RANGE-WIDE PHYLOGEOGRAPHY OF THE FOUR-TOED SALAMANDER (HEMIDACTYLIUM SCUTATUM): OUT OF APPALACHIA AND INTO THE GLACIAL AFTERMATH

Timothy A. Herman

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2009

Committee:

Juan Bouzat, Advisor

Christopher Phillips

Karen Root ii ABSTRACT

Juan Bouzat, Advisor

Due to its limited vagility, deep ancestry, and broad distribution, the four-toed salamander (Hemidactylium scutatum) is well suited to track biogeographic patterns across eastern North America. The range of the monotypic genus Hemidactylium is highly disjunct in its southern and western portions, and even within contiguous portions is highly localized around pockets of preferred nesting habitat. Over 330 Hemidactylium genetic samples from 79 field locations were collected and analyzed via mtDNA sequencing of the cytochrome oxidase 1 gene

(co1). Phylogenetic analyses showed deep divergences at this marker (>10% between some haplotypes) and strong support for regional monophyletic clades with minimal overlap. Patterns of haplotype distribution suggest major river drainages, both ancient and modern, as boundaries to dispersal. Two distinct allopatric clades account for all sampling sites within glaciated areas of

North America yet show differing patterns of recolonization. High levels of haplotype diversity were detected in the southern Appalachians, with several members of widely ranging clades represented in the region as well as other unique, endemic, and highly divergent lineages.

Bayesian divergence time analyses estimated the common ancestor of all living Hemidactylium included in the study at roughly 8 million years ago, with the most basal splits in the species confined to the Blue Ridge Mountains. This pattern of radiation from the southern Appalachians parallels that of the “Out of Appalachia” hypothesis of the geographic origin of the lungless salamanders, and lends further support to the importance of this region as a generator of biodiversity in eastern North America.

iii

Female four-toed salamander (Hemidactylium scutatum) attending eggs in Sphagnum moss, Walton County, Florida. iv ACKNOWLEDGMENTS

First and foremost I would like to thank my wife, Maria Herman, for the many long hours she spent driving through the night and hunched over moss, as well as her seemingly interminable patience, which allowed me to accomplish this daunting project. I would also like to thank Jeremy Ross for his generous contributions towards driving, collecting samples, and assistance in the laboratory.

I thank J. Corser and K. Enge for providing assistance in the field, tissue samples, and collecting localities. I thank R. Bonett, Z. Felix, J. Gilhen, and G. Lipps for providing tissue samples for analysis and help with collecting locations. I thank many who helped with sample collection in the field: K. Bekker, B. Bogaczyk, J. Boundy, C. Carpenter, R. Chalmers, B.

Gasdorf, I. Guenther, K. Hamed, T. Majure, K. McGrath, P. Moler, J. Petranka, C. Phillips, B.

Roller, V. Schneider, J. Settles, K. Stanford, E. Timpe, and L. Williams. I also thank many more who assisted with identifying collecting localities: R. Altig, S. Bennett, A. Braswell, A. Breisch,

J. Briggler, C. Brune, C. Camp, M. Eliott, J. Gardner, W. Gibbons, J. Gillingham, S. Graham, C.

Guyer, C. Hall, S. Hall, J. Harrison, A. Hebda, D. Hipes, J. Hohman, K. Irwin, J. Jensen, T.

Johnson, R. Jones, S. Kilpatrick, T. Mann, J. MacGregor, R. Montanucci, K. Morris, T. Pauley,

R. Pfingsten, S. Roble, A. Sanders, M. Sasser, D. Saugey, D. Stevenson, M. Sisson, J. Skeen, J.

Taylor, S. Trauth, T. Walsh, and C. Wilson. I thank my father, Michael Herman, for assisting with a portion of travel, and the Toledo Naturalists’ Association for partial funding of labwork through the Harold F. Mayfield Grant. I thank my committee members and R. Bonett for helpful comments on the manuscript. Finally I thank the Toledo Zoo for professional development funding and R. Andrew Odum and the rest of the zoo’s Department of Herpetology for accommodating my many collecting trips. v TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

METHODS ...... 6

Sampling ...... 6

DNA Extraction, Amplification, and Sequencing ...... 7

Phylogeographic Analysis ...... 8

RESULTS ...... 14

Sequence Variation ...... 14

Genetic Structure and Patterns of Distribution ...... 14

Mismatch Distributions and Tests of Recent Expansion ...... 22

Mantel Tests of Barriers...... 24

Divergence Time Estimates ...... 25

DISCUSSION ...... 27

Conservation Implications ...... 38

Conclusions ...... 39

REFERENCES ...... 42

APPENDICES ....…………………………………………………………………… 49

1 Number of samples and haplotypes/clades detected at each collecting

locality included in the study ...... 50

2 Results of Tajima’s D and Fu’s Fs tests...... 52

3 Haplotype networks of individual clades ...... 53

4 Topographic relief map of the southern Appalachian Mountains, vi showing major physiographic features and proposed hydrological

barriers to dispersal discussed in the text ...... 56

5 Review of co-distributed taxa with phylogeographic patterns

concordant with those resolved in Hemidactylium ...... 57

LIST OF FIGURES

Figure Page

1 Documented range of Hemidactylium scutatum, showing the 79 sampling

locations included in this study...... 5

2 Bayesian 50% majority rule consensus phylogram generated from 77 unique

ingroup co1 haplotype sequences and 2 outgroup sequences...... 15

3 Map of geographic distribution of major clades and subclades as defined by

phylogenetic analyses...... 17

4 Haplotype networks generated by TCS overlaid on the distribution of

Hemidactylium scutatum ...... 21

5 Observed pairwise mismatch distributions for all samples together, and for

Clades A through E ...... 23

6 Generalized map of phylogeographic patterns of expansion resolved in

Hemidactylium scutatum...... 28 vii LIST OF TABLES

Table Page

1 Distribution of haplotype and nucleotide diversity and haplotypes

among clades and number of sites and samples where representatives

from each clade were detected ...... 16

2 Mean uncorrected genetic distance [SE] -- between clades (below diagonal),

and within clades (on diagonal, in boldface type)...... 18

3 Results of partial Mantel tests of hypothesized geographic barriers to

dispersal ...... 24

4 Divergence time estimates for selected clades and subclades generated by

the Bayesian coalescent approach in BEAST ...... 26

1 INTRODUCTION

The forests, mountains, and waterways of eastern North America are renowned for their biodiversity. Many taxa have undergone extensive radiations in this region that occur nowhere else on earth, such as radiations of over 330 crayfish species in the family Cambaridae (Crandall and Buhay 2008), nearly 200 darter species in the subfamily Etheostomatinae (FishBase 2009;

Jelks et al. 2008), and 293 mussel species in the order Unionoida (MUSSEL Project 2009).

Similarly, eastern North America is the global center of diversity for living salamanders (order

Caudata), with 7 of the 10 living families represented in the region. Of these, the Plethodontidae

(Lungless salamanders) is the most diverse, currently containing 383 (67%) of the world’s described caudates. Since Wilder and Dunn first published on the subject in 1920 it has been suggested that the origin of all plethodontids resides in the southern Appalachian Mountains, a scenario known as the “Out of Appalachia” hypothesis. Though the precise mode and cause of the loss of lungs has been contested in recent years (Ruben and Boucot 1989; Beachy and Bruce

1992; Ruben et al. 1993), the geographic location of the evolution of this family has been largely accepted.

Several plethodontid genera are endemic to the eastern United States and four of these,

Phaeognathus, Haideotriton, Stereochilus, and Hemidactylium, are monotypic. Moreover,

Phaeognathus, Haideotriton, and Stereochilus are all localized in distribution to specific habitats in the southeastern United States. In contrast, Hemidactylium scutatum, the four-toed salamander, ranges across one of the broadest geographical extents of any plethodontid (Figure

1). This distribution, from Nova Scotia to northern Florida in the east, and west to Minnesota,

Oklahoma, and Louisiana, is regarded as widely contiguous throughout New England and the eastern United States, but highly disjunct in the southern and western portions of its range. 2 However, even in portions of the range considered contiguous, Hemidactylium populations tend to be highly localized and tightly associated with specific habitats. This patchy distribution, in conjunction with the species’ cryptic nature and need for shallow, fishless, and typically mossy wetlands within intact hardwood forests for reproduction (Petranka 1998), has resulted in fourteen states and provinces giving the four-toed salamander special conservation status

(Chalmers and Loftin 2006).

Phylogenetic placement of Hemidactylium within the family has been contested in several publications in recent years, ranging from sister taxa of the entire Plethodontidae (Macey

2005), to sister taxa of the Bolitoglossinae – Spelerpinae clade (Chippindale et al. 2004), to the most deeply rooted taxa of the Bolitoglossinae (Mueller et al. 2004). The Bolitoglossinae is currently the most diverse subfamily of all the salamanders, containing 261 of the 578 described salamander species (AmphibiaWeb 2009). Consistent among all studies is that Hemidactylium was resolved as deeply rooted in the family, representing the sister taxa to a very high proportion of the described extant caudate species (45.2-67.5%). Placement in all studies is corroborated by the extant distribution of Hemidactylium scutatum if the “Out of Appalachia” hypothesis of the origin of the Plethodontidae is accepted (Wilder and Dunn 1920). If Mueller’s placement of

Hemidactylium as a deeply branching taxa within the Bolitoglossinae is accepted, it would be the sole member of this clade east of the Rocky Mountains, making the evolutionary history of the taxon of particular interest among the caudate fauna of eastern North America. Furthermore,

Bayesian and penalized likelihood analyses of complete mitochondrial genomes have generated an age estimate of the Hemidactylium lineage at 107-108 million years old (Mueller 2006), placing the ancestry of this genus among the most ancient of the extant plethodontid taxa.

Prior to this study, no genetic, morphological, habitat or behavioral studies have been 3 conducted in which Hemidactylium have been the subject of rigorous comparison throughout its extensive range (Petranka 1998). Indeed, it has been noted in the past that even basic comparisons of populations for variation in life history or habitat use are lacking (Lannoo 1998).

