ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah Acta Herpetologica 6(2): 175-208, 2011 Ecological associations and genetic divergence in Black-bellied Salamanders (Desmognathus quadramaculatus) of the Southern Appalachian Mountains Jessica A. Wooten1, Leslie J. Rissler2 1Department of Biology, The University of Findlay, Findlay, OH 45840, USA. 2 Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487, USA. Corre- sponding author. E-mail: Rissler@as.ua.edu Submitted on: 2011, 7th March; revised on: 2011, 31th July; accepted on: 2011, 09th October. Abstract. The discovery and subsequent description of cryptic biodiversity is often chal- lenging, especially for groups that have undergone rapid lineage accumulation in the relatively recent past. Even without formal descriptions, understanding genetic diversity patterns as they relate to underlying ecological or historical processes can be important for conservation. The dusky salamanders of the genus Desmognathus, with 20 described species, comprise the second largest genus of plethodontid salamanders in the eastern United States. However, due to the presence of high genetic diversity and relatively few morphological synapomorphies, the number of species is likely to increase. For the three nominal species within the D. quadramaculatus species complex, including D. quad- ramaculatus, D. folkertsi, and D. marmoratus, we used a portion of the mitochondrial genome and nuclear markers in the form of amplified fragment length polymorphisms (AFLP) to uncover spatial patterns of genetic diversity. Within D. quadramaculatus and D. marmoratus, we uncovered four well-supported lineages with the mitochondrial sequences; phylogeographic patterns were not congruent with the AFLP data. Both sets of markers identified a clear isolation by stream distance. Using multiple regressions, we found that historical river drainages and terrestrial ecoregions explained the phylogeo- graphic patterning we observed for D. quadramaculatus. Keywords. Salamanders, Desmognathus quadramaculatus, Desmognathus marmora- tus, AFLP, streams, Desmognathus folkertsi, mtDNA, ecoregions. INTRODUCTION In light of the constant threat of anthropogenic habitat alteration (Davis et al., 2008) and the ubiquity of cryptic biodiversity (Bickford et al., 2006), the discovery and subse- quent descriptions of unique evolutionary lineages are central to understanding the full 176 J.A. Wooten and L.J. Rissler extent of biodiversity on Earth. However, the discovery and subsequent description of cryptic species is challenging, especially for organismal groups that have experienced rapid lineage accumulation in the recent past (Kozak et al., 2006; Wiens et al., 2006) and that lack obvious morphological differentiation. This is certainly true in lungless salamanders (Plethodontidae) where many species boundaries and evolutionary relationships have been concealed by the absence of obvious morphological synapomorphies (Wake, 1966; Larson et al., 1981; Wake et al., 1983; Carr, 1996; Adams and Rohlf, 2000; Highton and Peabody, 2000; Chippindale et al., 2004; Mueller et al., 2004; Wiens et al., 2006; Tilley et al., 2008). Salamanders are a challenging group for which to delimit species boundaries and uncover cryptic biodiversity. Many salamander taxa exhibit extensive intraspecific variation in traits, such as color and pattern. At the same time, they exhibit extreme interspecific mor- phological similarity, which is usually coupled with high levels of genetic diversity (Wake, 1966, 1991; Wake et al., 1983; Highton and Peabody, 2000; Wake and Jockusch, 2000; Gao and Shubin, 2003; Mueller et al., 2004; Kozak et al., 2006; Wiens et al., 2006; Beamer and Lamb, 2008; Tilley et al., 2008). The Plethodontidae is the most species-rich family of sala- manders containing approximately 68% of all known species (amphibiaweb.org), with new species being described regularly because of extreme morphological similarities and wide- spread homoplasy (e.g., Tilley and Mahoney, 1996; García-París and Wake, 2000; Jockusch et al., 2001; Camp et al., 2002; McCranie et al., 2005; Hanken et al., 2007). The dusky salamanders (Desmognathus) comprise one of the most species-rich genera within Plethodontidae (Vieites et al., 2007), with the hotspot of biodiversity in the Appala- chian Mountains (Kozak et al., 2006; Wiens et al., 2006; Vieites et al., 2007). Most desmog- nathan species are restricted to, or are associated with, swift-flowing mountain streams in the Appalachian Mountains of eastern North America. Many recent studies agree that the 20 currently recognized species may be a significant underestimation of the full extent of the biodiversity within the genus (Kozak et al., 2005; Beamer and Lamb, 2008), and the number of recognized species will likely increase due to the presence of extensive morphological sta- sis, rampant homoplasy, and high levels of genetic diversity among and within groups. To complicate modern-day taxonomy, studies have revealed a more complex and intertwined evolutionary history for many species within the genus Desmognathus (Titus and Larson, 1996; Rissler and Taylor, 2003; Kozak et al., 2005; Jones et al., 2006; Beamer and Lamb, 2008; Tilley et al., 2008), especially for D. marmoratus and D. quadramaculatus (Titus and Larson, 1996; Rissler and Taylor, 2003; Jackson, 2005; Jones et al., 2006). Interestingly, these stud- ies have suggested that some populations of D. quadramaculatus are more closely related to populations of D. marmoratus, a wholly aquatic and relatively rare salamander species (Titus and Larson, 1996; Rissler and Taylor, 2003; Jackson, 2005; Jones et al., 2006) with a limited geographic distribution. However, to date, there have been no published studies focusing exclusively on the phylogeography of D. quadramaculatus across its range. The use of nuclear markers in plethodontid salamander species, with the exception of allozymes, is uncommon for studies of fine-scaled population structure, although sequenc- ing nuclear genes is becoming more common. That said, amplified fragment length poly- morphism (AFLP; Vos et al., 1995) is a PCR-based, anonymous dominant nuclear-marker technique that has been extensively used to investigate intraspecific phylogeographic pat- terns, uncover new species, and delimit species boundaries in many groups (Shaw, 2002; Seman et al., 2003; Wang et al., 2003; Creer et al., 2004; Finn et al., 2006; Mendelson and Simons, 2006; Garoia et al., 2007; Kinkead et al., 2007; Nicolè et al., 2007), especially in 177Ecology and genetic of Black-bellied Salamanders plants, (e.g., Hoarau et al., 2001; Garcia et al., 2004; Albach et al., 2006; Agrimonti et al., 2007; Andrade et al., 2007; Assefa et al., 2007; Nicolè et al., 2007), microbes and fungi (e.g., Vos et al., 1995; Blears et al., 1998; Bensch and Åkesson, 2005), and invertebrates (Wilding et al., 2001; Shaw, 2002; Carisio et al., 2004; Mock et al., 2004; Pizzo et al., 2006). Studies that use AFLP in vertebrate taxa are limited (Bensch and Åkesson, 2005), but this number has increased over the past few years (Ogden and Thorpe, 2002; Wang et al., 2003; Makowsky et al., 2008). At present, there are only a few known studies that involve the use of AFLP in salamander taxa (Voss and Shaffer, 1997; Curtis and Taylor, 2003; Riberon et al., 2004; Lowe et al., 2006; Whitlock et al., 2006; Jehle et al., 2007; Kinkead et al., 2007; Milá et al., 2010), even though AFLP is a useful marker that can be used to investigate population structure and genetic variability at the species level (Mueller and Wolfenbarger, 1999). The goals of our study were to use both mitochondrial and AFLP data to: 1) address the phylogeographic and genetic patterns across populations of D. quadramaculatus, and 2) assess the spatial patterns of divergence, specifically testing the role of drainages (i.e., current versus historical river drainages) as barriers of gene exchange. MATERIALS AND METHODS The study taxon Desmognathus quadramaculatus is a species complex comprised of at least three nominal species, including, D. quadramaculatus (Black-bellied Salamander), D. folkertsi (Dwarf Black-bel- lied Salamander), and D. marmoratus (Shovel-nosed Salamander). Desmognathus quadramaculatus exhibits the largest geographic range of the three and is a semi-aquatic species that can be found in swift-flowing, montane streams extending from southern West Virginia southward through the Great Smoky and Unicoi Mountains, and into the Blue Ridge escarpment of western North Car- olina. This species reaches its southern-most geographic limit in the upper Piedmont of northern Georgia (Jensen et al., 2008). Desmognathus folkertsi and D. marmoratus both exhibit relatively small geographic rang- es and are often syntopic with that of D. quadramaculatus. Desmognathus folkertsi is a recently described, semi-aquatic species (Camp et al. 2002) known from northern Georgia and abutting areas of the Carolinas; whereas, D. marmoratus is an aquatic species that has a larger geographic distribution extending from southern Virginia, south along the Blue Ridge Mountains to northern Georgia. Desmognathus folkertsi appears to represent a cohesive, monophyletic taxon (Jackson, 2005; Wooten 2007; Wooten et al., 2010). However, D. marmoratus, as it is currently recognized, consists of several lineages and may be a paraphyletic taxon (Jackson, 2005; Jones et al., 2006; Kozak et al., 2006). Jones et al. (2006) suggested the recognition of southernmost populations of D. marmoratus as a separate species (D. aureatus). Sampling locations We examined 281 individuals from 56 populations D. quadramaculatus from the southern Appalachian Mountains in the eastern United States, as well as closely related species, including D. monticola (n = 15), D. folkertsi (n = 23), and D. marmoratus (n = 8; Fig. 1). Detailed information on locality, museum voucher number, Genbank accession number, and