It is estimated that a large proportion of amino acid substitutions in Drosophila have been fixed by natural selection, and as organisms are faced with an ever-changing array of pathogens and parasites to which they must adapt, we have investigated the role of parasite-mediated selection as a likely cause. To quantify the effect, and to identify which genes and pathways are most likely to be involved in the host–parasite arms race, we have re-sequenced population samples of 136 immunity and 287 position-matched non-immunity genes in two species of Drosophila. Using these data, and a new extension of the McDonald-Kreitman approach, we estimate that natural selection fixes advantageous amino acid changes in immunity genes at nearly double the rate of other genes. We find the rate of adaptive evolution in immunity genes is also more variable than other genes, with a small subset of immune genes evolving under intense selection. These genes, which are likely to represent hotspots of host–parasite coevolution, tend to share similar functions or belong to the same pathways, such as the antiviral RNAi pathway and the IMD signalling pathway. These patterns appear to be general features of immune system evolution in both species, as rates of adaptive evolution are correlated between the D. melanogaster and D. simulans lineages. In summary, our data provide quantitative estimates of the elevated rate of adaptive evolution in immune system genes relative to the rest of the genome, and they suggest that adaptation to parasites is an important force driving molecular evolution.

An absolute timescale for evolution is essential if we are to associate evolutionary phenomena, such as adaptation or speciation, with potential causes, such as geological activity or climatic change. Timescales in most phylogenetic studies use geologically dated fossils or phylogeographic events as calibration points, but more recently, it has also become possible to use experimentally derived estimates of the mutation rate as a proxy for substitution rates. The large radiation of drosophilid taxa endemic to the Hawaiian islands has provided multiple calibration points for the Drosophila phylogeny, thanks to the "conveyor belt" process by which this archipelago forms and is colonized by species. However, published date estimates for key nodes in the Drosophila phylogeny vary widely, and many are based on simplistic models of colonization and coalescence or on estimates of island age that are not current. In this study, we use new sequence data from seven species of Hawaiian Drosophila to examine a range of explicit coalescent models and estimate substitution rates. We use these rates, along with a published experimentally determined mutation rate, to date key events in drosophilid evolution. Surprisingly, our estimate for the date for the most recent common ancestor of the genus Drosophila based on mutation rate (25–40 Ma) is closer to being compatible with independent fossil-derived dates (20–50 Ma) than are most of the Hawaiian-calibration models and also has smaller uncertainty. We find that Hawaiian-calibrated dates are extremely sensitive to model choice and give rise to point estimates that range between 26 and 192 Ma, depending on the details of the model. Potential problems with the Hawaiian calibration may arise from systematic variation in the molecular clock due to the long generation time of Hawaiian Drosophila compared with other Drosophila and/or uncertainty in linking island formation dates with colonization dates. As either source of error will bias estimates of divergence time, we suggest mutation rate estimates be used until better models are available.

Bright-red colors in vertebrates are commonly involved in sexual, social, and interspecific signaling [1-8] and are largely produced by ketocarotenoid pigments. In land birds, ketocarotenoids such as astaxanthin are usually metabolically derived via ketolation of dietary yellow carotenoids [9, 10]. However, the molecular basis of this gene-environment mechanism has remained obscure. Here we use the yellowbeak mutation in the zebra finch (Taeniopygia guttata) to investigate the genetic basis of red coloration. Wild-type ketocarotenoids were absent in the beak and tarsus of yellowbeak birds. The yellowbeak mutation mapped to chromosome 8, close to a cluster of cytochrome P450 loci (CYP2J2-like) that are candidates for carotenoid ketolases. The wild-type zebra finch genome was found to have three intact genes in this cluster: CYP2J19A, CYP2J19B, and CYP2J40. In yellowbeak, there are multiple mutations: loss of a complete CYP2J19 gene, a modified remaining CYP2J19 gene (CYP2J19(yb)), and a non-synonymous SNP in CYP2J40. In wild-type birds, CYP2J19 loci are expressed in ketocarotenoid-containing tissues: CYP2J19A only in the retina and CYP2J19B in the beak and tarsus and to a variable extent in the retina. In contrast, expression of CYP2J19(yb) is barely detectable in the beak of yellowbeak birds. CYP2J40 has broad tissue expression and shows no differences between wild-type and yellowbeak. Our results indicate that CYP2J19 genes are strong candidates for the carotenoid ketolase and imply that ketolation occurs in the integument in zebra finches. Since cytochrome P450 enzymes include key detoxification enzymes, our results raise the intriguing possibility that red coloration may be an honest signal of detoxification ability.

Supplementary information: 4 tables, 6 figuresSupplementary materials: Two Excel files for GWAS, eQTL and eigenGWAS, and summary of gene expression analysis. One .mpg file that contains videos of motile sperm of alternative karyomorphs. 2Sperm competition is an important selective force in many organisms. As a result, sperm have evolved to be among the most diverse cells in the animal kingdom. However, the relationship between sperm morphology, sperm motility and fertilisation success is only partially understood. The extent to which between-male variation is heritable is largely unknown, and remarkably few studies have investigated the genetic architecture of sperm traits, especially sperm morphology. Here we use high-density genotyping and gene expression profiling to explore the considerable sperm trait variation that exists in the zebra finch Taeniopygia guttata.We show that nearly all of the genetic variation in sperm morphology is caused by an inversion polymorphism on the Z chromosome acting as a 'supergene'. These results provide a striking example of two evolutionary genetic predictions. First, that in species where females are the heterogametic sex, genetic variation affecting sexually dimorphic traits will accumulate on the Z chromosome. Second, recombination suppression at the inversion allows beneficial dominant alleles to become fixed on whichever haplotype they first arise, without being exchanged onto other haplotypes. Finally, we show that the inversion polymorphism will be stably maintained by heterozygote advantage, because heterozygous males have the fastest and most successful sperm.Sperm are perhaps the most diverse cells in the animal kingdom, with enormous morphological variation between taxa, between species, between males and within an ejaculate 1 .Considerable interest in sperm diversity has arisen following the realisation that sperm competition (post-copulatory sexual selection) is a powerful selective force in many organisms 2 , and that sperm morphology has co-evolved with female reproductive tract morphology 3 . The zebra finch is a model species for studies of sperm biology. Sperm length is repeatable within an ejaculate, yet variable between different males; most morphological traits (head, midpiece, tail and total length) are highly heritable 4 . Furthermore, there is a documented phenotypic and genetic correlation between morphology and sperm swimming velocity ('motility') 5 . In artificially selected lines, pronounced differences in total sperm length are apparent after just three generations of divergent selection, and males with long sperm have the greatest probability of fertilisation success in sperm competition 3 trials 6 . Additionally, the zebra finch has its genome sequenced, assembled and annotated 7 , and so the toolkit to explore the genetics of phenotypic variation is available.In this study we set out to understand the genetic architecture of sperm morphology and motility in the zebra finch. Our aim was to combine genome wide association mapping with analyses of ...