DSpace at VNU: Molecular phylogenetics of the mud and musk turtle family Kinosternidae

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Accepted Manuscript Molecular phylogenetics of the mud and musk turtle family Kinosternidae John B Iverson, Minh Le, Colleen Ingram PII: DOI: Reference: S1055-7903(13)00251-0 http://dx.doi.org/10.1016/j.ympev.2013.06.011 YMPEV 4635 To appear in: Molecular Phylogenetics and Evolution Received Date: Revised Date: Accepted Date: 25 November 2012 15 May 2013 18 June 2013 Please cite this article as: Iverson, J.B., Le, M., Ingram, C., Molecular phylogenetics of the mud and musk turtle family Kinosternidae, Molecular Phylogenetics and Evolution (2013), doi: http://dx.doi.org/10.1016/j.ympev 2013.06.011 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Molecular phylogenetics of the mud and musk turtle family Kinosternidae a* b,c,d d John B Iverson , Minh Le , Colleen Ingram a Department of Biology, Earlham College, Richmond, Indiana 47374 United States Faculty of Environmental Sciences, Hanoi University of Science, 334 Nguyen Trai Road, Hanoi, Vietnam c Centre for Natural Resources and Environmental Studies, Vietnam National University, 19 Le Thanh Tong Street, Hanoi, Vietnam d Department of Herpetology, American Museum of Natural History, Central Park West at 79th Street, New York 10024 United States [Present address: Department of Biology, University of Virginia, Charlottesville, Virginia 22904 United States] b ABSTRACT The turtle family Kinosternidae comprises 25 living species of mud and musk turtles confined to the New World Previous attempts to reconstruct a phylogenetic history of the group have employed morphological, isozyme, and limited mitochondrial DNA sequence data, but have not been successful in producing a well-resolved phylogeny With tissues from every recognized species and most subspecies, we sequenced three mitochondrial (cyt b, 12S, 16S) and three nuclear markers (C-mos, RAG1, RAG2) Our analyses revealed the existence of three wellresolved clades within the Kinosterninae (aged > 22 mya), only two of which have been named: Sternotherus and Kinosternon We here describe the third clade as a new genus The evolutionary relationships among most species were well resolved, although those belonging to the K scorpioides species group will require more extensive geographic and genetic sampling Divergence time estimates and ancestral area reconstructions permitted the development of the first rigorous hypothesis of the zoogeographic history of the group, including support for three separate dispersals into South America, at least two of which preceded the closure of the Panamanian portal Key words: Claudius, DNA, evolution, Kinosternon, Staurotypus, Sternotherus *Corresponding author Phone 765-983-1045; FAX +1 765 983 1497 E-mail address: johni@earlham.edu (J B Iverson) Iverson, Le, and Ingram Introduction The turtle family Kinosternidae includes 25 recognized extant aquatic to semiaquatic species (38 taxa including subspecies) distributed in the New World from Canada to Argentina (Iverson, 1992a; TTWG, 2012) Although the greatest living diversity is in Mesoamerica (Iverson, 1992a, 1992b), fossil taxa are most diverse in the Eocene and Oligocene of Wyoming and South Dakota (Hutchison, 1991) Two subfamilies, the Staurotypinae (including the genera Staurotypus and Claudius) and the Kinosterninae (including Kinosternon and Sternotherus), have been generally recognized as distinct, with some authors elevating them to the family level (Bickham and Carr, 1983; Vetter, 2005) Referred to as “mud” or “musk” turtles, members of this family are small, secretive, malodorous, and generally non-descript (Bonin et al., 2006; Schilde, 2001; Vetter, 2005) As a result of this combination of traits, recognition of the living species diversity in the family has been delayed compared to most other turtle families For example, ten species (40% of total) and subspecies (of 13 total) have been described since 1922 (TTWG, 2012), with one or two species described each decade since then Furthermore, because of a paucity of meristic characters and the existence of significant (though drab) color variation within and among populations, species boundaries have often been difficult to establish (e.g., Bourque, 2012c; Iverson, 2010; Lamb and Lovich, 1990; Serb et al., 2001) Nonetheless, undescribed taxonomic