Phylogenomics and the rise of the angiosperms.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods1,2. A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome3,4. Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins5-7. However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes8. This 15-fold increase in genus-level sampling relative to comparable nuclear studies9 provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade.

Global Brassicaceae phylogeny based on filtering of 1,000-gene dataset.

The mustard family (Brassicaceae) is a scientifically and economically important family, containing the model plant Arabidopsis thaliana and numerous crop species that feed billions worldwide. Despite its relevance, most phylogenetic trees of the family are incompletely sampled and often contain poorly supported branches. Here, we present the most complete Brassicaceae genus-level family phylogenies to date (Brassicaceae Tree of Life or BrassiToL) based on nuclear (1,081 genes, 319 of the 349 genera; 57 of the 58 tribes) and plastome (60 genes, 265 genera; all tribes) data. We found cytonuclear discordance between the two, which is likely a result of rampant hybridization among closely and more distantly related lineages. To evaluate the impact of such hybridization on the nuclear phylogeny reconstruction, we performed five different gene sampling routines, which increasingly removed putatively paralog genes. Our cleaned subset of 297 genes revealed high support for the tribes, whereas support for the main lineages (supertribes) was moderate. Calibration based on the 20 most clock-like nuclear genes suggests a late Eocene to late Oligocene origin of the family. Finally, our results strongly support a recently published new family classification, dividing the family into two subfamilies (one with five supertribes), together representing 58 tribes. This includes five recently described or re-established tribes, including Arabidopsideae, a monogeneric tribe accommodating Arabidopsis without any close relatives. With a worldwide community of thousands of researchers working on Brassicaceae and its diverse members, our new genus-level family phylogeny will be an indispensable tool for studies on biodiversity and plant biology.

A Hyb-Seq phylogeny of Boechera and related genera using a combination of Angiosperms353 and Brassicaceae-specific bait sets.

PREMISE: Although Boechera (Boechereae, Brassicaceae) has become a plant model system for both ecological genomics and evolutionary biology, all previous phylogenetic studies have had limited success in resolving species relationships within the genus. The recent effective application of sequence data from target enrichment approaches to resolve the evolutionary relationships of several other challenging plant groups prompted us to investigate their usefulness in Boechera and Boechereae. METHODS: To resolve the phylogeny of Boechera and closely related genera, we utilized the Hybpiper pipeline to analyze two combined bait sets: Angiosperms353, with broad applicability across flowering plants; and a Brassicaceae-specific bait set designed for use in the mustard family. Relationships for 101 samples representing 81 currently recognized species were inferred from a total of 1114 low-copy nuclear genes using both supermatrix and species coalescence methods. RESULTS: Our analyses resulted in a well-resolved and highly supported phylogeny of the tribe Boechereae. Boechereae is divided into two major clades, one comprising all western North American species of Boechera, the other encompassing the eight other genera of the tribe. Our understanding of relationships within Boechera is enhanced by the recognition of three core clades that are further subdivided into robust regional species complexes. CONCLUSIONS: This study presents the first broadly sampled, well-resolved phylogeny for most known sexual diploid Boechera. This effort provides the foundation for a new phylogenetically informed taxonomy of Boechera that is crucial for its continued use as a model system.

An updated classification of the Brassicaceae (Cruciferae).

Based on recent achievements in phylogenetic studies of the Brassicaceae, a novel infrafamilial classification is proposed that includes major improvements at the subfamilial and supertribal levels. Herein, the family is subdivided into two subfamilies, Aethionemoideae (subfam. nov.) and Brassicoideae. The Brassicoideae, with 57 of the 58 tribes of Brassicaceae, are further partitioned into five supertribes, including the previously recognized Brassicodae and the newly established Arabodae, Camelinodae, Heliophilodae, and Hesperodae. Additional tribus-level contributions include descriptions of the newly recognized Arabidopsideae, Asperuginoideae, Hemilophieae, Schrenkielleae, and resurrection of the Chamireae and Subularieae. Further detailed comments on 17 tribes in need of clarifications are provided.

Precipitation is the main axis of tropical plant phylogenetic turnover across space and time.