Vicariant populations such as those in Missouri, Illinois, Indiana, and western Ohio are thought to be relicts of glacial activity and climatic change in recent geological history, isolated for tens of thousands to millions of years (Conant 1960; Phillips et al. 1999; Daniel 1989). However, in some populations, isolation may extend back further in time, as in the case of the geologically ancient Blue Ridge and Ouachita Mountains and along the Gulf Coast – ecosystems beyond the reach of recent glacial exterminations of northern biota. Such an ancient lineage, coupled with the complex geographic and climatic factors surrounding the taxon’s evolutionary history, make

Hemidactylium a fascinating candidate for phylogeographic study, as other authors have indicated in the past (Frost 2000).

The patchy distribution and deep genetic history suggest that the preferred habitat of

Hemidactylium was once more widespread and contiguous in North America. Indeed,

Hemidactylium has been suggested to have a periglacial distribution (Thurow 1997), being broadly distributed across the extent of both the Illinoian and Wisconsinan events. The species reaches high abundances in the shallow marshy wetlands of glacial origin across the upper

Midwest and northeastern United States. Additionally though, Hemidactylium persists south of the glacial maximum in many widespread populations distributed throughout diverse and varied topography. Southern localities of Hemidactylium are very poorly known and thought to be extremely disjunct and uncommon. Several records of the species exist from the Ozark and

Ouachita Mountains of Arkansas and Missouri, though fewer than 10 animals had been reported from Oklahoma, Louisiana, Mississippi, the Florida panhandle, or South Carolina prior to this 4 study. In contrast, records of Hemidactylium exist from several counties in Alabama and

Georgia (Mount 1975; Elliott 2008).

Here we investigate the phylogeography of Hemidactylium scutatum, with a comprehensive comparison of genetic relationships among populations across the entire range of the species. The objectives of this study were to quantify the diversity within the widely dispersed and isolated populations of Hemidactylium, establish patterns of relatedness among these populations, and finally identify potential extrinsic factors that may have generated these observed patterns of genetic connectivity and distribution. Relationships among these populations may help to clarify the barriers to dispersal and routes of expansion, which determined the current extent of the species’ distribution. The history of Hemidactylium scutatum is investigated in the biogeographical context of the “Out of Appalachia” hypothesis for the evolutionary origin of this taxon. Overall distributional patterns and relationships among major clades provided insights into the deepest history of the species, the southern disjunct populations informing early pathways of expansion and isolation, and the northern

Hemidactylium elucidating the patterns of rapid and recent expansion following the retreat of the last glacial advance. In addition to utility in a biogeographical context, we hope that this data on the relationships of the many isolated locales might guide conservation efforts to protect and manage such cryptic and understudied populations. 5

Figure 1 Documented range of Hemidactylium scutatum, showing the 79 sampling locations included in this study. 6 METHODS

Sampling

Tissue samples from Hemidactylium scutatum were collected from 2004-2007 at 79 sites located throughout the range of the species in eastern North America, with a sampling area spanning approximately 17 degrees latitude and over 33 degrees of longitude (Figure 1).

Sampling sites were chosen in an attempt to evenly sample the known range of the species, while utilizing existing distributional records to facilitate the success of collecting trips. In some cases, additional sites were added to resolve geographic boundaries of monophyletic groups, or to improve resolution when multiple divergent haplotypes were detected in relatively close geographic proximity. As a result of the latter, sampling locales reached a higher density in the southern Appalachian Mountains than elsewhere in the range, and conversely were at a lower density in the recently glaciated northern extent of Hemidactylium’s distribution. Due to the cryptic nature of Hemidactylium throughout much of the year, sampling efforts were focused on locating female salamanders tending nests. Nesting date varies with the onset of warmer spring temperatures, a pattern that was found to hold true with both increasing latitude (see Herman and

Enge 2007) and altitude. Collections of samples from nesting females ranged from 18 February in Mississippi to 5 June in Minnesota. The estimation of nesting date was a very effective method of detection for Hemidactylium, resulting in successfully locating the species in several states with fewer than a dozen animals found historically and with no records of reproduction

(Herman, in prep.). For a complete list of collections see Appendix 1.

Material for genetic analysis was collected primarily in the form of complete tails. The natural ability of the four-toed salamander to drop its tail voluntarily (caudal autotomy) when threatened with predation, and to fully regenerate this lost tail, was utilized to collect samples 7 non-lethally from adult and subadult animals (Bowling Green State University IACUC protocol

#04-007). The salamanders were immediately released to the exact location where found following tissue sample collection, and no animals were removed from the field. In cases where no metamorphosed animals were encountered, developing fertile eggs were collected for genetic material. Samples were collected from 295 animals and 40 eggs (total n=335) at 79 sites selected across the entire range of the four-toed salamander (Figure 1), with a median of 5 samples collected per site (mean: 4.24, range: 1-10, s.d.: 1.94). Of these, 320 were collected in the field by TAH, and 15 were collected and donated by other herpetologists throughout the country (see Acknowledgements). All tissues were preserved individually in 95% ethanol for transportation to the laboratory and long-term storage. GPS coordinates were taken for collection localities using Garmin eTrex Legend or GPSMap 60CSx handheld units (Garmin

International, Inc., Olathe, Kansas). GPS and photographic voucher data of salamanders, nests, and habitat were taken for each individual and all data was distributed to the state or federal agency with jurisdiction over the wildlife at each sampling locale.

DNA extraction, amplification, and sequencing

Total DNA was extracted from all samples utilizing standard Proteinase K digestion followed by phenol-chloroform-isoamyl alcohol and salt-precipitation techniques (Chomczynski and Sacchi 1987). Quality and quantity of DNA extractions were assessed through 1% agarose gel electrophoresis. Primers previously published by Hebert et al. (2003), LCO1490 (5' GGT

CAA CAA ATC ATA AAG ATA TTG G 3') and a modification of HCO2198 (5' TAA ACT

TCA GGG TGA CCA AAA AAT YA 3') with the replacement of C with Y at the 3' end, were utilized to amplify ~708 bp of the mitochondrial gene cytochrome oxidase 1 (co1). For genetic comparison of Hemidactylium populations, the co1 gene was selected due to its suitably high rate 8 of variability in the species, its performance at accurately resolving intraspecific phylogenies

(Mueller 2006), and its proposed use as a “genetic barcode” to delineate species (Hebert et al.

2004; Vences et al. 2005). Amplification reactions were prepared in 25 L reaction volumes using approximately 50 ng of template DNA, 80 M dNTPs, 0.4 M of each primer, 2.0 mM

MgCl2, 1 unit of Taq, and 1X Taq buffer (Promega, Madison, WI). The PCR amplification profile included an initial denaturation at 94C for 3 min, 40 cycles of denaturation at 94C for

45 seconds, annealing at 51.5C for 45 seconds, and extension at 72C for 120 seconds, followed by one final 10 minute extension step at 75C. PCR products were then checked on

1% agarose gel, ethanol precipitated, and resuspended in sterile deionized water for direct sequencing.

Purified PCR products were directly sequenced using BigDye version 3.1 dye-terminator sequencing chemistry electrophoresed on an ABI-377 automated gene sequencer (Applied

Biosystems, Foster City, CA). Sequencing reactions were carried out in 5 L volumes, with 15-

20 ng of purified PCR product DNA, 1.0 L of BigDye sequencing reagent, 1X BigDye sequencing buffer, and 10 M primer. All samples were sequenced with the forward primer

LCO1490, and all unique haplotypes, all ambiguous sequences, and at a minimum one sample per sampling locality were re-sequenced with the reverse primer HCO2198. The sequencing profile included an initial denaturation at 96C for 3 min, 30 cycles of denaturation at 96C for

30 seconds, annealing at 50C for 15 seconds, and extension at 60C for 4 minutes, followed by a final cooling to 4C to stop all reactions.

Phylogeographic Analysis

Mitochondrial co1 sequences were manually aligned using BioEdit version 7.0.5.3 (Hall 9 2005). Sequences were checked and corrected for miscalls, and ambiguous and uneven ends were trimmed, resulting in a 576 bp segment used for phylogenetic analyses. The number of variable, conserved, and parsimony informative sites as well as maximum and average uncorrected sequence divergence within and among major clades were calculated using MEGA

4.0. In addition, overall levels of genetic diversity (i.e., nucleotide, and haplotype diversity) were estimated using Arlequin 3.11 (Excoffier et al. 2005). To visualize mutational pathways linking populations, a haplotype network based on statistical parsimony was generated using

TCS 1.2.1 (Clement et al. 2000). The confidence limit for creating connections between haplotypes was set to 95% (10 mutational steps).

To evaluate the relationships among the detected haplotypes from sampled populations, phylogenies were generated using both maximum parsimony and Bayesian inference methods

(Felsenstein 2004). Cytochrome oxidase 1 sequences from Batrachoseps wrightorum and

Eurycea bislineata were used as outgroups in reconstructing phylogenies. All unique haplotype sequences plus outgroups were analyzed using maximum parsimony (MP) with 100 bootstrap replicates in PAUP* version 4.0b10 (Swofford 1998). Parsimony analysis was conducted using heuristic searches of the phylogenetic space and tree-bisection-reconnection branch swapping, with starting trees obtained via ten replicates of random stepwise sequence addition. The

MulTrees option was enabled with MaxTrees set to 100,000 and the steepest descent option not in effect.

MrModeltest 2.2 (Nylander 2004) was used to select the best fit model of base pair substition for the dataset based on hierarchical likelihood ratio testing (hLRT) and the Akaike

Information Criterion (AIC). The MrModeltest output from both hLRT and AIC indicated

GTR+I+ (“rates=invgamma” setting in MrBayes) as the model that best conformed to the co1 10 data. Bayesian analysis was run using MrBayes version 3.1.2 (Ronquist and Huelsenbeck

2003). Simulations were executed with 1 cold and 5 heated chains and prior probability density was left to the default flat Dirichlet model. Each chain was started with a randomly selected tree.

