Flower Size as an Honest Signal in Royal Irises (Iris Section Oncocyclus, Iridaceae).

Flower traits, such as flower size or color changes, can act as honest signals indicating greater rewards such as nectar; however, nothing is known about shelter-rewarding systems. Large flowers of Royal irises offer overnight shelter as a reward to Eucera bees. A black patch might signal the entrance to the tunnel (shelter) and, together with the flower size, these might act as honest signals. We hypothesize that larger flowers and black patches indicate larger tunnels, and larger tunnels will increase pollinator visits, enhancing the plants' reproductive success. We measured seven species in a controlled environment and two species from three natural populations varying in flower size. Fruit and seed sets were assessed in these natural populations. We found a positive correlation between the flower, patch size, and tunnel volume, suggesting that the flowers and patch size act as honest signals, both under controlled conditions and in the wild. However, in natural populations, this positive relationship and its effect on fitness was population-specific. Flower size increased the fitness in YER I. petrana, and interactions between flower/patch size and tunnel size increased the fitness in YER and I. atropurpurea NET populations. This suggests that the honesty of the signal is positively selected in these two populations. This study supports the hypothesis that pollinator-mediated selection leads to the honest signaling of flower advertisement.

Eco-Geography and Phenology Are the Major Drivers of Reproductive Isolation in the Royal Irises, a Species Complex in the Course of Speciation.

The continuous nature of speciation implies that different species are found at different stages of divergence, from no- to complete reproductive isolation. This process and its underlying mechanisms are best viewed in incipient species. Moreover, the species complex can offer unique insight into how reproductive isolation (RI) has evolved. The royal irises (Iris section Oncocyclus) are a young group of species in the course of speciation, providing an ideal system for speciation study. We quantified pre- and post-zygotic reproductive barriers between the eight Israeli species of this complex and estimated the total RI among them. We tested for both pre-pollination and post-pollination reproductive barriers. Pre-pollination barriers, i.e., eco-geographic divergence and phenological differentiation were the major contributors to RI among the Iris species. On the other hand, post-pollination barriers, namely pollen-stigma interactions, fruit set, and seed viability had negligible contributions to total RI. The strength of RI was not uniform across the species complex, suggesting that species may have diverged at different rates. Overall, this study in a young, recently diverged group of species provides insight into the first steps of speciation, suggesting a crucial role of the pre-zygotic barriers.

De novo transcriptome characterization of Iris atropurpurea (the Royal Iris) and phylogenetic analysis of MADS-box and R2R3-MYB gene families.

The Royal Irises (section Oncocyclus) are a Middle-Eastern group of irises, characterized by extremely large flowers with a huge range of flower colors and a unique pollination system. The Royal Irises are considered to be in the course of speciation and serve as a model for evolutionary processes of speciation and pollination ecology. However, no transcriptomic and genomic data are available for these plants. Transcriptome sequencing is a valuable resource for determining the genetic basis of ecological-meaningful traits, especially in non-model organisms. Here we describe the de novo transcriptome assembly of Iris atropurpurea, an endangered species endemic to Israel's coastal plain. We sequenced and analyzed the transcriptomes of roots, leaves, and three stages of developing flower buds. To identify genes involved in developmental processes we generated phylogenetic gene trees for two major gene families, the MADS-box and MYB transcription factors, which play an important role in plant development. In addition, we identified 1503 short sequence repeats that can be developed for molecular markers for population genetics in irises. This first reported transcriptome for the Royal Irises, and the data generated, provide a valuable resource for this non-model plant that will facilitate gene discovery, functional genomic studies, and development of molecular markers in irises, to complete the intensive eco-evolutionary studies of this group.

Foliar Endophytic Fungi Inhabiting an Annual Grass Along an Aridity Gradient.

Mutualistic fungi are known to increase plant tolerance to abiotic and biotic stress. Therefore, it is expected that along aridity gradients the diversity and composition of symbiotic fungal community will be associated with climate. We examined the diversity of foliar endophytic fungi, inhabiting an annual grass, growing in three different climates (arid, Mediterranean, and wet Mediterranean) along the Israeli aridity gradient. Among the identified endophyte taxa, some were unique to each site, some were common to the two sites located in the extremes of the gradient, but none was common to all sites. Although most fungal endophyte taxa identified were not related to stress adaptation, we detected two that are considered to benefit plants by mitigating stress: Cladosporium and Trichoderma. Cladosporium is highly osmotolerant, frequently found in saline environments. Trichoderma is a biocontrol agent, frequently found in mesic environments. These findings support the hypothesis that species composition of foliar endophytic fungi is associated with stress adaptation of plants.

