Roman T. Kellenberger

Transposable elements play a pivotal role in genome evolution and phenotypic variation in numerous eukaryotic species ¹ . Helitrons, a recently identified category of transposons, remain poorly understood in terms of epigenetic regulation and real-time mobilization in plants ^2,3 . Here our study reveals that reduced DNA methylation combined with heat stress promotes the mobilization of the Xuan – Feng Helitron family in wheat. Activation is marked by transcription, extrachromosomal circular DNA formation and novel somatic insertions. Genetic segregation and heterologous reconstitution establish Feng8 as the autonomous driver of the Xuan – Feng family mobilization. These findings represent a step forward in the study of active Helitrons and their potential biological functions as well as their role in genome dynamics and their potential use in crop breeding.

Plant sexual deception, the floral mimicry of female insects to attract mate-searching males for pollination, is a long-studied reproductive strategy with a poorly understood genomic basis. Here, we assembled the genomes of a sexually deceptive, a semi-deceptive, and a derived nondeceptive floral form of the South African daisy Gorteria diffusa to chromosome-scale and near-chromosome scale, respectively. We located several previously identified genes involved in the development of deceptive floral traits, including tandem duplications of GdbHLH and GdMYBSG6 transcription factors regulating the complex coloration of sexually deceptive floral structures. Using additional genotyping-by-sequencing data of six G. diffusa floral forms, we further identified several large inverted genomic segments with a high fixation index (FST), which seem to play a role in maintaining the distinct identity of some floral forms in zones of secondary contact. Finally, genome synteny analyses revealed that the genome of the derived nondeceptive floral form is contracted and shows signs of recent genome-wide deletion of long terminal repeat retrotransposons. Our results provide insight into the genomic elements underlying plant sexual deception as well as some of the structural genomic differences between sexually deceptive and nondeceptive floral forms.

Reproductive isolation is mediated by ecological, phenotypic, and genetic barriers. Where such barriers are not upheld, hybridisation can occur, revealing insight about the formation of species. Here, we provide an integrative investigation of hybrid formation between the orchids Gymnadenia conopsea and G. rhellicani and potential isolating barriers in zones of overlap in the European Alps. We examine opportunities for hybridisation by quantifying hybrid frequency and pollinator assemblages of parent species. Next, we characterise floral phenotypes and genetic parentage of hybrids to examine how genetic inheritance of traits shapes ecological interactions, and thus fitness, of hybrids. We find that pollinator assemblages of parents overlap partially, consistent with observed rare hybrids (G. conopsea and G. rhellicani . No evidence of backcrossing or introgression was found, but direction of pollen transfer varied across sites. Additionally, hybrids were heterozygous for parental alleles at two loci impacting anthocyanin production, suggesting hybrids’ intermediate floral colour reflects inheritance of these alleles. Finally, we find little support that ploidy variation or hybrid seed infertility explains the absence of advanced generations of hybrids. Thus, we suggest that floral isolation represents an important isolating mechanism between G. conopsea and G. rhellicani in limiting the formation of uncommon, unfit hybrids and shaping species boundaries.

Summary: Gorteria diffusa has elaborate petal spots that attract pollinators through sexual deception, but how G. diffusa controls spot development is largely unknown. Here, we investigate how pigmentation is regulated during spot formation. We determined the anthocyanin composition of G. diffusa petals and combined gene expression analysis with protein interaction assays to characterise R2R3‐MYBs that likely regulate pigment production in G. diffusa petal spots. We found that cyanidin 3‐glucoside pigments G. diffusa ray floret petals. Unlike other petal regions, spots contain a high proportion of malonylated anthocyanin. We identified three subgroup 6 R2R3‐MYB transcription factors (GdMYBSG6‐1,2,3) that likely activate the production of spot pigmentation. These genes are upregulated in developing spots and induce ectopic anthocyanin production upon heterologous expression in tobacco. Interaction assays suggest that these transcription factors regulate genes encoding three anthocyanin synthesis enzymes. We demonstrate that the elaboration of complex spots in G. diffusa begins with the accumulation of malonylated pigments at the base of ray floret petals, positively regulated by three paralogous R2R3‐MYB transcription factors. Our results indicate that the functional diversification of these GdMYBSG6s involved changes in the spatial control of their transcription, and modification of the duration of GdMYBSG6 gene expression contributes towards floral variation within the species.