river drainage are presented in Appendix 1. Voucher specimens were euthanized in using ethyl 3-aminobenzoate methanesulfonate 178 J.A. Wooten and L.J. Rissler (MS 222; Sigma-Aldrich) at a concentration of 2 g / L. An approximately 1 cm piece of tail tissue was removed for genetic analysis and frozen (-80oC) in the permanent tissue archive at The University of Alabama Herpetology Collection (UAHC). Specimens were then fixed in 10% formalin and preserved in 70% ethanol. All specimens were deposited in the UAHC stored at The University of Alabama, Tuscaloosa or in the Appalachian State Herpetology Collection (APPSU), Boone, North Carolina. Mitochondrial DNA extraction and sequencing Whole genomic DNA was extracted from approximately 5 mm of tail tissue using the DNeasy tissue-kit protocol for animal tissues (Qiagen, Valencia, CA). Amplification of the 12S rRNA and a portion of the valine transfer tRNA regions of the mitochondrial genome was completed on all individuals by using primers B and G from Titus and Larson (1996) and following a modified meth- odology of Rissler and Taylor (2003). This portion of the mitochondrial genome has been shown to be a highly informative marker for discriminating intra- and interspecific relationships in Desmog- nathus (Titus and Larson, 1996; Crespi et al., 2003; Rissler and Taylor, 2003; Rissler et al., 2004). ExoSapIT (U.S. Biochemicals Corp., Cleveland, OH) at 37 oC for 45 min and 80 oC for 15 min was used to purify the PCR products. Cycle-sequencing was completed in forward and reverse directions on the purified PCR prod- ucts using BigDye Terminator (Applied Biosystems, Foster City, CA), followed by purification with Sephadex G-50 (Sigma Aldrich Corp., St. Louis, MO), and visualized on an ABI 3100 automated DNA sequencer. Additional mtDNA sequences were retrieved from Genbank for D. wrighti (out- group), D. quadramaculatus, D. marmoratus, D. aeneus, and D. monticola, and these accession num- bers are listed in Appendix 2. Collapse 1.2 (http://darwin.uvigo.es/software/collapse.html) was used to remove any identical haplotypes before phylogenetic analyses. Amplified fragment length polymorphism Our AFLP procedures were modified from the protocol by Voss et al. (1995) and Blears et al. (1998). Digestion reactions in 20 µl volumes were performed on whole genomic DNA with a concen- tration of 25 ng / µl using the following cocktail per reaction: 2 µl EcoRI 10X restriction buffer, 1.0 µl EcoRI, 0.2 µl MSE, and 12 µl H2O. Following a 5 h incubation period at 37 oC, 20 µl of ligation mix- ture consisting of 12 µl H2O, 4 µl 10X T4 DNA ligase buffer, 1.5 µl of each double-stranded adapt- er pair (75 pmol), and 1.0 µl of T4 ligase was added to the digestion solution. The double-stranded adaptor pairs were constructed from the following complementary single-stranded oligonucleotides: EcoRI adaptor: 5’-CTC GTA GAC TGC GTA CC-3’ AND 5’-AAT TGG TAC GCA TAC-3’. Ligation was performed for 10 h at 16 oC. Following ligation, each solution was diluted with 160 µl H2O. Two increasingly selective PCR amplifications of the ligated DNA were performed. For pre- selective amplification, 10µl of the diluted restriction-ligation product was used as a template and added to 40 µl preselective amplification solution consisting of: 5 µl 10X PCR buffer, 1.3 µl of each preselective primer (15 pmol), 4.0 µl dNTP (10 mM), 1.0 µl formamide, 2.5 µl MgCl2 (25 mM), 24.75 µl H2O, and 0.5 µl Taq DNA polymerase. Four preselective primer combinations were used including: EcoRI + AC / MSE + CA; EcoRI + CA / MSE + CA; EcoRI + CA / MSE + CG; and EcoRI + CA / MSE + GC. The PCR cycling conditions were a preliminary 72 oC extension for 60 sec fol- lowed by 20 cycles of 94 oC for 50 sec, 56 oC for 60 sec, and 72 oC for 120 sec. Preselective solutions were diluted with 125 µl of H2O following PCR cycling. For selective PCR, 5 µl of diluted preselective solution was added to 20 µl of selective PCR solution which consisted of 0.5 µl formamide, 10 µl H2O, 2.5 µl 10X PCR buffer, 3 µl MgCl2 (25 mM), 3 µl dNTP (10 mM), 1.5 µl (0.5 pmol) of the flourophore (6-FAM)-labeled EcoRI primer, 1.5 µl (25 pmol) of the unlabeled MSE selective primer, and 0.5 µl Taq DNA polymerase. The PCR 179Ecology and genetic of Black-bellied Salamanders cycling conditions were: a touchdown cycle of 94 oC for 50 sec, 57 oC for 60 sec, and 72 oC for 120 sec, followed by 20 cycles of 94 oC for 50 sec, 56 oC for 60 sec, and 72oC for 120 sec. The 20 cycles were followed by a final extension at 72 oC for 10 min. Three combinations of selective primer combinations were chosen including: EcoRI + CAA / MSE + CA; EcoRI + CAA / MSE + CG; and EcoRI + CAA / MSE + GC from a primer screening procedure in another study (Wooten and Tolley-Jordan, 2009). When combined, these combinations produced between 450 and 555 well-defined bands per sample, distributed widely across the 60-350 scoring window. The selective amplification products were purified using fine Sephadex G-50 (Sig- ma-Aldrich Corp., St. Louis, MO). We loaded 1.5 µl of purified product per sample along with 0.5 µl GeneScan-500 ROX ladder (Perkin-Elmer) into 96-well plates and samples were analyzed using an automated ABI 3100 with GeneScan software. Electropherograms were imported into GeneMarker v1.6 for further analyses. Phylogenetic reconstruction – mtDNA and AFLP Approximately 600 bp of the 12S rRNA and valine transfer tRNA regions were sequenced. Forward and reverse sequences were assembled, and any ambiguous sites were verified using Sequencher 4.6 (Gene Codes, Ann Arbor, MI). Clustal W procedure in BioEdit v7.0.9 (http://www. mbio.ncsu.edu/BioEdit/bioedit.html) was used to align all sequences. The alignment was manually corrected. The sequences were sufficiently similar to allow manual correction following alignment without restrictions based upon the gap positions in the hypothesized loop regions of the 12S rRNA sequences. We used D. wrighti as the outgroup in all phylogenetic reconstructions; D. wrighti is cur- rently accepted as the basal member of the genus Desmognathus (Titus and Larson, 1996; Rissler and Taylor, 2003; Jones et al., 2006; Tilley et al., 2008). Phylogenetic relationships among haplotypes using mtDNA sequences were estimated using maximum likelihood and Bayesian procedures. For the Bayesian procedure, the model of sequence evolution that best fit the mtDNA sequence data was determined using mrmodeltest v2.2 (http:// www.abc.se/~nylander). mrbayes v3.1 was then used to conduct a Bayesian phylogenetic analysis (Huelsenbeck and Ronquist, 2001) using the TrN + Γ model of evolution. Six heated Markov chains were run for 10×106 generations, sampling every 1000 generations for a total of 10,000 samples. Three replicate searches were conducted to verify that the analyses were optimal by producing simi- lar topologies and ln-likelihood scores (Huelsenbeck and Bollback, 2001). The burn-in procedure was used to eliminate topologies generated before the ln-likelihood was stabilized. Posterior prob- abilities were estimated as the proportion of trees sampled after the burn-in procedure containing each of the observed bipartitions (Larget and Simon, 1999). For the maximum-likelihood procedure, modeltest v3.7 (Posada and Crandall, 1998) was used to estimate the model of sequence evolution. Garli v0.951 (Zwickl, 2006) was used to recon- struct phylogenetic relationships among haplotypes using the maximum-likelihood procedure with the TrN + Γ model of evolution. Statistical support for each branch was assessed using bootstrap analysis with 1000 replications in Garli v0.951 (Zwickl, 2006). For AFLP analyses, each primer combination was used to create a unique template to normalize the data across the different runs, and this step standardized the calling of peaks across the four dif- ferent primer combinations using GeneMarker v1.6 (SoftGenetics, State College, Pennsylvania). Peaks were called between 60-350bps, and these data were entered into a binary matrix as discrete variables (1 for presence and 0 for absence). The binary matrix from each primer combination was combined into one data set and analyzed as phenotypes. A Jaccard dissimilarity matrix was generated in R Pack- age v4.0 (Casgrain and Legendre, 2001) from the combined binary matrix. A phylogram was gener- ated in paup* (Swofford, 2002) using the minimum evolutionary procedure. Statistical support for each branch was assessed using both neighbor-joining and parsimony analyses with 1000 replications in paup* (Swofford, 2002). For analyses, Hardy-Weinberg equilibrium was assumed for the D. quad- 180 J.A. Wooten and L.J. Rissler ramaculatus, D. folkertsi, and D. marmoratus populations, and we assumed that each peak (i.e., frag- ment) represented a unique sequence (Zhivotovsky, 1999; Kinkead et al., 2007; Measey et al., 2007). Genetic diversity indices Patterns of genetic diversity within and across lineages and river drainage basins were inves- tigated using DnaSP v4.0 (Rozas and Rozas, 1999; Rozas et al., 2003) for populations of D. quadra- maculatus. For the three nominal species in the D. quadramaculatus complex, haplotype diversity (Nei, 1987), sequence diversity (k; Tajima, 1983), nucleotide diversity (p; Nei, 1987), the number of mutations per site (θ; Nei, 1987), and the total number of mutations (h; Nei, 1987) were calculated to measure sequence diversity. In order to test predictions made by the neutral theory of molecular evolution (Kimura, 1983) and for evidence of population-expansion events, we calculated Tajima’s D (Tajima, 1989), Fu and Li’s D* test statistic (Fu and Li, 1993), Fu and Li’s F* test statistic (Fu and Li, 1993) and Fu’s Fs statistic (Fu, 1997). For AFLP data, we used Structure v2.2 (Pritchard et al., 2000; Falush et al., 2003; Falush et al., 2007), which is a clustering method that assigns individuals to populations using multilocus geno- type data (Pritchard et al., 2000; Falush et al., 