Charles O. Hale

Summary: Grasses (Poaceae) dominate terrestrial ecosystems and sustain global food security, yet the genomic principles enabling their repeated adaptation to extreme environments remain unresolved. Combining dense phylogenomic sampling, global environmental data, and genomic large language models (gLLMs), we characterize the mutational targets underlying environmental shift across 687 genomes from 561 species spanning 17 climate zones. We identify 17-30 phylogenetically independent transitions into extreme temperature, water, and soil environments, accompanied by convergent shifts in genome-scale molecular properties, including the Nitrogen-to-Carbon balance and the biosynthetic cost of the proteome. Our gLLMs-informed phylogenetic mixed modeling framework identifies 330 genes that repeatedly associated with distinct axes of environmental variation, highlighting the importance of protein modification and localization within extracellular and organellar compartments. Overlaying independent convergent selection tests identifies 27 high-confidence candidates for further characterization. Together, our results show that grass adaptation is canalized by layered constraints at genome-wide and gene-specific scales, producing predictable evolutionary trajectories.

The growing availability of genomes from non-model organisms offers new opportunities to identify functional loci underlying trait variation through comparative genomics. While cis-regulatory regions drive much of phenotypic evolution, linking them to specific functions remains challenging. We identified 514 cis-regulatory motifs enriched in regulatory regions of five diverse grass species, with 73% consistently enriched across all, suggesting a deeply conserved regulatory code. Leveraging 57 new contig-level genome assemblies, we then quantified shared occupancy of specific motif instances within gene-proximal regions across 589 grass species, revealing widespread gain and loss over evolutionary time. Shared occupancy declined rapidly over the first few million years of divergence, yet ∼50% of motif instances were shared back to the origin of grasses ∼100 million years ago. We used phylogenetic mixed models to identify motif gains and losses associated with ecological niche transitions. Our models revealed significant environmental associations across 1282 motif–orthogroup combinations, including convergent gains of HSF/GARP motifs at an alpha-N-acetylglucosaminidase gene associated with occurrence in temperate environments. Our findings support a “stable motifs, variable binding sites” model in which cis-regulatory evolution involves turnover of thousands of individual binding site instances while largely preserving transcription factors’ binding preferences. Our results highlight the potential of comparative genomics and phylogenetic mixed models to reveal the genetic basis of complex traits.

Plants adapt to environmental variation both by evolving divergent trait means and by plastically adjusting trait expression in response to local conditions. While these dual strategies are essential for persistence in diverse environments, there are still outstanding questions about how they interact and vary across closely related species. For plants, water availability is a particularly important selective force that shapes species distributions, selects for growth habit and life history strategy, and can dictate individuals' plastic expressions of trait values and reproductive success. Here, we use ecological niche modeling, field soil characterization, and a controlled dry‐down experiment to understand how geographic distribution and evolutionary background among three closely related Phlox wild flower species and their F1 hybrids explain their responses to water availability. We infer that the species occupy distinct niches that diverge along a primary axis of water availability and soil moisture. Each species has a distinct growth habit that does not match broad predictions of divergence in response to water availability. Nevertheless, we find that all the species show a significant morphological response to controlled soil dry down with reduced biomass, smaller leaves, and fewer flowers, as would be predicted in a response to drought. We find that Phlox drummondii , which occupies intermediate habitats, exhibits the strongest plastic response to water limitation, despite it not having the broadest environmental niche. Additionally, most hybrids involving P. drummondii display intermediate phenotypes in both wet and dry treatments, while hybrids between P. cuspidata and P. roemeriana show phenotypes consistent with hybrid vigor. These results challenge the hypothesis that species from broader environments evolve greater plasticity. Instead, the most plastic species did not have the broadest niche, suggesting plasticity and niche breadth may evolve independently.

