Phylogeographic Divergence
Our recent phylogenomic dataset sampled 59 individuals and populations and estimated four geographic lineages (Pyron et al. 2022), but with fewer samples outside of the Blue Ridge mountains and including only 233 loci. Previous mitochondrial datasets indicated substantial population divergence in the species (Beamer and Lamb 2020; Pyron et al. 2020), with uncorrected ‘p’ distances in the COI barcode locus of 1.4­–6.3%. Here, we recover three lineages rather than four, corresponding to two major mountain ranges (Cohutta and Nantahala) and the expansive Piedmont and associated populations. Accounting for IBD when estimating population structure in conStruct suggests only two major clusters (Piedmont versus montane). Overall, lineages of Seepage Salamander are deeply divergent (~1.2Ma, ~4.7% mitochondrially), but exhibit extensive admixture both at their contact zones and deep into the core geographic range of each set of populations. The between-lineage Fst values border on “meaningful” significance (usually considered ~0.15) for population differentiation (Hartl and Clark 2007), but generally fall outside or on the low end of the “grey zone” of genomic divergence (Fst >0.2,Da >0.01) indicative of speciation across a wide variety of animals (Roux et al. 2016). Recent migration rates inferred from Fst and estimated by GADMA are >1 migrant/generation between all three lineages, suggesting relatively high and constant rates of gene flow through time.
The overall relationship between geographic and genetic distance between individuals, populations, and lineages matches the classic expectation of a “Case IV” scenario resulting from a lack of regional equilibrium (Hutchison and Templeton 1999). This pattern is typically driven by differing scale-dependent effects of gene flow and drift influencing population structure (Twyford et al. 2020), where migration has greater impacts over shorter distances and drift predominates past a threshold determined in part by habitat connectivity and migration range (van Strien et al. 2015). The lack of between-lineage IBD likely reflects the long-term impact of mountains and refugia in structuring local populations, explaining the presence of very close yet very different populations in the Blue Ridge. This also rejects IBA, as the strength of between-population IBD is less than within-population, indicating no acceleration of genetic divergence due to local adaptation in the Blue Ridge and Piedmont (Freedman et al. 2023). Correspondingly, the relatively recent expansion of the Piedmont lineages during the late Pleistocene explains the weakness of IBD within the most widely distributed lineage, as “Case I” dynamics (i.e., “pure” IBD) require relatively long timescales to become apparent. This again underscores the dynamic nature of these processes, and the attendant capacity of related patterns to shift over time.
The RDA results confirm the hypothesis that there is some apparent degree of ecological adaptation related to temperature and precipitation differences between montane and Piedmont populations (Miranda et al. 2023), but they are not abundant in the genome. Furthermore, there is a non-zero but limited degree of correlated adaptive divergence along ecological gradients and related phenotypic axes. Robustness in terms of size, limb length, and head shape (length and width) is likely related to both desiccation tolerance in warmer environments (Baken et al. 2020) and differing relative pressures of terrestriality (Ledbetter and Bonett 2019). That these variables show some degree of correlated adaptive differentiation between montane and Piedmont environments is not surprising, but it does not result in overall phenotypic divergence between lineages or apparently act to limit hybridization and gene flow. We consider our limited number of predictors in both analyses to offer a tradeoff between potential false positives and false negatives (Forester et al. 2018). Our sampling is not detailed enough to perform cluster-specific analyses (Carvalho et al. 2021), but a better-annotated molecular dataset could facilitate this in the future to detect microgeographic variation and adaptation.
The SDMs along with other natural-history observations (Harrison 1992; Graham et al. 2012) reinforce the strong influence of Level IV Ecoregions on the distribution of this species, particularly outside of the Blue Ridge Mountains. Similar results were seen in the approximately co-distributed congener Desmognathus cheaha (Pyron et al. 2023). We interpret this to indicate floristic or other associations (edaphic, hydrological, etc.) with seepage environments not captured by climatic variation and liable to change dramatically over ecological timescales (A. Lee-Yaw et al. 2022). Our fieldwork underscores the extreme microhabitat specificity involved in locating this species, but we have not yet pinpointed the exact determinants of what constitutes suitable habitat. For this reason, it is difficult to test hypotheses about historical distributions with paleoclimatic modeling given the lack of a mechanistic model and historical data layers that incorporate the relevant factors, which are still mostly unknown. Similarly, we did not consider modern estimated effective migration surfaces (Petkova et al. 2016) to be particularly relevant for investigating these historical processes, which shift rapidly through space and time.
Consequently, we hypothesize that structure in this species arises primarily from stabilizing rather than diversifying ecomorphological selection. This results from a high degree of ecological specificity to a highly specific microhabitat consisting of headwater seepages, moist leaf litter, and various moss species. These salamanders are almost never observed outside of this exact ecosystem, typically within the range of a few meters around spring heads or ravine streams. Yet, increases in suitable habitat during periods of cooling and expansion out of refugia appear to drive rapid geographic occupancy across a large expanse of the Piedmont and associated ecoregions, which is quickly fragmented during interglacial periods. This adaptive stasis and limited dispersal both drives IBD in geographically proximate areas but contributes to lineage cohesion across climatic cycles when these locally adapted lineages are brought into contact out of climatically proximate refugia during glacial cycles. Similar processes appear to be operating in other salamander systems in Mexico (Velo-Antón et al. 2013), suggesting analogous montane processes linking tropical and temperate dynamics in landscape genetics and lineage formation. This is essentially an extension of Janzen’s hypothesis (Janzen 1967; Muñoz and Bodensteiner 2019; Wishingrad and Thomson 2023), wherein behavior, ecology, and phenotype interact to drive local adaptation, constrain ecomorphological divergence, and promote lineage cohesion. Interestingly in this case, these processes ultimately appear to foster phylogeographic diversification while constraining speciation trajectories.