Habitat preference and flowering-time variation contribute to reproductive isolation between diploid and autotetraploid Anacamptis pyramidalis.

Tetraploid lineages are typically reproductively isolated from their diploid ancestors by post-zygotic isolation via triploid sterility. Nevertheless, polyploids often also exhibit ecological divergence that could contribute to reproductive isolation from diploid ancestors. In this study, we disentangled the contribution of different forms of reproductive isolation between sympatric diploid and autotetraploid individuals of the food-deceptive orchid Anacamptis pyramidalis by quantifying the strength of seven reproductive barriers: three prepollination, one post-pollination prezygotic and three post-zygotic. The overall reproductive isolation between the two cytotypes was found very high, with a preponderant contribution of two prepollination barriers, that is phenological and microhabitat differences. Although the contribution of post-zygotic isolation (triploid sterility) is confirmed in our study, these results highlight that prepollination isolation, not necessarily involving pollinator preference, can represent a strong component of reproductive isolation between different cytotypes. Thus, in the context of polyploidy as quantum speciation, that generates reproductive isolation via triploid sterility, ecological divergence can strengthen the reproductive isolation between cytotypes, reducing the waste of gametes in low fitness interploidy crosses and thus favouring the initial establishment of the polyploid lineage. Under this light, speciation by polyploidy involves ecological processes and should not be strictly considered as a nonecological form of speciation.

How to survive on Mediterranean coastal cliffs: tolerance to seawater in early life-cycle stages in Brassica incana Ten. (Brassicaceae).

Mediterranean coastal cliffs are reservoirs of plant biodiversity, hosting vulnerable plant species particularly exposed to the risk of local extinction due to extreme abiotic conditions and climate changes. Therefore, studies aiming to understand the tolerance of cliff plant species to abiotic stresses are important to predict their long-time persistence or to highlight inherent threats. We used an integrative approach including anatomical, physiological and phenotypic analyses on (a) seeds, (b) cotyledons of seedlings; and (c) young plants to assess whether the cliff species Brassica incana, can tolerate exposure to different seawater (SW: 25%, 50% and 100%) concentrations during the early stages of its life cycle. Seeds could germinate when exposed to up to 50% SW. Seeds did not germinate in 100% SW, but could resume germination after washing with freshwater. Seed germination rate also decreased with increasing SW concentration. Exposure to SW decreased stomatal size and stomatal index of cotyledons and caused long-lasting and severe damage to the photochemical reactions of photosynthesis. Photochemistry was also sensitive to SW in young plants, but the effect was lower than in cotyledons. This may involve a remodulation of chloroplast dimensions and activation of cellular metabolism. However, photochemical reactions limited photosynthesis at100% SW even after recovery from SW exposure. Our data show that B. incana has strong tolerance to seawater and shows clear signs of halophytic adaptation. Whilst seeds and juvenile plants are able to withstand SW, the seedling stage appears to be more sensitive.

Pollinator shifts between Ophrys sphegodes populations: might adaptation to different pollinators drive population divergence?

Local adaptation to different pollinators is considered one of the possible initial stages of ecological speciation as reproductive isolation is a by-product of the divergence in pollination systems. However, pollinator-mediated divergent selection will not necessarily result in complete reproductive isolation, because incipient speciation is often overcome by gene flow. We investigated the potential of pollinator shift in the sexually deceptive orchids Ophrys sphegodes and Ophrys exaltata and compared the levels of floral isolation vs. genetic distance among populations with contrasting predominant pollinators. We analysed floral hydrocarbons as a proxy for floral divergence between populations. Floral adoption of pollinators and their fidelity was tested using pollinator choice experiments. Interpopulation gene flow and population differentiation levels were estimated using AFLP markers. The Tyrrhenian O. sphegodes population preferentially attracted the pollinator bee Andrena bimaculata, whereas the Adriatic O. sphegodes population exclusively attracted A. nigroaenea. Significant differences in scent component proportions were identified in O. sphegodes populations that attracted different preferred pollinators. High interpopulation gene flow was detected, but populations were genetically structured at species level. The high interpopulation gene flow levels independent of preferred pollinators suggest that local adaptation to different pollinators has not (yet) generated detectable genome-wide separation. Alternatively, despite extensive gene flow, few genes underlying floral isolation remain differentiated as a consequence of divergent selection. Different pollination ecotypes in O. sphegodes might represent a local selective response imposed by temporal variation in a geographical mosaic of pollinators as a consequence of the frequent disturbance regimes typical of Ophrys habitats.

Is floral divergence sufficient to maintain species boundaries upon secondary contact in Mediterranean food-deceptive orchids?

