A vertebrate adaptive radiation is assembled from an ancient and disjunct spatiotemporal landscape.

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.

Few Fixed Variants between Trophic Specialist Pupfish Species Reveal Candidate Cis-Regulatory Alleles Underlying Rapid Craniofacial Divergence.

Investigating closely related species that rapidly evolved divergent feeding morphology is a powerful approach to identify genetic variation underlying variation in complex traits. This can also lead to the discovery of novel candidate genes influencing natural and clinical variation in human craniofacial phenotypes. We combined whole-genome resequencing of 258 individuals with 50 transcriptomes to identify candidate cis-acting genetic variation underlying rapidly evolving craniofacial phenotypes within an adaptive radiation of Cyprinodon pupfishes. This radiation consists of a dietary generalist species and two derived trophic niche specialists-a molluscivore and a scale-eating species. Despite extensive morphological divergence, these species only diverged 10 kya and produce fertile hybrids in the laboratory. Out of 9.3 million genome-wide SNPs and 80,012 structural variants, we found very few alleles fixed between species-only 157 SNPs and 87 deletions. Comparing gene expression across 38 purebred F1 offspring sampled at three early developmental stages, we identified 17 fixed variants within 10 kb of 12 genes that were highly differentially expressed between species. By measuring allele-specific expression in F1 hybrids from multiple crosses, we found that the majority of expression divergence between species was explained by trans-regulatory mechanisms. We also found strong evidence for two cis-regulatory alleles affecting expression divergence of two genes with putative effects on skeletal development (dync2li1 and pycr3). These results suggest that SNPs and structural variants contribute to the evolution of novel traits and highlight the utility of the San Salvador Island pupfish system as an evolutionary model for craniofacial development.

Ecological divergence in sympatry causes gene misexpression in hybrids.

Ecological speciation occurs when reproductive isolation evolves as a byproduct of adaptive divergence between populations. Selection favouring gene regulatory divergence between species could result in transgressive levels of gene expression in F1 hybrids that may lower hybrid fitness. We combined 58 resequenced genomes with 124 transcriptomes to identify patterns of hybrid gene misexpression that may be driven by adaptive regulatory divergence within a young radiation of Cyprinodon pupfishes, which consists of a dietary generalist and two trophic specialists-a molluscivore and a scale-eater. We found more differential gene expression between closely related sympatric specialists than between allopatric generalist populations separated by 1,000 km. Intriguingly, 9.6% of genes that were differentially expressed between sympatric species were also misexpressed in F1 hybrids. A subset of these genes were in highly differentiated genomic regions and enriched for functions important for trophic specialization, including head, muscle and brain development. These regions also included genes that showed evidence of hard selective sweeps and were significantly associated with oral jaw length-the most rapidly diversifying skeletal trait in this radiation. Our results indicate that divergent ecological selection in sympatry can contribute to hybrid gene misexpression which may act as a reproductive barrier between nascent species.

Hybrid gene misregulation in multiple developing tissues within a recent adaptive radiation of Cyprinodon pupfishes.

Genetic incompatibilities constitute the final stages of reproductive isolation and speciation, but little is known about incompatibilities that occur within recent adaptive radiations among closely related diverging populations. Crossing divergent species to form hybrids can break up coadapted variation, resulting in genetic incompatibilities within developmental networks shaping divergent adaptive traits. We crossed two closely related sympatric Cyprinodon pupfish species-a dietary generalist and a specialized molluscivore-and measured expression levels in their F1 hybrids to identify regulatory variation underlying the novel craniofacial morphology found in this recent microendemic adaptive radiation. We extracted mRNA from eight day old whole-larvae tissue and from craniofacial tissues dissected from 17-20 day old larvae to compare gene expression between a total of seven F1 hybrids and 24 individuals from parental species populations. We found 3.9% of genes differentially expressed between generalists and molluscivores in whole-larvae tissues and 0.6% of genes differentially expressed in craniofacial tissue. We found that 2.1% of genes were misregulated in whole-larvae hybrids whereas 19.1% of genes were misregulated in hybrid craniofacial tissues, after correcting for sequencing biases. We also measured allele specific expression across 15,429 heterozygous sites to identify putative compensatory regulatory mechanisms underlying differential expression between generalists and molluscivores. Together, our results highlight the importance of considering misregulation as an early indicator of genetic incompatibilities in the context of rapidly diverging adaptive radiations and suggests that compensatory regulatory divergence drives hybrid gene misregulation in developing tissues that give rise to novel craniofacial traits.

The behavioral origins of novelty: did increased aggression lead to scale-eating in pupfishes?

Behavioral changes in a new environment are often assumed to precede the origins of evolutionary novelties. Here, we examined whether an increase in aggression is associated with a novel scale-eating trophic niche within a recent radiation of Cyprinodon pupfishes endemic to San Salvador Island, Bahamas. We measured aggression using multiple behavioral assays and used transcriptomic analyses to identify differentially expressed genes in aggression and other behavioral pathways across 3 sympatric species in the San Salvador radiation (generalist, snail-eating specialist, and scale-eating specialist) and 2 generalist outgroups. Surprisingly, we found increased behavioral aggression and differential expression of aggression-related pathways in both the scale-eating and snail-eating specialists, despite their independent evolutionary origins. Increased behavioral aggression varied across both sex and stimulus context in both species. Our results indicate that aggression is not unique to scale-eating specialists. Instead, selection may increase aggression in other contexts such as niche specialization in general or mate competition. Alternatively, increased aggression may result from indirect selection on craniofacial traits, pigmentation, or metabolism-all traits which are highly divergent, exhibit signs of selective sweeps, and are affected by aggression-related genetic pathways which are differentially expressed in this system. In conclusion, the evolution of a novel predatory trophic niche within a recent adaptive radiation does not have clear-cut behavioral origins as previously assumed, highlighting the multivariate nature of adaptation and the complex integration of behavior with other phenotypic traits.

Parallel evolution of gene expression between trophic specialists despite divergent genotypes and morphologies.

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.

The Rate of Evolution of Postmating-Prezygotic Reproductive Isolation in Drosophila.

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.

Novel Candidate Genes Underlying Extreme Trophic Specialization in Caribbean Pupfishes.

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.