Pleiotropic effects of a supergene underlying male alternative reproductive tactics

Strong competition for mates can result in pronounced sexual dimorphism and the evolution of alternative reproductive tactics. Analogous to sex chromosomes that provide the genetic substrate for divergent selection between males and females, supergenes, such as chromosomal inversions, can provide the genetic substrate for divergent selection between alternative reproductive tactics (ARTs). This is particularly the case, when inversion genes are functionally associated with steroid metabolism. The phenotypic effects of supergene variants underlying ARTs have been described mainly in adult males, because it is in them that phenotypic variation is commonly most striking. However, through genetic or hormonal pleiotropy, supergene variants may also affect other life stages or the female phenotype. Identification of such pleiotropic effects is essential for understanding the evolution and maintenance of genetic polymorphisms, since fitness is a composite trait that involves survival and reproductive success in both sexes. For my thesis, I examined the pleiotropic effects of supergene variants underlying male ARTs in ruffs (Calidris pugnax). Adult ruff males exhibit alternative reproductive tactics with an autosomal inversion polymorphism determining three reproductive morphs. Lek-forming Independents have the largest body size and high testosterone levels, displaying and non-aggressive Satellites are slightly smaller and have low testosterone levels, and Faeders mimic females, are discretely smaller and have also low testosterone levels. Independents are the ancestral morph, whereas Satellites and Faeders are both inversion carriers, however, of distinct inversion haplotypes. Since the inversion is autosomal, the three morphs exist also in females. In my thesis, I first examine the effects of supergene variants during ontogeny on steroid metabolism, growth, and the development of flight ability in male and female ruffs. Second, I examine how supergene variants may affect reproduction in females. In my first chapter, I focused on the role of sex steroids coordinating the developmental trajectories that lead to the different reproductive phenotypes in ruffs. I first examined variation in circulating sex steroid concentrations during juvenile development between morphs and sexes. Then I examined variation in gene expression in embryos. For the gene expression analysis, I used a set of candidate genes involved in sex steroid metabolism and examined variation between morphs and sexes in two tissues: neural tissue of the social behavioural network and livers. We found profound regulatory changes in steroid metabolism between morphs, but not between sexes. Independents had higher variances in testosterone concentrations and lower variances in androstenedione concentrations compared to the inversion morphs. In neural tissue, inversion morphs overexpressed HSD17B2, a gene that is located inside the inversion region and responsible for converting testosterone to androstenedione. Our results suggest that HSD17B2 has a key role in mediating steroid metabolism between morphs and that testosterone synthesis is an important mechanism facilitating variation in developmental trajectories between ancestral and inversion morphs. To assess fitness consequences of the supergene variants during ontogeny, it is first necessary to examine when and how phenotypic variation develops. In my second chapter, I focused on the development of variation in body size between sexes and morphs. I examined growth trajectories of body mass, tarsus length and bill length from hatching to fledging and identified time-periods of growth variation between sexes and morphs. We found that 60-100% of the adult variation developed until fledging. In general, the larger sex/morph grew more quickly and reached its maximal absolute growth rate later than the smaller sex/morph. The time-periods with highest variation in growth occurred always shortly after the larger sex/morph reached its maximal growth rate. Maximal growth rates were reached first in tarsus and bill lengths, and then in body mass. These results show that phenotypic variation in body size between sexes and morphs starts to develop already in chicks. They provide a platform for future studies to relate variation in growth to selective pressures. In many bird species, the development of wings associated with the acquisition of flight ability is crucial in reducing juvenile´s vulnerability to predation. Therefore, variation in fledging age between sexes or morphs may have fitness consequences, which would be relevant for balancing selection. In my third chapter, I examined variation in fledging age and wing development between sexes and morphs. Between sexes, females fledged earlier and had faster wing development than males. Within both sexes, Faeders fledged earlier than Independents or Satellites. Within males, wings developed faster in Faeders than in Satellites or Independents. However, within females, wings developed slower in Faeders than in the other two morphs. Our findings suggest that early fledging provides females with an advantage as they may escape ground predators faster than do the males. Similar differences between morphs suggest an advantage for Faeders over the other two morphs. Although in ruffs the supergene variants have the most flamboyant consequences for male phenotypes, they may also affect female reproduction. In chapter four, I first focused on reproductive success in females. I examined variation in laying rate, egg size, yolk steroid concentrations, embryo survival and chick survival. We found that Faeder females laid fewer eggs, smaller eggs and their eggs had higher androstenedione concentrations than eggs from Independents or Satellites. Reproductive success of Faeder females was further reduced by higher offspring mortality. Until hatching, survival was most likely compromised by variation in egg composition and after hatching by smaller egg size. Overall, Faeder females produced substantially fewer fledglings than Independents or Satellites. Based on these results, I then explored whether and how variation in survival or male fertilization success could compensate for the observed variation in female reproductive success. I found that relatively high fertilization success of Faeder males is required for the persistence of the Faeder haplotype, because full compensation through morph-biased survivorship alone is unrealistic. These results suggest that the Faeder allele is sexually antagonistic and that the low reproductive success of Faeder females may benefit Faeder males through negative frequency dependent selection. In conclusion, the supergene variants underlying male ARTs in ruffs have a variety of pleiotropic effects. During ontogeny, morphs of both sexes differed in androgen metabolism, growth rates and the development of flight ability. In adult females, the Faeder allele show multiple antagonistic pleiotropic effects on egg production. These effects reduce the reproductive success of Faeder females to such an extent that the persistence of the Faeder haplotype is maintained by a relatively high fertilization success of Faeder males. This thesis highlights the importance of broadening the research focus in ARTs beyond adult males to understand their evolution and maintenance.

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