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Thermal-cycling parameters required an initial denaturation at 94°C for 2 min, followed by 40 cycles of 94°C (30 s), 48–53°C (30 s), and 72°C (30 s), and a final extension of 72°C for 5 min.All amplified products were purified using EXOSap-IT (USB Corporation, Cleveland, OH) and all sequencing reactions were performed at the University of Florida DNA Sequencing Core Laboratory (Gainesville, Florida) using ABI Prism Big Dye Terminator cycle sequencing protocols (Applied Biosystems, Foster City, CA) as described in Light and Reed (2009).Although morphometric analyses were unable to differentiate the subspecies, phylogenetic analyses of molecular data indicated that the 4 currently recognized subspecies of that do not correspond to the currently recognized subspecies, but whose geographic limits instead coincide with major geographical features in the southern United States and northern Mexico.The Southern Coahuila filter-barrier (Durango and Coahuila), the Deming Plains (New Mexico), and the Balcones Escarpment (Texas) likely have acted as intermittent physical barriers to gene flow among the distinct mitochondrial clades, which we recognize as subspecies within represents a disjunct distribution in central Mexico, ranging from southern Coahuila and Durango to Hidalgo (Fig. The geographic distribution of the species ranges farther east and north (including colder continental zones of the northern Great Plains) than any other species in the genus.2008; Fa and Morales 1993; Guevara-Chumacero et al. North America is a continent riddled with barriers that are known to limit gene flow within taxa (Jezkova et al.2009; Kerhoulas and Arbogast 2010; Mc Knight 2005; Riddle 1995; Riddle and Hafner 2006; Riddle et al. Whether these barriers cause genetic differences among subspecies or populations of C. Herein, we use mitochondrial DNA (mt DNA) from both fresh tissue and museum specimens as well as morphological data from museum specimens to provide an assessment of phylogeographic variation within .—Sixty-three specimens from 62 localities were examined in the mt DNA genetic analyses (16 of these specimens were obtained from museum study skins [Fig.
Phylogenetic analyses were conducted on all data sets using neighbor-joining, maximum-parsimony, maximum-likelihood, and Bayesian approaches in PAUP* version 4.0b10 (Swofford 2003); Randomized Axelerated Maximum-Likelihood (RAx ML—Stamatakis 2006); and Mr Bayes version 3.1.2 (Ronquist and Huelsenbeck 2003).Thermal-cycling parameters for ND2 required an initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C (30 s), 50°C (30 s), and 65°C (90 s), and a final extension of 65°C for 5 min.Polymerase chain reactions for skin snips from museum study skins were performed in 25-μl reaction volumes using 4 μl of Mg Cl, 2.5 μl of 10X buffer, 2 μl of deoxynucleoside triphosphate, 2 μl of 5 M betaine, 1 μl of each primer (at 10 m M), and 0.125 μl of r Taq DNA Polymerase (Ta Ka Ra, Mountain View, CA).More than 60 years ago, Glass (1947) addressed morphological and geographic variation within C.in light of its long taxonomic history: since the original description in 1858, 7 different names have been proposed for populations of this species (e.g., Allen 1894; Elliot 1903; Merriam 1889; Osgood 1900).
All genetic samples were obtained as tissue loans from natural history museums ( Appendix I)..—Mitochondrial DNA was extracted using the DNeasy Tissue Kit or the QIAamp DNA Mini Kit (QIAGEN Inc., Valencia, California) according to manufacturer's instructions; skin snips from museum study skins were presoaked in a IX phosphate-buffered saline buffer solution for 24 h prior to extraction process.