Male-biased recombination at chromosome ends in a songbird revealed by precisely mapping crossover positions.

Recombination plays a crucial role in evolution by generating novel haplotypes and disrupting linkage between genes, thereby enhancing the efficiency of selection. Here, we analyze the genomes of 12 great reed warblers (Acrocephalus arundinaceus) in a 3-generation pedigree to identify precise crossover positions along the chromosomes. We located more than 200 crossovers and found that these were highly concentrated toward the telomeric ends of the chromosomes. Apart from this major pattern in the recombination landscape, we found significantly higher frequencies of crossovers in genic compared with intergenic regions, and in exons compared with introns. Moreover, while the number of recombination events was similar between the sexes, the crossovers were located significantly closer to the ends of paternal compared with maternal chromosomes. In conclusion, our study of the great reed warbler revealed substantial variation in crossover frequencies within chromosomes, with a distinct bias toward the sub-telomeric regions, particularly on the paternal side. These findings emphasize the importance of thoroughly screening the entire length of chromosomes to characterize the recombination landscape and uncover potential sex-biases in recombination.

The rate of W chromosome degeneration across multiple avian neo-sex chromosomes.

When sex chromosomes evolve recombination suppression, the sex-limited chromosome (Y/W) commonly degenerate by losing functional genes. The rate of Y/W degeneration is believed to slow down over time as the most essential genes are maintained by purifying selection, but supporting data are scarce especially for ZW systems. Here, we study W degeneration in Sylvioidea songbirds where multiple autosomal translocations to the sex chromosomes, and multiple recombination suppression events causing separate evolutionary strata, have occurred during the last ~ 28.1-4.5 million years (Myr). We show that the translocated regions have maintained 68.3-97.7% of their original gene content, compared to only 4.2% on the much older ancestral W chromosome. By mapping W gene losses onto a dated phylogeny, we estimate an average gene loss rate of 1.0% per Myr, with only moderate variation between four independent lineages. Consistent with previous studies, evolutionarily constrained and haploinsufficient genes were preferentially maintained on W. However, the gene loss rate did not show any consistent association with strata age or with the number of W genes at strata formation. Our study provides a unique account on the pace of W gene loss and reinforces the significance of purifying selection in maintaining essential genes on sex chromosomes.

Consequences of partially recessive deleterious genetic variation for the evolution of inversions suppressing recombination between sex chromosomes1.

The evolution of suppressed recombination between sex chromosomes is widely hypothesized to be driven by sexually antagonistic selection (SA), where tighter linkage between the sex-determining gene(s) and nearby SA loci is favored when it couples male-beneficial alleles to the proto-Y chromosome, and female-beneficial alleles to the proto-X. Although difficult to test empirically, the SA selection hypothesis overshadows several alternatives, including an incomplete but often-repeated "sheltering" hypothesis which suggests that expansion of the sex-linked region (SLR) reduces the homozygous expression of deleterious mutations at selected loci. Here, we use population genetic models to evaluate the consequences of partially recessive deleterious mutational variation for the evolution of otherwise neutral chromosomal inversions expanding the SLR on proto-Y chromosomes. Both autosomal and SLR-expanding inversions face a race against time: lightly-loaded inversions are initially beneficial, but eventually become deleterious as they accumulate new mutations, after which their chances of fixing become negligible. In contrast, initially unloaded inversions eventually become neutral as their deleterious load reaches the same equilibrium as non-inverted haplotypes. Despite the differences in inheritance and indirect selection, SLR-expanding inversions exhibit similar evolutionary dynamics to autosomal inversions over many biologically plausible parameter conditions. Differences emerge when the population average mutation load is quite high; in this case large autosomal inversions that are lucky enough to be mutation-free can rise to intermediate to high frequencies where selection in homozygotes becomes important (Y-linked inversions never appear as homozygous karyotypes); conditions requiring either high mutation rates, highly recessive deleterious mutations, weak selection, or a combination thereof.

