Species-tree topology impacts the inference of ancient whole-genome duplications across the angiosperm phylogeny.

PREMISE: The history of angiosperms is marked by repeated rounds of ancient whole-genome duplications (WGDs). Here we used state-of-the-art methods to provide an up-to-date view of the distribution of WGDs in the history of angiosperms that considers both uncertainty introduced by different WGD inference methods and different underlying species-tree hypotheses. METHODS: We used the distribution synonymous divergences (Ks) of paralogs and orthologs from transcriptomic and genomic data to infer and place WGDs across two hypothesized angiosperm phylogenies. We further tested these WGD hypotheses with syntenic inferences and Bayesian models of duplicate gene gain and loss. RESULTS: The predicted number of WGDs in the history of angiosperms (~170) based on the current taxon sampling is largely similar across different inference methods, but varies in the precise placement of WGDs on the phylogeny. Ks-based methods often yield alternative hypothesized WGD placements due to variation in substitution rates among lineages. Phylogenetic models of duplicate gene gain and loss are more robust to topological variation. However, errors in species-tree inference can still produce spurious WGD hypotheses, regardless of method used. CONCLUSIONS: Here we showed that different WGD inference methods largely agree on an average of 3.5 WGD in the history of individual angiosperm species. However, the precise placement of WGDs on the phylogeny is subject to the WGD inference method and tree topology. As researchers continue to test hypotheses regarding the impacts ancient WGDs have on angiosperm evolution, it is important to consider the uncertainty of the phylogeny as well as WGD inference methods.

A de novo long-read genome assembly of the sacred datura plant (Datura wrightii) reveals a role of tandem gene duplications in the evolution of herbivore-defense response.

The sacred datura plant (Solanales: Solanaceae: Datura wrightii) has been used to study plant-herbivore interactions for decades. The wealth of information that has resulted leads it to have potential as a model system for studying the ecological and evolutionary genomics of these interactions. We present a de novo Datura wrightii genome assembled using PacBio HiFi long-reads. Our assembly is highly complete and contiguous (N50 = 179Mb, BUSCO Complete = 97.6%). We successfully detected a previously documented ancient whole genome duplication using our assembly and have classified the gene duplication history that generated its coding sequence content. We use it as the basis for a genome-guided differential expression analysis to identify the induced responses of this plant to one of its specialized herbivores (Coleoptera: Chrysomelidae: Lema daturaphila). We find over 3000 differentially expressed genes associated with herbivory and that elevated expression levels of over 200 genes last for several days. We also combined our analyses to determine the role that different gene duplication categories have played in the evolution of Datura-herbivore interactions. We find that tandem duplications have expanded multiple functional groups of herbivore responsive genes with defensive functions, including UGT-glycosyltranserases, oxidoreductase enzymes, and peptidase inhibitors. Overall, our results expand our knowledge of herbivore-induced plant transcriptional responses and the evolutionary history of the underlying herbivore-response genes.

Complementing model species with model clades.

Model species continue to underpin groundbreaking plant science research. At the same time, the phylogenetic resolution of the land plant Tree of Life continues to improve. The intersection of these two research paths creates a unique opportunity to further extend the usefulness of model species across larger taxonomic groups. Here we promote the utility of the Arabidopsis thaliana model species, especially the ability to connect its genetic and functional resources, to species across the entire Brassicales order. We focus on the utility of using genomics and phylogenomics to bridge the evolution and diversification of several traits across the Brassicales to the resources in Arabidopsis, thereby extending scope from a model species by establishing a "model clade". These Brassicales-wide traits are discussed in the context of both the model species Arabidopsis thaliana and the family Brassicaceae. We promote the utility of such a "model clade" and make suggestions for building global networks to support future studies in the model order Brassicales.

GOgetter: A pipeline for summarizing and visualizing GO slim annotations for plant genetic data.

Premise: The functional annotation of genes is a crucial component of genomic analyses. A common way to summarize functional annotations is with hierarchical gene ontologies, such as the Gene Ontology (GO) Resource. GO includes information about the cellular location, molecular function(s), and products/processes that genes produce or are involved in. For a set of genes, summarizing GO annotations using pre-defined, higher-order terms (GO slims) is often desirable in order to characterize the overall function of the data set, and it is impractical to do this manually. Methods and Results: The GOgetter pipeline consists of bash and Python scripts. From an input FASTA file of nucleotide gene sequences, it outputs text and image files that list (1) the best hit for each input gene in a set of reference gene models, (2) all GO terms and annotations associated with those hits, and (3) a summary and visualization of GO slim categories for the data set. These output files can be queried further and analyzed statistically, depending on the downstream need(s). Conclusions: GO annotations are a widely used "universal language" for describing gene functions and products. GOgetter is a fast and easy-to-implement pipeline for obtaining, summarizing, and visualizing GO slim categories associated with a set of genes.

