Genetic underpinnings of arthropod community distributions in Populus trichocarpa.

Community genetics seeks to understand the mechanisms by which natural genetic variation in heritable host phenotypes can encompass assemblages of organisms such as bacteria, fungi, and many animals including arthropods. Prior studies that focused on plant genotypes have been unable to identify genes controlling community composition, a necessary step to predict ecosystem structure and function as underlying genes shift within plant populations. We surveyed arthropods within an association population of Populus trichocarpa in three common gardens to discover plant genes that contributed to arthropod community composition. We analyzed our surveys with traditional single-trait genome-wide association analysis (GWAS), multitrait GWAS, and functional networks built from a diverse set of plant phenotypes. Plant genotype was influential in structuring arthropod community composition among several garden sites. Candidate genes important for higher level organization of arthropod communities had broadly applicable functions, such as terpenoid biosynthesis and production of dsRNA binding proteins and protein kinases, which may be capable of targeting multiple arthropod species. We have demonstrated the ability to detect, in an uncontrolled environment, individual genes that are associated with the community assemblage of arthropods on a host plant, further enhancing our understanding of genetic mechanisms that impact ecosystem structure.

Evolution of a ZW sex chromosome system in willows.

Transitions in the heterogamety of sex chromosomes (e.g., XY to ZW or vice versa) fundamentally alter the genetic basis of sex determination, however the details of these changes have been studied in only a few cases. In an XY to ZW transition, the X is likely to give rise to the W because they both carry feminizing genes and the X is expected to harbour less genetic load than the Y. Here, using a new reference genome for Salix exigua, we trace the X, Y, Z, and W sex determination regions during the homologous transition from an XY system to a ZW system in willow (Salix). We show that both the W and the Z arose from the Y chromosome. We find that the new Z chromosome shares multiple homologous putative masculinizing factors with the ancestral Y, whereas the new W lost these masculinizing factors and gained feminizing factors. The origination of both the W and Z from the Y was permitted by an unexpectedly low genetic load on the Y and this indicates that the origins of sex chromosomes during homologous transitions may be more flexible than previously considered.

Phylogenomics reveals patterns ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives.

Despite the economic, ecological, and scientific importance of the genera Salix L. (willows) and Populus L. (poplars, cottonwoods, and aspens) Salicaceae, we know little about the sources of differences in species diversity between the genera and of the phylogenetic conflict that often confounds estimating phylogenetic trees. Salix subgenera and sections, in particular, have been difficult to classify, with one recent attempt termed a 'spectacular failure' due to a speculated radiation of the subgenera Vetrix and Chamaetia. Here we use targeted sequence capture to understand the evolutionary history of this portion of the Salicaceae plant family. Our phylogenetic hypothesis was based on 787 gene regions and identified extensive phylogenetic conflict among genes. Our analysis supported some previously described subgeneric relationships and confirmed polyphyly of others. Using an fbranch analysis we identified several cases of hybridization in deep branches of the phylogeny, which likely contributed to discordance among gene trees. In addition, we identified a rapid increase in diversification rate near the origination of the Vetrix-Chamaetia clade in Salix. This region of the tree coincided with several nodes that lacked strong statistical support, indicating a possible increase in incomplete lineage sorting due to rapid diversification. The extraordinary level of both recent and ancient hybridization in both Salix and Populus have played important roles in the diversification and diversity in these two genera.

Structural variation of a sex-linked region confers monoecy and implicates GATA15 as a master regulator of sex in Salix purpurea.

The Salicaceae, including Populus and Salix, are dioecious perennials that utilize different sex determination systems. This family provides a useful system to better understand the evolution of dioecy and sex chromosomes. Here, a rare monoecious genotype of Salix purpurea, 94003, was self- and cross-pollinated and progeny sex ratios were used to test hypotheses on possible mechanisms of sex determination. To delimit genomic regions associated with monoecious expression, the 94003 genome sequence was assembled and DNA- and RNA-Seq of progeny inflorescences was performed. Based on alignments of progeny shotgun DNA sequences to the haplotype-resolved monoecious 94003 genome assembly and reference male and female genomes, a 1.15 Mb sex-linked region on Chr15W was confirmed to be absent in monecious plants. Inheritance of this structural variation is responsible for the loss of a male-suppressing function in what would otherwise be genetic females (ZW), resulting in monoecy (ZWH or WWH ), or lethality, if homozygous (WH WH ). We present a refined, two-gene sex determination model for Salix purpurea, mediated by ARR17 and GATA15 that is different from the single-gene ARR17-mediated system in the related genus Populus.

