Grace A. Burgin

The large size and high repetitive content of many plant genomes have hindered elucidation of how the fundamental processes of evolution give rise to speciation. Here we present chromosome-level assemblies of 6 gigabase genomes for four closely related Phlox wildflower species. We generate extensive population genetic data for the three well-studied annual species P. drummondii, P. cuspidata, and P. roemeriana, including structural variation from whole-genome long-read resequencing data. The whole-genome assemblies reveal extensive differences in amount and distribution of genetic variation within and between species, reflective of differences in life-history strategies, mating systems, and edaphic specialization. The population genetic data exposes a history of widespread and consistent gene flow between all three annual Phlox species throughout their divergence and speciation. Our unmasking of structural variants and repetitive elements exposes rich and dynamic forms of genetic variation that show strong phylogenetic signal and pervasive patterns of gene flow. Our results lay a foundation for untangling the highly complex genomes of plants to make advancements in our understanding of the processes of speciation and divergence.

Self-incompatibility (SI) describes a widespread collection of genetic mechanisms in flowering plants used to specifically recognize and reject self-pollen. These mechanisms are fundamental to plant sexual reproduction and offer valuable insight into the molecular basis of cell–cell communication and self-recognition more broadly. Here, we leverage an independent evolution of SI in the lineage containing Phlox (Polemoniaceae) to identify the gene causing self-pollen recognition which we name Phlox drummondii Pistil Identity Receptor Kinase (PdPIRK) . Recognition of self-pollen associates with a single genomic region containing the Phlox S -locus. We generate predictions regarding how S -loci must function and evolve to identify a single candidate gene within this S -associated region. This gene, PdPIRK , is highly and specifically expressed in the pistil and has exceptionally high polymorphism maintained by negative frequency-dependent selection, two hallmarks of self-pollen recognition genes. Functional validation with gene silencing confirms that PdPIRK is necessary for self-incompatibility, and we further demonstrate allele-specific activity, confirming its role in self-pollen recognition per se. PdPIRK encodes a G-type lectin receptor-like kinase, which is a member of the same gene family as SRK , the gene controlling self-pollen recognition in the distantly related Brassicaceae. Our findings suggest the presence of genetic constraints or paths of least resistance governing how S -loci evolve and add to our understanding of the diverse molecular mechanisms through which organisms achieve self-recognition.

Root exudation, the export of soluble carbon compounds from living plant roots into soil, is an important pathway for soil carbon formation, but high rates of exudation can also induce rapid soil organic matter decomposition – a phenomenon known as the priming effect. Long-term soil warming associated with climate change could alter exudation rates and impact soil microbes by changing soil carbon chemistry. We hypothesized that warming-induced changes to exudation rate combined with direct effects of long-term warming on soil microbial communities would regulate the microbial priming effect. We tested this hypothesis with an artificial root exudate experiment using intact soil cores from a long-term soil warming experiment in a temperate forest. We found that chronic soil warming did not alter soil carbon formation from exudates, but did reduce the exudate-induced priming effect; exudation caused greater soil carbon loss in unwarmed than warmed soils. We used DNA stable isotope probing with 16S ribosomal RNA gene and shotgun metagenomic sequencing to determine whether long-term warming affected which microbes consume 13carbon-labeled artificial exudates. We found significant differences in bacterial community composition and relative gene abundances of 13carbon-enriched compared to natural abundance DNA. Both soil bacterial community composition and specific enzyme-coding gene families were strongly correlated with soil carbon priming in unwarmed treatments, but these effects were absent in warmed treatments. Our results suggest that the root exudate-induced priming effect is mediated by microbial biomass, community structure, and gene abundance, and that chronic warming reduces the priming effect by altering these microbial variables.

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.

Self-incompatibility (SI) describes a widespread collection of genetic mechanisms in flowering plants used to specifically recognize and reject self-pollen. These mechanisms are fundamental to plant sexual reproduction and offer valuable insight into the molecular basis of cell-cell communication and self-recognition more broadly. Here, we leverage an independent evolution of SI in the lineage containing Phlox (Polemoniaceae) to characterize a novel gene causing self-pollen recognition which we name Phlox drummondii Pistil Identity Receptor Kinase ( PdPIRK ). Recognition of self-pollen associates with a single genomic region containing the Phlox S -locus. We generate predictions regarding how S -loci must function and evolve to identify a single candidate gene within this S -associated region. This gene, PdPIRK , is highly and specifically expressed in the pistil and has exceptionally high polymorphism maintained by negative frequency dependent selection, two hallmarks of self-pollen recognition genes. Functional validation with gene silencing confirms that PdPIRK is necessary for self-incompatibility, and we further demonstrate allele specific activity, confirming its role in self-pollen recognition per se. PdPIRK encodes a G-type lectin receptor-like kinase, which is a member of the same gene family as SRK , the gene controlling self-pollen recognition in the distantly related Brassicaceae. Our findings suggest the presence of genetic constraints or paths of least resistance governing how S -loci evolve and add to our understanding of the diverse molecular mechanisms through which organisms achieve self-recognition.

