The dimorphic diaspore model Aethionema arabicum (Brassicaceae): Distinct molecular and morphological control of responses to parental and germination temperatures.

Plants in habitats with unpredictable conditions often have diversified bet-hedging strategies that ensure fitness over a wider range of variable environmental factors. A striking example is the diaspore (seed and fruit) heteromorphism that evolved to maximize species survival in Aethionema arabicum (Brassicaceae) in which external and endogenous triggers allow the production of two distinct diaspores on the same plant. Using this dimorphic diaspore model, we identified contrasting molecular, biophysical, and ecophysiological mechanisms in the germination responses to different temperatures of the mucilaginous seeds (M+ seed morphs), the dispersed indehiscent fruits (IND fruit morphs), and the bare non-mucilaginous M- seeds obtained by pericarp (fruit coat) removal from IND fruits. Large-scale comparative transcriptome and hormone analyses of M+ seeds, IND fruits, and M- seeds provided comprehensive datasets for their distinct thermal responses. Morph-specific differences in co-expressed gene modules in seeds, as well as in seed and pericarp hormone contents, identified a role of the IND pericarp in imposing coat dormancy by generating hypoxia affecting ABA sensitivity. This involved expression of morph-specific transcription factors, hypoxia response and cell wall-remodeling genes, as well as altered abscisic acid (ABA) metabolism, transport, and signaling. Parental temperature affected ABA contents and ABA-related gene expression and altered IND pericarp biomechanical properties. Elucidating the molecular framework underlying the diaspore heteromorphism can provide insight into developmental responses to globally changing temperatures.

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.

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.

'Root of all success': Plasticity in root architecture of invasive wild radish for adaptive benefit.

Successful plant establishment in a particular environment depends on the root architecture of the seedlings and the extent of edaphic resource utilization. However, diverse habitats often pose a predicament on the suitability of the fundamental root structure of a species that evolved over a long period. We hypothesized that the plasticity in the genetically controlled root architecture in variable habitats provides an adaptive advantage to worldwide-distributed wild radish (Raphanus raphanistrum, Rr) over its close relative (R. pugioniformis, Rp) that remained endemic to the East Mediterranean region. To test the hypothesis, we performed a reciprocal comparative analysis between the two species, growing in a common garden experiment on their native soils (Hamra/Sandy for Rr, Terra Rossa for Rp) and complementary controlled experiments mimicking the major soil compositions. Additionally, we analyzed the root growth kinetics via semi-automated digital profiling and compared the architecture between Rr and Rp. In both experiments, the primary roots of Rr were significantly longer, developed fewer lateral roots, and showed slower growth kinetics than Rp. Multivariate analyses of seven significant root architecture variables revealed that Rr could successfully adapt to different surrogate growth conditions by only modulating their main root length and number of lateral roots. In contrast, Rp needs to modify several other root parameters, which are very resource-intensive, to grow on non-native soil. Altogether the findings suggest an evo-devo adaptive advantage for Rr as it can potentially establish in various habitats with the minimal tweak of key root parameters, hence allocating resources for other developmental requirements.

Corrigendum: Evolution of tandem repeats is mirroring post-polyploid cladogenesis in Heliophila (Brassicaceae).

[This corrects the article DOI: 10.3389/fpls.2020.607893.].

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.

Nested whole-genome duplications coincide with diversification and high morphological disparity in Brassicaceae.

Angiosperms have become the dominant terrestrial plant group by diversifying for ~145 million years into a broad range of environments. During the course of evolution, numerous morphological innovations arose, often preceded by whole genome duplications (WGD). The mustard family (Brassicaceae), a successful angiosperm clade with ~4000 species, has been diversifying into many evolutionary lineages for more than 30 million years. Here we develop a species inventory, analyze morphological variation, and present a maternal, plastome-based genus-level phylogeny. We show that increased morphological disparity, despite an apparent absence of clade-specific morphological innovations, is found in tribes with WGDs or diversification rate shifts. Both are important processes in Brassicaceae, resulting in an overall high net diversification rate. Character states show frequent and independent gain and loss, and form varying combinations. Therefore, Brassicaceae pave the way to concepts of phylogenetic genome-wide association studies to analyze the evolution of morphological form and function.

Morphologically and physiologically diverse fruits of two Lepidium species differ in allocation of glucosinolates into immature and mature seed and pericarp.

