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Molecular Phylogeographic Methods Reveal the Identity and Origin of a Dusky Salamander (Genus Desmognathus)

Population in Southern Illinois, USADonald B. Shepard1,4, Nicholus Ledbetter2, Amber L. Anderson2,

and Andrew R. Kuhns3

1School of Biological Sciences, Louisiana Tech University, P.O. Box 3179, Ruston, Louisiana 71272, USA2Department of Biology, University of Central Arkansas, 201 Donaghey Avenue, LSC 180, Conway,

Arkansas 72035, USA3Illinois Natural History Survey, Prairie Research Institute, University of Illinois, 1816 South Oak Street,

Champaign, Illinois 61820, USA4Corresponding author, e-mail: [email protected]

Abstract.—Introduced species can negatively affect natural communities and ecosystems through interactions with native species. Dusky salamanders (genus Desmognathus) are commonly collected from the wild and used as fish-ing bait, which can result in release outside their native population or beyond the limits of the range of the species. Desmognathus conanti is the only species of the genus native to Illinois, where it occurs in Pulaski County in the extreme southern tip of the state. In 1986, a population of Desmognathus was discovered at Jug Spring, Johnson County, about 32 km north of previously known Illinois populations. We generated mitochondrial DNA sequence data for Jug Spring Desmognathus and D. conanti from Pulaski County, and combined them with DNA sequences from GenBank to determine the species identity and geographic origin of Jug Spring Desmognathus. Our analy-ses confirmed the species identity of Pulaski County D. conanti and showed that Jug Spring Desmognathus are D. fuscus, a species that ranges throughout the eastern U.S. but is not previously known from Illinois. Jug Spring Desmognathus were most closely related to haplotypes of D. fuscus from the Cumberland Plateau of Tennessee, pointing to this region as the likely source of the Jug Spring population. The impacts of the introduced D. fuscus on the Jug Spring ecosystem are unknown, but their presence may negatively affect invertebrates and other salaman-ders occupying the spring and adjacent habitats. We recommend the population be monitored and that surveys be conducted to determine if this introduced species is expanding its range.

Key Words.—amphibian conservation; bait-bucket introduction; Batrachochytrium salamandrivorans (Bsal); forensic herpetology; introduced species; spring lizard

Introduction

Introduced species can have profound impacts on the integrity of natural communities and ecosystems through competitive and/or predatory interactions with native species (Parker et al. 1999; Mooney and Cleland 2001; Vilà et al. 2011). An important first step in man-aging introduced species is accurately identifying them as non-native. This is relatively straightforward when introduced species are easy to discriminate from native species and the introduced species is clearly outside its native range. However, it can be problematic when in-troduced species are difficult to distinguish morphologi-cally from native species and/or may represent a natural disjunct population (Bonett et al. 2007). In such cases, phylogeographic analysis of DNA sequences is an effec-tive method for confirming suspected species introduc-tions and identifying their geographic origins (Scheffer and Lewis 2001; Hebert and Cristescu 2002; McDowall 2008; Fitzpatrick et al. 2012).

Herpetological Conservation and Biology 11:434–450.Submitted: 23 March 2016; Accepted: 4 October 2016; Published: 16 December 2016.

Dusky salamanders (genus Desmognathus) are com-mon inhabitants of streams and riparian habitats in the eastern United States (U.S.), reaching their peak diver-sity in the southern Appalachian Mountains (Petranka 1998; Bonett et al. 2007). In the southeastern U.S., Des-mognathus and other salamanders collectively referred to as spring lizards are commonly (and legally) collected from the wild then used or sold as fishing bait (Martof 1953; Jensen and Waters 1999; Copeland et al. 2009). Salamanders collected as fishing bait may be transport-ed outside their native population or beyond the limits of the range of the species (Martof 1953). This human-mediated dispersal can result in gene flow among geo-graphically distant populations or the founding of a new extralimital population when individuals escape or are discarded from bait buckets (Martof 1953). For ex-ample, extralimital introductions of Western Tiger Sala-manders (Ambystoma mavortium) in California and Seal Salamanders (Desmognathus monticola) in Arkansas are thought to have occurred via the bait industry and

Copyright © 2016. Donald B. ShepardAll Rights Reserved.

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Shepard et al.—Identity and origin of introduced dusky salamanders.

Figure 1. Map of southern Illinois showing locations of native and introduced populations of Desmognathus with inset of eastern United States showing study area (outlined in box) and Putnam County, Tennessee.

bait buckets of fisherman (Bonett et al. 2007; Johnson et al. 2011). Using molecular phylogeographic methods, researchers identified the Great Plains region (Kansas, New Mexico, Oklahoma, Texas) as the source of the California introductions (Johnson et al. 2011) and north-ern Georgia as the source of the Arkansas introduction (Bonett et al. 2007).

The genus Desmognathus currently comprises 21 recognized species; however, several of these are com-posed of multiple morphologically cryptic but geneti-cally divergent lineages that likely warrant recognition as distinct species (Kozak et al. 2005; Beamer and Lamb 2008; AmphibiaWeb. 2015. AmphibiaWeb: Informa-tion on amphibian biology and conservation. Available from http://AmphibiaWeb.org/ [Accessed 15 December 2015]). Only one species, D. conanti (Spotted Dusky Salamander), is native to Illinois, USA, where it is known from Pulaski County in the extreme southern tip of the state along the Ohio River (Rossman 1958; Smith 1961; Brandon and Huheey 1979; Phillips et al. 1999; Fig. 1). In 1986, a population of Desmognathus was discovered at Jug Spring, near Dutchman Lake, John-son County, approximately 32 km (about 20 mi) north of the nearest Pulaski County locality (Moeller 1994;

Fig. 1). Dutchman Lake is a popular fishing locale and Desmognathus there occur in spring-fed streams along Dutchman Creek near a parking lot at the end of a road aptly named, Fishing Hole Lane.