diversity is suspected to exist (Webb, 1984; Iverson, unpublished) The family Kinosternidae also exhibits a stunning diversity of life history traits when compared to other turtle families It ranges from north temperate to tropical habitats, and from rain forest to grasslands to desert (Bonin et al., 2006) It includes totally aquatic to semiterrestrial species, with adult carapace lengths of 10 to 38 cm (Bonin et al., 2006), and femaledominated to male-dominated sexual size dimorphism (Ceballos et al 2103) At least one species exhibits close to the maximum skeletal mass relative to body mass among all vertebrates (Iverson, 1984) Some species have a greatly reduced plastron, whereas others have a plastron so extensive as to completely close the shell (Hutchison, 1991) The group includes members capable of submerged, fully aquatic respiration (Belkin, 1968), and others capable of estivating underground for up to two years (Rose, 1980) Some species produce a single clutch in the spring, others nest multiple times in the summer, and others nest nearly year-round (Iverson, 2010), with clutches ranging from one or two relatively huge eggs to ten or more relatively tiny eggs (Iverson, 1999) Embryonic development is direct in some species, whereas others exhibit early embryonic diapause and/or late embryonic estivation, with incubation times from 56 to over 366 days (Ewert, 1991) Finally, sex determination in the family ranges from genetic (with sex chromosomes) to temperature-dependent (Ewert et al., 2004) Unfortunately, understanding the evolution of these diverse traits has been impeded by the lack of a well-resolved phylogeny for the group Published phylogenetic hypotheses to date have been based on morphology (Iverson, 1991), protein electromorph data (Iverson, 1991; Seidel et al., 1986), and small segments of the mitochondrial genome, using limited taxonomic sampling (Iverson, 1998; Serb et al., 2001) None employed complete taxonomic sampling or nuclear markers, nor applied modern phylogenetic methods Hence, the phylogenetic structure for the family is not well-resolved (e.g., see Iverson, 1998) To solve this deficiency, we sequenced mitochondrial (cyt b, 12S, and 16S) and nuclear (C-mos, RAG1, and RAG2) markers from representatives of every recognized species and most subspecies in this family (a total of 34 samples) Contemporary phylogenetic methods were used to test previous hypotheses regarding the relationships among the known Iverson, Le, and Ingram kinosternid turtles and to direct future research in clarifying cryptic diversity in the family In addition, the recovered phylogeny was calibrated using known fossils to permit a reconstruction of the zoogeographic history of the family, particularly the timing of its multiple dispersals into South America Materials and methods 2.1 Sampling and laboratory methods All 25 species of the family Kinosternidae were included in the study (Table S1) Outgroup polarity was provided by the sister family Dermatemydidae (Barley et al., 2010; Krenz et al., 2005; Near et al., 2005) We sequenced three regions of the mitochondrial genome, the complete cytochrome b (cyt b) sequence, and the partial 12S and 16S rRNA genes (1958 total aligned bp) We also sequenced three nuclear fragments of the C-mos, RAG1, and RAG2 genes (2553 total aligned bp) Primers used for this study are listed in Table S2 DNA was extracted from tissues and blood samples using the DNeasy kit (QIAGEN, Valencia, CA, USA) following manufacturer’s instructions for animal tissues We also extracted DNA from bone samples using the procedures described in Le et al (2007) PCR volume consisted of 30µl (9µl of water, 2µl of each primers, 15µl of HotStar Taq Master Mix (QIAGEN, Valencia, CA, USA), and 2µl of diluted DNA) PCR conditions for the mitochondrial genes were: 95°C for 15 to activate the Taq; with 42 cycles at 95°C for 30 s, 45°C for 45 s, 72°C for 60 s; and a final extension of 72°C for Nuclear DNA was amplified using the same PCR conditions, while the annealing temperatures were 52°C for RAG1 and the second fragment of RAG2, 56°C for the first fragment of RAG2, and 58°C for C-mos PCR products were visualized using electrophoresis through a 2% low melting-point agarose gel (NuSieve GTG, FMC Biopolymers) stained with ethidium bromide and/or Safe DNA (SYBR®) PCR products were cleaned using PerfectPrep® PCR Cleanup 96 