Two parallel analyses were run for 4,000,000 generations, and sampled every 100th generation for a total of 40001 trees. After 4,000,000 generations, standard deviation between runs had dropped to 0.007491 and convergence was first checked by graphically visualizing plots of the ln-likelihood values of run samples against generation time. Stationarity was confirmed by examination of trace plots using Tracer 1.4 (Rambaut and Drummond 2006). The first 25% of

Markov chain samples (n=10,000) were discarded as burn-in, resulting in a total of 30,001 sample trees for inclusion in the posterior distribution. The Bayesian phylogenetic tree was constructed from the majority-rule consensus of the samples in the posterior distribution.

Long Branch Attraction (LBA) is a phenomenon whereby distant outgroups are preferentially connected to long ingroup branches due to an increased probability of identical base pair mutations that are not synapomorphic (see Bergsten 2005 for a review of LBA). LBA can result in incorrect placement of outgroup rooting, which may distort phylogenetic topology, most frequently encountered in parsimony analysis. Due to the lack of closely related extant taxa that could be used as outgroups of Hemidactylium, LBA was considered as a potential confounder to accurate reconstruction of the co1 phylogeny. To test for LBA, three methods outlined by Bergsten (2005) were implemented. First, MP analysis was rerun with the removal of outgroup taxa to check for changes in ingroup topology. Second, MP analysis were run with the inclusion of outgroup taxa and the removal of long-branched ingroup taxa to which outgroup taxa are rooted in the MP analysis of the completed dataset. Finally, concordance in topology and outgroup placement in the MP phylogeny was checked against the topology from 11 phylogenetic methods that incorporate branch length (Bayesian analysis utilizing the

GTR+I+ model). Though these methods do not completely exclude the possibility of LBA, a topology which is consistent across all tests is less likely to be a result of LBA (Bergsten 2005).

To compare the relative degree to which major lineages have differentiated, genetic variation within and among clades was evaluated using MEGA 4.0 (Tamura et al. 2007). Major clades resolved by the MP and Bayesian analyses were compared to calculate mean uncorrected genetic distance (nucleotide p-distance) both within and between clades. Both haplotype and nucleotide diversity within and among clades were also calculated using Arlequin 3.11

(Excoffier et al. 2005).

Once the relationships between haplotypes were established by phylogenetic analysis, the resulting clades were evaluated to understand potential factors explaining their geographic distribution. To test for recent population expansions, Tajima’s D (Tajima 1989) and Fu’s Fs

(Fu 1997) tests were also run in Arlequin on the major clades resolved in phylogenetic analyses.

These tests detect deviations from selective neutrality, indicating a population that is not at equilibrium. Significantly negative values for both D and Fs indicate an excess of young haplotypes expected from a recent demographic expansion. Pairwise mismatch distributions as well as distributions expected under a simulated null model of continuous expansion were generated using Arlequin 3.11 for all ingroup sequences and for each subset of sequences within major clades resolved. These observed and simulated distributions were compared to test significant goodness-of-fit via sum of squared deviation (SSD). A significant p-value for SSD indicates that mutational patterns are not attributable to a sudden demographic expansion and suggests that some extrinsic variable(s) may be affecting populations (Schneider and Excoffier

1999). 12 Phylogeographic breaks between southern clades were tested for significance using similar methodology to that reported by Lemmon et al. (2007) and Martinez-Solano et al. (2007).

Seven hypothesized barriers, based on the presence of modern rivers and inferred ancient river drainages, were chosen given their significance as phylogeographic breaks in prior studies of other taxa (see Soltis et al. 2006), as well as their inferred significance based on the distributional patterns of major clades resolved in this genetic analysis. These included the Arkansas River,

Mississippi River embayment, Black Warrior + Tombigbee Rivers, Coosa + Alabama Rivers,

Apalachicola + Chattahoochee Rivers. For populations within the Blue Ridge, the Little

Tennessee + Toxaway + Keowee Rivers and French Broad River were tested. For each hypothesized barrier test, three matrices were generated for a subset of sampling sites spanning the barrier. In most cases, all representatives of monophyletic clades or relevant regional subclades adjacent to the barrier in question were included in the test. For the Apalachicola +

Chattahoochee test, members of the clades tested from the Blue Ridge were excluded, due to their location north of the headwaters of this drainage system. These matrices were constructed to compare genetic distances, geographic distances, and a binary comparative matrix of location on the same side (0) or on opposite sides (1) of the geographic feature in question (Lemmon et al. 2007; Martinez-Solano et al. 2007). Geographic distance matrices were generated using GPS coordinates collected in the field entered into Geographic Distance Matrix Generator v1.2.1

[Ersts (n.d.), http://biodiversityinformatics.amnh.org/open_source/gdmg] to generate distance measures that factor the curvature of the Earth’s surface into the path traveled (great circle distance). The genetic distance matrices were then tested against the binary matrices of relative barrier position and geographic distance to test for significant correlation using a partial Mantel test (Mantel 1967; Smouse et al. 1986). The use of a partial Mantel test allows two variables to 13 be tested for correlation (genetic distance vs. geographic distance, genetic distance vs. barrier) while simultaneously taking into account the effect of the third variable matrix. This analysis of barriers was conducted in the program XLSTAT (Addinsoft, Paris, France), using the Pearson correlation coefficient and 10,000 random replicates to generate a p-value.

Finally, divergence time estimates were generated for all major clades and a number of statistically supported subclades within the resultant co1 phylogeny to place the evolutionary changes observed in Hemidactylium in a historical context. The time to most recent common ancestor (TMRCA) was calculated using a Bayesian Markov-chain Monte Carlo (MCMC) analysis implemented in the program BEAST 1.4.8 (Drummond and Rambaut 2007). XML input files were generated using the program BEAUti (distributed with BEAST software) including the Batrachoseps wrightorum sequence as an outgroup. Analyses utilized a GTR+I+ model of base pair substitution (as indicated by MrModeltest output described above) and a relaxed molecular clock with an uncorrelated lognormal distribution. The average mutation rate was set to 0.0109406 substitution/site/million years. This rate (S.D. = 0.0011705) was calculated by Mueller’s (2006) study as the average mutation rate of co1 in the Hemidactylium +

Batrachoseps clade since their divergence (unpub. data). The analysis was run under three different root height constraints: unconstrained, constrained to 64-107 m.y. [95% confidence interval of TMRCA of Hemidactylium and Batrachoseps calculated by Vieites et al. (2007)], and constrained to 90-109 m.y. [95% confidence interval of TMRCA of Hemidactylium and

Batrachoseps calculated by Mueller (2006)]. All three scenarios were run using a Bayesian skyline coalescent tree prior with 10 groups. Analyses were run for 10,000,000 generations, and sampled every 1000. Output files were examined using Tracer 1.4 to check for stationarity of the sampling distribution. 14 RESULTS

Sequence Variation

Seventy-seven unique co1 haplotypes (Genbank accession numbers: GQ150399-

GQ150475) were detected among 335 samples from 79 sampling locations, with no more than 3 different haplotypes detected at any given collection site. Excluding outgroups, 115 of 576 characters were parsimony-informative, 440 characters were conserved across all taxa, and 21 represented singletons. No insertions or deletions were detected among ingroup or outgroup taxa. The total number of haplotypes (n=77) was very close to the total number of sampling sites

(n=79), however these values were not indicative of an equitable distribution of haplotypes, with some being widespread among several sites, and others endemic to only one. Uncorrected sequence divergences between distinct haplotypes ranged from 0.17% to 10.42% (mean 4.26%).

Estimates of genetic diversity for the complete sample revealed a haplotype diversity of 0.962

(s.d.=0.005) and nucleotide diversity of 0.038 (s.d.=0.018) (Table 1). Overall, the sequence data showed a pattern of high diversity to the south, with by far the highest diversity centralized in the southern Appalachian Mountains and lower diversity in the northern portions of the range, both in number of haplotypes and divergences between haplotypes (Table 1).

Genetic Structure and Patterns of Distribution

Phylogenies generated using maximum parsimony and Bayesian probability were highly concordant. Bayesian and MP analyses both resolved 5 major monophyletic groups (clades B-F, see Figure 2) with strong statistical support, and Bayesian analysis resolved a sixth less strongly supported clade (A, pp=0.78) comprising a complex and widespread radiation event. These six clades varied dramatically in their distributions. Two clades (A and D) are widely dispersed, and constitute roughly 90% of the geographic extent of the known range of Hemidactylium. 15

Figure 2. Bayesian 50% majority rule consensus phylogram generated from 77 unique ingroup co1 haplotype sequences and two outgroup sequences. Letter codes next to haplotype numbers refer to the collecting locality at which the sample was obtained (see Appendix 1). A plus symbol (+) next to this locality code indicates that the haplotype was detected at more than one location. Open circles denote nodes resolved by maximum parsimony analysis with >50% bootstrap probability. Numbers above each node are Bayesian posterior probabilities, while those below each node are MP bootstrap values. Letters A through F and italicized names delineate major clades and subclades referred to in the text. 16 These two clades are clearly subdivided further into several statistically supported subclades

correlated in distribution with specific geographic features (Southwest, Teays, Ouachita,

Northeast, Ozark, Midwest; Figures 2-3). Conversely, clades E and F are the most divergent, yet

most restricted in distribution. All clades are distributed allopatrically with the exception of the

Teays subclade of clade A (see Figure 3). This subclade occurs sympatrically with three other

haplotypes, two from clade A, one from clade D, at three different sites on the periphery of its

distribution. The relationships among clades A-D are not clearly resolved with the co1 mtDNA

marker in this study, resulting in a polytomy at their base in both MP and Bayesian analyses.

Clade A contained the highest diversity, with 33 haplotypes, while clade F contained only 3

(Table 1). Gene diversity varied among clades, from a low of 0.77 for clade D to a high of 0.93

in the widespread and divergent clade A (Table 1). Mean within clade genetic distances

(uncorrected p-nucleotide) ranged from 0.23 to 3.16%, while between clades ranged from 3.77 to

9.46% (Table 2). Mean genetic distance to outgroups ranged from 19.73 to 21.54%.

Table 1. Distribution of haplotype and nucleotide diversity and haplotypes among clades and number of sites and samples where representatives from each clade were detected.

