May 2016 – Tutorial Genomics, Ecology, Evolution, etc

Genetic sex determination, i. e. the determination of sexual phenotypes by the effect of sex-determining genes, is found in the majority of vertebrates. Sex determination genes have evolved multiple times independently and can be located on different chromosomes. Depending on whether the presence of the sex determining region (SDR) determines female or male sex, genetic systems of sex determination are called ZW or XY systems respectively and the sex which is heterozygous for the SDR is called the heterogametic sex. Lower fitness in the heterogametic sex has long been observed in interspecific hybrids in a wide range of animal and even plant species, an observation called Haldane’s rule. In this paper the authors find a similar pattern in (non-hybrid) tetrapod species: by comparing the adult sex ratio in XY and ZW systems in 344 tetrapod species, they find that the ASR is skewed towards the homogametic sex (towards females in an XY system and towards males in a ZW system).

This observation is based on a dataset containing known genetic sex determination systems and adult sex ratios (ASRs) of species across the vertebrate phylogeny. Within amphibians and reptiles (in which both XY and ZW systems are found), the authors show that ASRs in ZW systems are significantly more male biased than in XY systems and that the proportion of species with male-biased ASRs is greater in ZW than in XY systems. Furthermore these observations hold true for the combined dataset of amphibians, reptiles, mammals (which have a conserved XY-system and male-biased ASRs), and birds (which have a conserved ZW system and female-biased ASRs).

It is important to test whether these observations are actually caused by the GSD or whether there are other factors, which could systematically influence ASR:

– ASRs could be influenced by body size and breeding latitude through correlated life history traits like development, growth and reproductive ecology.

– Differences in body size and dispersal between sexes can lead to differences in mortality which influence ASRs.

The authors account for potential effects of sex-biased dispersal, body size, breeding latitude and sexual size dimorphism in a phylogenetically corrected multi-predictor analysis. Although they do find a significant correlation between sexual size dimorphism and ASR as well as between sex-biased dispersal and ASR, the effect of the GSD remains significant in all cases. Because the dataset for sex-biased dispersal is limited to 32 species in total, which is less than 10% of the number of species in the complete dataset, it is not included in the main multi-predictor model.

Another important factor is the effect of phylogenetic relatedness between species: The effects of GSDs on ASRs of more closely related species are more likely to be correlated due to shared genetic and phenotypic traits.

To account for this, phylogenetic corrections, which are based on composite phylogenies of different tetrapod groups, are applied. As these composite phylogenies don’t include branch length information, different methods are used to assign arbitrary branch lengths, which has surprisingly little effect on the results. Two different methods are applied to account for phylogenetic relatedness across samples: Phylogenetic generalized least squares (PGLS) models to test for differences in ASRs between XY and ZW taxa and Pagel’s discreet method (PDM) to test the fit of dependent and independent models of transitions in ASR bias and GSD. As the second model implies, the number of transitions between GSDs should be more important than the phylogenetic relatedness between species. The author’s claim to take this into account by rerunning their analyses while reducing three large groups with a known shared sexual system (mammals, birds and snakes) to a single datapoint, resulting in unchanged significant differences in ASRs between GSDs.

I wonder whether it would also make a difference to reduce further groups, which share non-independent evolution of SDRs, to single datapoints. For example this dataset includes five species of lizards from the family Lacertidae, which are assumed to share a conserved GSD (Rovatsos et al. 2016) and 9 lizard species of the genus Anolis included in the dataset are likely to share a common sex chromosome system (Gamble et al. 2014). Furthermore in many amphibians and reptiles nothing is known about synteny across sex chromosomes and it is likely that a rigorous reduction of GSDs with common ancestry into single datapoints would reduce the number of independent observations and thus statistical power.

However, the number of relevant datapoints in amphibians is fairly limited anyway: Amphibian species with an XY sex determination system show no significant ASR bias (or even a slight male bias after phylogenetic correction). Thus the observed effect within amphibians relies on data for only 11 species with a ZW system.There are good reasons to be careful when making general conclusions from this dataset:

Sex reversal is common in some amphibian species, which could bias the observed ASRs. Furthermore, although the authors claim to have included only species with known GSDs, the GSD for amphibians with homomorphic, microscopically indistinguishable sex chromosomes is difficult to determine and frequent subject of scientific dissent.

