Es that had been then mapped to the reference assemblies to assess diversity inside and amongst the 3 lineages. For these six samples, we hand-captured and collected samples from wild desert tortoises in the following 3 internet sites in 2013: two Sinaloan G. morafkai from the Reserva La Sierrita, Sierra de Alamos, Sonora, Mexico (Fig. 1, internet site ALS; tropical deciduous forest); two Sonoran G. morafkai from the Rancho San Judas north of Hermosillo, Sonora, Mexico (Fig. 1, web-site RSJ; Sonoran desertscrub/plains of Sonora); and two G. agassizii from Trout Canyon west of Las Vegas bordering the Eastern Mojave and Northeastern Mojave recovery units (USFWS 2011), Clark County, Nevada (Fig. 1, web site TC; Mojave desertscrub with Larrea tridentata and Yucca brevifolia). For RNA sample collection, we obtained <1 mL whole blood via brachial or subcarapacial venipuncture and mixed it with a greater than 50 volume RNA lysis/binding buffer from the Ambion RNAqueous kit (Life Technologies, Thermo Fisher Scientific Inc., Grand Island, NY). Samples were immediately put on ice and then transferred to liquid nitrogen for storage within 4 h of collection. All RNA samples were verified as being of the assumed lineage (and not hybrids) with subsequent DNA analyses using 25 microsatellite loci and mtDNA (Edwards and Berry 2013).DNA sequencingWe sequenced a 1109 base pair (bp) portion of mitochondrial DNA (ND3, tRNAarg, ND4L, and part of ND4) following Edwards (2003) and Murphy et al. (2007). Some individuals sequenced for this locus had been used in previous studies (Murphy et al. 2007; Edwards et al. 2012, 2015a, 2015b), including the same fragment for G. flavomarginatus (Edwards et al. 2014). We assessed optimal PCR conditions for each primer pair under 72 possible conditions by varying temperatureSamplesPhylogenetic analyses employed 82 DNA samples collected from 38 sites previously used in other studies (Edwards et al. 2004, 2010, 2015a, 2015b; Murphy et al. 2007). These represented samples from across the range of the desert tortoises (Table 1; Fig. 1). In addition, we obtained two samples of G. berlandieri from a private collection and two samples of G. flavomarginatus collected in Durango, Mexico (Morafka et al. 1994). The RNA-seq data gathering used nine individuals in three flowcell lanes on the Illumina HiSeq platform. We dedicated two lanes to high-coverage sequencing of three individuals, one for each of the following lineages: a captive individual of G. agassizii in Arizona that originated in California (Moj_A haplotype); a captive individual of Sonoran?2016 The Authors. Ecology and Evolution published by John Wiley Sons Ltd.Speciation in GopherusT. Edwards et al.from 52 to 64 and MgCl2 concentration from 1.0 to 4.5 mmol/L. PCR amplifications for Sanger sequencing used 30 lL reaction volumes containing 0.2 lmol/L of each primer, 10 mmol/L Tris Cl (pH 8.3), 0.2 mmol/L of each dNTP, 0.4 units of Platinum Taq (Life Technologies, Thermo Fisher Scientific Inc.), 5.0 mmol/L KCl, 10 ng of genomic DNA, and locus-specific amounts of MgCl2 (Table 2). PCR began with an initial 3 min denaturation at 94 , followed by 35 cycles of 30 s at 94 , 30 s at the locus-specific annealing temperature (Table 2), and 90 s at 72 , followed by 3 min incubation at 72 . We PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21175589 submitted PCR solution for the University of Arizona Genetics Core for DNA sequencing in each MedChemExpress Z-IETD-FMK forward and reverse directions and followed common protocols for the 3730XL DNA Analyzer (Applied Biosystems, Foster C.