Reproductive isolation is an intrinsic aspect of species formation. For that reason, the identification of the precise isolating traits, and the rates at which they evolve, is crucial to understanding how species originate and persist. Previous work has measured the rates of evolution of prezygotic and postzygotic barriers to gene flow, yet no systematic analysis has studied the rates of evolution of postmating-prezygotic (PMPZ) barriers. We measured the magnitude of two barriers to gene flow that act after mating occurs but before fertilization. We also measured the magnitude of a premating barrier (female mating rate in nonchoice experiments) and two postzygotic barriers (hybrid inviability and hybrid sterility) for all pairwise crosses of all nine known extant species within the melanogaster subgroup. Our results indicate that PMPZ isolation evolves faster than hybrid inviability but slower than premating isolation. Next, we partition postzygotic isolation into different components and find that, as expected, hybrid sterility evolves faster than hybrid inviability. These results lend support for the hypothesis that, in Drosophila, reproductive isolation mechanisms (RIMs) that act early in reproduction (or in development) tend to evolve faster than those that act later in the reproductive cycle. Finally, we tested whether there was evidence for reinforcing selection at any RIM. We found no evidence for generalized evolution of reproductive isolation via reinforcement which indicates that there is no pervasive evidence of this evolutionary process. Our results indicate that PMPZ RIMs might have important evolutionary consequences in initiating speciation and in the persistence of new species.

To investigate the origins and stages of vertebrate adaptive radiation, we reconstructed the spatial and temporal histories of adaptive alleles underlying major phenotypic axes of diversification from the genomes of 202 Caribbean pupfishes. On a single Bahamian island, ancient standing variation from disjunct geographic sources was reassembled into new combinations under strong directional selection for adaptation to the novel trophic niches of scale-eating and molluscivory. We found evidence for two longstanding hypotheses of adaptive radiation: hybrid swarm origins and temporal stages of adaptation. Using a combination of population genomics, transcriptomics, and genome-wide association mapping, we demonstrate that this microendemic adaptive radiation of novel trophic specialists on San Salvador Island, Bahamas experienced twice as much adaptive introgression as generalist populations on neighboring islands and that adaptive divergence occurred in stages. First, standing regulatory variation in genes associated with feeding behavior (prlh, cfap20, and rmi1) were swept to fixation by selection, then standing regulatory variation in genes associated with craniofacial and muscular development (itga5, ext1, cyp26b1, and galr2) and finally the only de novo nonsynonymous substitution in an osteogenic transcription factor and oncogene (twist1) swept to fixation most recently. Our results demonstrate how ancient alleles maintained in distinct environmental refugia can be assembled into new adaptive combinations and provide a framework for reconstructing the spatiotemporal landscape of adaptation and speciation.

The genetic changes responsible for evolutionary transitions from generalist to specialist phenotypes are poorly understood. Here we examine the genetic basis of craniofacial traits enabling novel trophic specialization in a sympatric radiation of Cyprinodon pupfishes endemic to San Salvador Island, Bahamas. This recent radiation consists of a generalist species and two novel specialists: a small-jawed "snail-eater" and a large-jawed "scale-eater." We genotyped 12 million single nucleotide polymorphisms (SNPs) by whole-genome resequencing of 37 individuals of all three species from nine populations and integrated genome-wide divergence scans with association mapping to identify divergent regions containing putatively causal SNPs affecting jaw size-the most rapidly diversifying trait in this radiation. A mere 22 fixed variants accompanied extreme ecological divergence between generalist and scale-eater species. We identified 31 regions (20 kb) containing variants fixed between specialists that were significantly associated with variation in jaw size which contained 11 genes annotated for skeletal system effects and 18 novel candidate genes never previously associated with craniofacial phenotypes. Six of these 31 regions showed robust signs of hard selective sweeps after accounting for demographic history. Our data are consistent with predictions based on quantitative genetic models of adaptation, suggesting that the effect sizes of regions influencing jaw phenotypes are positively correlated with distance between fitness peaks on a complex adaptive landscape.

Parallel evolution of gene expression commonly underlies convergent niche specialization, but parallel changes in expression could also underlie divergent specialization. We investigated divergence in gene expression and whole‐genome genetic variation across three sympatric Cyprinodon pupfishes endemic to San Salvador Island, Bahamas. This recent radiation consists of a generalist and two derived specialists adapted to novel niches: a scale‐eating and a snail‐eating pupfish. We sampled total mRNA from all three species at two early developmental stages and compared gene expression with whole‐genome genetic differentiation among all three species in 42 resequenced genomes. Eighty percent of genes that were differentially expressed between snail‐eaters and generalists were up or down regulated in the same direction between scale‐eaters and generalists; however, there were no fixed variants shared between species underlying these parallel changes in expression. Genes showing parallel evolution of expression were enriched for effects on metabolic processes, whereas genes showing divergent expression were enriched for effects on cranial skeleton development and pigment biosynthesis, reflecting the most divergent phenotypes observed between specialist species. Our findings reveal that even divergent niche specialists may exhibit convergent adaptation to higher trophic levels through shared genetic pathways. This counterintuitive result suggests that parallel evolution in gene expression can accompany divergent ecological speciation during adaptive radiation.

21 22Reproductive isolation (RI) is an intrinsic aspect of species, as described in the Biological 23 Species Concept. For that reason, the identification of the precise traits and mechanisms 24 of RI, and the rates at which they evolve, is crucial to understanding how species 25 originate and persist. Nonetheless, precise measurements of the magnitude of 26 reproductive isolation are rare. Previous work has measured the rates of evolution of 27 prezygotic and postzygotic barriers to gene flow, yet no systematic analysis has carried 28 out the study of the rates of evolution of postmating-prezygotic (PMPZ) barriers. We 29 systematically measured the magnitude of two barriers to gene flow that act after mating 30 occurs but before zygotic fertilization and also measured a premating (female mating rate 31 in nonchoice experiments) and two postzygotic barriers (hybrid inviability and hybrid 32 sterility) for all pairwise crosses of species within the Drosophila melanogaster 33 subgroup. Our results indicate that PMPZ isolation evolves faster than hybrid inviability 34 but slower than premating isolation. We also describe seven new interspecific hybrids in 35 the group. Our findings open up a large repertoire of tools that will enable researchers to 36 manipulate hybrids and explore the genetic basis of interspecific differentiation, 37 reproductive isolation, and speciation.