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Abstract Changes in gene expression underlie most phenotypic differences among closely related species. While previous studies in model systems have identified conserved genes and pathways involved in craniofacial evolution, less is known about gene expression differences associated with craniofacial divergence in rapidly evolving species. Here, we investigate craniofacial-specific gene expression in a nascent adaptive radiation of Cyprinodon pupfishes endemic to San Salvador Island, Bahamas, which includes 3 trophic specialists with highly divergent craniofacial morphologies (two scale-eaters and a molluscivore) derived from an ancestral Caribbean-wide generalist. We compared gene expression in the most morphologically divergent craniofacial region with the relatively conserved caudal region across 5 Cyprinodon species and 9 populations. We focused on the hatchling stage, the earliest developmental stage at which craniofacial differences among species are evident. Our approach revealed a large proportion of differentially expressed genes (DEGs) found exclusively in the craniofacial region of the specialists only. By intersecting these specialist-specific craniofacial-exclusive genes with genomic regions harboring fixed single-nucleotide variants under selection in the specialists, we identified 14 candidate genes. We confirmed novel craniofacial expression for 2 of these candidates, pycr3 and atp8a1, genes not previously associated with craniofacial development or function, in hatchlings using in situ mRNA hybridization and observed species-specific differences in the pharyngeal arches and craniofacial muscles, respectively. Our findings demonstrate how an “evolutionary mutant” model can reveal novel gene expression patterns, highlighting the power of integrating tissue-species transcriptomics with speciation genomics to identify novel regulators of craniofacial evolution. 

Abstract Adaptive radiation results in part from ecological opportunity in a new environment, but it is unclear how pre-existing constraints in the founding population may limit this process. Genetic lines of least resistance, and by proxy morphological variance, are known to limit adaptive divergence, but ecological variance is rarely investigated. Here we test whether ecological or morphological lines of least resistance in generalist populations may have constrained the directions of species divergence in two independent Caribbean adaptive radiations ofCyprinodonpupfishes. We find almost universal congruence between the major multivariate dimensions of intraspecific craniofacial and dietary variance within generalist populations and the major axes of interspecific divergence within each adaptive radiation. This is surprising given that we document unique trophic specialists within each radiation, including a bivalve-specialist, zooplanktivore, molluscivore/ostracod-specialist, and scale-eating specialist, while nearly all generalist populations were observed to feed rarely on these same resources. We conclude that pre-existing genetic constraints within each founding generalist population, resulting in dimensions of greater ecological and morphological variance, may partially constrain and predict the directions of species divergence and dietary specialization during adaptive radiation. We also provide a new framework for examining ecological lines of least resistance. 

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage usingin situhybridization chain reaction (HCR). We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions. 

Abstract One of the most exceptional adaptations to extreme drought is found in the sister group to tetrapods, the lungfishes (Dipnoi), which can aestivate inside a mucus cocoon for multiple years at reduced metabolic rates with complete cessation of ingestion and excretion. However, the function of the cocoon tissue is not fully understood. Here we developed a new more natural laboratory protocol for inducing aestivation in the West African lungfish,Protopterus annectens,and investigated the structure and function of the cocoon. We used electron microscopy and imaging of live tissue-stains to confirm that the inner and outer layers of the paper-thin cocoon are composed primarily of living cells. However, we also repeatedly observed extensive bacterial and fungal growth covering the cocoon and found no evidence of anti-microbial activity in vitro againstE. colifor the cocoon tissue in this species. This classroom discovery-based research, performed during a course-based undergraduate research experience course (CURE), provides a robust laboratory protocol for investigating aestivation and calls into the question the function of this bizarre vertebrate adaptation. 

Abstract Understanding the extent of parallelism across adaptive radiations remains a central problem in evolutionary biology. We used whole-genome resequencing of 123 individuals to compare an adaptive radiation ofCyprinodonpupfishes in Laguna Chichancanab, Mexico, to an independent radiation of San Salvador Island (SSI) pupfishes in the Bahamas, and assess the repeatability of adaptive genetic architecture, sources of adaptive variation, and stages of selection. Despite rapid craniofacial divergence of trophic specialists within 8-15 kya, only two candidate genes (3.6%) were shared between Caribbean radiations. Although adaptive introgression played a major role on SSI, we found minimal evidence of adaptive introgression in Chichancanab, likely due to geographic isolation of this inland lake. Instead, de novo mutations provided a substantial source of adaptive variation (30.6%) for the endemic zooplanktivore, 15.3 times higher than the endemic scale-eater on SSI. We found strong evidence that selection occurred in stages, first on regulatory and standing genetic variation, then on de novo and nonsynonymous mutations, parallel to SSI. We then verified that adaptive variants near vision-development and spermatogenesis genes, uniquely within Chichancanab, are consistent with greater visual acuity and divergent sperm morphology in the zoooplanktivore. Consistent with extensive adaptive de novo mutations inWNT10Aand rapid diversification of tooth size in the zooplanktivore, we found that experimental inhibition of this pathway in generalists results in narrower teeth. We conclude that de novo mutations, not introgression, can drive rapid adaptive radiations in isolated environments.