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 polygenic or simple genetic basis and if variation in compatibility localizes to the genomic locus causing self-pollen recognition (the “ S -locus”). Phlox drummondii (Polemoniaceae) has been a model system for exploring mating system evolution and expresses extensive range-wide variation in the SI response. 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 variation in SI, 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. Through these crossing experiments, we demonstrate that P. drummondii has a sporophytic SI system, suggesting that an independent evolution of SI occurred in the lineage containing Phlox . 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 three 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 underlying genetic architecture upon which selection could act to drive this frequent and evolutionarily significant transition.

Characterizing the mechanisms of reproductive isolation (RI) between lineages is key to determining how new species are formed and maintained. In flowering plants, interactions between the reproductive organs of the flower –the pollen and the pistil– serve as the last barrier to reproduction before fertilization. As such, these pollen-pistil interactions are both complex and important for determining a suitable mate. Here, we test whether differences in style length (a part of the pistil) generate a postmating prezygotic mechanical barrier between five species of perennial Phlox wildflowers with geographically overlapping distributions. We perform controlled pairwise reciprocal crosses between three species with long styles and two species with short styles to assess crossing success (seed set). We find that heterospecific seed set is broadly reduced compared to conspecific cross success and reveal a striking asymmetry in heterospecific crosses between species with different style lengths. To determine the mechanism underlying this asymmetric reproductive isolating barrier we assess pollen tube growth in vivo and in vitro. We demonstrate that pollen tubes of short-styled species do not grow long enough to reach the ovaries of long-styled species. We find that short-styled species also have smaller pollen and that both within and between species pollen diameter is highly correlated with pollen tube length. Our results support the hypothesis that the small pollen of short-styled species lacks resources to grow pollen tubes long enough to access the ovaries of the long-styled species, resulting in an asymmetrical, mechanical barrier to reproduction. Such reproductive isolating mechanisms, combined with additional pollen-pistil incompatibilities, may be particularly important for closely related species in geographic proximity that share pollinators.

In flowering plants, pollen-pistil interactions can serve as an important barrier to reproduction between species. As the last barrier to reproduction before fertilization, interactions between these reproductive organs are both complex and important for determining a suitable mate. Here, we test whether differences in style length generate a post-mating prezygotic mechanical barrier between five species of perennial Phlox wildflowers with geographically overlapping distributions. We perform controlled pairwise reciprocal crosses between three species with long styles and two species with short styles to assess crossing success (seed set). We find that heterospecific seed set is broadly reduced compared to conspecific cross success and reveal a striking asymmetry in heterospecific crosses between species with different style lengths. To determine the mechanism underlying this asymmetric reproductive isolating barrier we assess pollen tube growth in vitro and in vivo . We demonstrate that pollen tubes of short-styled species do not grow long enough to reach the ovaries of long-styled species. We find that short-styled species also have smaller pollen and that both within and between species pollen diameter is highly correlated with pollen tube length. Our results support the hypothesis that the small pollen of short-styled species lacks resources to grow pollen tubes long enough to access the ovaries of the long-styled species, resulting in an asymmetrical, mechanical barrier to reproduction. Such mechanisms, combined with additional pollen-pistil incompatibilities, may be particularly important for closely related species in geographic proximity that share pollinators.

Premise: A central goal of pollination biology is to connect plants with the identity of their pollinator(s). While predictions based on floral syndrome traits are extremely useful, direct observation can reveal further details of a species' pollination biology. The wildflower Phlox drummondii has a floral syndrome consistent with pollination by Lepidoptera. We tested this prediction using empirical data.Methods: We observed each step of pollination in P. drummondii. First, we observed 55.5 h of floral visitation across the species range. We used temporal pollinator exclusion to determine the contribution of diurnal and nocturnal pollination to reproductive output. We then quantified P. drummondii pollen transfer by the dominant floral visitor, Battus philenor. Finally, we tested the effect of B. philenor visitation on P. drummondii reproduction by quantifying fruit set following single pollinator visits.Results: Battus philenor is the primary pollinator of P. drummondii. Pollination is largely diurnal, and we observed a variety of lepidopteran visitors during the diurnal period. However, B. philenor was the most frequent visitor, representing 88.5% of all observed visits. Our results show that B. philenor is an extremely common visitor and also an effective pollinator by demonstrating that individuals transfer pollen between flowers and that a single visit can elicit fruit set.Conclusions: Our data are consistent with the prediction of lepidopteran pollination and further reveal a single butterfly species, B. philenor, as the primary pollinator. Our study demonstrates the importance of empirical pollinator observations, adds to our understanding of pollination mechanics, and offers a specific case study of butterfly pollination.