The association between haemosporidian infection and non-breeding moult location in great reed warblers revisited by combining feather stable isotope profiles and geolocator data.

Stable isotope analysis provides valuable insights into the ecology of long-distance migratory birds during periods spent away from a specific study site. In a previous study, Swedish great reed warblers (Acrocephalus arundinaceus) infected with haemosporidian parasites differed in feather isotope ratios compared to non-infected birds, suggesting that infected and non-infected birds spent the non-breeding season in different locations or habitats. Here, we use a novel dataset comprising geolocator data, isotopes, and haemosporidian infection status of 92 individuals from four Eurasian populations to investigate whether parasite transmission varies with geography or habitats. We found that the probability of harbouring Plasmodium and Leucocytozoon parasites was higher in birds moulting in the eastern region of the non-breeding grounds. However, no geographic pattern occurred for Haemoproteus infections or overall infection status. In contrast to the previous study, we did not find any relationship between feather isotope ratios and overall haemosporidian infection for the entire current dataset. Plasmodium-infected birds had lower feather δ15N values indicating that they occupied more mesic habitats. Leucocytozoon-infected birds had higher feather δ34S values suggesting more coastal sites or wetlands with anoxic sulphate reduction. As the composition and prevalence of haemosporidian parasites differed between the old and the current dataset, we suggest that the differences might be a consequence of temporal dynamics of haemosporidian parasites. Our results emphasize the importance of replicating studies conducted on a single population over a restricted time period, as the patterns can become more complex for data from wider geographical areas and different time periods.

RecView: an interactive R application for locating recombination positions using pedigree data.

BACKGROUND: Recombination reshuffles alleles at linked loci, allowing genes to evolve independently and consequently enhancing the efficiency of selection. This makes quantifying recombination along chromosomes an important goal for understanding how selection and drift are acting on genes and chromosomes. RESULTS: We present RecView, an interactive R application and its homonymous R package, to facilitate locating recombination positions along chromosomes or scaffolds using whole-genome genotype data of a three-generation pedigree. RecView analyses and plots the grandparent-of-origin of all informative alleles along each chromosome of the offspring in the pedigree, and infers recombination positions with either of two built-in algorithms: one based on change in the proportion of the alleles with specific grandparent-of-origin, and one on the degree of continuity of alleles with the same grandparent-of-origin. RecView handles multiple offspring and chromosomes simultaneously, and all putative recombination positions are reported in base pairs together with an estimated precision based on the local density of informative alleles. We demonstrate RecView using genotype data of a passerine bird with an available reference genome, the great reed warbler (Acrocephalus arundinaceus), and show that recombination events can be located to specific positions. CONCLUSIONS: RecView is an easy-to-use and highly effective application for locating recombination positions with high precision. RecView is available on GitHub ( https://github.com/HKyleZhang/RecView.git ).

Evidence of Site-Specific and Male-Biased Germline Mutation Rate in a Wild Songbird.

Germline mutations are the ultimate source of genetic variation and the raw material for organismal evolution. Despite their significance, the frequency and genomic locations of mutations, as well as potential sex bias, are yet to be widely investigated in most species. To address these gaps, we conducted whole-genome sequencing of 12 great reed warblers (Acrocephalus arundinaceus) in a pedigree spanning 3 generations to identify single-nucleotide de novo mutations (DNMs) and estimate the germline mutation rate. We detected 82 DNMs within the pedigree, primarily enriched at CpG sites but otherwise randomly located along the chromosomes. Furthermore, we observed a pronounced sex bias in DNM occurrence, with male warblers exhibiting three times more mutations than females. After correction for false negatives and adjusting for callable sites, we obtained a mutation rate of 7.16 × 10-9 mutations per site per generation (m/s/g) for the autosomes and 5.10 × 10-9 m/s/g for the Z chromosome. To demonstrate the utility of species-specific mutation rates, we applied our autosomal mutation rate in models reconstructing the demographic history of the great reed warbler. We uncovered signs of drastic population size reductions predating the last glacial period (LGP) and reduced gene flow between western and eastern populations during the LGP. In conclusion, our results provide one of the few direct estimates of the mutation rate in wild songbirds and evidence for male-driven mutations in accordance with theoretical expectations.

