Insertion-Deletion Events Are Depleted in Protein Regions with Predicted Secondary Structure.

A fundamental goal in evolutionary biology and population genetics is to understand how selection shapes the fate of new mutations. Here, we test the null hypothesis that insertion-deletion (indel) events in protein-coding regions occur randomly with respect to secondary structures. We identified indels across 11,444 sequence alignments in mouse, rat, human, chimp, and dog genomes and then quantified their overlap with four different types of secondary structure-alpha helices, beta strands, protein bends, and protein turns-predicted by deep-learning methods of AlphaFold2. Indels overlapped secondary structures 54% as much as expected and were especially underrepresented over beta strands, which tend to form internal, stable regions of proteins. In contrast, indels were enriched by 155% over regions without any predicted secondary structures. These skews were stronger in the rodent lineages compared to the primate lineages, consistent with population genetic theory predicting that natural selection will be more efficient in species with larger effective population sizes. Nonsynonymous substitutions were also less common in regions of protein secondary structure, although not as strongly reduced as in indels. In a complementary analysis of thousands of human genomes, we showed that indels overlapping secondary structure segregated at significantly lower frequency than indels outside of secondary structure. Taken together, our study shows that indels are selected against if they overlap secondary structure, presumably because they disrupt the tertiary structure and function of a protein.

Shared E-Scooter Trajectory Analysis During the COVID-19 Pandemic in Austin, Texas.

By March of 2020, most cities worldwide had enacted stay-at-home public health orders to slow the spread of COVID-19. Restrictions on nonessential travel had extensive impacts across the transportation sector in the short term. This study explores the effects of COVID-19 on shared e-scooters by analyzing route trajectory data in the pre- and during-pandemic periods in Austin, TX, from a single provider. Although total shared e-scooter trips decreased during the pandemic, partially owing to vendors pulling out of the market, this study found average trip length increased, and temporal patterns of this mode did not meaningfully change. A count model of average daily trips by road segment found more trips on segments with sidewalks and bus stops during the pandemic than beforehand. More trips were observed on roads with lower vehicle miles traveled and fewer lanes, which might suggest more cautious travel behavior since there were fewer trips in residential neighborhoods. Stay-at-home orders and vendor e-scooter rebalancing operations inherently influence and can limit trip demand, but the unique trajectory data set and analysis provide cities with information on the road design preferences of vulnerable road users.

Safe streets for some: A review of local active transportation responses across the U.S. during the COVID-19 pandemic.

Introduction & research objectives: The COVID-19 pandemic significantly disrupted daily travel. This paper contrasts 51 US cities' responses, namely street reallocation criteria and messaging related to physical activity (PA) and active transportation (AT) during the early months of the pandemic. This study can be utilized by cities for aiding in the creation of locally responsive policies that acknowledge and remedy a lack of safe active transportation. Methods: A content analysis review was conducted of city orders and documents related to PA or AT for the largest city by population in all 50 US states and the District of Columbia. Authoritative documents issued from each city's public health declaration (ca. March 2020) to September 2020 were reviewed. The study obtained documents from two crowdsourced datasets and municipal websites. Descriptive statistics were used to compare policies and strategies, with a focus on reallocation of street space. Results: A total of 631 documents were coded. Considerable variation existed in city responses to COVID-19 that impacted PA and AT. Most cities' stay-at-home orders explicitly permitted outdoor PA (63%) and many encouraged PA (47%). As the pandemic continued, 23 cities (45%) had pilot programs that reallocated street space for non-motorized road users to recreate and travel. Most cities explicitly mentioned a rationale for the programs (e.g., to provide space for exercise (96%) and to alleviate crowding or provide safe AT routes (57%)). Cities used public feedback to guide placement decisions (35%) and several welcomed public input to adjust initial actions. Geographic equity was a criterion in 35% of programs and 57% considered inadequately sized infrastructure in decision-making. Conclusions: If cities want to emphasize AT and the health of their citizens, safe access to dedicated infrastructure needs to be prioritized. More than half of study cities did not instate new programs within the first 6 months of the pandemic. Cities should study peer responses and innovations to inform and create locally responsive policies that can acknowledge and remedy a lack of safe AT.

