Materials and Methods
Sampling. We collected adult salamanders of A. barbouriand A. texanum from Ohio, Indiana, and Kentucky. Study sites were
selected from outside the proposed zone of sympatry, from typicalA. barbouri and A. texanum breeding habitats, i.e., stream
and vernal pool, respectively, and previously confirmed as harboringA. barbouri or A. texanum mtDNA, respectively (Figure 1).
We collected a total of 18 males and 18 females of A. barbourifrom 3 localities and a total of 18 males and 18 females of A.
texanum were collected from 4 localities (Figure 1). For both species,
only unspent adults in breeding condition were selected (i.e., females
with swollen abdomens and males with swollen cloacas). We also
considered direction of movement. If an adult was seen moving away from
a stream or vernal pool, they were typically not collected. Assessing
movement direction was especially important for A. barbouri , as
they are not explosive breeders, i.e., their breeding season for a
particular population, may last months, whereas A. texanum may
only last a few days, and the risk of collecting a spent adult was
greater than that of A. texanum (Petranka 1984). We began
collection for both species on the same night, but techniques differed
between A. barbouri and A. texanum sites. For A.
texanum , 30.5 m x 0.6 m sections of tarp drift fences were installed
along vernal pools, with pitfall traps placed every 6 m. Conversely, we
collected A. barbouri crossing roads or trails running parallel
to streams. We transported animals to an environmental chamber and
placed with either a conspecific or a heterospecific individual of the
opposite sex within 48 hours.
Breeding and Egg development. We used a nested experimental
design, considering genotype crossings as a fixed factor, and resulting
eggs/larval clutches from each pair as a nested random factor within
each crossing. Four treatments of genotype crossings were made for adult
pairs (Figure 2). Each pair was randomly assigned to a 90 L aquarium,
resulting in nine pairs per treatment, for a total of 36 aquaria. Prior
to salamander introductions, we filled aquaria with 54.5 L of water,
with 3.78 L coming from each field site and the rest being dechlorinated
water. Field site water inoculations occurred to spur natural algal
growth in aquaria, as algae have been shown to have a mutualistic
relationship with some Ambystoma species (Graham et al. 2014).
Additionally, field site water might be important for scent-cued site
fidelity (Gamble et al. 2007). Light cycle was set to a 12hr/12hr
day/night cycle. We placed two large, flat limestone rocks in each tank
to provide typical oviposition location of A. barbouri . Along
with the rocks, we anchored plastic aquatic plants to the bottom of each
tank to mimic typical A. texanum oviposition location of grasses
or plants. Once pairings were placed in aquaria, we checked each pair
multiple times per day for deposited spermatophores, active
spermatophore uptake by females, and oviposition. Oviposition was
considered an indication of successful breeding, and the effect of the
four pairing treatments on breeding proportion was determined using
Fisher’s exact test. We recorded oviposition location, which included
plants, rocks, or scattered throughout aquaria. We also used Fisher’s
exact test to determine oviposition location differences between the
pairings. After six days, we removed adult pairings from the
aquaria.
We reared the eggs in the breeding experiment aquaria, with no water
change, filtration, or cleaning of aquaria occurring prior to or during
egg stage to avoid disruption (personal communication, Ruth Marcec
Greaves). Oxygen was provided using air pumps and air stones placed in
the corner of each aquarium. The temperature throughout the egg stage
increased from 12°C to 15°C, approximating average outdoor temperatures
at collection localities. We calculated egg development times (i.e., the
difference in days between oviposition date and hatch date) using the
median hatch date of a clutch, as single clutches typically took up to
10 days to complete hatching. Given the difficulty of photographing all
eggs without disruption of development, the maximum value of either egg
number or larval number was used to measure clutch size.
We collected larvae from breeding tanks for 10 consecutive days
following the first hatching and placed them into 13.6 L holding tubs
filled with dechlorinated water and separated by clutch. At the end of
the 10-day collection period, larvae in holding tubs were photographed
and counted using the OpenCV python package and ImageJ software (Bradski
2000, Schneider et al. 2012). For larval length measurements, at the end
of the 10-day collection period, clutches were photographed with a ruler
as a reference. Three clutches of each genotype crossing were randomly
selected, and within each clutch, 15 random larvae were selected by
using the random point selector tool, and their length measured using
ImageJ. Statistical effects of pairing groups on egg development time,
larval count, and initial larval sizes, were determined using one-way
ANOVAs.