Many species of Desmognathus are undifferentiated morphologically and can only be identified positively using genetic data (Tilley and Mahoney 1996; Bonett 2002; Beamer and Lamb 2008). Thus, it was difficult to determine the species identity of Jug Spring Desmog-nathus and ascertain whether they were native or intro-duced. Using allozyme gel electrophoresis, Moeller (1994) compared Jug Spring Desmognathus to 17 popu-lations of D. conanti and D. fuscus from Illinois, Indi-ana, Kentucky, Ohio, and Tennessee, USA. He found that Jug Spring Desmognathus were more similar genet-ically to D. conanti than to D. fuscus, but they were not closely related to any of the populations of D. conanti that he sampled, including those from Pulaski County, Illinois (Moeller 1994). The retention of dorsal spots after metamorphosis and the presence of an orange post-ocular stripe may help to distinguish D. conanti from D. fuscus, but these characters are not consistently diagnos-tic and vary ontogenetically and geographically (Ross-man 1958; Bonett 2002).

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Herpetological Conservation and Biology

At the time of the study on Jug Spring Desmognathus (Moeller 1994), D. conanti and D. fuscus were thought to be sister taxa; both were formerly considered subspecies of D. fuscus (Rossman 1958). However, we now have strong evidence from mitochondrial DNA (mtDNA) that these species are not sister taxa and that each spe-cies is actually composed of multiple, genetically diver-gent, phylogeographic lineages (Titus and Larson 1996; Kozak et al. 2005; Beamer and Lamb 2008). Because Moeller (1994) only compared Jug Spring Desmogna-thus to populations of D. conanti and D. fuscus, and he found that Jug Spring salamanders were not closely re-lated to any of the populations of D. conanti that he sam-pled, it is possible that Jug Spring Desmognathus are neither D. conanti nor D. fuscus, but are instead a dif-ferent species of Desmognathus. Thus, Moeller (1994) provided evidence that Jug Spring Desmognathus might be introduced, but he was unable to resolve the species identity and geographic origin of the population. Here, we use phylogeographic analysis of mtDNA sequences to determine the species identity and geographic origin of Jug Spring Desmognathus. Our results are important not only for documenting the biodiversity of Illinois, but also for the management of native species, especially D. conanti, which is listed as Endangered in Illinois (Illi-nois Endangered Species Protection Board 2015).

Materials and Methods

Data collection.—We collected tissue samples for DNA analysis from 10 Jug Spring Desmognathus on 13 March 2013. We searched for salamanders by turning over rocks in and around streams and caught salaman-ders by hand. For each individual, we clipped the last approximately 5 mm of the tail tip and placed it in a uniquely labeled 1.5 ml tube of 95% ethanol. We re-corded GPS coordinates (37.4889N, 88.9145W) and released salamanders where they were captured. We obtained a tissue sample of D. conanti from Pulaski County, Illinois, USA (37.1313N, 89.2043W; 18 July 2003), from the Illinois Natural History Survey Am-phibian and Reptile Frozen Tissue Collection (INHS Tissue 1532). Data for these samples are included in Appendices 1 and 2.

In the lab, we extracted genomic DNA from each sample using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, California, USA) following the protocol of the manufacturer. We quantified DNA yield using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA) and standardized DNA concentrations to 10–25 ng/μl. We used polymerase chain reaction (PCR) to amplify the mitochondrially encoded NADH dehydrogenase subunit 2 (ND2) gene and a portion of the cytochrome c oxidase I (COX1)

gene. For ND2, we used the PCR primers METf.6 (AAGCTTTCGGGCCCATACC) and COIr.8 (GCTAT-GTCTGGGGCTCCAATTAT) and followed the ther-mocycler protocol from Kozak et al. (2005). For se-quencing ND2, we used METf.6 and a newly designed internal primer (Desmog_ND2_391F: TCAACAT-GACAAAARCTTGCAC). For COX1, we used the primers cox1F (CGGCCACTTTACCYRTGATAATY-ACTCG) and cox1R (GTATTAAGATTTCGGTCTGT-TAGAAGTAT) for PCR and sequencing and followed the thermocycler protocol from Beamer and Lamb (2008). We verified PCR products using gel electropho-resis and cleaned them using ExoSap-IT (Affymetrix, Santa Clara, California, USA). Sequencing reactions and automated sequencing were performed by Eurofins Genomics (Louisville, Kentucky, USA) using BigDye Terminator chemistry and an ABI3730xl 96-capillary sequencer (Applied Biosystems, Foster City, California, USA). We edited chromatograms by eye to verify base calls and assembled contigs from forward and reverse sequences using Geneious v.7.1.5 (Kearse et al. 2012).