High-resolution mapping reveals hotspots and sex-biased recombination in Populus trichocarpa.

Fine-scale meiotic recombination is fundamental to the outcome of natural and artificial selection. Here, dense genetic mapping and haplotype reconstruction were used to estimate recombination for a full factorial Populus trichocarpa cross of 7 males and 7 females. Genomes of the resulting 49 full-sib families (N = 829 offspring) were resequenced, and high-fidelity biallelic SNP/INDELs and pedigree information were used to ascertain allelic phase and impute progeny genotypes to recover gametic haplotypes. The 14 parental genetic maps contained 1,820 SNP/INDELs on average that covered 376.7 Mb of physical length across 19 chromosomes. Comparison of parental and progeny haplotypes allowed fine-scale demarcation of cross-over regions, where 38,846 cross-over events in 1,658 gametes were observed. Cross-over events were positively associated with gene density and negatively associated with GC content and long-terminal repeats. One of the most striking findings was higher rates of cross-overs in males in 8 out of 19 chromosomes. Regions with elevated male cross-over rates had lower gene density and GC content than windows showing no sex bias. High-resolution analysis identified 67 candidate cross-over hotspots spread throughout the genome. DNA sequence motifs enriched in these regions showed striking similarity to those of maize, Arabidopsis, and wheat. These findings, and recombination estimates, will be useful for ongoing efforts to accelerate domestication of this and other biomass feedstocks, as well as future studies investigating broader questions related to evolutionary history, perennial development, phenology, wood formation, vegetative propagation, and dioecy that cannot be studied using annual plant model systems.

Repeated turnovers keep sex chromosomes young in willows.

BACKGROUND: Salicaceae species have diverse sex determination systems and frequent sex chromosome turnovers. However, compared with poplars, the diversity of sex determination in willows is poorly understood, and little is known about the evolutionary forces driving their turnover. Here, we characterized the sex determination in two Salix species, S. chaenomeloides and S. arbutifolia, which have an XY system on chromosome 7 and 15, respectively. RESULTS: Based on the assemblies of their sex determination regions, we found that the sex determination mechanism of willows may have underlying similarities with poplars, both involving intact and/or partial homologs of a type A cytokinin response regulator (RR) gene. Comparative analyses suggested that at least two sex turnover events have occurred in Salix, one preserving the ancestral pattern of male heterogamety, and the other changing heterogametic sex from XY to ZW, which could be partly explained by the "deleterious mutation load" and "sexually antagonistic selection" theoretical models. We hypothesize that these repeated turnovers keep sex chromosomes of willow species in a perpetually young state, leading to limited degeneration. CONCLUSIONS: Our findings further improve the evolutionary trajectory of sex chromosomes in Salicaceae species, explore the evolutionary forces driving the repeated turnovers of their sex chromosomes, and provide a valuable reference for the study of sex chromosomes in other species.

Abundance of Major Cell Wall Components in Natural Variants and Pedigrees of Populus trichocarpa.

The rapid analysis of biopolymers including lignin and sugars in lignocellulosic biomass cell walls is essential for the analysis of the large sample populations needed for identifying heritable genetic variation in biomass feedstocks for biofuels and bioproducts. In this study, we reported the analysis of cell wall lignin content, syringyl/guaiacyl (S/G) ratio, as well as glucose and xylose content by high-throughput pyrolysis-molecular beam mass spectrometry (py-MBMS) for >3,600 samples derived from hundreds of accessions of Populus trichocarpa from natural populations, as well as pedigrees constructed from 14 parents (7 × 7). Partial Least Squares (PLS) regression models were built from the samples of known sugar composition previously determined by hydrolysis followed by nuclear magnetic resonance (NMR) analysis. Key spectral features positively correlated with glucose content consisted of m/z 126, 98, and 69, among others, deriving from pyrolyzates such as hydroxymethylfurfural, maltol, and other sugar-derived species. Xylose content positively correlated primarily with many lignin-derived ions and to a lesser degree with m/z 114, deriving from a lactone produced from xylose pyrolysis. Models were capable of predicting glucose and xylose contents with an average error of less than 4%, and accuracy was significantly improved over previously used methods. The differences in the models constructed from the two sample sets varied in training sample number, but the genetic and compositional uniformity of the pedigree set could be a potential driver in the slightly better performance of that model in comparison with the natural variants. Broad-sense heritability of glucose and xylose composition using these data was 0.32 and 0.34, respectively. In summary, we have demonstrated the use of a single high-throughput method to predict sugar and lignin composition in thousands of poplar samples to estimate the heritability and phenotypic plasticity of traits necessary to develop optimized feedstocks for bioenergy applications.