Bouteloua is a PACMAD grass genus that dominates much of the central and southern North American prairies and plains, where species such as sideoats grama ( B. curtipendula ), blue grama ( B. gracilis ), and black grama ( B. eriopoda ) are both ecologically dominant and among the most valuable forage grasses of arid rangelands. Yet, genomic resources for the genus remain limited. We generated PacBio HiFi draft assemblies for B. curtipendula (653 Mb, N50 25.5 Mb), B. gracilis (808 Mb, N50 4.1 Mb), and B. eriopoda (1.67 Gb, N50 1.0 Mb). All assemblies showed >98.5% BUSCO completeness. Helixer predicted 83,877-215,536 gene models per assembly. However, syntenic copy numbers (2 in B. curtipendula , 3 in B. gracilis , and 6 in B. eriopoda ) indicate that at least two species are polyploid, and the inflated gene model counts likely reflect homeologous copies and fragmented predictions rather than true gene numbers. panEDTA identified 25-40% transposable-element content, dominated by LTR retrotransposons. These assemblies provide foundational genomic references for Bouteloua and support comparative studies across PACMAD grasses.

Flowering plants display extensive variation in selfing rate, a trait with significant ecological and evolutionary consequences. Many species use genetic mechanisms to recognize and reject self-pollen (termed self-incompatibility or SI), and the loss of SI is one of the most common evolutionary transitions among flowering plants. Despite the ubiquity of transitions to self-compatibility (SC), little is known about the genetic architecture through which SC evolves. Specifically, it is important to determine if SC has a simple or polygenic basis and if variation localizes to the self-pollen recognition locus (the “S-locus”). Phlox drummondii (Polemoniaceae) is a model system for exploring mating system evolution and expresses range-wide variation in SI. Here, we investigate the genetic architecture of SC variants segregating within this otherwise SI species. Using multiple independent crosses, we uncover numerous QTLs associated with intraspecific SI variation, consistent with a polygenic genetic architecture. While some QTLs overlap across mapping experiments, other QTLs are unique, suggesting that multiple genetic routes to SC exist. We demonstrate that P. drummondii has a sporophytic SI system, revealing an independent evolution of SI within the Phlox lineage. We map this novel S-locus and find that the genomic region containing the S-locus is associated with intraspecific variation in SI in one of the 3 mapping populations. Although further work is necessary to clarify the conditions under which quantitative variation in SI represents a transitional pathway to complete SC, our study reveals the genetic architecture upon which selection could act to drive this frequent and evolutionarily significant transition.

SUMMARY: Nitrogen (N) fertilization in crop production significantly impacts ecosystems, often disrupting natural plant–microbe–soil interactions and causing environmental pollution. This study tested the hypothesis that diverse species adapting independently to various environments might exhibit a wide range of rhizosphere nutrient management strategies, and some of them may be conducive to an efficient N economy for crops. We analyzed the N cycle in the rhizospheres of 36 Andropogoneae grass species related to maize and sorghum and observed significant phylogenetic variation among their impacts on N availability and losses. All three annual species examined, including sorghum and maize, function as N ‘Conservationists’, reducing soil nitrification potential and conserving NH₄⁺. In contrast, seven of the assayed perennial species enhance nitrification and leaching (‘Leachers’). Four other species exhibit similar nitrification stimulation effects but limited NO₃⁻ losses (‘Nitrate Keepers’). We complemented the controlled phenotypic evaluation with an evolutionary‐ecological analysis of the same species. We identified several soil characteristics associated with the phylogenetic variation in rhizosphere N dynamics across grasses and highlighted the crucial roles of a few transporter genes in soil N management and utilization. In addition to the ecological and genetic insights, these findings offer valuable guidelines for future maize breeding efforts to enhance agricultural N efficiency and sustainability.

Maize (Zea mays L.) is the world's most productive grain crop and a cornerstone of global food supply. However, in temperate agricultural systems, maize exhibits 2 key anomalies. First, as a tropical species, maize cannot be planted in the cold conditions of early spring when light and natural soil nitrogen are available, resulting in a shorter growing season and creating a seasonal mismatch between nitrogen accessibility and demand. Second, maize kernel protein is a major nitrogen sink, driving fertilizer demand because of the scale of cultivation. This inefficient mismatch stems from modern maize's uses and the modest nutritional value of storage proteins. To address these anomalies, we established the Circular Economy that Reimagines Corn Agriculture initiative. Our vision requires advances in 3 research areas: (ⅰ) developing cold and frost tolerance during germination and early growth to enable the use of spring nitrogen and light resources; (ⅱ) reducing nitrogen allocation to grain by reducing low-quality storage proteins and developing alternative nitrogen sinks; and (ⅲ) stabilizing soil nitrogen by enhancing biological nitrification inhibition. We present blueprints for a nitrogen-efficient, cold-tolerant maize designed to utilize the full growing season, enabling farmers in temperate regions to fully leverage maize's C4 photosynthesis, reduce fertilizer inputs, increase yields, and minimize environmental impact.

