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Dewlap Color in the Hispaniolan Bark Anole: A Sexually Dimorphic Trait Sex Linked Through Chromosomal Fusions
Longo Hollanda de Mello, Pietro
Longo Hollanda de Mello, Pietro
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Abstract
Gene flow is expected to prevent the buildup of genetic differences between interbreeding populations, even at relatively low levels. How populations can diverge despite gene flow is still a key question in evolutionary biology. Cline theory predicts that loci under divergent selection across an environmental gradient will accumulate in regions of reduced recombination. Heteromorphic sex chromosomes often have lower recombination rates than autosomes. In XY systems, regions of the Y that have degenerated become unable to form chiasmata and thus recombine with its homologous X chromosome. Consequently, not only the Y, but also regions of the X that have become hemizygous do not recombine when in males, reducing the average recombination rate of both Y and X chromosomes relative to autosomes. The impact of sex-linkage goes beyond a reduction in recombination rate. Hemizygous regions have their recessive and partially recessive alleles exposed to selection when in the heterogametic sex. Therefore, selection can act more effectively on these recessive loci when they are sex linked then when they are in autosomes. My goal in this dissertation was to test whether genes under divergent selection across an environmental cline are disproportionately represented in sex chromosomes. Anole lizards are part of a textbook example of an adaptive radiation. Males in this genus have extensible and colorful throatfans called dewlaps. Dewlap color is determined by multiple interacting color cells (chromatophores), and the pigment and structural colors they produce. Males use this secondary sex trait in both intraspecific and interspecific communication. Dewlap color in the Hispaniolan Bark anole, Anolis distichus, is hypothesized to be under divergent selection for signal optimization across an environmental gradient up the Barahona Mountains, in Southern Hispaniola. Populations with orange and yellow dewlaps come in contact across a narrow contact zone in the slopes of the Barahona Mountains where specimens with intermediate color phenotypes are abundant. Previous studies based on genome-wide markers suggested that despite the abrupt change of color across the contact zone, these populations show very low population structure. This creates a unique opportunity for identifying candidate genes for color through transcriptomic comparisons of orange and yellow skin, as well as genome-wide association studies and bulk segregant analyses based on dewlap color using specimens from the contact zone. To test the hypothesis of disproportional sex linkage of dewlap color genes in A. distichus, I first identify in Chapter 1 candidate sex-linked scaffolds in A. distichus. Anolis distichus is part of clade of anoles in which autosomes have recently fused to sex chromosomes, forming neo-sex chromosomes. Therefore, I use synteny mapping between A. distichus to the already assembled A. carolinensis and A. sagrei genomes to identify scaffolds in the A. distichus assembly are syntenic to the ancient X and Y chromosomes in anoles. Next, I use whole genome shotgun (WGS) sequencing and double digest reduced representation DNA (ddRAD) to confirm the hypothesis of sex linkage based on synteny mapping and to test the hypothesis that regions of the A. distichus sex chromosomes that were formerly autosomal have started to degenerate since their fusion to the ancient X and Y sex chromosomes of anoles. By comparing patterns of heterozygosity, depth of coverage between male and female reads, and by identifying aligned reads containing male exclusive k-mers I show that A. distichus assembly has a neo-X and a neo-Y chromosome, and that the degeneration of the neo-Y is not only apparently ongoing but also following the classic model of sex chromosome evolution proposed by Charlesworth. Interestingly, multiple autosome-to-sex-chromosome fusions have occurred in the ancestral of A. distichus. Consequently, different regions of the neo-X and the neo-Y have different levels of divergence. This is also the case in the closely related A. sagrei, although with different chromosome-to-sex-chromosome fusions. As at least three instances of autosome-to-sex-chromosome fusions have taken place within anoles, and as dosage compensation is known to occur in Anolis, this genus appears to be an ideal system for studying the evolution of sex chromosome degeneration and dosage compensation in vertebrates. In Chapter 2 we use transcriptomic comparisons