Melica as an emerging model system for comparative studies in temperate Pooideae grasses.

BACKGROUND AND AIMS: Pooideae grasses contain some of the world's most important crop and forage species. Although much work has been conducted on understanding the genetic basis of trait diversification within a few annual Pooideae, comparative studies at the subfamily level are limited by a lack of perennial models outside 'core' Pooideae. We argue for development of the perennial non-core genus Melica as an additional model for Pooideae, and provide foundational data regarding the group's biogeography and history of character evolution. METHODS: Supplementing available ITS and ndhF sequence data, we built a preliminary Bayesian-based Melica phylogeny, and used it to understand how the genus has diversified in relation to geography, climate and trait variation surveyed from various floras. We also determine biomass accumulation under controlled conditions for Melica species collected across different latitudes and compare inflorescence development across two taxa for which whole genome data are forthcoming. KEY RESULTS: Our phylogenetic analyses reveal three strongly supported geographically structured Melica clades that are distinct from previously hypothesized subtribes. Despite less geographical affinity between clades, the two sister 'Ciliata' and 'Imperfecta' clades segregate from the more phylogenetically distant 'Nutans' clade in thermal climate variables and precipitation seasonality, with the 'Imperfecta' clade showing the highest levels of trait variation. Growth rates across Melica are positively correlated with latitude of origin. Variation in inflorescence morphology appears to be explained largely through differences in secondary branch distance, phyllotaxy and number of spikelets per secondary branch. CONCLUSIONS: The data presented here and in previous studies suggest that Melica possesses many of the necessary features to be developed as an additional model for Pooideae grasses, including a relatively fast generation time, perenniality, and interesting variation in physiology and morphology. The next step will be to generate a genome-based phylogeny and transformation tools for functional analyses.

Testing the chilling- before drought-tolerance hypothesis in Pooideae grasses.

Temperate Pooideae are a large clade of economically important grasses distributed in some of the Earth's coldest and driest terrestrial environments. Previous studies have inferred that Pooideae diversified from their tropical ancestors in a cold montane habitat, suggesting that above-freezing cold (chilling) tolerance evolved early in the subfamily. By contrast, drought tolerance is hypothesized to have evolved multiple times independently in response to global aridification that occurred after the split of Pooideae tribes. To independently test predictions of the chilling-before-drought hypothesis in Pooideae, we assessed conservation of whole plant and gene expression traits in response to chilling vs. drought. We demonstrated that both trait responses are more similar across tribes in cold as compared to drought, suggesting that chilling responses evolved before, and drought responses after, tribe diversification. Moreover, we found significantly more overlap between drought and chilling responsive genes within a species than between drought responsive genes across species, providing evidence that chilling tolerance genes acted as precursors for the novel acquisition of increased drought tolerance multiple times independently, partially through the cooption of chilling responsive genes.

Major niche transitions in Pooideae correlate with variation in photoperiodic flowering and evolution of CCT domain genes.

The external cues that trigger timely flowering vary greatly across tropical and temperate plant taxa, the latter relying on predictable seasonal fluctuations in temperature and photoperiod. In the grass family (Poaceae) for example, species of the subfamily Pooideae have become specialists of the northern temperate hemisphere, generating the hypothesis that their progenitor evolved a flowering response to long days from a short-day or day-neutral ancestor. Sampling across the Pooideae, we found support for this hypothesis, and identified several secondary shifts to day-neutral flowering and one to short-day flowering in a tropical highland clade. To explain the proximate mechanisms for the secondary transition back to short-day-regulated flowering, we investigated the expression of CCT domain genes, some of which are known to repress flowering in cereal grasses under specific photoperiods. We found a shift in CONSTANS 1 and CONSTANS 9 expression that coincides with the derived short-day photoperiodism of our exemplar species Nassella pubiflora. This sets up the testable hypothesis that trans- or cis-regulatory elements of these CCT domain genes were the targets of selection for major niche shifts in Pooideae grasses.

Flowering time runs hot and cold.

