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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.

Plants, fungi and algae are important components of global biodiversity and are fundamental to all ecosystems. They are the basis for human well-being, providing food, materials and medicines. Specimens of all three groups of organisms are accommodated in herbaria, where they are commonly referred to as botanical specimens.The large number of specimens in herbaria provides an ample, permanent and continuously improving knowledge base on these organisms and an indispensable source for the analysis of the distribution of species in space and time critical for current and future research relating to global biodiversity. In order to make full use of this resource, a research infrastructure has to be built that grants comprehensive and free access to the information in herbaria and botanical collections in general. This can be achieved through digitization of the botanical objects and associated data.The botanical research community can count on a long-standing tradition of collaboration among institutions and individuals. It agreed on data standards and standard services even before the advent of computerization and information networking, an example being the Index Herbariorum as a global registry of herbaria helping towards the unique identification of specimens cited in the literature.In the spirit of this collaborative history, 51 representatives from 30 institutions advocate to start the digitization of botanical collections with the overall wall-to-wall digitization of the flat objects stored in German herbaria. Germany has 70 herbaria holding almost 23 million specimens according to a national survey carried out in 2019. 87% of these specimens are not yet digitized. Experiences from other countries like France, the Netherlands, Finland, the US and Australia show that herbaria can be comprehensively and cost-efficiently digitized in a relatively short time due to established workflows and protocols for the high-throughput digitization of flat objects.Most of the h

Understanding how and why rates of evolutionary diversification vary is a key issue in evolutionary biology, ecology, and biogeography. Evolutionary rates are the net result of interacting processes summarized under concepts such as adaptive radiation and evolutionary stasis. Here, we review the central concepts in the evolutionary diversification literature and synthesize these into a simple, general framework for studying rates of diversification and quantifying their underlying dynamics, which can be applied across clades and regions, and across spatial and temporal scales. Our framework describes the diversification rate (d) as a function of the abiotic environment (a), the biotic environment (b), and clade‐specific phenotypes or traits (c); thus, d ~ a,b,c. We refer to the four components (a–d) and their interactions collectively as the “Evolutionary Arena.” We outline analytical approaches to this framework and present a case study on conifers, for which we parameterize the general model. We also discuss three conceptual examples: the Lupinus radiation in the Andes in the context of emerging ecological opportunity and fluctuating connectivity due to climatic oscillations; oceanic island radiations in the context of island formation and erosion; and biotically driven radiations of the Mediterranean orchid genus Ophrys. The results of the conifer case study are consistent with the long‐standing scenario that low competition and high rates of niche evolution promote diversification. The conceptual examples illustrate how using the synthetic Evolutionary Arena framework helps to identify and structure future directions for research on evolutionary radiations. In this way, the Evolutionary Arena framework promotes a more general understanding of variation in evolutionary rates by making quantitative results comparable between case studies, thereby allowing new syntheses of evolutionary and ecological processes to emerge

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.

Sexually deceptive orchids of the genus Ophrys may rapidly evolve by adaptation to pollinators. However, understanding of the genetic basis of potential changes and patterns of relationships is hampered by a lack of genomic information. We report the complete plastid genome sequences of Ophrys iricolor and O. sphegodes, representing the two most species-rich lineages of the genus Ophrys. Both plastomes are circular DNA molecules (146754 bp for O. sphegodes and 150177 bp for O. iricolor) with the typical quadripartite structure of plastid genomes and within the average size of photosynthetic orchids. 213 Simple Sequence Repeats (SSRs) (31.5% polymorphic between O. iricolor and O. sphegodes) were identified, with homopolymers and dipolymers as the most common repeat types. SSRs were mainly located in intergenic regions but SSRs located in coding regions were also found, mainly in ycf1 and rpoC2 genes. The Ophrys plastome is predicted to encode 107 distinct genes, 17 of which are completely duplicated in the Inverted Repeat regions. 83 and 87 putative RNA editing sites were detected in 25 plastid genes of the two Ophrys species, all occurring in the first or second codon position. Comparing the rate of nonsynonymous (dN) and synonymous (dS) substitutions, 24 genes (including rbcL and ycf1) display signature consistent with positive selection. When compared with other members of the orchid family, the Ophrys plastome has a complete set of 11 functional ndh plastid genes, with the exception of O. sphegodes that has a truncated ndhF gene. Comparative analysis showed a large co-linearity with other related Orchidinae. However, in contrast to O. iricolor and other Orchidinae, O. sphegodes has a shift of the junction between the Inverted Repeat and Small Single Copy regions associated with the loss of the partial duplicated gene ycf1 and the truncation of the ndhF gene. Data on relative genomic coverage and validation by PCR indicate the presence, with a different ratio, of

