Unique genetic signatures of local adaptation over space and time for diapause, an ecologically relevant complex trait, in Drosophila melanogaster.

Organisms living in seasonally variable environments utilize cues such as light and temperature to induce plastic responses, enabling them to exploit favorable seasons and avoid unfavorable ones. Local adapation can result in variation in seasonal responses, but the genetic basis and evolutionary history of this variation remains elusive. Many insects, including Drosophila melanogaster, are able to undergo an arrest of reproductive development (diapause) in response to unfavorable conditions. In D. melanogaster, the ability to diapause is more common in high latitude populations, where flies endure harsher winters, and in the spring, reflecting differential survivorship of overwintering populations. Using a novel hybrid swarm-based genome wide association study, we examined the genetic basis and evolutionary history of ovarian diapause. We exposed outbred females to different temperatures and day lengths, characterized ovarian development for over 2800 flies, and reconstructed their complete, phased genomes. We found that diapause, scored at two different developmental cutoffs, has modest heritability, and we identified hundreds of SNPs associated with each of the two phenotypes. Alleles associated with one of the diapause phenotypes tend to be more common at higher latitudes, but these alleles do not show predictable seasonal variation. The collective signal of many small-effect, clinally varying SNPs can plausibly explain latitudinal variation in diapause seen in North America. Alleles associated with diapause are segregating in Zambia, suggesting that variation in diapause relies on ancestral polymorphisms, and both pro- and anti-diapause alleles have experienced selection in North America. Finally, we utilized outdoor mesocosms to track diapause under natural conditions. We found that hybrid swarms reared outdoors evolved increased propensity for diapause in late fall, whereas indoor control populations experienced no such change. Our results indicate that diapause is a complex, quantitative trait with different evolutionary patterns across time and space.

A small survey of introduced Zaprionus indianus (Diptera: Drosophilidae) in orchards of the eastern United States.

The African fig fly, Zaprionus indianus (Gupta), is a generalist fruit fly that typically breeds in decaying fruits from over 70 plant species. The species has spread globally from its native range in tropical Africa, becoming an invasive pest on ripening figs in Brazil. First reported in the United States in 2005 in Florida, Z. indianus has since been documented as far north as Canada and is hypothesized to recolonize northwards from southern refugia each year. We sampled drosophilid communities over the growing season at 2 orchards in Virginia from 2020 to 2022 and 11 orchards along the East Coast during the fall of 2022 to quantify the abundance of Z. indianus relative to other drosophilids across locations, seasons, and fruit crops. Massachusetts had the northernmost population, with no Z. indianus detected in Maine and no correlation between latitude and relative abundance. Variation in Z. indianus relative abundance was high between nearby orchards and abundance was higher on peaches relative to apples within orchards. Comparisons of seasonal abundance curves between 2 Virginia orchards showed similar dynamics across years with individuals first detected around July and becoming absent around December, with peaks in late summer and mid-fall. The variation in seasonal and latitudinal abundance shown here highlights a need for broader sampling to accurately characterize the range, spread, and environmental tolerances of Z. indianus in North America.

An intronic enhancer of Bmp6 underlies evolved tooth gain in sticklebacks.

Threespine stickleback fish offer a powerful system to dissect the genetic basis of morphological evolution in nature. Marine sticklebacks have repeatedly invaded and adapted to numerous freshwater environments throughout the Northern hemisphere. In response to new diets in freshwater habitats, changes in craniofacial morphology, including heritable increases in tooth number, have evolved in derived freshwater populations. Using a combination of quantitative genetics and genome resequencing, here we fine-mapped a quantitative trait locus (QTL) regulating evolved tooth gain to a cluster of ten QTL-associated single nucleotide variants, all within intron four of Bone Morphogenetic Protein 6 (Bmp6). Transgenic reporter assays revealed this intronic region contains a tooth enhancer. We induced mutations in Bmp6, revealing required roles for survival, growth, and tooth patterning. Transcriptional profiling of Bmp6 mutant dental tissues identified significant downregulation of a set of genes whose orthologs were previously shown to be expressed in quiescent mouse hair stem cells. Collectively these data support a model where mutations within a Bmp6 intronic tooth enhancer contribute to evolved tooth gain, and suggest that ancient shared genetic circuitry regulates the regeneration of diverse vertebrate epithelial appendages including mammalian hair and fish teeth.

