Expanding the Triangle of U: Comparative analysis of the Hirschfeldia incana genome provides insights into chromosomal evolution, phylogenomics and high photosynthesis-related traits.

BACKGROUND AND AIMS: The Brassiceae tribe encompasses many economically important crops and exhibits high intraspecific and interspecific phenotypic variation. After a shared whole-genome triplication (WGT) event (Br-α, ~15.9 million years ago), differential lineage diversification and genomic changes contributed to an array of divergence in morphology, biochemistry, and physiology underlying photosynthesis-related traits. Here, the C3 species Hirschfeldia incana is studied as it displays high photosynthetic rates under high-light conditions. Our aim was to elucidate the evolution that gave rise to the genome of H. incana and its high-photosynthesis traits. METHODS: We reconstructed a chromosome-level genome assembly for H. incana (Nijmegen, v2.0) using nanopore and chromosome conformation capture (Hi-C) technologies, with 409Mb in size and an N50 of 52Mb (a 10× improvement over the previously published scaffold-level v1.0 assembly). The updated assembly and annotation was subsequently employed to investigate the WGT history of H. incana in a comparative phylogenomic framework from the Brassiceae ancestral genomic blocks and related diploidized crops. KEY RESULTS: Hirschfeldia incana (x=7) shares extensive genome collinearity with Raphanus sativus (x=9). These two species share some commonalities with Brassica rapa and B. oleracea (A genome, x=10 and C genome, x=9, respectively) and other similarities with B. nigra (B genome, x=8). Phylogenetic analysis revealed that H. incana and R. sativus form a monophyletic clade in between the Brassica A/C and B genomes. We postulate that H. incana and R. sativus genomes are results of hybridization or introgression of the Brassica A/C and B genome types. Our results might explain the discrepancy observed in published studies regarding phylogenetic placement of H. incana and R. sativus in relation to the "Triangle of U" species. Expression analysis of WGT retained gene copies revealed sub-genome expression divergence, likely due to neo- or sub-functionalization. Finally, we highlighted genes associated with physio-biochemical-anatomical adaptive changes observed in H. incana which likely facilitate its high-photosynthesis traits under high light. CONCLUSIONS: The improved H. incana genome assembly, annotation and results presented in this work will be a valuable resource for future research to unravel the genetic basis of its ability to maintain a high photosynthetic efficiency in high-light conditions and thereby improve photosynthesis for enhanced agricultural production.

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.

The Impact of Chromosomal Rearrangements in Speciation: From Micro- to Macroevolution.

Chromosomal rearrangements (CRs) have been known since almost the beginning of genetics. While an important role for CRs in speciation has been suggested, evidence primarily stems from theoretical and empirical studies focusing on the microevolutionary level (i.e., on taxon pairs where speciation is often incomplete). Although the role of CRs in eukaryotic speciation at a macroevolutionary level has been supported by associations between species diversity and rates of evolution of CRs across phylogenies, these findings are limited to a restricted range of CRs and taxa. Now that more broadly applicable and precise CR detection approaches have become available, we address the challenges in filling some of the conceptual and empirical gaps between micro- and macroevolutionary studies on the role of CRs in speciation. We synthesize what is known about the macroevolutionary impact of CRs and suggest new research avenues to overcome the pitfalls of previous studies to gain a more comprehensive understanding of the evolutionary significance of CRs in speciation across the tree of life.

Synteny Identifies Reliable Orthologs for Phylogenomics and Comparative Genomics of the Brassicaceae.

Large genomic data sets are becoming the new normal in phylogenetic research, but the identification of true orthologous genes and the exclusion of problematic paralogs is still challenging when applying commonly used sequencing methods such as target enrichment. Here, we compared conventional ortholog detection using OrthoFinder with ortholog detection through genomic synteny in a data set of 11 representative diploid Brassicaceae whole-genome sequences spanning the entire phylogenetic space. Then, we evaluated the resulting gene sets regarding gene number, functional annotation, and gene and species tree resolution. Finally, we used the syntenic gene sets for comparative genomics and ancestral genome analysis. The use of synteny resulted in considerably more orthologs and also allowed us to reliably identify paralogs. Surprisingly, we did not detect notable differences between species trees reconstructed from syntenic orthologs when compared with other gene sets, including the Angiosperms353 set and a Brassicaceae-specific target enrichment gene set. However, the synteny data set comprised a multitude of gene functions, strongly suggesting that this method of marker selection for phylogenomics is suitable for studies that value downstream gene function analysis, gene interaction, and network studies. Finally, we present the first ancestral genome reconstruction for the Core Brassicaceae which predating the Brassicaceae lineage diversification ∼25 million years ago.

The Gynandropsis gynandra genome provides insights into whole-genome duplications and the evolution of C4 photosynthesis in Cleomaceae.