Evidence suggests that anthropogenically-mediated global warming results in accelerated flowering for many plant populations. However, the fact that some plants are late flowering or unaffected by warming, underscores the complex relationship between phase change, temperature, and phylogeny. In this review, we present an emerging picture of how plants sense temperature changes, and then discuss the independent recruitment of ancient flowering pathway genes for the evolution of ambient, low, and high temperature-regulated reproductive development. As well as revealing areas of research required for a better understanding of how past thermal climates have shaped global patterns of plasticity in plant phase change, we consider the implications for these phenological thermal responses in light of climate change.

Spatial variation in high temperature-regulated gene expression predicts evolution of plasticity with climate change in the scarlet monkeyflower.

A major way that organisms can adapt to changing environmental conditions is by evolving increased or decreased phenotypic plasticity. In the face of current global warming, more attention is being paid to the role of plasticity in maintaining fitness as abiotic conditions change over time. However, given that temporal data can be challenging to acquire, a major question is whether evolution in plasticity across space can predict adaptive plasticity across time. In growth chambers simulating two thermal regimes, we generated transcriptome data for western North American scarlet monkeyflowers (Mimulus cardinalis) collected from different latitudes and years (2010 and 2017) to test hypotheses about how plasticity in gene expression is responding to increases in temperature, and if this pattern is consistent across time and space. Supporting the genetic compensation hypothesis, individuals whose progenitors were collected from the warmer-origin northern 2017 descendant cohort showed lower thermal plasticity in gene expression than their cooler-origin northern 2010 ancestors. This was largely due to a change in response at the warmer (40°C) rather than cooler (20°C) treatment. A similar pattern of reduced plasticity, largely due to a change in response at 40°C, was also found for the cooler-origin northern versus the warmer-origin southern population from 2017. Our results demonstrate that reduced phenotypic plasticity can evolve with warming and that spatial and temporal changes in plasticity predict one another.

Convergent evolution of the annual life history syndrome from perennial ancestors.

Despite most angiosperms being perennial, once-flowering annuals have evolved multiple times independently, making life history traits among the most labile trait syndromes in flowering plants. Much research has focused on discerning the adaptive forces driving the evolution of annual species, and in pinpointing traits that distinguish them from perennials. By contrast, little is known about how 'annual traits' evolve, and whether the same traits and genes have evolved in parallel to affect independent origins of the annual syndrome. Here, we review what is known about the distribution of annuals in both phylogenetic and environmental space and assess the evidence for parallel evolution of annuality through similar physiological, developmental, and/or genetic mechanisms. We then use temperate grasses as a case study for modeling the evolution of annuality and suggest future directions for understanding annual-perennial transitions in other groups of plants. Understanding how convergent life history traits evolve can help predict species responses to climate change and allows transfer of knowledge between model and agriculturally important species.

CCDC22 and CCDC93, two potential retriever-interacting proteins, are required for root and root hair growth in Arabidopsis.

Endomembrane trafficking is essential for plant growth and often depends on a balance between secretory and endocytic pathways. VPS26C is a component of the retriever complex which has been shown to function in the recycling of integral plasma membrane proteins in human cell culture and is part of a core retriever complex in Arabidopsis that is required for root hair growth. In this work, we report a characterization of the Arabidopsis homologues of CCDC22 and CCDC93, two additional proteins required for retriever function in humans. Phylogenetic analysis indicates that CCDC22 (AT1G55830) and CCDC93 (AT4G32560) are single copy genes in plants that are present across the angiosperms, but like VPS26C, are absent from the grasses. Both CCDC22 and CCDC93 are required for root and root hair growth in Arabidopsis and localize primarily to the cytoplasm in root epidermal cells. Previous work has demonstrated a genetic interaction between VPS26C function and a VTI13-dependent trafficking pathway to the vacuole. To further test this model, we characterized the vti13 ccdc93 double mutant and show that like vps26c, ccdc93 is a suppressor of the vti13 root hair phenotype. Together this work identifies two new proteins essential for root and root hair growth in plants and demonstrate that the endosomal pathway(s) in which CCDC93 functions is genetically linked to a VTI13-dependent trafficking pathway to the vacuole.

Variation in climatic tolerance, but not stomatal traits, partially explains Pooideae grass species distributions.