To determine the species identity and geographic origin of Jug Spring Desmognathus, we sequenced ND2 for one individual and COX1 for all 10 individuals. We also sequenced ND2 and COX1 for our sample of D. conanti from Pulaski County, Illinois. We combined our ND2 sequences with a data set of ND2 sequences for Desmognathus from Kozak et al. (2005) and Martin et al. (2016). Together these data comprised 1038 base-pairs (bp) for 106 terminal taxa and included representatives of all currently recognized species as well as genetically divergent phylogeographic lineages within some spe-cies that may represent morphologically cryptic species (Appendix 1). We combined our COX1 sequences with a data set of COX1 sequences for Desmognathus from Beamer and Lamb (2008) and Hibbitts et al. (2015). To-gether these data comprised 551 bp for 102 terminal taxa and included representatives of most recognized species (13 of 21) as well as extensive geographic sampling of genetic variation within several wide-ranging species (e.g., D. conanti, D. fuscus) in the southeastern U.S. (Appendix 2). We used both ND2 and COX1 because the ND2 data set has better taxonomic coverage and bet-ter geographic sampling west of the Appalachians in the Ohio River basin whereas COX1 has better geographic sampling in the Atlantic and Gulf coastal plains of the USA. Because the vertebrate mitochondrial genome is inherited as a single haploid unit with no recombination, mitochondrial genes are completely linked. Thus, ND2 and COX1 will share the same history.

Data analysis.—For both ND2 and COX1, we aligned sequences in Geneious using the MUSCLE al-gorithm and visually inspected alignments to verify an

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open reading frame. We included ND2 sequence of Pha-eognathus hubrichti (AY728233) and COX1 sequence of D. quadramaculatus (EU311649) from GenBank to use as outgroups in phylogenetic analyses (Mueller et al. 2004; Kozak et al. 2009). We used PartitionFinder (Lanfear et al. 2012) to determine the best partitioning scheme and substitution models for each alignment. Based on the Bayesian Information Criterion, the best partitioning scheme for the ND2 alignment was by co-don position with the best substitution models being HKY + Γ + I for first and second positions and GTR + Γ for third positions. The best partitioning scheme for the COX1 alignment was also by codon position with the best substitution models being K80 + Γ + I for first positions, F81 for second positions, and GTR + Γ for third positions.

We inferred phylogenetic relationships using Bayes-ian Inference in MrBayes v.3.2 (Ronquist et al. 2012). For both ND2 and COX1, we conducted two inde-pendent searches in MrBayes v.3.2 consisting of three heated and one cold chain for five million generations, retaining every 1,000th sample. We applied the appropri-ate substitution model to each codon position, unlinked substitution model parameters across partitions, linked branch lengths of partitions and scaled them relative to each other, and used default priors. We assessed burn-in within runs and convergence between runs by viewing plots of likelihood and parameter values in Tracer v.1.6 (Rambaut et al. 2014). We also ensured that Effective Sample Size (ESS) for all parameters was > 200 (Drum-mond and Bouckaert 2015). We discarded trees sampled before burn-in (20%), combined post-burn-in trees from the two independent runs, and generated a 50% majority rule consensus phylogram. We determined the species identity of Jug Spring Desmognathus to be the species (or clade) with which it shares a sister-taxon relationship in the ND2 and COX1 phylogenies. Relationships were considered to have high support when the Bayesian pos-terior probability (Bpp) of nodes was ≥ 0.95.

For both ND2 and COX1, we used MEGA v.5 (Tamura et al. 2011) to calculate uncorrected pairwise sequence divergence (p-distance) between Jug Spring Desmognathus and its sister taxon as well as between Jug Spring Desmognathus and Pulaski County D. conan-ti. We inferred the most likely geographic origin of Jug Spring Desmognathus to be the area from which its sis-ter taxon was sampled. Because geographic sampling differed between ND2 and COX1 phylogenies, we used the level of sequence divergence to identify which of the two hypothesized geographic origins is more likely. We recognize that ND2 and COX1 may have different substitution rates (Mueller 2006) so levels of sequence divergence may not be directly comparable. However, we can use the sequence divergence between Jug Spring

Desmognathus and Pulaski County D. conanti to gauge the relative rates of ND2 and COX1.

Finally, we constructed a phylogenetic network in SplitsTree4 (Huson and Bryant 2006) using all ND2 sequences of the species identified as conspecific with Jug Spring Desmognathus. We used uncorrected (p) genetic distances and the NeighborNet method (Bryant and Moulton 2004; Huson and Bryant 2006). Phylo-genetic networks may provide additional insight about relationships among haplotypes that are not revealed by a phylogenetic tree, and may be especially informative when trying to determine the geographic origin of an introduced population (Huson and Bryant 2006; Bonett et al. 2007; Johnson et al. 2011). With a single locus, as in our case, a split network allows us to focus on re-lationships of haplotypes within the species in question while also conveying the uncertainty in those relation-ships (Huson and Bryant 2006).

Results

Both ND2 and COX1 phylogenies identify Jug Spring Desmognathus as D. fuscus with high support (Bpp = 1.0 for ND2 and 0.99 for COX1; Figs. 2 and 3). The 551 bp-region of COX1 we sequenced was identical for all 10 Jug Spring individuals (Fig. 3). As expected, both phylogenies place Pulaski County D. conanti in a clade with other D. conanti and most closely related to populations from western Tennessee (Bpp = 1.0 for ND2; Fig. 2) and western Kentucky (Bpp = 0.90 for COX1; Fig. 3).