The genetic architecture of novel trophic specialists: larger effect sizes are associated with exceptional oral jaw diversification in a pupfish adaptive radiation.

The genetic architecture of adaptation is fundamental to understanding the mechanisms and constraints governing diversification. However, most case studies focus on loss of complex traits or parallel speciation in similar environments. It is still unclear how the genetic architecture of these local adaptive processes compares to the architecture of evolutionary transitions contributing to morphological and ecological novelty. Here, we identify quantitative trait loci (QTL) between two trophic specialists in an excellent case study for examining the origins of ecological novelty: a sympatric radiation of pupfishes endemic to San Salvador Island, Bahamas, containing a large-jawed scale-eater and a short-jawed molluscivore with a skeletal nasal protrusion. These specialized niches and trophic traits are unique among over 2000 related species. Measurements of the fitness landscape on San Salvador demonstrate multiple fitness peaks and a larger fitness valley isolating the scale-eater from the putative ancestral intermediate phenotype of the generalist, suggesting that more large-effect QTL should contribute to its unique phenotype. We evaluated this prediction using an F2 intercross between these specialists. We present the first linkage map for pupfishes and detect significant QTL for sex and eight skeletal traits. Large-effect QTL contributed more to enlarged scale-eater jaws than the molluscivore nasal protrusion, consistent with predictions from the adaptive landscape. The microevolutionary genetic architecture of large-effect QTL for oral jaws parallels the exceptional diversification rates of oral jaws within the San Salvador radiation observed over macroevolutionary timescales and may have facilitated exceptional trophic novelty in this system.

A 190 base pair, TGF-ß responsive tooth and fin enhancer is required for stickleback Bmp6 expression.

The ligands of the Bone Morphogenetic Protein (BMP) family of developmental signaling molecules are often under the control of complex cis-regulatory modules and play diverse roles in vertebrate development and evolution. Here, we investigated the cis-regulatory control of stickleback Bmp6. We identified a 190bp enhancer ~2.5 kilobases 5' of the Bmp6 gene that recapitulates expression in developing teeth and fins, with a core 72bp sequence that is sufficient for both domains. By testing orthologous enhancers with varying degrees of sequence conservation from outgroup teleosts in transgenic reporter gene assays in sticklebacks and zebrafish, we found that the function of this regulatory element appears to have been conserved for over 250 million years of teleost evolution. We show that a predicted binding site for the TGFß effector Smad3 in this enhancer is required for enhancer function and that pharmacological inhibition of TGFß signaling abolishes enhancer activity and severely reduces endogenous Bmp6 expression. Finally, we used TALENs to disrupt the enhancer in vivo and find that Bmp6 expression is dramatically reduced in teeth and fins, suggesting this enhancer is necessary for expression of the Bmp6 locus. This work identifies a relatively short regulatory sequence that is required for expression in multiple tissues and, combined with previous work, suggests that shared regulatory networks control limb and tooth development.

Two developmentally temporal quantitative trait loci underlie convergent evolution of increased branchial bone length in sticklebacks.

In convergent evolution, similar phenotypes evolve repeatedly in independent populations, often reflecting adaptation to similar environments. Understanding whether convergent evolution proceeds via similar or different genetic and developmental mechanisms offers insight towards the repeatability and predictability of evolution. Oceanic populations of threespine stickleback fish, Gasterosteus aculeatus, have repeatedly colonized countless freshwater lakes and streams, where new diets lead to morphological adaptations related to feeding. Here, we show that heritable increases in branchial bone length have convergently evolved in two independently derived freshwater stickleback populations. In both populations, an increased bone growth rate in juveniles underlies the convergent adult phenotype, and one population also has a longer cartilage template. Using F2 crosses from these two freshwater populations, we show that two quantitative trait loci (QTL) control branchial bone length at distinct points in development. In both populations, a QTL on chromosome 21 controls bone length throughout juvenile development, and a QTL on chromosome 4 controls bone length only in adults. In addition to these similar developmental profiles, these QTL show similar chromosomal locations in both populations. Our results suggest that sticklebacks have convergently evolved longer branchial bones using similar genetic and developmental programmes in two independently derived populations.

Strength of enemy release from parasitoids is context-dependent in the invasive African Fig Fly, Zaprionus indianus.