The morphology and physiology of diaspores play crucial roles in determining the fate of seeds in unpredictable habitats. In some genera of the Brassicaceae different types of diaspores can be found. Lepidium appelianum produces non-dormant seeds within indehiscent fruits while in L. campestre dormant seeds are released from dehiscent fruits. We investigated whether the allocation of relevant defence compounds into different tissues in different Lepidium species may be related to the diverse dispersal strategy (indehiscent and dehiscent) and seed physiology (non-dormant and dormant). Total glucosinolate concentration and composition were analysed in immature and mature seeds and pericarps of L. appelianum and L. campestre using high-performance liquid chromatography. Moreover, for comparison, transgenic RNAi L. campestre lines were used that produce indehiscent fruits due to silencing of LcINDEHISCENCE, the INDEHISCENCE ortholog of L. campestre. Total glucosinolate concentrations were lower in immature compared to mature seeds in all studied Lepidium species and transgenic lines. In contrast, indehiscent fruits of L. appelianum maintained their total glucosinolate concentration in mature pericarps compared to immature ones, while in dehiscent L. campestre and in indehiscent RNAi-LcIND L. campestre a significant decrease in total glucosinolate concentrations from immature to mature pericarps could be detected. Indole glucosinolates were detected in lower abundance than the other glucosinolate classes (aliphatic and aromatic). Relatively high concentrations of 4-methoxyindol-3-ylmethyl glucosinolate were found in mature seeds of L. appelianum compared to other tissues, while no indole glucosinolates were detected in mature diaspores of L. campestre. The diaspores of the latter species may rather depend on aliphatic and aromatic glucosinolates for long-term protection. The allocation patterns of glucosinolates correlate with the morpho-physiologically distinct fruits of L. appelianum and L. campestre and may be explained by the distinct dispersal strategies and the dormancy status of both species.

Evolution of Tandem Repeats Is Mirroring Post-polyploid Cladogenesis in Heliophila (Brassicaceae).

The unigeneric tribe Heliophileae encompassing more than 100 Heliophila species is morphologically the most diverse Brassicaceae lineage. The tribe is endemic to southern Africa, confined chiefly to the southwestern South Africa, home of two biodiversity hotspots (Cape Floristic Region and Succulent Karoo). The monospecific Chamira (C. circaeoides), the only crucifer species with persistent cotyledons, is traditionally retrieved as the closest relative of Heliophileae. Our transcriptome analysis revealed a whole-genome duplication (WGD) ∼26.15-29.20 million years ago, presumably preceding the Chamira/Heliophila split. The WGD was then followed by genome-wide diploidization, species radiations, and cladogenesis in Heliophila. The expanded phylogeny based on nuclear ribosomal DNA internal transcribed spacer (ITS) uncovered four major infrageneric clades (A-D) in Heliophila and corroborated the sister relationship between Chamira and Heliophila. Herein, we analyzed how the diploidization process impacted the evolution of repetitive sequences through low-coverage whole-genome sequencing of 15 Heliophila species, representing the four clades, and Chamira. Despite the firmly established infrageneric cladogenesis and different ecological life histories (four perennials vs. 11 annual species), repeatome analysis showed overall comparable evolution of genome sizes (288-484 Mb) and repeat content (25.04-38.90%) across Heliophila species and clades. Among Heliophila species, long terminal repeat (LTR) retrotransposons were the predominant components of the analyzed genomes (11.51-22.42%), whereas tandem repeats had lower abundances (1.03-12.10%). In Chamira, the tandem repeat content (17.92%, 16 diverse tandem repeats) equals the abundance of LTR retrotransposons (16.69%). Among the 108 tandem repeats identified in Heliophila, only 16 repeats were found to be shared among two or more species; no tandem repeats were shared by Chamira and Heliophila genomes. Six "relic" tandem repeats were shared between any two different Heliophila clades by a common descent. Four and six clade-specific repeats shared among clade A and C species, respectively, support the monophyly of these two clades. Three repeats shared by all clade A species corroborate the recent diversification of this clade revealed by plastome-based molecular dating. Phylogenetic analysis based on repeat sequence similarities separated the Heliophila species to three clades [A, C, and (B+D)], mirroring the post-polyploid cladogenesis in Heliophila inferred from rDNA ITS and plastome sequences.

Naturally-primed life strategy plasticity of dimorphic Aethionema arabicum facilitates optimal habitat colonization.

