Disrupted Gene Networks in Subfertile Hybrid House Mice.

The Dobzhansky-Muller (DM) model provides a widely accepted mechanism for the evolution of reproductive isolation: incompatible substitutions disrupt interactions between genes. To date, few candidate incompatibility genes have been identified, leaving the genes driving speciation mostly uncharacterized. The importance of interactions in the DM model suggests that gene coexpression networks provide a powerful framework to understand disrupted pathways associated with postzygotic isolation. Here, we perform weighted gene coexpression network analysis to infer gene interactions in hybrids of two recently diverged European house mouse subspecies, Mus mus domesticus and M. m. musculus, which commonly show hybrid male sterility or subfertility. We use genome-wide testis expression data from 467 hybrid mice from two mapping populations: F2s from a laboratory cross between wild-derived pure subspecies strains and offspring of natural hybrids captured in the Central Europe hybrid zone. This large data set enabled us to build a robust consensus network using hybrid males with fertile phenotypes. We identify several expression modules, or groups of coexpressed genes, that are disrupted in subfertile hybrids, including modules functionally enriched for spermatogenesis, cilium and sperm flagellum organization, chromosome organization, and DNA repair, and including genes expressed in spermatogonia, spermatocytes, and spermatids. Our network-based approach enabled us to hone in on specific hub genes likely to be influencing module-wide gene expression and hence potentially driving large-effect DM incompatibilities. A disproportionate number of hub genes lie within sterility loci identified previously in the hybrid zone mapping population and represent promising candidate barrier genes and targets for future functional analysis.

Further resolution of the house mouse (Mus musculus) phylogeny by integration over isolation-with-migration histories.

BACKGROUND: The three main subspecies of house mice, Mus musculus castaneus, Mus musculus domesticus, and Mus musculus musculus, are estimated to have diverged ~ 350-500KYA. Resolution of the details of their evolutionary history is complicated by their relatively recent divergence, ongoing gene flow among the subspecies, and complex demographic histories. Previous studies have been limited to some extent by the number of loci surveyed and/or by the scope of the method used. Here, we apply a method (IMa3) that provides an estimate of a population phylogeny while allowing for complex histories of gene exchange. RESULTS: Results strongly support a topology with M. m. domesticus as sister to M. m. castaneus and M. m. musculus. In addition, we find evidence of gene flow between all pairs of subspecies, but that gene flow is most restricted from M. m. musculus into M. m. domesticus. Estimates of other key parameters are dependent on assumptions regarding generation time and mutation rate in house mice. Nevertheless, our results support previous findings that the effective population size, Ne, of M. m. castaneus is larger than that of the other two subspecies, that the three subspecies began diverging ~ 130 - 420KYA, and that the time between divergence events was short. CONCLUSIONS: Joint demographic and phylogenetic analyses of genomic data provide a clearer picture of the history of divergence in house mice.

Niche filling slows the diversification of Himalayan songbirds.

Speciation generally involves a three-step process--range expansion, range fragmentation and the development of reproductive isolation between spatially separated populations. Speciation relies on cycling through these three steps and each may limit the rate at which new species form. We estimate phylogenetic relationships among all Himalayan songbirds to ask whether the development of reproductive isolation and ecological competition, both factors that limit range expansions, set an ultimate limit on speciation. Based on a phylogeny for all 358 species distributed along the eastern elevational gradient, here we show that body size and shape differences evolved early in the radiation, with the elevational band occupied by a species evolving later. These results are consistent with competition for niche space limiting species accumulation. Even the elevation dimension seems to be approaching ecological saturation, because the closest relatives both inside the assemblage and elsewhere in the Himalayas are on average separated by more than five million years, which is longer than it generally takes for reproductive isolation to be completed; also, elevational distributions are well explained by resource availability, notably the abundance of arthropods, and not by differences in diversification rates in different elevational zones. Our results imply that speciation rate is ultimately set by niche filling (that is, ecological competition for resources), rather than by the rate of acquisition of reproductive isolation.

Divergence patterns of genic copy number variation in natural populations of the house mouse (Mus musculus domesticus) reveal three conserved genes with major population-specific expansions.

