Discussion
Divergence in reproduction-related traits has been documented in
congeneric species of salamanders and includes shifts in reproductive
phenology (Burkhart 2017), in habitat selection (Caspers et al. 2015,
Kieren et al. 2018, Weitere et al. 2004), or in clutch and egg sizes
(Petranka 1987). Previous research suggests that these traits can
diverge rapidly and can be coupled with genetic differentiation
indicative of local adaptation (Evans et al. 2020, Weitere et al. 2004).
This is consistent with our experiment as all measured traits remained
well differentiated between A. barbouri and A. texanumwhen bred and raised under a common treatment and supports previous
comparative studies (Sih and Maurer 1996). This suggests genetically
determined differentiation between lineages rather than plasticity in
response to different environmental conditions these species occupy.
The two species exhibited a different strategy in egg deposition,
generally consistent with literature descriptions (Petranka and Straus
1989). Ambystoma barbouri females prioritized attaching their
eggs to the undersides of rocks; however, once the available rocks were
expended, they attached their eggs to vegetation (Figure 4). A.
barbouri produced larger and fewer eggs per clutch, and the attachment
of those eggs to rocks and plants was very firm and difficult to
manually remove from the surface. Ambystoma texanum females used
artificial plants as an oviposition site, and once all available
vegetation was expended, eggs were scattered loosely throughout the
aquarium (Figure 4). Ambystoma texanum produced smaller eggs but
larger clutches which were laid in small clumps, lacked firm attachment,
and would rapidly fall off the vegetation if disturbed. Upon
oviposition, the A. barbouri eggs had a longer embryonic stage
and larvae were bigger upon hatching but took a shorter time to
metamorphosis and the metamorphs were smaller in comparison withA. texanum metamorphs.
These aforementioned traits are likely under selective pressure in the
respective breeding habitats of the two species. Small streams, the
preferred breeding habitat of A. barbouri , are generally more
prone to strong flooding events in the early spring when eggs are
deposited, and desiccation in the late spring and early summer when
larvae are developing (Eng et al. 2016, Holomuzki 1991, Johnson et al.
2009, Petranka and Sih 1986, Petranka and Sih 1987). Therefore, we would
expect A. barbouri to have adaptations to resist flowing streams
and flooding events, such as the laying of eggs under rocks with strong
adhesion and a longer egg development time to avoid larvae washing
downstream in fish inhabited sections of streams (Petranka and Sih
1986). This strategy has been seen in other stream breeding salamanders
and may adhere to the safe harbor hypothesis (Nussbaum 1987, Shine
1978). Larger initial larval size and shorter larval stage may then
serve to cope with early stream desiccation -in contrast to more slowly
desiccating vernal pools-, via faster metamorphosis as seen in other
amphibians with rapidly desiccating ephemeral habitats (Nussbaum 1987).
Additionally, access to available prey is critical for fast larval
development and metamorphosis, and a larger body size has been shown to
increase capture rates of large prey (Takagi and Miyashita 2019).
Although additional exploration of specific A. barbouri stream
invertebrate assemblages is necessary, streams have been generally
documented as having high invertebrate populations and low zooplankton
concentrations (Holomuzki 1991, Wan Maznah et al. 2018). Hatching at a
larger size may therefore give newly hatched A. barbouri access
to the large invertebrate prey and facilitate quick metamorphosis
(Mcwilliams 1989, Mcwilliams and Bachmann 1989, Smith and Petranka 1987,
Takagi and Miyashita 2019, Nussbaum 1987).
The larvae of A. barbouri also maintained darker pigmentation
than those of A. texanum (Figure 3.). Larval pigmentation is also
likely under selective pressure, with A. barbouri likely coming
under higher ultraviolet (UV) stress than A. texanum due to
shallowness of first-order streams inhabited by A. barbouri and
higher levels of UV penetration (Storfer et al. 1999). Previous studies
have noted the adaptive advantage of the darker coloration of A.
barbouri larvae living in shallow streams with high levels of UV
radiation as opposed to deeper, darker vernal pools inhabited byA. texanum larvae (Garcia et al. 2003, Garcia et al. 2004, Garcia
and Sih 2003, Storfer et al. 1999).
The selective pressures faced by stream breeding A. barbouri are
in contrast with the vernal pools occupied by A. texanum , which
do not tend to experience flooding events resulting in strong currents,
often maintain water well into summer, and typically have plentiful
zooplankton populations, as reflected in the diet of young A.
texanum (Gamble and Mitsch 2009, McWilliams and Bachmann 1989).
Selection against A. texanum traits in stream habitats might
therefore be quite strong. If a female A. texanumoviposits in a stream on either vegetation or on loose substrate, the
eggs may then be easily carried away by flooding events, and the newly
hatched larvae may not have small enough prey available to them. Our
results suggest that populations of A. barbouri and A.
texanum are under a number of strong selection pressures to oviposit in
the correct breeding habitats.
