Using RNA-seq to characterize pollen-stigma interactions for pollination studies.

Insects are essential for the reproduction of pollinator-dependent crops and contribute to the pollination of 87% of wild plants and 75% of the world's food crops. Understanding pollen flow dynamics between plants and pollinators is thus essential to manage and conserve wild plants and ensure yields are maximized in food crops. However, the determination of pollen transfer in the field is complex and laborious. We developed a field experiment in a pollinator-dependent crop and used high throughput RNA sequencing (RNA-seq) to quantify pollen flow by measuring changes in gene expression between pollination treatments across different apple (Malus domestica Borkh.) cultivars. We tested three potential molecular indicators of successful pollination and validated these results with field data by observing single and multiple visits by honey bees (Apis mellifera) to apple flowers and measured fruit set in a commercial apple orchard. The first indicator of successful outcrossing was revealed via differential gene expression in the cross-pollination treatments after 6 h. The second indicator of successful outcrossing was revealed by the expression of specific genes related to pollen tube formation and defense response at three different time intervals in the stigma and the style following cross-pollination (i.e. after 6, 24, and 48 h). Finally, genotyping variants specific to donor pollen could be detected in cross-pollination treatments, providing a third indicator of successful outcrossing. Field data indicated that one or five flower visits by honey bees were insufficient and at least 10 honey bee flower visits were required to achieve a 25% probability of fruit set under orchard conditions. By combining the genotyping data, the differential expression analysis, and the traditional fruit set field experiments, it was possible to evaluate the pollination effectiveness of honey bee visits under orchards conditions. This is the first time that pollen-stigma-style mRNA expression analysis has been conducted after a pollinator visit (honey bee) to a plant (in vivo apple flowers). This study provides evidence that mRNA sequencing can be used to address complex questions related to stigma-pollen interactions over time in pollination ecology.

Pollen tube growth from multiple pollinator visits more accurately quantifies pollinator performance and plant reproduction.

Pollination services from animals are critical for both crop production and reproduction in wild plant species. Accurately measuring the relative contributions of different animal taxa to pollination service delivery is essential for identifying key pollinators. However, widely used measures of pollinator effectiveness (e.g., single visit pollen deposition) may be inaccurate where plant reproduction is strongly constrained by pollen quality. Here, we test the efficacy of single and multiple pollinator visits for measuring pollinator performance in a model plant species (apple, Malus domestica Borkh) that is strongly limited by pollen quality. We determined pollination success using a suite of measures (pollen deposition, pollen tube growth, fruit and seed set) from single and multiple pollinator visits. We found that pollen deposition from a single pollinator visit seldom resulted in the growth of pollen tubes capable of eliciting ovule fertilisation and never resulted in fruit or seed production. In contrast, multiple pollinator visits frequently initiated the growth of pollen tubes capable of ovule fertilisation and often led to fruit and seed production. Our findings suggest that single visit pollen deposition may provide a poor measure of pollinator performance when linked to reproductive success of plant species that are constrain by pollen quality. Alternatively, pollen tube growth from single and multiple pollinator visits can provide a measure of pollinator performance that is more closely linked to plant reproduction.

Evaluating the taxa that provide shared pollination services across multiple crops and regions.

Many pollinator species visit multiple crops in multiple regions, yet we know little about their pollination service provisioning at local and regional scales. We investigated the floral visitors (n = 13,200), their effectiveness (n = 1718 single visits) and response to landscape composition across three crops avocado, mango and macadamia within a single growing region (1 year), a single crop (3 years) and across different growing regions in multiple years. In total, eight wild visitor groups were shared across all three crops. The network was dominated by three pollinators, two bees (Apis mellifera and Tetragonula spp.) and a fly, Stomorhina discolor. The visitation network for the three crops was relatively generalised but with the addition of pollen deposition data, specialisation increased. Sixteen managed and wild taxa were consistently present across three years in avocado, yet their contribution to annual network structure varied. Node specialisation (d') analyses indicated many individual orchard sites across each of the networks were significantly more specialised compared to that predicted by null models, suggesting the presence of site-specific factors driving these patterns. Identifying the taxa shared across multiple crops, regions and years will facilitate the development of specific pollinator management strategies to optimize crop pollination services in horticultural systems.