Sequence-based molecular phylogenetics and phylogeography of the American box turtles (Terrapene spp.) with support from DNA barcoding Bradley T. Martin a,⇑ , Neil P. Bernstein b , Roger D. Birkhead c , Jim F. Koukl a , Steven M. Mussmann d , John S. Placyk Jr. a a Department of Biology, The University of Texas at Tyler, Tyler, TX 75799, USA b Deptartment of Natural and Applied Sciences, Mount Mercy University, Cedar Rapids, IA 52402, USA c Alabama Science in Motion, Auburn University, Auburn, AL 36849, USA d Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA article info Article history: Received 31 December 2012 Accepted 8 March 2013 Available online 20 March 2013 Keywords: Box turtle DNA barcoding Molecular phylogenetics Phylogeography Terrapene Emydidae abstract The classification of the American box turtles (Terrapene spp.) has remained enigmatic to systematists. Previous comprehensive phylogenetic studies focused primarily on morphology. The goal of this study was to re-assess the classification of Terrapene spp. by obtaining DNA sequence data from a broad geo- graphic range and from all four recognized species and 11 subspecies within the genus. Tissue samples were obtained for all taxa except for Terrapene nelsoni klauberi . DNA was extracted, and the mitochondrial DNA (mtDNA) cytochrome b (Cytb) and nuclear DNA (nucDNA) glyceraldehyde-3-phosphate-dehydrogenase (GAPD) genes were amplified via polymerase chain reaction and sequenced. In addition, the mtDNA gene commonly used for DNA barcoding (cytochrome oxidase c subunit I; COI) was amplified and sequenced to calculate pairwise percent DNA sequence divergence comparisons for each Terrapene taxon. The sequence data were analyzed using maximum likelihood and Bayesian phylogenetic inference, a molecular clock, AMOVAs, SAMOVAs, haplotype networks, and pairwise percent sequence divergence comparisons. Terra- pene carolina mexicana and T. c. yucatana formed a monophyletic clade with T. c. triunguis , and this clade was paraphyletic to the rest of T. carolina . Terrapene ornata ornata and T. o. luteola lacked distinction phy- logenetically, and Terrapene nelsoni was confirmed to be the sister taxon of T. ornata . Terrapene c. major , T. c. bauri , and Terrapene coahuila were not well resolved for some of the analyses. The DNA barcoding results indicated that all taxa were different species (>2% sequence divergence) except for T. c. triunguis – T. c. mexicana and T. o. ornata – T. o. luteola . The results suggest that T. c. triunguis should be elevated to species status (Terrapene mexicana ), and mexicana and yucatana should be included in this group as sub- species. In addition, T. o. ornata and T. o. luteola should not be considered separate subspecies. The DNA barcoding data support these recommended taxonomic revisions. Because conservation efforts are typi- cally species-based, these results will be important for facilitating successful conservation management strategies. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction The American box turtle genus, Terrapene (Chelonia, Emydidae), includes four primarily terrestrial New World species, the eastern (T. carolina ; Linnaeus, 1758), ornate (T. ornata ; Agassiz, 1857), spot- ted (T. nelsoni ; Stejneger, 1925), and Coahuilan (T. coahuila ; Schmidt and Owens, 1944) box turtles, characterized, in part, by their ability to tightly close their plastron via a single moveable hinge (plastral shell kinesis). With the exception of T. coahuila , each species is further divided into multiple subspecies including the eastern (T. c. carolina ), three-toed (T. c. triunguis ), Mexican (T. c. mexicana), Yucatan (T. c. yucatana ), Gulf Coast (T. c. major ), Florida (T. c. bauri ), and the purportedly extinct giant (T. c. putnami ) box turtles for T. carolina ; the ornate (T. o. ornata ) and desert (T. o. lut- eola) box turtles for T. ornata ; and the northern spotted (T. n. klau- beri) and southern spotted (T. n. nelsoni ) box turtles for T. nelsoni . While their current classification is generally accepted based on morphological data and geographic distributions, the use of molec- ular data in understanding the evolutionary history of the group has been limited, as the systematic studies that have included Ter- rapene have (1) focused on higher level intergeneric classification, (2) failed to include all taxa within Terrapene (e.g., all species and subspecies), or (3) been limited in sample sizes and/or geographic sampling (Bickham et al., 1996; Butler et al., 2011; Feldman and 1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.03.006 ⇑ Corresponding author. Present address: Department of Biological Sciences, The University of Arkansas, Fayetteville, AR 72701, USA. Fax: +1 903 723 1095. E-mail addresses: bmartin@patriots.uttyler.edu, btm002@email.uark.edu (B.T. Martin). Molecular Phylogenetics and Evolution 68 (2013) 119–134 Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev