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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. 

Abstract Populations may adapt to similar environments via parallel or non‐parallel genetic changes, but the frequency of these alternative mechanisms and underlying contributing factors are still poorly understood outside model systems. We used QTL mapping to investigate the genetic basis of highly divergent craniofacial traits between the scale‐eater (Cyprinodon desquamator) and molluscivore (C. brontotheroides) pupfish adapting to two different hypersaline lake environments on San Salvador Island, Bahamas. We lab‐reared F2 scale‐eater x molluscivore intercrosses from two different lake populations, estimated linkage maps, scanned for significant QTL for 29 skeletal and craniofacial traits, female mate preference, and sex. We compared the location of QTL between lakes to quantify parallel and non‐parallel genetic changes. We detected significant QTL for six craniofacial traits in at least one lake. However, nearly all shared QTL loci were associated with a different craniofacial trait within each lake. Therefore, our estimate of parallel evolution of craniofacial genetic architecture could range from one out of six identical trait QTL (low parallelism) to five out of six integrated trait QTL (high parallelism). We suggest that pleiotropy and trait integration can affect estimates of parallel evolution, particularly within rapid radiations. We also observed increased adaptive introgression in shared QTL regions, suggesting that gene flow contributed to parallel evolution. Overall, our results suggest that the same genomic regions may contribute to parallel adaptation across integrated suites of craniofacial traits, rather than specific traits, and highlight the need for a more expansive definition of parallel evolution. 

ABSTRACT The physical interactions between organisms and their environment ultimately shape diversification rates, but the contributions of biomechanics to evolutionary divergence are frequently overlooked. Here, we estimated a performance landscape for biting in an adaptive radiation of Cyprinodon pupfishes, including scale-biting and molluscivore specialists, and compared performance peaks with previous estimates of the fitness landscape in this system. We used high-speed video to film feeding strikes on gelatin cubes by scale eater, molluscivore, generalist and hybrid pupfishes and measured bite dimensions. We then measured five kinematic variables from 227 strikes using the SLEAP machine-learning model. We found a complex performance landscape with two distinct peaks best predicted gel-biting performance, corresponding to a significant non-linear interaction between peak gape and peak jaw protrusion. Only scale eaters and their hybrids were able to perform strikes within the highest performance peak, characterized by larger peak gapes and greater jaw protrusion. A performance valley separated this peak from a lower performance peak accessible to all species, characterized by smaller peak gapes and less jaw protrusion. However, most individuals exhibited substantial variation in strike kinematics and species could not be reliably distinguished by their strikes, indicating many-to-many mapping of morphology to performance. The two performance peaks observed in the lab were partially consistent with estimates of a two-peak fitness landscape measured in the wild, with the exception of the new performance peak for scale eaters. We thus reveal a new bimodal non-linear biomechanical model that connects morphology to performance to fitness in a sympatric radiation of trophic niche specialists. 

Abstract Mutation rates vary by three orders of magnitude across eukaryotes. The drift-barrier hypothesis proposes that drift overwhelms selection for lower mutation rates in small populations, leading to higher mutation rates over time due to gradual accumulation of mutator alleles. Here, we test this hypothesis in the smallest long-term isolated population in the world, the critically endangered Devils Hole pupfish (Cyprinodon diabolis). We estimated germline mutation rates in embryonic lethal and adult populations using autozygous segments caused by recent inbreeding events. Our estimate, 8.09 x 10^-9per base pair per generation, is significantly higher than the average rate for actinopterygian fishes of 5.97 × 10⁻⁹(95% CI = 4.39 × 10⁻⁹- 7.55 × 10⁻⁹) and is lower than expected but still consistent with predictions from the drift-barrier hypothesis of 1.23 x 10^-8(95% CI = 7.81 × 10⁻⁹– 1.93 × 10⁻⁸), based on a recent meta-analysis of vertebrate mutation rates by Bergeron et al. (2023). We find that embryonic lethal individuals have a higher mutation rate than mature adults, potentially reflecting a segregating lethal mutator allele or damage to the cellular environment during embryo death. We also analyzed the mutational spectra of germline mutations and find that spectra between embryonic lethal and mature adults were similar, as is the spectra in Devils Hole pupfish and other fishes, despite differences in environmental temperature and oxygen stresses. Mutation rates in this critically endangered species provide new insights at one extreme into the mechanisms driving mutation rate variation across vertebrates. 

Abstract Adaptive radiations are striking examples of rapid speciation along ecological lines. In adaptive radiations, fast rates of lineage diversification often pair with rapid rates of morphological diversification. Such diversification has often been documented through the lens of ecological drivers, overlooking the intrinsic structural constraints that may also have a key role in configuring patterns of trait diversification. Covariation within and between traits has been hypothesized to govern the axes of trait evolution, either by increasing the degree of covariation between traits (i.e. integration), which promotes morphological coordination, or by strengthening the degree of covariation within traits (i.e. modularity), which allows organisms to explore novel trait combinations and different regions of morphospace. Here, we study the modularity of the skull within an adaptive radiation of pupfishes that is endemic to San Salvador Island, Bahamas. This radiation exhibits divergent craniofacial morphologies, including generalist, snail-eating specialist, and scale-eating specialist species. We assessed morphological disparity, integration strength, and modularity patterns across the sympatric San Salvador Island pupfish radiation, lab-reared hybrids, and closely related outgroup species. Our findings revealed an unexpected uniformity in the pattern of modularity across diverse species, supporting a five-module functional hypothesis comprising the oral jaw, pharyngeal jaw, neurocranium, hyoid apparatus, and hyomandibula. Despite this conserved modularity pattern, all species exhibited weak but significantly varying strengths of overall between-module integration and significant disparity across all cranial regions. Our results suggest rapid morphological diversification can occur even with conserved patterns of modularity. We propose that broadscale patterns of modularity are more conserved while between-module associations are more evolvable between species. 

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.