Analyzing the processes that determine whether species boundaries are maintained on secondary contact may shed light on the early phase of speciation. In Anacamptis morio and Anacamptis longicornu, two Mediterranean orchid sister-species, we used molecular and morphological analyses, together with estimates of pollination success and experimental crosses, to assess whether floral isolation can shelter the species' genomes from genetic admixture on secondary contact. We found substantial genetic and morphological homogenization in sympatric populations in combination with an apparent lack of postmating isolation. We further detected asymmetric introgression in the sympatric populations and an imbalance in cytotype representation, which may be due either to a difference in flowering phenology or else be a consequence of cytonuclear incompatibilities. Estimates of genetic clines for markers across sympatric zones revealed markers that significantly deviated from neutral expectations. We observed a significant correlation between spur length and reproductive success in sympatric populations, which may suggest that directional selection is the main cause of morphological differentiation in this species pair. Our results suggest that allopatric divergence has not led to the evolution of sufficient reproductive isolation to prevent genomic admixture on secondary contact in this orchid species pair.

Do native and invasive herbivores have an effect on Brassica rapa pollination?

Mutualistic (e.g. pollination) and antagonistic (e.g. herbivory) plant-insect interactions shape levels of plant fitness and can have interactive effects. By using experimental plots of Brassica rapa plants infested with generalist (Mamestra brassicae) and specialised (Pieris brassicae) native herbivores and with a generalist invasive (Spodoptera littoralis) herbivore, we estimated both pollen movement among treatments and the visiting behaviour of honeybees versus other wild pollinators. Overall, we found that herbivory has weak effects on plant pollen export, either in terms of inter-treatment movements or of dispersion distance. Plants infested with the native specialised herbivore tend to export less pollen to other plants with the same treatment. Other wild pollinators preferentially visit non-infested plants that differ from those of honeybees, which showed no preferences. Honeybees and other wild pollinators also showed different behaviours on plants infested with different herbivores, with the former tending to avoid revisiting the same treatment and the latter showing no avoidance behaviour. When taking into account the whole pollinator community, i.e. the interactive effects of honeybees and other wild pollinators, we found an increased avoidance of plants infested by the native specialised herbivore and a decreased avoidance of plants infested by the invasive herbivore. Taken together, our results suggest that herbivory may have an effect on B. rapa pollination, but this effect depends on the relative abundance of honeybees and other wild pollinators.

Pollen transfer efficiency and its effect on inflorescence size in deceptive pollination strategies.

Pollination systems differ in pollen transfer efficiency, a variable that may influence the evolution of flower number. Here we apply a comparative approach to examine the link between pollen transfer efficiency and the evolution of inflorescence size in food and sexually deceptive orchids. We examined pollination performance in nine food-deceptive, and eight sexually deceptive orchids by recording pollen removal and deposition in the field. We calculated correlations between reproductive success and flower number (as a proxy for resources allocated during reproductive process), and directional selection differentials were estimated on flower number for four species. Results indicate that sexually deceptive species experience decreased pollen loss compared to food-deceptive species. Despite producing fewer flowers, sexually deceptive species attained levels of overall pollination success (through male and female function) similar to food-deceptive species. Furthermore, a positive correlation between flower number and pollination success was observed in food-deceptive species, but this correlation was not detected in sexually deceptive species. Directional selection differentials for flower number were significantly higher in food compared to sexually deceptive species. We suggest that pollination systems with more efficient pollen transfer and no correlation between pollination success and number of flowers produced, such as sexual deception, may allow the production of inflorescences with fewer flowers that permit the plant to allocate fewer resources to floral displays and, at the same time, limit transpiration. This strategy can be particularly important for ecological success in Mediterranean water-deprived habitats, and might explain the high frequency of sexually deceptive species in these specialised ecosystems.

Does facilitating pollinator learning impede deceptive orchid attractiveness? A multi-approach test of avoidance learning.

It has often been proposed that nectarless deceptive orchid species exploit naïve pollinators in search of food before they learn to avoid their flowers, and that intraspecific floral trait polymorphism, often noted in this plant group, could prolong the time needed for learning, thus increasing orchid reproductive success. We tested the importance of avoidance learning in a European deceptive orchid, Anacamptis morio, which has been reported to have a highly variable fragrance bouquet among individuals. We used an indirect approach, i.e. we facilitated pollinators' ability to learn to avoid A. morio by adding anisaldehyde to selected inflorescences, a scent compound that is easily perceived by the natural pollinators and produced in large quantities by the closely related, nectar producing Anacamptis coriophora, a species that shares pollinator species with A. morio. In a series of three experiments (in artificial arrays, in natural populations and in bumblebee behavioural observations), we consistently found no difference either of reproductive success of or visitation rates to scent-added versus control inflorescences. We also found that the decrease of reproductive success over time in artificial populations of this deceptive species was not as important as expected. Together, these data suggest that pollinators do not fully learn to avoid deceptive inflorescences, and that pollinator avoidance behaviour alone may explain the lower reproductive success usually found in deceptive orchids. We discuss the possible explanations for this pattern in deceptive orchids, particularly in relation to pollinator cognition and learning abilities. Lastly, in light of our results, the potential for higher average reproductive success in deceptive orchids with high phenotypic variability driven by avoidance learning thus appears to be challenged.