What Maintains Flower Colour Variation within Populations?

Natural selection acts on phenotypic trait variation. Understanding the mechanisms that create and maintain trait variation is fundamental to understanding the breadth of diversity seen on Earth. Flower colour is among the most conspicuous and highly diverse traits in nature. Most flowering plant populations have uniform floral colours, but a minority exhibit within-population colour variation, either discrete (polymorphic) or continuous. Colour variation is commonly maintained by balancing selection through multiple pollinators, opposing selection regimes, or fluctuating selection. Variation can also be maintained by heterozygote advantage or frequency-dependent selection. Neutral processes, or a lack of selection, may maintain variation, although this remains largely untested. We suggest several prospective research directions that may provide insight into the evolutionary drivers of trait variation.

All the Colors of the Rainbow: Diversification of Flower Color and Intraspecific Color Variation in the Genus Iris.

Floral color plays a key role as visual signaling and is therefore of great importance in shaping plant-pollinator interactions. Iris (Iridaceae), a genus comprising over 300 species and named after the Greek goddess of the colorful rainbow, is famous for its dazzling palette of flower colors and patterns, which vary considerably both within and among species. Despite the large variation of flower color in Iris, little is known about the phylogenetic and ecological contexts of floral color. Here, we seek to resolve the evolution of flower color in the genus Iris in a macroevolutionary framework. We used a phylogenetic analysis to reconstruct the ancestral state of flower color and other pollination-related traits (e.g., the presence of nectar and mating system), and also tracked the evolution of color variation. We further explored weather floral trait transitions are better explained by environmental or pollinator-mediated selection. Our study revealed that the most recent common ancestor likely had monomorphic, purple flowers, with a crest and a spot on the fall. The flowers were likely insect-pollinated, nectar-rewarding, and self-compatible. The diversity of floral traits we see in modern irises, likely represents a trade-off between conflicting selection pressures. Whether shifts in these flower traits result from abiotic or biotic selective agents or are maintained by neutral processes without any selection remains an open question. Our analysis serves as a starting point for future work exploring the genetic and physiological mechanisms controlling flower coloration in the most color-diverse genus Iris.

Plants' ability to sense and respond to airborne sound is likely to be adaptive: reply to comment by Pyke et al.

Ecol. Lett. 22, 2019, 1483 demonstrated, for the first time, a rapid response of a plant to the airborne sounds of pollinators. Pyke et al. argue that this response is unlikely to be adaptive. Here we clarify some misunderstandings, and demonstrate the potential adaptive value using theoretical modelling and field observations.

Increased sugar concentration in response to a wide range of pollinator sounds can be adaptive for the plant: answer to Raguso et al.

In Veits et al., we showed that flowers respond to a range of pollinator sounds by increased nectar sugar concentration. Here we clarify that (1) our argument is relevant to most pollinators, and not limited to bees (2) specifically, bees do access Oenothera Drumondii nectar in this area.

Flowers respond to pollinator sound within minutes by increasing nectar sugar concentration.

Can plants sense natural airborne sounds and respond to them rapidly? We show that Oenothera drummondii flowers, exposed to playback sound of a flying bee or to synthetic sound signals at similar frequencies, produce sweeter nectar within 3 min, potentially increasing the chances of cross pollination. We found that the flowers vibrated mechanically in response to these sounds, suggesting a plausible mechanism where the flower serves as an auditory sensory organ. Both the vibration and the nectar response were frequency-specific: the flowers responded and vibrated to pollinator sounds, but not to higher frequency sound. Our results document for the first time that plants can rapidly respond to pollinator sounds in an ecologically relevant way. Potential implications include plant resource allocation, the evolution of flower shape and the evolution of pollinators sound. Finally, our results suggest that plants may be affected by other sounds as well, including anthropogenic ones.

Pollinator-mediated selection on floral size and tube color in Linum pubescens: Can differential behavior and preference in different times of the day maintain dimorphism?

