Journal of Biogeography (J. Biogeogr.) (2017) 44, 1665–1678

ORIGINAL Biogeographical evidence for common ARTICLE vicariance and rare dispersal in a southern Appalachian harvestman (, cavicolens) Marshal Hedin1,* and Maureen McCormack1,2

1Department of Biology, San Diego State ABSTRACT University, San Diego, CA 92182-4614, USA, Aim Species or higher taxa that are obviously dispersal-limited, but which 2Wisconsin State Lab of Hygiene, University occupy large geographical distributions, represent a biogeographical paradox. of Wisconsin-Madison, Madison, WI 53706, USA Dispersal must have happened, likely under special and infrequent environ- mental conditions, but details have been lost to history. The overarching goal of our research is to understand the details of a ‘common vicariance, rare dis- persal’ biogeographical history in a widespread but habitat-specialized harvest- man species () with a southern Appalachian centre of distribution.

Location Eastern North America, southern Appalachians. Methods We assessed cryptic speciation using mitochondrial and nuclear gene DNA sequence data, testing alternative delimitation hypotheses using multi- species coalescent analyses. We also tested whether riverine barriers are associ- ated with mitochondrial genealogical structuring, focusing on multiple rivers in the southern Blue Ridge physiographical province. Finally, we conducted popu- lation genetic analyses to assess female-based range expansion out of the south- ern Blue Ridge. Results Genetic analyses suggest a large number of species-level lineages within S. cavicolens, although we prefer a more conservative three-species hypothesis. These putative species are geographically cohesive and allopatric (Ozarks, Cumberland Plateau, southern Blue Ridge), with the Blue Ridge spe- cies including multiple divergent mitochondrial haplogroups. Several genealogi- cal breaks in the Blue Ridge species coincide with riverine barriers, separating mostly allopatric mitochondrial lineages. Contrasting with evidence for con- strained gene flow and vicariance, two Blue Ridge haplogroups reveal extensive range expansion both northwards and westwards, resulting in the widespread distribution of closely related haplotypes, and occasional sympatry of dispersive haplotypes. Main Conclusions Hidden beneath the apparently widespread distribution of a single species is a history of old vicariance separating geographically disjunct cryptic species. How these lineages came to occupy such disparate geographies is illustrated by dynamics within the Blue Ridge species, where both in situ vicariance and long-distance dispersal have shaped a ‘common vicariance, rare dispersal’ biogeographical history. *Correspondence: Marshal Hedin, Department Keywords of Biology, San Diego State University, San competitive exclusion, cryptic species, glacial refugia, long-distance dispersal, Diego, CA 92182-4614, USA. E-mail: [email protected] riverine barrier, short-range endemism

ª 2017 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 1665 doi:10.1111/jbi.12973 M. Hedin and M. McCormack