Pigmentation. We measured natural pigmentation in larvae of
each genotype early in development. Due to different hatching times of
each genotype, hatching times of clutches within genotypes were averaged
and pigmentation was measured 27 days post-average hatching date. We
randomly selected three clutches from each genotype and took a random
sample of six larvae per clutch taken, for a total sample size of 18
larvae per genotype (Figure 2). We placed each clutch sample into a
glass petri dish, with enough water to cover each larva. The dishes were
set onto stainless steel and covered with aluminum foil to prevent
pigmentation change due to background color and laboratory lights
(Garcia et al. 2003, Garcia and Sih 2003). We held larvae in these
conditions for 24 hours prior to imaging (Garcia et al. 2003, Garcia and
Sih 2003). Photographs were taken with a Zeiss Discovery V12 microscope
installed with an AxioCam MRc5, under LED light, with 10 ms of exposure
and 9.8x zoom. Images were exported as uncompressed BigTiff files and
cropped into 250x420 pixel rectangles using Adobe Illustrator 2022. RGB
values for each pixel were extracted and then averaged to produce a
single grayscale value for each larva. This grayscale value was then
divided by 3 to create a grayscale range between zero and one, zero
being black and one being white. The effect of genotype on pigmentation
(grayscale values) was determined using one-way ANOVA, with clutch
included as a nested variable within each genotype.
Laboratory and Mesocosm Fitness Experiments. The same
experimental design used in the breeding tanks was applied to our
laboratory and mesocosm settings, using genotype crossing as fixed
factor and clutch as a random factor nested within genotype (Figure 2).
We transferred larvae from laboratory holding tubs to mesocosm or
laboratory enclosures for fitness proxy experiments. Three clutches were
randomly selected from each genotype, and six random samples of 15
larvae were taken from each clutch. From each clutch, three replicates
were assigned to 90 L aquaria and the other three to mesocosms. In
summary, we used 36 aquaria and 36 mesocosms, with three 15-larvae
replicates per clutch, and three clutches per genotype crossing (Figure
2). Experiments taking place in both laboratory and mesocosm were
identical in concept and experimental design, but differed in
environmental conditions, larval density, and prey availability and
type. Larvae of each genotype were then reared to metamorphosis and
number surviving to metamorphosis, time to metamorphosis, and size at
metamorphosis were measured as proxies for fitness (Earl and Whiteman
2015, Semlitsch et al. 1988).
The laboratory setup was similar to the breeding experiment. Thirty-six
90 L aquaria were filled with 54.5 L of dechlorinated water and placed
in an environmental chamber with 12 hr/12 hr day/night cycle, and a
gradual increase in temperature from 15-22°C to mimic natural
environmental conditions. Laboratory husbandry included daily feedings
of ad libitum brine shrimp. The mesocosm experiment arrangement
consisted of 36 polyethylene stock tanks (1.85 m in diameter; 1,480-L
volume), filled with water and 1 kg of deciduous leaves and inoculated
with zooplankton. To deter predation upon larvae in mesocosms, we
fastened a 0.15-cm fiberglass screen-mesh lid on the top of each
mesocosm. We visually confirmed the presence of zooplankton in all
experimental mesocosms prior to larval inoculation. Twice a week, tanks
were checked until the first metamorph was seen. After the first
metamorphosis, daily checks of mesocosms occurred each morning to
protect metamorphs from desiccation. Upon metamorphosis, which was
defined by a full reabsorption of the gills, larvae were removed from
enclosures by netting, the date was recorded, individual photographs
were taken using a Sony DSCW800 camera, and SVL measured using ImageJ
software. Genotype effect on size at metamorphosis, time to
metamorphosis, and survival to metamorphosis of larvae raised in
laboratory aquaria and mesocosms were determined using one-way ANOVA,
where clutch was a nested, random factor. All statistical analyses were
conducted using R software version 4.0.2 (R Core Team, 2020). Post-hoc
t-tests were conducted for each one-way ANOVA. To correct for type I
error inflation associated with multiple comparisons, all post-hoc
t-test p-values were first run without correction, combined into a
vector, and then corrected together using the Benjamini-Hochberg
procedure (Supp Table 1.).