Complex petal spot formation in the Beetle Daisy (Gorteria diffusa) relies on spot-specific accumulation of malonylated anthocyanin regulated by paralogous GdMYBSG6 transcription factors.

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.

The evolution of flower colour.

Flowers are the most commonly seen colourful elements of the natural world, and in this primer we explain the evolution of their spectacular range of colours. To understand flower colour, we first explain what colour is and how a flower can have different colours in the eyes of different observers. We briefly introduce the molecular and biochemical basis of flower colour, which is primarily based on well-characterised pigment synthesis pathways. We then consider the evolution of flower colour over four timescales - its origin and deep evolution, its macroevolution, its microevolution and finally, the recent effects of human behaviour on flower colour and its evolution. Because flower colour is so evolutionarily labile, and at the same time so striking to the human eye, it is an exciting subject for current and future research efforts.

Multiple gene co-options underlie the rapid evolution of sexually deceptive flowers in Gorteria diffusa.

Gene co-option, the redeployment of an existing gene in an unrelated developmental context, is an important mechanism underlying the evolution of morphological novelty. In most cases described to date, novel traits emerged by co-option of a single gene or genetic network. Here, we show that the integration of multiple co-opted genetic elements facilitated the rapid evolution of complex petal spots that mimic female bee-fly pollinators in the sexually deceptive South African daisy Gorteria diffusa. First, co-option of iron homeostasis genes altered petal spot pigmentation, producing a color similar to that of female pollinators. Second, co-option of the root hair gene GdEXPA7 enabled the formation of enlarged papillate petal epidermal cells, eliciting copulation responses from male flies. Third, co-option of the miR156-GdSPL1 transcription factor module altered petal spot placement, resulting in better mimicry of female flies resting on the flower. The three genetic elements were likely co-opted sequentially, and strength of sexual deception in different G. diffusa floral forms strongly correlates with the presence of the three corresponding morphological alterations. Our findings suggest that gene co-options can combine in a modular fashion, enabling rapid evolution of novel complex traits.

Comparative Genomics of the Waterfowl Innate Immune System.

Animal species differ considerably in their ability to fight off infections. Finding the genetic basis of these differences is not easy, as the immune response is comprised of a complex network of proteins that interact with one another to defend the body against infection. Here, we used population- and comparative genomics to study the evolutionary forces acting on the innate immune system in natural hosts of the avian influenza virus (AIV). For this purpose, we used a combination of hybrid capture, next- generation sequencing and published genomes to examine genetic diversity, divergence, and signatures of selection in 127 innate immune genes at a micro- and macroevolutionary time scale in 26 species of waterfowl. We show across multiple immune pathways (AIV-, toll-like-, and RIG-I -like receptors signalling pathways) that genes involved genes in pathogen detection (i.e., toll-like receptors) and direct pathogen inhibition (i.e., antimicrobial peptides and interferon-stimulated genes), as well as host proteins targeted by viral antagonist proteins (i.e., mitochondrial antiviral-signaling protein, [MAVS]) are more likely to be polymorphic, genetically divergent, and under positive selection than other innate immune genes. Our results demonstrate that selective forces vary across innate immune signaling signalling pathways in waterfowl, and we present candidate genes that may contribute to differences in susceptibility and resistance to infectious diseases in wild birds, and that may be manipulated by viruses. Our findings improve our understanding of the interplay between host genetics and pathogens, and offer the opportunity for new insights into pathogenesis and potential drug targets.

Low-Input High-Molecular-Weight DNA Extraction for Long-Read Sequencing From Plants of Diverse Families.

Long-read DNA sequencing technologies require high molecular weight (HMW) DNA of adequate purity and integrity, which can be difficult to isolate from plant material. Plant leaves usually contain high levels of carbohydrates and secondary metabolites that can impact DNA purity, affecting downstream applications. Several protocols and kits are available for HMW DNA extraction, but they usually require a high amount of input material and often lead to substantial DNA fragmentation, making sequencing suboptimal in terms of read length and data yield. We here describe a protocol for plant HMW DNA extraction from low input material (0.1 g) which is easy to follow and quick (2.5 h). This method successfully enabled us to extract HMW from four species from different families (Orchidaceae, Poaceae, Brassicaceae, Asteraceae). In the case of recalcitrant species, we show that an additional purification step is sufficient to deliver a clean DNA sample. We demonstrate the suitability of our protocol for long-read sequencing on the Oxford Nanopore Technologies PromethION® platform, with and without the use of a short fragment depletion kit.

Demographic consequences of heterogeneity in conspecific density dependence among mast-fruiting tropical trees.

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.