inferred based on the combination of geographical distribu- INTRODUCTION tion, time-calibrated phylogenies and algorithmic biogeo- Short-range endemic (SRE) taxa, defined as species or graphical analyses that indirectly imply dispersal higher taxa with naturally small geographical distributions (Schonhofer€ et al., 2013). (Harvey, 2002; Harvey et al., 2011; Keith & Hedin, 2012), This paper focuses on a single wide-ranging Sabacon present a biogeographical paradox. SRE taxa are often dis- species (S. cavicolens Packard, 1884) from the eastern Uni- tributed as arrays of parapatric or allopatric taxa, consis- ted States. This taxon is related to species from southern tent with a biogeographical history dominated by low Europe, with the lineage that ultimately led to S. cavicolens dispersal and vicariance. However, the larger encompassing hypothesized to have dispersed to eastern North America geographical distribution of such allo- or parapatric arrays near the Eocene-Oligocene transition (Schonhofer€ et al., implies some level of dispersal during the history of the 2013). Sabacon cavicolens occurs most commonly in moist, lineage (e.g. Garcıa-Vazquez & Ribera, 2016). An illustra- rocky habitats in the richly forested mountains of the tive example is the North American spider genus Hypochi- southern Appalachians, but also includes more northern lus, with five species in the southern Appalachians, two populations in previously glaciated regions (e.g. Wisconsin, species in the southern Rockies and three species in mon- Michigan, Maine, etc.), and disjunct populations in the tane California. All Hypochilus species are habitat-specia- Ozark highlands (Fig. 1; Shear, 1975). We hypothesize that lized, short-range endemics, where allopatry prevails even S. cavicolens displays a ‘common vicariance, rare dispersal’ within montane regions (Hedin, 2001). Moreover, available biogeographical history, and as such, represents a micro- genetic data for individual species suggests extreme popula- cosm of biogeographical patterns observed more broadly tion genetic fragmentation (Hedin & Wood, 2002; Keith & within Sabacon. However, because the timeframe is more Hedin, 2012). Overall, vicariance clearly dominates the his- recent, inferred patterns provide more direct and thus tory of this SRE taxon. But how do we explain the overall more powerful evidence for biogeographical processes. For larger geographical distribution in California, or in Appala- example intraspecific phylogeographic data can be used to chia, or in North America? Biogeographers are left to infer more directly infer demographic expansion events; such historical dispersal based on indirect evidence, sometimes demographic information is lost in comparisons at deeper despite a complete lack of evidence for modern-day dis- phylogenetic levels. persal abilities in such taxa. This is a problem with Geographical fragmentation has promoted SRE specia- ancient and rare dispersal – dispersal must have happened, tion in many terrestrial lineages that occupy the mountains likely under special environmental conditions, but details of the southern Appalachians. Vertebrate examples are have been lost to history. numerous in salamanders (e.g. Weisrock & Larson, 2006; Biogeographical patterns observed in the harvestman Kozak & Wiens, 2010; Herman & Bouzat, 2016), whereas genus Sabacon seem consistent with a history dominated by examples include other harvestmen (Thomas & vicariance, with occasional rare dispersal. Sabacon includes Hedin, 2008; Hedin & Thomas, 2010), spiders (Hedin, over 50 described species known from disjunct geographical 1997, 2001; Hendrixson & Bond, 2005; Keith & Hedin, centres in the Northern Hemisphere (North America, west- 2012; Hedin et al., 2015) and others. Also, intraspecific ern Europe, eastern Siberia, Nepal, Korea and Japan; see phylogeographical fragmentation is evident within more Schonhofer€ et al., 2013; Martens, 2015). Sabacon prefer wide-ranging species, with many taxa including divergent moist, cool microhabitats, in habitats such as caves, upland populations on distinct massifs, separated by lowland river- forests under rocks or woody debris and under rocks near ine barriers. For example a conspicuous lowland riverine streams (summarized in Schonhofer€ et al., 2013). It appears barrier is the Asheville Basin, which includes the French that physiological constraints to cryophilic microhabitats Broad River. This relatively dry lowland is unsuitable for restrict gene flow, and higher level phylogenetic results are habitat-specialized upland species, and many phylogenetic consistent with this assertion. Larger phylogenetic clades and phylogeographic studies have implicated this barrier correspond to geographical centres of endemism on differ- (e.g. Thomas & Hedin, 2008; Kozak & Wiens, 2010; Hedin ent continents, each of these centres with arrays of closely et al., 2015). More generally, Thomas & Hedin (2008) related SRE taxa occurring in allopatry or parapatry. How- hypothesized that multiple riverine barriers in the southern ever, interwoven in this vicariance-dominated history is evi- Blue Ridge physiographical province may act to promote dence for rare, long-distance dispersal. Because harvestmen divergence, a pattern also seen in salamanders (Herman & are not known to be phoretic (Giribet & Sharma, 2015), Bouzat, 2016). this dispersal must have occurred via intrinsic short-range Climatic stability through time, and in particular a lack of movements over hundreds or thousands of generations. In glaciation in the southernmost mountains during the Pleis- Sabacon, an apparent pulse of trans-continental dispersal tocene, has also promoted high diversity in the southern events is temporally coincident with special environmental Blue Ridge. However, many lineages have ultimately conditions of the ‘icehouse’ Eocene-Oligocene transition expanded out of these refugial mountains (reviewed in Soltis (Ivany et al., 2000; Schonhofer€ et al., 2013). These ancient et al., 2006; Herman & Bouzat, 2016), with genetic studies dispersal events occurred in multiple lineages, and are revealing patterns consistent with post-glacial range

1666 Journal of Biogeography 44, 1665–1678 ª 2017 John Wiley & Sons Ltd Biogeographical history of Appalachian Sabacon

Figure 1 Map of sampled locations for Sabacon cavicolens. Upper left inset shows overall geographical sample, highlighting distributions for Ozarks, Cumberland, and southern Blue Ridge genetic clades. Main map shows sample locations for mitochondrial subclades within southern Blue Ridge Sabacon. Numbers correspond to geographical locations, as in Appendix S1. Circled numbers correspond to sites with Clinch Mountain-Bullpen syntopy; site 74 (square box) includes Clinch Mountain-Cumberland syntopy. Approximate location of relevant regional rivers shown. [Colour figure can be viewed at wileyonlinelibrary.com] expansion from a southern Appalachian centre of distribu- rare dispersal’ biogeographical history at relatively shallow tion. These expansion events involve lineages that have time-scales. rapidly dispersed northwards to previously glaciated regions, but there is also evidence for expansion westwards and even MATERIALS AND METHODS southwards (Walker et al., 2009; Emerson et al., 2010; Herman & Bouzat, 2016). Taxon sampling and sequence data collection The research presented here addresses three primary questions. Based on a three-gene concatenated analysis for Specimens were hand-collected or collected with an aspira- a limited sample, Schonhofer€ et al. (2013) suggested that S. tor from under rocks and woody debris, typically along cavicolens includes multiple cryptic species. We use a larger streams. Specimens destined for molecular work were pre- specimen sample with mitochondrial and five nuclear genes served in 100% EtOH and stored at À80 °C. Genomic to formally assess cryptic speciation, testing alternative DNA was extracted from leg tissues using the Qiagen delimitation hypotheses using multispecies coalescent DNeasy kit, per manufacturer’s instructions; the remaining (MSC) analyses. Finding cryptic species would indicate specimen was retained as a voucher in the SDSU Terres- more regional endemism than the current single-species trial collection. The complete sample includes implies. Second, we use a dense geographical 168 specimens from 88 locations, with denser sampling in sample from the southern Blue Ridge to explicitly test the southern Blue Ridge (Fig. 1; see Appendix S1 in Sup- whether riverine barriers have resulted in phylogeographical porting Information). The sample covers the known south- fragmentation in Sabacon. Finally, we seek evidence for ern, western and north-eastern distributional limits of S. range expansion from the southern Blue Ridge. Overall, our cavicolens, but lacks north-western samples (e.g. Michigan, goal is to understand the details of a ‘common vicariance, Wisconsin, etc.; Shear, 1975).

