Summary Evolutionary arms‐races between plants and insect herbivores have long been proposed to generate key innovations such as plant toxins and detoxification mechanisms that can drive diversification of the interacting species. A novel front‐line of plant defence is the killing of herbivorous insect eggs. We test whether an egg‐killing plant trait has an evolutionary basis in such a plant–insect arms‐race. Within the crucifer family (Brassicaceae), some species express a hypersensitive response (HR)‐like necrosis underneath butterfly eggs (Pieridae) that leads to eggs desiccating or falling off the plant. We studied the phylogenetic distribution of this trait, its egg‐killing effect on and elicitation by butterflies, by screening 31 Brassicales species, and nine Pieridae species. We show a clade‐specific induction of strong, egg‐killing HR‐like necrosis mainly in species of the Brassiceae tribe including Brassica crops and close relatives. The necrosis is strongly elicited by pierid butterflies that are specialists of crucifers. Furthermore, HR‐like necrosis is linked to PR1 defence gene expression, accumulation of reactive oxygen species and cell death, eventually leading to egg‐killing. Our findings suggest that the plants’ egg‐killing trait is a new front on the evolutionary arms‐race between Brassicaceae and pierid butterflies beyond the well‐studied plant toxins that have evolved against their caterpillars.

The genus Aethionema is a sister-group to the core-group of the Brassicaceae family that includes Arabidopsis thaliana and the Brassica crops. Thus, Aethionema is phylogenetically well-placed for the investigation and understanding of genome and trait evolution across the family. We aimed to improve the quality of the reference genome draft version of the annual species Aethionema arabicum Second, we constructed the first Ae. arabicum genetic map. The improved reference genome and genetic map enabled the development of each other. We started with the initially published genome (version 2.5). PacBio and MinION sequencing together with genetic map v2.5 were incorporated to produce the new reference genome v3.0. The improved genome contains 203 MB of sequence, with approximately 94% of the assembly made up of called (non-gap) bases, assembled into 2,883 scaffolds (with only 6% of the genome made up of non-called bases (Ns)). The N50 (10.3 MB) represents an 80-fold increase over the initial genome release. We generated a Recombinant Inbred Line (RIL) population that was derived from two ecotypes: Cyprus and Turkey (the reference genotype. Using a Genotyping by Sequencing (GBS) approach, we generated a high-density genetic map with 749 (v2.5) and then 632 SNPs (v3.0) was generated. The genetic map and reference genome were integrated, thus greatly improving the scaffolding of the reference genome into 11 linkage groups. We show that long-read sequencing data and genetics are complementary, resulting in an improved genome assembly in Ae. arabicum They will facilitate comparative genetic mapping work for the Brassicaceae family and are also valuable resources to investigate wide range of life history traits in Aethionema.

The predominantly South-African plant genus Pelargonium L'Hér. (Geraniaceae) displays remarkable morphological diversity, several basic chromosome numbers as well as high levels of organelle genomic rearrangements, and represents the 7th largest Cape Floristic Region clade. In this study, we reconstructed a phylogenetic tree based on 74 plastome exons and nuclear rDNA ITS regions for 120 species, which represents 43% taxon coverage for Pelargonium. We also performed a dating analysis to examine the timing of the major radiations in the genus. Phylogenetic analyses of nucleotide, amino acid, and ITS alignments confirmed the previously-documented subgeneric split into five main clades ((C1,C2),(B(A1,A2))) although clade only A1 received low bootstrap support. Using calibration evidence from a range of sources the Pelargonium crown age was estimated to be 9.7 My old, much younger than previous estimates for the genus but similar to recent studies of other Cape Floristic lineages that are part of both Fynbos and Succulent Karoo biomes.

The genus Aethionema is sister to the core Brassicaceae (including Arabidopsis thaliana ) and thus has an important evolutionary position for comparative analyses. Aethionema arabicum (Brassicaceae) is emerging as a model to understand the evolution of various traits. We generated transcriptome data for seven Ae. arabicum genotypes across the species range including Cyprus, Iran and Turkey. Combined flow cytometry and single nucleotide polymorphism (SNP) analyses identified distinct tetraploid (Iranian) and diploid populations (Turkish/Cypriot). The Turkish and Cypriot lines had a higher genome-wide genetic diversity than the Iranian lines. However, one genomic region contained genes with a higher diversity in the Iranian than the Turkish/Cypriot lines. Sixteen percent of the genes in this region were chaperonins involved in protein folding. Additionally, an analysis of glucosinolate profiles, chemical defence compounds of the Brassicaceae, showed a difference in diversity of indolic glucosinolates between the Iranian and Turkish/Cypriot lines. We showed that different Ae. arabicum individuals have different ploidy levels depending on their location (Iranian versus Turkish/Cypriot). Moreover, these differences between the populations are also shown in their defence compounds.

