Philipp M. Schlüter

Fleshy fruits have evolved diverse traits to attract seed dispersers in response to frugivore behavior and sensory capacities. Fruit scent has been suggested to signal ripeness and nutritional quality, yet the volatile components involved and the information they convey remain poorly understood. It is unknown which information is encoded in fruit scent, whether plants actively synthesize these signals, and thus whether scent constitutes an evolved communication system shaping seed-dispersal interactions. Aliphatic esters, chemicals whose odor is often described as “fruity,” are abundant in some ripe fruits, especially those dispersed by animals which tend to rely on their sense of smell for fruit selection. Moreover, they have been argued to be associated with sugar content, potentially rendering them an honest signal and hence a hotspot of animal-plant chemical communication. We investigated whether aliphatic esters indicate fruit quality honestly and represent an adaptive trait shaped by disperser identity. Using 13 fig species ( Ficus spp.; Moraceae) in Madagascar, we quantified seed dispersal by multiple animals using a quantitative ecological network. We then quantified chemical signals and nutritional rewards using thermal desorption gas chromatography-mass spectrometry (TD-GCMS) and high-performance liquid chromatography (HPLC), and used genome-guided transcriptome assembly to identify the candidate genes responsible for ester signaling. Our results show that (a) aliphatic esters occur more frequently in species dispersed primarily by olfactory-oriented mammals than in those dispersed by visually oriented birds; (b) ester abundance correlates positively with soluble sugar across species only in mammal-dispersed species, indicating an honest signal that is activated only when ecologically relevant; and (c) putative AAT gene revealing elevated expression associated with the increased abundance of aliphatic esters, sugars in single mammal-dispersed taxon. Together, these findings support the hypothesis that fruits have evolved to utilize the biochemical link between esters (signals) and sugars (rewards) to provide honest signals to seed dispersers. Significance Statement: Fruit–frugivore communication is a critical component of ecological systems as it allows animals to find and identify ripe fruit, thus facilitating seed dispersal, plant reproduction, forest regeneration, and animal community maintenance. Chemical communication via fruit scent remains underexplored, particularly regarding the information encoded, active signal synthesis, and adaptive significance. Using a model system of wild figs from Madagascar, our study suggests that fruits have evolved to utilize aliphatic esters, a group of chemicals commonly described as “fruity” by human observers, to signal sugar levels to seed-dispersing animals. In species that rely on lemurs, which tend to rely strongly on their sense of smell, ester levels were higher than in bird-dispersed species, where visual signals are more prominent. Further, in lemur-dispersed species ester levels were correlated with sugar, indicating that they are reliably signaling fruit quality. Linking these patterns to elevated expression of the gene mediating ester biosynthesis, we also identify a molecular mechanism potentially under selection. These findings establish fruit scent as an adaptive communication system and reveal how plant chemicals evolve under mammalian selection.

Telling species apart using DNA sequence data plays a key role in understanding, monitoring, and managing biodiversity. However, plant species discrimination is often difficult due to the complex nature of plant species boundaries. To inform future strategies for DNA-based identification of plants using the nuclear genome and to gain fundamental insights into the genomic nature of differences between plant species, we conducted a large-scale analysis mining data from 151 studies. Of the 1713 multiple-sampled species evaluated, 1202 resolved as monophyletic (70.2%). We then assessed the density of species-specific SNPs (SSSNPs) in the DNA sequence data - of the 462 species from 27 genera assessed in detail, there was a median density of 193 SSSNPs per Mb and 412 species (89.2%) had at least one SSSNP. Randomly sub-sampling the SNP data showed an asymptote in species discrimination with around 3000 randomly selected SNPs. Finally, we undertook a resampling of 6 target-capture datasets and showed that 1-9 pre-selected loci provided equivalent levels of species discrimination compared to hundreds of nuclear loci. These findings provide an important quantitative assessment of the genomic nature of differences between plant species and provide foundations for the development of enhanced approaches for high-resolution DNA-based plant species discrimination.

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.

Pollinator-driven evolution of floral traits is thought to be a major driver of angiosperm speciation and diversification. Ophrys orchids mimic female insects to lure male pollinators into pseudocopulation. This strategy, called sexual deception, is species-specific, thereby providing strong premating reproductive isolation. Identifying the genomic architecture underlying pollinator adaptation and speciation may shed light on the mechanisms of angiosperm diversification. Here, we report the 5.2 Gb chromosome-scale genome sequence of Ophrys sphegodes. We find evidence for transposable element expansion that preceded the radiation of the O. sphegodes group, and for gene duplication having contributed to the evolution of chemical mimicry. We report a highly differentiated genomic candidate region for pollinator-mediated evolution on chromosome 2. The Ophrys genome will prove useful for investigations into the repeated evolution of sexual deception, pollinator adaptation and the genomic architectures that facilitate evolutionary radiations.

Pollinator-driven evolution of floral traits is thought to be a major driver of angiosperm speciation and diversification. The bee orchids (genus Ophrys) mimic their pollinators’ female pheromone, shape and colour to lure male pollinators into pseudocopulation. This strategy, called sexual deception, is highly species-specific, thereby providing strong premating reproductive isolation. Identifying the genomic architecture underlying pollinator adaptation and speciation may shed light on the mechanisms of angiosperm diversification. We report the 5.2 Gb chromosome-scale genome sequence of Ophrys sphegodes and show that chromosomal rearrangements took place in the Ophrys lineage. We find evidence for a transposable element expansion event that preceded the radiation of the O. sphegodes group, and for gene duplication having contributed to the evolution of chemical mimicry. Moreover, one region on chromosome 2 is highly differentiated between O. sphegodes and other Ophrys species, indicating a candidate genomic region for pollinator-mediated evolution.

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.

Interactions among neighbours can alter demography and traits of commingled species via adaptation or plasticity in phenotypic expression, and understanding these two mechanisms in diverse communities is important for determining the ecological and evolutionary consequences of plant–plant interactions. We reciprocally transplanted perennial species (Arenaria armerina and Festuca indigesta) among patches of two foundation shrub species and open ground to assess whether origin microsite (defined as the spatially distinct abiotic and biotic conditions associated with the two shrubs and open ground) determines germination, recruitment and growth that, in turn, promotes fine‐scale distribution of species among microsites. In addition, we tested the effect of origin microsite on traits, competitive ability, drought tolerance and outlier loci to assess whether origin microsite conditions drove differences in traits, strategies and adaptive loci. Germination was consistently greater for seeds planted back into their origin microsite relative to seeds sourced from foreign microsites, although this effect was weakened for recruitment. Plant growth was best in open sites regardless of origin microsite. In the greenhouse, A. armerina had conserved traits within origin microsite but distinct trait values among origins, specifically plants from the most productive microsite (e.g. sufficient light, high nutrients and improved water availability) had distinct trait values. Festuca indigesta had conserved trait responses among microsites while within microsite, individuals had significant trait plasticity to different environmental conditions. The combined field and greenhouse results suggest that fine‐scale distributions are supported by local adaptation among microsites of A. armerina and phenotypic plasticity of F. indigesta. Synthesis. Adaptation or plasticity in phenotypic expression has different implications for demographic rate and persistence of species in changing environments. Local adaptation to neighbours suggests that reductions in foundation species diversity could concomitantly lead to reduced genetic diversity of commingled species while a plastic response indicates a more robust and broad response to changing climatic and biotic conditions.

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: theLupinusradiation 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 genusOphrys. 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.