Experimental design and sample collection
Parental coral colonies of A. tenuis and A. loripes were
collected from Trunk Reef (18°35′S,
146°80′E), central Great Barrier Reef in November 2015 and crossed in
the laboratory to form two F1 hybrid and two parental purebred offspring
groups (see Figure 2, Chan et al., 2018 for detailed crossing protocol
and experimental design). Briefly, parental colonies were kept and
spawned under ambient conditions. Egg-sperm bundles of individual
parental colonies were collected and separated with a 100 µm filter. A
mixed sperm solution with equal quantity of sperm from each conspecific
colony was used to fertilize eggs from the other species in the cross to
produce the hybrids, and to fertilize conspecific eggs to produce the
purebreds. The abbreviation of the offspring groups throughout this
study are: TT (purebred A. tenuis ), TL (hybrid), LT (hybrid) and
LL (purebred A. loripes ), where the maternal parent is listed
prior to the paternal parent in a genetic cross by convention (Miller et
al., 2012). For example, “TL” is a hybrid formed by crossing A.
tenuis eggs with A. loripes sperm.
Embryos were reared to planula
stage and settled onto ceramic plugs under ambient conditions five days
post-spawning. Settled recruits were randomly and evenly distributed
across two treatment conditions: ambient conditions (27ºC and 415 ppmp CO2) and elevated conditions (ambient +1 °C and
685 ppm p CO2). There were 12 replicate tanks per
treatment and each tank contained 20 ceramic plugs of each of the four
offspring group (i.e., each offspring group had 12 x 20 = 240 ceramic
plugs per treatment). For the elevated conditions, recruits were ramped
at a rate of +2 ºC and +~50ppm a day until they arrived
at the targeted conditions. Given the predicted sea surface temperature
(SST) increase in coral reefs ranges from ~ 1.4 and
~3.6 °C by the year 2100 (under RCP 2.6 and 8.5
respectively and relative to pre-Industrial period) (Bindoff et al.,
2019), an elevated temperature of +1 ºC to present day ambient
temperature reflects a realistic scenario that will likely occur in the
coming decades. Note that present day SST has already increase by
~0.9°C since pre-industrial time (Bindoff et al., 2019).
Coral recruits were reared under treatment conditions in filtered
seawater for seven months at the National Sea Simulator of the
Australian Institute of Marine Science. A microalgal diet supplement was
supplied to the corals daily and their fitness traits and associated
microbial communities were examined. To mimic the natural environment as
closely as possible, the experimental conditions followed diurnal and
annual temperature variations of Davies Reef (18.83° S, 147.63° E),
which is a reef near the collection sites of the parental colonies. At
the end of the seven-month experiment, recruits from three tanks of each
treatment were randomly selected for sampling. Due to the small size
(and therefore low RNA quantity) of individual recruits, multiple
recruits of the same offspring group from the same tank were pooled to
form one sample. Each pooled sample contained 30 coral polyps. RNA
pooling was considered appropriate as the purpose of this study was to
examine population-level rather than individual-level differences
(Davies et al., 2016; Kendziorski et al., 2003). Three pooled samples
per offspring group per treatment were collected, except only one sample
was available for purebred A. tenuis (TT) under elevated
conditions due to high mortality (Table S1). Samples were snap-frozen in
liquid nitrogen and stored at -80 °C until RNA extraction.