Ryan Andrew Folk

Nitrogen-fixing symbioses, particularly those occurring in root nodules, are among the most consequential mutualisms in natural and agricultural systems and represent a globally important source of bioavailable nitrogen. Despite their importance, patterns of diversity and composition among diazotrophic symbionts—and the processes structuring those patterns in natural systems—remain poorly resolved, with competing hypotheses emphasizing ecological, or phylogenetic constraints on host–symbiont associations. Here, using a broad survey of nodulating plants from the southeastern United States, we examine how diazotrophic symbiont communities vary across host plant phylogeny, habitat context, and geographic origin. We find that host phylogeny is the primary determinant of symbiont composition, outweighing effects of fine-scale taxonomic identity. Symbiont associations are therefore structured mainly at deeper phylogenetic levels, consistent with phylogenetically constrained, or “fuzzy,” host specificity. Likewise, nodule community diversity—potentially reflecting variation in host control over infection—differs primarily among higher-level clades rather than among closely related taxa. Habitat context also shapes nodule communities, but its influence is secondary and most evident in undisturbed environments. As well, nonnative legumes harbor distinct symbiont assemblages despite occupying similar habitats, whereas distantly related legume clades share symbionts across habitats, highlighting interactions among phylogeny, ecology, and geographic history. Overall, our results show that host phylogeny exerts the strongest influence on nodule microbial communities, likely reflecting evolutionary divergence in symbiotic function across major host lineages.

Many agricultural soils are deficient in key macronutrients needed for healthy plant development. Relying on highly water-soluble commercial fertilizers for long durations can be costly and environmentally harmful. This study investigates a phosphorus-loaded Mg/Fe layered double hydroxide (LDH) dispersed on Douglas fir biochar (Mg/Fe-LDH biochar) as a controlled-release fertilizer and evaluates its impact on bush bean ( Phaseolus vulgaris L .) growth. Emphasizing sustainability, the work integrates controlled-release fertilizers, biochar, and LDH modification to enhance nutrient use efficiency and mitigate environmental runoff. Mg/Fe-LDH was directly synthesized on biochar via a co-precipitation approach, loaded the composite with phosphate by anion exchange, and characterized the material using elemental analysis, N ₂ Brunauer-Emmett-Teller (BET) determinations surface area analysis, and x-ray photoelectron spectroscopy to confirm successful LDH modification on Douglas fir biochar, and high surface area with accessible active sites. The synthesis yielded a stable P-Mg/Fe-LDH biochar with enhanced dispersibility and phosphate-buffering capacity, enabling controlled-release fertilization. In greenhouse experiments, bush beans grown with the P-Mg/Fe-LDH biochar exhibited improved growth metrics, including increased yield (beans fresh weight of 31.7 g), biomass (plant dry weight of 6.3 g), plant height (32.8 cm), and improved nutrient uptakes (1.88 mg (P) g ^-1 ) at 100.88 kg (P ₂ O ₅ ) ha ^-1 compared with unfertilized controls and conventional P fertilizers, indicating efficient, controlled-release phosphate delivery and sustained nutrient availability. The results demonstrate that integrating LDH-modified biochar can enhance P uptake and plant growth while reducing leaching losses. Overall, this study highlights the strategic significance of combining biochar, layered double hydroxides, and controlled-release formulations to advance sustainable nutrient management and improve crop performance in agroecosystems. The findings offer a promising pathway for environmentally conscious fertilizer design and soil amendment strategies that align with global goals for resource efficiency and food security. Graphical

Dalbergia (Leguminosae) is a pantropical genus that serves as an ideal system for studying pantropical biogeography and diversification. However, limited taxon sampling and molecular data have hindered the resolution of intrageneric relationships and understanding of its evolutionary history. Here, we reconstruct a densely sampled phylogeny of Dalbergia based on hybrid capture of 89 low‐copy nuclear loci, including 98 species spanning all major biogeographic regions and most previously recognized taxonomic clades. Phylogenetic analyses using concatenated and coalescent approaches support the monophyly of Dalbergia and consistently identify six major clades, including a distinct and newly identified Africa‐Madagascar lineage (clade E) with potential taxonomic significance. Our results propose merging two of its sections, sect. Dalbergia and sect. Selenolobium , and expanding sect. Ecastaphyllum to include some African species. Topological incongruences observed between concatenated and coalescent trees, together with the high gene tree conflict at certain nodes, are consistent with localized effects of hybridization and/or incomplete lineage sorting. Divergence dating and model‐based biogeographic analyses support a Neotropical origin of Dalbergia in the middle Eocene (~39 Ma), followed by repeated long‐distance dispersal events from Africa to Asia, Neotropics, and Madagascar, and back into the Neotropics. An early diversification burst in Africa during the late Oligocene–early Miocene (~27–18 Ma) preceded the genus′s intercontinental expansion. Diversification rates were high across all four regions, with Madagascar showing the highest speciation rate. These macroevolutionary dynamics coincided with Eocene–Miocene climatic changes and habit shifts. This study provides a robust phylogenetic framework for Dalbergia , refines its temporal and biogeographic history, and illustrates how dispersal, climatic change, and lineage‐specific diversification have interacted to generate pantropical biodiversity.