Early natural historians-Comte de Buffon, von Humboldt, and De Candolle-established environment and geography as two principal axes determining the distribution of groups of organisms, laying the foundations for biogeography over the subsequent 200 years, yet the relative importance of these two axes remains unresolved. Leveraging phylogenomic and global species distribution data for Mimosoid legumes, a pantropical plant clade of c. 3500 species, we show that the water availability gradient from deserts to rain forests dictates turnover of lineages within continents across the tropics. We demonstrate that 95% of speciation occurs within a precipitation niche, showing profound phylogenetic niche conservatism, and that lineage turnover boundaries coincide with isohyets of precipitation. We reveal similar patterns on different continents, implying that evolution and dispersal follow universal processes.

The best of both worlds: Combining lineage-specific and universal bait sets in target-enrichment hybridization reactions.

PREMISE: Researchers adopting target-enrichment approaches often struggle with the decision of whether to use universal or lineage-specific probe sets. To circumvent this quandary, we investigate the efficacy of a simultaneous enrichment by combining universal probes and lineage-specific probes in a single hybridization reaction, to benefit from the qualities of both probe sets with little added cost or effort. METHODS AND RESULTS: Using 26 Brassicaceae libraries and standard enrichment protocols, we compare results from three independent data sets. A large average fraction of reads mapping to the Angiosperms353 (24-31%) and Brassicaceae (35-59%) targets resulted in a sizable reconstruction of loci for each target set (x̄ ≥ 70%). CONCLUSIONS: High levels of enrichment and locus reconstruction for the two target sets demonstrate that the sampling of genomic regions can be easily extended through the combination of probe sets in single enrichment reactions. We hope that these findings will facilitate the production of expanded data sets that answer individual research questions and simultaneously allow wider applications by the research community as a whole.

A draft genome and transcriptome of common milkweed (Asclepias syriaca) as resources for evolutionary, ecological, and molecular studies in milkweeds and Apocynaceae.

Milkweeds (Asclepias) are used in wide-ranging studies including floral development, pollination biology, plant-insect interactions and co-evolution, secondary metabolite chemistry, and rapid diversification. We present a transcriptome and draft nuclear genome assembly of the common milkweed, Asclepias syriaca. This reconstruction of the nuclear genome is augmented by linkage group information, adding to existing chloroplast and mitochondrial genomic resources for this member of the Apocynaceae subfamily Asclepiadoideae. The genome was sequenced to 80.4× depth and the draft assembly contains 54,266 scaffolds ≥1 kbp, with N50 = 3,415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. A total of 14,474 protein-coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. Scaffolds covering 75 Mbp of the Asclepias assembly formed 11 linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosomes show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. The progesterone 5ß-reductase gene family, a key component of cardenolide production, is likely reduced in Asclepias relative to other Apocynaceae. The genome and transcriptome of common milkweed provide a rich resource for future studies of the ecology and evolution of a charismatic plant family.

Resolving the backbone of the Brassicaceae phylogeny for investigating trait diversity.

The Brassicaceae family comprises c. 4000 species including economically important crops and the model plant Arabidopsis thaliana. Despite their importance, the relationships among major lineages in the family remain unresolved, hampering comparative research. Here, we inferred a Brassicaceae phylogeny using newly generated targeted enrichment sequence data of 1827 exons (> 940 000 bases) representing 63 species, as well as sequenced genome data of 16 species, together representing 50 of the 52 currently recognized Brassicaceae tribes. A third of the samples were derived from herbarium material, facilitating broad taxonomic coverage of the family. Six major clades formed successive sister groups to the rest of Brassicaceae. We also recovered strong support for novel relationships among tribes, and resolved the position of 16 taxa previously not assigned to a tribe. The broad utility of these phylogenetic results is illustrated through a comparative investigation of genome-wide expression signatures that distinguish simple from complex leaves in Brassicaceae. Our study provides an easily extendable dataset for further advances in Brassicaceae systematics and a timely higher-level phylogenetic framework for a wide range of comparative studies of multiple traits in an intensively investigated group of plants.

PacBio-Based Mitochondrial Genome Assembly of Leucaena trichandra (Leguminosae) and an Intrageneric Assessment of Mitochondrial RNA Editing.