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generating Sanger sequence data for five nuclear genes (see Mitochondrial gene tree and species discovery Appendix S1). These data were supplemented with transcrip- analyses tome data from a single sample (see Appendix S2). Nuclear Mitochondrial cytochrome oxidase I (COI) sequences were genes included ribosomal 28S and elongation factor one- generated for the complete specimen sample, using PCR pri- alpha (EF1-a) exon, used previously in species-level harvest- mers and conditions summarized in Appendix S2. A single man studies (e.g. Derkarabetian et al., 2011; Schonhofer€ COI sequence was also extracted from a transcriptome (see et al., 2013), plus three novel nuclear genes generated from Appendix S2). Twelve ingroup COI sequences were previ- comparisons of Sabacon transcriptomes (Table 1; see ously reported (Schonhofer€ et al., 2013). PCR products were Appendix S2). PCR conditions and primers are summarized purified via polyethylene glycol (PEG) precipitation, and in Appendix S2. PCR products were purified via PEG precip- Sanger sequenced at the SDSU Microchemical Core Facility. itation or Millipore plates, and Sanger sequenced at SDSU or Sequence contigs were assembled and edited using Macrogen USA. All matrices were assembled, edited and Sequencher 4.5 (Gene Codes Corporation, Ann Arbor, MI, aligned manually in Geneious. EF1-a data from four indi- USA), and manually aligned using Geneious Pro 7.1.7. viduals included a single heterozygous site per sequence, (http://www.geneious.com, Kearse et al., 2012). which were phased manually. Heterozygous nuclear A mitochondrial gene tree was reconstructed using maxi- sequences for the three novel nuclear genes were bioinfor- mum likelihood implemented in the RAxML_GUI matically phased to alleles using the software program Phase (Stamatakis, 2006, 2014; Silvestro & Michalak, 2012). Analy- 2.1.1 (Stephens et al., 2001; Stephens & Donnelly, 2003). ses included a thorough bootstrap analysis (1000 bootstrap SeqPhase (Flot, 2010) was first used to convert matrices for replicates) followed by multiple inferences (100) on align- input into Phase, with Phase analyses then conducted using ments. Sequences were partitioned by codon, with a default settings (phase threshold = 90%, 100 iterations, thin- GTR_Gamma model applied to each partition. Mitochon- ning interval = 1, burn-in = 100). drial trees were rooted using sequences from the Ozarks lin- To summarize overall patterns of nuclear gene divergence, eage, following the root placement of Schonhofer€ et al. a NeighborNet network was constructed in SplitsTree4 (2013) based on Bayesian analyses of concatenated nuclear (Huson & Bryant, 2006). This network was based on multi- and mitochondrial data. The mitochondrial RAxML gene genic nuclear genetic distances among individuals, calculated tree was used as input in bPTP species delimitation analyses, using Pofad 1.05 (Joly & Bruneau, 2006). Pofad analyses implemented on the bPTP server (http://species.h-its.org/ptp/ were conducted using both standardized (all individual ; Zhang et al., 2013). PTP implements a model assuming matrices given the same weight) and non-standardized matri- gene tree branch lengths generated by two independent Pois- ces (more variable matrices with greater weight); individual son process classes (within- and among-species substitution gene distance matrices were calculated using Kimura 2-para- events) to delimit putative species. bPTP analyses were run meter (K2P) distances in mega 6.06 (Tamura et al., 2013). for 100,000 Markov chain Monte Carlo (MCMC) genera- Bayes factor delimitation (BFD; Grummer et al., 2014) tions, with a thinning of 100 and burn-in of 0.1. was used to statistically distinguish among alternative species delimitation models. This method compares the marginal likelihoods of competing hypotheses (e.g. species tree models Nuclear species validation analyses with sequences assigned to differing numbers of lineages), RAxML and bPTP analyses reveal multiple, highly divergent and chooses the model that best explains the data based on COI lineages, perhaps corresponding to cryptic species (see calculation of BFs (Grummer et al., 2014). Following COI Results; also Schonhofer€ et al., 2013). To further assess this bPTP and nuclear Pofad results, Sabacon lineages were split possibility we subsampled 20 individuals from 19 locations, or combined to represent three alternative species

Table 1 Eastern Sabacon gene data.

Primer combo Aligned length/no. Substitution name sequences model PI sites Nucleotide diversity Annotation

COI 954/168 266 0.081 28S 1149/17 HKY+I+G 17 0.006 EF1-a 666/21 K80+I 18 0.010 SAB A1_A2 551/31 K80+I 18 0.008 Homologous to Ixodes protein ISCW001223, Mid1-interacting protein SAB A9_A10 777/29 K80+I 26 0.008 Homologous to Ixodes protein ISCW005133, Zinc finger protein SAB B5_B6 504/27 K80+I 47 0.028 Homologous to Ixodes protein ISCW010225, Conserved hypothetical protein

Note: Parsimony informative sites and nucleotide diversity values calculated using mega (Tamura et al., 2013).