Integrative genomics reveals paths to sex dimorphism in Salix purpurea L.

Sex dimorphism and gene expression were studied in developing catkins in 159 F2 individuals from the bioenergy crop Salix purpurea, and potential mechanisms and pathways for regulating sex development were explored. Differential expression, eQTL, bisulfite sequencing, and network analysis were used to characterize sex dimorphism, detect candidate master regulator genes, and identify pathways through which the sex determination region (SDR) may mediate sex dimorphism. Eleven genes are presented as candidates for master regulators of sex, supported by gene expression and network analyses. These include genes putatively involved in hormone signaling, epigenetic modification, and regulation of transcription. eQTL analysis revealed a suite of transcription factors and genes involved in secondary metabolism and floral development that were predicted to be under direct control of the sex determination region. Furthermore, data from bisulfite sequencing and small RNA sequencing revealed strong differences in expression between males and females that would implicate both of these processes in sex dimorphism pathways. These data indicate that the mechanism of sex determination in Salix purpurea is likely different from that observed in the related genus Populus. This further demonstrates the dynamic nature of SDRs in plants, which involves a multitude of mechanisms of sex determination and a high rate of turnover.

Sexual dimorphism in the dioecious willow Salix purpurea.

PREMISE: The evolution of sex chromosomes is driven by sexual dimorphism, yet it can be challenging to document sexually dimorphic traits in dioecious plant species. At the genetic level, sexual dimorphism can be identified through sequence variation between females and males associated with sexually antagonistic traits and different fitness optima. This study aims to examine sexual dimorphism for 26 traits in three populations of Salix purpurea (a diversity panel and F1 and F2 populations) and determine the effect of the traits on biomass yield, a key trait in Salix bioenergy crops across multiple years, locations, and under manipulated growth conditions. METHODS: Sexual dimorphism was evaluated for morphological, phenological, physiological, and wood composition traits in a diversity panel of unrelated S. purpurea accessions and in full-sib F1 and F2 families produced through controlled cross pollinations and grown in replicated field trials. RESULTS: We observed sexual dimorphism in the timing of development for several traits that were highly predictive of biomass yield across three populations of S. purpurea. Across all populations and years surveyed, males had significantly shallower branching angle. Male plants highly predictive of biomass yield across three populations of S. purpurea also accumulated more nitrogen under fertilizer amendment as measured by SPAD in the diversity panel and had greater susceptibility to the rust fungus Melampsora americana in the F2 family. Allometric modelling of biomass yield showed an effect of sex and of location on the interaction between yield and stem height. CONCLUSIONS: These results provide evidence of sexual dimorphism for certain traits in S. purpurea that may be involved in sex chromosome evolution.

Characterization of Salix nigra floral insect community and activity of three native Andrena bees.

Salix nigra (black willow) is a widespread tree that hosts many species of polylectic hymenopterans and oligolectic bees of the genus Andrena. The early flowering of S. nigra makes it an important nutritive resource for arthropods emerging from hibernation. However, since S. nigra is dioecious, not all insect visits will lead to successful pollination. Using both visual observation and pan-trapping, we characterized the community of arthropods that visited S. nigra flowers and assessed differences among male and female trees as well as the chemical and visual drivers that influenced community composition across 3 years. We found that male trees consistently supported higher diversity of insects than female trees and only three insect species, all Andrena spp., consistently visited both sexes. Additionally, Andrena nigrae, which was the only insect that occurred more on female than male flowers, correlated strongly to volatile cues. This suggests that cross-pollinators cue into specific aspects of floral scent, but diversity of floral visitors is driven strongly by visual cues of yellow male pollen. Through time, the floral activity of two Andrena species remained stable, but A. nigrae visited less in 2017 when flowers bloomed earlier than other years. When native bee emergence does not synchronize with bloom, activity appears to be diminished which could threaten species that subsist on a single host. Despite the community diversity of S. nigra flowers, its productivity depends on a small fraction of species that are not threatened by competition, but rather rapidly changing conditions that lead to host-insect asynchrony.

