Sequence-based molecular phylogenetics and phylogeography of the American box turtles (Terrapene spp.) with support from DNA barcoding.

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 geographic 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. Terrapene 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 phylogenetically, 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 subspecies. 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 typically species-based, these results will be important for facilitating successful conservation management strategies.

Immunoperoxidase in the interpretation of discordant histologic and urease findings for Helicobacter pylori.

Azure A and methylene blue ("Diff-Quik," DQ) and tissue urease (U) tests are popular methods to diagnose Helicobacter pylori. These tests usually correlate well but sometimes produce discordant results. This study evaluates the DQ and U tests by comparing them with the immunoperoxidase reference method to resolve discordant results. DQ and U tests were performed on gastric biopsies. Results were tabulated as DQ(+)/U(+), DQ(+)/U(-), DQ(-)/U(+), and DQ(-)/U(-). Cases that were DQ(+)/U(+) were recorded as positive and not tested with immunoperoxidase. Cases that had discordant DQ/U results were tested by immunoperoxidase to resolve the discordance. Cases which were negative for both DQ/U were evaluated by immunoperoxidase to confirm the validity of DQ(1-)/U(-). The groups were compared with concordant results (DQ(1-)/U(-) group) and immunoperoxidase versus discordant DQ/U results and immunoperoxidase. There were 56 gastric biopsy specimens. Among all cases, 6 were DQ(+)/U(+). Of the remaining 50 cases, 38 were concordant DQ(-)/U(-), whereas 12 showed discordant DQ/U results. All 38 concordant DQ(-)/U(-) specimens were confirmed negative, 11 discordant DQ/U cases were confirmed negative, and 1 DQ(+)/U(-) specimen was confirmed positive by immunoperoxidase. Comparison of concordant versus discordant results was not statistically significant (P=0.10). Among all discordant DQ and U, 11/12 (92%) were confirmed negative by immunoperoxidase. Thus, both concordant negative results and discordant results can be considered negative. Such interpretation of discordant results might prevent unnecessary additional procedures or treatment.