1668 Journal of Biogeography 44, 1665–1678 ª 2017 John Wiley & Sons Ltd Biogeographical history of Appalachian Sabacon delimitation hypotheses (Table 2). A *beast species tree (in- Based on COI gene tree patterns, four analyses were con- ferred using *beast 1.8.2, Drummond et al., 2012) was esti- ducted to examine the relationship between genetic (patris- mated for each alternative hypothesis, using nuclear only tic) distance and putative barriers (Little Tennessee, matrices, without outgroups. *beast analyses were per- Tuckaseegee, French Broad; Table 3), while controlling for formed using 10 million generations, with data saved every geographical distance. Using a randomly sampled single hap- 1000 generations; the first 20% of each run was discarded as lotype per geographical location, phylogenetic patristic dis- burn-in. For each hypothesis three *beast replicates were tances were calculated from a COI RAxML tree using the conducted to ensure convergence, further assessed using ‘adephylo’package (Jombart & Dray, 2008) in R (2014). For effective sample size (ESS) values with Tracer 1.6 (Rambaut each test a binary river barrier response matrix was assem- et al., 2014). Substitution models were chosen with Parti- bled, with locations on the same versus opposite sides of a tionFinder 1.1.1 (Lanfear et al., 2012), using linked branch potential barrier scored as 0 versus 1 respectively (see lengths, beast models of molecular evolution, BIC model Table 3). Geographical distance matrices were calculated selection and greedy searches (Table 1). Because almost all using the Geographic Distance Matrix Generator (http:// nucleotide variation occurred at third positions for nuclear biodiversityinformatics.amnh.org/open_source/gdmg/). Partial protein coding genes, a single model was applied to each mantel tests were calculated using the ‘vegan’ package (Oksa- gene. Marginal likelihoods were estimated using path-sam- nen et al., 2015) in R using 10,000 permutations to evaluate pling (PS, Lartillot & Philippe, 2006) and stepping-stone (SS, significance. Xie et al., 2011) methods, with 100 path steps, a chain length of 100,000 generations and likelihoods saved every 100 gen- Range expansion of mitochondrial subclades erations. Marginal likelihood estimates (MLE) were averaged across replicate runs to generate a single PS and SS value for Standard nucleotide summary statistics were calculated for each hypothesis. Bayes factors were calculated by taking the southern Blue Ridge COI subclades (see Results), including difference between the log of the best MLE and the log of intra- and intergroup K2P distances, number of haplotypes other MLEs and multiplying each result by two [i.e. (h), number of segregating sites (S) and nucleotide diversity

2 9 (ÀlnHypA ÀÀlnHypB)]. Following Kass & Raftery (p). All values were calculated in mega (Tamura et al.,

(1995), BF values exceeding 10 were considered as ‘decisive’ 2013). Tajima’s D (Tajima, 1989) and Fu’s Fs (Fu, 1997) support for a hypothesis. were used to detect deviations from demographic equilib- rium, where an excess of low-frequency polymorphisms (cor- responding to negative values) indicates demographic Test for riverine barriers expansion (or non-neutral molecular evolution). Significance The significance of mitochondrial phylogeographical breaks was assessed in Arlequin 3.5.2.2 (Excoffier & Lischer, 2010) across modern-day riverine barriers was tested using partial using 50,000 coalescent simulations. Mismatch distributions Mantel tests (e.g. Kozak et al., 2006; Lemmon et al., 2007). were used to test for spatial expansion (Excoffier, 2004;

Table 2 Alternative species delimitation hypotheses tested using BFD.

Average marginal likelihoods Hypothesis Distinct species (total in parentheses) (path sampling/stepping stone) Motivation

H1 Ozarks, Cumberland, Chunky Gal_1, À7017.69/À7019.1 COI RaxML clades + conservative Chunky Gal_2, Joyce Kilmer, interpretation of bPTP northern Georgia, Bullpen, Clinch Mountain (8)

H2 Ozarks, Cumberland, southern Montane (3) À7036.05/À7037.37 Conservative COI RaxML, nuclear POFAD H3 Ozarks, Appalachians (2) À7087.49/À7089.15 Conservative biogeographical hypothesis

Table 3 Results of partial Mantel tests for riverine barriers.

Clade/Barrier RP-value Details

Within Joyce Kilmer – role of little Tennessee 0.1937 0.0278 Populations 8–20 vs. 21–25 on opposite sides of barrier Within Bullpen – role of Tuckaseegee 0.9237 0.0001 Populations (26, 27, 31, 32) vs. (28–30, 33–43) on opposite sides of barrier Bullpen/Joyce Kilmer – role of little Tennessee 0.3044 0.0001 Populations 8–20 vs. 21–43 on opposite sides of barrier Bullpen/Clinch – role of French Broad 0.8208 0.0001 Populations (26–28, 31–43) vs. (29, 30, 46–80) on opposite sides of barrier

Notes: See Fig. 1 for map of locations. All tests involving Bullpen Clade excluded populations 44–50, part of ‘Bullpen expansion clade’ (i.e. not found in southern Blue Ridge).