plate (Eppendorf Scientific Inc., Hamburg, Germany) and cycle sequenced 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 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) 2.2 Phylogenetic analyses DNA sequences were edited, checked for ambiguities, and aligned using Geneious v5.4 (Drummond et al., 2011) For coding regions, alignments were refined by eye to translated sequences to confirm reading frame conservation and checked for premature stop 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 partitioned into first, second, and third position jModelTest 2.1.1 (Darriba et al., 2012) was used to determine the appropriate model of sequence evolution for each partition for all model-based analyses The best models were GTR + G + I for the mtDNA regions 16S 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 second positions of C-mos; GTR for the third position of C-mos and first and second positions of RAG1; GTR + I for the third position of RAG1; HKY+G for the first and second positions of RAG2; and HKY for the third position of RAG2 For the ML analyses, four independent searches were performed on the concatenated mtDNA, nuDNA, and combined datasets with partitions modeled Iverson, Le, and Ingram separately All other parameters of GARLI were left at the default settings The four independent searches were compared to confirm that the heuristic searches were converging on the same likelihood and topology, and the topology with the highest likelihood value was considered the best tree 100 non-parametric ML bootstrap replicates were examined to determine support for each node Bayesian posterior probabilities were calculated using the Metropolis-coupled Markov chain Monte Carlo (MC3) sampling approach in MrBayes v3.01 (Ronquist et al., 2012) Four independent searches were performed for each dataset; each search consisted of a cold chain and heated chains All searches started with random trees and uniform prior probabilities for all possible trees For all datasets, the original Markov chains were run for X107 generations and trees were sampled every 10000 generations To determine that stationarity had been reached, we compared both the fluctuating values of the likelihood from the four independent searches using TRACER v1.4 (Rambaut and Drummond, 2007) and convergence rates of posterior split probabilities and branch lengths using AWTY (Nylander et al., 2008) The “burn-in” value was conservatively set at 1,000; the first 1,000 (10,000,000 generations) trees were eliminated from the approximation of posterior probabilities The trees retained from each run were combined and a 50% majority rule consensus tree was produced The ML trees from each independent GARLI and MrBayes search were compared The likelihood scores from the “best tree” recovered using GARLI and the ML tree from each MrBayes search were optimized using PAUP* for comparison of ML and topology testing Uncorrected p distances between each sample pair were calculated for the cyt b sequence 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 implemented in the program BEAST v.1.7.5 (Drummond and Rambaut, 2007), using the subroutine BEAUti v1.7.5 to set the analysis parameters The model of evolution for partitions that fit GTR were conservatively set to HKY This was only after initial runs using GTR had low ESS values for prior and posterior, with one of the relative rates in each of the GTR modeled partition going to zero Reducing the model to HKY fixed this issue without any change in results Two calibration points were used The minimum age for the divergence between the Dermatemydidae and Kinosternidae was constrained to 74.8 mya, based on stem kinosternid fossils from the lower third of the Kaiparowits Formation (Brinkman and Rodriguez De La Rosa, 2006; Brinkman et al in press; Hutchison et al in press; Knauss et al., 2011) Hutchison (1991) described the oldest known kinosternine fossils from the early Eocene (Lysitian), and suggested the divergence of the subfamilies Kinosterninae and Staurotypinae at between 56 and 65 mya For our analysis we constrained the minimum age of the Kinosterninae/Staurotypinae split to 53 mya based on Koch and Morrill (2000) In each analysis, the Markov chain was run for 100 million generations and sampled every 10,000 Convergence to stationarity was checked using TRACER v 1.5 (Rambaut and Drummond, 2007) for each search