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.

Cold-regulated VRN1 and VRN3 expression is consistent with an early origin of vernalization responsiveness in the temperate Pooideae grasses.

Summary: Semelparous annual plants flower a single time during their 1‐yr life cycle, investing much of their energy into rapid reproduction. By contrast, iteroparous perennial plants flower multiple times over several years, and partition their resources between reproduction and persistence. To which extent evolutionary transitions between life‐cycle strategies are internally constrained at the developmental, genetic and phylogenetic level is unknown. Here we study the evolution of life‐cycle strategies in the grass subfamily Pooideae and test if transitions between them are facilitated by evolutionary precursors. We integrate ecological, life‐cycle strategy and growth data in a phylogenetic framework. We investigate if growth traits are candidates for a precursor. Species in certain Pooideae clades are predisposed to evolve annuality from perenniality, potentially due to the shared inheritance of specific evolutionary precursors. Seasonal dry climates, which have been linked to annuality, were only able to select for transitions to annuality when the precursor was present. Allocation of more resources to above‐ground rather than below‐ground growth is a candidate for the precursor. Our findings support the hypothesis that only certain lineages can respond quickly to changing external conditions by switching their life‐cycle strategy, likely due to the presence of evolutionary precursors.

Accumulating evidence is suggesting more frequent tropical-to-temperate transitions than previously thought. This raises the possibility that biome transitions could be facilitated by precursor traits. A wealth of ecological, genetic, and physiological evidence suggests overlap between drought and frost stress responses, but the origin of this overlap, i.e., the evolution of these responses relative to each other, is poorly known. Here, we test whether adaptation to frost and/or severe winters in grasses (Poaceae) was facilitated by ancestral adaptation to drought. We used occurrence patterns across Köppen–Geiger climate zones to classify species as drought, frost, and/or winter tolerant, followed by comparative analyses. Ancestral state reconstructions revealed different evolutionary trajectories in different clades, suggesting both drought-first and frost-first scenarios. Explicit simultaneous modelling of drought and frost/winter tolerance provided some support for correlated evolution, but suggested higher rates of gain of frost/winter tolerance in drought-sensitive rather than drought-tolerant lineages. Overall, there is limited support across grasses as a whole that drought tolerance acted as an evolutionary precursor to frost or severe winter tolerance. Different scenarios in different clades is consistent with present-day grasses being either cold or drought specialists, possibly as a consequence of trade-offs between different stress tolerance responses.

Flowering time is a carefully regulated trait controlled primarily through the action of the central genetic regulator, FLOWERING LOCUS T (FT). Recently it was demonstrated that a microRNA, miR5200, targets the end of the second exon of FT under short-day photoperiods in the grass subfamily Pooideae, thus preventing FT transcripts from reaching threshold levels under non-inductive conditions. Pooideae are an interesting group in that they rapidly diversified from the tropics into the northern temperate region during a major global cooling event spanning the Eocene-Oligocene transition. We hypothesize that miR5200 photoperiod-sensitive regulation of Pooideae flowering time networks assisted their transition into northern seasonal environments. Here, we test predictions derived from this hypothesis that miR5200, originally found in bread wheat and later identified in Brachypodium distachyon, (1) was present in the genome of the Pooideae common ancestor, (2) is transcriptionally regulated by photoperiod, and (3) is negatively correlated with FT transcript abundance, indicative of miR5200 regulating FT. Our results demonstrate that miR5200 did evolve at or around the base of Pooideae, but only acquired photoperiod-regulated transcription within the Brachypodium lineage. Based on expression profiles and previous data, we posit that the progenitor of miR5200 was co-regulated with FT by an unknown mechanism.