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Excoffier & Lischer, 2010), considering both sum of Joyce Kilmer and Bullpen (Figs 1–3); pairwise average K2P squared deviations (SSD) statistics and the raggedness distances among these subclades are relatively high, ranging index (Harpending, 1994), with statistical significance from 6% to 13% (Table 4). Sequences from two additional assessed in Arlequin based on parametric bootstraps southern populations form a grade that we refer to as (99,000 replicates). Chunky Gal. Within the southern Blue Ridge clade, geo- Bayesian skyline plots (BSPs) were also used to detect graphically adjacent Joyce Kilmer and Bullpen are COI sister changes in effective population size, potentially associated clades, occupying the intermediate geographical region with range expansion (Ho & Shapiro, 2011). These analyses between disjunct northern Georgia and Clinch Mountain were conducted using beast 1.8.3 (Drummond et al., 2012), clades (Fig. 1). focusing on three well-sampled Blue Ridge COI subclades (see Results). Models of molecular evolution were chosen Nuclear evidence for cryptic species using PartitionFinder. beast analyses were conducted using a lognormal uncorrelated relaxed clock model, piece- Mitochondrial bPTP analyses suggest up to twelve putative wise-constant Coalescent: Bayesian Skyline tree prior (Drum- cryptic species within S. cavicolens (Figs 2 & 3). These corre- mond et al., 2005), with MCMC chain lengths (> 20 spond to the primary clades and subclades outlined above, million) set to achieve high ESS values. A COI clock rate was some of which are further split in bPTP analyses (e.g. each specified based on a well calibrated arthropod rate, using a Chunky Gal branch corresponds to a putative species, etc.; normal prior with a ucld.mean of 0.0178 Æ 0.0019 (Papado- Figs 2 & 3). This high number of distinct mitochondrial lin- poulou et al., 2010; Table 4). This mitochondrial rate has eages is not reflected in the nuclear standardized-distances provided realistic divergence time estimates in other harvest- Pofad results, which more conservatively indicate three men (DiDomenico & Hedin, 2016), but we acknowledge the groups congruent with three primary mitochondrial clades caveats involved in applying a universal rate in this particular (Ozarks, Cumberland Plateau, southern Blue Ridge; Fig. 4a). system. All beast analyses were replicated to insure consis- Pofad results using non-standardized distances are similar tent results. (not shown). Individuals from different southern Blue Ridge mitochondrial subclades do not form distinct clusters in nuclear Pofad networks (Fig. 4a). RESULTS Three alternative hypotheses were tested with nuclear-only Unique mitochondrial sequences and unphased nuclear DNA matrices using BFD (Table 2). Average marginal likelihood sequences have been submitted to GenBank (see values are highest for the eight species model using both PS Appendix S1). Sequence lengths, number of parsimony infor- and SS methods, with BF values indicating ‘decisive’ support mative sites, sequence evolution models and diversity statis- over alternative three-species and two-species hypotheses tics are summarized in Table 1. (Table 2). Likewise, BF values indicate ‘decisive’ support for a three-species hypothesis over the two-species hypothesis. Because the three-species hypothesis is clearly supported by Mitochondrial clades independent analyses (Figs 2 & 4a), we prefer this more con- Three primary geographical clades are recovered in COI servative hypothesis; the *beast tree corresponding to this RAxML analyses (Figs 1 & 2). These include an Ozarks result is shown in Fig. 4b. An argument as to why BFD might clade, a Cumberland Plateau clade (including a location near potentially over-split lineages is presented in the Discussion. the type locality of S. cavicolens), and a broadly distributed southern Blue Ridge clade with an apparent centre of diversi- Riverine barriers fication in the Blue Ridge of the southern Appalachians. Four subclades are found within the southern Blue Ridge Several genealogical breaks both within and among southern clade, informally named Clinch Mountain, northern Georgia, Blue Ridge COI subclades suggest a possible role for rivers as

Table 4 Nucleotide summary statistics for Blue Ridge COI subclades.

COI subclade noGA CG JK CM BP nS p

Northern Georgia 0.005 ––––10 12 0.0045 Chunky Gal 0.112 0.03 ––– 4 47 0.0288 Joyce Kilmer 0.131 0.06 0.015 ––32 57 0.0144 Clinch Mountain 0.082 0.117 0.128 0.028 – 61 120 0.0269 Bullpen 0.134 0.069 0.076 0.125 0.018 54 91 0.0177 Bullpen expansion – –––– 9 33 0.0115

Notes: Number of base substitutions per site, averaged over all sequence pairs within groups (on diagonal, italics), and between groups (lower tri- angle), conducted using K2P model. Chunky Gal populations treated as a clade. n = Number of sequences, S = segregating sites, p = nucleotide diversity. All values calculated in mega.