Summary: Gorteria diffusa has elaborate petal spots that attract male bee-fly pollinators through sexual deception but the genetic basis of G. diffusa petal spot development is currently unknown. Here we investigate the regulation of pigmentation during spot formation. We used UHPLC-MS/MS to determine the anthocyanin composition of spots and background pigmentation in G. diffusa . Combining gene expression analysis with protein interaction assays we characterised three R2R3-MYB genes regulating anthocyanin production in G. diffusa spots. We found that cyanidin 3-glucoside pigments G. diffusa ray floret petals. Unlike other petal regions, spots contain a high proportion of malonylated anthocyanin. We identified three paralogous subgroup 6 R2R3-MYB transcription factors that activate the production of petal spot pigmentation. The corresponding genes are upregulated in developing spots and induce ectopic anthocyanin production upon heterologous expression in tobacco. EMSAs and dual luciferase assays suggest that these transcription factors regulate genes encoding three anthocyanin synthesis enzymes: anthocyanidin synthase (GdANS), dihydroflavonol reductase (GdDFR) and malonyl transferase (GdMAT1), accounting for the spot-specific production of malonylated pigments. Here we provide the first molecular characterisation of G. diffusa spot development, showing that the elaboration of complex spots begins with accumulation of malonylated pigments at the base of ray floret petals, positively regulated by three subgroup 6 R2R3-MYB transcription factors.

The morphologically diverse daisy species Gorteria diffusa employs varying levels of sexually deceptive pollination. The species comprises at least fifteen spatially and phenotypically discrete floral morphotypes that are associated with a range of pollination strategies, from generalism to highly specialised sexual deception involving visual mimicry of females of the bee-fly Megapalpus capensis . However, the pattern of evolution of the unique floral traits in this lineage remains unknown because the phylogenetic history of the closely related floral morphotypes has proved unresolvable using traditional approaches. Here we apply genotyping-by-sequencing (GBS), a reduced representation sequencing technology that has significantly increased the tractability of phylogenetic problems involving recent radiations, to the recalcitrant phylogenetic problem of Gorteria across its South African distribution. Population genomic analyses show that individuals group according to morphotype, irrespective of geographic proximity, highlighting the distinctiveness of the morphotypes at the genetic level. We resolve the phylogenetic history of the closely related morphotypes, demonstrating that they are mostly well supported monophyletic entities that are grouped into at least three distinct geographically separated clades. Our results suggest that both incomplete lineage sorting and introgression across geographical clades have previously hindered reconstruction of the phylogeny of this species complex that has diversified rapidly during the Quaternary. Sexual deception is a phylogenetically derived pollination strategy within the complex that evolved at least twice, and was likely achieved by sequential evolution of a set of floral traits that in combination elicit sexual responses from the bee-fly pollinator. While insight into the evolution of sexual deception has been limited by strong phylogenetic conservatism of this strategy in other plant lineages, our results both provide the framework, and confirm the utility of G. diffusa , for further understanding the genetic pathways and selective pressures underlying the complex phenotypes required to exploit insect mating behaviour for pollination.

The role of conspecific density dependence (CDD) in the maintenance of species richness is a central focus of tropical forest ecology. However, tests of CDD often ignore the integrated effects of CDD over multiple life stages and their long-term impacts on population demography. We combined a 10-year time series of seed production, seedling recruitment and sapling and tree demography of three dominant Southeast Asian tree species that adopt a mast-fruiting phenology. We used these data to construct individual-based models that examine the effects of CDD on population growth rates ( λ ) across life-history stages. Recruitment was driven by positive CDD for all species, supporting the predator satiation hypothesis, while negative CDD affected seedling and sapling growth of two species, significantly reducing λ . This negative CDD on juvenile growth overshadowed the positive CDD of recruitment, suggesting the cumulative effects of CDD during seedling and sapling development has greater importance than the positive CDD during infrequent masting events. Overall, CDD varied among positive, neutral and negative effects across life-history stages for all species, suggesting that assessments of CDD on transitions between just two stages (e.g. seeds seedlings or juveniles mature trees) probably misrepresent the importance of CDD on population growth and stability.

Maintenance of polymorphism by overdominance (heterozygote advantage) is a fundamental concept in evolutionary biology. In most examples known in nature, overdominance is a result of homozygotes suffering from deleterious effects. Here we show that overdominance maintains a non-deleterious polymorphism with black, red and white floral morphs in the Alpine orchid Gymnadenia rhellicani. Phenotypic, metabolomic and transcriptomic analyses reveal that the morphs differ solely in cyanidin pigments, which are linked to differential expression of an anthocyanidin synthase (ANS) gene. This expression difference is caused by a premature stop codon in an ANS-regulating R2R3-MYB transcription factor, which is heterozygous in the red colour morph. Furthermore, field observations show that bee and fly pollinators have opposite colour preferences; this results in higher fitness (seed set) of the heterozygous morph without deleterious effects in either homozygous morph. Together, these findings demonstrate that genuine overdominance exists in nature.