Plasticity in plant dispersal traits can maximise the ability of a plant species to survive in stressful environments during colonization. Aethionema arabicum (Brassicaceae) is a dimorphic annual species that is hypothesized to survive stressful conditions during colonization due to adaptive plasticity in life-phase (vegetative vs sexual) and fruit morph (dehiscent [DEH] vs indehiscent fruits [IND]). We tested for adaptive plasticity in life-phase and fruit morphs along laboratory environmental stress gradients found in the natural habitats of Ae. arabicum. We considered optimal environmental conditions (750-2000 m above sea level) to be those that resulted in the following fitness parameters: higher biomass and a higher total number of fruits compared to stressful habitats. We found evidence of plasticity in life-phase and fruit-morph along a stressful environmental gradient. High hydrothermal stress proportionally increased the number of dehiscent morphs and non-dormant seeds germinating in autumn. This offsets natural phenology towards dry and cold winter (less hydrothermal stress), yielding fewer fruits that dehisce in the next generation. We conclude that the plastic responses of Ae. arabicum to natural stress gradients constitute a strategy of long-term adaptive benefits and favouring potential pathways of colonisation of the optimal habitat.

Dispersal biophysics and adaptive significance of dimorphic diaspores in the annual Aethionema arabicum (Brassicaceae).

Heteromorphic diaspores (fruits and seeds) are an adaptive bet-hedging strategy to cope with spatiotemporally variable environments, particularly fluctuations in favourable temperatures and unpredictable precipitation regimes in arid climates. We conducted comparative analyses of the biophysical and ecophysiological properties of the two distinct diaspores (mucilaginous seed (M+ ) vs indehiscent (IND) fruit) in the dimorphic annual Aethionema arabicum (Brassicaceae), linking fruit biomechanics, dispersal aerodynamics, pericarp-imposed dormancy, diaspore abscisic acid (ABA) concentration, and phenotypic plasticity of dimorphic diaspore production to its natural habitat and climate. Two very contrasting dispersal mechanisms of the A. arabicum dimorphic diaspores were revealed. Dehiscence of large fruits leads to the release of M+ seed diaspores, which adhere to substrata via seed coat mucilage, thereby preventing dispersal (antitelechory). IND fruit diaspores (containing nonmucilaginous seeds) disperse by wind or water currents, promoting dispersal (telechory) over a longer range. The pericarp properties confer enhanced dispersal ability and degree of dormancy on the IND fruit morph to support telechory, while the M+ seed morph supports antitelechory. Combined with the phenotypic plasticity to produce more IND fruit diaspores in colder temperatures, this constitutes a bet-hedging survival strategy to magnify the prevalence in response to selection pressures acting over hilly terrain.

Two-tier morpho-chemical defence tactic in Aethionema via fruit morph plasticity and glucosinolates allocation in diaspores.

Fruit dimorphism and the production of glucosinolates (GSLs) are two specific life history traits found in the members of Brassicales, which aid to optimize seed dispersal and defence against antagonists, respectively. We hypothesized that the bipartite dispersal strategy demands a tight control over the production of fruit morphs with expectedly differential allocation of defensive anticipins (GSLs). In dimorphic Aethionema, herbivory by Plutella xylostella at a young stage triggered the production of more dehiscent (seeds released from fruit) than indehiscent fruit morphs (seeds enclosed within persistent pericarp) on the same plant upon maturity. Total GSL concentrations were highest in the mature seeds of dehiscent fruits from Aethionema arabicum and Aethionema saxatile among the different ontogenetic stages of the diaspores. Multivariate analyses of GSL profiles indicated significantly higher concentrations of specific indole GSLs in the diaspores, which require optimal defence after dispersal (i.e., seeds of dehiscent and fruit/pericarp of indehiscent fruit). Bioassays with a potentially coinhabitant fungus, Aspergillus quadrilineatus, support the distinct defensive potential of the diaspores corresponding to their GSL allocation. These findings indicate a two-tier morpho-chemical defence tactic of Aethionema via better protected fruit morphs and strategic provision of GSLs that optimize protection to the progeny for survival in nature.

Hybridization-facilitated genome merger and repeated chromosome fusion after 8 million years.

The small genus Ricotia (nine species, Brassicaceae) is confined to the eastern Mediterranean. By comparative chromosome painting and a dated multi-gene chloroplast phylogeny, we reconstructed the origin and subsequent evolution of Ricotia. The ancestral Ricotia genome originated through hybridization between two older genomes with n = 7 and n = 8 chromosomes, respectively, on the Turkish mainland during the Early Miocene (c. 17.8 million years ago, Ma). Since then, the allotetraploid (n = 15) genome has been altered by two independent descending dysploidies (DD) to n = 14 in Ricotia aucheri and the Tenuifolia clade (2 spp.). By the Late Miocene (c. 10 Ma), the latter clade started to evolve in the most diverse Ricotia core clade (6 spp.), the process preceded by a DD event to n = 13. It is noteworthy that this dysploidy was mediated by a unique chromosomal rearrangement, merging together the same two chromosomes as were merged during the origin of a fusion chromosome within the paternal n = 7 genome c. 20 Ma. This shows that within a time period of c. 8 Myr genome evolution can repeat itself and that structurally very similar chromosomes may originate repeatedly from the same ancestral chromosomes by different pathways (end-to-end translocation versus nested chromosome insertion).