# samples # locations # haplotypes Haplotype Diversity (SD) Nucleotide Diversity (SD)

All Samples 335 79 77 0.962 (+/- 0.005) 0.0376 (+/- 0.0184) A-NE/SW 131 33 33 0.932 (+/- 0.010) 0.0193 (+/- 0.0097) B-AL+FL 39 8 11 0.887 (+/- 0.027) 0.0114 (+/- 0.0061) C-SBR+GA 33 11 13 0.928 (+/- 0.019) 0.0132 (+/- 0.0070) D-Midwest 112 24 12 0.774 (+/- 0.031) 0.0068 (+/- 0.0038) E-SC 15 3 5 0.810 (+/- 0.059) 0.0280 (+/- 0.0148) F-Blount 5 1 3 0.800 (+/- 0.164) 0.0021 (+/- 0.0018)

17

Figure 3. Map of geographic distribution of major clades and subclades as defined by phylogenetic analyses. The dashed white line indicates the southern limit of Pleistocene glaciations and outlines the unglaciated Driftless Area of the upper Mississippi River Valley. 18

Table 2. Mean uncorrected genetic gistance [SE] -- between clades (below diagonal), and within clades (on diagonal, in boldface type).

A-NE/SW B-AL+FL C-SBR+GA D-Midwest E-SC F-Blount outgroups A-NE/SW 0.0207 [0.0031] B-AL+FL 0.0494 [0.0072] 0.0136 [0.0028] C-SBR+GA 0.0377 [0.0058] 0.0496 [0.0072] 0.0154 [0.0027] D-Midwest 0.0401 [0.0058] 0.0593 [0.0079] 0.0392 [0.0063] 0.0105 [0.0023] E-SC 0.0596 [0.0073] 0.0745 [0.0088] 0.0600 [0.0076] 0.0600 [0.0078] 0.0316 [0.0046] F-Blount 0.0807 [0.0100] 0.0946 [0.0110] 0.0813 [0.0102] 0.0794 [0.0101] 0.0832 [0.0094] 0.0023 [0.0016] outgroups 0.2045 [0.0111] 0.2154 [0.0117] 0.2055 [0.0115] 0.2029 [0.0118] 0.2042 [0.0107] 0.1973 [0.0115] 0.2014 [0.0148]

Clade A as defined by the Bayesian analysis is comprised of the remainder of ingroup

taxa not included within the 5 more strongly supported clades. Within clade A are several

subclades strongly supported by both Bayesian and MP analyses with clearly defined geographic

distributions (see Figure 2: Ouachita, SW, Teays, NE subclades) as well as several divergent

lineages found only at single sampling localities in the south-central Appalachians (Unicoi,

Sevier, Avery, Scott). The taxa within clade A are distributed in two disparate geographic

regions in the northeast and southwest of Hemidactylium’s current range, effectively bisected by

the distributions of clades B through F. Within these two regions the members of clade A are

contiguously distributed, however neither the Bayesian nor MP analyses resolved the

northeastern or southwestern haplotypes as monophyletic with the co1 gene. The post-glacial

northeast is populated by three haplotypes within clade A, two of which vary by a single base

pair and are found from the northern Blue Ridge of Virginia to the coastal plain and north to

Nova Scotia. The third haplotype, varying by 9 base pairs, was also detected at two sites in

North Carolina. Bayesian analysis placed these three haplotypes into a single monophyletic

group, here referred to as the Northeast subclade, while MP did not resolve the relationship

between the two more divergent northeast haplotypes. 19 Clade B is distributed from Dugger Mountain in northeastern Alabama throughout central and southern Alabama and along the Florida panhandle. Clade B spans the Apalachicola

River south of the Chattahoochee/Flint River confluence, though both MP and Bayesian analysis resolved the populations east of the Apalachicola as a monophyletic group within the clade.

Clade B also spans the Alabama + Coosa Rivers with representative haplotypes detected in Bibb

Co., Alabama.

The southern Blue Ridge, as well as most of Georgia, is populated by representatives of clade C. Southern Blue Ridge samples included populations in Tennessee, North Carolina,

South Carolina, and Georgia. Clade C spans the upper Chattahoochee River, but was only detected to the southeast of the Ridge and Valley province, east of the middle/lower

Chattahoochee, and southwest of the Savannah River.

Amongst the most widely dispersed lineages, clade D ranges broadly across the southern

Cumberland Plateau and to the north and west of Hemidactylium’s distribution. A discrete monophyletic subclade of this lineage is responsible for recolonizing the entirety of the glaciated region of the upper Midwestern United States (“Midwest” subclade, see Figure 2). Though these postglacial populations are widely dispersed and highly disjunct, variation between members of the Midwest group consists of no more than two base pair mutations at three variable sites within the subclade. Similarly, a single subclade of D, varying by only a single base pair, was detected at all sampling sites in the Ozark uplift. The most divergent haplotypes in this clade were found at sites on the southern Cumberland Plateau. Clade D spans a number of major rivers, which would suggest an age of the clade to be greater than that portion of the river, or at least a course change in the river’s history, which allowed for movement across. Such rivers include the

Tennessee River near Chattanooga, the Mississippi River near the Ozark uplift, the Missouri 20 River, and the Ohio River. In the case of the Missouri and Ohio rivers, identical haplotypes were found on both sides.

Clade E, containing members with the highest within-clade genetic distances (mean uncorrected p-nucleotide distance = 0.0316) and highest nucleotide diversity (0.028), was detected at three sites to the east of the Blue Ridge. This clade appears to be restricted in distribution to some of the very few known sites in South Carolina, and an area of North

Carolina near the French Broad River. No clear geographic boundaries to this clade are evident from the limited number of sampling locations where the species could be found in the region.

The most geographically restricted lineage, clade F, was detected at only one site and is likely limited to two adjacent stream drainages on the western edge of the Great Smoky

Mountains. This clade represents the most highly divergent co1 lineage detected in this study, resolved as the sister group to all other Hemidactylium and the rooting location with outgroup taxa in both MP and Bayesian analyses with complete support (bp=100, pp=1.00).

Tests for LBA were conducted by separate removal of both outgroup sequences, removal of clade F sequences with inclusion of outgroups, and removal of clades E and F with inclusion of outgroups. These three datasets were then independently analyzed with maximum parsimony and all resulting topologies and rooting locations were consistent with those of the complete dataset. Additionally, MP and Bayesian trees generated were both rooted with the outgroup in the identical location with high support. These results indicate a low probability that the branching topology of major clades was a result of LBA by the highly divergent groups.

The connection of haplotypes into mutational networks with 95% confidence resulted in the formation of nine separate networks, which were largely congruent with the separations between major clades identified in the Bayesian and MP phylogenies (Figure 4). Clades B, C, D, 21

Figure 4. Haplotype networks generated by TCS overlaid on the distribution of Hemidactylium scutatum. Solid black lines represent a single mutation, while small white circles represent transitional haplotypes not detected in the sample. The area of haplotype circles is proportional to the number of individuals in which the sequence was detected. Haplotype h71 (in white) required two additional steps beyond the 95% connection limit to be linked to the rest of Clade A (indicated by dashed line). Note that Clade E consists of three separate networks due to high within-clade genetic distances. Individual networks labeled with haplotype numbers are presented in Appendix 2. 22 and F formed separate networks consistent with the phylogenetic groupings. The many subclades of clade A were also linked into a single haplotype network, with the exception of haplotype 71. The placement of this divergent lineage from Sevier County, Tennessee, was not strongly supported in either MP or Bayesian analyses, and required an additional 2 steps beyond the 10 allowed for 95% confidence in order to be linked to the network comprised of the rest of clade A (Figure 4 and Appendix 3). The most notable difference between the phylogenetic trees and the haplotype network was the division of clade E into three separate networks. Due to the high divergences between populations in clade E, a total of 21 steps were required to link these haplotypes into a single network, well beyond the 95% cutoff. Forty-one steps were required to link all clades into a single haplotype network.

Mismatch Distributions and Tests of Recent Expansion

Pairwise mismatch distributions for all samples together and clades A through E are presented in Figure 5 with the expected distributions under continuous expansion overlaid.

Clade F was excluded from the analysis due to the extremely limited number of samples (n=5).

The combined dataset’s distribution, as well as that of several of the clades, appears to be multimodal, indicating a population which is in equilibrium or subdivided. However, the mismatch distribution for all samples, as well as that of clade C (southern Blue Ridge + Georgia) and clade D (Midwest) did not vary significantly from the model of continuous demographic expansion. P-values for the sum of squared deviation (SSD) for clades A (NE/SW), B (AL+FL), and E (SC) were significant (p0.05) indicating that a sudden demographic expansion cannot explain the observed patterns of variation in these clades.

Results of Tajima’s D test and Fu’s Fs were not significant for any individual clade (see

Appendix 2). However, for the complete dataset including all ingroup samples, Fu’s Fs was 23

Pairwise Mismatch Distribution (all samples)

0.08

0.07 SSD=0.005 0.06 p=0.273 τ=21.430 0.05

0.04

frequency 0.03

0.02

0.01

0 0 5 10 15 20 25 30 35 40 45 50 55 60 # base pair differences

Clade A - NE/SW Clade B-AL/FL Clade C-GA 0.16 0.3 0.16

0.14 0.14 SSD=0.012 0.25 SSD=0.062 SSD=0.017 0.12 p=0.009 p=0.001 0.12 p=0.227 0.2 0.1 τ=13.357 τ=7.656 0.1 τ=9.645

0.08 0.15 0.08

0.06 0.06

frequency 0.1

frequency frequency 0.04 0.04 0.05 0.02 0.02 0 0 0 0 2 4 6 8 10 12 14 16 18 20 22 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 # pairwise differences # pairwise differences # pairwise differences

Clade D-Midwest Clade E-SC 0.25 0.2 SSD=0.037 0.18 SSD=0.082 0.2 0.16 p=0.284 0.14 p=0.000 0.15 τ=8.418 0.12 τ=24.492 0.1

0.1 0.08 frequency frequency 0.06 0.05 0.04 0.02 0 0 0 1 2 3 4 5 6 7 8 9 10 11 0 2 4 6 8 10 12 14 16 18 20 22 24 26 # pairwise differences # pairwise differences

Figure 5. Observed pairwise mismatch distributions for all samples together, and for Clades A through E. Clade F was omitted due to small sample size. Overlain on the mismatch histrogram is the expected distribution under a simulated null model of continuous expansion. The high p-values of all samples together, Clade C, and Clade D indicate that these mismatch distributions fit the model of a sudden demographic expansion. 24 significantly negative (FS=-23.51652, p=0.012), consistent with an overall demographic population expansion.