In the ND2 phylogeny, Jug Spring Desmognathus are most closely related to D. fuscus from the Cumber-land Plateau of Tennessee (Bpp = 1.0; Fig. 2). In the COX1 phylogeny, Jug Spring Desmognathus are sister to a widely distributed clade of D. fuscus comprising populations from North Carolina to Massachusetts and westward to Indiana and Kentucky (Bpp = 0.99; Fig. 3). Sequence divergence in ND2 between Jug Spring Desmognathus and D. fuscus from the Cumberland Pla-teau of Tennessee (Putnam County) is 0.87% whereas sequence divergence in COX1 between Jug Spring Des-mognathus and populations of D. fuscus from its sister clade (North Carolina, Virginia, West Virginia, Massa-chusetts, Indiana, Kentucky) averages 6.13%. In com-parison, sequence divergence between Jug Spring Des-mognathus and Pulaski County D. conanti is 10.67% for ND2 and 8.35% for COX1, indicating a slightly higher rate of evolution in ND2 (i.e., more substitutions per unit time). The ND2 phylogenetic network shows Jug Spring Desmognathus and D. fuscus from the Cumber-land Plateau of Tennessee (Putnam and Morgan coun-ties) clustered together at the end of a long edge, far separated from other haplotypes (Fig. 4).


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Figure 2. Bayesian 50% consensus phylogram for species of Desmognathus based on analysis of ND2 mitochondrial DNA sequences in MrBayes. Taxa are labeled by species, county, and state. Nodes are labeled with Bayesian posterior probabilities (Bpp); asterisks (*) indicate Bpp = 100%. Jug Spring D. sp. and Illinois D. conanti are shown in bold.

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Figure 3. Bayesian 50% consensus phylogram for species of Desmognathus based on analysis of COX1 mitochondrial DNA sequences in MrBayes. Taxa are labeled by species, county, and state. Nodes are labeled with Bayesian posterior probabilities (Bpp); asterisks (*) indicates Bpp = 100%. Jug Spring D. sp. and Illinois D. conanti are shown in bold.

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Discussion

We found that Jug Spring Desmognathus are D. fus-cus, a species that ranges throughout much of the east-ern U.S., but has not been recorded previously from Il-linois (Phillips et al. 1999). Desmognathus conanti, a species broadly distributed across the southern U.S., is native to Illinois and our sample from Pulaski County confirmed the identity of this population. The western range limit of D. fuscus occurs in southeastern Indiana, central Kentucky, and north-central Tennessee, although the species extends along the Cumberland River into western Kentucky where it forms a contact zone with D. conanti (Bonett 2002; AmphibiaWeb. 2015. op. cit.). Jug Spring D. fuscus are thus outside the known native distribution of D. fuscus. If Jug Spring D. fuscus rep-resent an isolated remnant population, as opposed to an extralimital introduction, then we would predict signifi-cant genetic divergence from other populations of D. fuscus. This appeared to be the case in analyses with COX1, but analyses with ND2, which had better sam-pling throughout the Ohio River basin, showed that Jug Spring D. fuscus are closely related to and only slightly divergent from populations of D. fuscus from the Cum-berland Plateau of Tennessee. The portion of COX1 that we sequenced was also invariable among the 10 Jug Spring Desmognathus we examined, which would be predicted by a founder effect and serves as additional support that the population is introduced (Fitzpatrick et al. 2012). Similarly, Bonett et al. (2007) found that D. monticola introduced to the Ozarks of northwestern

Arkansas were invariable in COX1 (515 bp for seven individuals). Natural populations of Desmognathus are often composed of multiple closely related mtDNA hap-lotypes (Tilley et al. 2008; Wooten et al. 2010; Hibbitts et al. 2015).

The phylogenetic placement of Jug Spring D. fus-cus and the low level of sequence divergence (0.87% in ND2) from D. fuscus of the Cumberland Plateau of Ten-nessee point to this geographic region as the likely source of the Jug Spring introduction. Our results for COX1 were inconclusive concerning the geographic origin of Jug Spring D. fuscus, but this data set lacked samples from the Cumberland Plateau (Beamer and Lamb 2008). Sequence divergence in COX1 between Jug Spring D. fuscus and other populations of D. fuscus was consider-ably higher (mean 6.13%) than sequence divergence in ND2 between Jug Spring D. fuscus and D. fuscus from the Cumberland Plateau (0.87%). Because ND2 had a higher rate of evolution than COX1, a difference in rates cannot explain the disparity in sequence divergence ob-served between Jug Spring D. fuscus and their sister tax-on in each phylogeny. Instead, this disparity is primarily due to variation in geographic sampling and the absence of closely related haplotypes in the COX1 data set. The level of ND2 sequence divergence between Jug Spring D. fuscus and D. fuscus from Putnam County, Tennessee (0.87%) is less than the sequence divergence between Cumberland Plateau populations of D. fuscus from Put-nam and Morgan counties (1.65%), which are separated by about 64 km (Ron Bonett and Ken Kozak, unpubl. data). Species of Desmognathus often show a strong

Figure 4. Phylogenetic network based on uncorrected (p) genetic distances between ND2 haplotypes of Desmognathus fuscus. Haplotypes are labeled by their geographic origin (County, State) and Jug Spring D. sp. is shown in bold.

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pattern of Isolation-by-Distance, meaning that genetic distance and geographic distance between populations are positively correlated (Tilley 2016). Based on this, we estimate that the source population of Jug Spring D. fuscus is fairly close (< 64 km) to the Putnam County, Tennessee locality.