Demographic history inference and the polyploid continuum.

Polyploidy is an important generator of evolutionary novelty across diverse groups in the Tree of Life, including many crops. However, the impact of whole-genome duplication depends on the mode of formation: doubling within a single lineage (autopolyploidy) versus doubling after hybridization between two different lineages (allopolyploidy). Researchers have historically treated these two scenarios as completely separate cases based on patterns of chromosome pairing, but these cases represent ideals on a continuum of chromosomal interactions among duplicated genomes. Understanding the history of polyploid species thus demands quantitative inferences of demographic history and rates of exchange between subgenomes. To meet this need, we developed diffusion models for genetic variation in polyploids with subgenomes that cannot be bioinformatically separated and with potentially variable inheritance patterns, implementing them in the dadi software. We validated our models using forward SLiM simulations and found that our inference approach is able to accurately infer evolutionary parameters (timing, bottleneck size) involved with the formation of auto- and allotetraploids, as well as exchange rates in segmental allotetraploids. We then applied our models to empirical data for allotetraploid shepherd's purse (Capsella bursa-pastoris), finding evidence for allelic exchange between the subgenomes. Taken together, our model provides a foundation for demographic modeling in polyploids using diffusion equations, which will help increase our understanding of the impact of demography and selection in polyploid lineages.

Applying Machine Learning to Classify the Origins of Gene Duplications.

Nearly all lineages of land plants have experienced at least one whole-genome duplication (WGD) in their history. The legacy of these ancient WGDs is still observable in the diploidized genomes of extant plants. Genes originating from WGD-paleologs-can be maintained in diploidized genomes for millions of years. These paleologs have the potential to shape plant evolution through sub- and neofunctionalization, increased genetic diversity, and reciprocal gene loss among lineages. Current methods for classifying paleologs often rely on only a subset of potential genomic features, have varying levels of accuracy, and often require significant data and/or computational time. Here, we developed a supervised machine learning approach to classify paleologs from a target WGD in diploidized genomes across a broad range of different duplication histories. We collected empirical data on syntenic block sizes and other genomic features from 27 plant species each with a different history of paleopolyploidy. Features from these genomes were used to develop simulations of syntenic blocks and paleologs to train a gradient boosted decision tree. Using this approach, Frackify (Fractionation Classify), we were able to accurately identify and classify paleologs across a broad range of parameter space, including cases with multiple overlapping WGDs. We then compared Frackify with other paleolog inference approaches in six species with paleotetraploid and paleohexaploid ancestries. Frackify provides a way to combine multiple genomic features to quickly classify paleologs while providing a high degree of consistency with existing approaches.

Dynamic genome evolution in a model fern.

The large size and complexity of most fern genomes have hampered efforts to elucidate fundamental aspects of fern biology and land plant evolution through genome-enabled research. Here we present a chromosomal genome assembly and associated methylome, transcriptome and metabolome analyses for the model fern species Ceratopteris richardii. The assembly reveals a history of remarkably dynamic genome evolution including rapid changes in genome content and structure following the most recent whole-genome duplication approximately 60 million years ago. These changes include massive gene loss, rampant tandem duplications and multiple horizontal gene transfers from bacteria, contributing to the diversification of defence-related gene families. The insertion of transposable elements into introns has led to the large size of the Ceratopteris genome and to exceptionally long genes relative to other plants. Gene family analyses indicate that genes directing seed development were co-opted from those controlling the development of fern sporangia, providing insights into seed plant evolution. Our findings and annotated genome assembly extend the utility of Ceratopteris as a model for investigating and teaching plant biology.

Genome size evolution in the diverse insect order Trichoptera.

BACKGROUND: Genome size is implicated in the form, function, and ecological success of a species. Two principally different mechanisms are proposed as major drivers of eukaryotic genome evolution and diversity: polyploidy (i.e., whole-genome duplication) or smaller duplication events and bursts in the activity of repetitive elements. Here, we generated de novo genome assemblies of 17 caddisflies covering all major lineages of Trichoptera. Using these and previously sequenced genomes, we use caddisflies as a model for understanding genome size evolution in diverse insect lineages. RESULTS: We detect a ∼14-fold variation in genome size across the order Trichoptera. We find strong evidence that repetitive element expansions, particularly those of transposable elements (TEs), are important drivers of large caddisfly genome sizes. Using an innovative method to examine TEs associated with universal single-copy orthologs (i.e., BUSCO genes), we find that TE expansions have a major impact on protein-coding gene regions, with TE-gene associations showing a linear relationship with increasing genome size. Intriguingly, we find that expanded genomes preferentially evolved in caddisfly clades with a higher ecological diversity (i.e., various feeding modes, diversification in variable, less stable environments). CONCLUSION: Our findings provide a platform to test hypotheses about the potential evolutionary roles of TE activity and TE-gene associations, particularly in groups with high species, ecological, and functional diversities.