Accurate determination of genotypic variance of cell wall characteristics of a Populus trichocarpa pedigree using high-throughput pyrolysis-molecular beam mass spectrometry.

BACKGROUND: Pyrolysis-molecular beam mass spectrometry (py-MBMS) analysis of a pedigree of Populus trichocarpa was performed to study the phenotypic plasticity and heritability of lignin content and lignin monomer composition. Instrumental and microspatial environmental variability were observed in the spectral features and corrected to reveal underlying genetic variance of biomass composition. RESULTS: Lignin-derived ions (including m/z 124, 154, 168, 194, 210 and others) were highly impacted by microspatial environmental variation which demonstrates phenotypic plasticity of lignin composition in Populus trichocarpa biomass. Broad-sense heritability of lignin composition after correcting for microspatial and instrumental variation was determined to be H2 = 0.56 based on py-MBMS ions known to derive from lignin. Heritability of lignin monomeric syringyl/guaiacyl ratio (S/G) was H2 = 0.81. Broad-sense heritability was also high (up to H2 = 0.79) for ions derived from other components of the biomass including phenolics (e.g., salicylates) and C5 sugars (e.g., xylose). Lignin and phenolic ion abundances were primarily driven by maternal effects, and paternal effects were either similar or stronger for the most heritable carbohydrate-derived ions. CONCLUSIONS: We have shown that many biopolymer-derived ions from py-MBMS show substantial phenotypic plasticity in response to microenvironmental variation in plantations. Nevertheless, broad-sense heritability for biomass composition can be quite high after correcting for spatial environmental variation. This work outlines the importance in accounting for instrumental and microspatial environmental variation in biomass composition data for applications in heritability measurements and genomic selection for breeding poplar for renewable fuels and materials.

Sex determination through X-Y heterogamety in Salix nigra.

The development of non-recombining sex chromosomes has radical effects on the evolution of discrete sexes and sexual dimorphism. Although dioecy is rare in plants, sex chromosomes have evolved repeatedly throughout the diversification of angiosperms, and many of these sex chromosomes are relatively young compared to those found in vertebrates. In this study, we designed and used a sequence capture array to identify a novel sex-linked region (SLR) in Salix nigra, a basal species in the willow clade, and demonstrated that this species has XY heterogamety. We did not detect any genetic overlap with the previously characterized ZW SLRs in willows, which map to a different chromosome. The S. nigra SLR is characterized by strong recombination suppression across a 2 MB region and an excess of low-frequency alleles, resulting in a low Tajima's D compared to the remainder of the genome. We speculate that either a recent bottleneck in population size or factors related to positive or background selection generated this differential pattern of Tajima's D on the X and autosomes. This discovery provides insights into factors that may influence the evolution of sex chromosomes in plants and contributes to a large number of recent observations that underscore their dynamic nature.

A General Model to Explain Repeated Turnovers of Sex Determination in the Salicaceae.

Dioecy, the presence of separate sexes on distinct individuals, has evolved repeatedly in multiple plant lineages. However, the specific mechanisms by which sex systems evolve and their commonalities among plant species remain poorly understood. With both XY and ZW sex systems, the family Salicaceae provides a system to uncover the evolutionary forces driving sex chromosome turnovers. In this study, we performed a genome-wide association study to characterize sex determination in two Populus species, P. euphratica and P. alba. Our results reveal an XY system of sex determination on chromosome 14 of P. euphratica, and a ZW system on chromosome 19 of P. alba. We further assembled the corresponding sex-determination regions, and found that their sex chromosome turnovers may be driven by the repeated translocations of a Helitron-like transposon. During the translocation, this factor may have captured partial or intact sequences that are orthologous to a type-A cytokinin response regulator gene. Based on results from this and other recently published studies, we hypothesize that this gene may act as a master regulator of sex determination for the entire family. We propose a general model to explain how the XY and ZW sex systems in this family can be determined by the same RR gene. Our study provides new insights into the diversification of incipient sex chromosomes in flowering plants by showing how transposition and rearrangement of a single gene can control sex in both XY and ZW systems.