PREMISE OF THE STUDY:Previous phylogenetic studies employing molecular markers have yielded various insights into the evolutionary history across Brassicales, but many relationships between families remain poorly supported or unresolved. A recent phylotranscriptomic approach utilizing 1155 nuclear markers obtained robust estimates for relationships among 14 of 17 families. Here we report a complete family-level phylogeny estimated using the plastid genome.METHODS:We conducted phylogenetic analyses on a concatenated data set comprising 44,926 bp from 72 plastid genes for species distributed across all 17 families. Our analysis includes three additional families, Tovariaceae, Salvadoraceae, and Setchellanthaceae, that were omitted in the previous phylotranscriptomic study.KEY RESULTS:Our phylogenetic analyses obtained fully resolved and strongly supported estimates for all nodes across Brassicales. Importantly, these findings are congruent with the topology reported in the phylotranscriptomic study. This consistency suggests that future studies could utilize plastid genomes as markers for resolving relationships within some notoriously difficult clades across Brassicales. We used this new phylogenetic framework to verify the placement of the At-α event near the origin of Brassicaceae, with median date estimates of 31.8 to 42.8 million years ago and restrict the At-β event to one of two nodes with median date estimates between 85 to 92.2 million years ago. These events ultimately gave rise to novel chemical defenses and are associated with subsequent shifts in net diversification rates.CONCLUSIONS:We anticipate that these findings will aid future comparative evolutionary studies across Brassicales, including selecting candidates for whole-genome sequencing projects.

The biochemical defense of plants can change during their life-cycle and impact herbivore feeding and plant fitness. The annual species Aethionema arabicum is part of the sister clade to all other Brassicaceae. Hence, it holds a phylogenetically important position for studying crucifer trait evolution. Glucosinolates (GS) are essentially Brassicales-specific metabolites involved in plant defense. Using two Ae. arabicum accessions (TUR and CYP) we identify substantial differences in glucosinolate profiles and quantities between lines, tissues and developmental stages. We find tissue specific side-chain modifications in aliphatic GS: methylthioalkyl in leaves, methylsulfinylalkyl in fruits, and methylsulfonylalkyl in seeds. We also find large differences in absolute glucosinolate content between the two accessions (up to 10-fold in fruits) that suggest a regulatory factor is involved that is not part of the quintessential glucosinolate biosynthetic pathway. Consistent with this hypothesis, we identified a single major multi-trait quantitative trait locus controlling total GS concentration across tissues in a recombinant inbred line population derived from TUR and CYP. With fine-mapping, we narrowed the interval to a 58 kb region containing 15 genes, but lacking any known GS biosynthetic genes. The interval contains homologs of both the sulfate transporter SULTR2;1 and FLOWERING LOCUS C. Both loci have diverse functions controlling plant physiological and developmental processes and thus are potential candidates regulating glucosinolate variation across the life-cycle of Aethionema. Future work will investigate changes in gene expression of the candidates genes, the effects of GS variation on insect herbivores and the trade-offs between defense and reproduction.

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.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.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.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.

Understanding how plants cope with changing habitats is a timely and important topic in plant research. Phenotypic plasticity describes the capability of a genotype to produce different phenotypes when exposed to different environmental conditions. In contrast, the constant production of a set of distinct phenotypes by one genotype mediates bet hedging, a strategy that reduces the temporal variance in fitness at the expense of a lowered arithmetic mean fitness. Both phenomena are thought to represent important adaptation strategies to unstable environments. However, little is known about the underlying mechanisms of these phenomena, partly due to the lack of suitable model systems. We used phylogenetic and comparative analyses of fruit and seed anatomy, biomechanics, physiology, and environmental responses to study fruit and seed heteromorphism, a typical morphological basis of a bet-hedging strategy of plants, in the annual Brassicaceae species Aethionema arabicum Our results indicate that heteromorphism evolved twice within the Aethionemeae, including once for the monophyletic annual Aethionema clade. The dimorphism of Ae. arabicum is associated with several anatomic, biomechanical, gene expression, and physiological differences between the fruit and seed morphs. However, fruit ratios and numbers change in response to different environmental conditions. Therefore, the life-history strategy of Ae. arabicum appears to be a blend of bet hedging and plasticity. Together with the available genomic resources, our results pave the way to use this species in future studies intended to unravel the molecular control of heteromorphism and plasticity.