and compared across all runs The search results were summarized using 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 were reconstructed using both the Statistical Dispersal-Vicariance Analysis (S-DIVA) and the Bayesian Binary method (BBM) implemented in RASP (Reconstruct Ancestral State in Phylogenies: Yu et al., 2011) We used a Iverson, Le, and Ingram time-calibrated phylogeny that included representatives from all 25 species of Kinosternidae and a single outgroup (Dermatemys) We coded eleven geographical areas: A -South America, B Central America (south of Mesoamerica, Honduras to Panama), C - Mesoamerica (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 Although more fine scale coding is possible, we find this simplistic matrix more appropriate for the questions at hand, rather than making data overly complex and over-parameterized, exceeding their explanatory value Because most sampled taxa occupied only one or two areas, and only one occupied three (leucostomum), we set the maximum number of areas for reconstruction in both S-DIVA and BBM at three Results 3.1 Phylogenetic analyses The final data matrix contained 4511 aligned characters, and among the six markers, cyt b and 16S were the most phylogenetially informative (Table 1) Phylogenies estimated for each locus were variable in the amount of resolution; mitochondrial gene trees were resolved with support while the nuclear gene trees showed variable and poor resolution distributed across the tree (not shown) Phylogenies based on the combined nuclear data also demonstrated minimal resolution (e.g., Fig S1), whereas those based on the combined mitochondrial sequences were almost fully resolved (e.g., Fig S2) and nearly identical to those based on the combined nuclear and mitochondrial data set While these datasets varied in the amount of phylogenetic information, comparisons not reveal any major conflict; areas that were not completely congruent among trees were restricted to nodes involving short branches and/or weak bootstrap support, and therefore we focused on the results from the total concatenated dataset The four different analyses (ML and BI, codon and non-codon-based) recovered almost identical topologies, differing only in node support values (Fig 1) The two previously recognized subfamilies, Staurotypinae and Kinosterninae (TTWG, 2012), were recovered as monophyletic in all analyses (ML bootstrap [BP] = 100%, BI posterior probability [PP] = 100%; only codon-based support values reported in the text, but see Fig 1) Within the Kinosterninae, three major clades were resolved with high support (ML BP = 8997%, BI PP = 100%), although the relationship among the three clades was not well resolved (ML BP 99%) Mesoamerica is hypothesized as the ancestral area for the extant Kinosterninae (Node 65, 84%) followed by the divergence of the Mesoamerican clade (the leucostomum group; Node 42, 65%) from the ancestor of the remaining Kinosternon plus Sternotherus which is hypothesized to have dispersed into the Southeastern USA (Node 64, marginal probability = 87%) S-DIVA (Fig S3) reconstructed the ancestral areas for these nodes with much larger distributions, with support distributed across a number of combinations including large areas of the current distribution of the Kinosternidae This is not unexpected due to the nature of the different methods; S-DIVA maximizes vicariance and minimizes dispersal/extinction leading to a preference for larger ancestral areas (Yu et al., 2011) At almost every node, BBM showed strong support for a much Iverson, Le, and Ingram smaller geographic area, typically one to at most three areas, while S-DIVA increased the number of areas from the tips to the base of the tree, with the basal node reconstructed as potentially including nearly the entire geographic range the Kinosternidae Predicted ancestral areas for the BBM analyses were not impacted by the maximum number of areas chosen (i.e., three, six, or twelve; latter not shown), whereas ancestral areas were strongly influenced by the number of areas for S-DIVA, increasing to the maximal setting (whether three, six or twelve; latter not shown) Discussion 4.1 Taxonomic implications Our analysis confirmed the monophyly of the two previously recognized clades within of the family Kinosternidae, one including the genera Staurotypus and Claudius, and the other including Sternotherus