Moeller (1994) provided evidence that Jug Spring Desmognathus were introduced, but his ability to posi-tively identify the species and geographic origin was hampered by limited sampling (11 populations of D. conanti and six populations of D. fuscus). He found that Jug Spring Desmognathus were not closely related to any of the populations that he sampled, but they were more similar genetically to D. conanti than D. fuscus (Moeller 1994). However, Moeller (1994) sampled only six populations of D. fuscus from southern Ohio, south-ern Indiana, and northern Kentucky. Our phylogenetic and network analyses with ND2 showed that Cumber-land Plateau populations of D. fuscus form a genetically divergent lineage apart from other D. fuscus, including those from Kentucky and Ohio. Bonett et al. (2007) emphasized the importance of adequate sampling of ge-netic diversity across the range of a species when using molecular-based methods to determine whether a popu-lation is introduced and whence it came. Moeller (1994) lacked samples from the Cumberland Plateau of Tennes-see, which proved critical for determining the origin of Jug Spring D. fuscus in our study. The importance of sampling is further exemplified by our more ambiguous results based on COX1 compared to ND2, of which only the latter included samples from the Cumberland Pla-teau of Tennessee. The distribution of this lineage of D. fuscus (fuscus A clade of Kozak et al. (2005)) is not well established and more sampling in the region would help to bolster our conclusions.

The impacts of the introduced D. fuscus on the Jug Spring ecosystem are unknown. Species of Desmogna-thus are predators of invertebrates and other salaman-ders, and their presence may negatively affect naïve native fauna inhabiting the spring and cave. Peck and Lewis (1978) did not document any threatened or en-dangered invertebrates in Jug Spring Cave during their surveys of subterranean invertebrate fauna of Illinois, but Cave Salamanders (Eurycea lucifuga) occur within the cave system (Matt Niemiller, pers. comm.). West-ern Tiger Salamanders (Ambystoma mavortium) intro-duced into California have been shown to have deleteri-ous effects on native California Tiger Salamanders (A. californiense) through displacement and hybridization (Riley et al. 2003; Ryan et al. 2009). Desmognathus fus-cus and D. conanti hybridize where their ranges contact in western Kentucky (Bonett 2002), but Jug Spring D. fuscus are about 32 km from the nearest locality for D. conanti in Illinois. Furthermore, the area between these

populations largely consists of agricultural and wetland habitats, which would impede dispersal and gene flow. In the 30 y since the discovery of the Jug Spring Des-mognathus, no additional localities along Dutchman Creek have been reported, but we are unaware of any targeted surveys. Therefore, we recommend that sur-veys be conducted along Dutchman Creek to ensure that the introduced population of D. fuscus is not expanding its range from Jug Spring. The population should also be monitored periodically and potential impacts on na-tive species should be assessed.

Using salamanders as fishing bait is legal in many states, including Tennessee, the identified source of Jug Spring D. fuscus. Other than limits on the species and number of individuals that can be collected, most states have few regulations on this practice (Nanjappa and Con-rad 2011). Transportation of salamanders outside their native population may facilitate the spread of pathogens, yet most states do not require disease/pathogen testing for exported or imported animals or those used as bait (Picco and Collins 2008; Nanjappa and Conrad 2011). In response to threats posed by the fungal pathogen, Ba-trachochytrium salamandrivorans (Bsal), the U.S. Fish and Wildlife Service (2016) recently listed 201 species of salamanders (67 species native to the U.S.) as Injuri-ous Wildlife under the Lacey Act, which prohibits im-portation and interstate transport. Species of the genera Desmognathus and Eurycea are commonly used as bait, but remain unregulated by this ruling due to a lack of data (Copeland et al. 2009; U.S. Fish and Wildlife Ser-vice 2016). If species of these genera are eventually found to be susceptible to Bsal or to be potential carriers of the fungus, then interstate transport would likewise be prohibited under federal law with individual states regulating transport within their own borders. Regard-less of whether this happens, the practice of using sala-manders as bait warrants increased regulation given the multitude of potentially negative impacts. To minimize these impacts without impinging greatly on individual fishermen, we recommend that the use and sale of sala-manders as bait be restricted to the drainage basin (HUC 4 or 6) from which the salamanders were collected. Many species of salamanders commonly used as bait in the eastern U.S. are associated with streams and genetic data indicate that population connectivity in these spe-cies is largely determined by drainage patterns (Jones et al. 2006; Kozak et al. 2006; Kuchta et al. 2016). Be-cause salamander populations within the same drainage basin are more likely to be linked by dispersal naturally, any human-mediated dispersal within a basin would likely have little to no impact.

Genetic data allow identification of species when morphology is uninformative and provide a means to determine the geographic origin of introduced species

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as well as trace their invasion route (Liu et al. 2006; Baker 2008; McDowall 2008; Guillemaud et al. 2010; Huffman and Wallace 2012). In addition to generating genetic data from populations under study, research-ers also frequently need reference genetic data from other species or populations for comparison (McDowall 2008). The availability of reference genetic data and the geographic scale of sampling can limit the success of these approaches (Bonett et al. 2007). For these reasons, publicly available genetic databases (e.g., GenBank) are invaluable resources. However, for the utility of these databases to be maximized, researchers not only need to submit their data, but it is imperative that they provide locality information or make it easy to connect genetic and locality data in their publications (Pope et al. 2015). The fact that our study would not have been possible without the genetic and locality data made available by other researchers underscores this point.

Acknowledgments.—We thank Ethan Kessler and Cody Roden for help with fieldwork and we thank Chris Phillips (INHS) for loaning a tissue sample of Desmog-nathus conanti from Pulaski County, Illinois. We also thank Scott Ballard for alerting us to Moeller’s Mas-ter’s thesis at SIU-Carbondale and Matt Niemiller for information on the fauna of Jug Spring Cave. We thank Toby Hibbitts and Gary Voelker for providing DNA sequences of Texas Desmognathus conanti from their study. Finally, we thank Ron Bonett, Ken Kozak, Ben Lowe, and Steve Tilley for providing additional tissues, DNA sequences, and/or locality data for some species of Desmognathus. Field collections were made under per-mit from the Illinois Department of Natural Resources and under University of Illinois IACUC protocol 14000. Funding was provided by the University of Central Ar-kansas.

