Fig 1.
(A) Sampling sites in Great Smoky Mountains National Park (boundary data from the National Park Service [29]). Contour lines represent 250-m elevation intervals, with the thickest line at 1,000 m to indicate the approximate lower range limit of both species. Darker shading indicates higher elevation. (B) Illustrations of study species (used with permission of J. M. Fleming, University of Tennessee, 29 Sep 2025) drawn to relative scale for large adults. (C) Location of study area within southeastern North America. All maps are original, created by the authors using the free and open source QGIS (http://www.qgis.org) and R [30,31] with public domain data from the USGS National Map (https://www.usgs.gov/3d-elevation-program/) and the National Park Service [29].
Table 1.
Performance of the final target capture kit for 13 samples sequenced for the second test.
Fig 2.
Performance of the preliminary target capture kit across the Plethodontidae.
Proportion of reads from first capture run that were kept (final mapped) or discarded because they were optical duplicates, supplementary (mapped to more than one locus), singleton (read missing mate pair), or unmapped. Number on each bar indicates the percentage of reads that were retained after processing (i.e., final mapped). N and S after D. quadramaculatus refer to the northern and southern clades, respectively.
Fig 3.
Correlations between individual genetic distance and geographic distance.
P-values for Desmognathus wrighti (left) and Plethodon jordani (right) as estimated from Mantel randomization tests with 9999 replicates. Red trend lines were fitted with least squares regression.
Fig 4.
Strength of evidence for different numbers of ancestral populations.
Results of cross entropy estimates for each of 100 replicates for different numbers of ancestral populations of Desmognathus wrighti (left) and Plethodon jordani (right) with the sNMF algorithm [45]. Better fit models have lower entropy.
Fig 5.
Population structure of Desmognathus wrighti.
(A) Map of sampling locations shaded according to ancestry estimates from the admixture model with k = 2 fitted using sNMF [45]. Locations with more than one individual are shown as black dots with individual shaded circles arranged symmetrically around them. (B) PCoA of the pairwise genetic distance matrix, also shaded according to the admixture model with K = 2 fitted via sNMF. (C) Barplots of ancestry estimates according to the admixture model in STRUCTURE. Individuals in the barplots are sorted by longitude (west to east) and ellipses connected by lines illustrate corresponding groups of individuals across the graphs. D. wrighti illustration used with permission of J. M. Fleming, University of Tennessee, 29 Sep 2025. Original map created by the authors using the free and open source QGIS (http://www.qgis.org) with public domain data from the USGS National Map (https://www.usgs.gov/3d-elevation-program/) and the National Park Service [29].
Fig 6.
Population structure of Plethodon jordani.
(A) Map of sampling locations shaded according to ancestry estimates from the admixture model with k = 2 fitted via sNMF [45]. Locations with more than one individual are shown as black dots with individual shaded circles arranged symmetrically around them. (B) PCoA of the pairwise genetic distance matrix, also shaded according to the admixture model with K = 2 fitted using sNMF. (C) Barplots of ancestry estimates according to the admixture model in STRUCTURE. Individuals in the barplots are sorted by longitude (west to east) and ellipses connected by lines illustrate corresponding groups of individuals across the graphs. P. jordani illustration used with permission of J. M. Fleming, University of Tennessee, 29 Sep 2025. Original map created by the authors using the free and open source QGIS (http://www.qgis.org) with public domain data from the USGS National Map (https://www.usgs.gov/3d-elevation-program/) and the National Park Service [29].