and Kinosternon The fossil record dates the divergence of these two clades at >54 mya (see Section 2.3) Given 1) that the other North American turtle subfamilies date from only 34 mya (emydids) to 52 mya (geoemydids; Spinks and Shaffer, 2009), 2) that other speciose cryptodiran turtle families date from ca 52 to 70 mya (Testudinidae and Geoemydidae), 60 to 90 mya (Emydidae), and 100-129 mya (Trionychidae)(Lourenỗo et al., 2012; Spinks and Shaffer, 2009; Wang et al., 2012), and 3) that the two subfamilies Staurotypinae and Kinosterninae are 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 recognizing these two clades as separate families (Staurotypidae and Kinosternidae; Table 3) Our analysis also resolved three relatively old (22-25 my), distinctly monophyletic clades within the restricted Kinosternidae (Fig 2) The age of these clades exceeds the estimated ages of most of the recognized genera in the other primarily North American radiation (the Emydidae) for which data are available For example, Martin et al (2013) provided the following estimates: Actinemys and Emys (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 between members of these three kinosternid clades range from 10.3 to 12.3% (Table 1), generally exceeding distances found among species in the same genus (reviewed by VargasRamirez et al., 2010) The three clades are also differentiated by at least their carination and life style patterns (Fig 1; see also below) These data argue that the three kinosternid clades merit recognition as genera, but only two of the clades have previously been so named (Sternotherus and Kinosternon) Because members of the third radiation have been known since at least 1831, but the distinction of that clade has been unrecognized and undiagnosed until now, we here describe this “hidden”, early, tropical radiation of the turtle subfamily Kinosterninae as a distinct genus (Table 3) Family Kinosternidae Agassiz 1857 Tribe Kinosternini Hutchison 1991 Cryptochelys gen nov Synonymy: Kinosternon Duméril and Bibron (in Duméril and Duméril 1851:17 (in part)[and nearly all subsequent authors] Iverson, Le, and Ingram Etymology: From the Greek, kruptos (cryptic, hidden) and chelus (tortoise, turtle) The genus is feminine, requiring a feminine suffix for adjectival species names Type species: Kinosternon leucostomum Duméril and Bibron (in Duméril 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 Bibron in Duméril and Duméril 1851) Diagnosis: Kinosternid (sensu stricto) turtles lacking an entoplastron (present in Baltemys and Xenochelys), with reduced carination (basically unicarinate; usually tricarinate in Baltemys, Xenochelys, Sternotherus, and Kinosternon, though nearly acarinate in some in the latter genus), a reduced neural series (typically five bones, all posteriorly symmetric; six in C creaseri) not in contact with the nuchal bone (usually six with neural contact in other kinosternids; Iverson, 1988b), the presence of clasping organs on the posterior crus and thigh (except absent in C acuta and C creaseri; also present in Sternotherus, but absent in many Kinosternon), the anterior end of the anterior musk duct groove reaching only to the anterior half of the third peripheral (unknown for dunni; reaching to the second peripheral in Sternotherus and most Kinosternon, and to the first peripheral in Baltemys and Xenochelys), a gular scute of intermediate width (much narrower in Sternotherus and usually broader in Kinosternon; see Appendix in Iverson, 1991), and distinctive mitochondrial DNA Phylogenetic definition: All members of the Kinosternini more closely related to Cryptochelys leucostoma than to Sternotherus odoratus or Kinosternon flavescens Distribution: Atlantic versant of Mexico, Central, and extreme northwestern South America, and Pacific versant of South America from Panama to northern Peru Fossil history: Langebartel (1953) reported post-Pleistocene remains on the Yucatan that may represent C creaseri, and Cadena et al (2007) described kinosternid fragments from the late Pleistocene of Colombia that likely represent C leucostoma (see 4.3 below) 4.2 Phylogenetics The relationships among the species of Cryptochelys have previously been obscure, primarily because of the unavailability of tissues from rare taxa (especially angustipons, dunni, and creaseri) Iverson (1988a) first proposed that acuta and creaseri were