Supergenes and social organization in a bird species

ResearchBlogging.org

 

 

 

Cindy Dupuis, Xinji Li, Casper van der Kooi

 

The development of new molecular mechanisms and next generation sequencing techniques have advanced our knowledge on the genetic basis underlying phenotypic polymorphism. Over the coarse of recent years, scientific studies have documented large genomic regions with drastic phenotypic effects, the so-called supergenes. A supergene is a set of genes on the same chromosome that exhibit close genetic linkage and thus inherits as one unit.

The evolution of a supergene requires that multiple loci with complementary effects become linked (i.e. they are genetically clustered and recombination between the loci is suppressed) and that optimal alleles at the linked loci are combined. Genetic clustering of different loci can occur when, via mutation, an adaptive interaction between two closely placed loci is created. In addition, gene duplications or translocations that generate a series of (novel) complementary genes can give rise to supergenes. The probability of a recombination event occurring in between loci depends on various factors. The chance of a recombination event occurring in between two loci will be small when the loci are located closely together, as the chance of a recombination event in between two loci generally decreases with physical distance between the loci. Given the large size of supergenes, additional mechanisms seem, nonetheless, important. This can, for instance, be maintained via structural differences, such as inversions, between the supergene and their homologous chromosomal region.

An interesting example of a supergene in an invertebrate is the case documented by Purcell et al. (2014). They documented a large, nonrecombining region that is association with social organisation in an ant species. The nonrecombining region was found to largely constitute one chromosome and was hence aptly called the ‘social chromosome’. They find a structurally similar region with similar effects in another ant species, however the regions exhibit no homology, suggesting parallel evolution of the social chromosome. Examples of vertebrates social systems determined by supergenes are, to our knowledge, unknown.

Two recent articles (Küpper et al., 2016; Lamichhancy et al., 2016) revealed a single supergene controlling alternative male mating tactics in the ruff (Philomachus pugnax). The studies were carried out independently by two research groups, but reach almost the same conclusions. The ruff (Philomachus pugnax) is a lekking wader known for the great diversity in the male plumage color and behavioral polymorphism. Three types of males can be distinguished; these types are characterized by differences in territoriality and behavior that are highly correlated with differences in nuptial plumage and body size. Predominantly dark-colored Independent males are most common (80-95% of males), these males defend small territories on a lek. Smaller, lighter colored Satellite males (5-20%) are non-territorial and less strict to a particular lek. Satellite males make use of – and are largely tolerated by – the residences of Independent males. The third type are the Faeder males, which are very rare (<1% of males). Faeder males lack male display, are small and resemble the unornamented females; however, they have disproportionately large testes.

Previous studies using pedigrees of large, captive populations showed that reproductive polymorphism follows a single-locus autosomal pattern of inheritance (Lank et al., 1995; Lank et al., 2013). The dominant Faeder allele controls development into Faeder males, whereas the Satelllite allele (that is dominant to Independent) controls development into Satellite or Independent males. Ekblom et al. (2012) studied the nucleotide sequence variation and gene expression in ornamental feathers from 5 Independent and 6 Satellites males using transcriptome sequencing. No significant expression divergence of pre-identified coloration candidate genes was found, but many genetic markers showed nucleotide differentiation between the two morphs. Later, Farrell et al. (2013) used linkage analysis and comparative mapping to locate the Faeder locus, and found linkage to microsatellite markers on avian chromosome 11 that included the Melanocortin-1 receptor (MC1R) gene, a strong candidate in alternative male morph determination, because it is considered to be important in plumage coloration.

Using the captive population that was previously phenotyped, Küpper et al. now set out to determine the genomic structure of the existing morph divergence in P. pugnax. The first step in their analysis was to generate and annotate the full genome for one Independent male. Followingly, the authors identified SNPs in the population using RAD sequencing. More than one million SNPs could be distinguished, and Faeder and Satellites could be mapped to a genetic map based on 3’948 SNPs. Interestingly, both morphs mapped to the same region on chromosome 11, but exhibited clear structural differences. This was corroborated by a GWAS analysis on 41 unrelated Satellite, Independant and Faeder males from a natural population.