Chromosome-Scale Genome Assembly of Gilia yorkii Enables Genetic Mapping of Floral Traits in an Interspecies Cross.

Substantial morphological variation in land plants remains inaccessible to genetic analysis because current models lack variation in important ecological and agronomic traits. The genus Gilia was historically a model for biosystematics studies and includes variation in morphological traits that are poorly understood at the genetic level. We assembled a chromosome-scale reference genome of G. yorkii and used it to investigate genome evolution in the Polemoniaceae. We performed QTL (quantitative trait loci) mapping in a G. yorkii×G. capitata interspecific population for traits related to inflorescence architecture and flower color. The genome assembly spans 2.75 Gb of the estimated 2.80-Gb genome, with 96.7% of the sequence contained in the nine largest chromosome-scale scaffolds matching the haploid chromosome number. Gilia yorkii experienced at least one round of whole-genome duplication shared with other Polemoniaceae after the eudicot paleohexaploidization event. We identified QTL linked to variation in inflorescence architecture and petal color, including a candidate for the major flower color QTL-a tandem duplication of flavanol 3',5'-hydroxylase. Our results demonstrate the utility of Gilia as a forward genetic model for dissecting the evolution of development in plants including the causal loci underlying inflorescence architecture transitions.

Underwater CAM photosynthesis elucidated by Isoetes genome.

To conserve water in arid environments, numerous plant lineages have independently evolved Crassulacean Acid Metabolism (CAM). Interestingly, Isoetes, an aquatic lycophyte, can also perform CAM as an adaptation to low CO2 availability underwater. However, little is known about the evolution of CAM in aquatic plants and the lack of genomic data has hindered comparison between aquatic and terrestrial CAM. Here, we investigate underwater CAM in Isoetes taiwanensis by generating a high-quality genome assembly and RNA-seq time course. Despite broad similarities between CAM in Isoetes and terrestrial angiosperms, we identify several key differences. Notably, Isoetes may have recruited the lesser-known 'bacterial-type' PEPC, along with the 'plant-type' exclusively used in other CAM and C4 plants for carboxylation of PEP. Furthermore, we find that circadian control of key CAM pathway genes has diverged considerably in Isoetes relative to flowering plants. This suggests the existence of more evolutionary paths to CAM than previously recognized.

Modelling of gene loss propensity in the pangenomes of three Brassica species suggests different mechanisms between polyploids and diploids.

Plant genomes demonstrate significant presence/absence variation (PAV) within a species; however, the factors that lead to this variation have not been studied systematically in Brassica across diploids and polyploids. Here, we developed pangenomes of polyploid Brassica napus and its two diploid progenitor genomes B. rapa and B. oleracea to infer how PAV may differ between diploids and polyploids. Modelling of gene loss suggests that loss propensity is primarily associated with transposable elements in the diploids while in B. napus, gene loss propensity is associated with homoeologous recombination. We use these results to gain insights into the different causes of gene loss, both in diploids and following polyploidization, and pave the way for the application of machine learning methods to understanding the underlying biological and physical causes of gene presence/absence.

Animal chromosome counts reveal a similar range of chromosome numbers but with less polyploidy in animals compared to flowering plants.

Understanding the mechanisms that underlie chromosome evolution could provide insights into the processes underpinning the origin, persistence and evolutionary tempo of lineages. Here, we present the first database of chromosome counts for animals (the Animal Chromosome Count database, ACC) summarizing chromosome numbers for ~15,000 species. We found remarkable a similarity in the distribution of chromosome counts between animals and flowering plants. Nevertheless, the similarity in the distribution of chromosome numbers between animals and plants is likely to be explained by different drivers. For instance, we found that while animals and flowering plants exhibit similar frequencies of speciation-related changes in chromosome number, plant speciation is more often related to changes in ploidy. By leveraging the largest data set of chromosome counts for animals, we describe a previously undocumented pattern across the Tree of Life-animals and flowering plants show remarkably similar distributions of haploid chromosome numbers.

Analysis of the Coptis chinensis genome reveals the diversification of protoberberine-type alkaloids.

