Evolution of Cold Acclimation and Its Role in Niche Transition in the Temperate Grass Subfamily Pooideae

Abstract

The grass subfamily Pooideae dominates the grass floras in cold temperate regions and has evolved complex physiological adaptations to cope with extreme environmental conditions like frost, winter, and seasonality. One such adaptation is cold acclimation, wherein plants increase their frost tolerance in response to gradually falling temperatures and shorter days in the autumn. However, understanding how complex traits like cold acclimation evolve remains a major challenge in evolutionary biology. Here, we investigated the evolution of cold acclimation in Pooideae and found that a phylogenetically diverse set of Pooideae species displayed cold acclimation capacity. However, comparing differential gene expression after cold treatment in transcriptomes of five phylogenetically diverse species revealed widespread species-specific responses of genes with conserved sequences. Furthermore, we studied the correlation between gene family size and number of cold-responsive genes as well as between selection pressure on coding sequences of genes and their cold responsiveness. We saw evidence of protein-coding and regulatory sequence evolution as well as the origin of novel genes and functions contributing toward evolution of a cold response in Pooideae. Our results reflect that selection pressure resulting from global cooling must have acted on already diverged lineages. Nevertheless, conservation of cold-induced gene expression of certain genes indicates that the Pooideae ancestor may have possessed some molecular machinery to mitigate cold stress. Evolution of adaptations to seasonally cold climates is regarded as particularly difficult. How Pooideae evolved to transition from tropical to temperate biomes sheds light on how complex traits evolve in the light of climate changes.

Figure 1.

The Pooideae phylogeny, present temperature data for focal species, and paleotemperature. A, Schematic phylogeny of the study species. The species phylogeny and dates are inferred from Schubert et al. (2018), except for B. sylvaticum, which was not included in the study. Placement and divergence date for B. sylvaticum are thus approximate. B, The range of the minimum temperature of the coldest month (WorldClim v1.4 data set, Bioclim variable 6, 2.5 km² resolution [Hijmans et al., 2005]) of the species geographical distribution (Global Biodiversity Information Facility, 2018). C, Oxygen isotope ratios as a proxy for historical global temperature (Zachos et al., 2001; Mudelsee et al., 2014). The transition between the Eocene and the Oligocene is shaded. Mya, Million years ago.

Figure 2.

Frost tolerance after cold acclimation. Box plot representation shows the regrowth of nine acclimated and nonacclimated Pooideae species after exposure to three freezing temperatures (−4°C, −8°C, and −12°C). Regrowth is scored on a scale from 0 to 9, where 0 is dead and 9 is undamaged. Significant differences in regrowth between acclimated and nonacclimated plants are indicated by asterisks (***, P ≤ 0.001; **, P ≤ 0.01; and *, P ≤ 0.05).

Figure 3.

GO enrichment and positive selection in branch-specific cold-responsive genes. A, GO enrichment analysis of HCOGs that were differentially expressed (DEGs) in all species (Pooideae base [PB]), only in species after N. stricta split off (early split [ES]), or only in B. distachyon and H. vulgare (late split [LS]). Significant differences are indicated by asterisks (*, P < 0.05; **, P < 0.01; and ***, P < 0.005, Fisher’s exact test). GO enrichments are shown for up- and down-regulated DEGs, not distinguishing between short- and long-term responses. Both the number of annotated genes and the number of annotations are indicated for each set of branch-specific DEGs. B, Positive selection at different stages in Pooideae evolution. The circles and numbers represent the HCOG gene trees that were tested for positive selection at each split. The inner blue circle and numbers below the branches represent HCOGs with branch-specific differential expression (i.e. genes that were cold responsive exclusively in the species under the respective branch), whereas the outer circles and numbers above the branches represent all other HCOGs. The purple and red pie-chart slices represent the proportions of HCOGs (first number) with positive selection (P < 0.05) among the total number of tested HCOGs (second number). The P values indicate the overrepresentation of positive selection among the branch-specific DEGs (hypergeometric test).

Figure 4.

Summary of phylogenetic analyses and gene expression experiments. Emergence of protein motifs and expansion of gene families important for cold acclimation are displayed on a schematic Pooideae species tree. The right-hand section depicts expression profiles of de novo transcripts in response to long- and short-term cold treatment of the respective species in the species tree. Expression patterns are approximated from the literature for L. perenne (CBF [Tamura and Yamada, 2007], WCS19/COR14 [Oishi et al., 2010; Ergon et al., 2016], and FST [Hisano et al., 2008; Paina et al., 2014]) and O. sativa (DHN8 [Lee et al., 2005], DHN13 [Aguan et al., 1991], and ctCOR-like [Maruyama et al., 2014]).