Derived woodiness—the evolution of woody growth from herbaceous ancestors—has arisen hundreds of times across flowering plants, yet the environmental conditions associated with its repeated evolution remain poorly understood. Here, we analyse woodiness evolution in the mustard family (Brassicaceae; ~4,150 species) using a time-calibrated phylogeny of 2,927 species, including 374 of the 385 known woody species, together with global growth-form and climatic niche data. We infer 231 independent origins of woodiness alongside 176 reversals to herbaceousness, indicating that woodiness evolves repeatedly but remains evolutionarily unstable. Although woody species are enriched on islands, most occur on the mainland, where woodiness is consistently associated with drought and reduced frost. Correlated-evolution analyses reveal that drought is associated primarily with the persistence of woodiness, whereas reduced frost is associated with gains of woodiness and increased frost with its loss. These findings identify distinct climatic associations with the gain, persistence, and loss of woody growth forms.

PREMISE:Advances in transcriptomic and reduced representation genomic sequencing are deepening our understanding of how hybridization, reticulation, and environmental variation impact species diversification and genome size. Leucaena is a useful system for exploring the genomic basis of allopatric and allopolyploid speciation events and the effect of environmental pressures on genome size across 30° of latitude. We investigate phylogenetic relationships, the roles of polyploidy and hybridization in speciation, and genome size evolution in the genus. METHODS:Using newly generated RNA-sequencing data for Leucaena, we applied reference-guided and de novo phylotranscriptomics to reconstruct nuclear and organellar phylogenies. We then used comparative genome sizes from 252 samples and phylogenetic methods to investigate genome size evolution broadly and the impacts of environmental variables specifically. RESULTS:The phylogenetic results supported cladogenetic, rather than reticulate/hybrid, origins for most of the 19 paleotetraploid species. By contrast, gene tree data supported hybrid origins for octoploid Leucaena. The ancestral paleotetraploid genome size (1.52 pg/2 C) is relatively conserved among the paleotetraploids, rejecting our hypothesis associated with environmental variables significantly impacting genome sizes. CONCLUSIONS:The phylogenetic results illustrate the complex interplay of intrinsic and external factors that impact speciation, including ancient whole-genome duplication (WGD), cladogenesis, secondary contact, and allopolyploidy. A weak relationship between genome size and environmental variables suggests that other factors, including paleotetraploid genomic stability, have constrained genome size variation following a WGD 16+ million years ago. The findings are consistent with a small but growing number of studies identifying groups with ancient WGDs that resist diploidization associated with gene and DNA loss.

Sterile insect technique is a method to control insect pest populations by sterilizing males with ionizing radiation. However, radiation sickness lowers the fitness of sterilized males. In this study, we investigate impacts of ionizing radiation on gene transcription, specifically alternative splicing events in irradiated male Aedes aegypti mosquitoes. We compared RNA sequencing data from mosquitoes irradiated with a single standard X-ray dose of 50 Grey and un-irradiated control mosquitoes using the Multivariate Analysis of Transcript Splicing computational tool. We found that radiation exposure caused alternative splicing events in 197 genes that are involved in a variety of biological processes including the Hippo and Notch cell signaling pathways. Our results suggest that radiation damage produced by ionizing radiation can alter the splicing of genes involved in important biological functions in male Ae. aegypti mosquitoes. These findings identify several new leads for new projects aimed at understanding the impact of radiation-induced alternative splicing on mosquito fitness and improving sterile insect technique by the development of radio-resistant mosquito strains.