Copy number variation represents a major source of genetic divergence, yet the evolutionary dynamics of genic copy number variation in natural populations during differentiation and adaptation remain unclear. We applied a read depth approach to genome resequencing data to detect copy number variants (CNVs) ≥1 kb in wild-caught mice belonging to four populations of Mus musculus domesticus. We complemented the bioinformatics analyses with experimental validation using droplet digital PCR. The specific focus of our analysis is CNVs that include complete genes, as these CNVs could be expected to contribute most directly to evolutionary divergence. In total, 1863 transcription units appear to be completely encompassed within CNVs in at least one individual when compared to the reference assembly. Further, 179 of these CNVs show population-specific copy number differences, and 325 are subject to complete deletion in multiple individuals. Among the most copy-number variable genes are three highly conserved genes that encode the splicing factor CWC22, the spindle protein SFI1, and the Holliday junction recognition protein HJURP. These genes exhibit population-specific expansion patterns that suggest involvement in local adaptations. We found that genes that overlap with large segmental duplications are generally more copy-number variable. These genes encode proteins that are relevant for environmental and behavioral interactions, such as vomeronasal and olfactory receptors, as well as major urinary proteins and several proteins of unknown function. The overall analysis shows that genic CNVs contribute more to population differentiation in mice than in humans and may promote and speed up population divergence.

Contributions of protein-coding and regulatory change to adaptive molecular evolution in murid rodents.

The contribution of regulatory versus protein change to adaptive evolution has long been controversial. In principle, the rate and strength of adaptation within functional genetic elements can be quantified on the basis of an excess of nucleotide substitutions between species compared to the neutral expectation or from effects of recent substitutions on nucleotide diversity at linked sites. Here, we infer the nature of selective forces acting in proteins, their UTRs and conserved noncoding elements (CNEs) using genome-wide patterns of diversity in wild house mice and divergence to related species. By applying an extension of the McDonald-Kreitman test, we infer that adaptive substitutions are widespread in protein-coding genes, UTRs and CNEs, and we estimate that there are at least four times as many adaptive substitutions in CNEs and UTRs as in proteins. We observe pronounced reductions in mean diversity around nonsynonymous sites (whether or not they have experienced a recent substitution). This can be explained by selection on multiple, linked CNEs and exons. We also observe substantial dips in mean diversity (after controlling for divergence) around protein-coding exons and CNEs, which can also be explained by the combined effects of many linked exons and CNEs. A model of background selection (BGS) can adequately explain the reduction in mean diversity observed around CNEs. However, BGS fails to explain the wide reductions in mean diversity surrounding exons (encompassing ~100 Kb, on average), implying that there is a substantial role for adaptation within exons or closely linked sites. The wide dips in diversity around exons, which are hard to explain by BGS, suggest that the fitness effects of adaptive amino acid substitutions could be substantially larger than substitutions in CNEs. We conclude that although there appear to be many more adaptive noncoding changes, substitutions in proteins may dominate phenotypic evolution.

Use of a natural hybrid zone for genomewide association mapping of craniofacial traits in the house mouse.

The identification of the genes involved in morphological variation in nature is still a major challenge. Here, we explore a new approach: we combine 178 samples from a natural hybrid zone between two subspecies of the house mouse (Mus musculus domesticus and Mus musculus musculus), and high coverage of the genome (~ 145K SNPs) to identify loci underlying craniofacial shape variation. Due to the long history of recombination in the hybrid zone, high mapping resolution is anticipated. The combination of genomes from subspecies allows the mapping of both, variation within subspecies and inter-subspecific differences, thereby increasing the overall amount of causal genetic variation that can be detected. Skull and mandible shape were measured using 3D landmarks and geometric morphometrics. Using principal component axes as phenotypes, and a linear mixed model accounting for genetic relatedness in the mapping populations, we identified nine genomic regions associated with skull shape and 10 with mandible shape. High mapping resolution (median size of significant regions = 148 kb) enabled identification of single or few candidate genes in most cases. Some of the genes act as regulators or modifiers of signalling pathways relevant for morphological development and bone formation, including several with known craniofacial phenotypes in mice and humans. The significant associations combined explain 13% and 7% of the skull and mandible shape variation, respectively. In addition, a positive correlation was found between chromosomal length and proportion of variation explained. Our results suggest a complex genetic architecture for shape traits and support a polygenic model.