Solar heating may explain extreme diel flight altitude changes in migrating birds.

Great reed warblers, Acrocephalus arundinaceus,1 and great snipes, Gallinago media,2 exhibit a diel cycle in flight altitudes-flying much higher during the day than the night-when performing migratory flights covering both night and day. One hypothesis proposed to explain this behavior is that the birds face additional heating by solar radiation during daytime and hence must climb to very high, and thus also very cold, altitudes to avoid overheating during daytime flights.1,2 Yet, solar heat gain in birds has been shown to drastically decrease with wind speed,3,4 and the quantitative heating effect by solar radiation on a bird flying with an airspeed of 10 m/s or more is unknown. We analyzed temperature data from multisensor data loggers (MDLs)5,6 placed without direct exposure to solar radiation on great reed warblers (the logger covered by feathers on the back) and great snipes (the logger on the leg, covered from the sun by the tail). We found that logger temperatures were significantly higher (5.9°C-8.8°C in great reed warblers and 4.8°C-5.4°C in great snipes) during the day than during the night in birds flying at the same altitudes (and thus also the same expected ambient air temperatures). These results strongly indicate that the heat balance of the flying birds is indeed affected by solar radiation, which is in accordance with the hypothesis that solar radiation is a key factor causing the remarkable diel cycles in flight altitude observed in these two long-distance migrant bird species.1,2.

The evolutionary maintenance of ancient recombining sex chromosomes in the ostrich.

Sex chromosomes have evolved repeatedly across the tree of life and often exhibit extreme size dimorphism due to genetic degeneration of the sex-limited chromosome (e.g. the W chromosome of some birds and Y chromosome of mammals). However, in some lineages, ancient sex-limited chromosomes have escaped degeneration. Here, we study the evolutionary maintenance of sex chromosomes in the ostrich (Struthio camelus), where the W remains 65% the size of the Z chromosome, despite being more than 100 million years old. Using genome-wide resequencing data, we show that the population scaled recombination rate of the pseudoautosomal region (PAR) is higher than similar sized autosomes and is correlated with pedigree-based recombination rate in the heterogametic females, but not homogametic males. Genetic variation within the sex-linked region (SLR) (π = 0.001) was significantly lower than in the PAR, consistent with recombination cessation. Conversely, genetic variation across the PAR (π = 0.0016) was similar to that of autosomes and dependent on local recombination rates, GC content and to a lesser extent, gene density. In particular, the region close to the SLR was as genetically diverse as autosomes, likely due to high recombination rates around the PAR boundary restricting genetic linkage with the SLR to only ~50Kb. The potential for alleles with antagonistic fitness effects in males and females to drive chromosome degeneration is therefore limited. While some regions of the PAR had divergent male-female allele frequencies, suggestive of sexually antagonistic alleles, coalescent simulations showed this was broadly consistent with neutral genetic processes. Our results indicate that the degeneration of the large and ancient sex chromosomes of the ostrich may have been slowed by high recombination in the female PAR, reducing the scope for the accumulation of sexually antagonistic variation to generate selection for recombination cessation.

Sex matters: predator presence induces sexual dimorphism in a monomorphic prey, from stress genes to morphological defences.