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Figure 2 COI RAxML gene tree, rooted using Ozarks clade. Clades or groups named as in text; phylogenetic detail for Bullpen and Clinch Mountain subclades shown in Fig. 3. Bootstrap values shown only for primary clades. Arrowhead designates site with syntopy; black ovals indicate clades or sequences supported as putative species in bPTP analyses. Clades impacted by potential riverine barriers named. Location numbers as in Appendix S1. [Colour figure can be viewed at wileyonlinelibrary.com] barriers to female-based gene flow. The Little Tennessee plays for Bullpen and Clinch Mountain populations found outside a potential role within the Joyce Kilmer subclade, and of the southern Blue Ridge. Closely related haplotypes in a between the Joyce Kilmer and Bullpen subclades; the Tuck- weakly supported Bullpen ‘expansion’ subclade (Fig. 3) are aseegee plays a potential role within the Bullpen subclade; disjunct from remaining Bullpen locations; included in this the French Broad plays a role between Clinch and Bullpen geographically dispersed lineage are samples from northern subclades (Figs 1–3). The influence of all of these potential Alabama and southern Tennessee (sites 45, 46), northeastern barriers is statistically significant using partial Mantel tests Tennessee (sites 47, 48, 50) and southern Illinois (site 44; (Table 3). Fig. 1). Illinois sequences are ~1% divergent from other Bull- pen sequences, whereas the Bullpen subclade is on average > 7% divergent from samples in the neighbouring Joyce Kil- Syntopy and expansion of mitochondrial lineages mer subclade (Table 4). This pattern of highly dispersed Although allopatry of mitochondrial clades or subclades is samples showing minimal genetic divergence is also found in the rule, there were five instances where multiple specimens the Clinch Mountain subclade, although disparate samples sampled from a locality carry haplotypes belonging to two here do not form a cohesive mitochondrial lineage (Fig. 3). separate COI lineages (Figs 1–3). In four instances, haplo- A sample from New Hampshire (site 80) is approximately types belonging to Bullpen and Clinch Mountain subclades 880 km from related populations, but NH haplotypes are are syntopic (Fig. 3). Most of these locations are in the < 1% divergent from related Clinch Mountain haplotypes. Appalachian Valley and Ridge physiographical province Tajima’s D values are not statistically significant, whereas north-east of the French Broad River, with one exception in values for three subclades are significant for Fu’s Fs south-central Tennessee (Fig. 1, site 46). At the remaining (Table 5). This pattern is potentially consistent with a higher locality (site 74 – Cave Springs Recreation Area), members sensitivity of Fu’s Fs to population expansion (Fu, 1997; of Clinch Mountain and Cumberland Plateau clades are syn- Ramırez-Soriano et al., 2008). Mismatch distributional analy- topic. It is notable that all instances of syntopy are outside ses for the Clinch subclade did not converge. Mismatch dis- of the southern Blue Ridge. tributions for three other subclades are unimodal, with non- Qualitative patterns of low genetic divergence over large significant SSD values indicating observed data consistent geographical distances are consistent with spatial expansion with the spatial expansion model of Arlequin (Table 5);

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Figure 3 COI RAxML gene tree for Bullpen and Clinch Mountain subclades. Bootstrap values shown only for primary lineages. Arrowheads designate locations with syntopy; black ovals indicate clades or sequences supported as species in bPTP analyses. Clades impacted by potential riverine barriers named. Location numbers as in Appendix S1. [Colour figure can be viewed at wileyonlinelibrary.com] these subclades show small raggedness values, also consistent MSC analyses and formal hypothesis testing, and these analy- with expansion. Bayesian Skyline Plots for Clinch, Bullpen ses conservatively support the existence of three distinct spe- and Joyce Kilmer all indicate increases in population effective cies in this complex. We hypothesize that the Cumberland sizes (see Appendix S3), with temporally older demographic clade corresponds to S. cavicolens, as our sampled Red River increases in Clinch and Bullpen consistent with hypothesized Gorge population is geographically close to the type locality. range expansion. This implies that both the Ozarks and southern Blue Ridge lineages need to be described as new taxa, which is planned for a separate manuscript. Whether these three separate lin- DISCUSSION eages exhibit morphological divergence remains unclear. In Sabacon, male characters are most diagnostic, but males for Cryptic species the S. cavicolens complex are rare in collections. Shear (1975) The possibility of cryptic species in S. cavicolens was argued illustrated minor differences in male chelicerae and pedipal- in Schonhofer€ et al. (2013), based on concatenated analyses pal patellas for specimens from Ferne Clyffe IL (Bullpen sub- of three genes. We further examined this possibility using clade), Mt Mitchell NC (Clinch Mountain subclade) and

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Figure 4 (a) NeighborNet network reconstructed using standardized Pofad nuclear distances. Colours for specimens from southern Blue Ridge mitochondrial subclades as in Figs 2 & 3. (b) Nuclear-only *beast species tree estimated for three-species hypothesis, with posterior probability values at nodes. Tree files from three separate *beast runs were combined using LogCombiner, and a maximum clade credibility tree (burnin = 3000) was produced using TreeAnnotator. [Colour figure can be viewed at wileyonlinelibrary.com]

Vermont (Clinch Mountain subclade). Adult males are cur- and bPTP results. However, nuclear Pofad results do not rently unknown for the Ozarks and Cumberland clades clearly indicate more than three lineages. Although it is (holotype S. cavicolens specimen is a female), but given the possible that genome-scale (e.g. RADSeq) or more rapidly differences observed in males within the southern Blue Ridge evolving nuclear genes will help resolve additional taxa clade, we predict that more substantive diagnostic differences within the southern Blue Ridge radiation, it may also be will exist in males from these obvious ‘cryptic’ species. that BFD is over-splitting the complex. This potential Nuclear-only BFD results actually support more than over-splitting occurs because of the panmixia assumption three species, consistent with the mitochondrial gene tree of the MSC model, which unrealistically assumes that