BACKGROUND AND AIMS: Grasses in subfamily Pooideae live in some of the world's harshest terrestrial environments, from frigid boreal zones to the arid windswept steppe. It is hypothesized that the climate distribution of species within this group is driven by differences in climatic tolerance, and that tolerance can be partially explained by variation in stomatal traits. METHODS: We determined the aridity index (AI) and minimum temperature of the coldest month (MTCM) for 22 diverse Pooideae accessions and one outgroup, and used comparative methods to assess predicted relationships for climate traits versus fitness traits, stomatal diffusive conductance to water (gw) and speed of stomatal closure following drought and/or cold. KEY RESULTS: Results demonstrate that AI and MTCM predict variation in survival/regreening following drought/cold, and gw under drought/cold is positively correlated with δ 13C-measured water use efficiency (WUE). However, the relationship between climate traits and fitness under drought/cold was not explained by gw or speed of stomatal closure. CONCLUSIONS: These findings suggest that Pooideae distributions are at least partly determined by tolerance to aridity and above-freezing cold, but that variation in tolerance is not uniformly explained by variation in stomatal traits.

Insights into the evo-devo of plant reproduction using next-generation sequencing approaches.

The development of plant model organisms has traditionally been analyzed using resource-heavy, tailored applications that are not easily transferable to distantly related non-model taxa. Thus, our understanding of plant development has been limited to a subset of traits, and evolutionary studies conducted most effectively either across very wide [e.g. Arabidopsis thaliana and Oryza sativa (rice)] or narrow (i.e. population level) phylogenetic distances. As plant biologists seek to capitalize on natural diversity for crop improvement, enhance ecosystem functioning, and better understand plant responses to climate change, high-throughput and broadly applicable forms of existing molecular biology assays are becoming an invaluable resource. Next-generation sequencing (NGS) is increasingly becoming a powerful tool in evolutionary developmental biology (evo-devo) studies, particularly through its application to understanding trait evolution at different levels of gene regulation. Here, I review some of the most common and emerging NGS-based methods, using exemplar studies in reproductive plant evo-devo to illustrate their potential.

Understanding Past, and Predicting Future, Niche Transitions based on Grass Flowering Time Variation.

Since their origin in the early Cretaceous, grasses have diversified across every continent on Earth, with a handful of species (rice [Oryza sativa], maize [Zea mays], and wheat [Triticum aestivum]) providing most of the caloric intake of contemporary humans and their livestock. The ecological dominance of grasses can be attributed to a number of physiological innovations, many of which contributed to shifts from closed to open habitats that incur daily (e.g. tropical mountains) and/or seasonal extremes in temperature (e.g. temperate/continental regions) and precipitation (e.g. tropical savannas). In addition to strategies that allow them to tolerate or resist periodically stressful environments, plants can adopt escape behaviors by modifying the relative timing of distinct development phases. Flowering time is one of these behaviors that can also act as a postzygotic barrier to reproduction and allow temporal partitioning of resources to promote coexistence. In this review, we explore what is known about the phylogenetic pattern of flowering control in grasses, and how this relates to broad- and fine-scale niche transitions within the family. We then synthesize recent findings on the genetic basis of flowering time evolution as a way to begin deciphering why certain aspects of flowering are seemingly so conserved, and what the implications of this are for future adaptation under climate change.

Implications of region-specific gene expression for development of the partially fused petunia corolla.

Angiosperm petal fusion (sympetaly) has evolved multiple times independently and is associated with increased specificity between plants and their pollinators. To uncover developmental genetic changes that might have led to the evolution of sympetaly in the asterid core eudicot genus Petunia (Solanaceae), we carried out global and fine-scale gene expression analyses in different regions of the corolla. We found that, despite several similarities with the choripetalous model species Arabidopsis thaliana in the proximal-distal transcriptome, the Petunia axillaris fused and proximal corolla tube expresses several genes that in A. thaliana are associated with the distal petal region. This difference aligns with variation in petal shape and fusion across ontogeny of the two species. Moreover, differential gene expression between the unfused lobes and fused tube of P. axillaris petals revealed three strong candidate genes for sympetaly based on functional annotation in organ boundary specification. Partial silencing of one of these, the HANABA TARANU (HAN)-like gene PhGATA19, resulted in reduced fusion of Petunia hybrida petals, with silencing of both PhGATA19 and its close paralog causing premature plant senescence. Finally, detailed expression analyses for the previously characterized organ boundary gene candidate NO APICAL MERISTEM (NAM) supports the hypothesis that it establishes boundaries between most P. axillaris floral organs, with the exception of boundaries between petals.