Inducible defences allow prey to increase survival chances when predators are present while avoiding unnecessary costs in their absence. Many studies report considerable inter-individual variation in inducible defence expression, yet what underlies this variation is poorly understood. A classic vertebrate example of a predator-induced morphological defence is the increased body depth in crucian carp (Carassius carassius), which reduces the risk of predation from gape-size limited predators. Here, we report that among-individual variation in morphological defence expression can be linked to sex. We documented sexual dimorphism in lakes in which crucian carp coexisted with predators, where females showed shallower relative body depths than males, but not in a predator-free lake. When exposing crucian carp from a population without predators to perceived predation risk in a laboratory environment (presence/absence of pike, Esox lucius), we found that males expressed significantly greater morphological defence than females, causing sexual dimorphism only in the presence of predators. We uncovered a correlative link between the sex-specific inducible phenotypic response and gene expression patterns in major stress-related genes (POMC, MC3R, and MC4R). Together, our results highlight that sex-specific responses may be an important, yet underappreciated, component underlying inter-individual differences in the expression of inducible defences, even in species without pronounced sexual dimorphism.

Animal tracing with sulfur isotopes: Spatial segregation and climate variability in Africa likely contribute to population trends of a migratory songbird.

Climatic conditions affect animals but range-wide impacts at the population level remain largely unknown, especially in migratory species. However, studying climate-population relationships is still challenging in small migrants due to a lack of efficient and cost-effective geographic tracking method. Spatial distribution patterns of environmental stable isotopes (so called 'isoscapes') generally overcome these limitations but none of the currently available isoscapes provide a substantial longitudinal gradient in species-rich sub-Saharan Africa. In this region, sulphur (δ34 S) has not been sufficiently explored on a larger scale. We developed a δ34 S isoscape to trace animal origins in sub-Saharan Africa by coupling known-origin samples from tracked migratory birds with continental remotely sensed environmental data building on environment-δ34 S relationships using a flexible machine learning technique. Furthermore, we link population-specific nonbreeding grounds with interannual climatic variation that might translate to breeding population trends. The predicted δ34 S isotopic map featured east-west and coast-to-inland isotopic gradients and was applied to predict nonbreeding grounds of three breeding populations of Eurasian Reed Warblers Acrocephalus scirpaceus with two distinct migratory phenotypes. Breeding populations as well as migratory phenotypes exhibited large-scale segregation within the African nonbreeding range. These regions also differed substantially in the interannual climatic variation, with higher interannual variability in the eastern part of the range during 2001-2012. Over the same period, the eastern European breeding population seemed to have experienced a more steep decline in population size. The link between migratory patterns and large-scale climatic variability appears important to better understand population trajectories in many declining migratory animals. We believe animal tracing using sulphur isotopes will facilitate these efforts and offers manifold ecological and forensic applications in the biodiversity hotspot of sub-Saharan Africa.

A novel neo-sex chromosome in Sylvietta brachyura (Macrosphenidae) adds to the extraordinary avian sex chromosome diversity among Sylvioidea songbirds.

We report the discovery of a novel neo-sex chromosome in an African warbler, Sylvietta brachyura (northern crombec; Macrosphenidae). This species is part of the Sylvioidea superfamily, where four separate autosome-sex chromosome translocation events have previously been discovered via comparative genomics of 11 of the 22 families in this clade. Our discovery here resulted from analyses of genomic data of single species-representatives from three additional Sylvioidea families (Macrosphenidae, Pycnonotidae and Leiothrichidae). In all three species, we confirmed the translocation of a part of chromosome 4A to the sex chromosomes, which originated basally in Sylvioidea. In S. brachyura, we found that a part of chromosome 8 has been translocated to the sex chromosomes, forming a unique neo-sex chromosome in this lineage. Furthermore, the non-recombining part of 4A in S. brachyura is smaller than in other Sylvioidea species, which suggests that recombination continued along this region after the fusion event in the Sylvioidea ancestor. These findings reveal additional sex chromosome diversity among the Sylvioidea, where five separate translocation events are now confirmed.

Restricted X chromosome introgression and support for Haldane's rule in hybridizing damselflies.