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.

Divergent selection by pollinators can bring about strong reproductive isolation via changes at few genes of large effect. This has recently been demonstrated in sexually deceptive orchids, where studies (1) quantified the strength of reproductive isolation in the field; (2) identified genes that appear to be causal for reproductive isolation; and (3) demonstrated selection by analysis of natural variation in gene sequence and expression. In a group of closely related Ophrys orchids, specific floral scent components, namely n-alkenes, are the key floral traits that control specific pollinator attraction by chemical mimicry of insect sex pheromones. The genetic basis of species-specific differences in alkene production mainly lies in two biosynthetic genes encoding stearoyl–acyl carrier protein desaturases (SAD) that are associated with floral scent variation and reproductive isolation between closely related species, and evolve under pollinator-mediated selection. However, the implications of this genetic architecture of key floral traits on the evolutionary processes of pollinator adaptation and speciation in this plant group remain unclear. Here, we expand on these recent findings to model scenarios of adaptive evolutionary change at SAD2 and SAD5, their effects on plant fitness (i.e., offspring number), and the dynamics of speciation. Our model suggests that the two-locus architecture of reproductive isolation allows for rapid sympatric speciation by pollinator shift; however, the likelihood of such pollinator-mediated speciation is asymmetric between the two orchid species O. sphegodes and O. exaltata due to different fitness effects of their predominant SAD2 and SAD5 alleles. Our study not only provides insight into pollinator adaptation and speciation mechanisms of sexually deceptive orchids but also demonstrates the power of applying a modeling approach to the study of pollinator-driven ecological speciation.

BACKGROUND: Sexually deceptive orchids of the genus Ophrys mimic the mating signals of their pollinator females to attract males as pollinators. This mode of pollination is highly specific and leads to strong reproductive isolation between species. This study aims to identify candidate genes responsible for pollinator attraction and reproductive isolation between three closely related species, O. exaltata, O. sphegodes and O. garganica. Floral traits such as odour, colour and morphology are necessary for successful pollinator attraction. In particular, different odour hydrocarbon profiles have been linked to differences in specific pollinator attraction among these species. Therefore, the identification of genes involved in these traits is important for understanding the molecular basis of pollinator attraction by sexually deceptive orchids. RESULTS: We have created floral reference transcriptomes and proteomes for these three Ophrys species using a combination of next-generation sequencing (454 and Solexa), Sanger sequencing, and shotgun proteomics (tandem mass spectrometry). In total, 121 917 unique transcripts and 3531 proteins were identified. This represents the first orchid proteome and transcriptome from the orchid subfamily Orchidoideae. Proteome data revealed proteins corresponding to 2644 transcripts and 887 proteins not observed in the transcriptome. Candidate genes for hydrocarbon and anthocyanin biosynthesis were represented by 156 and 61 unique transcripts in 20 and 7 genes classes, respectively. Moreover, transcription factors putatively involved in the regulation of flower odour, colour and morphology were annotated, including Myb, MADS and TCP factors. CONCLUSION: Our comprehensive data set generated by combining transcriptome and proteome technologies allowed identification of candidate genes for pollinator attraction and reproductive isolation among sexually deceptive orchids. This includes genes for hydrocarbon and anthocyanin biosynthesis and re