2003), to discern between lineages of the D. quadra- maculatus species complex. We assigned individuals to five groups based upon the Bayesian phylo- gram to test for congruence between the phylogeny and the group placement using the AFLP frag- ments, a multiloci, dominant, nuclear markers, using Structure v2.2 (Pritchard et al., 2000; Falush et al., 2003; Falush et al., 2007). In Structure v2.2 (Pritchard et al., 2000; Falush et al., 2003; Falush et al., 2007), we ran 10,000 burn-in with 100,000 iterations for two – 10 (k) groups with the admixture ancestry model and the option of correlated allele frequencies between populations (Falush et al., 2003). We allowed Structure v2.2 to infer the degree of admixture alpha from the AFLP data. When alpha is close to zero, most individuals are from one population; whereas, when alpha is greater than one, individuals are admixed (Evanno et al., 2005). We set lambda, which was a parameter of the distribution of allelic frequencies, to one (Pritchard et al., 2000; Evanno et al., 2005). We completed a pilot run using the AFLP data, and we found that a burn-in of 1000 and MCMC of 10,000, with 10 replications for each value of k, was not sufficient. Because of this, we increased the burn-in to 10,000 and the MCMC to 100,000, with 10 replications for each value of k; we found that the likeli- hood values and the amount of variation for the likelihood of each k stabilized across replications. Finally, we chose the option of ancestdist, which calculates the 90% probability interval that a par- ticular individual would be assigned to a distinct group. For AFLP data, we used AFLP-SURV v1.0 (Vekemans, 2002; Vekemans et al., 2002) to esti- mate genetic diversity with dominant alleles using the Lynch and Milligan (1994) method. Statistical analysis of AFLP fragments was based on the assumption that the fragments act as diploid, domi- nant markers either being present (scored as 1) or absent (scored as 0). Nei’s genetic distance, which is the average, expected heterozygosity of the marker loci was used to estimate genetic diversity from the AFLP fragments. We assumed Hardy-Weinberg equilibrium to examine total gene diversity (Hj), mean gene diversity within populations (Hw), average gene diversity among populations (Hb), and Fst across the landscape (Vekemans, 2002; Vekemans et al., 2002). Because discrete genotypes are not generated from AFLP fragments, each fragment was treated as a single locus with two alleles (Blears et al., 1998). In AFLP-SURV, we chose the Bayesian non-uniform distribution with 1000 per- mutations for estimates of gene diversity. In addition, we constructed a multidimensional scaling (MDS) plot based on Jaccard’s dissimilarity measure (1-similarity; Gower 1971) using Statistica v.6 (StatSoft) and R Package v.4.0 (Casgrain and Legendre, 2001) to see if distinct clusters formed based on dissimilarity among populations or between D. folkertsi and D. quadramaculatus. In Arlequin v.3.11 (Excoffier et al., 2005), we calculated an AMOVA using the AFLP binary matrix for unique haplotypes and used the same scheme for assigning individuals to populations and groups as in the mtDNA analysis. Finally, we calculated Nei’s (1978) unbiased genetic distance (D) between lineages 181Ecology and genetic of Black-bellied Salamanders of D. folkertsi and again between D. folkertsi and D. quadramaculatus using the AFLP binary matrix in GeneAlEx 6.1 (Peakall and Smouse, 2006). Spatial partitioning of genetic variation Desmognathus quadramaculatus, like D. folkertsi, is a semi-aquatic salamander, and gene flow can occur via aquatic or terrestrial routes (Wooten et al., 2010). Therefore, we evaluated isolation by distance (Wright, 1943; Slatkin, 1993) using both land and aquatic routes of dispersal. We calculated distances among streams using Network Analyst in ArcGIS v9.0. An arbitrary value of 5,000,000 km was assigned to represent streams that were in different river drainages. Straight-line distances (km) via land were calculated for each individual using latitude and longitude between each geographic locality using the R package v4.0. We used Mantel tests in the R package v4.0 (Casgrain and Leg- endre, 2001) to test these associations. Analysis of molecular variance (AMOVA; Excoffier et al., 1992) was used to test the parti- tioning of genetic variation within and across streams and river drainage basins using Arlequin v3.1 (Excoffier et al., 2005). The AMOVA Φ statistics are equivalent to F statistics (Wright, 1931; Wright, 1965) and are determined to be significant based on at least 1000 nonparametric permutations of individuals, populations, or groups of populations. In our analyses, individuals were assigned to a stream and then grouped based on river drainage basin. We used multiple regression to examine whether patterns of genetic diversity were explained by the following phylogeographic breaks: 1) current taxonomic classification (i.e., D. folkertsi, D. quadramaculatus, and D. marmoratus), 2) current drainage patterns, 3) historical drainage pat- terns from the Pliocene (Kozak et al., 2006), 4) freshwater ecoregions (categorized according to sampling location and information available in Abell et al., 1999), and 5) terrestrial ecoregions (cat- egorized according to sampling location and the classification of temperate broad leaf and mixed forests ecoregions in Ricketts et al., 1999). To test these hypotheses, we generated uncorrected genet- ic distances for each mitochondrial haplotype calculated from paup* (Swofford, 2002). These val- ues were considered the dependent variable. For each of the five independent variables, characters were assigned based on geographic locality or taxonomic group, depending on the phylogeographic break, and a matrix of distances between all pairs of haplotypes was generated using R package v4.0 (Casgrain and Legendre, 2001). These matrices were imported into Permute! v3.4 (Casgrain, 2001); multiple-regressions were computed and 1000 permutations were completed (Legendre et al., 1994) to assess the probability that each break explains the patterns of genetic diversity observed. Back- wards regression was used to determine the importance of the independent variables. RESULTS Overall genetic variation For the nominal species within the D. quadramaculatus complex, 327 sequences rep- resenting 800 aligned bases were reported. For D. quadramaculatus, 201 unique haplo- types were identified from a total of 281 individuals and 56 sampling localities (Appendix 1). The TrN + Γ model of evolution was chosen from mrmodeltest v2.2 with nucleotide frequencies of A = 0.2828, C = 0.2201, G = 0.2025, and T = 0.2946; substitution model rate matrix: R(a) [A-C] = 1.0000, R(b) [A-G] = 2.5812, R(c) [A-T] = 1.0000, R(d) [C-G] = 1.0000, R(e) [C-T] = 2.1188, and R(f ) [G-T] = 1.0000; Γ distribution shape parameter equaled 0.4069. We reported bootstrap values of ≥ 70% (Hillis and Bull, 1993) and pos- 182 J.A. Wooten and L.J. Rissler terior probabilities ≥95% (Wilcox et al., 2002) to represent well-supported nodes for the phylogenetic hypothesis. For D. quadramaculatus, the mean number of AFLP fragments generated by each AFLP primer combination was 187.67, and this varied from 167 fragments for primer combination EcoRI + CAA / MseI + GC to 212 fragments for primer combination EcoRI + CAA / MseI + CG. When all AFLP fragments were considered, a total of 563 fragments were generated from three primer combinations. Of these, 536 were polymorphic for D. quadramaculatus from 38 populations. We tested for AFLP fragment-size homoplasy and found that there was no significant correlation between polymorphic AFLP fragment size and frequency of fragment occurrence (r = -0.089, P = 0.348; Vekemans et al., 2002). Phylogenetic analysis – mtDNA The Bayesian analysis and the maximum-likelihood phylograms yielded similar topol- ogies for D. quadramaculatus species complex and the outgroup. Because of this, we rep- resented the phylogenetic hypothesis of the D. quadramaculatus species complex with the Bayesian phylogram with the posterior probabilities and maximum-likelihood bootstrap values for common branches. We generated a 50% majority-rule consensus phylogram from the Bayesian analysis with a mean ln-likelihood = -15898.7 following a burn-in pro- cedure to remove 2600 topologies that were generated before stability of the ln-likelihood was established. Haplotypes within the D. quadramaculatus complex formed a well-supported mono- phyletic group when all three nominal species were included in the analysis (Figs. 2A and 2B). There were two major lineages recovered (Lineage 1 and 2) with strong statistical sup- port; Lineage 1 was comprised of D. folkertsi, which formed a monophyletic group, and Lineage 2 was composed of D. quadramaculatus and D. marmoratus (Fig. 2A). Within Lin- eage 2, several well-supported lineages were formed; two lineages were found south of the French Broad River (Fig. 2A; Lineage 2A and 2B) and two lineages were found north of the French Broad River (Fig. 2B; Lineages 2C and 2D). Lineage 2A was comprised of both nominal species D. quadramaculatus and D. marmoratus; whereas; Lineage 2B consisted only of D. quadramaculatus. Both Lineages 2A and 2B consisted of individuals from Geor- gia, South Carolina, and southwestern North Carolina. The northern lineage (Lineage 2C and 2D) consisted of D. quadramaculatus and D. marmoratus individuals from West Vir- ginia, Virginia, and northern Tennessee. The population from northern Tennessee (Fig. 1, Map Locality 5) formed a monophyletic, well-supported clade; this population is undergo- ing further research. Lineage 2D consisted of a single population in North Carolina (Fig. 1, Map Locality 13); this population formed a well-supported and basal lineage to Lineage 2C. However, because Lineage 2D consisted of a single population in an isolated geographic area, surveys for D. quadramaculatus populations are underway in surrounding streams. Phylogenetic analysis – AFLP Desmognathus folkertsi and D. quadramaculatus formed well-supported, distinct line- ages when the AFLP data were used, but within the species, no clear geographic patterns were found (data not shown). Desmognathus marmoratus individuals were nested within 183Ecology and genetic of Black-bellied Salamanders D. quadramaculatus for the AFLP, but these nodes were not well-supported. Furthermore, the MDS plot did not reveal any distinct groups for D. quadramaculatus, D. folkertsi, or D. marmoratus. Fig. 1. Study localities in the southern Appalachian Mountians. Sampling localities spanned parts of West Virginia, Virginia, North Carolina, Tennessee, South Carolina, and Georgia. 