Gynandropsis gynandra (Cleomaceae) is a cosmopolitan leafy vegetable and medicinal plant, which has also been used as a model to study C4 photosynthesis due to its evolutionary proximity to C3 Arabidopsis (Arabidopsis thaliana). Here, we present the genome sequence of G. gynandra, anchored onto 17 main pseudomolecules with a total length of 740 Mb, an N50 of 42 Mb and 30,933 well-supported gene models. The G. gynandra genome and previously released genomes of C3 relatives in the Cleomaceae and Brassicaceae make an excellent model for studying the role of genome evolution in the transition from C3 to C4 photosynthesis. Our analyses revealed that G. gynandra and its C3 relative Tarenaya hassleriana shared a whole-genome duplication event (Gg-α), then an addition of a third genome (Th-α, +1×) took place in T. hassleriana but not in G. gynandra. Analysis of syntenic copy number of C4 photosynthesis-related gene families indicates that G. gynandra generally retained more duplicated copies of these genes than C3T. hassleriana, and also that the G. gynandra C4 genes might have been under positive selection pressure. Both whole-genome and single-gene duplication were found to contribute to the expansion of the aforementioned gene families in G. gynandra. Collectively, this study enhances our understanding of the polyploidy history, gene duplication and retention, as well as their impact on the evolution of C4 photosynthesis in Cleomaceae.

Recent speciation associated with range expansion and a shift to self-fertilization in North American Arabidopsis.

The main processes classically evoked for promoting reproductive isolation and speciation are geographic separation reducing gene flow among populations, divergent selection, and chance genomic change. In a case study, we present evidence that the additional factors of climate change, range expansion and a shift in mating towards inbreeding can initiate the processes leading to parapatric speciation. At the end of the last Pleistocene glaciation cycle, the North American plant Arabidopsis lyrata expanded its range and concomitantly lost its reproductive mode of outcrossing multiple times. We show that in one of the newly colonized areas, the self-fertilizing recolonization lineage of A. lyrata gave rise to selfing A. arenicola, which expanded its range to subarctic and arctic Canada and Greenland, while the parental species remained restricted to temperate North America. Despite the vast range expansion by the new species, mutational load did not increase, probably because of selfing and quasi-clonal selection. We conclude that such peripheral parapatric speciation combined with range expansion and inbreeding may be an important but so far overlooked mode of speciation.

Parallel reduction in flowering time from de novo mutations enable evolutionary rescue in colonizing lineages.

Understanding how populations adapt to abrupt environmental change is necessary to predict responses to future challenges, but identifying specific adaptive variants, quantifying their responses to selection and reconstructing their detailed histories is challenging in natural populations. Here, we use Arabidopsis from the Cape Verde Islands as a model to investigate the mechanisms of adaptation after a sudden shift to a more arid climate. We find genome-wide evidence of adaptation after a multivariate change in selection pressures. In particular, time to flowering is reduced in parallel across islands, substantially increasing fitness. This change is mediated by convergent de novo loss of function of two core flowering time genes: FRI on one island and FLC on the other. Evolutionary reconstructions reveal a case where expansion of the new populations coincided with the emergence and proliferation of these variants, consistent with models of rapid adaptation and evolutionary rescue.

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.

Genomic Blocks in Aethionema arabicum Support Arabideae as Next Diverging Clade in Brassicaceae.

The tribe Aethionemeae is sister to all other crucifers, making it a crucial group for unraveling genome evolution and phylogenetic relationships within the crown group Brassicaceae. In this study, we extend the analysis of Brassicaceae genomic blocks (GBs) to Aethionema whereby we identified unique block boundaries shared only with the tribe Arabideae. This was achieved using bioinformatic methods to analyze synteny between the recently updated genome sequence of Aethionema arabicum and other high-quality Brassicaceae genome sequences. We show that compared to the largely conserved genomic structure of most non-polyploid Brassicaceae lineages, GBs are highly rearranged in Aethionema. Furthermore, we detected similarities between the genomes of Aethionema and Arabis alpina, in which also a high number of genomic rearrangements compared to those of other Brassicaceae was found. These similarities suggest that tribe Arabideae, a clade showing conflicting phylogenetic position between studies, may have diverged before diversification of the other major lineages, and highlight the potential of synteny information for phylogenetic inference.

Lineage-specific adaptation to climate involves flowering time in North American Arabidopsis lyrata.

Adaptation to local climatic conditions is commonly found within species, but whether it involves the same intraspecific genomic variants is unknown. We studied this question in North American Arabidopsis lyrata, whose current distribution is shaped by post-glacial range expansion from two refugia, resulting in two distinct genetic clusters covering comparable climatic gradients. Using pooled whole-genome sequence data of 41 outcrossing populations, we identified loci associated with three niche-determining climatic variables in the two clusters and compared these outliers. Little evidence was found for parallelism in climate adaptation for single nucleotide polymorphisms (SNPs) and for genes with an accumulation of outlier SNPs. Significantly increased selection coefficients supported them as candidates of climate adaptation. However, the fraction of gene ontology (GO) terms shared between clusters was higher compared to outlier SNPs and outlier genes, suggesting that selection acts on similar pathways but not necessarily the same genes. Enriched GO terms involved responses to abiotic and biotic stress, circadian rhythm and development, with flower development and reproduction being among the most frequently detected. In line with GO enrichment, regulators of flowering time were detected as outlier genes. Our results suggest that while adaptation to environmental gradients on the genomic level are lineage-specific in A. lyrata, similar biological processes seem to be involved. Differential loss of standing genetic variation, probably driven by genetic drift, can in part account for the lack of parallel evolution on the genomic level.