Understanding the mechanisms underlying the success of biological invasions is essential to employ effective prediction and management strategies. The escape from natural enemies in invaded regions (enemy release hypothesis) and increased competitive ability are hallmarks of invasive species; however, these two processes are rarely studied within the same context. Here, we examined the effect of enemy release on the competition outcomes of a successful invasive insect pest in North America, the African fig fly (Zaprionus indianus). Parasitoid wasps such as Leptopilina heterotoma that parasitize drosophilid larvae may seek out established species with known host suitability over a novel species, so we hypothesized Z. indianus may have low susceptibility to parasitoids, giving them a competitive advantage over co-occurring drosophilids. We tested this hypothesis by comparing the adult emergence rates from Z. indianus larvae reared alone or in competition with Drosophila hydei or D. simulans larvae in the presence and absence of parasitoid wasps. These interactions might be influenced by larval density, so we tested competitive interactions under low and high larval densities. At low larval densities, Z. indianus emerged at equal rates to D. hydei but outcompeted D. simulans, and these outcomes were not affected by parasitoids. However, at high densities, the addition of parasitoids shifted competition outcomes in favor of Z. indianus, suggesting enemy release provides a competitive advantage under some circumstances. These results indicate that the strength of enemy release in Z. indianus is widely dependent on contextual factors such as density and competitor species. Further investigation of how these results apply to field environments could offer insight into how Z. indianus alters ecosystems and how productive biological control may limit the spread of Z. indianus.

Limited population structure but signals of recent selection in introduced African Fig Fly (Zaprionus indianus) in North America.

Invasive species have devastating consequences for human health, food security, and the environment. Many invasive species adapt to new ecological niches following invasion, but little is known about the early steps of adaptation. Here we examine population genomics of a recently introduced drosophilid in North America, the African Fig Fly, Zaprionus indianus. This species is likely intolerant of subfreezing temperatures and recolonizes temperate environments yearly. We generated a new chromosome-level genome assembly for Z. indianus. Using resequencing of over 200 North American individuals collected over four years in temperate Virginia, plus a single collection from subtropical Florida, we tested for signatures of recolonization, population structure, and adaptation within invasive populations. We show founding populations are sometimes small and contain close genetic relatives, yet temporal population structure and differentiation of populations is mostly absent across recurrent recolonization events. Although we find limited signals of genome-wide spatial or temporal population structure, we identify haplotypes on the X chromosome that are repeatedly differentiated between Virginia and Florida populations. These haplotypes show signatures of natural selection and are not found in African populations. We also find evidence for several large structural polymorphisms segregating within North America populations and show X chromosome evolution in invasive populations is strikingly different from the autosomes. These results show that despite limited population structure, populations may rapidly evolve genetic differences early in an invasion. Further uncovering how these genomic regions influence invasive potential and success in new environments will advance our understanding of how organisms evolve in changing environments.

A cosmopolitan inversion facilitates seasonal adaptation in overwintering Drosophila.

Fluctuations in the strength and direction of natural selection through time are a ubiquitous feature of life on Earth. One evolutionary outcome of such fluctuations is adaptive tracking, wherein populations rapidly adapt from standing genetic variation. In certain circumstances, adaptive tracking can lead to the long-term maintenance of functional polymorphism despite allele frequency change due to selection. Although adaptive tracking is likely a common process, we still have a limited understanding of aspects of its genetic architecture and its strength relative to other evolutionary forces such as drift. Drosophila melanogaster living in temperate regions evolve to track seasonal fluctuations and are an excellent system to tackle these gaps in knowledge. By sequencing orchard populations collected across multiple years, we characterized the genomic signal of seasonal demography and identified that the cosmopolitan inversion In(2L)t facilitates seasonal adaptive tracking and shows molecular footprints of selection. A meta-analysis of phenotypic studies shows that seasonal loci within In(2L)t are associated with behavior, life history, physiology, and morphological traits. We identify candidate loci and experimentally link them to phenotype. Our work contributes to our general understanding of fluctuating selection and highlights the evolutionary outcome and dynamics of contemporary selection on inversions.

Spatial and temporal variation in abundance of introduced African fig fly (Zaprionus indianus) (Diptera: Drosophilidae) in the eastern United States.