One example for this is Bufo viridis. The ASR of B. viridis is strongly male biased (0.70), and the GSD is supposed to be a ZW system based on the entry from www.treeofsex.org. However, the claim that B. viridis is female heterogametic is based on a single study, which detected that all seven females examined in a single Moldavian population were heterozygous for a chromosomal inversion. Such a pattern has never been found in any other green toad population, but instead multiple sex linked genetic markers have been developed, which show male-heterogametic segregation patterns in crosses from different B. viridis populations as well as in the closely related species B. siculus, B. balearicus and B. variabilis (Stöck et al. 2011). In my opinion it would be more appropriate to assign B. viridis to species with XY system, which would result in a decrease in the overall differences in ASRs between both groups.

Possible reasons for the effect of the sex-determination system on adult sex ratios

In general, a skewed adult sex ratio can have two different reasons: a skewed gametic sex ratio or higher mortality of one sex resulting in different sex ratios in adults. In more detail six potential not mutually exclusive explanations of how the GSD could bias adult sex ratios are proposed and discussed:

– Sexual selection in males could increase mortality.

This would be expected to result in a bias towards females in XY and ZW systems and cannot explain male biased ASRs in ZW systems.

– Recessive deleterious mutations on X/Z chromosomes or Y/W specific deleterious mutations.

Recombination suppression on sex chromosomes leads to degeneration of the sex-linked region on Y /W chromosomes, which can result in adverse fitness effects caused by either deleterious mutations on the Y/W, or deleterious recessive mutations on the hemizygous part of the X/Z chromosome.

Based on a population genetic model they develop, the authors claim that the accumulation of deleterious mutations may not be enough to cause the observed adult sex-ratio bias. However, they admit that many of their parameter estimates are very crude and results may vary when other factors are taken into account, like large differences in the rate of deleterious mutations.

The number of deleterious mutations is expected to increase with increasing sex chromosome differentiation and degeneration. Sex chromosome differentiation in tetrapods spans a wide range from completely homomorphic sex chromosomes in many lizards and amphibians but also in some families of snakes and birds to complete loss of the Y chromosome in some mammals. It would thus be interesting to look if there is an association between variable sex chromosome degeneration and skews in the ASR within groups with homologous sex chromosomes.

– Imperfect dosage compensation.

In the heterogametic sex, genes located in the hemizygous region of the X/Z chromosome are present in only one functional copy. In order to reach similar expression levels as in the homogametic sex, the expression of these genes has to be increased. However, research has shown that not all genes are upregulated in the same way and as a result many sex chromosomal genes have a lower expression levels in the heterogametic than in the homogametic sex.

This explanation is unlikely to result in a general pattern across tetrapods, because there are different mechanisms of dosage compensation in vertebrates: mammals deactivate one X chromosome in females to compensate for gene loss on the Y chromosome, while birds show incomplete dosage compensation on a gene-by-gene basis. Since one X is deactivated in the homogametic sex in mammals, we would expect to find sex-specific fitness differences based on dosage compensation only for non-mammals.

– Meiotic drive:

Meiotic drive systems are genetic variants, which favor their own transmission by distorting sex ratios at meiosis. The authors point out, that the observed skews in ASR are unlikely to be caused by meiotic drive, because the sex ratio at birth does not predict the adult sex ratio in mammals and birds. However, there is little information on sex ratio at birth in reptiles or amphibians. Furthermore, a better measure for the effect meiotic drive would be the gametic sex ratio, since the sex ratio may be already skewed at birth due to sex-specific differences in embryonic mortality.

– More rapid degeneration of X and Y chromosomes during lifetime:

The author’s propose, that the Y/W may be more affected by further degeneration during lifetime (for example by increased telomere shortening or loss of epigenetic marks). To my knowledge this is rather speculative, as I am not aware of any results supporting this hypothesis.

– Sexually antagonistic selection:

Loci, which are only beneficial to one sex, but may be detrimental to the other are expected to accumulate on sex chromosomes. In an XY-system, male beneficial loci are expected to be found in linkage disequilibrium with the SDR, which ensures that they are exclusively transmitted to males. The positive fitness effects of these Y/W-linked sexually antagonistic mutations would thus result in a postive skew towards the heterogametic sex (although the evolution of recombination suppression may introduce further degeneration of the Y/W chromosome, which can be detrimental). Furthermore, the authors develop a model for sexually antagonistic selection of loci located on X/Z chromosomes and come to the conclusion, that there are no robust generalizations about the direction of the skew of the adult sex ratio resulting from these loci.