Reconstructions of vascular plant mitochondrial genomes (mt-genomes) are notoriously complicated by rampant recombination that has resulted in comparatively few plant mt-genomes being available. The dearth of plant mitochondrial resources has limited our understanding of mt-genome structural diversity, complex patterns of RNA editing, and the origins of novel mt-genome elements. Here, we use an efficient long read (PacBio) iterative assembly pipeline to generate mt-genome assemblies for Leucaena trichandra (Leguminosae: Caesalpinioideae: mimosoid clade), providing the first assessment of non-papilionoid legume mt-genome content and structure to date. The efficiency of the assembly approach facilitated the exploration of alternative structures that are common place among plant mitochondrial genomes. A compact version (729 kbp) of the recovered assemblies was used to investigate sources of mt-genome size variation among legumes and mt-genome sequence similarity to the legume associated root holoparasite Lophophytum. The genome and an associated suite of transcriptome data from select species of Leucaena permitted an in-depth exploration of RNA editing in a diverse clade of closely related species that includes hybrid lineages. RNA editing in the allotetraploid, Leucaena leucocephala, is consistent with co-option of nearly equal maternal and paternal C-to-U edit components, generating novel combinations of RNA edited sites. A preliminary investigation of L. leucocephala C-to-U edit frequencies identified the potential for a hybrid to generate unique pools of alleles from parental variation through edit frequencies shared with one parental lineage, those intermediate between parents, and transgressive patterns.

Deciphering the sexual diploid members of the Boechera suffrutescens complex (Brassicaceae, Boechereae).

Boechera is a model genus that is of particular interest for understanding apomixis due to the presence of numerous apomictic diploid lineages that are tightly correlated with hybridisation events. Boechera includes many narrowly distributed endemics and apomictic hybrid lineages that obscure morphological boundaries amongst taxa. In this study, we focus on the Boechera suffrutescens complex, a phylogenetically well-supported but taxonomically complex north-western United States clade whose diploid species currently include the widespread B. suffrutescens and two narrowly distributed serpentine endemics, B. constancei and B. rollei. Using a 15-locus microsatellite dataset, we infer ploidy and sexual vs. apomictic reproduction for all individuals and then assess species limits for all sexual diploid samples. Our results support the recognition of B. rollei and B. constancei as distinct species and reveal three divergent sexual diploid lineages within B. suffrutescens sensu lato. The latter three lineages exhibit geographic, genetic and morphological coherence and consequently warrant recognition at the species rank. These include Boechera suffrutescens s.s., which is restricted to Idaho and eastern Oregon, Boechera botulifructa, a newly described species distributed along the Cascade Mountain Province from Lassen County, California north to Deschutes County, Oregon and the heretofore dismissed species Boechera duriuscula (basionym ≡ Arabis duriuscula), which occurs along the Sierra Nevada Province from Plumas County southwards to Fresno County, California. Our data also reveal substructure in B. constancei that is likely attributable to the highly fragmented distribution of its serpentine habitat. This refined taxonomic framework for the B. suffrutescens complex enhances Boechera as a model system, adds to our knowledge of speciation in edaphically extreme environments and provides information on ongoing conservation efforts for these taxa.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus.

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall. The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics. In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Coupled enhancer and coding sequence evolution of a homeobox gene shaped leaf diversity.

Here we investigate mechanisms underlying the diversification of biological forms using crucifer leaf shape as an example. We show that evolution of an enhancer element in the homeobox gene REDUCED COMPLEXITY (RCO) altered leaf shape by changing gene expression from the distal leaf blade to its base. A single amino acid substitution evolved together with this regulatory change, which reduced RCO protein stability, preventing pleiotropic effects caused by its altered gene expression. We detected hallmarks of positive selection in these evolved regulatory and coding sequence variants and showed that modulating RCO activity can improve plant physiological performance. Therefore, interplay between enhancer and coding sequence evolution created a potentially adaptive path for morphological evolution.

Mimosoid legume plastome evolution: IR expansion, tandem repeat expansions, and accelerated rate of evolution in clpP.

The Leguminosae has emerged as a model for studying angiosperm plastome evolution because of its striking diversity of structural rearrangements and sequence variation. However, most of what is known about legume plastomes comes from few genera representing a subset of lineages in subfamily Papilionoideae. We investigate plastome evolution in subfamily Mimosoideae based on two newly sequenced plastomes (Inga and Leucaena) and two recently published plastomes (Acacia and Prosopis), and discuss the results in the context of other legume and rosid plastid genomes. Mimosoid plastomes have a typical angiosperm gene content and general organization as well as a generally slow rate of protein coding gene evolution, but they are the largest known among legumes. The increased length results from tandem repeat expansions and an unusual 13 kb IR-SSC boundary shift in Acacia and Inga. Mimosoid plastomes harbor additional interesting features, including loss of clpP intron1 in Inga, accelerated rates of evolution in clpP for Acacia and Inga, and dN/dS ratios consistent with neutral and positive selection for several genes. These new plastomes and results provide important resources for legume comparative genomics, plant breeding, and plastid genetic engineering, while shedding further light on the complexity of plastome evolution in legumes and angiosperms.

