Mismatch between pollen and pistil size causes asymmetric mechanical reproductive isolation across Phlox species

Characterizing the mechanisms of reproductive isolation between lineages is key to determining how new species are formed and maintained. In flowering plants, interactions between the reproductive organs of the flower—the pollen and the pistil—serve as the last barrier to reproduc- tion before fertilization. As such, these pollen–pistil interactions are both complex and important for determining a suitable mate. Here, we test whether differences in style length (a part of the pistil) generate a postmating prezygotic mechanical barrier between five species of perennial Phlox wildflowers with geographically overlapping distributions. We perform controlled pairwise reciprocal crosses between three species with long styles and two species with short styles to assess crossing success (seed set). We find that the heterospecific seed set is broadly reduced compared to conspecific cross success and reveal a striking asymmetry in heterospecific crosses between species with different style lengths. To determine the mechanism underlying this asymmetric reproductive isolating barrier, we assess pollen tube growth in vivo and in vitro. We demonstrate that pollen tubes of short-styled species do not grow long enough to reach the ovaries of long-styled species. We find that short- styled species also have smaller pollen and that both within- and between-species pollen diameter is highly correlated with pollen tube length. Our results support the hypothesis that the small pollen of short-styled species lacks resources to grow pollen tubes long enough to access the ovaries of the long-styled species, resulting in an asymmetrical, mechanical barrier to reproduction. Such reproductive isolating mechanisms, combined with additional pollen–pistil incompatibilities, may be particularly important for closely related species in geographic proximity that share pollinators.