A Dead Gene Walking: Convergent Degeneration of a Clade of MADS-Box Genes in Crucifers.

Genes are "born," and eventually they "die." These processes shape the phenotypic evolution of organisms and are hence of great biological interest. If genes die in plants, they generally do so quite rapidly. Here, we describe the fate of GOA-like genes that evolve in a dramatically different manner. GOA-like genes belong to the subfamily of Bsister genes of MIKC-type MADS-box genes. Typical MIKC-type genes encode conserved transcription factors controlling plant development. We show that ABS-like genes, a clade of Bsister genes, are indeed highly conserved in crucifers (Brassicaceae) maintaining the ancestral function of Bsister genes in ovule and seed development. In contrast, their closest paralogs, the GOA-like genes, have been undergoing convergent gene death in Brassicaceae. Intriguingly, erosion of GOA-like genes occurred after millions of years of coexistence with ABS-like genes. We thus describe Delayed Convergent Asymmetric Degeneration, a so far neglected but possibly frequent pattern of duplicate gene evolution that does not fit classical scenarios. Delayed Convergent Asymmetric Degeneration of GOA-like genes may have been initiated by a reduction in the expression of an ancestral GOA-like gene in the stem group of Brassicaceae and driven by dosage subfunctionalization. Our findings have profound implications for gene annotations in genomics, interpreting patterns of gene evolution and using genes in phylogeny reconstructions of species.

Fruit fracture biomechanics and the release of Lepidium didymum pericarp-imposed mechanical dormancy by fungi.

The biomechanical and ecophysiological properties of plant seed/fruit structures are fundamental to survival in distinct environments. Dispersal of fruits with hard pericarps (fruit coats) encasing seeds has evolved many times independently within taxa that have seed dispersal as their default strategy. The mechanisms by which the constraint of a hard pericarp determines germination timing in response to the environment are currently unknown. Here, we show that the hard pericarp of Lepidium didymum controls germination solely by a biomechanical mechanism. Mechanical dormancy is conferred by preventing full phase-II water uptake of the encased non-dormant seed. The lignified endocarp has biomechanically and morphologically distinct regions that serve as predetermined breaking zones. This pericarp-imposed mechanical dormancy is released by the activity of common fungi, which weaken these zones by degrading non-lignified pericarp cells. We propose that the hard pericarp with this biomechanical mechanism contributed to the global distribution of this species in distinct environments.

Anatolian origins and diversification of Aethionema, the sister lineage of the core Brassicaceae.

PREMISE OF THE STUDY: The Irano-Turanian region harbors three biodiversity hotspots and ∼25% of Brassicaceae species are endemic to the region. Aethionema (∼61 species) is the sister lineage to the core Brassicaceae and occurs mainly in the Irano-Turanian region. The evolutionary important position of Aethionema makes it an ideal reference for broader comparative genetics and genomics. To understand the evolution of Aethionema, and for a broader understanding of crucifer evolution, a time-calibrated phylogenetic tree and biogeographical history of the genus is needed. METHODS: Seventy-six plastome coding regions and nuclear rDNA genes, mainly from herbarium material, covering 75% of all Aethionema species, were used to resolve a time-calibrated Aethionema phylogeny. The different clades were characterized based on four morphological characters. The ancestral area of Aethionema was estimated with historical biogeographical analyses. KEY RESULTS: Three well-supported major clades within Aethionema were resolved. The ancestral area reconstruction and divergence-time estimates are consistent with major dispersal events during the Pliocene from the Anatolian Diagonal. CONCLUSIONS: We find that most Aethionema lineages originated along the Anatolian Diagonal, a floristic bridge connecting the east to the west, during the Pliocene. The dispersal of Aethionema correlates with the local geological events, such as the uplift of the Anatolian and Iranian plateaus and the formation of the major mountain ranges of the Irano-Turanian region. Knowing the paleo-ecological context for the evolution of Aethionema, in addition to the other lineages of Brassicaceae, facilitates our broader understanding for trait evolution and species diversification across the Brassicaceae.