Evidence for pervasive adaptive protein evolution in wild mice.

The relative contributions of neutral and adaptive substitutions to molecular evolution has been one of the most controversial issues in evolutionary biology for more than 40 years. The analysis of within-species nucleotide polymorphism and between-species divergence data supports a widespread role for adaptive protein evolution in certain taxa. For example, estimates of the proportion of adaptive amino acid substitutions (alpha) are 50% or more in enteric bacteria and Drosophila. In contrast, recent estimates of alpha for hominids have been at most 13%. Here, we estimate alpha for protein sequences of murid rodents based on nucleotide polymorphism data from multiple genes in a population of the house mouse subspecies Mus musculus castaneus, which inhabits the ancestral range of the Mus species complex and nucleotide divergence between M. m. castaneus and M. famulus or the rat. We estimate that 57% of amino acid substitutions in murids have been driven by positive selection. Hominids, therefore, are exceptional in having low apparent levels of adaptive protein evolution. The high frequency of adaptive amino acid substitutions in wild mice is consistent with their large effective population size, leading to effective natural selection at the molecular level. Effective natural selection also manifests itself as a paucity of effectively neutral nonsynonymous mutations in M. m. castaneus compared to humans.

Long-term balancing selection at the blood group-related gene B4galnt2 in the genus Mus (Rodentia; Muridae).

Recent surveys of the human genome have highlighted the significance of balancing selection in relation to understanding the evolutionary origins of disease-associated variation. Cis-regulatory variation at the blood group-related glycosyltransferase B4galnt2 is associated with a phenotype in mice that closely resembles a common human bleeding disorder, von Willebrand disease. In this study, we have performed a survey of the 5' flanking region of the B4galnt2 gene in several Mus musculus subspecies and Mus spretus. Our results reveal a clear pattern of trans-species polymorphism and indicate that allele classes conferring alternative tissue-specific expression patterns have been maintained for >2.8 My in the genus Mus. Furthermore, analysis of B4galnt2 expression patterns revealed the presence of an additional functional class of alleles, supporting a role for gastrointestinal phenotypes in the long-term maintenance of expression variation at this gene.

Positive and negative selection in murine ultraconserved noncoding elements.

There are many more selectively constrained noncoding than coding nucleotides in the mammalian genome, but most mammalian noncoding DNA is subject to weak selection, on average. One of the most striking discoveries to have emerged from comparisons among mammalian genomes is the hundreds of noncoding elements of more than 200 bp in length that show absolute conservation among mammalian orders. These elements represent the tip of the iceberg of a much larger class of conserved noncoding elements (CNEs). Much evidence suggests that CNEs are selectively constrained and not mutational cold-spots, and there is evidence that some CNEs play a role in the regulation of development. Here, we quantify negative and positive selection acting in murine CNEs by analyzing within-species nucleotide variation and between-species divergence of CNEs that we identified using a phylogenetically independent comparison. The distribution of fitness effects of new mutations in CNEs, inferred from within-species polymorphism, suggests that CNEs receive a higher number of strongly selected deleterious mutations and many fewer nearly neutral mutations than amino acid sites of protein-coding genes or regulatory elements close to genes. However, we also show that CNEs experience a far higher proportion of adaptive substitutions than any known category of genomic sites in murids. The absolute rate of adaptation of CNEs is similar to that of amino acid sites of proteins. This result suggests that there is widespread adaptation in mammalian conserved noncoding DNA elements, some of which have been implicated in the regulation of crucially important processes, including development.

Genome-wide analysis of alternative splicing evolution among Mus subspecies.

Alternative splicing, the combination of different exons to produce a variety of transcripts from a single gene, contributes enormously to transcriptome diversity in mammals, and the majority of genes encode alternatively spliced products. Previous research comparing mouse, rat and human has shown that a significant proportion of splice forms are not conserved across species, suggesting that alternative transcripts are an important source of evolutionary novelty. Here, we studied the evolution of alternative splicing in the early stages of species divergence in the house mouse. We sequenced the testis transcriptomes of three Mus musculus subspecies and Mus spretus using Illumina technology. On the basis of a genome-wide analysis of read coverage differences among subspecies, we identified several hundred candidate alternatively spliced regions. We conservatively estimate that 6.5% of testis-expressed genes show alternative splice differences between at least one pair of M. musculus subspecies, a proportion slightly higher than the proportion of genes differentially expressed among subspecies. These results suggest that differences in both the structure and abundance of transcripts contribute to early transcriptome divergence.

