Introduction
The process of speciation is frequently accompanied by divergence in
life history traits, due to different environmental pressures acting on
the newly-forming species (Abbott et al. 2013, Doellman et al. 2018).
Divergence in these phenotypic traits can be induced by environmental
conditions or can be genetically determined (Fischer et al. 2016, Le
Sage et al. 2021, Sabino-Pinto et al. 2019). Genetically-determined
traits that increase population fitness of a species in its environment
are called adaptive traits, and the process by which trait phenotypes
and underlying genotypes increase in frequency in a population is
referred to as local adaptation (Hereford 2009, Hoban et al. 2016,
Savolainen et al. 2013). Adaptive traits may enforce the process of
speciation as hybrids often exhibit intermediate values of such traits,
which are associated with lower fitness of the hybrids in either
parental habitat (Barton 2001, Delmore and Irwin 2014, Hatfield and
Schluter 1999, McGee et al. 2015, Seehausen 2004). On the other hand,
phenotypic plasticity, allows the expression of different phenotypes
from a single genotype due to differing environmental stimuli (Benard
2004, Fitzpatrick 2012, Michimae 2006, Pfennig et al. 2010, Van Buskirk
2009). Plastic traits generally do not decrease fitness of hybrids,
which can lead to genetic homogenization of the newly-forming species
and hinder lineage divergence (Abbott et al. 2013, Gow et al. 2006,
Nonaka et al. 2015, Seehausen 2006, Taylor et al. 2006). As both plastic
and genetically determined traits have an effect on speciation and play
a role in the ability of populations to thrive in their environments, it
is crucial to understand if traits delimiting closely related species
are plastic or genetic in nature (Campbell‐Staton et al. 2018, Corl et
al. 2018). Such knowledge is central to our ability to 1) delimit
species and conservation management units, 2) inform management
strategies with respect to conservation priorities and reintroductions,
and 3) better understand the magnitude of environmental change that any
population can tolerate.
Ambystoma barbouri and A. texanum are considered two
separate, closely related salamander species (Kraus and Petranka 1989).
The two salamanders are generally differentiated by traits associated
with their breeding behavior. Ambystoma barbouri is typically a
stream breeding salamander, which attaches its eggs in a single layer
underneath flat (typically limestone) rocks of first and second order
streams (Drayer et al. 2020, Holomuzki 1991, Kats and Sih 1992,
Niemiller et al. 2009). Ambystoma texanum is typically a vernal
pool breeder that deposits eggs in small clumps on submerged vegetation,
as is typical for the genus Ambystoma (Petranka and Sih 1987,
Pfingsten et al. 2013). The differences in reproductive traits between
them have been used to delimitate their ranges, as the two species are
morphologically similar (Kraus and Petranka 1989 but see Petranka 1989
on differences in tooth morphology). Based on this delimitation, the two
species are generally considered parapatric. Ambystoma
barbouri ’s range extends from the Nashville basin to central
Ohio (Figure 1). Due to its small range, A. barbouri has been
listed as near-threatened in Tennessee and has recently been placed
under a Species Status Assessment for its full range. In contrast to the
limited range of A. barbouri , A. texanum inhabits an
expansive range from central Texas to northern Ohio and southern
Michigan (Figure 1).
The reproductive and larval traits such as oviposition location, egg
size, clutch size, and larval pigmentation that differentiate A.
barbouri from A. texanum (Petranka 1982, Petranka and Sih 1987,
Venesky and Parris 2009) seem to exhibit a parapatric distribution. In
some locations an abrupt switch in reproductive traits can be observed
within kilometers, with no intermediate forms observed (Kraus and
Petranka 1989). For example, the area running from western Kentucky to
central Ohio represents a presumed zone of sympatry, as breeding
habitats are not clearly differentiated and both species can be found in
non-traditional habitats such as roadside ditches (Pfingsten et al.
2013). Intermediate forms of reproduction-related traits from this
sympatric zone, such as clutch size and larval size, have not been
documented in the literature
(Petranka 1982).
The genetic structure of the two species is also complex. In some parts
of their distribution, the geographic boundary between stream- versus
pool-related reproductive traits seem to correspond well with the
geographic distribution of two divergent mitochondrial DNA clades (Bi
and Bogart 2010, Denton et al. 2014, Eastman et al. 2009, Williams et
al. 2013). However, this pattern is not exclusive, as signals of
mitochondrial introgression and mito-nuclear discordance have been
documented, with some A. barbouri mtDNA genomes present withinA. texanum range and breeding habitat (Denton et al. 2014). These
results also suggest that the two taxa might not be reproductively
isolated but that selection pressures associated with differing breeding
habitats might have resulted in species-specific or population-specific
adaptations. This raises a question to what extent the
reproduction-related traits are adaptive or plastic, whether barriers to
hybridization exist between the two species, and whether potential
hybrids exhibit inferior fitness.
The
objective of this research was two-fold. First, we compare
reproduction-related traits of A. barbouri and A.
texanum females, mated with either conspecific or heterospecific males
in laboratory settings. We hypothesized that the traits associated with
their distinct reproductive strategies/habitats, such as egg deposition
location or clutch size, are genetically-determined rather
than phenotypically plastic and represent adaptations to the species’
different breeding habitats. If true, we expected that the females of
the two species would maintain their distinct reproductive traits when
bred under a common treatment (Richter-Boix et al., 2013). Second, we
compared life-history traits of hybrid versus nonhybrid offspring (Hall,
1999). We hypothesized that hybridization has negative consequences
which would potentially explain the geographically abrupt turnover of
reproductive and developmental traits observed in the A.
barbouri / A. texanum sympatric zone. If hybrid inferiority was
present, we expected to find one of the following: 1) breeding between
heterospecific pairs to be less frequent than breeding between
conspecific pairs, 2) the viability of hybrid eggs or larvae to be
reduced, or 3) hybrid offspring to exhibit intermediate phenotypes in
comparison to non-hybrid offspring making them less suitable for either
parental habitat.