Chinese goldthread (Coptis chinensis Franch.), a member of the Ranunculales, represents an important early-diverging eudicot lineage with diverse medicinal applications. Here, we present a high-quality chromosome-scale genome assembly and annotation of C. chinensis. Phylogenetic and comparative genomic analyses reveal the phylogenetic placement of this species and identify a single round of ancient whole-genome duplication (WGD) shared by the Ranunculaceae. We characterize genes involved in the biosynthesis of protoberberine-type alkaloids in C. chinensis. In particular, local genomic tandem duplications contribute to member amplification of a Ranunculales clade-specific gene family of the cytochrome P450 (CYP) 719. The functional versatility of a key CYP719 gene that encodes the (S)-canadine synthase enzyme involved in the berberine biosynthesis pathway may play critical roles in the diversification of other berberine-related alkaloids in C. chinensis. Our study provides insights into the genomic landscape of early-diverging eudicots and provides a valuable model genome for genetic and applied studies of Ranunculales.

The contributions from the progenitor genomes of the mesopolyploid Brassiceae are evolutionarily distinct but functionally compatible.

The members of the tribe Brassiceae share a whole-genome triplication (WGT), and one proposed model for its formation is a two-step pair of hybridizations producing hexaploid descendants. However, evidence for this model is incomplete, and the evolutionary and functional constraints that drove evolution after the hexaploidy are even less understood. Here, we report a new genome sequence of Crambe hispanica, a species sister to most sequenced Brassiceae. Using this new genome and three others that share the hexaploidy, we traced the history of gene loss after the WGT using the Polyploidy Orthology Inference Tool (POInT). We confirm the two-step formation model and infer that there was a significant temporal gap between those two allopolyploidizations, with about a third of the gene losses from the first two subgenomes occurring before the arrival of the third. We also, for the 90,000 individual genes in our study, make parental subgenome assignments, inferring, with measured uncertainty, from which of the progenitor genomes of the allohexaploidy each gene derives. We further show that each subgenome has a statistically distinguishable rate of homoeolog losses. There is little indication of functional distinction between the three subgenomes: the individual subgenomes show no patterns of functional enrichment, no excess of shared protein-protein or metabolic interactions between their members, and no biases in their likelihood of having experienced a recent selective sweep. We propose a "mix and match" model of allopolyploidy, in which subgenome origin drives homoeolog loss propensities but where genes from different subgenomes function together without difficulty.

Patterns and Processes of Diploidization in Land Plants.

Most land plants are now known to be ancient polyploids that have rediploidized. Diploidization involves many changes in genome organization that ultimately restore bivalent chromosome pairing and disomic inheritance, and resolve dosage and other issues caused by genome duplication. In this review, we discuss the nature of polyploidy and its impact on chromosome pairing behavior. We also provide an overview of two major and largely independent processes of diploidization: cytological diploidization and genic diploidization/fractionation. Finally, we compare variation in gene fractionation across land plants and highlight the differences in diploidization between plants and animals. Altogether, we demonstrate recent advancements in our understanding of variation in the patterns and processes of diploidization in land plants and provide a road map for future research to unlock the mysteries of diploidization and eukaryotic genome evolution.

Pilot RNA-seq data from 24 species of vascular plants at Harvard Forest.

PREMISE: Large-scale projects such as the National Ecological Observatory Network (NEON) collect ecological data on entire biomes to track climate change. NEON provides an opportunity to launch community transcriptomic projects that ask integrative questions in ecology and evolution. We conducted a pilot study to investigate the challenges of collecting RNA-seq data from diverse plant communities. METHODS: We generated >650 Gbp of RNA-seq for 24 vascular plant species representing 12 genera and nine families at the Harvard Forest NEON site. Each species was sampled twice in 2016 (July and August). We assessed transcriptome quality and content with TransRate, BUSCO, and Gene Ontology annotations. RESULTS: Only modest differences in assembly quality were observed across multiple k-mers. On average, transcriptomes contained hits to >70% of loci in the BUSCO database. We found no significant difference in the number of assembled and annotated transcripts between diploid and polyploid transcriptomes. DISCUSSION: We provide new RNA-seq data sets for 24 species of vascular plants in Harvard Forest. Challenges associated with this type of study included recovery of high-quality RNA from diverse species and access to NEON sites for genomic sampling. Overcoming these challenges offers opportunities for large-scale studies at the intersection of ecology and genomics.

Chromosome-scale inference of hybrid speciation and admixture with convolutional neural networks.