Evidence of a largely staminal origin for the Jaltomata calliantha (Solanaceae) floral corona.

BACKGROUND: Understanding the evolution of novel features requires homology assessments at different levels of biological organization. In flowering plants, floral coronas that play various roles in plant-pollinator interactions have evolved multiple times independently, but are highly variable in their final position and overall morphology. Coronas of the Solanaceae species Jaltomata calliantha are found between the corolla and stamens, adjacent to the gynoecium, and form cups that house copious amounts of their characteristic blood red nectar. To test the hypothesis that J. calliantha coronas evolved as an outgrowth of stamens and therefore have staminal identity, we assessed their development, floral organ identity gene expression, and cellular morphology. RESULTS: Jaltomata calliantha coronas emerge after the initiation of all conventional floral organs on the abaxial side of the proximally modified stamens and then expand medially and laterally to form nectar cups. Overlapping expression of the B-class organ identity genes JcAPETALA3 and both JcPISTILLATA/GLOBOSA orthologs (JcGLO1 and JcGLO2), and the C-class-like gene JcAGAMOUS1-like, unites the stamens and corona. Epidermal cell shape also connects the adaxial surface of coronas and petals, and the stamen base, with remaining floral organs showing divergent cell types. CONCLUSIONS: Our data, based on multiple lines of evidence, support a largely staminal origin for J. calliantha coronas. However, since slightly enlarged stamen bases are found in Jaltomata species that lack coronas, and J. calliantha stamen bases share cell types with petals, we hypothesize that stamen bases recruited part of the petal identity program prior to fully expanding into a corona.

Successive evolutionary steps drove Pooideae grasses from tropical to temperate regions.

Angiosperm adaptations to seasonally cold climates have occurred multiple times independently. However, the observation that less than half of all angiosperm families are represented in temperate latitudes suggests internal constraints on the evolution of cold tolerance/avoidance strategies. Similar to angiosperms as a whole, grasses are primarily tropical, but one major clade, subfamily Pooideae, radiated extensively within temperate regions. It is posited that this Pooideae niche transition was facilitated by an early origin of long-term cold responsiveness around the base of the subfamily, and that a set of more ancient pathways enabled evolution of seasonal cold tolerance. To test this, we compared transcriptome-level responses of disparate Pooideae to short-/long-term cold and with those previously known in the subtropical grass rice (Ehrhartoideae). Analyses identified several highly conserved cold-responsive 'orthogroups' within our focal Pooideae species that originated successively during the diversification of land plants, predominantly via gene duplication. The majority of conserved Pooideae cold-responsive genes appear to have ancient roles in stress responses, with most of the orthogroups also being sensitive to cold in rice. However, a subgroup of genes was likely co-opted de novo early in the Pooideae. These results highlight a plausible stepwise evolutionary trajectory for cold adaptations across Pooideae.

Heterochronic developmental shifts underlie floral diversity within Jaltomata (Solanaceae).

BACKGROUND: Heterochronic shifts during mid- to late stages of organismal development have been proposed as key mechanisms generating phenotypic diversity. To determine whether late heterochronic shifts underlie derived floral morphologies within Jaltomata (Solanaceae)-a genus whose species have extensive and recently evolved floral diversity-we compared floral development of four diverse species (including an ambiguously ancestral or secondarily derived rotate, two putatively independently evolved campanulate, and a tubular morph) to the ancestral rotate floral form, as well as to an outgroup that shares this ancestral floral morphology. RESULTS: We determined that early floral development (< 1 mm bud diameter, corresponding to completion of organ whorl initiation) is very similar among all species, but that different mature floral forms are distinguishable by mid-development (> 1 mm diameter) due to differential growth acceleration of corolla traits. Floral ontogeny among similar mature rotate forms remains comparable until late stages, while somewhat different patterns of organ growth are found between species with similar campanulate forms. CONCLUSIONS: Our data suggest shared floral patterning during early-stage development, but that different heterochronic shifts during mid- and late-stage development contribute to divergent floral traits. Heterochrony thus appears to have been important in the rapid and repeated diversification of Jaltomata flowers.

