witha more extensive plastron, dispersed westward and apparentlyadapted to the increased seasonal aridity in North America at thattime (Hay, 1908:39; Zachos et al., 2001; as evidenced by the signif-icant transition in general turtle diversity from primarily aquatic toprimarily terrestrial; Hutchison, 1982, 1992, 2005). The thirdgroup (Cryptochelys) remained tropical and dispersed into CentralAmerica (i.e., to the new Chortis Block; Savage, 2002).

During the warming and drying of the early Miocene (20–25 mya; Woodburne, 2004), the Mesoamerican Cryptochelys cladeis hypothesized to have dispersed northward and southward andthe ancestral range divided (node 5; ca. 18 mya) by an unknownvicariant event into a northern element (to become herrerai, crea-seri, and acuta) and a southern element (leucostoma/dunni/angusti-pons). This was likely the same event that accounted for thedivergence of the 17 mya fossil Staurotypus moschus in Panamafrom its Mesoamerican congeners (Cadena et al., 2012). From Cen-tral America, Cryptochelys twice dispersed into South America,once by C. leucostoma and once by ancestral C. dunni (Fig. 2). Unfor-tunately, timing of the dispersal of the first clade will require fu-ture extensive geographic sampling across the range of C.leucostoma. However, based on our chronogram (Fig. 2), the seconddispersal must have occurred by about 8 mya (Node 7), well beforethe closure of the Panamanian portal (ca. 2.5–4 mya; Coates andObando, 1996; Iturralde-Vinent, 2006; Iturralde-Vinent and Mac-Phee, 1999; Kirby et al., 2008; Woodburne, 2004). It is noteworthythat an increasing list of reptile and amphibian taxa (among oth-ers; Cody et al., 2010) show evidence of dispersal to South Americain the mid- to late Miocene (before portal closure): Rhinoclemmys,

20–22 mya (Le and McCord, 2008); Bolitiglossa, 18 mya (Hankenand Wake, 1982); Lachesis, 6–18 mya (Zamudio and Greene,1997); Apalone, >5 mya (Head et al., 2006); and others might alsohave done so (e.g., the turtles Chelydra, Trachemys, and Cryptochelysleucostoma).

Within Sternotherus (node 11; Fig 2), one species (odoratus)evolved as an aquatic habitat generalist (perhaps primarily north-ern in distribution), whereas the others (minor/carinatus) adaptedto the permanent rivers and streams of the Gulf Coast of southeast-ern North America. Based primarily on the mammalian fauna,Webb et al. (1981) reported that the ‘‘Gulf Coast seems to havebeen a separate biotic province during most of the Miocene’’, andthe carinatus/minor complex apparently diverged there during thattime. One member of that group (carinatus, in the western Gulf) be-came the most specialized with its high, tent-like carapace. Theeastern forms retained lower, basically tricarinate shells (node13), with the population in the upper Black Warrior River in Ala-bama (depressus) only recently evolving its very low shell. Curi-ously, the oldest fossil record for Sternotherus is only ca. 7 mya(Bourque, 2011).

The ancestral restricted genus Kinosternon is hypothesized tohave originated in eastern North America between 17 and22 mya, but must have dispersed westward as far as southwesternNorth America (Sonora, according to the BBM) by 14–17 mya, be-cause fossil fragments of Kinosternon ranging in age from ca. 14to 18 mya are known from Delaware, extreme northern Florida,Nebraska, and New Mexico (Bourque, 2012a, 2012b; Holman,1998; Hutchison, 1991). This ancestor must have been flavescens-like in morphology and ecology (e.g., reduced carination and semi-aquatic), given the retention of those traits in both descendentclades within Kinosternon (baurii, subrubrum, flavescens, arizonense,and durangoense; Iverson, 1991; Serb et al. 2001), and the presenceof that general morphology in the two New Mexico Miocene spe-cies, K. pojoaque (ca. 14 mya; Bourque, 2012a) and K. skullridges-cens (ca. 15 mya; Bourque, 2012b).

Within the restricted genus Kinosternon, because of a vicariantevent ca. 17 mya (node 14; perhaps the continued faulting of theBasin and Range province (Woodburne, 2004) coupled with warm,dry conditions in the early Miocene), the clade in the central andeastern USA (the ‘‘subrubrum group’’, also including flavescens, bau-rii, and steindachneri) diverged from the clade of other Kinosternonin the southwestern USA and northern and central Mexico.Although the zoogeographic history of the latter clade was poorlyresolved in both the RASP analyses, the scorpioides group appar-ently dispersed via the Pacific coast into southeastern Mexico,through Central America, into sympatry with Cryptochelys, andeventually into South America.