184 J.A. Wooten and L.J. Rissler Statistical analyses of genetic data Haplotypes within the D. quadramaculatus species complex exhibited typical to high levels of genetic diversity. Desmognathus folkertsi exhibited the lowest level of genetic diversity among the three nominal species with a k = 1.16%; whereas, D. quadramaculatus and D. marmoratus exhibited high levels of diversity with a k = 12.02% and k = 20.08%, respectively (Table 1). Both D. quadramaculatus and D. marmoratus exhibited Tajima’ D, Fu and Li’s D*, Fu and Li’s F*, and Fu’s F statistics were not significant; however, these same measures for D. folkertsi were significantly negative (Table 1). This suggests that pop- ulations of D. quadramaculatus and D. marmoratus have been established for a long peri- od of time and have not experienced recent range expansions. However, for D. folkertsi, the phylogeny had shallower branches than expected under the neutral model of evolu- tion, which is consistent with a recent range expansion. When the data were partitioned by river drainage for D. quadramaculatus, the Cat- awba River drainage exhibited the highest levels of diversity k = 184.164; whereas, the Fig. 2. (part 2a) Bayesian phylogram using the 12S rRNA portion of the mitochondrial genome with the maximum-likelihood model of evolution TrN + G for unique haplotypes of D. quadramaculatus, D. folkertsi, and D. marmoratus. Lineage 1 is D. folkertsi. Lineage 2A is basically D. quadramaculatus and D. marmoratus that occur in southern areas including northern Georgia, North Carolina, and southern Ten- nessee. Lineage 2B contains D. quadramaculatus from populations of Georgia, South Carolina, and North Carolina. (part 2b) Northern populations of D. quadramaculatus. Lineage 2C contains populations from Virginia, West Virginia, North Carolina, and Tennessee. Lineage 2C-3 is a clade in Tennessee (see text). Only support values on major nodes are given (bootstrap values from 1000 replicates). See Appendix 1 and 2 for more detail on specific localities and samples. 185Ecology and genetic of Black-bellied Salamanders Tennessee, Savannah, New, Chattahoochee, and Coosa/Tallapoosa River drainages exhib- ited moderate levels of genetic diversity, k = 140.599, k =163.422, k = 112.131, k = 70.099, and k = 156.990, respectively, for mtDNA (Table 2). Only 4.47% of the genetic diversi- ty occurred among drainages but 90.37% of the genetic diversity was partitioned within drainages (Table 3). This pattern is indicative of gene flow across the landscape, either by natural or human-mediated means. The expected heterozygosity values (Hj) calculated using the AFLP data for the two main mitochondrial lineages within the D. quadramaculatus species complex, Lineage 1 (D. folkertsi) and Lineage 2 (D. quadramaculatus plus D. marmoratus) were equal to 0.241 and 0.229, respectively (Table 4). To calculate the expected heterozygosity values (Hj) in only D. quadramaculatus, individuals were divided into two groups using lineages derived from the mitochondrial topology (i.e., Lineages 2A and 2B and Lineages 2C and 2D). Group 1 included D. quadramaculatus individuals from Lineages 2A and 2B, and Group 2 included D. quadramaculatus individuals from Lineages 2C and 2D. Group 1 and Group 2 Table 1. Comparative summary statistics of the mitochondrial sequence variation in Desmognathus qua- dramaculatus, D. folkertsi, and D. marmoratus. π: nucleotide diversity per site with Jukes and Cantor cor- rection; k: mean number of nucleotide differences; η: total number of mutations; θ: the amount of varia- tion expected at each nucleotide site if there is neutral evolution. D. quadramaculatus D. folkertsi D. marmoratus Haplotype diversity ± SD 0.950 ± 0.008 0.822 ± 0.083 0.978 ± 0.054 π ± SD 0.230 ± 0.0154 0.012 ± 0.004 0.333 ± 0.039 K 96.184 6.866 160.667 η 439 61 335 θ per site 0.177 0.028 0.245 Tajima’s D 0.967 (P > 0.10) -2.305 (P < 0.01) 1.785 (0.10 > P > 0.05) Fu and Li’s D* -1.551 (P > 0.10) -2.956 (P < 0.05) 1.166 (P > 0.10) Fu and Li’s F* -0.342 (P > 0.10) -3.227 (P < 0.02) 1.494 (0.05 < P < 0.10) Fu’s Fs statistic 18.63 -2.401 3.569 Table 2. Comparative summary statistics of the 12S rRNA mitochondrial sequence variation in Desmog- nathus quadramaculatus partitioned by river drainage. Symbols as in Table 1. Tennessee (n = 64) Coosa/ Tallapoosa (n = 21) Chattahoochee (n = 14) Savannah (n = 10) New River (n = 10) Catawba (n = 11) Haplotype diversity ± SD 0.977 ± 0.010 0.948 ± 0.031 1.000 ± 0.027 1.000 ± 0.045 0.878 ± 0.025 1.000 ± 0.039 π ± SD 0.227 ± 0.022 0.284 ± 0.035 0.128 ± 0.006 0.283 ± 0.069 0.2013 ± 0.031 0.307 ± 0.056 k 140.599 156.990 74.099 163.422 112.131 184.164 η 538 369 407 388 375 401 θ per site 0.224 0.186 0.220 0.238 0.141 0.228 186 J.A. Wooten and L.J. Rissler exhibited expected heterozygosity (Hj) values equal to 0.222 and 0.237, respectively. There was little genetic differentiation among study sites of D. quadramaculatus with a total gene diversity (Ht) equal to 0.236 and an Fst equal to 0.028 (Table 5). Analysis of molecular variance (AMOVA) for the AFLP data using three populations (Lineage 1, Group 1, and Group 2; see description above) and two groups derived from the maximum-likelihood topology (Fig. 2; Lineage 1 and Lineage 2) supported the mtD- NA conclusions that most of the variation (88.44%) is found within the populations (ΦCT). Table 3. Partition of genetic variation for mitochondrial sequences in Desmognathus quadramaculatus determined by AMOVA. d.f. Sum of squares Variance component Percentage of variation Among groups (lineages*) 1 640.945 3.893 5.160 Among population (drainages**) Within groups 3 440.933 3.376 4.470 Within populations 188 12821.412 68.199 90.370 Total 192 13903.290 75.468 100.000 * Lineages (Fig. 2). ** Five major river drainage basins including New River, Catawba, Tennessee, Chattahoochee, Coosa/Tallapoosa, Savannah were used. Table 4. Genetic diversity derived from AFLP data partitioned by lineage inferred from the mitochondrial genome for Desmognathus quadramaculatus, D. folkertsi, and D. marmoratus. n Number of loci % Polymorphic loci Hj* ± SE Lineage 1 (D. folkertsi) 10 563 71.4 0.241 ± 0.008 Lineage 2A + 2B (D. quadramaculatus and D. marmoratus)** 27 563 68.4 0.222 ± 0.007 Lineage 2C + 2D (D. quadramaculatus and D. marmoratus)*** 31 563 71.9 0.237 ± 0.007 *Hj: Nei’s gene diversity; expected heterozygosity under Hardy-Weinberg genotypic proportions. ** southern populations. *** northern populations Table 5. Genetic diversity derived from AFLP data computed over all populations for Desmognathus qua- dramaculatus. Hw: mean Nei’s gene diversity; mean within-population expected heterozygosity under Har- dy-Weinberg genotypic proportions; Ht: total gene diversity; Hb: Nei’s Dst; average gene diversity among populations in excess of that observed within populations; Fst: Wright’s fixation index; proportion of total gene diversity that occurs among as opposed to within populations. n Hw ± SE Ht ± SE Hb ± SE Fst ± SE 58 0.2293 ± 0.0074 0.2358 ± 0.0013 0.0066 ± 0.0001 0.0279 ± 0.0312 187Ecology and genetic of Black-bellied Salamanders The proportion of the variation explained by among groups (ΦSC) and among populations within groups (ΦST) was small, comprising only 5.94% and 5.62%, respectively (Table 6). Nei’s D value between the Lineage 1 and Lineage 2 (Fig. 2) in the D. quadramaculatus spe- cies complex was 0.028, and that between D. quadramaculatus and D. folkersi was 0.0542. The optimal number of populations (k) computed in Structure v2.2 (Pritchard et al., 2000; Falush et al., 2003; Falush et al., 2007) using the AFLP fragment data was three; however, those three groups were not congruent with the three studied taxa. The estimat- ed probability of three populations (ln P(D)) was equal to –16557.7, with a variance of ln P(D) equal to 1048.5, α = 0.2552, and Fst = 0.3317. Desmognathus folkertsi was placed into the first inferred cluster 43.3% of the time, more often than in either the second or third inferred cluster. Desmognathus quadramaculatus and D. marmoratus were grouped into the first inferred cluster 64.0% of the time, and 10.9% and 25.0% for the second and third inferred clusters, respectively. Testing phylogeographic breaks We examined the impact of river drainages and ecoregions on phylogeographic pat- terns in D. quadramaculatus. We found individuals of D. quadramaculatus to be isolated Table 6. Partition of genetic variation in Desmognathus quadramaculatus determined by AMOVA using AFLP fragment data. d.f. Sum of squares Variance component Percentage of variation Among groups (species*) 1 231.951 5.264 5.940 Among populations (lineages**) within groups 1 222.043 4.978 5.620 Within populations 65 5094.329 78.374 88.440 Total 67 5548.323 88.616 100.000 * D. folkertsi and D. quadramaculatus only Table 7. Multiple regression tests of genetic distance, historical and modern river drainage connections, freshwater and terrestrial ecoregions assignment based on geographic locality, and species group based upon uncorrected genetic distance and current taxonomic assignment. Matrix b P Species-level group* 0.101 0.003 Current drainage basin -0.049 0.085 Historical drainage basin -0.108 0.037 Freshwater ecoregions 0.002 0.474 Terrestrial ecoregions 0.116 0.043 * D. quadramaculatus, D. folkertsi, or D. marmoratus 188 J.A. Wooten and L.J. Rissler by stream (r = 0.335, P < 0.0001) and straight-line (r = 259.526, P = 0.001) distance using the mitochondrial sequences. However, when using the AFLP fragment data, D. quadra- maculatus populations were only isolated by stream distance (r = 0.135, P = 0.024). We found that current taxonomic species, Pliocene river drainage basin, and terrestrial ecore- gions explained the observed phylogeographic pattern in D. quadramaculatus (R2 = 0.017; P = 0.013; Table 7). Neither current river drainage basin nor freshwater ecoregions helped to explain the phylogenetic patterns in D. quadramaculatus (Table 7). DISCUSSION Lineages and population structure – mitochondrial sequences The phylogenetic analysis within the D. quadramaculatus species complex generated from the mitochondrial sequences revealed two major lineages, Lineage 1 and Lineage 2 (Fig. 2). Lineage 1 consisted of only D. folkertsi; Lineage 2 consisted of both northern and southern populations of D. quadramaculatus and D. marmoratus. The lineages of D. mar- moratus formed well-supported branches within D. quadramaculatus. Within Lineage 2, which contained both D. quadramaculatus and D. marmoratus, four well-supported lineages were uncovered (Fig. 2). Overall, Lineage 2 was separated geographically, with distinct northern (Lineage 2C and 2D) and southern (Lineage 2A and 2B) lineages. One lineage of particular interest is a single population of D. qua- dramaculatus found in North Carolina (Lineage 2D) which formed a well-supported and basal relationship to D. quadramaculatus in Lineage 2C (Fig. 2). Because this basal relationship was formed using individuals only from an isolated, single population located north of the French Broad River (Fig. 1, Map Locality 13), future research is underway to investigate surrounding geographic areas to reveal the phylogenetic pat- terning in this area. Overall, Lineage 2C appears to be a polytomy with few well-sup- ported branches, which may be indicative of a recent population expansion. Within Lineage 2C, a single population from Tennessee (Fig. 1, Map Locality 5) appears to be a unique evolutionary lineage that is well nested within Lineage 2C and is of future research interest (Fig. 2). Although our sample size was limited for D. marmoratus due to drought condi- tions during sampling years, the inclusion of these samples in our analyses allowed us to make some comparisons with D. quadramaculatus. Our mitochondrial sequence data suggests that southern D. marmoratus populations are nested within southern D. quad- ramaculatus lineages (See Lineage 2A) with strong statistical support (Fig. 2). However, due to a small sample size for southern D. marmoratus and the fact that we only used one portion of the mitochondrial genome to discern these fine-scale genetic patterns, our results for the relationship between southern D. quadramaculatus and southern D. marmoratus do not corroborate the published phylogenetic relationships that suggest a sister relationship between southern populations of D. marmoratus with D. folkertsi (Jackson, 2005; Jones et al., 2006). For northern populations of D. marmoratus (Lineage 2C), we uncovered a complex, intertwined relationship between D. marmoratus and D. quadramaculatus, where popu- 189Ecology and genetic of Black-bellied Salamanders lations of D. marmoratus are more closely related to populations of D. quadramaculatus, rather than to populations of itself. These results corroborate the findings of other authors, and indicate that D. marmoratus, especially northern populations, may not be an exclu- sive species in relation to D. quadramaculatus (Titus and Larson, 1996; Rissler and Taylor, 2003; Jackson, 2005; Jones et al., 2006). Past research using mitochondrial sequence evidence revealed that D. marmoratus and D. quadramaculatus are not monophyletic (Titus and Larson, 1996; Rissler and Taylor, 2003; Jones et al., 2006); our data revealed similar phylogenetic patterns. Voss et al. (1995) found that of 16 allozyme loci, eight were fixed between populations of D. marmoratus north and south of the Eastern Continental Divide. In an unpublished master’s thesis, Jackson (2005) revealed that D. quadramaculatus and D. marmoratus from populations in the northern part of the geographic range were nested together when phylogenetic analy- sis was completed using three portions of the mitochondrial genome (cytochrome b, ND4, and 12S) and a nuclear gene (GAPDH). More research that includes additional and strate- gic population sampling is needed to tease apart these complex evolutionary relationships among D. folkertsi, D. quadramaculatus, and D. marmoratus. Lineages and population structure – AFLP fragments Our study is one of only a few that used AFLPs to investigate population genetics in plethodontid salamanders (Lowe et al., 2006; Wooten, 2007; Wooten et al., 2010). Our AFLP fragment data were sufficient to differentiate between D. folkertsi and D. quadra- maculatus, but were not able to distinguish between D. marmoratus and D. quadramac- ulatus. The results from the AFLP data and the mitochondrial data were not congruent, indicating that these markers may not be suitable for fine-scale phylogenetic analysis in desmognathan taxa. Regardless, future taxonomic revisions will likely be necessary, because nether D. marmoratus or D. quadramaculatus do not appear to be monophyl- etic lineages. Genetic diversity among populations Patterns of genetic diversity can often be explained by historical drainage connections for many freshwater species, including fishes and amphibians (Mayden, 1988; Burridge et al., 2006; Jones et al., 2006; Kozak et al., 2006). For D. quadramaculatus populations, we found that there was as much genetic variance explained within a single stream as there was between streams or drainages. Semi-aquatic salamander species, including D. folkertsi, D. quadramaculatus, and D. monticola, are typically used as fish bait, particularly for fish- ing for black bass (genus Micropterus), and, therefore, bait-bucket release has been used to explain phylogenetic and disjunct geographic range patterns in some taxa (Martof, 1953; Jensen and Waters, 1999; Bonett et al., 2007). Bait-bucket release may explain some of the genetic partitioning among populations of these and other salamander species (Wooten et al., 2010); however, fine-scale population studies and phylogenetic analysis may reveal that some of these populations are disjunct, relict populations that are isolated and not associ- ated with bait-bucket release (Camp and Wooten, ms under review). 190 J.A. Wooten and L.J. Rissler Phylogeographic breaks and phylogeography Current river drainages do not significantly explain the phylogenetic patterns that are observed in the D. quadramaculatus species complex. In fact, our data suggest that his- torical drainages have played a more important role in shaping these patterns. This is not surprising considering that modern drainages are composites of historical drainages and there is a strong association between historic drainage patterns and phylogenetic relation- ships (Kozak et al., 2006; Jones et al., 2006). In our analyses, individuals from the upper Savannah River drainage form sister relationships with individuals from the lower Ten- nessee River drainage (Fig. 2; quadramaculatus 78-81). In addition, individuals from the Catawba River drainage form relationships with individuals from the New River drainage; it has been hypothesized that the Catawba River was once part of the New River drain- age system (Map Localities 8-10; Jones et al., 2006) and this would explain the observed phylogenetic patterning. It is also possible that the observed phylogeographic patterns are a result of movement among and between populations, across both aquatic and terrestri- al routes. However, it is unknown how far individuals of the D. quadramaculatus species complex move across either route; it is difficult to estimate dispersal rates and distances (Milá et al., 2010) and these data are absent for most species (Grant et al., 2010). That said, we do know, however, it is not unlikely that during times of flooding that these sala- manders can use terrestrial routes for dispersal, augmenting gene flow among populations (Camp and Wooten, ms under review). We also found that the phylogenetic patterns can be explained by breaks in terrestrial, but not freshwater ecoregions. This may help support the idea that salamanders in the D. quadramaculatus complex exhibit phylogenetic pat- terns due to limited dispersal abilities across terrestrial terrain without the aid of flooding, but not freshwater ecoregions, where the salamanders can move along the waterways for dispersal, which may help to stabilize their populations (Grant et al., 2010). In D. quadramaculatus, the patterns of genetic diversity across the landscape have been shaped by historical drainage patterns that have occurred throughout the history of the Appalachian Mountains. Because of the complex topology, including the presence of large rivers and valleys and steep topological relief, many barriers to gene flow exist; phylogeographic patterns of D. quadramaculatus reflect this isolation. We found that pale- odrainage patterns influence the genetic diversity of D. quadramaculatus more than cur- rent drainage patterns. These patterns are similar to those reported in Eurycea bislineata (Kozak et al. 2006), and support the idea that paleodrainages directly influence phyloge- netic patterns in many fish and salamander that are restricted or may rely on water as a means of dispersal. Results presented here further corroborate and extend previous mito- chondrial studies that suggest that historical river drainage patterns influence the phyloge- netic patterns of desmognathan species (Voss et al., 1995; Rissler and Taylor, 2003; Jack- son, 2005; Jones et al., 2006). CONCLUSIONS Undescribed species, parallel evolution, and morphological conservatism are factors that contribute to the complexity of desmognathan taxonomy (Jackson, 2005). Desmog- 191Ecology and genetic of Black-bellied Salamanders nathus quadramaculatus and D. marmoratus exhibited high levels of genetic diversity for desmognathan salamanders; however, this may be due to small sample size for D. marmo- ratus and the presence of undescribed species in D. quadramaculatus. The genetic diver- sity of D. quadramaculatus was not partitioned by current river drainage, but the phylo- geographic patterns may be explained, at least in part, by historical river drainage connec- tions. In addition, terrestrial ecoregions, rather than freshwater ecoregions, explained the genetic distances between individuals. This provides additional evidence that D. quadra- maculatus likely disperses via both terrestrial and aquatic pathways, with gene flow being enhanced through terrestrial routes, but constrained within drainages. Thus, future con- servation programs that may be developed to protect these salamanders should consider the terrestrial environment in addition to stream quality. In summary, more research is needed to tease out the complexly interwoven nature of the evolutionary history shared by these three salamander species. ACKNOWLEDGEMENTS This manuscript is a result of a chapter from the Ph.D. dissertation of Jessica Wooten under the direction of L. Rissler. H. Smith-Somerville, P. Harris, J. Lopez-Bautista, and C. Camp provid- ed comments on earlier versions of the manuscript. Many people helped in the field or obtained tissue for us, including S. Eagle, W. Van Devender, A. Van Devender, C. Camp, D. Beamer, M. Chadwick, C. Cox, S. Parker, J. Hodgson, D. Merritt, J. Humphries, J. Waldron, Z. Felix, B. Sut- ton, R. Makowsky, C. Makowsky, S. Fields, and W. Smith. W. Holznagel, L. Tolley-Jordan, and E. Toorens provided assistance with the laboratory work and AFLP fragment analysis. P. 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Li ne ag e Sp ec ie s M ap Lo ca lit y R iv er D ra in ag e C ou nt y St at e Lo ca lit y La tit ud e Lo ng itu de A cc es si on N um be r 1 Fo lk er ts i 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 14 41 33 1 Fo lk er ts i 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 14 41 34 1 Fo lk er ts i 44 Te nn es se e U ni on G A W es t F or k W ol f C re ek 34 .7 68 83 .9 46 EU 14 41 63 1 Fo lk er ts i 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 14 41 36 1 Fo lk er ts i 49 Te nn es se e U ni on G A H el to n C re ek 34 .7 49 83 .9 26 EU 14 41 48 1 Fo lk er ts i 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 14 41 35 1 Fo lk er ts i 35 C oo sa /T al la po os a G ilm er G A H ol ly C re ek 34 .7 95 84 .6 03 EU 14 41 46 1 Fo lk er ts i 44 Te nn es se e U ni on G A W es t F or k W ol f C re ek 34 .7 68 83 .9 46 EU 14 42 03 1 Fo lk er ts i 44 Te nn es se e U ni on G A W es t F or k W ol f C re ek 34 .7 68 83 .9 46 EU 14 42 04 1 Fo lk er ts i 43 Te nn es se e U ni on G A C oo pe r’s C re ek 34 .7 90 84 .0 31 EU 14 41 39 1 Fo lk er ts i 43 Te nn es se e U ni on G A C oo pe r’s C re ek 34 .7 90 84 .0 31 EU 14 41 41 1 Fo lk er ts i 32 Te nn es se e C la y N C M us kr at B ra nc h 35 .0 47 83 .6 09 EU 14 42 10 1 Fo lk er ts i 49 Te nn es se e U ni on G A C oo pe r’s C re ek 34 .7 49 83 .9 26 EU 14 41 40 1 Fo lk er ts i 49 Te nn es se e U ni on G A H el to n C re ek 34 .7 49 83 .9 26 EU 14 41 50 1 Fo lk er ts i 49 Te nn es se e U ni on G A H el to n C re ek 34 .7 49 83 .9 26 EU 14 41 52 1 Fo lk er ts i 49 Te nn es se e U ni on G A H el to n C re ek 34 .7 49 83 .9 26 EU 14 41 57 1 Fo lk er ts i 49 Te nn es se e U ni on G A H el to n C re ek 34 .7 49 83 .9 26 EU 14 41 51 1 Fo lk er ts i 43 Te nn es se e U ni on G A C oo pe r’s C re ek 34 .7 90 84 .0 31 EU 14 41 38 1 Fo lk er ts i 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 14 41 86 1 Fo lk er ts i 56 Sa va nn ah C la y N C Tr ib ut ar y of H ia w as se e R iv er 34 .7 60 84 .0 04 EU 14 41 45 1 Fo lk er ts i 49 Te nn es se e U ni on G A H el to n C re ek 34 .7 49 83 .9 26 EU 14 41 53 1 Fo lk er ts i 49 Te nn es se e U ni on G A H el to n C re ek 34 .7 49 83 .9 26 EU 14 41 54 1 Fo lk er ts i 20 Sa va nn ah Pi ck en s SC Ea st at oe C re ek 35 .0 51 82 .8 19 EU 14 42 21 2- A 1 m ar m or at us 50 Sa va nn ah R ab un G A M oc ca si n C re ek 34 .8 44 83 .5 87 EU 55 22 79 2- A 1 m ar m or at us 50 Sa va nn ah R ab un G A M oc ca si n C re ek 34 .8 44 83 .5 87 EU 59 91 25 2- A 1 m ar m or at us 53 C ha tt ah oo ch ee W hi te G A Sm ith ga ll W oo ds 34 .6 90 83 .7 70 EU 59 91 26 200 J.A. Wooten and L.J. Rissler Li ne ag e Sp ec ie s M ap Lo ca lit y R iv er D ra in ag e C ou nt y St at e Lo ca lit y La tit ud e Lo ng itu de A cc es si on N um be r 2- A 2 m ar m or at us 10 C at aw ba C al dw el l N C D ix on C re ek 36 .1 07 81 .7 82 EU 55 23 22 2- A 2 m ar m or at us 10 C at aw ba C al dw el l N C D ix on C re ek 36 .1 07 81 .7 82 EU 55 23 21 2- C 1 m ar m or at us 10 C at aw ba C al dw el l N C D ix on C re ek 36 .1 07 81 .7 82 EU 55 23 20 m ar m or at us 7 N ew R iv er W at au ga N C H ow ar d C re ek 36 .2 81 81 .7 21 EU 14 42 12 m on tic ol a 39 C oo sa /T al la po os a G ilm er G A H ol ly C re ek 34 .6 63 84 .4 41 EU 55 22 29 m on tic ol a 39 C oo sa /T al la po os a G ilm er G A O w lto w n C re ek 34 .6 63 84 .4 41 EU 55 22 44 m on tic ol a 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 22 72 m on tic ol a 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 23 42 m on tic ol a 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 22 73 m on tic ol a 39 C oo sa /T al la po os a G ilm er G A O w lto w n C re ek 34 .6 63 84 .4 41 EU 55 22 45 m on tic ol a 40 C oo sa /T al la po os a Lu m pk in G A N im be lw ill C re ek 34 .5 82 84 .1 84 EU 55 22 47 m on tic ol a 54 Te nn es se e H ab er sh am G A D em er es t 34 .5 60 83 .5 39 EU 55 22 51 m on tic ol a 49 Te nn es se e U ni on G A H el to n C re ek 34 .7 49 83 .9 26 EU 55 22 50 m on tic ol a 40 C oo sa /T al la po os a Lu m pk in G A N im be lw ill C re ek 34 .5 82 84 .1 84 EU 55 22 48 m on tic ol a 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 55 22 74 m on tic ol a 32 Te nn es se e C la y N C M us kr at B ra nc h 35 .0 47 83 .6 09 EU 14 42 07 m on tic ol a 32 Te nn es se e C la y N C M us kr at B ra nc h 35 .0 47 83 .6 09 EU 14 42 08 m on tic ol a 53 C ha tt ah oo ch ee W hi te G A Sm ith ga ll W oo ds 34 .6 90 83 .7 70 EU 55 22 80 m on tic ol a 39 C oo sa /T al la po os a G ilm er G A H ol ly C re ek 34 .6 63 84 .4 41 EU 14 41 75 2- A 1 qu ad ra m ac ul at us 35 C oo sa /T al la po os a M ur ra y G A La ur el C re ek 34 .7 95 84 .6 03 EU 55 22 30 2- A 1 qu ad ra m ac ul at us 39 C oo sa /T al la po os a M ur ra y G A O w lto w n C re ek 34 .6 63 84 .4 41 EU 55 22 32 2- A 1 qu ad ra m ac ul at us 35 C oo sa /T al la po os a M ur ra y G A La ur el C re ek 34 .7 95 84 .6 03 EU 55 22 31 2- A 1 qu ad ra m ac ul at us 40 C oo sa /T al la po os a Lu m pk in G A N im be lw ill C re ek 34 .5 82 84 .1 84 EU 55 22 34 2- A 1 qu ad ra m ac ul at us 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 22 33 2- A 1 qu ad ra m ac ul at us 10 C at aw ba C al dw el l N C D ix on C re ek 36 .1 07 81 .7 82 EU 55 23 16 2- A 2 qu ad ra m ac ul at us 56 Sa va nn ah C la y N C Tr ib ut ar y of H ia w as se e R iv er 34 .7 60 84 .0 04 EU 14 41 47 2- A 2 qu ad ra m ac ul at us 7 N ew R iv er W at au ga N C H ow ar d C re ek 36 .2 81 81 .7 21 EU 14 42 15 2- A 2 qu ad ra m ac ul at us 7 N ew R iv er W at au ga N C H ow ar d C re ek 36 .2 81 81 .7 21 EU 55 23 13 201Ecology and genetic of Black-bellied Salamanders Li ne ag e Sp ec ie s M ap Lo ca lit y R iv er D ra in ag e C ou nt y St at e Lo ca lit y La tit ud e Lo ng itu de A cc es si on N um be r 2- A 2 qu ad ra m ac ul at us 7 N ew R iv er W at au ga N C H ow ar d C re ek 36 .2 81 81 .7 21 EU 55 23 11 2- A 2 qu ad ra m ac ul at us 12 N ew R iv er Bu rk e N C St ee le ’s C re ek 35 .9 06 81 .8 29 EU 55 23 27 2- A 2 qu ad ra m ac ul at us 7 N ew R iv er W at au ga N C H ow ar d C re ek 36 .2 81 81 .7 21 EU 55 23 09 2- A 2 qu ad ra m ac ul at us 7 N ew R iv er W at au ga N C H ow ar d C re ek 36 .2 81 81 .7 21 EU 55 23 12 2- A 2 qu ad ra m ac ul at us 7 N ew R iv er W at au ga N C H ow ar d C re ek 36 .2 81 81 .7 21 EU 55 23 14 2- A 2 qu ad ra m ac ul at us 12 N ew R iv er Bu rk e N C St ee le ’s C re ek 35 .9 06 81 .8 29 EU 55 23 31 2- A 2 qu ad ra m ac ul at us 11 N ew R iv er A ve ry N C N or th H ar pe r C re ek 36 .0 07 81 .8 55 EU 55 23 37 2- A 2 qu ad ra m ac ul at us 11 N ew R iv er A ve ry N C N or th H ar pe r C re ek 36 .0 07 81 .8 55 EU 55 23 33 2- A 2 qu ad ra m ac ul at us 10 C at aw ba C al dw el l N C G re en M ou nt ai n C re ek 36 .1 07 81 .7 82 EU 55 23 23 2- A 2 qu ad ra m ac ul at us 11 N ew R iv er A ve ry N C N or th H ar pe r C re ek 36 .0 07 81 .8 55 EU 55 23 35 2- A 2 qu ad ra m ac ul at us 11 N ew R iv er A ve ry N C N or th H ar pe r C re ek 36 .0 07 81 .8 55 EU 55 23 36 2- A 2 qu ad ra m ac ul at us 12 N ew R iv er Bu rk e N C St ee le ’s C re ek 35 .9 06 81 .8 29 EU 55 23 28 2- A 2 qu ad ra m ac ul at us 12 N ew R iv er Bu rk e N C St ee le ’s C re ek 35 .9 06 81 .8 29 EU 55 23 29 2- A 2 qu ad ra m ac ul at us 11 N ew R iv er A ve ry N C N or th H ar pe r C re ek 36 .0 07 81 .8 55 EU 55 23 34 2- A 2 qu ad ra m ac ul at us 27 Te nn es se e G ra ha m N C Ta po co 35 .3 81 83 .9 01 EU 55 23 00 2- A 3 qu ad ra m ac ul at us 21 Sa va nn ah Pi ck en s SC W ild ca t C re ek 35 .0 37 82 .7 08 EU 55 22 37 2- A 3 qu ad ra m ac ul at us 23 Te nn es se e Ja ck so n N C Pu m pk in to w n C re ek 35 .2 51 83 .2 89 EU 55 22 95 2- A 3 qu ad ra m ac ul at us 22 Te nn es se e M ac on N C R ic km an C re ek 35 .2 73 83 .4 01 EU 55 23 03 2- A 3 qu ad ra m ac ul at us 22 Te nn es se e M ac on N C R ic km