Genome and metagenome sequencing: Using the human methyl-binding domain to partition genomic DNA derived from plant tissues.

PREMISE OF THE STUDY: Variation in the distribution of methylated CpG (methyl-CpG) in genomic DNA (gDNA) across the tree of life is biologically interesting and useful in genomic studies. We illustrate the use of human methyl-CpG-binding domain (MBD2) to fractionate angiosperm DNA into eukaryotic nuclear (methyl-CpG-rich) vs. organellar and prokaryotic (methyl-CpG-poor) elements for genomic and metagenomic sequencing projects. • METHODS: MBD2 has been used to enrich prokaryotic DNA in animal systems. Using gDNA from five model angiosperm species, we apply a similar approach to identify whether MBD2 can fractionate plant gDNA into methyl-CpG-depleted vs. enriched methyl-CpG elements. For each sample, three gDNA libraries were sequenced: (1) untreated gDNA, (2) a methyl-CpG-depleted fraction, and (3) a methyl-CpG-enriched fraction. • RESULTS: Relative to untreated gDNA, the methyl-depleted libraries showed a 3.2-11.2-fold and 3.4-11.3-fold increase in chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA), respectively. Methyl-enriched fractions showed a 1.8-31.3-fold and 1.3-29.0-fold decrease in cpDNA and mtDNA, respectively. • DISCUSSION: The application of MBD2 enabled fractionation of plant gDNA. The effectiveness was particularly striking for monocot gDNA (Poaceae). When sufficiently effective on a sample, this approach can increase the cost efficiency of sequencing plant genomes as well as prokaryotes living in or on plant tissues.

Leaf shape evolution through duplication, regulatory diversification, and loss of a homeobox gene.

In this work, we investigate morphological differences between Arabidopsis thaliana, which has simple leaves, and its relative Cardamine hirsuta, which has dissected leaves comprising distinct leaflets. With the use of genetics, interspecific gene transfers, and time-lapse imaging, we show that leaflet development requires the REDUCED COMPLEXITY (RCO) homeodomain protein. RCO functions specifically in leaves, where it sculpts developing leaflets by repressing growth at their flanks. RCO evolved in the Brassicaceae family through gene duplication and was lost in A. thaliana, contributing to leaf simplification in this species. Species-specific RCO action with respect to its paralog results from its distinct gene expression pattern in the leaf base. Thus, regulatory evolution coupled with gene duplication and loss generated leaf shape diversity by modifying local growth patterns during organogenesis.

Does hybridization drive the transition to asexuality in diploid Boechera?

Gametophytic apomixis is a common form of asexual reproduction in plants. Virtually all gametophytic apomicts are polyploids, and some view polyploidy as a prerequisite for the transition to apomixis. However, any causal link between apomixis and polyploidy is complicated by the fact that most apomictic polyploids are allopolyploids, leading some to speculate that hybridization, rather than polyploidy, enables apomixis. Diploid apomixis presents a rare opportunity to isolate the role of hybridization, and a number of diploid apomicts have been documented in the genus Boechera (Brassicaceae). Here, we present the results of a microsatellite study of 1393 morphologically and geographically diverse diploid individuals, evaluating the hypothesis that diploid Boechera apomicts are hybrids. This genus-wide dataset was made possible by the applicability of a core set of microsatellite loci in 69 of the 70 diploid Boechera species and by our ability to successfully genotype herbarium specimens of widely varying ages. With few exceptions, diploid apomicts exhibited markedly high levels of heterozygosity resulting from the combination of disparate genomes. This strongly suggests that most apomictic diploid Boechera lineages are of hybrid origin, and that the genomic consequences of hybridization allow for the transition to gametophytic apomixis in this genus.

The complex evolutionary dynamics of ancient and recent polyploidy in Leucaena (Leguminosae; Mimosoideae).