Plants adapt to environmental variation both by evolving divergent trait means and by plastically adjusting trait expression in response to local conditions. While these dual strategies are essential for persistence in diverse environments, how they interact and vary across closely related species is understudied. For plants, water availability is a particularly important selective force that shapes species distributions, selects for growth habit and life history strategy, and can dictate individuals’ plastic expressions of trait values and reproductive success. Here, we use ecological niche modeling, field soil collections, and a controlled drought experiment to test how biogeography and evolutionary history influence responses to limited water availability in three closely related Texas annual Phlox species and their F1 hybrids. We infer that the species occupy distinct niches that diverge along a primary axis of water availably and soil moisture. Each species has a distinct vegetative growth habit that does not match broad predictions of divergence in response to water availability. Nevertheless, we find that all the species show a significant morphological response to controlled soil dry down with reduced biomass, smaller leaves, and fewer flowers, as would be predicted in a response to drought. We find that Phlox drummondii , which occupies intermediate habitats, exhibits the strongest plastic response to water limitation, despite it not having the broadest environmental niche. Additionally, most hybrids involving P. drummondii display intermediate phenotypes in both wet and dry treatments, while hybrids between P. cuspidata and P. roemeriana show phenotypes consistent with hybrid vigor. These results challenge the hypothesis that species from broader environments evolve greater plasticity. Instead, the most plastic species did not have the broadest niche, suggesting plasticity and niche breadth may evolve independently.

Summary: Grasses in the PACMAD clade independently colonized cold environments from warm-climate ancestors, but whether their molecular responses to freezing reflect shared evolutionary solutions or lineage-specific innovations remains unknown. We used comparative proteomics to test whether protein-level cold responses show stronger cross-species conservation than previously observed at the transcript level. We quantified seasonal rhizome proteomes (winter vs summer) from five PACMAD species grown in a common garden exposed to sustained sub-zero temperatures, identified differentially abundant proteins, and compared fold-change magnitudes across species using orthogroup-based correlation analyses. We further examined LEA3 protein structure through hydropathy profiling and motif analysis. Shared cold-responsive proteins showed higher cross-species fold-change correlation ( ρ = 0.80) than background proteins ( ρ = 0.45), despite greater divergence in baseline abundance. LEA3 was the only ortholog elevated across all five species. Cold-tolerant species contained more tandem 11-mer repeats than the cold-sensitive maize, and two species accumulated multiple LEA3 paralogs, increasing total LEA3 abundance. Independent evolution of freezing tolerance in PACMAD grasses is governed by evolutionary constraints on protein-level response magnitude, reflecting the retention of an ancestral protective capacity. Structural divergence of LEA3 in maize suggests that transcriptional induction alone does not ensure freezing tolerance; functional protection likely requires intact motif architecture.

The growing availability of genomes from non-model organisms offers new opportunities to identify functional loci underlying trait variation through comparative genomics. While cis -regulatory regions drive much of phenotypic evolution, linking them to specific functions remains challenging. We identified 514 cis -regulatory motifs enriched in regulatory regions of five diverse grass species, with 73% consistently enriched across all, suggesting a deeply conserved regulatory code. We then quantified conservation of specific motif instances across 589 grass species, revealing widespread gain and loss over evolutionary time. Conservation declined rapidly over the first few million years of divergence, yet ∼50% of motif instances were conserved back to the origin of grasses ∼100 million years ago. Conservation patterns varied by gene class, with modestly higher conservation at transcription factor genes. To test for adaptive cis -regulatory changes, we used phylogenetic mixed models to identify motif gains and losses associated with ecological niche transitions. Our models revealed polygenic adaptation across 810 motif-orthogroup combinations, including convergent gains of HSF/GARP motifs at an Alpha-N-acetylglucosaminidase gene associated with adaptation to temperate environments. Our results support a “stable code, variable sites” model in which cis -regulatory evolution involves extensive turnover of individual binding site instances while largely preserving transcription factors’ binding preferences. Cis -regulatory changes at hundreds to thousands of genes appear to contribute to environmental adaptation. Our results highlight the potential of comparative genomics and phylogenetic mixed models to reveal the genetic basis of complex traits.