How (some) mammals lost their hair.

An approach that allows scientists to identify regions of the genome that evolved faster in hairless mammals reveals candidate genetic mechanisms that gave rise to hairlessness.

Complex genetics cause and constrain fungal persistence in different parts of the mammalian body.

Determining how genetic polymorphisms enable certain fungi to persist in mammalian hosts can improve understanding of opportunistic fungal pathogenesis, a source of substantial human morbidity and mortality. We examined the genetic basis of fungal persistence in mice using a cross between a clinical isolate and the lab reference strain of the budding yeast Saccharomyces cerevisiae. Employing chromosomally encoded DNA barcodes, we tracked the relative abundances of 822 genotyped, haploid segregants in multiple organs over time and performed linkage mapping of their persistence in hosts. Detected loci showed a mix of general and antagonistically pleiotropic effects across organs. General loci showed similar effects across all organs, while antagonistically pleiotropic loci showed contrasting effects in the brain vs the kidneys, liver, and spleen. Persistence in an organ required both generally beneficial alleles and organ-appropriate pleiotropic alleles. This genetic architecture resulted in many segregants persisting in the brain or in nonbrain organs, but few segregants persisting in all organs. These results show complex combinations of genetic polymorphisms collectively cause and constrain fungal persistence in different parts of the mammalian body.

Evolutionary, proteomic, and experimental investigations suggest the extracellular matrix of cumulus cells mediates fertilization outcomes†.

Studies of fertilization biology often focus on sperm and egg interactions. However, before gametes interact, mammalian sperm must pass through the cumulus layer; in mice, this consists of several thousand cells tightly glued together with hyaluronic acid and other proteins. To better understand the role of cumulus cells and their extracellular matrix, we perform proteomic experiments on cumulus oophorus complexes (COCs) in house mice (Mus musculus), producing over 24,000 mass spectra to identify 711 proteins. Seven proteins known to stabilize hyaluronic acid and the extracellular matrix were especially abundant (using spectral counts as an indirect proxy for abundance). Through comparative evolutionary analyses, we show that three of these evolve rapidly, a classic signature of genes that influence fertilization rate. Some of the selected sites overlap regions of the protein known to impact function. In a follow-up experiment, we compared COCs from females raised in two different social environments. Female mice raised in the presence of multiple males produced COCs that were smaller and more resistant to dissociation by hyaluronidase compared to females raised in the presence of a single male, consistent with a previous study that demonstrated such females produced COCs that were more resistant to fertilization. Although cumulus cells are often thought of as enhancers of fertilization, our evolutionary, proteomic, and experimental investigations implicate their extracellular matrix as a potential mediator of fertilization outcomes.

Male-derived copulatory plugs enhance implantation success in female Mus musculus.

Among a wide diversity of sexually reproducing species, male ejaculates coagulate to form what has been termed a copulatory plug. A number of functions have been attributed to copulatory plugs, including the inhibition of female remating and the promotion of ejaculate movement. Here we demonstrate that copulatory plugs also influence the likelihood of implantation, which occurs roughly 4 days after copulation in mice. Using a bead transfer method to control for differences in ejaculate retention and fertilization rates, we show that implantation rates significantly drop among females mated to genetically engineered males incapable of forming plugs (because they lack functional transglutaminase 4, the main enzyme responsible for its formation). Surprisingly, this result does not correlate with differences in circulating progesterone levels among females, an important hormone involved in implantation. We discuss three models that connect male-derived copulatory plugs to implantation success, including the hypothesis that plugs contribute to a threshold amount of stimulation required for females to become receptive to implantation.

The Evolution of Polymorphic Hybrid Incompatibilities in House Mice.