Gene Expression Networks Across Multiple Tissues Are Associated with Rates of Molecular Evolution in Wild House Mice.

Interactions between genes can influence how selection acts on sequence variation. In gene regulatory networks, genes that affect the expression of many other genes may be under stronger evolutionary constraint than genes whose expression affects fewer partners. While this has been studied for individual tissue types, we know less about the effects of regulatory networks on gene evolution across different tissue types. We use RNA-sequencing and genomic data collected from Mus musculus domesticus to construct and compare gene co-expression networks for 10 tissue types. We identify tissue-specific expression and local regulatory variation, and we associate these components of gene expression variation with sequence polymorphism and divergence. We found that genes with higher connectivity across tissues and genes associated with a greater number of cross-tissue modules showed significantly lower genetic diversity and lower rates of protein evolution. Consistent with this pattern, "hub" genes across multiple tissues also showed evidence of greater evolutionary constraint. Using allele-specific expression, we found that genes with cis-regulatory variation had lower average connectivity and higher levels of tissue specificity. Taken together, these results are consistent with strong purifying selection acting on genes with high connectivity within and across tissues.

Phenotypic plasticity and the evolution of a socially selected trait following colonization of a novel environment.

Novel selection pressures in new environments arise through two distinct processes. First, environmental conditions directly affect the fitness of different phenotypes. Second, phenotypic plasticity alters the distribution of phenotypes, thereby placing populations in new selective regimes. A small isolated population of dark-eyed juncos Junco hyemalis became established in San Diego, probably in the early 1980s and probably from the nearby mountains. The relatively mild coastal climate has resulted in an increase in both the mean and the variance of the length of time females breed each year, and this is assumed to be a result of phenotypic plasticity. The population has evolved reduced white in the tail. We studied contemporary patterns of selection on tail white, in the context of the altered breeding season length. Late-hatched nestlings had higher survival and were in better condition than early-hatched nestlings, but among survivors, late-hatched birds had less tail white. We suggest this reflects juvenile mortality favoring individuals with less tail white. In adults, we found weak sexual selection and no viability selection but positive selection on female tail white in association with fecundity. We argue that altered breeding season length had a major impact on patterns of selection and evolution in this population.

Extensive additivity of gene expression differentiates subspecies of the house mouse.

We have studied different subspecies of the house mouse and their reciprocal F(1) hybrids to estimate the within-locus mode of inheritance for subspecies differences in gene expression in three tissues (brain, liver, and testis) of male mice. This study investigates the mode of inheritance in crosses at a larger taxonomic distance than has been previously systematically investigated. We found the vast majority of transcripts to be additively expressed with only a few transcripts showing dominance or overdominance in expression, except for one direction of one cross, which showed large mis-expression in the testis. We suggest that, as time passes, more genes come to influence expression, and if there is no directional dominance, additivity becomes increasingly more likely, up to a threshold beyond which there is F(1) hybrid breakdown. Some previous studies on different organisms have found a large degree of dominance, commonly at shorter taxonomic differences. We surveyed these findings and show that the most consistent association exists between the amount of additivity detected in a study and the expression analysis method (in particular microarray platform), suggesting that at least some of the differences among studies might be methodological. Most studies agree with ours in that within-locus additivity seems to be general mode of inheritance for transcript expression. Differentially expressed transcripts identified in our screen among subspecies of house mice are candidate genes that could be involved in reproductive isolation between these subspecies.

Reduced X-linked diversity in derived populations of house mice.