an C re ek 35 .2 73 83 .4 01 EU 55 23 02 2- A 3 qu ad ra m ac ul at us 26 Sa va nn ah O co ne e SC Tr ib ut ar y of C ha tt og a R iv er 34 .8 08 83 .1 21 EU 55 23 55 2- A 3 qu ad ra m ac ul at us 26 Sa va nn ah O co ne e SC Tr ib ut ar y of C ha tt og a R iv er 34 .8 08 83 .1 21 EU 55 23 56 2- A 3 qu ad ra m ac ul at us 23 Te nn es se e Ja ck so n SC Pu m pk in to w n C re ek 35 .2 51 83 .2 89 EU 55 22 96 2- A 4 qu ad ra m ac ul at us 36 C oo sa /T al la po os a G ilm er N C R oc k C re ek 34 .7 81 84 .3 28 EU 55 22 60 2- A 4 qu ad ra m ac ul at us 36 C oo sa /T al la po os a G ilm er G A R oc k C re ek 34 .7 81 84 .3 28 EU 55 22 61 2- A 4 qu ad ra m ac ul at us 37 C ha tt ah oo ch ee G ilm er G A St an le y C re ek 34 .7 83 84 .3 03 EU 55 22 63 2- A 4 qu ad ra m ac ul at us 37 C ha tt ah oo ch ee G ilm er G A R oc k C re ek 34 .7 83 84 .3 03 EU 55 22 62 2- A 4 qu ad ra m ac ul at us 35 C oo sa /T al la po os a G ilm er G A H ol ly C re ek 34 .7 95 84 .6 03 EU 14 41 76 2- A 4 qu ad ra m ac ul at us 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 55 22 75 202 J.A. Wooten and L.J. Rissler Li ne ag e Sp ec ie s M ap Lo ca lit y R iv er D ra in ag e C ou nt y St at e Lo ca lit y La tit ud e Lo ng itu de A cc es si on N um be r 2- A 4 qu ad ra m ac ul at us 47 Te nn es se e U ni on G A H el to n C re ek 34 .7 48 83 .9 09 EU 14 41 93 2- A 4 qu ad ra m ac ul at us 47 Te nn es se e U ni on G A H el to n C re ek 34 .7 48 83 .9 09 EU 14 41 92 2- A 4 qu ad ra m ac ul at us 47 Te nn es se e U ni on G A H el to n C re ek 34 .7 48 83 .9 09 EU 14 41 89 2- A 4 qu ad ra m ac ul at us 43 Te nn es se e U ni on G A C oo pe r’s C re ek 34 .7 90 84 .0 31 EU 14 42 05 2- A 4 qu ad ra m ac ul at us 42 Te nn es se e U ni on G A Fl at C re ek 34 .7 48 84 .0 26 EU 14 41 97 2- A 4 qu ad ra m ac ul at us 43 Te nn es se e U ni on G A C oo pe r’s C re ek 34 .7 90 84 .0 31 EU 14 42 02 2- A 4 qu ad ra m ac ul at us 44 Te nn es se e U ni on G A W ol f C re ek 34 .7 68 83 .9 46 EU 55 22 81 2- A 4 qu ad ra m ac ul at us 28 Te nn es se e M on ro e T N Tu rk ey C re ek 35 .3 47 84 .1 93 EU 55 23 72 2- A 4 qu ad ra m ac ul at us 28 Te nn es se e M on ro e T N Tu rk ey C re ek 35 .3 47 84 .1 93 EU 55 23 71 qu ad ra m ac ul at us 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 55 22 77 qu ad ra m ac ul at us 33 Te nn es se e Po lk T N Bi g Lo st C re ek 35 .1 60 84 .4 69 EU 55 23 69 qu ad ra m ac ul at us 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 14 41 87 qu ad ra m ac ul at us 27 Te nn es se e G ra ha m N C Ta po co 35 .3 81 83 .9 01 EU 55 23 01 qu ad ra m ac ul at us 29 Te nn es se e M on ro e T N Tr ib ut ar y of T el lic o R iv er 35 .2 58 84 .0 90 EU 55 23 74 qu ad ra m ac ul at us 29 Te nn es se e M on ro e T N Tr ib ut ar y of T el lic o R iv er 35 .2 58 84 .0 90 EU 55 23 75 qu ad ra m ac ul at us 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 55 22 76 qu ad ra m ac ul at us 22 Te nn es se e M ac on N C R ic km an C re ek 35 .2 73 83 .4 01 EU 55 23 03 qu ad ra m ac ul at us 16 Te nn es se e Sw ai n N C N ol an d C re ek 36 .2 81 81 .7 21 EU 55 23 05 qu ad ra m ac ul at us 16 Te nn es se e Sw ai n N C N ol an d C re ek 36 .2 81 81 .7 21 EU 55 23 06 qu ad ra m ac ul at us 15 Te nn es se e Bl ou nt T N A br am ’s C re ek 35 .6 08 83 .9 36 EU 55 23 61 qu ad ra m ac ul at us 14 Te nn es se e Se vi er T N La ur el F al ls 35 .6 67 83 .5 97 EU 55 23 66 qu ad ra m ac ul at us 14 Te nn es se e Se vi er T N La ur el F al ls 35 .6 67 83 .5 97 EU 55 23 67 2- B 1 qu ad ra m ac ul at us 55 C ha tt ah oo ch ee R ab un G A N an cy to w n C re ek 34 .5 04 83 .4 81 EU 55 22 35 2- B 1 qu ad ra m ac ul at us 55 C ha tt ah oo ch ee R ab un G A N an cy to w n C re ek 34 .5 04 83 .4 81 EU 55 22 56 2- B 1 qu ad ra m ac ul at us 55 C ha tt ah oo ch ee R ab un G A N an cy to w n C re ek 34 .5 04 83 .4 81 EU 55 22 57 2- B 1 qu ad ra m ac ul at us 55 C ha tt ah oo ch ee R ab un G A N an cy to w n C re ek 34 .5 04 83 .4 81 EU 55 22 58 2- B 1 qu ad ra m ac ul at us 54 C ha tt ah oo ch ee H ab er sh am G A D em er es t 34 .5 60 83 .5 39 EU 55 22 52 2- B 1 qu ad ra m ac ul at us 54 C ha tt ah oo ch ee H ab er sh am G A D em er es t 34 .5 60 83 .5 39 EU 55 22 54 203Ecology and genetic of Black-bellied Salamanders Li ne ag e Sp ec ie s M ap Lo ca lit y R iv er D ra in ag e C ou nt y St at e Lo ca lit y La tit ud e Lo ng itu de A cc es si on N um be r 2- B 1 qu ad ra m ac ul at us 54 C ha tt ah oo ch ee H ab er sh am G A D em er es t 34 .5 60 83 .5 39 EU 55 22 53 2- B 1 qu ad ra m ac ul at us 24 Te nn es se e M ac on N C G le nn F al ls 35 .0 35 83 .2 37 EU 55 22 86 2- B 1 qu ad ra m ac ul at us 24 Te nn es se e M ac on N C G le nn F al ls 35 .0 35 83 .2 37 EU 55 22 87 2- B 1 qu ad ra m ac ul at us 22 Te nn es se e M ac on N C R ic km an C re ek 35 .2 73 83 .4 01 EU 55 23 04 2- B 1 qu ad ra m ac ul at us 25 Sa va nn ah O co ne e SC Tr ib ut ar y of C ha tt og a R iv er 34 .9 74 83 .1 10 EU 55 23 51 2- B 1 qu ad ra m ac ul at us 25 Sa va nn ah O co ne e SC Tr ib ut ar y of C ha tt og a R iv er 34 .9 74 83 .1 10 EU 55 23 52 2- B 1 qu ad ra m ac ul at us 23 Te nn es se e Ja ck so n N C Pu m pk in to w n C re ek 35 .2 51 83 .2 89 EU 55 22 94 qu ad ra m ac ul at us 26 Sa va nn ah O co ne e SC Tr ib ut ar y of C ha tt og a R iv er 34 .8 08 83 .1 21 EU 55 23 54 2- B 1 qu ad ra m ac ul at us 19 Te nn es se e Tr an sy lv an ia N C Lo ok in g G la ss F al ls 35 .2 89 82 .7 61 EU 55 22 88 2- B 1 qu ad ra m ac ul at us 23 Te nn es se e Ja ck so n N C Pu m pk in to w n C re ek 35 .2 51 83 .2 89 EU 55 22 97 2- B 1 qu ad ra m ac ul at us 21 Sa va nn ah Pi ck en s SC W ild ca t C re ek 35 .0 37 82 .7 08 EU 55 22 36 2- B 1 qu ad ra m ac ul at us 18 Te nn es se e H ay w oo d N C H un gr y C re ek 35 .3 68 82 .8 17 EU 55 22 89 2- B 1 qu ad ra m ac ul at us 18 Te nn es se e H ay w oo d N C H un gr y C re ek 35 .3 68 82 .8 17 EU 55 22 90 2- B 1 qu ad ra m ac ul at us 21 Sa va nn ah Pi ck en s SC W ild ca t C re ek 35 .0 35 83 .2 37 EU 55 23 53 2- B 1 qu ad ra m ac ul at us 20 Sa va nn ah Pi ck en s SC Ea st at oe C re ek 35 .0 51 82 .8 19 EU 55 23 50 2- B 2 qu ad ra m ac ul at us 40 C oo sa /T al la po os a Lu m pk in G A N im be lw ill C re ek 34 .5 82 84 .1 84 EU 55 22 46 2- B 2 qu ad ra m ac ul at us 37 C ha tt ah oo ch ee G ilm er G A St an le y C re ek 34 .7 83 84 .3 03 EU 55 22 64 2- B 2 qu ad ra m ac ul at us 41 Te nn es se e U ni on G A Su ch es C re ek 34 .7 01 84 .0 40 EU 14 42 06 2- B 2 qu ad ra m ac ul at us 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 14 41 88 2- B 2 qu ad ra m ac ul at us 51 Te nn es se e To w ns G A H ia w as se e R iv er 34 .8 24 83 .7 33 EU 55 22 78 2- B 2 qu ad ra m ac ul at us 52 C ha tt ah oo ch ee H ab er sh am G A Sp oi lc an e C re ek 34 .7 60 83 .7 52 EU 55 22 82 2- B 2 qu ad ra m ac ul at us 32 Te nn es se e C la y N C M us kr at B ra nc h 35 .0 47 83 .6 09 EU 14 42 09 2- B 3 qu ad ra m ac ul at us 40 C oo sa /T al la po os a Lu m pk in G A N im be lw ill C re ek 34 .5 82 84 .1 84 EU 55 22 49 2- B 3 qu ad ra m ac ul at us 44 Te nn es se e U ni on G A W es t F or k of W ol f C re ek 34 .7 68 83 .9 46 EU 14 41 71 2- B 3 qu ad ra m ac ul at us 54 C ha tt ah oo ch ee H ab er sh am G A D em er es t 34 .5 60 83 .5 39 EU 55 22 55 2- B 3 qu ad ra m ac ul at us 34 C oo sa /T al la po os a Fa nn in G A St ar C re ek 34 .8 71 84 .2 03 EU 55 22 70 2- B 3 qu ad ra m ac ul at us 43 Te nn es se e U ni on G A C oo pe r’s C re ek 34 .7 90 84 .0 31 EU 14 42 00 2- B 3 qu ad ra m ac ul at us 43 Te nn es se e U ni on G A C oo pe r’s C re ek 34 .7 90 84 .0 31 EU 14 41 99 204 J.A. Wooten and L.J. Rissler Li ne ag e Sp ec ie s M ap Lo ca lit y R iv er D ra in ag e C ou nt y St at e Lo ca lit y La tit ud e Lo ng itu de A cc es si on N um be r 2- B 3 qu ad ra m ac ul at us 34 C oo sa /T al la po os a Fa nn in G A St ar C re ek 34 .8 71 84 .2 03 EU 55 22 69 2- B 3 qu ad ra m ac ul at us 34 C oo sa /T al la po os a Fa nn in G A St ar C re ek 34 .8 71 84 .2 03 EU 55 22 71 2- B 3 qu ad ra m ac ul at us 44 Te nn es se e U ni on G A W es t F or k of W ol f C re ek 34 .7 68 83 .9 46 EU 14 41 67 2- B 3 qu ad ra m ac ul at us 44 Te nn es se e U ni on G A W es t F or k of W ol f C re ek 34 .7 68 83 .9 46 EU 14 41 68 2- B 3 qu ad ra m ac ul at us 44 Te nn es se e U ni on G A W es t F or k of W ol f C re ek 34 .7 68 83 .9 46 EU 14 41 70 2- B 3 qu ad ra m ac ul at us 47 Te nn es se e U ni on G A H el to n C re ek 34 .7 48 83 .9 09 EU 14 41 94 2- B 3 qu ad ra m ac ul at us 42 Te nn es se e U ni on G A Fl at C re ek 34 .7 48 84 .0 26 EU 14 41 96 2- B 3 qu ad ra m ac ul at us 44 Te nn es se e U ni on G A W es t F or k of W ol f C re ek 34 .7 68 83 .9 46 EU 14 41 69 2- B 3 qu ad ra m ac ul at us 43 Te nn es se e U ni on G A C oo pe r’s C re ek 34 .7 90 84 .0 31 EU 14 41 98 2- B 3 qu ad ra m ac ul at us 36 C oo sa /T al la po os a G ilm er G A R oc k C re ek 34 .7 81 84 .3 28 EU 55 22 59 2- B 3 qu ad ra m ac ul at us 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 23 48 2- B 3 qu ad ra m ac ul at us 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 23 44 qu ad ra m ac ul at us 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 23 49 qu ad ra m ac ul at us 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 23 43 qu ad ra m ac ul at us 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 23 45 qu ad ra m ac ul at us 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 23 47 qu ad ra m ac ul at us 38 C oo sa /T al la po os a G ilm er G A Bi g Tu rn ip to w n C re ek 34 .7 17 84 .3 90 EU 55 23 46 2- B 3 qu ad ra m ac ul at us 37 C ha tt ah oo ch ee G ilm er G A St an le y C re ek 34 .7 83 84 .3 03 EU 55 22 65 2- B 3 qu ad ra m ac ul at us 37 C ha tt ah oo ch ee G ilm er G A St an le y C re ek 34 .7 83 84 .3 03 EU 55 22 66 2- B 3 qu ad ra m ac ul at us 37 C ha tt ah oo ch ee G ilm er G A St an le y C re ek 34 .7 83 84 .3 03 EU 55 22 67 qu ad ra m ac ul at us 2 N ew R iv er N ic ho la s W V C ol lis on C re ek 38 .1 13 81 .1 