sister taxa, based on their parapatry and similar morphology and ecology On morphological grounds, Legler (1965) suggested that angustipons and dunni were sister taxa, and using combined morphology and preliminary mtDNA sequence data, Iverson (1998) first noted the close relationship of leucostoma and dunni, and of acuta and herrerai Our analysis is the first to clarify the relationships among the included taxa and the monophyly of this new genus However, more thorough geographic sampling is needed for the wide-ranging species leucostoma, since preliminary morphological data indicate the existence of undescribed variation (Berry, 1978; Iverson, unpublished) Within the genus Sternotherus the most primitive species has been hypothesized to be S carinatus (Zug, 1966) or S odoratus (Iverson, 1998; Tinkle, 1958) Our ML and BI analyses supported the latter, though with relatively low support indices (Fig 1), and our BEAST analysis placed odoratus as sister to carinatus (Fig 2) These data suggest that the divergence of odoratus, carinatus, and minor (including depressus) may have been nearly simultaneous S depressus was consistently (ML BP = 100%, BI PP = 100%) found to be sister to S m peltifer, from which it has long been assumed to have been derived (e.g., Iverson, 1977; Tinkle, 1958; Iverson, Le, and Ingram Walker et al., 1998b) The controversy concerning the recognition of depressus as a species or subspecies (Walker et al., 1998b) is still an open question The restricted genus Kinosternon comprises two well-supported clades, the “subrubrum group” (previously identified by Iverson, 1998; including, baurii, flavescens, and subrubrum [including steindachneri], for which the name Thyrosternum Agassiz 1857 could be applied as a subgenus), and the remaining species Within the former, the paraphyly of K subrubrum (as previously recognized; 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 conclusion that K durangoense and K arizonense, once considered subspecies of K flavescens because of obvious morphological similarity (Iverson, 1979a, 1979b, 1989b), are independent radiations and hence, separate species (Serb et al., 2001) With two exceptions, the relationships among the remaining members of the genus Kinosternon (sensu stricto) are not well resolved First, the “scorpioides group” (as defined by Berry et al., 1997, but excluding K alamosae and K chimalhuaca; Fig Node 24) is supported with high confidence (ML BP = 98%; BI PP = 100%), although the four currently recognized (parapatric) subspecies of K scorpioides were recovered as paraphyletic with respect to K integrum and K oaxacae in each analysis, with generally weak support (e.g., Fig 1) This suggests that K scorpioides likely represents a multispecies complex (see also Iverson, 2010), but much more complete geographic sampling will be necessary to clarify species boundaries in this clade Future work should also reconsider the validity of the South American taxa currently synonymized under K s scorpioides (TTWG, 2012): carajascensis da Cunha 1970 in central Brazil and (especially) the disjunct seriei Freiberg 1936 in Paraguay, Argentina, and Bolivia Second, samples of K integrum from Colima, Puebla, and Oaxaca were resolved (together) as monophyletic with complete support in all analyses Webb (1984) suggested that integrum is polytypic, but confirmation of that will require much more thorough geographic sampling, including comparisons with Pleistocene fossils from the Mexican Plateau (Cruz et al., 2009; Mooser, 1980) Iverson (1981, 1998) hypothesized that the morphologically similar, precisely parapatric K sonoriense and K hirtipes were sister taxa That relationship was resolved with reasonably high support in this study (ML BP = 75%; BI PP = 100%), although additional sampling across both of these wide-ranging taxa is needed, particularly given the subspecific variation in morphology that has been described in both (Iverson, 1981) The resolution of K alamosae and K chimalhuaca outside the K scorpioides group is an enigma All previous authors examining the group have concurred with their inclusion therein, and their close relationship 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 organs; Iverson, 1991) Since its description (Berry and Legler, 1980), K alamosae has been assumed to be most closely related to K integrum, despite its general external similarity to