Journal of Biogeography 44, 1665–1678 1673 ª 2017 John Wiley & Sons Ltd M. Hedin and M. McCormack population genetic structure does not exist within species frequently be found living at the edge of smaller order (Hedin et al., 2015). streams higher in drainage basins. It is also clear that speci- The conservative three-species hypothesis implies older mens in spatially expanding lineages can disperse across very vicariance and mostly exclusive allopatry of geographically large rivers (e.g. Tennessee River). In the southern Blue disparate taxa, found in the Ozarks, on the Cumberland Pla- Ridge where multiple allopatric lineages show breaks coinci- teau, and in the southern Blue Ridge. Many potential barri- dent with riverine barriers, rivers may not act as absolute ers to gene flow separate these regions, including large rivers physical barriers per se, but rather may reduce or filter levels (e.g. Mississippi and Tennessee Rivers) and landscape barri- of gene flow. We hypothesize that the interaction of compet- ers (e.g. low elevation habitats of the Appalachian Valley and itive exclusion (priority effects, Case et al., 2005), reflecting Ridge Province). If we accept the Ozarks root placement as ecological niche conservatism with close congeners on oppo- hypothesized in Schonhofer€ et al. (2013), then the overall sites sides of barriers, plus reduced gene flow largely main- biogeographical pattern in eastern Sabacon is similar to the tains lineage boundaries. If the southern Blue Ridge W>E>W pattern observed in phalangodid harvestmen subclades actually represent cryptic species, then reproductive (Hedin & Thomas, 2010), where in this case eastern Blue interference (low hybrid fitness) might also play a role in Ridge lineages have ‘escaped’ the mountains and dispersed maintaining lineage boundaries (Case et al., 2005). back westwards (and northwards). Expansion out of the Blue Ridge Riverine barriers in southern Appalachia Multiple analyses provide congruent evidence for demo- Clear evidence for phylogeographical fragmentation is graphic and spatial expansion for several Blue Ridge lineages, observed in the southern Blue Ridge species, which is parti- although both quantitative and spatial qualitative patterns tioned into divergent, mostly allopatric mitochondrial sub- are most compelling for Bullpen and Clinch Mountain sub- clades. Based on phylogeographical patterns found in the clades. Both lineages have apparent centres of distribution in harvestman Fumontana deprehendor, Thomas & Hedin the southern Blue Ridge, with expansions westwards and (2008) proposed that major rivers and intermontane basins northwards (Fig. 1). Informally, our field experience suggests in the southern Blue Ridge act as barriers to gene flow. Riv- that population densities are lower outside of the Blue Ridge, ers as barriers to gene flow have been hypothesized in other with populations more scattered in scarcer suitable micro- cryophilic harvestman taxa (Richart & Hedin, 2013). The habitats. We hypothesize that these lower densities, implying French Broad, Little Tennessee and Tuckaseegee Rivers have less congeneric competition, may have played a role in facili- been identified as potential barriers in several harvestmen tating lineage expansion. (Fumontana, Bishopella), beetle (Trechus) and salamander Northern distributional limits for eastern Sabacon remain taxa (Desmosgnathus, Plethodon) (Kane et al., 1990; Weisrock unknown, and future geographical sampling in the far north & Larson, 2006; Thomas & Hedin, 2008; Kozak & Wiens, is needed to determine if these populations represent the 2010) in the southern Blue Ridge. In most of these prior Clinch and/or Bullpen subclades, versus the Cumberland studies barriers were inferred qualitatively, with phylogenetic clade or perhaps a novel lineage. We hypothesize that Pleis- breaks in gene trees coinciding with proposed barriers. Here tocene glacial cycles have impacted spatial expansion dynam- we took a quantitative approach, and identified the Tuck- ics, consistent with an ‘out of Appalachia’ refugial model aseegee, Little Tennessee and French Broad rivers as signifi- observed in other taxa (Church et al., 2003; Soltis et al., cant barriers. The Hiwassee may also play a vicariance role 2006; Walker et al., 2009; Emerson et al., 2010; Herman & for the Northern Georgia subclade, although we did not for- Bouzat, 2016). Some of our sampled Sabacon populations mally test this hypothesis because of sparse geographical come from locations covered by Laurentide ice sheets at Last sampling adjacent to the Hiwassee (Fig. 1). Glacial Maximum (LGM) (e.g. southern New Hampshire), Exactly how rivers constrain gene flow, both historically and many species records from northern states (Wisconsin, and currently, remains unclear. This is particularly so given Michigan, Maine; Shear, 1975) are from locations covered by that Sabacon appear to prefer moist habitats and can LGM ice. Our BSP time estimates for population size

Table 5 Neutrality and mismatch results for Blue Ridge COI subclades.

COI subclade DP-value Fs P-value Spatial SSD P-value Raggedness index P-value

Northern Georgia 0.121 0.583 À2.377 0.069 0.009 0.867 0.022 0.968 Joyce Kilmer À0.215 0.557 À11.304 0.002 0.007 0.271 0.006 0.901 Clinch Mountain À0.137 0.520 À14.455 0.003 –– Bullpen À0.507 0.352 À15.248 0.001 0.004 0.800 0.006 0.745 Bullpen expansion À0.268 0.409 À1.096 0.223 0.034 0.474 0.086 0.595

Notes: D = Tajima’s D, F = Fu’s F. All values calculated in Arlequin.