Mantel Tests of Barriers

The results of partial Mantel tests conducted on hypothesized geographic barriers to dispersal are summarized in Table 3. In all but one of the seven barriers tested (French Broad

River), separation by the barrier was shown to be significantly correlated with the genetic distance between populations on either side. Similarly, the genetic distance was shown to be significantly correlated with geographic distance in all but one case (Arkansas River), suggesting isolation by distance (IBD) is occurring simultaneously. However, in all but one case where the barrier effects were significant (Apalachicola + Chattahoochee Rivers) r-values indicated a stronger correlation between genetic distance and the barrier than genetic distance and geographic distance.

Table 3. Results of partial Mantel tests of hypothesized geographic barriers to dispersal. Significant r-values (indicated in boldface type) indicate that the effects of the variable (barrier or geographic distance) are positively correlated with genetic distance. See Appendix 1 for key to site abbreviations.

Genetic Dist. vs. Genetic Dist. vs. Hypothesized Barrier Barrier > Geographic Dist. Sites Included Barrier Geographic Dist. r p-value r p-value ARCL, ARGA, ARMO, Arkansas R. 0.943 <0.0001 -0.023 0.5783 ARPO, MOBE, MODE, * MOWA, MOWY, OKMC ARGA, ARMO, ARPO, Mississippi Embayment 0.851 0.357 LAEF, MSAD, MSCH, <0.0001 <0.0001 * MSHI, MSLA, MSTI, MSWI, OKMC ALBI, ALCL, ALLE, ALTA, FLGA, FLLE, FLLI, FLWA, Black Warrior+Tombigbee R. 0.934 <0.0001 0.225 <0.0001 LAEF, MSAD, MSCH, * MSHI, MSLA, MSTI, MSWI, TNHE ALBI, ALCL, ALLE, ALTA, FLGA, FLLE, FLLI, FLWA, Coosa+Alabama R. 0.501 <0.0001 0.447 <0.0001 LAEF, MSAD, MSCH, * MSHI, MSLA, MSTI, MSWI, TNHE ALBI, ALCL, ALLE, ALTA, FLGA, FLLE, FLLI, FLWA, Apalachicola+Chattahoochee R. 0.256 <0.0001 0.337 <0.0001 GAEA, GAGR, GAHA, GAJA, GAPA

GAHA, NCAV, NCBU, Little TN+Toxaway+Keowee R. 0.490 0.348 NCCH, NCGR, NCMA, <0.0001 <0.0001 * SCOC, SCPI, TNBL, TNMC, TNMO, TNSE

NCAV, NCBU, NCSU, French Broad R. 0.065 0.0781 0.314 <0.0001 SCPI, TNBL, TNSE, TNSU, TNUN 25 Divergence Time Estimates

The results of Bayesian divergence time estimates generated a mean coalescent time for all Hemidactylium, which ranged from 7.363 to 8.197 million years, placing the ancestry of all living representatives of the species in the late Miocene (Table 4). Constraining the rootheight of

Hemidactylium + Batrachoseps to the Vietes et al. (2007) 95% confidence range (64-107 m.y.) increased the mean TMRCA estimates within Hemidactylium by an average of 3.6% over the estimates with an unconstrained rootheight. Constraining to the Mueller (2006) confidence range (90-109 m.y.) increased these values by an average of 6.0%, however neither of these constraints were significant, as the 95% confidence values for all three analyses overlapped broadly (Table 4). Common ancestry for major clades ranged from a low of ~230,000 years for the highly localized clade F, to roughly 1.7 million years for the widely distributed clade A, to a maximum of ~2.3 million years for the highly divergent clade E. Of the TMRCAs estimated, only the split of clade F from the rest of Hemidactylium, the common ancestor of clade E, and the common ancestor of clades A, B, C, and D predate the start of the Pleistocene, roughly 1.8 million years ago (Gibbard and Van Kolfschoten 2004). 26

Table 4. Divergence time estimates for selected clades and subclades generated by the Bayesian coalescent approach in BEAST. Times to most recent common ancestor (TMRCA) are presented in millions of years (m.y.) for each subset of the complete phylogeny. Three different analyses were performed: one with an unconstrained root height, and two with the root height constrained to the 95% confidence intervals of the divergence of Hemidactylium and Batrachoseps (see Methods).

TMRCA (95% C.I.) TMRCA (95% C.I.) TMRCA (95% C.I.) Clade Unconstrained Root Root height 64-107 m.y. Root height 90-109 m.y. Clade A 1.716 (1.167-2.402) 1.775 (1.229-2.466) 1.807 (1.154-2.469) Northeast 1.208 (0.580-1.972) 1.254 (0.554-2.022) 1.280 (0.572-2.084) Southwest 0.575 (0.300-0.896) 0.588 (0.326-0.896) 0.603 (0.314-0.911) Ouachita 0.369 (0.166-0.623) 0.379 (0.177-0.636) 0.392 (0.171-0.638) Teays 0.594 (0.286-0.952) 0.610 (0.271-0.967) 0.619 (0.289-1.016) Clade B 0.896 (0.521-1.307) 0.924 (0.524-1.357) 0.945 (0.552-1.396) Clade C 1.307 (0.774-1.954) 1.340 (0.790-1.984) 1.366 (0.780-2.058) Clade D 0.833 (0.462-1.262) 0.865 (0.472-1.293) 0.874 (0.491-1.339) Midwest 0.189 (0.082-0.313) 0.197 (0.091-0.334) 0.200 (0.086-0.337) Ozark 0.167 (0.025-0.327) 0.172 (0.020-0.328) 0.179 (0.013-0.345) Walden 0.161 (0.039-0.294) 0.168 (0.041-0.305) 0.171 (0.042-0.311) Clade E 2.258 (1.230-3.285) 2.399 (1.370-3.585) 2.469 (1.328-3.726) Clade F 0.232 (0.087-0.480) 0.233 (0.060-0.453) 0.238 (0.093-0.477) Clades A+B+C+D 3.216 (2.203-4.363) 3.332 (2.302-4.450) 3.432 (2.347-4.665) Hemidactylium scutatum 7.363 (4.619-10.378) 7.932 (4.974-11.506) 8.197 (4.562-12.196) Hemidactylium + Batrachoseps 49.339 (24.865-77.191) 74.023 (64.000-92.944) 99.760 (90.000-118.427) 27 DISCUSSION

The distribution of Hemidactylium scutatum across the diverse terrain of eastern North

America during a period of dramatic climatological change has had dramatic effects on the genetic makeup of the species. Varying levels of genetic differentiation among 6 major mitochondrial clades identified in this study are indicative of a highly subdivided and widespread species with populations subjected to a variety of evolutionary processes. The low diversity in the Midwest and Northeast is consistent with a rapid expansion recolonizing the vacant habitat revealed as Pleistocene glaciers retreated, while the higher diversity in the South suggests long term persistence in the region and relatively stable isolating mechanisms acting on the populations. Of significance both to the evolutionary history and the conservation of

Hemidactylium is the extremely high diversity of the southern Appalachian mountains, and in particular the Blue Ridge.

The distribution of haplotypes and major clades in Hemidactylium revealed the structure of routes of geographic and concordant demographic expansions in the species (Figure 6). An overall pattern was detected with the co1 marker that showed much higher genetic diversity in the southern Appalachian Mountains than anywhere else in the range (Figure 3, Appendix 4).

All major clades and most subclades have representatives in this region, primarily in the Blue

Ridge physiographic province. From here, clade A radiated to the northeast and southwest, clade

B and C radiated south, clade D radiated northwest, clade E radiated a short distance to the east, and clade F is endemic to the south-central Blue Ridge. This pattern, coupled with the presence of divergent endemic haplotypes from within clade A (h47, h48, h49, h71, h74, h75) and deeply rooted endemic clades (E and F) in the Blue Ridge, strongly suggest a placement of the mitochondrial ancestry of all living Hemidactylium in the Blue Ridge of the southern 28

Figure 6. Generalized map of phylogeographic patterns of expansion resolved in Hemidactylium scutatum. Labels on arrows refer to clades or subclades resolved in phylogenetic analyses followed by estimated TMRCA values in parentheses (in m.y.a.), indicating the sequential expansion across the southern United States. Numbered rivers correspond to barriers tested with partial Mantel tests: 1. Arkansas River, 2. Mississippi Embayment, 3. Black Warrior + Tombigbee R., 4. Coosa + Alabama R., 5. Apalachicola + Chattahoochee R., 6. Little Tennessee + Toxaway + Keowee R., 7. French Broad R. Colored shaded areas correspond to the distribution of major clades and subclades resolved in this study, with the exception of white, which is populated by several unique clade A haplotypes endemic to the southern Appalachians. The dashed white line indicates the southern limit of Pleistocene glaciations and outlines the unglaciated Driftless Area of the upper Mississippi River Valley. 29 Appalachians. A geographic origin of Hemidactylium in the southern Appalachians is not surprising given the widely accepted hypothesis first proposed by Wilder and Dunn (1920) placing the ancestry of the entirety of the Plethodontidae in this area. Plethodontid species and generic diversity is extremely high in the southern Appalachians, with dozens of endemic taxa.

However, Vieites et al. (2007) recently challenged this interpretation through modeling scenarios based on nuclear phylogenies, divergence time estimates, and paleogeographic reconstruction.