The African fig fly, Zaprionus indianus (Gupta), has spread globally from its native range in tropical Africa, becoming an invasive crop pest in select areas such as Brazil. Z. indianus was first reported in the United States in 2005 and has since been documented as far north as Canada. As a tropical species, Z. indianus is expected to have low cold tolerance, likely limiting its ability to persist at northern latitudes. In North America, the geographic regions where Z. indianus can thrive and seasonal fluctuations in its abundance are not well understood. The purpose of this study was to characterize the temporal and spatial variation in Z. indianus abundance to better understand its invasion of the eastern United States. We sampled drosophilid communities over the growing season at two orchards in Virginia from 2020-2022 and several locations along the East Coast during the fall of 2022. Virginia abundance curves showed similar seasonal dynamics across years with individuals first detected around July and becoming absent around December. Massachusetts was the northernmost population and no Z. indianus were detected in Maine. Variation in Z. indianus relative abundance was high between nearby orchards and across different fruits within orchards but was not correlated with latitude. Fitness of wild-caught females decreased later in the season and at higher latitudes. The patterns of Z. indianus abundance shown here demonstrate an apparent susceptibility to cold and highlight a need for systematic sampling to accurately characterize the range and spread of Z. indianus.

Chinese origin rhesus macaque major histocompatibility complex class I molecules promiscuously present epitopes from SIV associated with molecules of Indian origin; implications for immunodominance and viral escape.

The presentation of identical peptides by different major histocompatibility complex class I (MHC-I) molecules, termed promiscuity, is a controversial feature of T cell-mediated immunity to pathogens. The astounding diversity of MHC-I molecules in human populations, presumably to enable binding of equally diverse peptides, implies promiscuity would be a rare phenomenon. However, if it occurs, it would have important implications for immunity. We screened 77 animals for responses to peptides known to bind MHC-I molecules that were not expressed by these animals. Some cases of supposed promiscuity were determined to be the result of either non-identical optimal peptides or were simply not mapped to the correct MHC-I molecule in previous studies. Cases of promiscuity, however, were associated with alterations of immunodominance hierarchies, either in terms of the repertoire of peptides presented by the different MHC-I molecules or in the magnitude of the responses directed against the epitopes themselves. Specifically, we found that the Mamu-B*017:01-restricted peptides Vif HW8 and cRW9 were also presented by Mamu-A2*05:26 and targeted by an animal expressing that allele. We also found that the normally subdominant Mamu-A1*001:01 presented peptide Gag QI9 was also presented by Mamu-B*056:01. Both A2*05:26 and B*056:01 are molecules typically or exclusively expressed by animals of Chinese origin. These data clearly demonstrate that MHC-I epitope promiscuity, though rare, might have important implications for immunodominance and for the transmission of escape mutations, depending on the relative frequencies of the given alleles in a population.

Phenotypic plasticity, but not adaptive tracking, underlies seasonal variation in post-cold hardening freeze tolerance of Drosophila melanogaster.

In temperate regions, an organism's ability to rapidly adapt to seasonally varying environments is essential for its survival. In response to seasonal changes in selection pressure caused by variation in temperature, humidity, and food availability, some organisms exhibit plastic changes in phenotype. In other cases, seasonal variation in selection pressure can rapidly increase the frequency of genotypes that offer survival or reproductive advantages under the current conditions. Little is known about the relative influences of plastic and genetic changes in short-lived organisms experiencing seasonal environmental fluctuations. Cold hardening is a seasonally relevant plastic response in which exposure to cool, but nonlethal, temperatures significantly increases the organism's ability to later survive at freezing temperatures. In the present study, we demonstrate seasonal variation in cold hardening in Drosophila melanogaster and test the extent to which plasticity and adaptive tracking underlie that seasonal variation. We measured the post-cold hardening freeze tolerance of flies from outdoor mesocosms over the summer, fall, and winter. We bred outdoor mesocosm-caught flies for two generations in the laboratory and matched each outdoor cohort to an indoor control cohort of similar genetic background. We cold hardened all flies under controlled laboratory conditions and then measured their post-cold hardening freeze tolerance. Comparing indoor and field-caught flies and their laboratory-reared G1 and G2 progeny allowed us to determine the roles of seasonal environmental plasticity, parental effects, and genetic changes on cold hardening. We also tested the relationship between cold hardening and other factors, including age, developmental density, food substrate, presence of antimicrobials, and supplementation with live yeast. We found strong plastic responses to a variety of field- and laboratory-based environmental effects, but no evidence of seasonally varying parental or genetic effects on cold hardening. We therefore conclude that seasonal variation in post-cold hardening freeze tolerance results from environmental influences and not genetic changes.