Hybridization is a prevalent evolutionary process in plants and a potential driver of new species formation. Even though hybridization has been studied in many natural systems, the study of genomic signatures of past events of reticulation and hybridization was accelerated by the wide availability of high‐throughput sequencing methods. Similar to other species‐rich groups of plants, the occurrence of natural hybrids is widely documented in the sunflower family (Asteraceae), yet few studies have focused on ancient hybridization (that occurring between ancestral lineages), mainly due to the lack of comparative genomic data. With the recent increase in phylogenomic studies in Asteraceae made possible by family wide target‐capture probes, it has become clear that gene tree incongruence is widespread across the clade, likely reflective of complex speciation events in the past. One clade where natural hybridization is thought to be especially high is the subtribe Lychnophorinae of tribe Vernonieae, with ca. 145 species mostly endemic to the central Brazilian savannas and campos rupestres . Here, we for the first time propose a phylogenomic hypothesis for this subtribe and study the impacts of hybridization on its diversification. We newly sequenced 49 species (73 in total) with the Compositae1061 probe set and generated six phylogenetic trees based on different data treatments and using both multispecies pseudocoalescence and concatenated maximum likelihood analysis. To test whether a phylogenetic network, indicative of past and current reticulation events, better fits the data, we used a staggered sampling approach with SNaQ to overcome computational limitations. While overall our analyses confirm relationships recovered in previous analyses, we found five main foci of phylogenetic incongruence and hybridization across the tree. We explore how these reticulation events change our interpretation of some lineages in the subtribe, such as the Piptolepis pabstii/P. rosmarinifolia and the Lychnophora penninervia complex, further discussing potential taxonomic changes.

Species are the fundamental analytical units of evolutionary processes; thus evidence-based species delimitation is a crucial step for understanding species radiations. However, the task of delimiting species is particularly challenging in the context of a syngameon—a group of distinct, but closely related species that have incomplete reproductive isolation and frequently hybridize in nature. This problem is further exacerbated by the presence of cryptic species—species that are phenotypically distinct, though difficult to distinguish with gross morphology alone. Heuchera subsect. Heuchera comprises both clear and cryptic species within a syngameon that has seen study from morphological, experimental, and phylogenetic aspects. This group has long been recognized for its taxonomic complexity, namely two recognized hybrid zones with extreme morphological variation and persistent non-monophyly among parental populations. Here, we reassess species limits within Heuchera subsect. Heuchera , focusing on the hybrid complex between H. americana and H. richardsonii and adjacent H. americana populations. We use a multipronged approach with deep population-level sampling to 1) assess the genetic structure of 655 individuals across the geographic range of the H. americana group to identify genetic lineages and 2) assess the phenotypic diagnosability of these lineages. Despite extensive admixture and gene tree conflict, we find multiple cohesive lineages with diagnosable phenotypes. We recognize five species and three varieties within the H. americana group, one new and four resurrected. Our results demonstrate that even highly reticulate syngameons can be partitioned into meaningful taxonomic units with multiple lines of evidence.

The frequency of hybridization differs strongly across different taxa and areas of Earth, yet the environmental drivers of this variation remain poorly understood. Areas strongly influenced by Pleistocene glaciation have long been hypothesized to be rich in hybrid taxa through forcing previously allopatric species into contact and interrupting ecological barriers. Here, we revisit naturally occurring Heuchera americana × H. richardsonii in North America using deep population level sampling of nuclear and plastid genomes to re-evaluate the long-standing hypothesis that Pleistocene climate dynamics promoted hybridization in this system. An extensive population-level sampling scheme involving nuclear and plastid genetic data (729 individuals across 455 populations) reveals a more complex hybridization history than implied by previous representative phylogenetic sampling. Our analyses identify multiple independent chloroplast capture events since diversification within this complex, rather than a previously hypothesized single ancestral event. Strong geographic structure at higher-level plastid clades but little structure within subclades, together with the rarity of ancestral plastid genomes, suggests a unidirectional inheritance of the introgressed plastid genome rather than a simple geographic distribution of plastid haplotypes. Ancestral niche projections indicate that Pleistocene glacial maxima created a shared narrow eastern refugium east of the Mississippi River, enabling localized hybridization, while western refugia were larger and taxa probably allopatric. This contrast led to an east-west gradient in cytonuclear discord. Our results corroborate and refine a 90-year-old hypothesis of climate-mediated hybridization by demonstrating that historical gene flow was spatially restricted but hybrid genotypes now command a larger geographic area. Our study highlights the importance of deep population-level sampling for accurately reconstructing hybridization histories and underscores the catalytic role of climatic instability in general as a driver of hybridization in Heuchera , and likely many other taxa across the tree of life.