Their results indicated a widespread North American ancestor to the family with higher support for a western origin. In particular their assessment supported an Appalachian origin only for the

Spelerpine genera (Eurycea, Gyrinophilus, Pseudotrion, Sterochilus). In contrast, our findings place the ancestry of modern Hemidactylium, shown to be basal to the rest of the

Hemidactyliinae by Vieites et al. (2007), in the southern Appalachians as well. However, due to the recent TMRCA estimate for the living Hemidactylium (8 m.y.) relative to the divergence time estimates for the split from the western genus Batrachoseps (84 m.y. by Vieites et al. 2007, 108 m.y. by Mueller 2006), the possibility that Hemidactylium’s ancestor dispersed into the region prior to 8 m.y. cannot be refuted. The difficulty of inferring the deeper history of Hemidactylium is exacerbated by the lack of any closely related outgroup taxa to which a comparison can be made. These same difficulties have been encountered with other monotypic salamander genera in eastern North America (see Routman et al. 1994).

The distribution and relationships of clades and subclades revealed the patterns of early expansion which colonized southern habitats in the range of the species. Two major clades (B and C) and one subclade (Southwest of clade A) spread south and populated the Gulf Coast region. The varying level of genetic differentiation within these three groups, the lack of monophyly, and the TMRCA estimates all suggest that these expansions were not simultaneous, 30 but rather three separate expansion events from an Appalachian origin. These three lineages are comprised of widely scattered populations, with limits largely concordant with the courses of both ancient and modern rivers supported by the partial Mantel tests, and in several cases with r- values indicating the effects of the barrier are stronger than those of geographic distance (Table

3). Four of these rivers (Black Warrior, Chattahoochee, Coosa, Savannah) have maintained major portions of their courses since the early Tertiary (Swift et al. 1986), and are indicated as defining boundaries of these clades (Figure 6, Appendix 4). Their mean divergence times (Table

4) indicate a pattern of expansion in Hemidactylium spreading from east to west across the southern states, beginning with clade C spreading south out of the Blue Ridge (~1.3 m.y.a), followed by clade B (~0.9 m.y.a), and finally the Southwest group of clade A (~0.6 m.y.a.). This timeline continues even further to the west, with the common ancestor of the Ouachita subclades arising just under 0.4 million years ago (Figure 6).

The geographic distribution (within 44 km of the clade F site in the southern Blue Ridge), the high levels of gene and haplotype diversity relative to other clades (see Table 1), and the

TMRCA estimates suggest clade C to be an ancient clade within Hemidactylium. Eight of the thirteen different clade C haplotypes were detected at six adjacent sites in the Blue Ridge (see

Appendix 4). Such high diversity is expected given the complex and mountainous terrain across which these sites are distributed. Several river systems encompass this clade. Clade C’s hypothesized boundaries to the north and west (Little Tennessee + Tuckasegee + Keowee and

Chattahoochee + Apalachicola) were supported by the results of the partial Mantel tests. To the northeast, the Savannah River presents a likely barrier, as it has maintained its course since at least the early Tertiary (Swift et al 1986) and is located between the clade C sites and clade E to the north. The confluence of the Chattahoochee and Flint rivers in Georgia may also act as a 31 barrier to southward dispersal of the clade towards the Florida panhandle, isolating clades B and C.

Dugger Mountain, in Cleburne Co., Alabama, is the northernmost site where Clade B was detected. The uplifted terrain of this area lies just south of the low rolling hills of the Ridge and

Valley, and is considered by some geologists to be a southern extension of the Blue Ridge

(Murray 1961). Though insufficient samples were available in the area to test it, the upper Coosa

River and its headwaters are a potential boundary between clade B and the rest of the Blue Ridge to the northeast. The occurrence of clade B on either side of the Apalachicola River (south of the

Chattahoochee) is not surprising. The changing sea level (in excess of 80m above modern sea level) due to glacial cycles (Riggs 1984) would have resulted in various stages of inundation in the region, and the sandy coastal soils are conducive to drainage rearrangement. The

Apalachicola/Chattahoochee River is thought to have previously drained through the

Choctawhatchee valley (Puri and Vernon 1964), and a changing course of this major waterway would have allowed for dispersal across the modern lotic barriers of this region. The permeability of this river system to dispersal due to course changes is the likely cause of the genetic distance being more highly correlated to geographic distance than this barrier in the results of the partial Mantel test (Table 3).

Due to the limited sampling, the precise location of the phylogeographic boundary to the west of this clade is somewhat tenuous. Previous studies have indicated a phylogeographic break at the Mobile river basin (see Appendix 5) due in part to a long proposed ancient drainage, the “Appalachian River,” from the Appalachian Mountains directly into the Gulf of Mexico along this course (Hayes and Campbell 1894, 1900; Simpson 1900; Swift et al 1986; Mayden

1988). However, not all are in agreement with this hypothesis (see Starnes and Etnier 1986). 32 Evidence for a connection between the Tombigbee/Mobile and the Tennessee river systems has also been presented for freshwater mussel and fish communities (Simpson 1900; van der

Schalie 1939; Wall 1968; Mayden 1988; Phillips and Johnston 2004). It appears likely that there have been multiple connections between the two systems, at different locations, since the late

Cretaceous (Swift et al 1986; Mayden 1988). The data for Hemidactylium is supportive of a barrier extending through this region of western Alabama, and our data suggest that no single river may explain the pattern. Partial Mantel tests for barriers at both the Black Warrior +

Tombigbee drainages and the Coosa + Mobile drainages yielded significant correlation to genetic distance. However, at a site in Bibb Co., Alabama (west of the Coosa River), a clade B haplotype was detected in 5 samples, leading to a much higher correlation for the Black Warrior

+ Tombigbee system.

To the west of this river system is the most recently colonized region of the Gulf Coast based on level of co1 divergence. The Southwest subclade of clade A was detected across the area bound to the west by the Mississippi River embayment and to the east by the Mobile River and its tributaries, both of which were supported by partial Mantel test results. The lineage is likely bound to the northeast by the Lower Tennessee River. The east-west substructure detected within this southwest group may be the result of separation by the Pearl River valley. However, too few Hemidactylium populations are known from Mississippi to more clearly resolve this distributional pattern. Extensive surveys in the near future will likely discover more populations of Hemidactylium in this region, as three of the six Mississippi counties sampled for this study represented new distributional records for the species (Herman and Ross, in prep.). However, ongoing and rapid habitat modification due to extensive deforestation and pulpwood production has likely led to the extirpation of many populations in this area of the Gulf States. 33 Two separate dispersal events resulted in the colonization of the Interior Highlands of

Arkansas and Missouri, the only known populations of Hemidactylium west of the Mississippi

River (Figure 6). The Arkansas River forms a geographical divide, effectively separating the

Ozark and Ouachita uplifts of the Interior Highlands. Though the upland topography of this region was once contiguous, this major river likely attained its present course during the

Pleistocene (Quinn 1958). As evidenced by the results of the partial Mantel tests of the clades in the Interior Highlands, this river presents a major barrier to the dispersal of Hemidactylium in the region. No haplotypes from the Ouachita or Ozark subclades were found to span this barrier.

Observed patterns of haplotype distribution suggest two separate dispersal events across the

Mississippi River valley. Based on the level of divergence from the most closely related taxa, the southern event likely occurred first, and consisted of a member of clade A crossing the

Mississippi River south of its confluence with the Arkansas River, and led to the colonization of the southern Interior Highlands (Ouachita uplift). The northern event involved members of clade

D, crossing at a point north of the Arkansas River. Extant patterns of Hemidactylium distribution in the Ozarks are consistent with a crossing at Crowley’s Ridge or the unglaciated geology near the confluence of the Ohio and Mississippi rivers. Both crossing events are concordant with the distribution of other taxa in this region (see Appendix 5). Given the shallow divergences of these subclades in the context of the entire phylogeny, and their relationship to the Arkansas

River, both colonization events likely occurred during the Pleistocene. From the level of divergence of Ozark populations from those sampled across the entire postglacial Midwest, it appears unlikely that the Ozarks contained the refugium of Hemidactylium responsible for repopulating the western Great Lakes region following the most recent Pleistocene glaciation.

This hypothesis is further supported by the Bayesian phylogeny, which groups Ozark 34 populations most closely with those of the southern Cumberland Plateau (haplotypes h3 and h26, pp=0.98), rather than with the glaciated Midwest (Figure 2).

To the west of the Blue Ridge Mountains on the opposite side of the Great Appalachian

Valley lies the Cumberland Plateau (see Appendix 4 for a map of sampling and physiographic features in this region). Though no populations of Hemidactylium are known from within the valley in Tennessee (also known at the Ridge and Valley physiographic province), records are more or less contiguous along the eastern plateau, consisting of clade D’s northwestern expansion from the Appalachians. The southernmost populations on the Cumberland Plateau

(Marion Co., TN, Jackson Co., AL, and Walker Co., GA) provide some insight into the geological history of the region. In spite of their separation by Walden’s gorge and the modern course of the Tennessee River, the Walker Co. and Jackson Co. sites differ by only two of 576 bp and all three southern Cumberland sites are placed within clade D. From this distribution and the TMRCA estimate for populations on either side (Table 4), it appears that the Tennessee River did not shift to its modern course until very recently in the history of Hemidactylium, or the flow through Walden’s Gorge of the river was intermittent within the past 200,000 years. Either scenario provides further evidence for an Appalachian River or an alternative large-stream connection between the upper and lower Tennessee River.

Clade D continued to spread to the north and west from the Cumberland Plateau, colonizing the Ozarks and most recently the glaciated Midwest in the Great Lakes region. The

Midwest subclade of clade D and Northeast subclade of clade A account for the entire distributional extent of Hemidactylium formerly covered by the last glacial maximum. The

Northeast subclade ranges from western North Carolina up the Atlantic Coastal Plain to Nova

Scotia, and west across New York to northeastern Ohio. A zone of secondary contact between 35 these two subclades likely exists in north-central Ohio and possibly along the northern shores of the Great Lakes in southern Ontario. Similar patterns of limited genetic diversity, broadly distributed across glaciated regions, have been documented for many species (Hewitt 2000). The broad distribution of a very few haplotypes and rapid recolonization within a relatively short time frame following glaciation may be explained by the periglacial environmental conditions as the ice floes retreated. In addition to the likely abundance of suitable habitat for Hemidactylium as glaciers retreat (Thurow 1997), meltwater pulses due to increasing temperatures and breaching glacial dams would result in regular flooding of periglacial areas. These floods would greatly facilitate dispersal of relatively sedentary salamander species which undergo a larval stage by rapidly transporting the aquatic phase during flood events (larval drift). The mismatch distribution of clade D fit the null model, suggesting a demographic expansion necessary to repopulate the glaciated habitat following the retreat of the Laurentide ice sheet.

