How and When Does Outcrossing Occur in the Predominantly Selfing Species Medicago truncatula?

Empirical studies on natural populations of Medicago truncatula revealed selfing rates higher than 80%, but never up to 100%. Similarly, several studies of predominantly selfing species show variability in the level of residual outcrossing between populations and also between temporal samples of the same population. However, these studies measure global selfing rates at the scale of the population and we do not know whether there is intra-population variation and how outcrossing events are distributed, between genotypes, plants, flowers, or seeds. Theoretical studies predict the maintenance of residual outcrossing in highly selfing species due to environmental (e.g., pollen biology) and/or genetic determinants and decompositions of the variation in outcrossing rate using experimental data can be very informative to test these hypotheses. Here, we focus on one natural population of M. truncatula in order to describe precisely its mating system. In particular, we investigated the determinants of the selfing rate by testing for seasonal variations (environmental determinism) and variations between genotypes (genetic determinism). We measured selfing rates in maternal progenies from plants collected widely across a natural population. For each plant, we collected pods from flowers produced at the beginning and at the end of the flowering season to test for a seasonal variation in the outcrossing rate. For each collected offspring, we also estimated the likelihood that it was issued from a self-fertilization event and assessed the genetic component of variation of this mating system measure. We found a significant, albeit small, increase in outcrossing rate in progenies collected at the end [t(m) = 0.137 (SD = 0.025)] compared to those collected at the beginning [t(m) = 0.083 (0.016)] of the flowering season. A significant between genotypes variation in selfing rate was also detected, resulting in a heritability of 9% for the rate of residual outcrossing. Altogether, our work shows that despite a predominantly selfing reproductive mode, M. truncatula displays variation in residual outcrossing rate, and that this trait is likely under a complex determinism combining environmental and genetic factors. We discuss the evolutionary implications of our results for the population.