K arizonense (Iverson, 1989a) The identification of K chimalhuaca as sister to K hirtipes with very 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 nearest population of K hirtipes lies at least 100 km to the northeast, across at least two mountain ranges (with only integrum inhabiting the valley between them) Genetic sampling across the range of integrum may help explain the puzzling resolution of alamosae and chimalhuaca in our analyses Although it is clear that species (or genus) boundaries should not be based on such variable measures as uncorrected p distances, those values can still be useful as relative measures of Iverson, Le, and Ingram 13 character reconstruction studies will be necessary to evaluate the evolutionary history of these and other key kinosternid traits 4.5 Concluding thoughts Despite the resolution of many of the relationships among the kinosternids, and the establishment of a reasonable chronology of diversification, several key evolutionary questions remain Because most of the diversification within the genus Kinosternon (s.s.) occurred within the last 10 my (especially within the scorpioides group), we were not able to resolve their relationships with high confidence Doing so will require sampling additional genetic markers, as well as much more thorough sampling across the distributions of such wide-ranging taxa as K scorpioides, K integrum, and Cryptochelys leucostoma Such work is certain to uncover additional unappreciated variation in these groups Second, although the fossil record for Kinosternon (s.s.) dates from the Miocene (16-18 mya; see above), older fossil crown group kinosternids (to perhaps 22 mya) must exist But even more surprising is the lack of fossils older than my for the genus Sternotherus (Bourque, 2011) and only 0.5 my for the genus Cryptochelys (Cadena et al 2007) Significant fossils likely remain to be discovered for this group, particularly in the eastern USA and Mesoamerica 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 Lovich, 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 Reyes Velasco, 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 Brinkman and J Bourque P Meylan accompanied Iverson during field work in Mexico and American Southwest, and his expert field assistance was critical to early work Financial support for this project was provided by the American Philosophical Society, the American Museum of Natural History, the Joseph Moore Museum of Natural History at Earlham College, and Grant 106.15-2010.30 from Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) to ML Comments on early drafts of the manuscript by R Bour, J Bourque, S Pasachnik, M E Seidel, and two anonymous reviewers were greatly appreciated Appendix A Supplemental material Supplemental data can be found associated with this article can be found in the on-line version, at doi: xxxx Table S1 Origin of samples sequenced in this study with GenBank numbers Table S2 Primers used in this study Iverson, Le, and Ingram 14 Table S3 Compilation of uncorrected p distances between samples of kinosternid turtles based on cyt b sequence data Table S4 Estimates and variance of ages for each node as numbered in text Figure Fig S1 GARLI partitioned maximum likelihood tree for the Kinosternidae based on concatenated nuclear DNA data only Numbers at nodes are bootstrap support values Fig S2 GARLI partitioned maximum likelihood tree for the Kinosternidae based on concatenated mitochondrial DNA data only Numbers at nodes are bootstrap support values Fig S3 Ancestral distributions of extant kinosternid turtles reconstructed using the Statistical Dispersal-Vicariance Analysis (S-DIVA) method implemented in RASP (Reconstruct Ancestral State in Phylogenies) and color key to ancestral areas [COLOR- ON WEB ONLY] References Agassiz, L., 1857 Contributions to the Natural History of the United States of America Vol 1-2 Little, Brown and Co., Boston Barley, A.J., Spinks, P.Q., Thomson, R.C., Shaffer, H.B., 2010 Fourteen nuclear genes provide phylogenetic resolution for difficult 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of nodes are in Table S4 Calibration nodes are indicated by circled C P+Q = Pliocene + Quaternary Fig Graphical output from RASP (Reconstruct Ancestral State in Phylogenetics; see Section 2.4 for details) A) Reconstruction of ancestral distributions at each node based on 