Results
Interpopulation
differentiation
After filtering, 9,031 high quality SNPs remained from the larval
samples collected at ambient temperature across 68 individually
genotyped larvae from the five families (n = 7 – 20 for each). The five
families separated in multidimensional space, in which genetic variation
explained 43% of the variation between multilocus genotypes from the
five families (PC1-PC3: 25.8; 13.4; 4.1%) (Figure 1A). Families that
shared the same sire (Tijou 2A) roughly clustered together in Principle
Components Analysis (PCoA) space (WW1 and CW with WW2), whereas the
other two families were more divergent.
Analysis of population structure using Discriminant Analysis of
Principle Components (DAPC) further confirmed this pattern of clustering
between WW1 and CW along the first two discriminant functions. The 95%
confidence ellipses demonstrate that the dispersion of multilocus
genotypes also varied across the families, where WW2 shows the greatest
and WW1 shows the least variance. Cluster membership probabilities
calculated from DAPC analysis also showed flat distributions in
between-group and within-group allele variances, demonstrating that
admixture has occurred, especially between WW3, WC, WW1, and to a lesser
extent with WW2, and particularly CW.
Allele frequency profiles
The majority of loci were fixed at either 0 or 1. The average
distribution of loci in population purebred crosses was significantly
different compared to interpopulation crosses, with a greater abundance
of loci at intermediate frequencies in the interpopulational families
(compared to null distributions; Kolmogorov-Smirnov Two-Sample test,p = 2.1e-26 and 1.3e-21, Figure 2). This suggests that these
interpopulation hybrids have significantly greater genetic diversity
than within population purebreds. When individual purebred families were
analysed, the majority of the on average reduced diversity (e.g. lower
abundance of loci at intermediate frequencies) was attributed to crosses
WW1 and WW2, whereas WW3 demonstrated a similarly greater abundance of
loci at intermediate frequencies as the interpopulational crosses
(Supplementary Figure 1). WW1 and WW2 exhibited significantly different
distributions compared to the distribution of WC (p = 5.5e-15 and
2.4e-4, respectively) and CW (p = 1.2e-11 and 6.7e-4,
respectively) (Figure 2). In contrast, the distribution of loci in the
WW3 cross was not significantly different compared to WC (p =8.7e-2) or CW (p = 0.77). All families differed significantly
from expected, modelled HW frequencies when modelled again populations
of the same size (p = WW and WC: 1.03e-45; CW: 2.2e-46). Observed
heterozygosity (Ho), and alpha Diversity
(0Dα and2Dα at q = 0 and 2) was highest in the
interpopulation crosses (WC, CW) compared to the purebreds, with the
exception of WW2, which was also high (Table 1). Alpha Entropy at q = 1
was the highest in WC, WW2 and lowest in WW3.
QTL analysis
SNPs were correlated with the five families reared under ambient
temperature. The number of SNPs associated with these five families
varied depending on threshold cut-offs ranging from 2,224 SNPs (75%
quartile) to 87 SNPs at the stricter threshold value (99% quartile).
Twenty-six of the 87 SNPs (30%) could not be assigned annotations
within the A. digitifera genome, a common issue in coral and
symbiont ‘omics (Figure 3 inset, sensu (Barshis et al., 2013;
Peng et al., 2010; Shinzato et al., 2011; Sogin, Putnam, Anderson, &
Gates, 2016)).
In the 87 SNPs that contributed to the separation of crosses at ambient
temperatures, we identified proteins involved in a range of biological
processes, mainly immunity and stress, cell functioning and metabolism,
and calcification (Figure 3 inset). Stress related proteins included
lysosomal-trafficking regulator-like proteins, CEPU-1-like protein, E3
ubiquitin-protein ligase RNF213-like, spondin, NFX1-type zinc
finger-containing protein, NACHT, MAP/microtubule affinity and other
processes, NLRC3-like proteins. Proteins involved with collagen
production varied across families, as well as 2 dTDP-glucose
4,6-dehydratase-like and two sodium bicarbonate transporter-like
proteins. A host of kinase related proteins, including those involved in
cell functioning and transcription were detected.
PCoA loadings defined cut-offs for significant SNPs and hence assigned
proteins. Homeodomain-interacting proteins, NFX1-type zinc
finger-containing proteins and src proteins (Figure 3A), which are
broadly associated with transcription, immunity and stress, and cell
functioning were broadly positively related to the separation of
families WW2/WW1/CW and WW3 and WC along PCoA axis 1 (Figure 1A).
Alternatively, differences in cell functioning proteins were more
negatively associated with their separation. Proteins contributing to
the separation of WW2/WW3, WW1 and CW, and WC along PCoA2 were
associated with responses to UV radiation, and MAP proteins (associated
with cell functioning and immunity/stress, respectively) whereas
multiple proteins associated with immunity and transcription were
negatively associated with their separation.
Narrow-sense heritability
Population of origin (WW, WC, CW),
family cross identity (WW1, WW2, WW3, WC, CW) and symbiont type
significantly influenced juvenile responses to growth, bleaching and
survivorship at 27.5°C and 31°C (full summary in Quigley et al., 2020).
Specifically, juveniles with at least one parental coral sourced from
the northern GBR survived significantly better, grew more, and bleached
less at 31°C compared to other crosses, especially if infected with the
Symbiodiniaceae symbiont, D. trenchii .
Physiological data for juvenile
survival, growth, and bleaching were previously presented in Quigley et
al., 2020. These raw data were used to calculate Bayesian narrow-sense
heritability (h2) estimates for survival, bleaching,
and growth demonstrate that only survival of corals infected withC. goreaui was strongly affected by temperature (Figure 4,
Supplementary Table 1). Bleaching responses were generally associated
with low heritability estimates at both temperatures, although juveniles
associated with C. goreaui exhibited more moderate heritability
estimates. Heritable genotypic variation across families contributed
little to differences in growth rates at 27.5 and 31°C (Table 2). With
the other three Symbiodiniaceae taxa, h2 estimates
across the three traits varied in juveniles from the five families
infected at 27.5 and 31°C (Supplementary Figure 1, Supplementary Table
1).