PREMISE OF THE STUDY: The evolutionary history of Leucaena has been impacted by polyploidy, hybridization, and divergent allopatric species diversification, suggesting that this is an ideal group to investigate the evolutionary tempo of polyploidy and the complexities of reticulation and divergence in plant diversification. METHODS: Parsimony- and ML-based phylogenetic approaches were applied to 105 accessions sequenced for six sequence characterized amplified region-based nuclear encoded loci, nrDNA ITS, and four cpDNA regions. Hypotheses for the origin of tetraploid species were inferred using results derived from a novel species tree and established gene tree methods and from data on genome sizes and geographic distributions. RESULTS: The combination of comprehensively sampled multilocus DNA sequence data sets and a novel methodology provide strong resolution and support for the origins of all five tetraploid species. A minimum of four allopolyploidization events are required to explain the origins of these species. The origin(s) of one tetraploid pair (L. involucrata/L. pallida) can be equally explained by two unique allopolyploidizations or a single event followed by divergent speciation. CONCLUSIONS: Alongside other recent findings, a comprehensive picture of the complex evolutionary dynamics of polyploidy in Leucaena is emerging that includes paleotetraploidization, diploidization of the last common ancestor to Leucaena, allopatric divergence among diploids, and recent allopolyploid origins for tetraploid species likely associated with human translocation of seed. These results provide insights into the role of divergence and reticulation in a well-characterized angiosperm lineage and into traits of diploid parents and derived tetraploids (particularly self-compatibility and year-round flowering) favoring the formation and establishment of novel tetraploids combinations.

Phylogenetic and population genetic analyses of diploid Leucaena (Leguminosae; Mimosoideae) reveal cryptic species diversity and patterns of divergent allopatric speciation.

PREMISE OF THE STUDY: Leucaena comprises 17 diploid species, five tetraploid species, and a complex series of hybrids whose evolutionary histories have been influenced by human seed translocation, cultivation, and subsequent spontaneous hybridization. Here we investigated patterns of evolutionary divergence among diploid Leucaena through comprehensively sampled multilocus phylogenetic and population genetic approaches to address species delimitation, interspecific relationships, hybridization, and the predominant mode of speciation among diploids. METHODS: Parsimony- and maximum-likelihood-based phylogenetic approaches were applied to 59 accessions sequenced for six SCAR-based nuclear loci, nrDNA ITS, and four cpDNA regions. Population genetic comparisons included 1215 AFLP loci representing 42 populations and 424 individuals. RESULTS: Phylogenetic results provided a well-resolved hypothesis of divergent species relationships, recovering previously recognized clades of diploids as well as newly resolved relationships. Phylogenetic and population genetic assessments identified two cryptic species that are consistent with geography and morphology. CONCLUSIONS: Findings from this study highlight the importance and utility of multilocus data in the recovery of complex evolutionary histories. The results are consistent with allopatric divergence representing the predominant mode of speciation among diploid Leucaena. These findings contrast with the potential hybrid origin of several tetraploid species and highlight the importance of human translocation of seed to the origin of these tetraploids. The recognition of one previously unrecognized species (L. cruziana) and the elevation of another taxon (L. collinsii subsp. zacapana) to specific status (L. zacapana) is consistent with a growing number of newly diagnosed species from neotropical seasonally dry forests, suggesting these communities harbor greater species diversity than previously recognized.

Arabidopsis thaliana leaf form evolved via loss of KNOX expression in leaves in association with a selective sweep.

Morphological diversity is often caused by altered gene expression of key developmental regulators. However, the precise developmental trajectories through which morphologies evolved remain poorly understood. It is also unclear to what degree genetic changes contributing to morphological divergence were fixed by natural selection. Here we investigate these problems in the context of evolutionary developmental transitions that produced the simple unlobed leaf of the model species Arabidopsis thaliana. We demonstrate that A. thaliana leaf shape likely derived from a more complex lobed ancestral state that persists in extant Arabidopsis species. We also show that evolution of the unlobed leaf form in A. thaliana involved loss of expression of the knotted1-like homeobox gene SHOOTMERISTEMLESS (STM) in leaves and that cis-regulatory divergence contributed to this process. Further, we provide evidence for a selective sweep at the A. thaliana STM locus, indicating that loss of STM expression in A. thaliana leaves may have been fixed by positive selection. In summary, our data provide key information as to when and how the characteristic leaf form of A. thaliana evolved.