Herbarium genomics is proving promising as next-generation sequencing approaches are well suited to deal with the usually fragmented nature of archival DNA. We show that routine assembly of partial plastome sequences from herbarium specimens is feasible, from total DNA extracts and with specimens up to 146 years old. We use genome skimming and an automated assembly pipeline, Iterative Organelle Genome Assembly, that assembles paired-end reads into a series of candidate assemblies, the best one of which is selected based on likelihood estimation. We used 93 specimens from 12 different Angiosperm families, 73 of which were from herbarium material with ages up to 146 years old. For 84 specimens, a sufficient number of paired-end reads were generated (in total 9.4 × 1012 nucleotides), yielding successful plastome assemblies for 74 specimens. Those derived from herbarium specimens have lower fractions of plastome-derived reads compared with those from fresh and silica-gel-dried specimens, but total herbarium assembly lengths are only slightly shorter. Specimens from wet-tropical conditions appear to have a higher number of contigs per assembly and lower N50 values. We find no significant correlation between plastome coverage and nuclear genome size (C value) in our samples, but the range of C values included is limited. Finally, we conclude that routine plastome sequencing from herbarium specimens is feasible and cost-effective (compared with Sanger sequencing or plastome-enrichment approaches), and can be performed with limited sample destruction.

Sheet 1 shows the transcript names of Aethionema arabicum, Tarenaya hassleriana and Arabidopsis thaliana and whether or not they are conserved by position or only by sequence across the different lineages. Sheet 2 shows the distances of the positionally conserved LncRNAs to the nearest end of A. thaliana chromosomes. (XLSX 22 kb)

Background: Long non-coding RNAs (LncRNAs) have been identified as gene regulatory elements that influence the transcription of their neighbouring protein-coding genes. The discovery of LncRNAs in animals has stimulated genome-wide scans for these elements across plant genomes. Recently, 6480 LincRNAs were putatively identified in Arabidopsis thaliana (Brassicaceae), however there is limited information on their conservation.Results: Using a phylogenomics approach, we assessed the positional and sequence conservation of these LncRNAs by analyzing the genomes of the basal Brassicaceae species Aethionema arabicum and Tarenaya hassleriana of the sister-family Cleomaceae. Furthermore, we generated transcriptomes for another three Aethionema species and one other Cleomaceae species to validate their transcriptional activity. We show that a subset of LncRNAs are highly diverged at the nucleotide level, but conserved by position (syntenic). Positionally conserved LncRNAs that are expressed neighbour important developmental and physiological genes. Interestingly, >65 % of the positionally conserved LncRNAs are located within 2.5 Mb of telomeres in Arabidopsis thaliana chromosomes.Conclusion: These results highlight the importance of analysing not only sequence conservation, but also positional conservation of non-coding genetic elements in plants including LncRNAs.

Many large and economically important plant groups (e.g. Brassicaceae, Poaceae, Asteraceae, Fabaceae and Solanaceae) have had ancient whole genome duplications (WGDs) occurring near or at the time of their origins, suggesting that WGD contributed to the origin of novel key traits and drove species diversification. However, these large clades show phylogenetic asymmetries with a species-rich crown group and a species-poor sister clade, suggesting significant 'lag-times' between WGDs and radiations. The species-poor sister groups share many key traits, but are often restricted to the hypothesized center of origin for the larger clade. Thus, the ultimate success of the crown group does not only involve the WGD and novel key traits, but largely subsequent evolutionary phenomena including later migration events, changing environmental conditions and/or differential extinction rates.