Contemporary hybrid zones act as natural laboratories for the investigation of species boundaries and may shed light on the little understood roles of sex chromosomes in species divergence. Sex chromosomes are considered to function as a hotspot of genetic divergence between species; indicated by less genomic introgression compared to autosomes during hybridization. Moreover, they are thought to contribute to Haldane's rule, which states that hybrids of the heterogametic sex are more likely to be inviable or sterile. To test these hypotheses, we used contemporary hybrid zones of Ischnura elegans, a damselfly species that has been expanding its range into the northern and western regions of Spain, leading to chronic hybridization with its sister species Ischnura graellsii. We analysed genome-wide SNPs in the Spanish I. elegans and I. graellsii hybrid zone and found (i) that the X chromosome shows less genomic introgression compared to autosomes, and (ii) that males are underrepresented among admixed individuals, as predicted by Haldane's rule. This is the first study in Odonata that suggests a role of the X chromosome in reproductive isolation. Moreover, our data add to the few studies on species with X0 sex determination system and contradict the hypothesis that the absence of a Y chromosome causes exceptions to Haldane's rule.

Weak population genetic structure in Eurasian spruce bark beetle over large regional scales in Sweden.

The Eurasian spruce bark beetle, Ips typographus, is a major pest, capable of killing spruce forests during large population outbreaks. Recorded dispersal distances of individual beetles are typically within hundreds of meters or a few kilometers. However, the connectivity between populations at larger distances and longer time spans and how this is affected by the habitat is less studied, despite its importance for understanding at which distances local outbreaks may spread. Previous population genetic studies in I. typographus typically used low resolution markers. Here, we use genome-wide data to assess population structure and connectivity of I. typographus in Sweden. We used 152 individuals from 19 population samples, distributed over 830 km from Strömsund (63° 46' 8″ N) in the north to Nyteboda (56° 8' 50″ N) in the south, to capture processes at a large regional scale, and a transect sampling design adjacent to a recent outbreak to capture processes at a smaller scale (76 km). Using restriction site-associated DNA sequencing (RADseq) markers capturing 1409-1997 SNPs throughout the genome, we document a weak genetic structure over the large scale, potentially indicative of high connectivity with extensive gene flow. No differentiation was detected at the smaller scale. We find indications of isolation-by-distance both for relative (F ST) and absolute divergence (Dxy). The two northernmost populations are most differentiated from the remaining populations, and diverge in parallel to the southern populations for a set of outlier loci. In conclusion, the population structure of I. typographus in Sweden is weak, suggesting a high capacity to disperse and establish outbreak populations in new territories.

Sex-limited chromosomes and non-reproductive traits.

Sex chromosomes are typically viewed as having originated from a pair of autosomes, and differentiated as the sex-limited chromosome (e.g. Y) has degenerated by losing most genes through cessation of recombination. While often thought that degenerated sex-limited chromosomes primarily affect traits involved in sex determination and sex cell production, accumulating evidence suggests they also influence traits not sex-limited or directly involved in reproduction. Here, we provide an overview of the effects of sex-limited chromosomes on non-reproductive traits in XY, ZW or UV sex determination systems, and discuss evolutionary processes maintaining variation at sex-limited chromosomes and molecular mechanisms affecting non-reproductive traits.

Assessment of phylogenetic approaches to study the timing of recombination cessation on sex chromosomes.

The evolution of sex chromosomes is hypothesized to be punctuated by consecutive recombination cessation events, forming "evolutionary strata" that ceased to recombine at different time points. The demarcation of evolutionary strata is often assessed by estimates of the timing of recombination cessation (tRC ) along the sex chromosomes, commonly inferred from the level of synonymous divergence or with species phylogenies at gametologous (X-Y or Z-W) sequence data. However, drift and selection affect sequences unpredictably and introduce uncertainty when inferring tRC . Here, we assess two alternative phylogenetic approaches to estimate tRC ; (i) the expected likelihood weight (ELW) approach that finds the most likely topology among a set of hypothetical topologies and (ii) the BEAST approach that estimates tRC with specified calibration priors on a reference species topology. By using Z and W gametologs of an old and a young evolutionary stratum on the neo-sex chromosome of Sylvioidea songbirds, we show that the ELW and BEAST approaches yield similar tRC estimates, and that both outperform two frequently applied approaches utilizing synonymous substitution rates (dS) and maximum likelihood (ML) trees, respectively. Moreover, we demonstrate that both ELW and BEAST provide more precise tRC estimates when sequences of multiple species are included in the analyses.