A Phylogenomic Analysis of the Floral Transcriptomes of Sexually Deceptive and Rewarding European Orchids, Ophrys and Gymnadenia.

The orchids (Orchidaceae) constitute one of the largest and most diverse families of flowering plants. They have evolved a great variety of adaptations to achieve pollination by a diverse group of pollinators. Many orchids reward their pollinators, typically with nectar, but the family is also well-known for employing deceptive pollination strategies in which there is no reward for the pollinator, in the most extreme case by mimicking sexual signals of pollinators. In the European flora, two examples of these different pollination strategies are the sexually deceptive genus Ophrys and the rewarding genus Gymnadenia, which differ in their level of pollinator specialization; Ophrys is typically pollinated by pseudo-copulation of males of a single insect species, whilst Gymnadenia attracts a broad range of floral visitors. Here, we present and describe the annotated floral transcriptome of Ophrys iricolor, an Andrena-pollinated representative of the genus Ophrys that is widespread throughout the Aegean. Furthermore, we present additional floral transcriptomes of both sexually deceptive and rewarding orchids, specifically the deceptive Ophrys insectifera, Ophrys aymoninii, and an updated floral transcriptome of Ophrys sphegodes, as well as the floral transcriptomes of the rewarding orchids Gymnadenia conopsea, Gymnadenia densiflora, Gymnadenia odoratissima, and Gymnadenia rhellicani (syn. Nigritella rhellicani). Comparisons of these novel floral transcriptomes reveal few annotation differences between deceptive and rewarding orchids. Since together, these transcriptomes provide a representative sample of the genus-wide taxonomic diversity within Ophrys and Gymnadenia (Orchidoideae: Orchidinae), we employ a phylogenomic approach to address open questions of phylogenetic relationships within the genera. Specifically, this includes the controversial placement of O. insectifera within the Ophrys phylogeny and the placement of "Nigritella"-type morphologies within the phylogeny of Gymnadenia. Whereas in Gymnadenia, several conflicting topologies are supported by a similar number of gene trees, a majority of Ophrys gene topologies clearly supports a placement of O. insectifera as sister to a clade containing O. sphegodes.

Emergence of a floral colour polymorphism by pollinator-mediated overdominance.

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.

Trans-generational inheritance of herbivory-induced phenotypic changes in Brassica rapa.

Biotic stress can induce plastic changes in fitness-relevant plant traits. Recently, it has been shown that such changes can be transmitted to subsequent generations. However, the occurrence and extent of transmission across different types of traits is still unexplored. Here, we assessed the emergence and transmission of herbivory-induced changes in Brassica rapa and their impact on interactions with insects. We analysed changes in morphology and reproductive traits as well as in flower and leaf volatile emission during two generations with leaf herbivory by Mamestra brassicae and Pieris brassicae and two subsequent generations without herbivory. Herbivory induced changes in all trait types, increasing attractiveness of the plants to the parasitoid wasp Cotesia glomerata and decreasing visitation by the pollinator Bombus terrestris, a potential trade-off. While changes in floral and leaf volatiles disappeared in the first generation after herbivory, some changes in morphology and reproductive traits were still measurable two generations after herbivory. However, neither parasitoids nor pollinators further discriminated between groups with different past treatments. Our results suggest that transmission of herbivore-induced changes occurs preferentially in resource-limited traits connected to plant growth and reproduction. The lack of alterations in plant-insect interactions was likely due to the transient nature of volatile changes.

Herbivore-Induced DNA Demethylation Changes Floral Signalling and Attractiveness to Pollinators in Brassica rapa.

Plants have to fine-tune their signals to optimise the trade-off between herbivore deterrence and pollinator attraction. An important mechanism in mediating plant-insect interactions is the regulation of gene expression via DNA methylation. However, the effect of herbivore-induced DNA methylation changes on pollinator-relevant plant signalling has not been systematically investigated. Here, we assessed the impact of foliar herbivory on DNA methylation and floral traits in the model crop plant Brassica rapa. Methylation-sensitive amplified fragment length polymorphism (MSAP) analysis showed that leaf damage by the caterpillar Pieris brassicae was associated with genome-wide methylation changes in both leaves and flowers of B. rapa as well as a downturn in flower number, morphology and scent. A comparison to plants with jasmonic acid-induced defence showed similar demethylation patterns in leaves, but both the floral methylome and phenotype differed significantly from P. brassicae infested plants. Standardised genome-wide demethylation with 5-azacytidine in five different B. rapa full-sib groups further resulted in a genotype-specific downturn of floral morphology and scent, which significantly reduced the attractiveness of the plants to the pollinator bee Bombus terrestris. These results suggest that DNA methylation plays an important role in adjusting plant signalling in response to changing insect communities.