of orange, yellow and white skin to identify candidate genes associated with dewlap color in A. distichus. Given the apparently continuous distribution of color phenotypes in the contact zone in Barahona, we hypothesized that differences in dewlap color in A. distichus are driven by cis-regulatory regions acting on genes from the carotenoid, pteridine or guanine pathways – the three gene pathways associated with the production of non-melanic pigments or structural elements in anoles. By comparing orange, yellow, and white skin using paired and unpaired experimental designs we were able to show that both the carotenoid and the pteridine pathways are upregulated in orange skin relative to yellow skin. Among the genes upregulated in orange skin relative to yellow skin are SCARB1, a gene known to play a role in the uptake of carotenoids from the blood stream, and BCO1, a gene responsible for the cleavage of beta carotene into two retinal molecules. Our transcriptomic results led us to propose two mechanisms for the establishment of color in A. distichus: i) That orange dewlap colors are the product of the accumulation of orange ketocarotenoids and the synthesis of red drosopterins, while yellow dewlap colors are the product of the accumulation of yellow xanthophylls and the synthesis of yellow pteridines; or ii) That orange dewlap colors are the product of the accumulation of a large amount of xanthophylls, in particular lutein, and the synthesis of red drosopterins, while yellow dewlap colors are the product of the accumulation of lower amount of xanthophylls and the synthesis yellow pteridines. Finally, in Chapter 3 we tested our hypothesis that loci associated with the three dimensions of color (hue, chroma, and brightness) in the dewlap are disproportionately represented in sex chromosomes. First, we performed a set of chromatographic and histological analysis to characterize the pigments and cellular structures behind the dewlap color in A. distichus. Next, we identified candidate loci responsible for dewlap color in A. distichus using a pair of complementary approaches: a genome-wide association study (GWAS) of 288 specimens from the contact zone, and a pool sequencing (PoolSeq) analysis of 100 specimens selected based on chroma. These analyses allowed to identify several loci that are either in the vicinity of genes aligned to the A. disitchus genome, or that intersected with intronic regions within them. None of our candidate loci overlapped with presumed coding regions of these genes, corroborating our hypothesis that differences in color are likely determined by cis-regulatory regions. Among the candidate genes from this approach are once again BCO1, as well as CPY2W1, a cytochrome P450 that belongs to a family whose members are known to play a role in the ketolation of carotenoids in amphibians and birds, and SLC16A10, a solute carrier involved in the transportation the thyroid hormone, wich plays a key role in the determination of color pattern in zebrafish. As we predicted, a disproportional number of loci associated with dewlap color are in the neo-Y chromosome of the A. distichus assembly. All significant loci are in a region of the neo-Y syntenic to the ancient anole autosome 11. The neo-Y in A. distichus shows signatures of depth of coverage and heterozygosity consistent with an assembly based on Y-linked reads from a region currently undergoing degeneration. Therefore, neo-sex linkage of dewlap color, a secondary sex trait, in A. distichus is a byproduct of an autosome-to-sex-chromosome fusion rather than being a consequence of independent transpositions of locally adaptive genes to regions of reduced recombination. We hypothesize that autosome-to-sex-chromosome fusions, such as the one we identified in A. distichus, creates opportunities for diversification for a period post-fusion. During this period, we expect recessive and partially recessive alleles that were originally segregating in the ancestral autosomal chromosome to become exposed to selection when in the heterogametic sex. Divergent selection then sorts distinct alleles between populations, leading to the establishment of polymorphism within the species. As neo-sex linkage might include tens to hundreds of genes, this process could potentially quickly lead to the establishment of new allelic combinations that could result in, for example, a color polymorphism across an environmental gradient.
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Date
2022-08-31
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University of Kansas
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Keywords
Genetics, Evolution & development, Anolis, Color, Divergent selection, Faster-X, Neo-sex chromosome