MHCtools - an R package for MHC high-throughput sequencing data: Genotyping, haplotype and supertype inference, and downstream genetic analyses in non-model organisms.

The major histocompatibility complex (MHC) plays a central role in the vertebrate adaptive immune system and has been of long-term interest in evolutionary biology. While several protocols have been developed for MHC genotyping, there is a lack of transparent and standardized tools for downstream analysis of MHC data. Here, we present the r package mhctools and demonstrate the use of its functions to (i) assist accurate MHC genotyping from high-throughput amplicon-sequencing data, (ii) infer functional MHC supertypes using bootstrapped clustering analysis, (iii) identify segregating MHC haplotypes from family data, and (iv) analyse functional and genetic distances between MHC sequences. We employed mhctools to analyse MHC class I (MHC-I) amplicon data of 559 great reed warblers (Acrocephalus arundinaceus). We identified 390 MHC-I alleles which clustered into 14 functional supertypes. A phylogenetic analysis and analyses of positive selection suggested that the MHC-I alleles belong to several distinct functional groups. We furthermore identified 107 segregating haplotypes among 116 families, and found substantial variation in diversity with 4-21 MHC-I alleles and 3-13 MHC-I supertypes per haplotype. Finally, we show that the great reed warbler haplotypes harboured combinations of MHC-I supertypes with greater functional divergence than observed in simulated populations of possible haplotypes, a result that is in accordance with the divergent allele advantage hypothesis. Our study demonstrates the power of mhctools to support genotyping and analysis of MHC in non-model species, which we hope will encourage broad implementation among researchers in MHC genetics and evolution.

Extreme variation in recombination rate and genetic diversity along the Sylvioidea neo-sex chromosome.

Recombination strongly impacts sequence evolution by affecting the extent of linkage and the efficiency of selection. Here, we study recombination over the Z chromosome in great reed warblers (Acrocephalus arundinaceus) using pedigree-based linkage mapping. This species has extended Z and W chromosomes ("neo-sex chromosomes") formed by a fusion between a part of chromosome 4A and the ancestral sex chromosomes, which provides a unique opportunity to assess recombination and sequence evolution in sex-linked regions of different ages. We assembled an 87.54 Mbp and 90.19 cM large Z with a small pseudoautosomal region (0.89 Mbp) at one end and the fused Chr4A-part at the other end of the chromosome. A prominent feature in our data was an extreme variation in male recombination rate along Z with high values at both chromosome ends, but an apparent lack of recombination over a substantial central section, covering 78% of the chromosome. The nonrecombining region showed a drastic loss of genetic diversity and accumulation of repeats compared to the recombining parts. Thus, our data emphasize a key role of recombination in affecting local levels of polymorphism. Nonetheless, the evolutionary rate of genes (dN/dS) did not differ between high and low recombining regions, suggesting that the efficiency of selection on protein-coding sequences can be maintained also at very low levels of recombination. Finally, the Chr4A-derived part showed a similar recombination rate as the part of the ancestral Z that did recombine, but its sequence characteristics reflected both its previous autosomal, and current Z-linked, recombination patterns.

Findzx: an automated pipeline for detecting and visualising sex chromosomes using whole-genome sequencing data.

