Assessing Male Reproductive Investment in Papaveraceae Using Flow Cytometry Reveals Lineage-Specific Trajectories to Pollen-to-ovule Ratio Reduction

Male reproductive investment, in particular pollen production, is a crucial and ecologically relevant component of a plant's phenotype and reproductive success. Its evolutionary trajectory, however, remains understudied, partly due to a lack of convenient methods to assess it. We developed a protocol for pollen quantification by flow cytometry and applied it to 107 flowers from 38 Papaveraceae species differing widely in floral traits (e.g., floral symmetry, stamen number), pollination syndromes (e.g., wind and insect pollination) and reproductive systems (e.g., degree of autogamy). We phylogenetically tested whether pollen number evolved in association with ovule, carpel, stamen and flower numbers per inflorescence, and if there were interacting effects between floral symmetry and/or self-compatibility with pollen and ovule production. Compared to manual counts, results using flow cytometry were similar, but much faster to obtain and more precise. Pollen and ovule numbers per flower varied > 39,000x and > 550x, respectively, among species. Pollen production correlated positively with ovule, carpel and stamen numbers. Lineage-specific trajectories to pollen-to-ovule ratio reduction (to values < 300) are observed. One involved increased female investment in ruderal species belonging to the subfamily Papaveroideae, while the other occurs through decreased male investment and is associated with the evolution of floral traits towards greater specialisation. The impact of reproductive systems on male and female investment is limited to ovule production in non-actinomorphic flowers. Taken together, these results revealed that the evolutionary associations between reproductive systems, floral traits, and pollen and ovule production are lineage-specific. Given the profound contrasts at the subfamily level of Papaveraceae, broader surveys across the diversity of flowering plants are clearly needed to better understand factors driving the evolution of reproductive investment. Such studies will certainly be facilitated by our new high-throughput pollen counting method outlined here. ### Competing Interest Statement The authors have declared no competing interest.