1674 Journal of Biogeography 44, 1665–1678 ª 2017 John Wiley & Sons Ltd Biogeographical history of Appalachian Sabacon increase in Bullpen and Clinch post-date the LGM (see Common vicariance, rare dispersal Appendix S3), but we emphasize that these time estimates rely upon a COI clock rate that may not strictly apply in Eastern Sabacon might well be characterized as ‘fragile’ – Sabacon. specimens collected from suitable microhabitats and placed Expansion out of the southern Blue Ridge westwards and in dry vials at room temperature die within minutes (pers. northwards has also been found in the harvestmen genus obs MH, MM.). How such moisture- and temperature-sen- Bishopella (Hedin & Thomas, 2010). In Bishopella, two sitive organisms can achieve larger geographical distribu- mitochondrial clades that bound the Asheville Basin appear tions, even on different continents, seems paradoxical. Our to have diversified within the southern Blue Ridge and sub- data and analyses for the Sabacon cavicolens complex sheds sequently expanded out onto the Cumberland Plateau. Spa- light on this paradox. We view dynamics uncovered within tial patterns in wood-feeding roaches are also strikingly the southern Blue Ridge lineage as a microcosm of those similar to Sabacon – a chromosomal race distributed mostly seen more broadly in eastern North America and the east of the French Broad in montane NC also extends northern Hemisphere, where cycles of common vicariance northeastwards into northern Virginia, and a chromosomal and uncommon long-distance dispersal interact to shape race from the central southern Blue Ridge includes disjunct biogeographical histories. populations in northern Alabama and Kentucky (Nalepa et al., 2002). Generally, these spatial expansions demonstrate ACKNOWLEDGEMENTS that habitat-specialized cryophilic arthropods can sometimes cross large river basins, perhaps in the context of competi- We thank Fred Coyle, Shahan Derkarabetian, Ryan Fawcett, tive release. Dalton Hedin, Lars Hedin, Robin Keith, Michael Lowder, Range expansion in Sabacon has resulted in the secondary Jordan Satler, Jeff Shultz, Jim Starrett and Steven Thomas contact and sympatry of previously allopatric lineages, always for their help with specimen collection. Bill Shear provided outside of the southern Blue Ridge (Fig. 1). This sympatry is specimens for morphological study. Jim Starrett helped with almost certainly more common than our limited geographic RNA extractions, and Dave Carlson assisted with transcrip- and specimen sample indicates. Without morphological dif- tome assembly. We gratefully thank Steve Perlaky for allow- ferences to diagnose sympatric lineages, both nuclear and ing us to borrow a vehicle while in the field, and thank mitochondrial data are needed to understand the nature of Fred Coyle and the Abbott family for providing excellent interactions associated with this secondary contact. Thus far, field accommodations. Research was funded by grants to we have not sampled sufficient nuclear data at enough syn- M.M. from the American Arachnological Society (Vincent topic sites to understand such interactions. Our results reveal Roth Fund for Systematic Research), and grants to M.H. one instance of mitonuclear discordance from south-western from the U.S. Fish and Wildlife Service (agreement VA (Sample OP658, site 74, Fig. 1), placed in the mitochon- 401814G013), California State University Program for Edu- drial Clinch Mountain clade, but in the nuclear Cumberland cation and Research in Biotechnology (CSUPERB), and the clade (Figs 3 & 4). We hypothesize that this discordance NSF (DEB 1354558). Shahan Derkarabetian, Casey Richart results from mitochondrial introgression of Clinch haplo- and two anonymous reviewers provided comments that types into the Cumberland species. This hypothesis is consis- improved the manuscript. tent with the easier movement of organellar genes across lineage boundaries, but inconsistent with theory and empiri- cal data showing that introgression is typically asymmetrical REFERENCES in directionality from resident to invading lineages (Currat Case, T.J., Holt, R.D., McPeek, M.A. & Keitt, T.H. (2005) et al., 2008; Toews & Brelsford, 2012). Future research to The community context of species’ borders: ecological and understand nuclear gene flow dynamics for additional cases evolutionary perspectives. Oikos, 108,28–46. of sympatry will provide robust tests of cryptic speciation Church, S., Kraus, J., Mitchell, J., Church, D. & Taylor, D. hypotheses. 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Waters, J.M., Fraser, C.I. & Hewitt, G.M. (2013) Founder Appendix S2 PCR conditions and marker development. takes all: density-dependent processes structure biodiver- Appendix S3 COI Bayesian skyline plots. sity. Trends in Ecology and Evolution, 28,78–85. Weisrock, D.W. & Larson, A. (2006) Testing hypotheses of speciation in the Plethodon jordani species complex with DATA ACCESSIBILITY allozymes and mitochondrial DNA sequence. Biological Journal of the Linnaean Society, 89,20–51. Aligned matrices have been deposited at Dryad (doi:10.5061/ Xie, W.G., Lewis, P.O., Fan, Y., Kuo, L. & Chen, M.H. dryad.mk32s). (2011) Improving marginal likelihood estimation for Baye- sian phylogenetic model selection. Systematic Biology, 60, BIOSKETCHES 150–160. Zhang, J., Kapli, P., Pavlidis, P. & Stamatakis, A. (2013) A The Hedin lab studies the evolution and diversity of arach- general species delimitation method with applications to nids, including spiders and harvestmen, with a biogeographi- phylogenetic placements. Bioinformatics, 29, 2869–2876. cal emphasis in the southern Appalachians. Author contributions: M.H. implemented the study design, SUPPORTING INFORMATION directed data collection, conducted analyses and wrote the manuscript; M.M. implemented the study design, collected Additional Supporting Information may be found in the specimens and DNA sequence data. online version of this article:

Appendix S1 Sample information, including GenBank Editor: Brent Emerson numbers.

1678 Journal of Biogeography 44, 1665–1678 ª 2017 John Wiley & Sons Ltd