44 EU 14 41 42 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 23 96 2- C 1 qu ad ra m ac ul at us 46 Te nn es se e U ni on G A U nn am ed tr ib ut ar y of N ot te ly R iv er 34 .7 50 83 .8 50 EU 55 22 68 2- C 1 qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 23 07 2- C 1 qu ad ra m ac ul at us 9 C at aw ba W at au ga N C G re en M ou nt ai n C re ek 36 .1 14 81 .7 78 EU 55 23 18 2- C 1 qu ad ra m ac ul at us 9 C at aw ba W at au ga N C M id dl e Fo rk a t P ay ne ’s Br an ch 36 .1 14 81 .7 78 EU 55 23 24 205Ecology and genetic of Black-bellied Salamanders Li ne ag e Sp ec ie s M ap Lo ca lit y R iv er D ra in ag e C ou nt y St at e Lo ca lit y La tit ud e Lo ng itu de A cc es si on N um be r qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 22 83 2- C 2 qu ad ra m ac ul at us 2 N ew R iv er N ic ho la s W V C ol lis on C re ek 38 .1 13 81 .1 44 EU 14 41 44 2- C 2 qu ad ra m ac ul at us 4 N ew R iv er G ile s VA La ur el B ra nc h 37 .3 92 80 .6 42 EU 55 23 82 2- C 2 qu ad ra m ac ul at us 2 N ew R iv er N ic ho la s W V C ol lis on C re ek 38 .1 13 81 .1 44 EU 55 22 41 2- C 2 qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 23 15 2- C 2 qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 23 32 2- C 2 qu ad ra m ac ul at us 3 N ew R iv er M er ce r W V U nn am ed tr ib ut ar y of B lu e- st on e R iv er 37 .3 77 81 .2 24 EU 55 24 06 2- C 2 qu ad ra m ac ul at us 4 N ew R iv er G ile s VA La ur el B ra nc h 37 .3 92 80 .6 42 EU 55 23 81 2- C 2 qu ad ra m ac ul at us 4 N ew R iv er G ile s VA La ur el B ra nc h 37 .3 92 80 .6 42 EU 55 23 79 2- C 2 qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 22 84 2- C 2 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 24 02 2- C 2 qu ad ra m ac ul at us 3 N ew R iv er M er ce r W V U nn am ed tr ib ut ar y of B lu e- st on e R iv er 37 .3 77 81 .2 24 EU 55 24 09 2- C 2 qu ad ra m ac ul at us 2 N ew R iv er N ic ho la s W V C ol lis on C re ek 38 .1 13 81 .1 44 EU 55 22 38 2- C 2 qu ad ra m ac ul at us 4 N ew R iv er G ile s VA La ur el B ra nc h 37 .3 92 80 .6 42 EU 55 23 83 2- C 2 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 24 03 2- C 2 qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 22 98 2- C 2 qu ad ra m ac ul at us 4 N ew R iv er G ile s VA La ur el B ra nc h 37 .3 92 80 .6 42 EU 55 23 80 2- C 2 qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 23 08 2- C 2 qu ad ra m ac ul at us 3 N ew R iv er M er ce r W V U nn am ed tr ib ut ar y of B lu e- st on e R iv er 37 .3 77 81 .2 24 EU 55 24 13 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 57 206 J.A. Wooten and L.J. Rissler Li ne ag e Sp ec ie s M ap Lo ca lit y R iv er D ra in ag e C ou nt y St at e Lo ca lit y La tit ud e Lo ng itu de A cc es si on N um be r 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 60 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 68 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 65 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 73 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 64 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 58 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 59 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 63 2- C 3 qu ad ra m ac ul at us 5 Te nn es se e Su lli va n T N H ar pe r’s C re ek 36 .4 75 82 .0 90 EU 55 23 70 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 23 94 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 24 01 qu ad ra m ac ul at us 3 N ew R iv er M er ce r W V U nn am ed tr ib ut ar y of B lu e- st on e R iv er 37 .3 77 81 .2 24 EU 55 24 10 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 24 00 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 24 04 qu ad ra m ac ul at us 2 N ew R iv er N ic ho la s W V C ol lis on C re ek 38 .1 13 81 .1 14 EU 55 22 40 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 23 98 qu ad ra m ac ul at us 4 N ew R iv er G ile s VA La ur el B ra nc h 37 .3 92 80 .6 42 EU 55 23 77 qu ad ra m ac ul at us 3 N ew R iv er M er ce r W V U nn am ed tr ib ut ar y of B lu e- st on e R iv er 37 .3 77 81 .2 24 EU 55 24 12 qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 22 85 qu ad ra m ac ul at us 4 N ew R iv er G ile s VA La ur el B ra nc h 37 .3 92 80 .6 42 EU 55 23 84 qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 23 38 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 23 99 qu ad ra m ac ul at us 3 N ew R iv er M er ce r W V U nn am ed tr ib ut ar y of B lu e- st on e R iv er 37 .3 77 81 .2 24 EU 55 24 11 207Ecology and genetic of Black-bellied Salamanders Li ne ag e Sp ec ie s M ap Lo ca lit y R iv er D ra in ag e C ou nt y St at e Lo ca lit y La tit ud e Lo ng itu de A cc es si on N um be r qu ad ra m ac ul at us 6 N ew R iv er H en de rs on N C W es t F or k of F re nc h Br oa d R iv er 36 .2 27 81 .4 35 EU 55 23 39 qu ad ra m ac ul at us 3 N ew R iv er M er ce r W V U nn am ed tr ib ut ar y of B lu e- st on e R iv er 37 .3 77 81 .2 24 EU 55 24 06 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 23 95 qu ad ra m ac ul at us 2 N ew R iv er N ic ho la s W V C ol lis on C re ek 38 .1 13 81 .1 44 EU 55 22 43 qu ad ra m ac ul at us 4 N ew R iv er G ile s VA La ur el B ra nc h 37 .3 92 80 .6 42 EU 55 23 76 qu ad ra m ac ul at us 1 N ew R iv er Fa ye tt e W V G la de C re ek 38 .1 91 80 .8 97 EU 55 23 97 qu ad ra m ac ul at us 2 N ew R iv er N ic ho la s W V C ol lis on C re ek 38 .1 13 81 .1 44 EU 55 22 39 qu ad ra m ac ul at us 3 N ew R iv er M er ce r W V U nn am ed tr ib ut ar y of B lu e- st on e R iv er 37 .3 77 81 .2 24 EU 55 24 14 qu ad ra m ac ul at us 3 N ew R iv er M er ce r W V U nn am ed tr ib ut ar y of B lu e- st on e R iv er 37 .3 77 81 .2 24 EU 55 24 14 qu ad ra m ac ul at us 9 C at aw ba W at au ga N C G re en M ou nt ai n C re ek 36 .1 14 81 .7 78 EU 55 23 19 qu ad ra m ac ul at us 7 N ew R iv er W at au ga N C H ow ar d C re ek 36 .2 81 81 .7 21 EU 14 42 14 qu ad ra m ac ul at us 2 N ew R iv er N ic ho la s W V C ol lis on C re ek 38 .1 13 81 .1 44 EU 55 22 42 qu ad ra m ac ul at us 7 N ew R iv er W at au ga N C H ow ar d C re ek 36 .2 81 81 .7 21 EU 14 42 13 qu ad ra m ac ul at us 9 C at aw ba W at au ga N C G re en M ou nt ai n C re ek 36 .1 14 81 .7 78 EU 55 23 17 qu ad ra m ac ul at us 8 C at aw ba W at au ga N C M id dl e Fo rk a t P ay ne ’s Br an ch 36 .1 85 81 .6 54 EU 55 23 25 qu ad ra m ac ul at us 8 C at aw ba W at au ga N C M id dl e Fo rk a t P ay ne ’s Br an ch 36 .1 85 81 .6 54 EU 55 23 26 qu ad ra m ac ul at us 12 N ew R iv er Bu rk e N C St ee le ’s C re ek 35 .9 06 81 .8 29 EU 55 23 30 2- D qu ad ra m ac ul at us 13 Te nn es se e M ad is on N C Tr ib ut ar y of B ig L au ra l C re ek 35 .9 83 82 .6 62 EU 55 22 91 2- D qu ad ra m ac ul at us 13 Te nn es se e M ad is on N C Tr ib ut ar y of B ig L au ra l C re ek 35 .9 83 82 .6 62 EU 55 29 2 2- D qu ad ra m ac ul at us 13 Te nn es se e M ad is on N C Tr ib ut ar y of B ig L au ra l C re ek 35 .9 83 82 .6 62 EU 55 29 3 *I nd iv id ua ls fr om lo ca lit ie s 17 , 3 0, 3 1, a nd 4 8 w er e de le te d w he n id en tic al h ap lo ty pe s w er e re m ov ed . V ou ch er n um be rs : 1 U A H C 1 58 24 , 2 U A H C 1 58 25 , 3 U A H C 1 58 27 , 4 U A H C 1 58 26 , 5 U A H C 1 58 29 , 6 U A H C 1 58 30 , 7 U A H C 1 58 32 , 8 U A H C 1 58 31 , 9 U A H C 15 81 9, 10 U A H C 1 58 21 , 1 1 U A H C 1 58 34 , 1 2 U A H C 1 58 45 , 1 3 A PP SU 2 54 02 , 1 4 A PP SU 2 54 03 , 1 5 U A H C 1 58 38 , 1 6 U A H C 1 58 33 , 1 7 U A H C 1 58 42 , 1 8 U A H C 1 58 44 , 19 A PP SU 2 45 04 , 2 0 U A H C 1 58 43 , 2 1 U A H C 1 56 52 , 2 2 U A H C 1 56 53 , 2 3 A PP SU 2 45 09 , 2 4 A PP SU 2 45 07 , 2 5 A PP SU 2 45 10 , 2 6 A PP SU 2 45 05 , 2 7 A PP SU 2 45 06 208 J.A. Wooten and L.J. Rissler Appendix 2. Individuals from Genbank used in the genetic analyses. Lineage Species Accession Number County State Citation monticola AF437369 Giles VA Rissler and Taylor 2003 monticola AF437362 Craig VA Rissler and Taylor 2003 monticola AF437353 Giles VA Rissler and Taylor 2003 monticola AY549660 Giles VA Rissler et. al. 2004 monticola AY549650 Craig VA Rissler et. al. 2004 monticola AF437351 Giles VA Rissler and Taylor 2003 monticola AY549666 Giles VA Rissler et. al. 2004 monticola AY549644 Craig VA Rissler et. al. 2004 monticola AY549657 Giles VA Rissler et. al. 2004 monticola AY549679 Unknown NC Rissler et. al. 2004 monticola AY549673 Montgomery VA Rissler et. al. 2004 monticola AF437369 Giles VA Rissler and Taylor 2003 2-C1 marmoratus AF437329 Caldwell NC Rissler and Taylor 2003 2-C3 marmoratus AF437336 Smyth VA Rissler and Taylor 2003 2-B1 quadramaculatus AF437409 Henderson NC Rissler and Taylor 2003 2-C1 quadramaculatus AF437331 Giles VA Rissler and Taylor 2003 2-C1 quadramaculatus AF437332 Smyth VA Rissler and Taylor 2003 2-C1 quadramaculatus AF437320 Giles VA Rissler and Taylor 2003 2-C1 quadramaculatus AF437325 Giles VA Rissler and Taylor 2003 2-C1 quadramaculatus AF437321 Giles VA Rissler and Taylor 2003 2-C1 quadramaculatus AF437322 Giles VA Rissler and Taylor 2003 2-C1 quadramaculatus AF437326 Giles VA Rissler and Taylor 2003 2-C1 quadramaculatus AF437324 Giles VA Rissler and Taylor 2003 2-C1 quadramaculatus AF437333 Giles VA Rissler and Taylor 2003 quadramaculatus AF437323 Giles VA Rissler and Taylor 2003 quadramaculatus AF437319 Giles VA Rissler and Taylor 2003 quadramaculatus AF437330 Giles VA Rissler and Taylor 2003 quadramaculatus AF437335 Giles VA Rissler and Taylor 2003 quadramaculatus AF437328 Giles VA Rissler and Taylor 2003 quadramaculatus AF437327 Giles VA Rissler and Taylor 2003 quadramaculatus AF437334 Giles VA Rissler and Taylor 2003 2-D quadramaculatus AF437337 Giles VA Rissler and Taylor 2003 aeneus AF437410 Graham NC Rissler and Taylor 2003 fuscus AF437405 Giles VA Rissler and Taylor 2003 fuscus AF437406 Giles VA Rissler and Taylor 2003 imitator AF437408 Unknown TN Rissler and Taylor 2003 wrighti AF437317 Macon NC Rissler and Taylor 2003 wrighti AY135559 Sevier TN Crespi et al. 2003