Diversity of flower traits is often proposed as the outcome of selection exerted by pollinators. Positive directional pollinator-mediated selection on floral size has been widely shown to reduce phenotypic variance. However, the underlying mechanism of maintaining within-population floral color polymorphism is poorly understood. Divergent selection, mediated by different pollinators or by both mutualists and antagonists, may create and maintain such polymorphism, but it has rarely been shown to result from differential behavior of one pollinator. We tested whether different behaviors of the same pollinators in morning and evening are associated with dimorphic floral trait in Linum pubescens, a Mediterranean annual plant that exhibits variable within-population frequencies of dark- and light-colored flower tubes. Usia bicolor bee-flies, the major pollinators of L. pubescens, are mostly feeding in the flower in the morning, while in the evening they are mostly visiting the flowers for mating. In 2 years of studying L. pubescens in a single large population in the Carmel, Israel, we found in one year that dark-centered flowers received significantly higher fraction of visits in the morning. Fitness was positively affected by number of visits, but no fitness differences were found between tube-color morphs, suggesting that both morphs have similar pollination success. Using mediation analysis, we found that flower size was under positive directional pollinator-mediated selection in both years, but pollinator behavior did not explain entirely this selection, which was possibly mediated also by other agents, such as florivores or a-biotic stresses. While most pollinator-mediated selection studies show that flower size signals food reward, in L. pubescens, it may also signal for mating place, which may drive positive selection. While flower size found to be under pollinator-mediated selection in L. pubescens, differential behavior of the pollinators in morning and evening did not seem to explain flower color polymorphism.

Effect of expanded variation in anther position on pollinator visitation to wild radish, Raphanus raphanistrum.

Background and Aims: Plant-pollinator interactions shape the evolution of flowers. Floral attraction and reward traits have often been shown to affect pollinator behaviour, but the possible effect of efficiency traits on visitation behaviour has rarely been addressed. Anther position, usually considered a trait that influences efficiency of pollen deposition on pollinators, was tested here for its effect on pollinator visitation rates and visit duration in flowers of wild radish, Raphanus raphanistrum . Methods: Artificial selection lines from two experiments that expanded the naturally occurring phenotypic variation in anther position were used. In one experiment, plant lines were selected either to increase or to decrease anther exsertion. The other experiment decreased anther dimorphism, which resulted in increased short stamen exsertion. The hypothesis was that increased exsertion would increase visitation of pollen foragers due to increased visual attraction. Another hypothesis was that exsertion of anthers above the corolla would interfere with nectar foragers and increase the duration of visit per flower. Key Results: In the exsertion selection experiment, increased exsertion of both short and long stamens resulted in an increased number of fly visits per plant, and in the dimorphism experiment bee visits increased with increased short stamen exsertion. The duration of visits of nectar feeders declined significantly with increasing long stamen exsertion, which was opposite to the hypothesis. Conclusions: Until now, anther position was considered to be an efficiency trait to enhance pollen uptake and deposition. Anther position in wild radish is shown here also to have an ecological significance in attracting pollen foragers. This study suggests an additional adaptive role for anther position beyond efficiency, and highlights the multiple ecological functions of floral traits in plant-pollinator interactions.

Reproductive isolation between populations of Iris atropurpurea is associated with ecological differentiation.

Background and Aims Speciation is often described as a continuous dynamic process, expressed by different magnitudes of reproductive isolation (RI) among groups in different levels of divergence. Studying intraspecific partial RI can shed light on mechanisms underlying processes of population divergence. Intraspecific divergence can be driven by spatially stochastic accumulation of genetic differences following reduced gene flow, resulting in increased RI with increased geographical distance, or by local adaptation, resulting in increased RI with environmental difference. Methods We tested for RI as a function of both geographical distance and ecological differentiation in Iris atropurpurea, an endemic Israeli coastal plant. We crossed plants in the Netanya Iris Reserve population with plants from 14 populations across the species' full distribution, and calculated RI and reproductive success based on fruit set, seed set and fraction of seed viability. Key Results We found that total RI was not significantly associated with geographical distance, but significantly increased with ecological distance. Similarly, reproductive success of the crosses, estimated while controlling for the dependency of each component on the previous stage, significantly reduced with increased ecological distance. Conclusions Our results indicate that the rise of post-pollination reproductive barriers in I. atropurpurea is more affected by ecological differentiation between populations than by geographical distance, supporting the hypothesis that ecological differentiation is predominant over isolation by distance and by reduced gene flow in this species. These findings also affect conservation management, such as genetic rescue, in the highly fragmented and endangered I. atropurpurea.

Are pollinators the agents of selection for the extreme large size and dark color in Oncocyclus irises?