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Donald B. Shepard is an Assistant Professor in the School of Biological Sciences at Louisiana Tech University, USA. His research employs molecular methods and geospatial tools to examine patterns of genetic variation within species, identify cryptic diversity, and understand the processes that drive ecological and evolutionary diversification of amphibians and reptiles. (Photographed by Alex Pyron).

Nicholus Ledbetter is currently a Ph.D. student at the University of Tulsa in Tulsa, Oklahoma, USA. He earned his B.S. at the University of Central Arkansas and was a participant in the NSF summer REU program at Kansas State University. His dissertation research focuses on population genetics of the Oklahoma Salamander (Eurycea tynerensis). (Photographed by Trevor Burton).

Amber L. Anderson is currently a M.S. student at the University of Central Arkansas in Conway, Arkansas, USA. She earned her B.S. from the University of Arkansas at Little Rock. Her thesis research is a multiyear mark-recapture study of the Ouachita Dusky Salamander (Desmognathus brimleyorum) that aims to identify the factors that influence dispersal and determine how dispersal contributes to population dynamics of semi-aquatic salamanders. (Photographed by Chris Robin-son).

Andrew R. Kuhns is the Herpetologist for the Biotic Survey and Assessment Program at the Illinois Natural History Survey within the Prairie Research Institute at the University of Illinois Urbana-Champaign, USA. He conducts surveys and habitat assessments for threatened and endan-gered amphibians and reptiles in areas scheduled for transportation improvements. Thus, he spends more time than the average person looking for and identifying road kill. As time and funding al-lows, he pursues independent research pertaining to the distribution, ecology, and conservation of amphibians and reptiles. (Photographed by Deb Maurer).


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Species Voucher#/Museum# County/Parish, State GenBank Accession#D. abditus JFBM17798 Cumberland, Tennessee KR732330D. aeneus KHK51 Gilmer, Georgia AY612342D. aeneus BTL237 Macon, North Carolina KR732331D. apalachicolae BTL238 Liberty, Florida KX764600D. apalachicolae KHK157 Liberty, Florida AY612373D. auriculatus BTL239 Wakulla, Florida KR732333D. brimleyorum RMB2173 Garland, Arkansa AY612420D. brimleyorum FC11578 LeFlore, Oklahoma AY612419D. brimleyorum RMB2518 Montgomery, Arkansas AY612423D. brimleyorum RMB2327 Nevada, Arkansas AY612422D. brimleyorum RMB2201 Polk, Arkansas AY612421D. brimleyorum MVZS14353 Scott, Arkansas AY612418D. carolinensis KHK72 Buncombe, North Carolina AY612368D. carolinensis KHK80 Buncombe, North Carolina AY612369D. carolinensis KHK118 McDowell, North Carolina AY612372D. carolinensis KHK103 Yancey, North Carolina AY612371D. carolinensis KHK85 Yancey, North Carolina AY612370D. conanti KHK8.399 Abbeville, South Carolina AY612381D. conanti ASU23228 Anderson, South Carolina AY612382D. conanti KHK163 Ballard, Kentucky AY612387D. conanti KHK662 Hardeman, Tennessee AY612386D. conanti KHK227 Henderson, Tennessee AY612388D. conanti ASU23805 Jasper, Mississippi AY612415D. conanti ASU23806 Jasper, Mississippi Kozak et al. 2005D. conanti RMB251 Lewis, Tennessee AY612389D. conanti RMB221 Limestone, Alabama AY612384D. conanti FLN1532 Pulaski, Illinois KX764602D. conanti TJR2470 Richmond, Georgia KX764601D. conanti RMB225 Tallapoosa, Alabama AY612383D. conanti RMB275 Tishomingo, Mississippi AY612385D. conanti RMB239 Washington, Louisiana AY612390D. folkertsi KHK340 Union, Georgia AY612351D. fuscus KHK503 Alleghany, North Carolina AY612408D. fuscus RMB630 Barbour, West Virginia AY612400D. fuscus KHK531 Bath, West Virginia AY612403D. fuscus RMB595 Belmont, Ohio AY612399D. fuscus RMB831 Bradford, Pennsylvania AY612396D. fuscus RMB720 Campbell, Virginia AY612402D. fuscus FC13580 Duplin, North Carolina AY612414D. fuscus KHK141 Fairfield, South Carolina AY612392D. fuscus KHK142 Fairfield, South Carolina AY612393D. fuscus MVZS12956 Franklin, Massachusetts AY612395

Appendix 1. Specimen and locality information for the ND2 data set with GenBank number or source for each sequence.