BACKGROUND: Sex chromosomes have evolved numerous times, as revealed by recent genomic studies. However, large gaps in our knowledge of sex chromosome diversity across the tree of life remain. Filling these gaps, through the study of novel species, is crucial for improved understanding of why and how sex chromosomes evolve. Characterization of sex chromosomes in already well-studied organisms is also important to avoid misinterpretations of population genomic patterns caused by undetected sex chromosome variation. RESULTS: Here we present findZX, an automated Snakemake-based computational pipeline for detecting and visualizing sex chromosomes through differences in genome coverage and heterozygosity between any number of males and females. A main feature of the pipeline is the option to perform a genome coordinate liftover to a reference genome of another species. This allows users to inspect sex-linked regions over larger contiguous chromosome regions, while also providing important between-species synteny information. To demonstrate its effectiveness, we applied findZX to publicly available genomic data from species belonging to widely different taxonomic groups (mammals, birds, reptiles, and fish), with sex chromosome systems of different ages, sizes, and levels of differentiation. We also demonstrate that the liftover method is robust over large phylogenetic distances (> 80 million years of evolution). CONCLUSIONS: With findZX we provide an easy-to-use and highly effective tool for identification of sex chromosomes. The pipeline is compatible with both Linux and MacOS systems, and scalable to suit different computational platforms.

Consequences of partially recessive deleterious genetic variation for the evolution of inversions suppressing recombination between sex chromosomes.

The evolution of suppressed recombination between sex chromosomes is widely hypothesized to be driven by sexually antagonistic selection (SA), where tighter linkage between the sex-determining gene(s) and nearby SA loci is favored when it couples male-beneficial alleles to the proto-Y chromosome, and female-beneficial alleles to the proto-X. Despite limited empirical evidence, the SA selection hypothesis overshadows several alternatives, including an incomplete but often-repeated "sheltering hypothesis" that suggests that expansion of the sex-linked region (SLR) reduces homozygous expression of partially recessive deleterious mutations at selected loci. Here, we use population genetic models to evaluate the consequences of deleterious mutational variation for the evolution of neutral chromosomal inversions expanding the SLR on proto-Y chromosomes. We find that SLR-expanding inversions face a race against time: lightly loaded inversions are initially beneficial, but eventually become deleterious as they accumulate new mutations, and must fix before this window of opportunity closes. The outcome of this race is strongly influenced by inversion size, the mutation rate, and the dominance coefficient of deleterious mutations. Yet, small inversions have elevated fixation probabilities relative to neutral expectations for biologically plausible parameter values. Our results demonstrate that deleterious genetic variation can plausibly drive recombination suppression in small steps and would be most consistent with empirical patterns of small evolutionary strata or gradual recombination arrest.

The genomic architecture of the passerine MHC region: High repeat content and contrasting evolutionary histories of single copy and tandemly duplicated MHC genes.

The major histocompatibility complex (MHC) is of central importance to the immune system, and an optimal MHC diversity is believed to maximize pathogen elimination. Birds show substantial variation in MHC diversity, ranging from few genes in most bird orders to very many genes in passerines. Our understanding of the evolutionary trajectories of the MHC in passerines is hampered by lack of data on genomic organization. Therefore, we assembled and annotated the MHC genomic region of the great reed warbler (Acrocephalus arundinaceus), using long-read sequencing and optical mapping. The MHC region is large (>5.5 Mb), characterized by structural changes compared to hitherto investigated bird orders and shows higher repeat content than the genome average. These features were supported by analyses in three additional passerines. MHC genes in passerines are found in two different chromosomal arrangements, either as single copy MHC genes located among non-MHC genes, or as tandemly duplicated tightly linked MHC genes. Some single copy MHC genes are old and putative orthologues among species. In contrast tandemly duplicated MHC genes are monophyletic within species and have evolved by simultaneous gene duplication of several MHC genes. Structural differences in the MHC genomic region among bird orders seem substantial compared to mammals and have possibly been fuelled by clade-specific immune system adaptations. Our study provides methodological guidance in characterizing complex genomic regions, constitutes a resource for MHC research in birds, and calls for a revision of the general belief that avian MHC has a conserved gene order and small size compared to mammals.