Pollinator-mediated selection is a major evolutionary driver of floral traits; yet, such selection has rarely been tested for floral extreme traits. The Oncocyclus irises have exceptionally large, dark-colored flowers, associated with night-sheltering pollination and heat reward by the dark flowers. We quantified phenotypic selection on stem length, floral size and color in two species of iris (Iris atropurpurea and I. haynei), using an experimental approach. We estimated selection gradients for both flowers open to natural pollination and for flowers receiving supplementary hand pollination, assuming that open-pollinated flowers are affected by all factors that could influence fitness, whereas supplementary pollination removes the possible influence of pollinators. We found evidence for pollinator-mediated selection to increase floral size and stem length in I. atropurpurea, but floral color in this species was not under pollinator-mediated selection. In I. haynei, no pollinator-mediated selection on any of the traits was detected. We conclude that the extreme floral size of I. atropurpurea has probably evolved as a result of pollinator behavior. Lack of such evidence for I. haynei and for the dark floral color in both species suggests that other non-pollinator agents are selecting for these prominent traits, or that phenotypic color variation in these irises is neutral.

The endangered Iris atropurpurea (Iridaceae) in Israel: honey-bees, night-sheltering male bees and female solitary bees as pollinators.

BACKGROUND AND AIMS: The coastal plain of Israel hosts the last few remaining populations of the endemic Iris atropurpurea (Iridaceae), a Red List species of high conservation priority. The flowers offer no nectar reward. Here the role of night-sheltering male solitary bees, honey-bees and female solitary bees as pollinators of I. atropurpurea is documented. METHODS: Breeding system, floral longevity, stigma receptivity, visitation rates, pollen loads, pollen deposition and removal and fruit- and seed-set were investigated. KEY RESULTS: The main wild pollinators of this plant are male eucerine bees, and to a lesser extent, but with the potential to transfer pollen, female solitary bees. Honey-bees were found to be frequent diurnal visitors; they removed large quantities of pollen and were as effective as male sheltering bees at pollinating this species. The low density of pollen carried by male solitary bees was attributed to grooming activities, pollen displacement when bees aggregated together in flowers and pollen depletion by honey-bees. In the population free of honey-bee hives, male bees carried significantly more pollen grains on their bodies. Results from pollen analysis and pollen deposited on stigmas suggest that inadequate pollination may be an important factor limiting fruit-set. In the presence of honey-bees, eucerine bees were low removal-low deposition pollinators, whereas honey-bees were high removal-low deposition pollinators, because they removed large amounts into corbiculae and deposited relatively little onto receptive stigmas. CONCLUSIONS: Even though overall, both bee taxa were equally effective pollinators, we suggest that honey-bees have the potential to reduce the amount of pollen available for plant reproduction, and to reduce the amount of resources available to solitary bee communities. The results of this study have potential implications for the conservation of this highly endangered plant species if hives are permitted inside reserves, where the bulk of Oncocyclus iris species are protected.

Review. Genetic exchange and the origin of adaptations: prokaryotes to primates.

Data supporting the occurrence of adaptive trait transfer (i.e. the transfer of genes and thus the phenotype of an adaptive trait through viral recombination, lateral gene transfer or introgressive hybridization) are provided in this review. Specifically, we discuss examples of lateral gene transfer and introgressive hybridization that have resulted in the transfer or de novo origin of adaptations. The evolutionary clades in which this process has been identified include all types of organisms. However, we restrict our discussion to bacteria, fungi, plants and animals. Each of these examples reflects the same consequence, namely that the transfer of genetic material, through whatever mechanism, may result in adaptive evolution. In particular, each of the events discussed has been inferred to impact adaptations to novel environmental settings in the recipient lineage.

The genetic architecture of reproductive isolation in Louisiana irises: pollination syndromes and pollinator preferences.

In animal-pollinated plants, pollinator preferences for divergent floral forms can lead to partial reproductive isolation. We describe regions of plant genomes that affect pollinator preferences for two species of Louisiana Irises, Iris brevicaulis and Iris fulva, and their artificial hybrids. Iris brevicaulis and I. fulva possess bee and bird-pollination syndromes, respectively. Hummingbirds preferred I. fulva and under-visited both I. brevicaulis and backcrosses toward this species. Lepidopterans preferred I. fulva and backcrosses toward I. fulva, but also under-visited I. brevicaulis and I. brevicaulis backcrosses. Bumblebees preferred I. brevicaulis and F1 hybrids and rarely visited I. fulva. Although all three pollen vectors preferred one or the other species, these preferences did not prevent visitation to other hybrid/parental classes. Quantitative trait locus (QTL) mapping, in reciprocal BC1 mapping populations, defined the genetic architecture of loci that affected pollinator behavior. We detected six and nine QTLs that affected pollinator visitation rates in the BCIb and BCIf mapping populations, respectively, with as many as three QTLs detected for each trait. Overall, this study reflects the possible role of quantitative genetic factors in determining (1) reproductive isolation, (2) the pattern of pollinator-mediated genetic exchange, and thus (3) hybrid zone evolution.