 

In order to characterize the genomic region more precisely, they conducted a whole genome sequencing of a small set of Independent, Satellite and Faeder males. They showed that the region on chromosome 11 was highly differentiated between Satellite and Faeder morphs and that this region contained a greater nucleotide variation compared to the adjacent regions. Using the reads orientation, they found clear evidence for an inversion of the chromosomal regions between the different morphs. Interestingly, they found that one breakpoint occurs within an essential gene, CENPN (encoding centromere protein N, recessive lethal), which implies that individuals homozygous for the inversion are not viable – an observation that is confirmed by breeding experiments. The authors also suggested a recombination event or gene conversion to have occurred between the Satellites and Independent alleles.

 

By comparing gene sequences among morphs, the authors discovered that 78% of the gene sequences were different between morphs, and that those differences had the potential to change the encoded protein. Among the divergent genes, some where found to be involved in hormonal production, like HSD17B2, an enzyme inactivating testosterone and estradiol. Varying specifically depending on the morph, this enzyme may alter steroid metabolism and explain partly why plumage patterns and behavior is different between morphs. The MC1R gene was also found within the altered genomic region. This gene is considered an important locus controlling color polymorphism, which could be at the source of the reduced melanin levels in satellites. The PLCG2 gene, which has been rearranged in Faeders, was found to be a candidate gene for the rather feminine appearance and non-aggressive behavior in Faeders. Presumably, this gene is part of a cascade leading to the development of the usual impressive plumage of other males morphs.

 

In a second article, Lamichhancy et al., 2016 studied a natural ruff population using whole-genome sequencing. They first established a high-quality reference genome assembly from an Independent male and conducted functional annotation based on both evidence data and de novo gene predictions. Then, whole-genome resequencing and SNP calling were performed for 15 Independent, 9 Satellite and 1 Faeder males. Their genome-wide screen for genetic divergence estimates (FST) between different male morphs identified a 4.5-Mb region, based on which Independents and Satellites could be phylogenetically clustered as distinct groups. Screening for structural variants identified a 4.5-Mb inversion in Satellites that perfectly overlapped with the differentiated region. In addition, PCR-based sequencing confirmed the positions of proximal and distal breakpoints and identified a 2,108-bp insertion of a repetitive sequence at the distal breakpoint. Diagnostic tests showed that Satellite males were heterozygous (S/I), while most Independent males were homozygous (I/I). They suggested the Independent allele to represent the ancestral state, which is consistent with the conserved synteny among birds.

The comparison between Faeder and Independent males showed that the genetic differentiation was equally strong across the same region, creating a mirror image of the differentiation pattern between Satellites and Independents. Accordingly, the region could be subdivided into two parts: region A where Satellite and Faeder chromosomes were closely related and less closely related to Independent, and region B where the Satellite and Independent loci were closer related and divergent from Faeder. Since an inversion is expected to reduce the amount of recombination within the region between the wild-type (I) and mutant alleles (either S or F), the disruption of the differentiation pattern might be considered the result of one or two recombination events between an Independent and a Faeder-like chromosome. The divergence time between the Independent allele and Satellite or Faeder alleles was estimated to be approximately 4 million years, using the nucleotide divergence and estimated mutation rates for birds. The last recombination event was estimated to occur 520,000 ± 20,000 years ago.

To better understand the genetic consequences of the inversion and relate it to the phenotypic variantion in male ruffs, the authors searched for candidate mutations amongst the genes in the inverted region. Mutations in several genes with important functions were found on Satellite and Faeder chromosomes, including the abovementioned CENPN, HSD17B2 and MC1R genes as well as and SDR42E1 (the latter one is important for the metabolism of sex hormones). Missense mutations in derived MC1R were found to be associated to the Satellite and Faeder alleles, hinting at a potential mechanism explaining the male plumage polymorphism during breeding season.

In conclusion, these two studies demonstrated presence of a genomic inversion that led to the evolution of a supergene. This supergene determines the complex phenotypic variation in male ruffs. These two papers contribute to our understanding of supergenes, complex phenotypes and social organization.

 

Küpper C, Stocks M, Risse JE, Dos Remedios N, Farrell LL, McRae SB, Morgan TC, Karlionova N, Pinchuk P, Verkuil YI, Kitaysky AS, Wingfield JC, Piersma T, Zeng K, Slate J, Blaxter M, Lank DB, & Burke T (2016). A supergene determines highly divergent male reproductive morphs in the ruff. Nature genetics, 48 (1), 79-83 PMID: 26569125

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