Contribution of multiple isolating barriers to reproductive isolation between a pair of phytophagous ladybird beetles

Reproductive isolation between species may often be attained by multiple isolating barriers, but the components are rarely studied in animal taxa. To elucidate the nature of multiple isolating barriers, we quantified the strength of three premating barriers, including ecologically based ones (seasonal, habitat, and sexual), two postmating-prehatching barriers (reduced egg hatchability and conspecific sperm precedence [CSP]), and one posthatching barrier, including four components of F(1) hybrid reduced fitness, between two phytophagous ladybird beetles, Henosepilachna vigintioctomaculata and H. pustulosa. We detected five positive barriers (habitat isolation, sexual isolation, reduced egg hatchability, CSP, and reduced egg hatchability in backcrosses of F(1) hybrids). None of these barriers entirely prevents gene exchange when it acts alone, but jointly they generate nearly complete reproductive isolation even between sympatric populations. Host fidelity contributed most strongly to reproductive isolation by reducing interspecific hybridization through several important types of ecological isolation, including microspatial, habitat, and seasonal isolation. The existence of multiple isolating barriers likely helps keep reproductive isolation stable and robust, by complementing changes in the strength of leaky barriers. This complementarity of multiple isolating barriers yields the concept of robustness of reproductive isolation, which is important when considering the long-term maintenance of species boundaries in coexisting species pairs.