The plant family Brassicaceae (or crucifers) is an economically important group that includes many food crops (e.g. cabbages and radishes), horticultural species (e.g. Draba, Iberis, Lunaria), and model plant species (particularly Arabidopsis thaliana). Because of the fundamental importance of A. thaliana to plant biology, it makes the Brassicaceae an ideal system for comparative genomics and to test wider evolutionary, ecological and speciation hypotheses. One such hypothesis is the ‘Whole Genome Duplication Radiation Lag Time’ (WGD-RLT) model for the role of polyploidy on the evolution of important plant families such as the Brassicaceae. The WGD-RLT model indicates a higher rate of diversification of a core-group compared to its sister group, due to a lag time after a whole genome duplication event that made it possible for novel traits or geo- or ecological events to increase the core groups diversification rate. Aethionema is the species-poor sister genus of the core Brassicaceae and hence is at an important comparative position to analyse trait and genomic evolution of the species-rich core group. Aethionema species occur mainly in the western Irano-Turanian region, which is concordantly the biodiversity hotspot of the Brassicaceae family. Moreover comparing Aethionema to the Brassicaceae core group can help us to understand and test the ‘WGD-RLT’ model. However to be able to do so we first need to know more about Aethionema. In this thesis, I investigated various levels of evolutionary change (from macro, to micro to trait evolution) within the genus Aethionema, with a major focus the emerging model species Aethionema arabicum. Next generation sequencing has made it possible to use the genomes of many species in a comparative framework. However, the formation of proteins and enzymes, and in the end the phenotype of the whole plant, relies on transcription from particular regions of the genome including genes. Hence, the transcriptome makes it possible to assess the functional parts of the genome. However, the functional part of the genome not only relies on the protein coding genes. Gene regulatory elements like promoters and long non-coding RNAs function as regulators of gene expression and hence are involved in increasing or decreasing transcription. In Chapter 2 I used the transcriptome of four different Aethionema species to understand the lineage specificity of these long non-coding RNAs. Moreover in a comparison with the Brassicaceae core group and Brassicaceae’s sister family the Cleomaceae I show that although the position of long non-coding RNAs can be conserved, their sequences do not have to be. Most of the Aethionema species occur in the Irano-Turanian region, a politically instable region, making it hard for scientist to collect from. However the natural history collections made throughout the last centuries are a great resource. Combing these collections with the newest sequencing techniques, e.g. next generation sequencing, have allowed me to infer the phylogeny of ~75% of the known Aethionema species in a time calibrated and historical biogeographical framework. Hence, I was able to establish that Aethionema species likely originated from the Anatolian Diagonal and that major geological events like the uplift of the Turkish and Iranian plateau have had a hand in their speciation (Chapter 3). To examine species-level processes I sequenced and analysed transcriptomes of eight Ae. arabicum accessions coming from Cyprus, Iran and Turkey to investigate population structure, genetic diversity and local adaptation (Chapter 4). The most prominent finding was a ploidy difference between the Iranian and Turkish/Cypriotic lines, whereby the former were (allo)tetraploid and the latter diploid. The tetraploid Iranian lines seem to have one set of alleles from the Turkish/Cypriotic gene-pool. However we do not know where the other alleles come from. In addition to the differences in ploidy level there are also differences in glucosinolate defence compounds between these two populations (Iranian vs Turkish/Cypriotic), with the Iranian lines lacking the diversity and concentration of indolic glucosinolates that the Turkish/Cypriotic lines have. This chapter serves as a good resource and starting point for future research in the region, maybe by using the natural history collections that are at hand. Glucosinolates (i.e. mustard oils) are mainly made by Brassicales species, with their highest structural diversity in the Brassicaceae. In Chapter 5, I examined two Ae. arabicum lines (CYP and TUR) and their recombinant inbred lines to assess glucosinolate composition in different tissues and throughout the plants development. The levels of glucosinolates in the leaves changed when Ae. arabicum went from vegetative to a reproductive state. Moreover, a major difference in glucosinolate content (up to 10-fold) between CYP and TUR indicates a likely regulatory pathway outside of the main glucosinolate biosynthesis pathway. Multi-trait and multi-environment QTL analyses based on leaves, reproductive tissues and seeds identified a single major QTL. Fine mapping this region reduced the interval to only fifteen protein coding genes, including the two most intriguing candidates: FLOWERING LOCUS C (FLC) and the sulphate transporter SULTR2;1. These findings show an interesting correlation between development and defence. Finally, Chapter 6 gives a final discussion of this thesis and its results. It brings the different topics together, put them in a bigger picture and look forward to new research possibilities.