Leucaena is a versatile legume shrub/tree used as tropical livestock forage and in timber industries, but it is considered a high environmental weed risk due to its prolific seed production and broad environmental adaptation. Interspecific crossings between Leucaena species have been used to create non-flowering or sterile triploids that can display reduced weediness and other desirable traits for broad use in forest and agricultural settings. However, assessing the success of the hybridisation process before evaluating the sterility of putative hybrids in the target environment is advisable. Here, RNA sequencing was used to develop breeding markers for hybrid parental identification in Leucaena. RNA-seq was carried out on 20 diploid and one tetraploid Leucaena taxa, and transcriptome-wide unique genetic variants were identified relative to a L. trichandra draft genome. Over 16 million single-nucleotide polymorphisms (SNPs) and 0.8 million insertions and deletions (indels) were mapped. These sequence variations can differentiate all species of Leucaena from one another, and a core set of about 75,000 variants can be genetically mapped and transformed into genotyping arrays/chips for the conduction of population genetics, diversity assessment, and genome-wide association studies in Leucaena. For genetic fingerprinting, more than 1500 variants with even allele frequencies (0.4–0.6) among all species were filtered out for marker development and testing in planta. Notably, SNPs were preferable for future testing as they were more accurate and displayed higher transferability within the genus than indels. Hybridity testing of ca. 3300 putative progenies using SNP markers was also more reliable and highly consistent with the field observations. The developed markers pave the way for rapid, accurate, and cost-effective diversity assessments, variety identification and breeding selection in Leucaena.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods(1,2). 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 genome(3,4). Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins(5-7). 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 genes(8). This 15-fold increase in genus-level sampling relative to comparable nuclear studies(9) 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.

Based on recent achievements in phylogenetic studies of the Brassicaceae, a novel infrafamilial classifica-tion 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 Bras-sicoideae, 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, Heliophilo-dae, 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.

Early natural historians-Comte de Buffon, von Humboldt, and De Candolle-established environment and ge-ography 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 precipi-tation niche, showing profound phylogenetic niche conservatism, and that lineage turnover boundaries coin-cide with isohyets of precipitation. We reveal similar patterns on different continents, implying that evolution and dispersal follow universal processes.

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.

Context The legume shrub/tree leucaena (Leucaena leucocephala (Lam.) de Wit.) is highly regarded as a cattle fodder, although it is considered an environmental weed in many parts of Australia. Aims We investigated the feasibility of developing a forage variety of triploid leucaena through interspecific hybridisation that is sterile (without seeds) as a strategy to mitigate its weed risk. Methods A conventional breeding strategy was used to create triploid hybrids from hand-crosses between tetraploid and diploid species of leucaena. Molecular markers were developed to confirm successful crosses and flow cytometry was used to confirm the ploidy level. The plants are being evaluated for flowering behaviour, seed and herbage production across two environmentally diverse sites at Carnarvon and Kununurra in northern Western Australia. Key results During 2018/19 and 2019/20, 2260 crosses between 45 different combinations of diploid females by tetraploid males and their reciprocals have created over 3000 putative triploid hybrid plants. This is the first time that triploids have successfully been created in Australia and 10 new parental combinations were created as a world-first. Conclusions A cohort of sterile triploid plants has been identified and selections will be made based on their edible biomass productivity, insect tolerance, capacity for regrowth after cutting, nutritive value and plant chemistry (including anti-methanogenic responses). A sterile leucaena variety will have application not only in Australian grazing systems but also in other tropical regions of the world. Implications A cost-effective technology for mass vegetative propagation of leucaena will be required for commercialisation of a sterile variety.

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.

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 published family phylogenies are incompletely sampled, generally contain massive polytomies, and/or show incongruent topologies between datasets. Here, we present the most complete Brassicaceae genus-level family phylogenies to date (Brassicaceae Tree of Life, or BrassiToL) based on nuclear (>1,000 genes, almost all 349 genera and 53 tribes) and plastome (60 genes, 79% of the genera, all tribes) data. We found cytonuclear discordance between nuclear and plastome-derived phylogenies, which is likely a result of rampant hybridisation among closely and more distantly related species, and highlight rogue taxa. To evaluate the impact of this rampant hybridisation on the nuclear phylogeny reconstruction, we performed four different sampling routines that increasingly removed variable data and likely paralogs. Our resulting cleaned subset of 297 nuclear genes revealed high support for the tribes, while support for the main lineages remained relatively low. Calibration based on the 20 most clock-like nuclear genes suggests a late Eocene to late Oligocene ‘icehouse origin’ of the family. Finally, we propose five new or re-established tribes, including the recognition of Arabidopsideae, a monotypic tribe to accommodate Arabidopsis . With a worldwide community of thousands of researchers working on this family, our new, densely sampled family phylogeny will be an indispensable tool to further highlight Brassicaceae as an excellent model family for studies on biodiversity and plant biology.