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Species Voucher#/Museum# County/Parish, State GenBank Accession#D. fuscus KHK314 Grayson, Virginia AY612409D. fuscus KHK570 Greene, Virginia KR826999D. fuscus RMB696 Guliford, North Carolina AY612394D. fuscus FLN8224 Johnson, Illinois KX764603D. fuscus KHK468 Lee, North Carolina AY612407D. fuscus KHK423 Menifee, Kentucky AY612398D. fuscus KHK429 Morgan, Tennessee AY612411D. fuscus RMB339 Oldham, Kentucky AY612397D. fuscus RMB716 Pittsylvania, Virginia AY612401D. fuscus RMB513 Putnam, Tennessee AY612410D. fuscus KHK557 Rockingham, Virginia AY612404D. fuscus KHK434 Rutherford, North Carolina AY612406D. fuscus KHK435 Rutherford, North Carolina Kozak et al. 2005D. fuscus RMB2331 Surry, North Carolina AY612412D. fuscus RMB2332 Surry, North Carolina AY612413D. fuscus RMB743 Watauga, North Carolina AY612405D. imitator WRK Jackson, North Carolina KX764604D. imitator KHK05 Sevier, Tennessee AY612343D. marmoratus KHK366 Caldwell, North Carolina AY612345D. marmoratus KHK18 Graham, North Carolina AY612344D. marmoratus CC44 Rabun, Georgia KR827000D. marmoratus KHK90 Yancey, North Carolina AY612346D. monticola KHK65 Buncombe, North Carolina AY612376D. monticola KHK782 Butler, Alabama AY612379D. monticola JFBM17794 Graham, North Carolina KX764605D. monticola KHK16 Graham, North Carolina AY612375D. monticola KHK60 Transylvania, North Carolina AY612377D. monticola KHK134 Unicoi, Tennessee AY612374D. monticola S13225 Westmoreland, Pennsylvania AY612378D. ochrophaeus RMB1224 Overton, Tennessee AY612367D. ochrophaeus WKS05 Tompkins, New York AY612366D. ocoee KHK31 Clay, North Carolina AY612353D. ocoee KHK44 Clay, North Carolina AY612354D. ocoee KHK154 Cocke, Tennessee AY612356D. ocoee KHK22 Graham, North Carolina AY612352D. ocoee RMB268 Jackson, Alabama AY612362D. ocoee KHK56 Jackson, North Carolina AY612361D. ocoee KHK266 Marion, Tennessee AY612360D. ocoee KHK53 Rabun, Georgia AY612358D. ocoee RMB2335 Rabun, Georgia AY612359D. ocoee KHK01 Sevier, Tennessee AY612355

Appendix 1 (continued). Specimen and locality information for the ND2 data set with GenBank number or source for each sequence.


447

Species Voucher#/Museum# County/Parish, State GenBank Accession#D. ocoee KHK62 Transylvania, North Carolina AY612357D. orestes KHK129 Avery, North Carolina AY612363D. orestes KHK305 Grayson, Virginia Kozak et al. 2005D. orestes KHK306 Grayson, Virginia AY612365D. orestes KHK140 Unicoi, Tennessee AY612364D. organi KHK73 Buncombe, North Carolina AY612341D. organi KHK310 Grayson, Virginia KR827001D. planiceps ST11008 Patrick, Virginia KR732337D. planiceps ST10865 Pittsylvania, Virginia KX764606D. quadramaculatus KHK593 Buncombe, North Carolina KR827003D. quadramaculatus KHK369 Caldwell, North Carolina AY612347D. quadramaculatus KHK52 Rabun, Georgia AY612350D. quadramaculatus RMB2329 Surry, North Carolina AY612349D. quadramaculatus KHK135 Unicoi, Tennessee AY612348D. quadramaculatus KHK499 Wilkes, North Carolina KR827002D. quadramaculatus CC14 KX764607D. santeetlah JFBM18737 Jackson, North Carolina KR732338D. santeetlah MVZS11775 Jackson, North Carolina AY612391D. welteri FC14355 Letcher, Kentucky AY612416D. welteri KHK414 Powell, Kentucky AY612417D. wrighti JFBM17195 Clay, North Carolina KX764608D. wrighti JFBM16100 Swain, North Carolina KR732339Phaeognathus hubrichti FC13612 Butler, Alabama AY728233

Appendix 1 (continued). Specimen and locality information for the ND2 data set with GenBank number or source for each sequence.

Species Voucher#/Museum# County/Parish, State GenBank Accession# D. apalachicolae DAB861 Leon, Florida EU311708 D. apalachicolae BTL238 Liberty, Florida KX764609 D. apalachicolae DAB218 Liberty, Florida EU311666 D. auriculatus DAB349 Baker, Florida EU311681 D. auriculatus DAB1385 Clinch, Georgia EU311650 D. auriculatus DAB348 Liberty, Georgia EU311680 D. auriculatus BTL239 Wakulla, Florida KX764610 D. brimleyorum FC11578 LeFlore, Oklahoma KX764611 D. brimleyorum RMB2327 Nevada, Arkansas KX764612 D. brimleyorum RMB2201 Polk, Arkansas KX764613 D. carolinensis DAB1105 Buncombe, North Caolina EU311642 D. carolinensis DAB946 Yancey, North Carolina EU311713 D. conanti DAB324 Amite, Mississippi EU311674 D. conanti DAB346 Baldwin, Alabama EU311678 D. conanti DAB252 Barnwell, South Carolina EU311668

Appendix 2. Specimen and locality information for the COX1 data set with GenBank number or source for each sequence.