Contrasting patterns of X-linked vs. autosomal diversity may be indicative of the mode of selection operating in natural populations. A number of observations have shown reduced X-linked (or Z-linked) diversity relative to autosomal diversity in various organisms, suggesting a large impact of genetic hitchhiking. However, the relative contribution of other forces such as population bottlenecks, variation in reproductive success of the two sexes, and differential introgression remains unclear. Here, we survey 13 loci, 6 X-linked and 7 autosomal, in natural populations of the house mouse (Mus musculus) subspecies complex. We studied seven populations of three different subspecies, the eastern house mouse M. musculus castaneus, the central house mouse M. m. musculus, and the western house mouse M. m. domesticus, including putatively ancestral and derived populations for each. All populations display lower diversity on the X chromosomes relative to autosomes, and this effect is most pronounced in derived populations. To assess the role of demography, we fit the demographic parameters that gave the highest likelihood of the data using coalescent simulations. We find that the reduction in X-linked diversity is too large to be explained by a simple demographic model in at least two of four derived populations. These observations are also not likely to be explained by differences in reproductive success between males and females. They are consistent with a greater impact of positive selection on the X chromosome, and this is supported by the observation of an elevated K(A) and elevated K(A)/K(S) ratios on the rodent X chromosome. A second contribution may be that the X chromosome less readily introgresses across subspecies boundaries.

Inferring the history of speciation in house mice from autosomal, X-linked, Y-linked and mitochondrial genes.

Patterns of genetic differentiation among taxa at early stages of divergence provide an opportunity to make inferences about the history of speciation. Here, we conduct a survey of DNA-sequence polymorphism and divergence at loci on the autosomes, X chromosome, Y chromosome and mitochondrial DNA in samples of Mus domesticus, M. musculus and M. castaneus. We analyzed our data under a divergence with gene flow model and estimate that the effective population size of M. castaneus is 200,000-400,000, of M. domesticus is 100,000-200,000 and of M. musculus is 60,000-120,000. These data also suggest that these species started to diverge approximately 500,000 years ago. Consistent with this recent divergence, we observed considerable variation in the genealogical patterns among loci. For some loci, all alleles within each species formed a monophyletic group, while at other loci, species were intermingled on the phylogeny of alleles. This intermingling probably reflects both incomplete lineage sorting and gene flow after divergence. Likelihood ratio tests rejected a strict allopatric model with no gene flow in comparisons between each pair of species. Gene flow was asymmetric: no gene flow was detected into M. domesticus, while significant gene flow was detected into both M. castaneus and M. musculus. Finally, most of the gene flow occurred at autosomal loci, resulting in a significantly higher ratio of fixed differences to polymorphisms at the X and Y chromosomes relative to autosomes in some comparisons, or just the X chromosome in others, emphasizing the important role of the sex chromosomes in general and the X chromosome in particular in speciation.

Comparison of algorithms for the analysis of Affymetrix microarray data as evaluated by co-expression of genes in known operons.

Oligonucleotide microarrays are an informative tool to elucidate gene regulatory networks. In order for gene expression levels to be comparable across microarrays, normalization procedures have to be invoked. A large number of methods have been described to correct for systematic biases in microarray experiments. The performance of these methods has been tested only to a limited extend. Here, we evaluate two different types of microarray analyses: (i) the same gene in replicate samples and (ii) different, but co-expressed genes in the same sample. The reliability of the latter analysis needs to be determined for the analysis of regulatory networks and our report is the first attempt to evaluate for the accuracy of different microarray normalization methods in this respect. Consistent with previous results we observed a large effect of the normalization method on the outcome of the expression analyses. Our analyses indicate that different normalization methods should be performed depending on whether a study is aiming to detect differential gene expression between independent samples or whether co-expressed genes should be identified. We make recommendations about the most appropriate method to use.

Using the Mus musculus hybrid zone to assess covariation and genetic architecture of limb bone lengths.

Two subspecies of the house mouse, Mus musculus domesticus and Mus musculus musculus, meet in a narrow contact zone across Europe. Mice in the hybrid zone are highly admixed, representing the full range of mixed ancestry from the two subspecies. Given the distinct morphologies of these subspecies, these natural hybrids can be used for genomewide association mapping at sufficiently high resolution to directly infer candidate genes. We focus here on limb bone length differences, which is of special interest for understanding the evolution of developmentally correlated traits. We used 172 first-generation descendants of wild-caught mice from the hybrid zone to measure the length of stylopod (humerus/femur), zeugopod (ulna/tibia) and autopod (metacarpal/metatarsal) elements in skeletal CT scans. We find phenotypic covariation between limb elements in the hybrids similar to patterns previously described in Mus musculus domesticus inbred strains, suggesting that the hybrid genotypes do not influence the covariation pattern in a major way. Mapping was performed using 143,592 SNPs and identified several genomic regions associated with length differences in each bone. Bone length was found to be highly polygenic. None of the candidate regions include the canonical genes known to control embryonic limb development. Instead, we are able to identify candidate genes with known roles in osteoblast differentiation and bone structure determination, as well as recently evolved genes of, as yet, unknown function.