The authors point out, that there is no clear support for any of these hypothesis. Further research could test the assumptions of some of these hypotheses: Recessive deleterious mutations on X/Z chromosomes or Y/W specific deleterious mutations, imperfect dosage compensation and sexually antagonistic selection are all related to sex chromosome degeneration and recombination suppression. Although it is difficult to comparatively quantify sex chromosome degeneration across species, more high quality sequences of sex chromosomes are becoming available and it may soon be possible to link sex chromosome degeneration on a gene level to sex specific fitness differences. A very crude proxy for this would be to include whether sex chromosomes are microscopically distinguishable (heteromorphic) or indistinguishable (homomorphic) in this analysis and test whether this explains significant variance in ASRs. Also further research could clarify whether there is a connection between ASR and sex ratio at birth or even better gametic sex ratio in amphibians or reptiles, which could be indicative of meiotic drive.

Conclusions

Overall, I am skeptical that comparing sexual systems as a simple binary character (male or female heterogametic) does adequately represent the diversity of tetrapod sex chromosome systems and I expect that fitness differences should be more related to sex chromosome degeneration than to the GSD itself. Although a significant proportion of the interspecific variation in ASRs is explained by the GSD in groups with variable sex determination systems, there are multiple possible confounding factors (like sex reversal, problems in determining GSDs, uncertainty of common ancestry of GSDs), which could easily lead to biases in the relatively small number of observations in these groups.

References:

Gamble T, Geneva AJ, Glor RE, Zarkower D (2014). Anolis sex chromosomes are derived from a single ancestral pair. Evolution.68(4):1027-41

Rovatsos M, Jasna V, Altmanova M, Johnson Pokorna M (2016). Conservation of sex chromosomes in lacertid lizards. Molecular Ecology.

Stöck M, Croll D, Dumas Z, Biollay S, Wang J, Perrin N (2011). A cryptic heterogametic transition revealed by sex-linked DNA markers in Palearctic green toads. Journal of Evolutionary Biology. 24:1064-1070

genomics / human

Identification of a large set of rare complete human knockouts

May 16, 2016 - by - Leave a Comment

High throughput genotyping and sequencing has led to the discovery of numerous sequence variants associated to human traits and diseases. An important type of variants involved are Loss of Function (LoF) mutations (frameshift indels, stop-gain and essential sites variants), which are predicted to completely disrupt the function of protein-coding genes. In case of Mendelian recessive diseases, for the condition to occur, the LoF variants must be biallelic, i.e. affecting both copies of a gene. The affected gene is then defined as “knockout”.

By studying the Icelandic population, authors aim to identify rare LoF mutations (Minor Allele Frequency, MAF < 2%) present in individuals participating in various disease projects. They then investigate at which frequency in the population these LoF mutations are homozygous (i.e. knockout) in the germline genome.

The Icelandic population Iceland is well-suited for genetic studies for three main reasons. The island was colonized by human population around the 9^th century by 8-20 thousand settlers. Since then the population grew to around 320’000 inhabitants today. The initial founder effect and rare genetic admixture make the Icelandic population a genetic isolate. In addition to an unusual genetic isolation, Iceland’s population benefits of a genealogical database containing family histories reaching centuries back in time, as well as a broad access to nationwide healthcare information.

These characteristics led to the development of large-scale genomic studies of Icelanders by de CODE Genetics. This biopharmaceutical company has published various studies, including this paper, related to genetic variants and diseases in Icelanders.

Loss of function mutation and rare complete knockouts Authors sequenced the whole genome of 2’626 Icelanders participating in various disease projects and identified variants in protein coding genes. These variants were annotated with the predicted impact that they have on the gene: LoF, moderate or low impact. A total of 6’795 LoF mutations in 4’924 genes were identified, with most of these variants (6’285) being rare (MAF < 2%).

The identified LoF variants were imputed into an additional 101’584 chip-genotyped and phased Icelanders, allowing the identification of the number of knockout genes in the population. Authors found that 1’485 previously identified LoF mutations (MAF <2%) are contributing to the knockout of 1’171 genes and that 8’041 individuals possess at least 1 of these knockout genes. Out of these 1’171 genes, 88 had been already linked by previous studies to conditions through a recessive mode of inheritance.