448

Species Voucher#/Museum# County/Parish, State GenBank Accession# D. conanti DAB345 Butler, Alabama EU311677 D. conanti DAB1387 Effingham, Georgia EU311651 D. conanti DAB647 Grant, Louisiana EU311699 D. conanti DAB646 Henderson, North Carolina EU311698 D. conanti ASU23806 Jasper, Mississippi KX764614 D. conanti DAB322 Jasper, Mississippi EU311672 D. conanti DAB438 Jasper, Mississippi EU311685 D. conanti DAB922 Lawrence, Alabama EU311712 D. conanti DAB222 Livingston, Kentucky EU311667 D. conanti TJH2756 Newton, Texas Hibbitts et al. 2015 D. conanti TJH2757 Newton, Texas Hibbitts et al. 2015 D. conanti TJH3262 Newton, Texas Hibbitts et al. 2015 D. conanti FLN1532 Pulaski, Illinois KX764615 D. conanti TJR2470 Richmond, Georgia KX764616 D. conanti TJH3266 Sabine, Texas Hibbitts et al. 2015 D. conanti TJH3269 Sabine, Texas Hibbitts et al. 2015 D. conanti TJH3270 Sabine, Texas Hibbitts et al. 2015 D. conanti DAB347 Santa Rosa, Florida EU311679 D. conanti TCWC94726 Tyler, Texas Hibbitts et al. 2015 D. conanti TJH2696 Tyler, Texas Hibbitts et al. 2015 D. conanti DAB435 Washington, Florida EU311684 D. conanti DAB323 Washington, Louisiana EU311673 D. conanti DAB867 Wayne, Georgia EU311709 D. conanti DAB868 Wayne, Georgia EU311710 D. conanti DAB321 West Feliciana, Louisiana EU311671 D. fuscus DAB881 Bamberg, South Carolina EU311711 D. fuscus DAB1039 Bath, Kentucky EU311640 D. fuscus DAB201 Beaufort, North Carolina EU311664 D. fuscus DAB637 Berkeley, South Carolina EU311695 D. fuscus DAB1485 Bladen, North Carolina EU311655 D. fuscus DAB508 Bladen, North Carolina EU311687 D. fuscus DAB1042 Bland, Virginia EU311641 D. fuscus DAB755 Burke, North Carolina EU311705 D. fuscus DAB715 Caldwell, North Carolina EU311702 D. fuscus DAB265 Calhoun, South Carolina EU311669 D. fuscus DAB1487 Carteret, North Carolina EU311656 D. fuscus DAB501 Colleton, South Carolina EU311686 D. fuscus DAB209 Craven, North Carolina EU311665 D. fuscus DAB414 Craven, North Carolina EU311682 D. fuscus DAB1505 Davidson, North Carolina EU311659 D. fuscus DAB1484 Davie, North Carolina EU311654

Appendix 2 (continued). Specimen and locality information for the COX1 data set with GenBank number or source for each sequence.


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Species Voucher#/Museum# County/Parish, State GenBank Accession# D. fuscus DAB1478 Duplin, North Carolina EU311653 D. fuscus FC13580 Duplin, North Carolina KX764617 D. fuscus DAB434 Edgecombe, North Carolina EU311683 D. fuscus DAB806 Florence, South Carolina EU311707 D. fuscus MVZ224931 Franklin, Massachusetts AY728227 D. fuscus DAB1506 Iredell, North Carolina EU311660 D. fuscus DAB1036 Jefferson, Indiana EU311639 D. fuscus FLN8223 Johnson, Illinois KX764619 D. fuscus FLN8224 Johnson, Illinois KX764621 D. fuscus FLN8225 Johnson, Illinois KX764622 D. fuscus FLN8226 Johnson, Illinois KX764620 D. fuscus FLN8227 Johnson, Illinois KX764618 D. fuscus FLN8228 Johnson, Illinois KX764623 D. fuscus FLN8229 Johnson, Illinois KX764624 D. fuscus FLN8230 Johnson, Illinois KX764625 D. fuscus FLN8231 Johnson, Illinois KX764626 D. fuscus FLN8232 Johnson, Illinois KX764627 D. fuscus DAB1488 Montgomery, North Carolina EU311657 D. fuscus DAB1496 Montgomery, North Carolina EU311658 D. fuscus DAB1545 New Hanover, North Carolina EU311663 D. fuscus DAB290 Pitt, North Carolina EU311670 D. fuscus DAB972 Pitt, North Carolina EU311719 D. fuscus DAB603 Randolph, West Virginia EU311694 D. fuscus DAB596 Rockingham, Virginia EU311692 D. fuscus DAB638 Scotland, North Carolina EU311696 D. fuscus DAB782 Scotland, North Carolina EU311706 D. fuscus DAB526 Watauga, North Carolina EU311689 D. fuscus DAB1517 Wilkes, North Carolina EU311662 D. monticola DAB642 Graham, North Carolina EU311697 D. monticola DAB1346 Lumpkin, Georgia EU311646 D. monticola — Monroe, Alabama AY549708 D. monticola — Monroe, Alabama AY549717 D. monticola DAB571 Monroe, Tennessee EU311690 D. monticola DAB954 Transylvania, North Carolina EU311717 D. monticola DAB1256 Union, Georgia EU311644 D. monticola DAB524 Watauga, North Carolina EU311688 D. ochrophaeus DAB602 Randolph, West Virginia EU311693 D. ocoee DAB1406 Douglas, Georgia EU311652 D. ocoee DAB1352 Lumpkin, Georgia EU311647 D. ocoee DAB1122 Macon, North Carolina EU311643 D. ocoee DAB951 Union, Georgia EU311715



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Species Voucher#/Museum# County/Parish, State GenBank Accession# D. orestes DAB719 Burke, North Carolina EU311703 D. orestes DAB739 Caldwell, North Carolina EU311704 D. quadramaculatus DAB1356 Madison, North Carolina EU311649 D. santeetlah DAB327 Graham, North Carolina EU311676 D. welteri DAB326 Harlan, Kentucky EU311675



Molecular Phylogeographic Methods Reveal the Identity and ... · native species. Dusky salamanders...

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