Demographic Models and Behavioral Assessments Uncover Distinct Species Histories in the Pseudocryptic Nudibranch Genus <i>Hermissenda</i>

The Pacific nudibranch <i>Hermissenda crassicornis</i> (sensu lato) is a well-known model organism in neuroscience. This species was recently split into three pseudocryptic species based on differences in genetics, morphology, and behavior. We used ddRADSeq data from 33 individuals (2354 loci) and coalescent isolation-with-migration models alongside forward simulations to estimate and evaluate the demographic history of the clade. We inferred (1) a novel phylogenetic tree topology with the North American species as sisters, (2) relatively old divergence times (0.55 and 1.29 mya), (3) a much larger population size in the southern species <i>H. opalescens</i>, and (4) no recent or current gene flow between the sympatric species <i>H. crassicornis</i> and <i>H. opalescens</i>. Then we examined behavioral differences between sympatric <i>H. crassicornis</i> and <i>H. opalescens</i> to characterize possible mechanisms promoting their reproductive isolation. Mating experiments showed that both <i>H. crassicornis</i> and <i>H. opalescens</i> display assortative mating, consistent with the existence of a prezygotic reproductive barrier. Our results support the splitting of <i>H. crassicornis</i> (sensu lato) and reinforce the need to reassess previous studies that used the species complex as a model organism.