Introduction
The presence of substantial population structure within many if not most
taxa is well-documented empirically (Slatkin 1987; Palumbi et al. 1997),
and delimiting species in such instances has become a focal topic in
systematics (Hey and Pinho 2012; Sukumaran and Knowles 2017). A variety
of processes can generate geographic genetic structure such as local
environmental adaptation and isolation by distance (Duminil et al. 2007;
Leffler et al. 2012), accumulating deep divergence between populations
over time and resulting in multiple phylogeographic lineages within
cohesive species (Rissler et al. 2006; Soltis et al. 2006). If these
divergences persist and are not resorbed (Rosenblum et al. 2012), the
eventual outcome is generally speciation (Dynesius and Jansson 2014),
wherein populations diverge sufficiently to acquire independent
evolutionary trajectories characterized by reproductive isolation and
low rates of hybridization (Singhal and Moritz 2013). Completed
speciation is often characterized by adaptive ecological, genetic, and
phenotypic differentiation that reinforce species boundaries via
reproductive isolation and selection against hybrids over time (Coyne
and Orr 2004).
In contrast, substantial population structure can originate within
species that is nonetheless held in check by high rates of gene flow
(Bohonak 1999) and is consequently not indicative of incipient,
incomplete, or ongoing speciation (see Huang and Knowles 2016), but is
instead a long-term, stable, or persistent endpoint on its own (Yang et
al. 2019). Furthermore, the degree and rate of population divergence may
or may not be correlated to rate of completed speciation, depending on
geography and connectivity (Kisel et al. 2010; Harvey et al. 2017;
Singhal et al. 2018b, 2022; Burbrink et al. 2023). Secondary contact can
even result in lineage fusion (Maier et al. 2019) and speciation
reversal (Kearns et al. 2022), with subsequent gain of structure (Frei
et al. 2022). Consequently, population structure may represent the
outcome of various processes yielding geographic genetic diversity
within species that nonetheless do not reflect speciation trajectories
(Cutter and Gray 2016; Sukumaran and Knowles 2017).
Genetic structure in species may therefore consist of divergence that is
neutral (e.g., isolation by barrier, distance, or hierarchy; Baptestini
et al. 2013) or adaptive (e.g., ecological selection resulting in
isolation by adaptation or environment; Nosil et al. 2008; Wang and
Bradburd 2014). Such isolation (Zbinden et al. 2022; Moreno-Contreras et
al. 2023) can consequently produce regional divergence that is bound
together by migration over time and space (Garnier et al. 2004; Sexton
et al. 2014). This may represent incomplete speciation (Nosil et al.
2009) or ephemeral differentiation that is ultimately resorbed
(Rosenblum et al. 2012). Protracted scenarios of speciation-like
outcomes can also occur wherein evolutionarily distinct lineages
nevertheless exhibit gene flow in primary or secondary contact (Smadja
and Butlin 2011; Pardo-Diaz et al. 2012; Burbrink et al. 2021). Complex
landscapes may therefore produce substantial local differentiation
(Brauer et al. 2018; Nali et al. 2020), interacting with climatic cycles
to produce recurring episodes of divergence (Fitzpatrick et al. 2009)
and secondary contact with ongoing gene flow (Schield et al. 2019;
O’Connell et al. 2021).
Consequently, deep intraspecific divergence of genetic lineages can be
generated by a variety of geographic and ecological processes that are
either i) part of a generalized trajectory of divergence towards
speciation, or ii) population structure that is nonetheless unified by
gene flow (Avise 2000; Coyne and Orr 2004). Therefore, demographic model
selection and tests of isolation-by-distance (Jackson et al. 2017) can
be instrumental for differentiating structure versus speciation
(Sukumaran and Knowles 2017). When the former is produced by
landscape-scale processes that are held in check by ongoing migration
(Seeholzer and Brumfield 2018), this is detectable by a variety of
methods (Carstens et al. 2022). In contrast, speciation is demonstrated
by reduced migration (below thresholds of isolation by distance or
environment) between ecologically, geographically, and phenotypically
divergent lineages (Burbrink et al. 2021).
Finally, integrated analyses of phenotype that test for the correlated
ecomorphological signature of phylogeographic lineage divergence are
also crucial for testing speciation (Zamudio et al. 2016), even in
putatively cryptic taxa (Singhal et al. 2018a). Consequently,
multi-modal analyses are needed to discern an accurate picture in many
cases (Bertl et al. 2018); the data and methods needed to test structure
versus speciation will often be the same as those needed to identify the
mode, mechanism, and process of ecological, geographic, genetic, and
phenotypic divergence (Pyron et al. 2023). The fundamental test is
therefore whether the processes driving the accumulation of geographic
genetic structure (e.g., IBD/IBE) are implicated in diversifying
ecomorphological selection promoting divergence between the incipient
phylogeographic lineages, or whether other mechanisms simultaneously
foster high levels of migration between these populations sufficient to
promote lineage cohesion and maintain species identity (Kisel et al.
2010; Seeholzer and Brumfield 2018; Freedman et al. 2023; Prates et al.
2023).
Here, we evaluate “structure” versus “speciation” in the Seepage
Salamander (Desmognathus aeneus ) using tests of
isolation-by-distance, automated demographic-model selection, species
distribution modeling, genotype-environment and -phenotype association,
and morphometric divergence. Seepage Salamanders are miniaturized
(~3–5cm total length), terrestrial, direct-developing
lungless salamanders (Plethodontidae) from the southeastern United
States, primarily from the Blue Ridge of Georgia and North Carolina
(Harrison 1992). Given their size, their home range and dispersal
abilities are miniscule, and they are therefore highly susceptible to
landscape-level genetic fragmentation over ecological timescales. They
also exhibit strong ecological specificity for seepage habitats
associated with the headwaters of high-gradient creeks and streams.
Interestingly, they are also found in analogues of this microhabitat at
low elevations in dispersed, isolated pockets across the Piedmont,
Southeastern Plains, Ridge and Valley, and Southwestern Appalachians in
central Alabama and western Georgia, some of which were not discovered
until the 2010’s (Graham et al. 2012).
Recent range- and genome-wide results indicate up to 6.3% mitochondrial
divergence between four deeply divergent but spatially adjacent
phylogenomic lineages with extensive admixture (Beamer and Lamb 2020;
Pyron et al. 2020, 2022). Given their broad but fragmented geographic
range, we suggest that Seepage Salamanders occupied a continuous
distribution across the southeastern U.S. during cooler glacial periods.
Similarly, we hypothesize that their microhabitat specificity results in
stabilizing ecological selection and species cohesion despite landscape
genetic variation. Finally, we expect morphometric similarity to reflect
this ecological uniformity. We find that the evolutionary history of the
group is characterized by deep lineage divergence that is
counterbalanced by episodes of increased migration and a lack of
phenotypic differentiation; “structure” rather than “speciation.” It
is difficult to pinpoint the exact nature of geographic genetic
diversity (e.g., IBD/IBE) given the complex ecological nature of their
microhabitat adaptations, apparently involving hydrological, edaphic,
floristic, and climatic interactions. Nevertheless, the frequency of
estimated migration and observed admixture in individuals suggests that
reinforcement is not operating to select against hybridization between
the genetically differentiated populations. Formal tests of the
dichotomy between structure and speciation should be employed when this
distinction is consequential in phylogeography.