Amazingly, this scorpioides group is currently distributed fromSonora and Tamaulipas in Mexico southward to Argentina. Andalthough our sampling of the group is incomplete geographically,our results suggest that this group dispersed from coastal Mexicoto at least northern South America (based on our Venezuelan scor-pioides sample) in only about 2 million years (compare nodes 21and 24), by ca. 7 mya, and well before the closure of the Panama-nian portal (references above). K. scorpioides was thus the thirdkinosternid clade to disperse into northern South America, whereit dispersed to the Atlantic versant (as did Trachemys; e.g. Iverson,1992a), while the genus Cryptochelys dispersed primarily to the Pa-cific versant (as did Chelydra; e.g., Iverson 1992a). The absence ofCryptochelys in Atlantic drainages beyond Colombia, and the gener-alized morphology and ecology of K. scorpioides, likely facilitatedthe latter’s very rapid dispersal southward across the Amazon ba-sin to the more seasonally dry conditions of Bolivia, Paraguay, andArgentina. Soon after this extensive dispersal (i.e., beyond node24), and despite the evident vagility of the ancestral form, tectonicand/or climatic events apparently isolated allopatric populations

J.B. Iverson et al. / Molecular Phylogenetics and Evolution 69 (2013) 929–939 937

along coastal Oaxaca (oaxacae), possibly coastal Colima/Jalisco(chimalhuaca; see above), the Central Valley of Chiapas (abaxillare),South America (scorpioides), and the remainder of Mesoamericaand Central America (cruentatum/albogulare), each of which di-verged at least morphologically. Clearly, the scorpioides group rep-resents the most successful and rapid radiation in this family, anddeserves much more complete genetic sampling to clarify its phy-logeny and taxonomy.

4.4. Morphological implications

Within the Kinosternidae, the plastron is much reduced (withreduced kinesis) in several taxa, including all Sternotherus, Crypt-ochelys herrerai, C. angustipons, and some populations of Kinoster-non hirtipes (Iverson, 1991). Bramble et al. (1984) hypothesizedthat Sternotherus and C. herrerai evolved first among the kinoster-nids, and that anterior plastral lobe kinesis was ancestral, withhindlobe kinesis evolving later as an adaptation to terrestriality.That scenario would require the independent evolution of dualkinesis at least twice in the family (in Cryptochelys and inKinosternon).

Second, although the lack of clasping organs has previouslybeen considered a synapomorphy defining the scorpioides group(Berry and Legler, 1980; Iverson, 1991), it seems more likely thatclasping organs were lost independently at least twice (in the C.acuta/creaseri clade and the K. scorpioides clade). In addition, theirabsence in alamosae and chimalhuaca, seemingly outside the lattergroup, may be additional independent evolutionary events. Thor-ough character reconstruction studies will be necessary to evaluatethe evolutionary history of these and other key kinosternid traits.

4.5. Concluding thoughts

Despite the resolution of many of the relationships among thekinosternids, and the establishment of a reasonable chronologyof diversification, several key evolutionary questions remain. Be-cause most of the diversification within the genus Kinosternon(s.s.) occurred within the last 10 my (especially within the scorpio-ides group), we were not able to resolve their relationships withhigh confidence. Doing so will require sampling additional geneticmarkers, as well as much more thorough sampling across the dis-tributions of such wide-ranging taxa as K. scorpioides, K. integrum,and Cryptochelys leucostoma. Such work is certain to uncover addi-tional unappreciated variation in these groups.

Second, although the fossil record for Kinosternon (s.s.) datesfrom the Miocene (16–18 mya; see above), older fossil crowngroup kinosternids (to perhaps 22 mya) must exist. But even moresurprising is the lack of fossils older than 7 my for the genusSternotherus (Bourque, 2011) and only 0.5 my for the genus Crypt-ochelys (Cadena et al. 2007). Significant fossils likely remain to bediscovered for this group, particularly in the eastern USA andMesoamerica.

Acknowledgments

Tissue samples were generously provided by T.S. Akre, R.J.Burke, J. Campbell, J.C. Carr, O. Victoria Castano, C.R. Etchberger,M. Ewert, M.J. Forstner, C.J. Franklin, M. Gaston, D. Gicca, D. Greene,S. Guzman, D.R. Jackson, M. Klemens, K.L. Krysko, B. Lamar, J.E. Lo-vich, R.E. Lovich, K. Marion, C. May, W.P. McCord, F. Medem, P.Meylan, P. Moler, S. Pasachnik, C. Phillips, S. Platt, S. Poulin, J. ReyesVelasco, E. Rickart, P. Rosen, J. Serb, E. Smith, N. Soule, P. Stone, P.Vander Schouw, T. Tuberville, W. Van Devender, T.R. Van Devender,and G. Weatherman. Critical literature was provided by D. Brink-man and J. Bourque. P. Meylan accompanied Iverson during fieldwork in Mexico and American Southwest, and his expert field

assistance was critical to early work. Financial support for this pro-ject was provided by the American Philosophical Society, theAmerican Museum of Natural History, the Joseph Moore Museumof Natural History at Earlham College, and Grant 106.15-2010.30from Vietnam’s National Foundation for Science and TechnologyDevelopment (NAFOSTED) to ML. Comments on early drafts ofthe manuscript by R. Bour, J. Bourque, S. Pasachnik, M.E. Seidel,and two anonymous reviewers were greatly appreciated.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2013.06.011.

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