Lipid-conjugated telomerase template antagonists sensitize resistant HER2-positive breast cancer cells to trastuzumab.

HER2 amplification in breast cancer is associated with a more aggressive disease, greater likelihood of recurrence, and decreased survival compared to women with HER2-negative breast cancer. Trastuzumab is a monoclonal antibody that inhibits HER2 activity, making this compound an important therapeutic option for patients with HER2-positive breast cancer. However, resistance to trastuzumab develops rapidly in a large number of breast cancer patients. The objective of this study was to determine whether GRN163L, a telomerase template antagonist currently in clinical trials for cancer treatment, can augment the effects of trastuzumab in breast cancer cells with HER2 amplification. GRN163L was effective in inhibiting telomerase activity and shortening telomeres in HER2-positive breast cancer cells. We show that GRN163L acts synergistically with trastuzumab in inhibiting HER2-positive breast cancer cell growth. More importantly, we show that GRN163L can restore the sensitivity of therapeutic-resistant breast cancer cells to trastuzumab. These findings implicate that telomerase template antagonists have potential use in the treatment of cancers that have developed resistance to traditional cancer therapy.

Quantitation of doxorubicin uptake, efflux, and modulation of multidrug resistance (MDR) in MDR human cancer cells.

P-glycoprotein (Pgp), a membrane transporter encoded by the MDR1 gene in human cells, mediates drug efflux from cells, and it plays a major role in causing multidrug resistance (MDR). Confocal microscopy was used to study in vitro and in vivo drug accumulation, net uptake and efflux, and MDR modulation by P-glycoprotein inhibitors in MDR1-transduced human MDA-MB-435mdr (MDR) cancer cells. The MDR cells were approximately 9-fold more resistant to the anticancer drug doxorubicin than their parental wild-type MDA-MB-435wt (WT) cells. Doxorubicin accumulation in the MDR cells was only 19% of that in the WT cells. The net uptake of doxorubicin in the nuclei of the MDR cells was 2-fold lower than that in the nuclei of the WT cells. Pgp inhibitors verapamil, cyclosporine A, or PSC833 increased doxorubicin accumulation in the MDR cells up to 79%, and it reversed drug resistance in these cells. In living animals, doxorubicin accumulation in MDA-MB-435mdr xenograft tumors was 68% of that in the wild-type tumors. Administration of verapamil, cyclosporine A, or PSC833 before doxorubicin treatment of the animals increased doxorubicin accumulation in the MDR tumors up to 94%. These studies have added direct in vitro and in vivo information on the capacity of the transporter protein Pgp to efflux doxorubicin and on the reversal of MDR by Pgp inhibitors in resistant cancer cells.

Embryonic origin and genetic basis of cave associated phenotypes in the isopod crustacean Asellus aquaticus.

Characteristics common to animals living in subterranean environments include the reduction or absence of eyes, lessened pigmentation and enhanced sensory systems. How these characteristics have evolved is poorly understood for the majority of cave dwelling species. In order to understand the evolution of these changes, this study uses an invertebrate model system, the freshwater isopod crustacean, Asellus aquaticus, to examine whether adult differences between cave and surface dwelling individuals first appear during embryonic development. We hypothesized that antennal elaboration, as well as eye reduction and pigment loss, would be apparent during embryonic development. We found that differences in pigmentation, eye formation, and number of segments of antenna II were all present by the end of embryonic development. In addition, we found that cave and surface hatchlings do not significantly differ in the relative size of antenna II and the duration of embryonic development. To investigate whether the regions responsible for eye and pigment differences could be genetically linked to differences in article number, we genotyped F2 hybrids for the four previously mapped genomic regions associated with eye and pigment differences and phenotyped these F2 hybrids for antenna II article number. We found that the region previously known to be responsible for both presence versus absence of pigment and eye size also was significantly associated with article number. Future experiments will address whether pleiotropy and/or genetic linkage play a role in the evolution of cave characteristics in Asellus aquaticus.