Evolution of the miR5200-FLOWERING LOCUS T flowering time regulon in the temperate grass subfamily Pooideae.

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.

Repeated and diverse losses of corolla bilateral symmetry in the Lamiaceae.

Background and Aims: Independent evolution of derived complex characters provides a unique opportunity to assess whether and how similar genetic changes correlate with morphological convergence. Bilaterally symmetrical corollas have evolved multiple times independently from radially symmetrical ancestors and likely represent adaptations to attract specific pollinators. On the other hand, losses of bilateral corolla symmetry have occurred sporadically in various groups, due to either modification of bilaterally symmetrical corollas in late development or early establishment of radial symmetry. Methods: This study integrated phylogenetic, scanning electron microscopy (SEM)-based morphological, and gene expression approaches to assess the possible mechanisms underlying independent evolutionary losses of corolla bilateral symmetry. Key Results: This work compared three species of Lamiaceae having radially symmetrical mature corollas with a representative sister taxon having bilaterally symmetrical corollas and found that each reaches radial symmetry in a different way. Higher core Lamiales share a common duplication in the CYCLOIDEA (CYC ) 2 gene lineage and show conserved and asymmetrical expression of CYC2 clade and RAD genes along the adaxial-abaxial floral axis in species having bilateral corolla symmetry. In Lycopus americanus , the development and expression pattern of La-CYC2A and La-CYC2B are similar to those of their bilaterally symmetrical relatives, whereas the loss of La-RAD expression correlates with a late switch to radial corolla symmetry. In Mentha longifolia , late radial symmetry may be explained by the loss of Ml-CYC2A , and by altered expression of two Ml-CYC2B and Ml-RAD genes . Finally, expanded expression of Cc-CYC2A and Cc-RAD strongly correlates with the early development of radially symmetrical corollas in Callicarpa cathayana . Conclusions: Repeated losses of mature corolla bilateral symmetry in Lamiaceae are not uncommon, and may be achieved by distinct mechanisms and various changes to symmetry genes, including the loss of a CYC2 clade gene from the genome, and/or contraction, expansion or alteration of CYC2 clade and RAD -like gene expression.

Evidence for an Early Origin of Vernalization Responsiveness in Temperate Pooideae Grasses.

The ability of plants to match their reproductive output with favorable environmental conditions has major consequences both for lifetime fitness and geographic patterns of diversity. In temperate ecosystems, some plant species have evolved the ability to use winter nonfreezing cold (vernalization) as a cue to ready them for spring flowering. However, it is unknown how important the evolution of vernalization responsiveness has been for the colonization and subsequent diversification of taxa within the northern and southern temperate zones. Grasses of subfamily Pooideae, including several important crops, such as wheat (Triticum aestivum), barley (Hordeum vulgare), and oats (Avena sativa), predominate in the northern temperate zone, and it is hypothesized that their radiation was facilitated by the early evolution of vernalization responsiveness. Predictions of this early origin hypothesis are that a response to vernalization is widespread within the subfamily and that the genetic basis of this trait is conserved. To test these predictions, we determined and reconstructed vernalization responsiveness across Pooideae and compared expression of wheat vernalization gene orthologs VERNALIZATION1 (VRN1) and VRN3 in phylogenetically representative taxa under cold and control conditions. Our results demonstrate that vernalization responsive Pooideae species are widespread, suggesting that this trait evolved early in the lineage and that at least part of the vernalization gene network is conserved throughout the subfamily. These results are consistent with the hypothesis that the evolution of vernalization responsiveness was important for the initial transition of Pooideae out of the tropics and into the temperate zone.