Speciation and phylogeography
Like most mygalomorphs, the Antrodiaetus unicolor species complex
has certain life history traits (i.e. long generation times, limited
dispersal capabilities, morphological conservatism) that drastically
influence population structure (Coyle, 1971). This in conjunction with
extensive molecular divergence would most likely indicate that the
populations have been isolated from gene flow for an extended period of
time, which would increase speciation potential (Barraclough, 2019).
While that seems to be the case when comparing A. microunicolor ,
A.unicolorA, and A.unicolorB, lineages within A.unicolorB remain
ambiguous.
Specifically, the STRUCTURE analyses for both All_M20 and several UniB
datasets recover conflicting clusters (Figures 3a & 4; also see 3RAD
section above). These clusters generated for all datasets were
incongruent with monophyletic groups, which might be reflective of the
use of unlinked SNPs for STRUCTURE versus using all variation for
phylogenetic inference. While VAE for UniB_M30 provides some evidence
of structure separating these lineages, there is still slight overlap
between them (Figure 4a). Derkarabetian et al. (2019) used STRUCTURE and
VAE for species delimitation, which revealed clear VAE and STRUCTURE
clusters agreeing with multiple lines of evidence (i.e., COI clades,
DAPC, morphology) for species-level divergence within the harvestmanMetanonychus , a group that is also known to have high population
structure, conservative somatic morphology, with allopatry between
species in the focal species group. Our study, however, includes a taxon
with sympatry/parapatry across multiple lineages in addition to being
morphologically homogeneous, with A. microunicolor being the
exception. Given the more recent divergence in the Antrodiaetus
unicolor species complex (~11.5 mya; Hendrixson &
Bond, 2007) compared to Metanonychus (~25 mya;
Derkarabetian et al., 2019), the underlying genetic patterns indicate
speciation may not necessarily be complete and/or as clearly reflected
in the VAE clusters.
Although the mixed cluster assignments of individuals suggest admixture
between populations is occurring, it could also indicate incomplete
lineage sorting (i.e., ancestral polymorphism) and/or recent range
expansion (Avise, 2009). In addition, the species tree generated in
ASTRAL (Supplemental Figure S2) showed considerable uncertainty for most
of the relationships within A. unicolor B indicating an
appreciable amount of gene tree discordance. There are several potential
reasons for gene tree/species tree discordance (incomplete lineage
sorting, reticulation, gene duplication, or horizontal gene transfer;
see Maddison, 1997) but among these, incomplete lineage sorting (ILS)
seems the most likely explanation due to the prevalence of ILS in taxa
with long generation times, large effective population sizes, and/or low
mutation rates (Degnan & Rosenberg, 2009). Additionally, mtDNA data in
previous analyses hinted at the possibility of a recent range expansion
(Hendrixson, unpublished data; see speciation scenarios below).
All spiders in this species complex appear to have similar habitat
requirements (Coyle, 1971; Hendrixson & Bond, 2005a). However,
morphology, behavior, and large-scale ecological (i.e., climatic) data
offered some plausible insight into potential adaptive divergence.
Morphological, geographical, and molecular data clearly demarcateA. microunicolor and A. unicolor A as distinct lineages,
yet the niche similarity tests between these two lineages reveals no
significantly divergent or conserved niches. Two of the background areas
(75km and 100km) approach significant niche divergence when comparingA. microunicolor occurrence points to the background area ofA. unicolor A but not vice versa. It is possible that niche
divergence important for speciation in this complex occurs in other
ecological variables not tested in our study; however, our clustering
analyses show minimal gene flow between these lineages and suggests a
speciation model in which reproductive isolation accumulated in
allopatry without much ecological differentiation (i.e., niche
divergence may not have been a large driver in the speciation process).
With subsequent sympatry between A. microunicolor and A.
unicolor B these lineages potentially maintained reproductive isolation
through premating barriers, specifically character displacement with
disparities in size and breeding periods (e.g., Bond and Sierwald 2002).
The other lineages, largely sympatric, seem morphologically and
behaviorally similar, thus we use ENM ecological data. A.
microunicolor + A. unicolor A and A. unicolor B niche
similarity analyses yielded conflicting results. The comparison ofA. microunicolor + A. unicolor A occurrences to the
background area of A. unicolor B would suggest niche divergence
(i.e., significantly more different than expected) whereas the
comparison of occurrences of A. unicolor B to the background area
of A. microunicolor + A. unicolor A suggests niche
conservatism (i.e., significantly more similar than expected). For niche
divergence, one possible explanation could be that A. unicolor B
is much more widespread than both A. microunicolor and A.
unicolor A and, therefore, has more potential for heterogeneous
environmental conditions in the habitat available to them (e.g., more
tolerance for lower elevation compared to A. microunicolor andA. unicolor A). Additionally, niche conservatism could be the
result of A. unicolor B’s distribution mostly overlapping A.
microunicolor and A. unicolor A’s distributions, suggesting thatA. unicolor B can not only easily inhabit the same environmental
conditions but also likely prefers similar habitats as A.
microunicolor and A. unicolor A.
The niche similarity analyses involving lineages within A.
unicolor B yielded no obvious results for either niche divergence or
niche conservatism. The niche similarity analyses for A.
unicolor B1 and A. unicolor B2 show that their niches are no more
different or similar than expected for any of the background areas
tested, although the 100km background area hinted at niche conservatism
(not significantly though). The Little Tennessee River may be a
potential geographic barrier between these two lineages facilitating
reproductive isolation, but not enough time has occurred for the
accumulation of neutral divergence indicative of allopatric speciation.