Genetic Dissection of a Supergene Implicates Tfap2a in Craniofacial Evolution of Threespine Sticklebacks.

In nature, multiple adaptive phenotypes often coevolve and can be controlled by tightly linked genetic loci known as supergenes. Dissecting the genetic basis of these linked phenotypes is a major challenge in evolutionary genetics. Multiple freshwater populations of threespine stickleback fish (Gasterosteus aculeatus) have convergently evolved two constructive craniofacial traits, longer branchial bones and increased pharyngeal tooth number, likely as adaptations to dietary differences between marine and freshwater environments. Prior QTL mapping showed that both traits are partially controlled by overlapping genomic regions on chromosome 21 and that a regulatory change in Bmp6 likely underlies the tooth number QTL. Here, we mapped the branchial bone length QTL to a 155 kb, eight-gene interval tightly linked to, but excluding the coding regions of Bmp6 and containing the candidate gene Tfap2a Further recombinant mapping revealed this bone length QTL is separable into at least two loci. During embryonic and larval development, Tfap2a was expressed in the branchial bone primordia, where allele specific expression assays revealed the freshwater allele of Tfap2a was expressed at lower levels relative to the marine allele in hybrid fish. Induced loss-of-function mutations in Tfap2a revealed an essential role in stickleback craniofacial development and show that bone length is sensitive to Tfap2a dosage in heterozygotes. Combined, these results suggest that closely linked but genetically separable changes in Bmp6 and Tfap2a contribute to a supergene underlying evolved skeletal gain in multiple freshwater stickleback populations.

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks.

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Partially repeatable genetic basis of benthic adaptation in threespine sticklebacks.

The extent to which convergent adaptation to similar ecological niches occurs by a predictable genetic basis remains a fundamental question in biology. Threespine stickleback fish have undergone an adaptive radiation in which ancestral oceanic populations repeatedly colonized and adapted to freshwater habitats. In multiple lakes in British Columbia, two different freshwater ecotypes have evolved: a deep-bodied benthic form adapted to forage near the lake substrate, and a narrow-bodied limnetic form adapted to forage in open water. Here, we use genome-wide linkage mapping in marine × benthic F2 genetic crosses to test the extent of shared genomic regions underlying benthic adaptation in three benthic populations. We identify at least 100 Quantitative Trait Loci (QTL) harboring genes influencing skeletal morphology. The majority of QTL (57%) are unique to one cross. However, four genomic regions affecting eight craniofacial and armor phenotypes are found in all three benthic populations. We find that QTL are clustered in the genome and overlapping QTL regions are enriched for genomic signatures of natural selection. These findings suggest that benthic adaptation has occurred via both parallel and nonparallel genetic changes.

Parallel developmental genetic features underlie stickleback gill raker evolution.

BACKGROUND: Convergent evolution, the repeated evolution of similar phenotypes in independent lineages, provides natural replicates to study mechanisms of evolution. Cases of convergent evolution might have the same underlying developmental and genetic bases, implying that some evolutionary trajectories might be predictable. In a classic example of convergent evolution, most freshwater populations of threespine stickleback fish have independently evolved a reduction of gill raker number to adapt to novel diets. Gill rakers are a segmentally reiterated set of dermal bones important for fish feeding. A previous large quantitative trait locus (QTL) mapping study using a marine × freshwater F2 cross identified QTL on chromosomes 4 and 20 with large effects on evolved gill raker reduction. RESULTS: By examining skeletal morphology in adult and developing sticklebacks, we find heritable marine/freshwater differences in gill raker number and spacing that are specified early in development. Using the expression of the Ectodysplasin receptor (Edar) gene as a marker of raker primordia, we find that the differences are present before the budding of gill rakers occurs, suggesting an early change to a lateral inhibition process controlling raker primordia spacing. Through linkage mapping in F2 fish from crosses with three independently derived freshwater populations, we find in all three crosses QTL overlapping both previously identified QTL on chromosomes 4 and 20 that control raker number. These two QTL affect the early spacing of gill raker buds. CONCLUSIONS: Collectively, these data demonstrate that parallel developmental genetic features underlie the convergent evolution of gill raker reduction in freshwater sticklebacks, suggesting that even highly polygenic adaptive traits can have a predictable developmental genetic basis.