Interspecific tests of allelism reveal the evolutionary timing and pattern of accumulation of reproductive isolation mutations.

Despite extensive theory, little is known about the empirical accumulation and evolutionary timing of mutations that contribute to speciation. Here we combined QTL (Quantitative Trait Loci) analyses of reproductive isolation, with information on species evolutionary relationships, to reconstruct the order and timing of mutations contributing to reproductive isolation between three plant (Solanum) species. To evaluate whether reproductive isolation QTL that appear to coincide in more than one species pair are homologous, we used cross-specific tests of allelism and found evidence for both homologous and lineage-specific (non-homologous) alleles at these co-localized loci. These data, along with isolation QTL unique to single species pairs, indicate that >85% of isolation-causing mutations arose later in the history of divergence between species. Phylogenetically explicit analyses of these data support non-linear models of accumulation of hybrid incompatibility, although the specific best-fit model differs between seed (pairwise interactions) and pollen (multi-locus interactions) sterility traits. Our findings corroborate theory that predicts an acceleration ('snowballing') in the accumulation of isolation loci as lineages progressively diverge, and suggest different underlying genetic bases for pollen versus seed sterility. Pollen sterility in particular appears to be due to complex genetic interactions, and we show this is consistent with a snowball model where later arising mutations are more likely to be involved in pairwise or multi-locus interactions that specifically involve ancestral alleles, compared to earlier arising mutations.