Gene expression analysis indicates extensive genotype-specific crosstalk between the conjugative F-plasmid and the E. coli chromosome.

BACKGROUND: Plasmids are an important component of the bacterial genome, but the crosstalk between genes encoded on the chromosome and on the plasmid is still poorly understood. RESULTS: We performed a large-scale survey for genes on the E. coli chromosome that are affected by the presence of the conjugative F-plasmid (crosstalk). The expression pattern of about 4% (107 genes) of the genes encoded by the chromosome was affected by the presence of the F-plasmid. Comparing two different Escherichia coli strains, MG1655 and DH5alpha, we found a strong host genotype-specific crosstalk of the host chromosome with the F-plasmid. About 88% of the genes affected by the presence of the F-plasmid showed a significant plasmid by host genotype interaction, i.e. the presence of the F-plasmid resulted in a different gene expression in the two host genotypes. Less than 12% of the genes showed an additive effect of gene expression, i.e. host genotype independent crosstalk between plasmid and host chromosome. CONCLUSION: We propose that epistatic effects also contribute to the maintenance of F-plasmids in natural populations.

The Mouse Solitary Odorant Receptor Gene Promoters as Models for the Study of Odorant Receptor Gene Choice.

BACKGROUND: In vertebrates, several anatomical regions located within the nasal cavity mediate olfaction. Among these, the main olfactory epithelium detects most conventional odorants. Olfactory sensory neurons, provided with cilia exposed to the air, detect volatile chemicals via an extremely large family of seven-transmembrane chemoreceptors named odorant receptors. Their genes are expressed in a monogenic and monoallelic fashion: a single allele of a single odorant receptor gene is transcribed in a given mature neuron, through a still uncharacterized molecular mechanism known as odorant receptor gene choice. AIM: Odorant receptor genes are typically arranged in genomic clusters, but a few are isolated (we call them solitary) from the others within a region broader than 1 Mb upstream and downstream with respect to their transcript's coordinates. The study of clustered genes is problematic, because of redundancy and ambiguities in their regulatory elements: we propose to use the solitary genes as simplified models to understand odorant receptor gene choice. PROCEDURES: Here we define number and identity of the solitary genes in the mouse genome (C57BL/6J), and assess the conservation of the solitary status in some mammalian orthologs. Furthermore, we locate their putative promoters, predict their homeodomain binding sites (commonly present in the promoters of odorant receptor genes) and compare candidate promoter sequences with those of wild-caught mice. We also provide expression data from histological sections. RESULTS: In the mouse genome there are eight intact solitary genes: Olfr19 (M12), Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466, Olfr1402; five are conserved as solitary in rat. These genes are all expressed in the main olfactory epithelium of three-day-old mice. The C57BL/6J candidate promoter of Olfr370 has considerably varied compared to its wild-type counterpart. Within the putative promoter for Olfr266 a homeodomain binding site is predicted. As a whole, our findings favor Olfr266 as a model gene to investigate odorant receptor gene choice.

Genomic resources for wild populations of the house mouse, Mus musculus and its close relative Mus spretus.

Wild populations of the house mouse (Mus musculus) represent the raw genetic material for the classical inbred strains in biomedical research and are a major model system for evolutionary biology. We provide whole genome sequencing data of individuals representing natural populations of M. m. domesticus (24 individuals from 3 populations), M. m. helgolandicus (3 individuals), M. m. musculus (22 individuals from 3 populations) and M. spretus (8 individuals from one population). We use a single pipeline to map and call variants for these individuals and also include 10 additional individuals of M. m. castaneus for which genomic data are publically available. In addition, RNAseq data were obtained from 10 tissues of up to eight adult individuals from each of the three M. m. domesticus populations for which genomic data were collected. Data and analyses are presented via tracks viewable in the UCSC or IGV genome browsers. We also provide information on available outbred stocks and instructions on how to keep them in the laboratory.