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.

C. Donovan Bailey, Jeremy E. Coate, Ashley N. Eagan, and Shannon C. K. Straub Department of Biology, New Mexico State University, Las Cruces, New Mexico 88001, USA Department of Biology, Reed College, Portland, Oregon 97202, USA Department of Biology, Utah Valley University, 800 W University Parkway, Orem, Utah 84058, USA Department of Biology, Hobart and William Smith Colleges, Geneva, New York 14456, USA

This study was conducted to explore specific chill models and the mechanisms underlying rhizome bud dormancy break in Polygonatum kingianum. Rhizome buds were subjected to various chilling temperatures for different duration and then transferred to warm conditions for germination and subsequent evaluation of their response to temperature and chilling requirements. A CUkingianum model was constructed to describe the contribution of low temperature to the chill unit, and it was suggested that 2.97°C was the optimum temperature and that 11.54°C was the upper limit for bud release. The CASkingianum model showed the relationship between chilling accumulation and sprouting percentage; therefore, rhizome bud development could be predicted through the model. Weighted correlation network analysis (WGCNA) of transcriptomic data of endo-, eco- and nondormant rhizome buds generated 33 gene modules, 6 of which were significantly related to bud sprouting percentage. In addition, 7 significantly matched transcription factors (TFs) were identified from the promoters of 17 "real" hub genes, and DAG2 was the best matched TF that bound to AAAG element to regulate gene expression. The current study is valuable for developing a highly efficient strategy for seedling cultivation and provides strong candidates for key genes related to rhizome bud dormancy in P. kingianum.

Keynote paper presented at the International Leucaena Conference, 1‒3 November 2018, Brisbane, Queensland, Australia. Strategies to breed sterile leucaena for Western Australia include plant breeding and biotechnology tools to generate sterile lines at both the tetraploid and triploid ploidy levels. For tetraploids, the main target species is the commercial Leucaena leucocephala, that is well known for its potential as a high-quality, productive and persistent forage. Gene editing technologies (CRISPR) will be utilized to edit out flowering genes and develop a non-flowering L. leucocephala. For triploids, the strategy is to cross tetraploid species (L. leucocephala and/or L. diversifolia) with diploid species to generate sterile triploid hybrids. The diploid parents will include species that have good forage attributes such as L. collinsii, L. macrophylla, L. shannonii and L. pulverulenta. Several of these triploid crosses have already been created by the Department of Primary Industries and Regional Development (Perth, Western Australia) and will be evaluated in the Kimberley and Pilbara regions of Western Australia for their agronomic performance and sterility. Vegetative propagation will be required for the tetraploid gene-edited non-flowering L. leucocephala. Triploids can either be vegetatively propagated, once generated, or generated via a seed production nursery.

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 = 3415 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 eleven 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 5β-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.

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.

Keynote paper presented at the International Leucaena Conference, 1‒3 November 2018, Brisbane, Queensland, Australia. Ancestral genome duplication, genomic diploidization, allopatric diploid speciation and recent allotetraploidy (hybrid tetraploid formation) have all contributed to the complex evolutionary history of the genus Leucaena Benth. (Leguminosae: Caesalpinioideae: mimosoid clade). This complexity makes Leucaena an exemplary group to investigate the impacts of these diverse mechanisms on plant speciation across time and space. Furthermore, this complex evolutionary history offers unique opportunities and challenges for translational applied research to improve the use of Leucaena in agroforestry, livestock production, soil stabilization and enrichment and biofuels. Here we review and synthesize historical and recent research on the evolutionary history of Leucaena and highlight the availability of new genomic data resources and tools.

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.