A potential caveat to our findings is transgenerational plasticity in
breeding adult traits (i.e., oviposition location, clutch size,
offspring size), where traits of offspring are determined by
environmental conditions experienced by their parents (Richter-Boix et
al. 2014). In our system, this would mean that environmental differences
during early development of adults (i.e., development in stream or
vernal pool) would affect their breeding behavior and traits of their
offspring (Bell and Hellmann 2019, Herman and Sultan 2011, Richter-Boix
et al. 2014). Although transgenerational plasticity can have effects on
traits such as the size and morphology of metamorphs, it typically
arises due to differences in environmental conditions (e.g., high versus
low food availability), which were identical for A. barbouri andA. texanum in this study. Additionally, transgenerational
plasticity does not influence complex reproductive and developmental
traits, as would be necessary in this system (Orizaola et al. 2016,
Richter-Boix et al. 2014). Therefore, we believe that the observed
differences in reproductive and developmental traits is the result of
differing selection pressures that these species face in their
respective environments.
Despite the trait differentiation between A. barbouri andA. texanum, the two species readily hybridized. The hybridization
between these two species has been suspected (Denton et al. 2014), but
had never been documented, bar one induced hybridization (Petranka
1987). In our experiment, hybridization readily occurred at similar
rates between conspecifics and heterospecifics. This is despite reported
differences in courtship between A. barbouri and A.
texanum , such as more cryptic courtship and generally less nudging
behavior by females towards males in A. barbouri in comparison
with A. texanum (Arnold 1972, Garton 1972, McWilliams 1992, Licht
and Bogart 1990, Petranka 1984, Wyman 1971).
We did not observe any immediate negative fitness consequences of
hybridization. The hybrid crosses did not exhibit reduced viability of
eggs or larvae, or reduced survival to metamorphosis. However, we
recognize that hybrid vigor is common (Birchler et al. 2010,
Cogălniceanu et al. 2020, Fitzpatrick and Shaffer 2007, Hotz et al.
1999, Nzau Matondo et al. 2007), and that fitness consequences, if any,
are more likely to manifest in natural habitats. Indeed, in species and
populations that exhibit divergent traits linked to their local
environments, hybrids are typically expected to have inferior fitness in
either parental habitat (Moser et al. 2016). Interestingly, offspring ofA. texanum females had very poor survival in mesocosms, despite
mesocosms closely mimicking non-flowing, high leaf litter, high
zooplankton environments (Kneitel and Lessin 2010). This is in
comparison to A. barbouri female offspring, which had very high
survival in mesocosms. We are uncertain of what may have caused this
result, but it may have been due to a difference between experimental
conditions and typical A. texanum habitat. These potential
differences include increased water hardness, higher larvae density
resulting in cannibalism or bullying, and higher UV radiation, as
mesocosms were placed in fields with no canopy cover. This result
warrants further investigation as its cause may be inhibiting A.
texanum female migration into the geographic range of A.
barbouri .
One mechanism that may lessen the negative impact of hybridization
between phenotypically divergent species is the presence of maternal
effect, where phenotypes of the offspring are more greatly influenced by
the mother than would be expected from its genotype (Pfennig and Martin
2009). Maternal effects have been noted in amphibians previously (Crespi
and Lessig 2004, Kaplan 1987, Pfennig and Martin 2009), especially when
it comes to initial egg/larval size and further larval development
influenced by the egg size (Bernardo 1996, Dziminski and Roberts 2006,
Kaplan 1985, Kaplan 1992, Komoroski et al. 1998). Our hybrids exhibited
a maternal effect in some traits associated with oviposition and
fitness, such as clutch size, initial larval size, pigmentation, and
survival to metamorphosis, especially in mesocosms (Figure 5).
Conversely, a paternal effect was only observed in size at
metamorphosis. We believe maternal effects are the result of selection
pressure as intermediate traits would exhibit inferior fitness in either
habitat as argued above. As the preferred oviposition substrate is
dependent on breeding location, females are likely to be under selective
pressure to have offspring which exhibit life history traits suited to
their oviposition location, rather than paternal genotype. This maternal
effect may further serve as an explanation to why hybridization between
these species appears to be present, but intermediary phenotypes are not
reported in the wild. Even if hybridization in the wild is readily
occurring, egg masses and larvae will still appear to be the “correct”
species in each breeding habitat. Selection strength on these traits,
however, might differ among populations and depend on local
environmental conditions. Indeed, our populations of both species came
from their respective “typical” habitats, where we would expect
selection pressures to be maximized. Other habitats where both species
have been observed, such as roadside ditches, may impose weaker or
different selection pressures on hybrid populations.
In summary, our results suggest that
genetic adaptation, rather than phenotypic plasticity, plays a larger
role in determining traits defining A. barbouri and A.
texanum . Despite the propensity for A. barbouri and A.
texanum to hybridize and the lack of fitness consequences in
experimental conditions, the divergent selective pressures and resulting
divergence in life history traits may be due to the two species being on
distinct evolutionary trajectories (Abbott et al. 2013). Maternal
effects on hybrid offspring may be impeding the divergence of these
salamanders but may also be reducing the negative impact of
hybridization. Further investigation into the possible presence of
intermediary traits in non-typical habitats, coupled with genomic
assessments should be conducted to fully understand the extent and
consequences of hybridization between these two species of
salamanders.