Hybrid speciation via inheritance of alternate alleles of parental isolating genes.

It is increasingly realized that homoploid hybrid speciation (HHS), which involves no change in chromosome number, is an important mechanism of speciation. HHS will likely increase in frequency as ecological and geographical barriers between species are continuing to be disrupted by human activities. HHS requires the establishment of reproductive isolation between a hybrid and its parents, but the underlying genes and genetic mechanisms remain largely unknown. In this study, we reveal by integrated approaches that reproductive isolation originates in one homoploid hybrid plant species through the inheritance of alternate alleles at genes that determine parental premating isolation. The parent species of this hybrid species are reproductively isolated by differences in flowering time and survivorship on soils containing high concentrations of iron. We found that the hybrid species inherits alleles of parental isolating major genes related to flowering time from one parent and alleles of major genes related to iron tolerance from the other parent. In this way, it became reproductively isolated from one parent by the difference in flowering time and from the other by habitat adaptation (iron tolerance). These findings and further modeling results suggest that HHS may occur relatively easily via the inheritance of alternate parental premating isolating genes and barriers.

A targeted sequence capture array for phylogenetics and population genomics in the Salicaceae.

PREMISE: The family Salicaceae has proved taxonomically challenging, especially in the genus Salix, which is speciose and features frequent hybridization and polyploidy. Past efforts to reconstruct the phylogeny with molecular barcodes have failed to resolve the species relationships of many sections of the genus. METHODS: We used the wealth of sequence data in the family to design sequence capture probes to target regions of 300-1200 bp of exonic regions of 972 genes. RESULTS: We recovered sequence data for nearly all of the targeted genes in three species of Populus and three species of Salix. We present a species tree, discuss concordance among gene trees, and present population genomic summary statistics for these loci. CONCLUSIONS: Our sequence capture array has extremely high capture efficiency within the genera Populus and Salix, resulting in abundant phylogenetic information. Additionally, these loci show promise for population genomic studies.

Genome-Wide Association Study of Wood Anatomical and Morphological Traits in Populus trichocarpa.

To understand the genetic mechanisms underlying wood anatomical and morphological traits in Populus trichocarpa, we used 869 unrelated genotypes from a common garden in Clatskanie, Oregon that were previously collected from across the distribution range in western North America. Using GEMMA mixed model analysis, we tested for the association of 25 phenotypic traits and nine multitrait combinations with 6.741 million SNPs covering the entire genome. Broad-sense trait heritabilities ranged from 0.117 to 0.477. Most traits were significantly correlated with geoclimatic variables suggesting a role of climate and geography in shaping the variation of this species. Fifty-seven SNPs from single trait GWAS and 11 SNPs from multitrait GWAS passed an FDR threshold of 0.05, leading to the identification of eight and seven nearby candidate genes, respectively. The percentage of phenotypic variance explained (PVE) by the significant SNPs for both single and multitrait GWAS ranged from 0.01% to 6.18%. To further evaluate the potential roles of candidate genes, we used a multi-omic network containing five additional data sets, including leaf and wood metabolite GWAS layers and coexpression and comethylation networks. We also performed a functional enrichment analysis on coexpression nearest neighbors for each gene model identified by the wood anatomical and morphological trait GWAS analyses. Genes affecting cell wall composition and transport related genes were enriched in wood anatomy and stomatal density trait networks. Signaling and metabolism related genes were also common in networks for stomatal density. For leaf morphology traits (leaf dry and wet weight) the networks were significantly enriched for GO terms related to photosynthetic processes as well as cellular homeostasis. The identified genes provide further insights into the genetic control of these traits, which are important determinants of the suitability and sustainability of improved genotypes for lignocellulosic biofuel production.

Sequencing and Analysis of the Sex Determination Region of Populus trichocarpa.

