Evolution of reinforced reproductive isolation barriers between sympatric populations of Neurospora crassa and N. intermedia

Reinforcement selection for stronger reproductive isolation barriers in hybrid matings is suspected when barriers are stronger for sympatric than for allopatric matings. Reinforcement selection can be distinguished from other causes of strengthened, sympatric barriers, because only reinforcement should produce stronger barriers specifically in the species and sex suffering the greater cost of hybridization. We investigated reinforcement in two species of the eukaryotic microbe Neurospora using three different methods. First we performed a biogeographic analysis of reproductive isolation barriers between N. crassa and N. intermedia. Second, we mated maternal N. crassa, first to allopatric or sympatric heterospecifics, and then to conspecifics, to reveal how different reproductive isolation barriers affect overall maternal fitness under multiple mating conditions. Third, we completed quantitative trait locus (QTL) mapping of N. crassa x N. intermedia fruitbody development and tested if the genetic architecture of fruitbody development in sympatric matings is consistent with evolution by positive natural selection. We find that reproductive isolation between N. crassa and N. intermedia is stronger in sympatry and that stronger barriers are associated with the species (N. crassa) and mating role (maternal) that suffer the greatest cost of hybridization. Reinforcement in plant and animal species usually involves premating barriers, but sympatric Neurospora matings tend to result in aborted fruitbodies, a postmating barrier. In sequential mating experiments, maternal N. crassa could not be fertilized by conspecifics after hybridizing with allopatric N. intermedia, but had normal conspecific fertility after abortion of sympatric hybrid fruitbodies, so that abortion of sympatric hybrid fruitbodies increased maternal fitness of N. crassa. We mapped 15 reproductive isolation QTL, including 10 loci affecting fruitbody development in N. crassa fertilized by sympatric N. intermedia. A QTL sign test on the distribution of locus effects revealed that the genetic architecture of abortive sympatric fruitbody development is consistent with evolution by natural selection, as it should be if this barrier evolved via a reinforcement mechanism. Our conclusion that reinforcement selection has acted on a postmating barrier in N. crassa demonstrates the necessity of correctly identifying affected barriers when studying microbial species.