Contribution of hybridization to the evolution of a derived reproductive strategy in ricefishes

Transitions between reproductive strategies are costly and involve major changes in life history, behaviour and morphology. Nevertheless, in Sulawesi ricefishes, pelvic-brooding evolved from transfer-brooding in two distantly related lineages within in the genera Adrianichthys and in Oryzias , respectively. Females of pelvic-brooding species carry their eggs attached to their belly until the fry hatches. Despite their phylogenetic distance, both pelvic-brooding lineages share a set of external morphological traits. A recent study found no direct gene-flow between pelvic-brooding lineages suggesting independent evolution of the derived reproductive strategy. It could, however, also be more complex. Pre-existing variation in an admixed population may enable the re-use of genetic variants when subjected to similar external selection pressure, resulting in similar phenotypes. We thus used a multi-species coalescent (MSC) model and D-statistics to identify gene-tree – species-tree incongruencies, to evaluate the evolution of pelvic-brooding with respect to inter-specific gene-flow not only between pelvic-brooding lineages, but between pelvic-brooding lineages and other Sulawesi ricefish lineages. We found a general network-like evolution in Sulawesi ricefishes and as previously reported, no gene-flow between the pelvic-brooding lineages. Instead, we found hybridization between the ancestor of pelvic-brooding Oryzias and the common ancestor of the four Oryzias species from Lake Poso, home of the pelvic-brooding Adrianichthys lineage. Further, indications of introgression were located within two confidence intervals of quantitative trait loci (QTL) associated with pelvic-brooding in O. eversi . We thus hypothesize that a mix of de novo mutations and (ancient) standing genetic variation shaped the evolution of pelvic-brooding. Significance statement The evolution of pelvic-brooding in Oryzias eversi (Beloniformes:Adrianichthyidae), was recently described to be independent from another pelvic-brooding ricefish lineage ( Adrianichthys ) from Sulawesi. We confirmed these results, and detected no gene flow between the two distantly related pelvic-brooding lineages. Instead, we found ancient gene flow from another Orzyias lineage into the pelvic-brooding Oryzias lineage. One of the previously described QTL for pelvic brooding overlaps with a region of high introgression signal. Therefore, we assume that not only de novo mutations contributed to the evolution of pelvic-brooding in Orzyias , but that introduced ancient genetic variation was likely also recruited for the evolution of this derived brooding strategy.