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 [COLOR IN PRINT AND ON WEB] Iverson, Le, and Ingram Table Variation among markers used in our phylogenetic analysis of kinosternid turtles Marker Total # characters # variable # informative cyt b 1085 471 355 12S 363 61 29 16S 510 141 108 C-mos 522 61 39 RAG1 872 56 23 RAG2 1159 90 35 Table Explicit taxonomic changes recommended in this study Former name Proposed name Staurotypinae Staurotypidae Kinosternon (leucostomum group) Cryptochelys K leucostomum C leucostoma K acutum C acuta K subrubrum steindachneri K steindachneri K scorpiodes abaxillare K abaxillare - 21 DERMATEMYDIDAE Kinostern non 66/62 Cryptochelys Dermatemys mawii Claudius angustatus STAUROTYPIDAE Staurotypus salvinii */98 aquatic/tricarinate q / Staurotypus triporcatus */* 73/73 entoplastron/GSD Kinosternon leucostomum leucostomum semiaquatic 88/94 unicarinate Kinosternon angustipons Kinosternon dunni 91/97 Kinosternon herrerai */* no hinge Kinosternon herrerai entoplastron/small PL entoplastron/small PL Ki Kinosternon t creaserii aquatic Kinosternon acutum */99 tricarinate Kinosternon acutum */* KINOSTERNIDAE Sternotherus odoratus 99/96 Sternotherus carinatus aquatic/tricarinate */* Sternotherus minor minor Sternotherus PL hinges/no entoplastron/TSD PL hinges/no entoplastron/TSD 44/56 Sternotherus minor peltifer 53/82 Sternotherus depressus 67/‐ Kinosternon baurii 61/51 Kinosternon subrubrum steindachneri 94/88 Kinosternon flavescens */* */99 Kinosternon subrubrum hippocrepis */* 93/89 Kinosternon arizonense */* semiaquatic Kinosternon durangoense reduced carination 91/91 Kinosternon alamosae 69/75 */* Kinosternon sonoriense ‐/* 98/98 Kinosternon chimalhuaca 98/87 69/75 */* Ki t hi ti hirtipes */98 Kinosternon ‐/99 Kinosternon scorpioides albogulare 62/‐ Kinosternon integrum 60/‐ Kinosternon integrum */98 */91 Kinosternon integrum */* */98 / 61/75 Kinosternon oaxacae Kinosternon scorpioides abaxillare 90/96 44/‐ Kinosternon scorpioides cruentatum 58/54 Kinosternon scorpioides scorpioides 49/‐ 0.03 0.03 Dermatemys mawii g Claudius angustatus Staurotypus triporcatus Staurotypus salvinii Kinosternon leucostomum leucostomum Kinosternon angustipons Kinosternon dunni Kinosternon herrerai Kinosternon herrerai Kinosternon creaseri Kinosternon acutum Kinosternon acutum Sternotherus carinatus Sternotherus odoratus Sternotherus minor minor Sternotherus depressus Sternotherus minor peltifer Kinosternon Ki t baurii b ii Kinosternon subrubrum steindachneri Kinosternon flavescens Kinosternon subrubrum hippocrepis Kinosternon durangoense Kinosternon arizonense Kinosternon alamosae Kinosternon sonoriense Kinosternon chimalhuaca Kinosternon hirtipes Kinosternon scorpioides albogulare Kinosternon integrum Kinosternon integrum Kinosternon integrum Kinosternon scorpioides scorpioides Kinosternon scorpioides p cruentatum Kinosternon scorpioides abaxillare Kinosternon oaxacae C C 11 13 12 10 16 15 17 14 19 18 22 20 21 23 24 80.0 70.0 Cretaceous 60.0 Paleocene 50.0 40.0 Eocene 30.0 Oligocene 20.0 10.0 Miocene 0.0 P + Q Table Summary of uncorrected p distances within and among proposed genera of kinosternid turtles based on the cyt b sequence data Mean distance of samples between genera appears 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) Cryptochelys 16.0 (15.1-17.3) Sternotherus 16.3 (16.1-16.5) Kinosternon 17.1 (15.7-18.2) 1.4 17.8 (17.0-18.3) 16.8 (15.6-17.6) 16.7 (14.9-17.7) 7.6 (1.2-10.6) 11.3 (10.0-13.2) 12.1 (9.0-14.3) 5.8 (0.0-8.3) 10.3 (8.7-12.4) 6.8 (2.0-9.8) - Iverson, Le, and Ingram Highlights We present a DNA-based phylogeny of the turtle Family Kinosternidae A fossil-calibrated chronogram is provided A previously unrecognized clade is described as a new genus A zoogeographic history of the family is hypothesized 22 ... States] b ABSTRACT The turtle family Kinosternidae comprises 25 living species of mud and musk turtles confined to the New World Previous attempts to reconstruct a phylogenetic history of the. .. including the genera Staurotypus and Claudius, and the other including Sternotherus and Kinosternon The fossil record dates the divergence of these two clades at >54 mya (see Section 2.3) Given 1) that... to evaluate the evolutionary history of these and other key kinosternid traits 4.5 Concluding thoughts Despite the resolution of many of the relationships among the kinosternids, and the establishment