The ages and sizes of a sex-determination region (SDR) are difficult to determine in non-model species. Due to the lack of recombination and enrichment of repetitive elements in SDRs, the quality of assembly with short sequencing reads is universally low. Unique features present in the SDRs help provide clues about how SDRs are established and how they evolve in the absence of recombination. Several Populus species have been reported with a male heterogametic configuration of sex (XX/XY system) mapped on chromosome 19, but the exact location of the SDR has been inconsistent among species, and thus far, none of these SDRs has been fully assembled in a genomic context. Here we identify the Y-SDR from a Y-linked contig directly from a long-read PacBio assembly of a Populus trichocarpa male individual. We also identified homologous gene sequences in the SDR of P. trichocarpa and the SDR of the W chromosome in Salix purpurea. We show that inverted repeats (IRs) found in the Y-SDR and the W-SDR are lineage-specific. We hypothesize that, although the two IRs are derived from the same orthologous gene within each species, they likely have independent evolutionary histories. Furthermore, the truncated inverted repeats in P. trichocarpa may code for small RNAs that target the homologous gene for RNA-directed DNA methylation. These findings support the hypothesis that diverse sex-determining systems may be achieved through similar evolutionary pathways, thereby providing a possible mechanism to explain the lability of sex-determination systems in plants in general.

Host plant genetic control of associated fungal and insect species in a Populus hybrid cross.

Plants employ a diverse set of defense mechanisms to mediate interactions with insects and fungi. These relationships can leave lasting impacts on host plant genome structure such as rapid expansion of gene families through tandem duplication. These genomic signatures provide important clues about the complexities of plant/biotic stress interactions and evolution. We used a pseudo-backcross hybrid family to identify quantitative trait loci (QTL) controlling associations between Populus trees and several common Populus diseases and insects. Using whole-genome sequences from each parent, we identified candidate genes that may mediate these interactions. Candidates were partially validated using mass spectrometry to identify corresponding QTL for defensive compounds. We detected significant QTL for two interacting fungal pathogens and three insects. The QTL intervals contained candidate genes potentially involved in physical and chemical mechanisms of host-plant resistance and susceptibility. In particular, we identified adjoining QTLs for a phenolic glycoside and Phyllocolpa sawfly abundance. There was also significant enrichment of recent tandem duplications in the genomic intervals of the native parent, but not the exotic parent. Tandem gene duplication may be an important mechanism for rapid response to biotic stressors, enabling trees with long juvenile periods to reach maturity despite many coevolving biotic stressors.

Pathways to sex determination in plants: how many roads lead to Rome?

The presence of thousands of independent origins of dioecy in angiosperms provides a unique opportunity to address the parallel evolution of the molecular pathways underlying unisexual flowers. Recent progress towards identifying sex determination genes has identified hormone response pathways, mainly associated with cytokinin and ethylene response pathways, as having been recruited multiple times independently to control unisexuality. Moreover, transcriptomics has begun to identify commonalities among intermediate sections of signal transduction pathways. These recent advances set the stage for development of a comparative evolutionary development research program to identify the shared and unique aspects of the genetic pathways of unisexual flower development in angiosperms.

Improved genome assembly provides new insights into genome evolution in a desert poplar (Populus euphratica).

Populus euphratica is well adapted to extreme desert environments and is an important model species for elucidating the mechanisms of abiotic stress resistance in trees. The current assembly of P. euphratica genome is highly fragmented with many gaps and errors, thereby impeding downstream applications. Here, we report an improved chromosome-level reference genome of P. euphratica (v2.0) using single-molecule sequencing and chromosome conformation capture (Hi-C) technologies. Relative to the previous reference genome, our assembly represents a nearly 60-fold improvement in contiguity, with a scaffold N50 size of 28.59 Mb. Using this genome, we have found that extensive expansion of Gypsy elements in P. euphratica led to its rapid increase in genome size compared to any other Salicaceae species studied to date, and potentially contributed to adaptive divergence driven by insertions near genes involved in stress tolerance. We also detected a wide range of unique structural rearrangements in P. euphratica, including 2,549 translocations, 454 inversions, 121 tandem and 14 segmental duplications. Several key genes likely to be involved in tolerance to abiotic stress were identified within these regions. This high-quality genome represents a valuable resource for poplar breeding and genetic improvement in the future, as well as comparative genomic analysis with other Salicaceae species.