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 (Chan et al., 2018) for detailed crossing protocol and
experimental design). 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. Recruits
settled onto ceramic plugs were randomly distributed across two
treatment conditions (n = 12 replicate tanks per treatment, n = 20
ceramic plugs per offspring group per tank): ambient conditions (27ºC
and 415 ppm p CO2) and elevated conditions
(ambient +1 °C and 685 ppm p CO2). 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 were measured. 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.