Inferring the frequency and mode of hybridization among closely related organisms is an important step for understanding the process of speciation and can help to uncover reticulated patterns of phylogeny more generally. Phylogenomic methods to test for the presence of hybridization come in many varieties and typically operate by leveraging expected patterns of genealogical discordance in the absence of hybridization. An important assumption made by these tests is that the data (genes or SNPs) are independent given the species tree. However, when the data are closely linked, it is especially important to consider their nonindependence. Recently, deep learning techniques such as convolutional neural networks (CNNs) have been used to perform population genetic inferences with linked SNPs coded as binary images. Here, we use CNNs for selecting among candidate hybridization scenarios using the tree topology (((P1 , P2 ), P3 ), Out) and a matrix of pairwise nucleotide divergence (dXY ) calculated in windows across the genome. Using coalescent simulations to train and independently test a neural network showed that our method, HyDe-CNN, was able to accurately perform model selection for hybridization scenarios across a wide breath of parameter space. We then used HyDe-CNN to test models of admixture in Heliconius butterflies, as well as comparing it to phylogeny-based introgression statistics. Given the flexibility of our approach, the dropping cost of long-read sequencing and the continued improvement of CNN architectures, we anticipate that inferences of hybridization using deep learning methods like ours will help researchers to better understand patterns of admixture in their study organisms.

Genes derived from ancient polyploidy have higher genetic diversity and are associated with domestication in Brassica rapa.

Many crops are polyploid or have a polyploid ancestry. Recent phylogenetic analyses have found that polyploidy often preceded the domestication of crop plants. One explanation for this observation is that increased genetic diversity following polyploidy may have been important during the strong artificial selection that occurs during domestication. In order to test the connection between domestication and polyploidy, we identified and examined candidate genes associated with the domestication of the diverse crop varieties of Brassica rapa. Like all 'diploid' flowering plants, B. rapa has a diploidized paleopolyploid genome and experienced many rounds of whole genome duplication (WGD). We analyzed transcriptome data of more than 100 cultivated B. rapa accessions. Using a combination of approaches, we identified > 3000 candidate genes associated with the domestication of four major B. rapa crop varieties. Consistent with our expectation, we found that the candidate genes were significantly enriched with genes derived from the Brassiceae mesohexaploidy. We also observed that paleologs were significantly more diverse than non-paleologs. Our analyses find evidence for that genetic diversity derived from ancient polyploidy played a key role in the domestication of B. rapa and provide support for its importance in the success of modern agriculture.

TagSeq for gene expression in non-model plants: A pilot study at the Santa Rita Experimental Range NEON core site.

PREMISE: TagSeq is a cost-effective approach for gene expression studies requiring a large number of samples. To date, TagSeq studies in plants have been limited to those with a high-quality reference genome. We tested the suitability of reference transcriptomes for TagSeq in non-model plants, as part of a study of natural gene expression variation at the Santa Rita Experimental Range National Ecological Observatory Network (NEON) core site. METHODS: Tissue for TagSeq was sampled from multiple individuals of four species (Bouteloua aristidoides and Eragrostis lehmanniana [Poaceae], Tidestromia lanuginosa [Amaranthaceae], and Parkinsonia florida [Fabaceae]) at two locations on three dates (56 samples total). One sample per species was used to create a reference transcriptome via standard RNA-seq. TagSeq performance was assessed by recovery of reference loci, specificity of tag alignments, and variation among samples. RESULTS: A high fraction of tags aligned to each reference and mapped uniquely. Expression patterns were quantifiable for tens of thousands of loci, which revealed consistent spatial differentiation in expression for all species. DISCUSSION: TagSeq using de novo reference transcriptomes was an effective approach to quantifying gene expression in this study. Tags were highly locus specific and generated biologically informative profiles for four non-model plant species.

Polyploids increase overall diversity despite higher turnover than diploids in the Brassicaceae.

Although polyploidy is widespread across the plant Tree of Life, its long-term evolutionary significance is still poorly understood. Here, we examine the effects of polyploidy in explaining the large-scale evolutionary patterns within angiosperms by focusing on a single family exhibiting extensive interspecific variation in chromosome numbers. We inferred ploidy from haploid chromosome numbers for 80% of species in the most comprehensive species-level chronogram for the Brassicaceae. After evaluating a total of 94 phylogenetic models of diversification, we found that ploidy influences diversification rates across the Brassicaceae. We also found that despite diversifying at a similar rate to diploids, polyploids have played a significant role in driving present-day differences in species richness among clades. Overall, in addition to highlighting the complexity in the evolutionary consequences of polyploidy, our results suggest that rare successful polyploids persist while significantly contributing to the long-term evolution of clades. Our findings further indicate that polyploidy has played a major role in driving the long-term evolution of the Brassicaceae and highlight the potential of polyploidy in shaping present-day diversity patterns across the plant Tree of Life.