Drought and hybrid ancestry influence adaptive evolution and
generate GEI
Results from our transcript
categorisation (Fig. 2) and co-expression module enrichment analyses
(Fig 4) support the second hypothesis of garden-specific trait
differentiation. Despite the more arid conditions at the warm garden,
the marginal effect of drought on population-level transcript
differentiation was significant only at the cold garden. The strong
impact of drought on population differentiation found here conforms with
previous studies conducted in P. strobiformis (Goodrich et
al ., 2016; Bucholz et al ., 2020; Menon et al ., 2021).
While drought was an important selective pressure in both gardens, its
effect may have been exaggerated at the cold garden because of
freeze-related rupturing of cell wells during the previous winter season
(Bachofen et al . 2015) leading to elevated susceptibility to
drought stress. Given the overlap in molecular pathways leading to
stress tolerance (Blödner et al. 2005) such carry-over effects or
cross-susceptibility may be common across long-lived plants, although
the opposite indicating higher tolerance to the second stressor has also
been noted (Kong & Henry, 2019).
The absence of association with freezing stress could be due to global
rather than local adaptation (Booker et al ., 2020) aided by
expression of variants introgressed from P. flexilis , strong
confounding between geography and freeze-related variables or compounded
effects of drought and freeze stress in the cold garden. Ongoing work
demonstrating higher post-winter survival in the cold garden when
compared to the warm garden (Moler ERV. 2020) as well as a significant
association between P. flexilis ancestry and survival in the cold
garden provide some evidence for garden-specific effect of introgressed
variants. At the network level, we noted very limited preservation
across gardens using our joint threshold (Table S3; Fig. 4). The modules
that were strongly preserved consisted of transcripts with low garden
specific population differentiation and were generally related to
metabolic processes that are likely essential for basic organismal
functioning. Overall, this highlights strong and consistent patterns of
GEI.
At the multivariate level, modules correlated with ancestry were often
also strongly correlated with survival (Table S3; Table 2).
Specifically, two QST enriched and weakly
preserved modules – ME24 (warm garden) and ME13 (cold garden) – were
strongly associated with P. flexilis ancestry, as well as with
survival, making the traits and the loci underlying them a key candidate
for further detailed studies of GEI (Fig. 4; Table 2). Furthermore, none
of the transcripts encompassing these modules were shared across the two
gardens. Since we sampled the transcriptomes of juvenile hybrid trees,
it is possible that some of the strongly differentiated traits
documented here are involved in post-zygotic isolating barriers (Lindtkeet al ., 2014; Zhao et al ., 2014). Overall associations
between ancestry and expression levels of the threeQST categories, however, were not significant.
There are at least two potential explanations for these results. First,
ancestry estimates were obtained from a ddRADseq dataset, which although
is representative of overall genomic ancestry, provides less coverage of
genic regions in species with large genomes such as pines (Parchmanet al ., 2018). Second, ancestry was obtained for maternal trees
and correlated with the maternal tree’s genetic values that were
estimated using only the surviving seedlings. Given that postzygotic
barriers are often expressed in seedling stage (Ogasawara & Okubo,
2009) resulting in high mortality, it is likely that our design could
not fully capture the impact of ancestry on maternal tree’s genetic
expression value as we only used the surviving seedlings without strong
incompatibilities.
Interestingly, the proportion of P. flexilis ancestry was
positively associated with survival across both gardens, but significant
only at the cold garden. Survival and ancestry remained uncorrelated to
population-level transcript abundances. These results thus provide only
partial support for the third hypothesis of hybrid ancestry interacting
with GEI and highlight the need for an in-depth evaluation mapping
expression difference between parentals and hybrid trees in novel
environments. Nevertheless, drawing from our previous work (Menonet al ., 2021) and the likely wider climatic tolerance of P.
flexilis (Windmuller-Campione & Long, 2016), we speculate that the
significant correlation between ancestry and survival only at the cold
garden is indicative of P. flexilis -like variants enhancing
survival in cooler environments, which aligns with the higher
latitudinal distribution of the species range of P. flexilis thanP. strobiformis .