Patterns of Hemidactylium recolonization in the Northeastern glaciated regions are not as clearly resolved as those of the Midwest. Five haplotypes of two separate lineages were detected from North Carolina to Nova Scotia, and west to northeastern Ohio in the Lake Erie drainage basin. The monophyly of these two lineages as sister taxa is weakly supported by Bayesian analysis and unsupported by MP. One lineage, including haplotypes h33, h34, and h35, is widely distributed across the glaciated northeast, as well down the Atlantic Coastal Plain and

Piedmont east of the Appalachians as far south as central Virginia. The other lineage (h54 and h55) was detected at two sites in North Carolina to the east of the Blue Ridge, as well as a site in

Pennsylvania near the edge of the last glacial maximum, and syntopically with h34 at one glaciated site in New York. All haplotypes indicate likely refugia in the east-central

Appalachian Mountains and/or along the Atlantic coastal plain, though their exact number and 36 location is not clear. The northeastern United States and Atlantic coastal plain is one of the few regions where existing records of Hemidactylium are abundant and widespread. An intensive sampling of these areas would allow for more precise mapping of the distribution of the two lineages, and may reveal if there is evidence for localized glacial refugia in the complex topography of the northern Appalachians.

The DNA mismatch distribution of clade A did not revealed a pattern of sudden demographic expansion, which would be expected following the postglacial recolonization of northestern regions. The fact that such a pattern was not resolved in the expansion tests for the postglacial Northeast samples (which would have undergone a similarly rapid expansion to that of clade D in the northwest) is likely an artifact of the diversity and extent of the habitats populated by members of clade A. The inclusion of such varied habitats as the Gulf Coastal plain and the Ouachita Mountains in the clade A mismatch analysis likely masked any signature of expansion imparted by Northeastern samples. A separate DNA mismatch analysis including only members of clade A restricted to the Northeastern subclade revealed a haplotype distribution consistent with a rapid demographic expansion following the Pleistocene glaciations

(S.S.D.=0.112, p=0.130; data not shown).

The effects of recent glaciation events were further revealed in one subclade of clade A, found in West Virginia, extreme southern Ohio, eastern Kentucky, western Virginia, and extreme northeastern Tennessee. This region defines roughly the proposed headwater drainage basin of the ancient Teays River, which was dammed and then buried under till in the middle Pleistocene by advancing glaciers (Tight 1903). When main stem of the river was blocked by the expanding ice sheet, a vast lake was formed (Lake Tight) covering large portions of southern Ohio, eastern

Kentucky, and western West Virginia, which eventually breached its banks to drain through the 37 modern course of the Ohio River (Ver Steeg 1946). After it drained many of the former streams and rivers within its basin changed course, dramatically altering drainage patterns in the region (Ver Steeg 1946). At only three of the 79 sites sampled in this study were members from more than one clade or subclade detected. All three of these sites are located at the periphery of the Teays river drainage system and include a member of the Teays subclade of clade A

(Appendix 1), suggesting that Pleistocene drainage rearrangements may have altered barriers to dispersal in the region. The divergence time estimates corroborate an origin of the Teays subclades following a major glaciation cycle in the mid-Pleistocene ending around 620,000 years ago (Gibbard and Van Kolfschoten 2004); however, identical haplotypes found spanning the

Ohio River in southern Ohio suggest a Wisconsinan interruption in this river’s course.

A broad diversity of taxa show similar patterns of distribution to that of Hemidactylium in portions of their range (see Appendix 5 and references therein). Primarily amphibian taxa but also some fish, reptiles, and small mammals have been the subject of comparable phylogeographic investigation across ranges which overlap that of the four-toed salamander. A review of the major phylogeographic barriers and patterns found in Hemidactylium and similar distributional parallels found in other taxa is summarized in Appendix 5. While some taxa exhibit intraspecific genetic trends that mirror those of this salamander, the distribution of several entire species corresponds to clades or subclades resolved in this study. In some cases, such as the ancient Appalachian drainage through the Mobile basin and the southern

Apalachicola River, barriers have shifted, manifesting themselves as semi-permeable clade boundaries or zones of integration between subspecies in other taxa. Specifically in the case of the Appalachian River, evidence for connectivity in aquatic organisms in the Tennessee and

Mobile drainages can be interpreted as evidence of a barrier to terrestrial species. Finally, the 38 specific history of different taxa in a region can result in varying genetic signatures from a barrier. For example, one species may occur with monophyletic clades on both sides of a river, with these clades sharing a common ancestry suggesting a prior permeability in the barrier (such as Eurycea multiplicata in the Interior Highlands, Bonnet and Chippendale 2004). In contrast, another co-distributed species may have monophyletic clades on either side of the same river, but each with different ancestries, suggesting the river has been an important reproductive barrier since the arrival of this species in the region (such as the pattern seen in Hemidactylium scutatum in the Interior Highlands).

Conservation Implications

Due to the extremely isolated and disjunct nature of this salamander’s distribution, over half of the states and provinces where it occurs have granted it special conservation status of endangered, threatened, or species of concern. Many nesting localities for this species are known only from the data collected in this study, some of which consist of isolated upland water features less than one meter in diameter. Populations in the southern portion of the species range are poorly documented. Considering the high proportion of genetic variability represented in these populations, relative to those in the northern portion of Hemidactylium’s range, future conservation efforts should be directed at these southern sites to improve our limited understanding of the distribution, abundance, and habitat preferences of the species in the southeastern United States. Further study of patterns of nuclear genetic variation, morphology, and differences in habitat use may indicate that some of these southern populations warrant protection as distinct evolutionarily significant units (ESUs) within Hemidactylium. Particularly in the Gulf States of Mississippi, Alabama, and Georgia, habitat destruction is ongoing due to land use conversion from hardwood forest to residential development and pine forest plantations. 39 It should be noted that finer resolution of many of the unresolved historical patterns of distribution, isolation, and dispersal in Hemidactylium is hindered not so much by collection of samples as it is by the extremely limited number of known extant localities for the species in extensive portions of its range. For instance, all but three known sites in the southern Blue Ridge were represented in the analysis presented here, and an intensive, though fruitless, search was conducted at the sites not included. The phylogenetic relationships resolved in this study indicate that the most genetically distinct populations are confined to these localites in the southern Appalachian Mountains. Further survey work for Hemidactylium at additional sites in the Blue Ridge during the nesting season are warranted, as several populations in the region

(particularly those comprising clades E and F) are the strongest candidates for being managed as distinct evolutionarily significant units (ESUs) warranting targeted conservation. States such as

Tennessee and North Carolina, which contain both common and rare lineages of Hemidactylium, could have a greater impact on the conservation of this species if their research and preservation efforts were focused on these Blue Ridge populations. Validation of decades old records and discovery of multiple new localities for the species were essential to achieve the level of sampling necessary for an accurate phylogeographic analysis. Support of basic distributional surveys in such poorly understood and rarely seen taxa is a vital component of informed study and formulation of conservation policy.

Conclusions

Hemidactylium scutatum is a species with a long and complex history, likely extending back nearly 8 million years. This study showed that the co1 mtDNA gene was an effective marker at resolving highly structured, geographically distinct monophyletic clades within this taxon. Results presented here suggest that the mitochondrial ancestry of the species likely 40 originated in the Blue Ridge of the southern Appalachian Mountains, with all modern descendants radiating out from this region. These results are therefore consistent with an “Out of

Appalachia” hypothesis for the origin of Hemidactylium scutatum, a species deeply rooted in the

Plethodontidae (Macey 2005; Mueller 2006; Vieites et al. 2007). The older and more divergent expansions spread south into Alabama, Georgia, and Florida, while more recent expansions involved subclades nested within major clades, detected at sites on the western periphery and northern extent of the distribution. Patterns of postglacial recolonization indicate that at least two refugia are responsible for the demographic expansion in habitats vacated by the retreating

Laurentide ice sheet following the most recent Pleistocene glaciation. Though subsequent recolonization appeared to be relatively straightforward in the Midwest, with a few closely related haplotypes detected just south of the glacial maximum distributed broadly across the region, the number and location of source populations in the northeast require further sampling to be more accurately resolved, and may involve northern refugia or a source population of mixed ancestry. The distribution of mitochondrial haplotypes in the central Appalachians indicates secondary contact between a subclade distributed across the ancient Teays river basin, and three other divergent haplotypes on the periphery. This pattern may be attributable to the reworking of major drainage patterns in the region during Pleistocene glaciation events.

Many genetic patterns resolved in Hemidactylium are concordant with those of co- distributed species at both intra- and interspecific levels. Phylogeographic breaks identified between mitochondrial clades in Hemidactylium have also been identified in several other terrestrial taxa to be significant obstacles to gene flow. These geographic features may provide a useful map of testable genetic barriers to guide future phylogeographic investigations of other terrestrial species in eastern North America. Patterns resolved in Hemidactylium lend additional 41 support to the importance of the southern Appalachian Mountains as a driving factor in the diversification of amphibians and many other taxa in eastern North America, warranting the utmost attention in our conservation efforts. Further nuclear DNA work on these collections of

Hemidactylium should help to resolve the ambiguous relationships among major clades and further clarify the timing and patterns of expansion in this species. Although a taxonomic revision of Hemidactylium, based solely on mitochondrial data, is not recommended at this time, additional research may reveal clade F as a candidate for elevation to full species status.