Numerous taxa in the southern Appalachians exhibit similar
phylogeographical patterns, identifying either the Asheville or Little
Tennessee River Basins as potential barriers to gene flow:Hypochilus araneomorph spiders (Keith & Hedin, 2012), harvestmenSabacon cavicolens (Hedin & McCormack, 2017) and Fumontana
deprehendor (Thomas & Hedin, 2008), Dasycerus carolinensisbeetles (Caterino & Langton-Myers, 2019), Narceus millipedes
(Walker et al., 2009), and plethodontid salamanders (Kozak & Wiens,
2010; Weisrock & Larson, 2006). Consequently, these lineages could
prospectively be considered distinct species after sufficient time and
divergence in allopatry. Additionally, the niche similarity analyses forA. unicolor B1 + A. unicolor B2 to A. unicolor B3,
while not consistently significant for all background areas, does show
signs of niche divergence. For the 100km and 75km background areasA. unicolor B3 occurrences were significantly different from the
background of A. unicolor B1 and A. unicolor B2 but not
quite significantly different for the reciprocal comparison. Also, the
50km background area hints at niche divergence for both comparisons. One
explanation could be that the ancestor of A. unicolor B lineages
had undergone range expansion where A. unicolor B3 later became
geographically isolated from A. unicolor B1 and A.
unicolor B2 for a significant period of time generating reproductive
isolation, with subsequent secondary contact potentially driving niche
divergence and, therefore, reinforcing reproductive isolation in
sympatry.
One caveat for using large-scale ecological data like these is the
potential for not having the resolution needed for detecting
microhabitat differences, which are often important for divergent
selection and driving speciation (Massatti & Knowles, 2014; Soberón,
2007). Mygalomorph spiders like Antrodiaetus are known for
habitat specialization at small scales, such as preferring north-facing
slopes, shaded ravines, and particular soil types (Hedin et al., 2015;
Starrett et al., 2018). Therefore, it is possible that we could be
overlooking significant microhabitat differences that potentially drive
niche divergence, with speciation following. Another potential issue
could be how we defined the background areas. Considering that these
spiders have low dispersal capabilities, it may be that the regions with
higher amounts of distance incorrectly show niche divergence due to
incorporating potentially uninhabitable environments and, therefore,
misinterpreting possible speciation mechanisms (Warren et al., 2008).
Species limits of the A. unicolor species complex
Species delimitation of the Antrodiaetus unicolor species complex
has long been challenging due to both morphological stasis and variation
across their distribution (Coyle, 1971; Hendrixson & Bond, 2005a). As
already discussed, A. microunicolor is unambiguously recognized
as a species based on morphological, behavioral, and genetic divergence
from A. unicolor . However, the now paraphyletic assemblage ofA. unicolor lineages lacks distinctive features that could be
used to distinguish them. Because no nomenclatural changes have been
made to elevate these lineages to species status, the species-level
diversity remains underestimated in this complex.
This study employed genome-wide SNPs for several clustering analyses and
niche-based distribution modeling to evaluate genetic and ecological
exchangeability, which further elucidated potential species boundaries.
Clustering analyses with all individuals present (All_M20) as well as
morphology and behavior coincide with the three species hypothesis
(A. microunicolor , A. unicolor A, and A. unicolor B).
Alternatively, the niche identity analyses and STRUCTURE/VAE analyses
for A. unicolor B individuals only (UniB_M30) support the five
species hypothesis (additional splitting of A. unicolor B into B1,
B2, and B3). Although it is possible that these lineages were once
allopatric and diverged before recent secondary contact in the Southern
Appalachians, the tentative interpretation of potential incipient
speciation events within A. unicolor B and less conservative niche
identity test results remain too ambiguous for confident species
delineation within the clade. Our integrative approach utilizing
morphological, behavioral, and a substantial amount of molecular
characters with ecological niche modeling provided ample evidence of an
additional cohesion species within the complex for a total of three
(A. microunicolor , A. unicolor A, and A. unicolor B),
which potentially warrants distinguishing these two genetically and
ecologically different lineages as separate species. In that case, one
lineage would be designated as the true name-bearing Antrodiaetus
unicolor and one with a new name. Considering that Hendrixson and Bond
(2005a) designated a neotype for Antrodiaetus unicolor from
Desoto State Park in northeastern Alabama and specimens included in this
study from the surrounding areas cluster together in the lineageA. unicolor B, this lineage should be considered the true
name-bearing Antrodiaetus unicolor. To our knowledge, none of the
existing available names (junior synonyms of A. unicolor sensu
lato) would be attributed to any of the A. unicolor A individuals
based on geography. As such a new name will need to be proposed forA. unicolor A.
Overall, our study demonstrates the efficacy of genomic-scale data for
recognizing cryptic species, suggesting that species delimitation with
one data type, whether one mitochondrial gene or morphology,
underestimates species diversity in taxa with low vagility and relative
morphological stasis. Incorporating multiple lines of evidence (i.e.,
morphological, behavioral, geographical, and ecological diversity) in
addition to genomic-scale data underscores the robustness of integrative
species delimitation approaches across all organismal diversity despite
differences in biological or ecological characteristics. We were able to
resolve species-level paraphyly within the A. unicolor complex by
delimiting an additional putative species despite morphological
homogeneity. In addition, our study highlights another instance of
cryptic speciation in the southern Appalachians, with phylogeographical
patterns contributing to our understanding of the processes generating
biodiversity in this rich, geologically and environmentally complex
region.