Little is known about environmental drivers of opportunities for hybridization, but its phylogenetic distribution across species and areas is heterogeneous, suggesting that ecological traits may play an important role in concert with postzygotic isolation. Because plant-pollinator interactions are responsible for gene flow in most plant species, differences in the mosaic landscape of plant-pollinator interactions could explain why some plants are particularly prone to hybridization. Prezygotic isolation is mediated by sometimes complex pollen presentation; conversely, conserved pollination strategies would lead to evolutionary constraints on pollinator assemblage divergence in the speciation process and therefore predict higher opportunities for gene flow, although this hypothesis has yet to be tested. The plant taxonomic tribe Heuchereae (Saxifragaceae) is a well-characterized system for pollinator interactions and particularly for floral scent, the primary pollinator attractant in the group. Floral volatile organic compounds (VOCs) in this clade are hypervariable at the population level and are thought to be responsible for pollination selectivity, leading to divergent pollinator assemblages. Observing a contrast of hybridizing and non-hybridizing species, the levels of attractant divergence may therefore predict levels of hybridization. We investigated pollination biology in the plant genus Heuchera , notable for frequent interspecific gene flow compared to tribal relatives, asking whether high rates of hybridization may be associated with low interspecific divergence of VOCs and the pollinator assemblages they shape, using as our system the hybrid zone between H. americana var. americana and H. richardsonii in the midwestern USA. We optimized a closed-space collection and GC-MS (gas chromatography-mass spectrometry) protocol to characterize VOCs in Heuchera flowers. To identify floral visitation and effective pollinators we conducted pollination observations at 40 Heuchera populations over the span of two field seasons. GC-MS data from 89 Heuchera specimens representing 69 populations suggests that classes of VOCs, and to a large extent individual compounds, are shared within the hybrid complex while other Heuchera that are not thought to hybridize with these species have distinct species-specific compounds. Pollination observations and metabarcoding of pollinator pollen loads confirm shared effective pollinators in the hybrid zone and between adjacent parental populations. Attractant and visitation data considered together suggest that conservatism of pollinator interactions may be a typical feature associated with frequent hybridizers, perhaps arising from developmental or biochemical constraints on prezygotic isolation, and more broadly that the macroevolution of isolation mechanisms may be predictive of natural hybridization rate.

The origin and phylogenetic distribution of symbiotic associations between nodulating angiosperms and nitrogen-fixing bacteria have long intrigued biologists. Recent comparative evolutionary analyses have yielded alternative hypotheses: a multistep pathway of independent gains and losses of root nodule symbiosis vs. a single gain followed by numerous losses. A detailed reconstruction of the history of genes involved in signaling between nitrogen-fixing bacteria and potential hosts, particularly lipo-chitooligosaccharide (LCO) signaling, is needed to distinguish between these hypotheses. LCO recognition by plants involves the Nod Factor Perception ( NFP ) gene family; in the legume model Medicago truncatula (Fabales), MtNFP is essential for establishing rhizobial symbiosis. Here, we document convergent evolution of NFP , indicating multiple origins of LCO-driven symbiosis. In contrast to previous models that explain the recruitment of NFP via a single duplication in the ancestor of the nitrogen-fixing clade, our phylogenomic and synteny results suggest this duplication does not span the entire clade. Tandem duplication in a common ancestor of Cucurbitales and Rosales resulted in the NFP1 and NFP2 groups. In contrast, the phylogenetically closest paralog of MtNFP is MtLYR1 , located on a different chromosome within a large syntenic block. All available data indicate that a large-scale duplication resulted in MtNFP and MtLYR1 , likely corresponding to a whole-genome duplication in an ancestor of subfamily Papilionoideae of Fabaceae. We show that MtNFP and the NFP2 -like group are not orthologous, indicating multiple independent gains of NFP -based LCO signaling. This molecular convergence provides a possible mechanism for multiple gains of root nodule symbiosis across the nitrogen-fixing clade.

How species boundaries are maintained among sympatric closely related species experiencing gene flow is a puzzling question in evolutionary biology. Although introgression is commonly documented, the dynamics and gene function of introgression have rarely been explored to probe why frequent introgression does not necessarily destroy species boundaries in sympatry. In this study, we employ whole‐genome resequencing data to examine introgression among five closely related species of Roscoea that coexist in a ‘sky island’ with seventeen distinct morphological traits. Our findings reveal that introgression has led to the phylogenomic discordance between nuclear and chloroplast genomes among these morphologically distinct species. Additionally, introgression is predominantly asymmetrical in both intensity and gene function, particularly concerning recombination. Notably, the majority of gene functions associated with introgressed loci are unrelated to reproductive processes. Our results suggest that closely related species with incomplete allele assortment can coexist despite theoretical predictions, highlighting the semipermeable nature of species boundaries as reproductive isolation develops. This provides a critical conceptual framework for understanding the interplay between introgression and species persistence. Our finding offers insights into how related sympatric species boundaries can be maintained in the face of frequent asymmetrical gene introgression.