Double transmission deficit of LoF variants Because knockout genes should be deleterious for an organisms, we expect a deficit of homozygous for these genes in the population due to embryonic/fetal, perinatal or juvenile lethality. To investigate whether such a deficit was present, authors calculated the transmission probability of LoF variants from parents to their offspring.

Under Mendelian inheritance, the expected percent of transmission of the LoF mutated gene from heterozygous parents to their offspring (i.e. double transmission) is of 25%. However, results show a statistically significant deficit in double transmission, the observed double transmission probability being of 23.6%.

The rare LoF mutations were ranked according to the Residual Variation Intolerance Score (RVIS) percentiles and essentiality score percentiles. Both measures attempt to classify genes according to their tolerance to functional variation, with the lowest rank corresponding to genes being more sensitive to mutations. As expected, the lowest double transmission rate was found for the most sensitive genes (first percentile), suggesting that a homozygous state of LoF mutation in these genes is deleterious.

Tissue specific expression of knockout genes Authors investigated if genes were more likely to be knockout when expressed in specific tissues. By retrieving the information from previous studies of the number of genes that are highly expressed in 1 or more – but not all – 27 tissues, they calculated the fraction of these genes that were knockout in each tissue. They found that the brain and placenta were the tissue with the lowest fraction of knockout genes (3.1% and 3.9%, respectively), and that in testis, small intestine and duodenum were observed the highest fraction of biallelic LoF mutations (5.8%, 6.4%, and 6.9% respectively).

Conclusion and Comments The characteristics of Icelandic population and the incredibly large sample size (~ 1/3 of the total population) allowed authors to identify a large number of new and rare LoF mutations. Part of these mutations was shown to contribute to the knockout of an unexpected large number of genes in an unexpected large number of people. This study is the first to shed a light on the astonishing number of knockout present in human populations. In addition, by investigating the transmission probability, a deficit in homozygous loss-of function offspring was identified, especially when LoF mutations affected essential genes. This result was expected because of the predicted deleterious effect of biallelic LoF mutations.

Besides the aforementioned interesting results of the paper, some aspects were slightly disappointing. First, I was expecting authors to focus more on the genotype-phenotype aspects. Even if they pinpoint a deficit in double transmission, suggesting deleterious consequences for the organism, authors did not discuss the function of the identified knockout genes and their effect on the phenotype. Second, the paper was not an easy read. Many results were only mentioned without additional information on the methods or data used, and it was sometimes difficult to link them with the main aim of the study. Additionally, figures were sometimes misleading because of different axis scales or incomplete legends.

Finally, authors suggested that important tissues, such as the brain, have a lesser number of knockout compared to other tissues, writing that “genes that are highly expressed in the brain are less often completely knocked out than other genes”. However, this result is questionable as we do not have any measure of the number of knockout genes that we expect to be expressed only by chance in the tissues. In other words, the brain could have a lower number of knockout genes expressed compared to other tissues only because the total number of expressed genes in the brain is lower. Therefore we do not know if the lower number of knockout genes in the brain is due to chance or to biological reasons.

Nevertheless, this study opens the door to understanding how many knockout genes occur without phenotypic consequences in humans, what are the genes function and essentiality, and the role of the environment in the buildup of phenotype. The classical search for genetic variants associated to a phenotype, as in GWAS studies, could be reversed by first identifying individuals with the same genetic variants and then precisely phenotyping them.

Sulem, P., Helgason, H., Oddson, A., Stefansson, H., Gudjonsson, S., Zink, F., Hjartarson, E., Sigurdsson, G., Jonasdottir, A., Jonasdottir, A., Sigurdsson, A., Magnusson, O., Kong, A., Helgason, A., Holm, H., Thorsteinsdottir, U., Masson, G., Gudbjartsson, D., & Stefansson, K. (2015). Identification of a large set of rare complete human knockouts Nature Genetics, 47 (5), 448-452 DOI: 10.1038/ng.3243

evolution / genomics / Uncategorized

Supergenes and social organization in a bird species

May 6, 2016May 6, 2016 - by - Leave a Comment

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 (F_ST) 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

Month: May 2016

The genetic sex-determination system predicts adult sex ratios in tetrapods

Identification of a large set of rare complete human knockouts

Supergenes and social organization in a bird species