42

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APPENDICES

50 Appendix 1. Number of samples and haplotypes/clades detected at each collecting locality included in the study.

Locale # of Code State County Samples Haplotypes Clade (Subclade) ALBI Alabama Bibb 5 h1 B ALCL Alabama Cleburne 3 h2 B ALJA Alabama Jackson 5 h3 D ALLE Alabama Lee 5 h4, h5 B ALTA Alabama Tallapoosa 5 h6, h7 B ARCL Arkansas Cleburne 1 h8 D (Ozark) ARGA Arkansas Garland 3 h9, h10 A (Ouachita) ARMO Arkansas Montgomery 3 h11 A (Ouachita) ARPO Arkansas Polk 4 h12, h13, h14 A (Ouachita) FLGA Florida Gadsden 5 h15, h16 B FLLE Florida Leon 5 h17 B FLLI Florida Liberty 1 h18 B FLWA Florida Walton 10 h19 B GAEA Georgia Early 5 h20 C GAGR Georgia Greene 3 h21, h22 C GAHA Georgia Habersham 3 h23 C GAJA Georgia Jasper 1 h24 C GAPA Georgia Paulding 1 h25 C GAWA Georgia Walker 5 h26 D ILJO Illinois Jo Daviess 7 h27, h28 D (Midwest) ILVE Illinois Vermilion 6 h27, h28 D (Midwest) KYAD Kentucky Adair 5 h28 D (Midwest) KYLA Kentucky Laurel 5 h28 D (Midwest) KYME Kentucky Menifee 5 h28, h29 D (Midwest) 3 h28, h30 D (Midwest), A KYPO Kentucky Powell (Teays) LAEF Louisiana East Feliciana Parish 5 h31, h32 A (Southwest) MDPR Maryland Prince Georges/Charles 10 h33, h34, h35 A (Northeast) MEYO Maine York 5 h34 A (Northeast) MICH Michigan Chippewa 7 h28 D (Midwest) MIGO Michigan Gogebic 5 h27 D (Midwest) MIMO Michigan Montcalm 10 h28, h36, h37 D (Midwest) MNIT Minnesota Itasca 2 h27 D (Midwest) MNPI Minnesota Pine 5 h27, h28 D (Midwest) MOBE Missouri Benton 5 h38 D (Ozark) MODE Missouri Dent 5 h38 D (Ozark) MOWA Missouri Warren 4 h38 D (Ozark) MOWY Missouri Wayne 5 h38 D (Ozark) MSAD Mississippi Adams 1 h39 A (Southwest) MSCH Mississippi Chickasaw 1 h40 A (Southwest) MSHI Mississippi Hinds 4 h41, h42, h43 A (Southwest) MSLA Mississippi Lauderdale 2 h44 A (Southwest) MSTI Mississippi Tishomingo 4 h45 A (Southwest) MSWI Mississippi Wilkinson 1 h46 A (Southwest) 51 NCAV North Carolina Avery 5 h47, h48, h49 A NCBU North Carolina Buncombe 5 h50, h51 E NCCH North Carolina Cherokee 4 h52 C NCGR North Carolina Graham 1 h52 C NCMA North Carolina Macon 3 h53 C NCOR North Carolina Orange 5 h54 A (Northeast) NCSU North Carolina Surry 4 h54, h55 A (Northeast) NSGU Nova Scotia Guysborough 5 h34 A (Northeast) NSHX Nova Scotia Halifax 1 h34 A (Northeast) NYCH New York Chautauqua 5 h33, h34, h35 A (Northeast) NYSA New York Saratoga 4 h34, h54 A (Northeast) OHAD Ohio Adams 1 h28 D (Midwest) OHAS Ohio Ashtabula 5 h33 A (Northeast) OHAT Ohio Athens 2 h28 D (Midwest) OHHE Ohio Henry 5 h28 D (Midwest) OHSC Ohio Scioto 5 h30 A (Teays) OHWI Ohio Williams 5 h27, h28 D (Midwest) OKMC Oklahoma McCurtain 5 h13 A (Ouachita) PAMO Pennsylvania Monroe 2 h54 A (Northeast) SCOC South Carolina Oconee 2 h56 C SCPI South Carolina Pickens 5 h57 E SCYO South Carolina York 5 h58, h59 E TNBL Tennessee Blount 5 h60, h61, h62 F TNHE Tennessee Henry 5 h45, h63 A (Southwest) TNMA Tennessee Marion 5 h64, h65 D TNMC Tennessee McMinn 5 h66, h67 C TNMG Tennessee Morgan 5 h68 D TNMO Tennessee Monroe 5 h69, h70 C TNSE Tennessee Sevier 5 h71 A TNSU Tennessee Sullivan 5 h72, h73 A (Teays) TNUN Tennessee Unicoi 5 h74 A 5 h30, h33 A (Teays), A VAAU Virginia Augusta (Northeast) VACH Virginia Chesterfield 1 h34 A (Northeast) VASC Virginia Scott 5 h75, h76 A, A (Teays) WVHA West Virginia Hardy 5 h30, h77 A (Teays) WVKA West Virginia Kanawha 5 h30 A (Teays)

52 Appendix 2. Results of Tajima’s D and Fu’s Fs tests. Significant values for Fu’s Fs for all samples indicated in boldface type.

Group Tajima’s D (p-value) Fu’s Fs (p-value) Clade A -0.07989 (0.53940) -1.46851 (0.40560) Clade B 0.24051 (0.66400) 1.69252 (0.77940) Clade C -0.01592 (0.55660) 0.38897 (0.60440) Clade D 0.43012 (0.73060) 1.19250 (0.72360) Clade E 2.10866 (0.99520) 9.17550 (0.99820) Clade F 1.45884 (0.95320) -0.18585 (0.26220) All samples 0.05046 (0.60500) -23.51652 (0.01160) 53

Appendix 3. Haplotype networks of individual clades.

Clade A 54

Clade B

Clade C 55

Clade D

Clade E Clade F 56 Appendix 4. Topographic relief map of the southern Appalachian Mountains, showing major physiographic features and proposed hydrological barriers to dispersal discussed in the text. Overlain are Hemidactylium sampling locations colored to indicate major clades and divergent Clade A haplotypes endemic to the region. The dashed blue line represents the hypothesized prehistoric connection between the upper Tennessee River and the Mobile River basin draining this region directly into the Gulf of Mexico. 57 Appendix 5. Review of co-distributed taxa with phylogeographic patterns concordant with those resolved in Hemidactylium.

Feature Concordant Taxa

Eurycea multiplicata (R. M. Bonett and Chippindale 2004), Plethodon serratus (Highton and Webster 1976), Plethodon caddoensis, P. fourchensis, P. kiamichi, P. Arkansas River ouachitae, P. sequoyah, P. angusticlavius, Desmognathus brimleyorum (Conant and Collins 1998) Sciurus niger, S. carolinensis (Moncrief 1993), Eumeces fasciatus (Howes et al. Mississippi 2006), Pantherophis obsoleta (Burbrink et al. 2000), Pantherophis guttata (Burbrink embayment 2002), Ambystoma maculatum (Zamudio and Savage 2003), Etheostoma caeruleum (Ray et al. 2006), Pseudacris (Lemmon et al. 2007) Sceloporus undulatus (Leaché and Reeder 2002), Diadophis punctatus (Fontanella et al. 2008), Coluber constrictor (Burbrink et al. 2008), Plethodon mississippi Mobile River basin (Highton et al. 1989), Lepomis gulosus (Bermingham and Avise 1986), Agkistrodon piscivorus, Lampropeltis getula, Sistrurus miliarius, Trachemys scripta (Conant and Collins 1998) fish (Wooten and Lydeard 1990; Scribner and Avise 1993; Bermingham and Avise 1986; Nedbal and Philipp 1994), salamanders (Donovan et al. 2000; Liu et al. 2006; Pauly et al. 2007), snakes (Burbrink et al 2000), alligator (Davis et al. 2002), turtles Apalachicola + (Walker et al. 1995; Roman et al. 1999; Osentoski and Lamb 1995), rodents (Avise Chattahoochee et al. 1979; Avise 1983), pine trees (Parker et al. 1997; Mylecraine et al. 2004), and Rivers pitcher plants (Godt and Hamrick 1998). See Soltis et al. (2006) for summary. Permeability of southern Apalachicola R. seen in Pseudacris crucifer (Austin et al. 2004), Pseudacris nigrita (Lemmon et al 2007), Pantherophis obsoleta (Burbrink et al 2002) Little Tenn. + Plethodon chlorobryonis, P. metcalfi, P. teyahalee (Highton et al. 1989; Highton and Toxaway + Keowee Peabody 2000), Desmognathus wrighti (Crespi et al. 2003) Rivers Desmognathus wrighti (Crespi et al. 2003), Desmognathus ocoee/carolinensis French Broad River (Tilley and Mahoney 1996), Plethodon teyahalee/cylindraceus (Highton and Peabody 2000), Aneides aeneus (Bernardo and Corser, in prep.) Ambystoma maculatum (Zamudio and Savage 2003), Ambystoma tigrinum (Church Separate et al. 2003), Eurycea bislineata (Kozak et al. 2006), Pseudacris crucifer (Austin et Eastern/Midwestern al. 2002), Rana pipiens (Hoffman and Blouin 2004), Pantherophis obsoleta glacial refugia (Burbrink et al. 2000), Blarina brevicauda (Brant and Orti 2002) Southern Diadophis punctatus (Fontanella et al. 2008), Sceloporus undulatus (Leaché and Mississippi River Reeder 2002), Desmognathus auriculatus, D. brimleyorum, D. conanti, Eurycea crossing quadridigitata, Plethodon kisatchie (Conant and Collins 1998) Ozark - Cryptobranchus alleganiensis, Eurycea longicauda, Eurycea lucifuga, Plethodon Cumberland dorsalis/angusticlavius, Hybopsis dissimilis, Typhlichthys subterraneus (Conant Plateau sister taxa 1960), Noturus exilis (Hardy et al. 2002) Cumberland taxa Aneides aeneus, Eurycea lucifuga, Gyrinophilus porphyriticus duryi, Pseudotriton distributed across montanus diastictus (Pfingsten and Matson 2003) Ohio River Teays River Gyrinophilus porphyriticus duryi (R. A. Brandon 1966), Eurycea bislineata complex associated taxa (Kozak et al. 2006), Plethodon kentucki, P. richmondi, Desmognathus welteri Ancient Tennessee freshwater mussels and fish (Simpson 1900; van der Schalie 1939; Wall 1968; Swift - Mobile River et al. 1986; Mayden 1988; Phillips and Johnston 2004), Sternotherus (Iverson 1977; connection Seidel and Lucchino 1981; Reynolds and Seidel 1983)