Resolving the mechanistic and genetic bases of reproductive barriers between species is essential to understanding the evolutionary forces that shape speciation. Intrinsic hybrid incompatibilities are often treated as fixed between species, yet there can be considerable variation in the strength of reproductive isolation between populations. The extent and causes of this variation remain poorly understood in most systems. We investigated the genetic basis of variable hybrid male sterility (HMS) between two recently diverged subspecies of house mice, Mus musculus domesticus and Mus musculus musculus We found that polymorphic HMS has a surprisingly complex genetic basis, with contributions from at least five autosomal loci segregating between two closely related wild-derived strains of M. m. musculus One of the HMS-linked regions on chromosome 4 also showed extensive introgression among inbred laboratory strains and transmission ratio distortion (TRD) in hybrid crosses. Using additional crosses and whole genome sequencing of sperm pools, we showed that TRD was limited to hybrid crosses and was not due to differences in sperm motility between M. m. musculus strains. Based on these results, we argue that TRD likely reflects additional incompatibilities that reduce hybrid embryonic viability. In some common inbred strains of mice, selection against deleterious interactions appears to have unexpectedly driven introgression at loci involved in epistatic hybrid incompatibilities. The highly variable genetic basis to F1 hybrid incompatibilities between closely related mouse lineages argues that a thorough dissection of reproductive isolation will require much more extensive sampling of natural variation than has been commonly utilized in mice and other model systems.

The baubellum is more developmentally and evolutionarily labile than the baculum.

Understanding the evolutionary forces that influence sexual dimorphism is a fundamental goal in biology. Here, we focus on one particularly extreme example of sexual dimorphism. Many mammal species possess a bone in their penis called a baculum. The female equivalent of this bone is called the baubellum and occurs in the clitoris, which is developmentally homologous to the male penis. To understand the potential linkage between these two structures, we scored baculum/baubellum presence/absence across 163 species and analyzed their distribution in a phylogenetic framework. The majority of species (N = 134) shared the same state in males and females (both baculum and baubellum present or absent). However, the baubellum has experienced significantly more transitions, and more recent transitions, so that the remaining 29 species have a baculum but not a well-developed baubellum. Even in species where both bones are present, the baubellum shows more ontogenetic variability and harbors more morphological variation than the baculum. Our study demonstrates that the baculum and baubellum are generally correlated across mammals, but that the baubellum is more evolutionarily and developmentally labile than the baculum. The accumulation of more evolutionary transitions, especially losses in the baubellum, as well as noisier developmental patterns, suggests that the baubellum may be nonfunctional, and lost over time.

Evolutionary expansion of DNA hypomethylation in the mammalian germline genome.

DNA methylation in the germline is among the most important factors influencing the evolution of mammalian genomes. Yet little is known about its evolutionary rate or the fraction of the methylome that has undergone change. We compared whole-genome, single-CpG DNA methylation profiles in sperm of seven species-human, chimpanzee, gorilla, rhesus macaque, mouse, rat, and dog-to investigate epigenomic evolution. We developed a phylo-epigenetic model for DNA methylation that accommodates the correlation of states at neighboring sites and allows for inference of ancestral states. Applying this model to the sperm methylomes, we uncovered an overall evolutionary expansion of the hypomethylated fraction of the genome, driven both by the birth of new hypomethylated regions and by extensive widening of hypomethylated intervals in ancestral species. This expansion shows strong lineage-specific aspects, most notably that hypomethylated intervals around transcription start sites have evolved to be considerably wider in primates and dog than in rodents, whereas rodents show evidence of a greater trend toward birth of new hypomethylated regions. Lineage-specific hypomethylated regions are enriched near sets of genes with common developmental functions and significant overlap across lineages. Rodent-specific and primate-specific hypomethylated regions are enriched for binding sites of similar transcription factors, suggesting that the plasticity accommodated by certain regulatory factors is conserved, despite substantial change in the specific sites of regulation. Overall our results reveal substantial global epigenomic change in mammalian sperm methylomes and point to a divergence in trans-epigenetic mechanisms that govern the organization of epigenetic states at gene promoters.

Whole exome sequencing of wild-derived inbred strains of mice improves power to link phenotype and genotype.

The house mouse is a powerful model to dissect the genetic basis of phenotypic variation, and serves as a model to study human diseases. Despite a wealth of discoveries, most classical laboratory strains have captured only a small fraction of genetic variation known to segregate in their wild progenitors, and existing strains are often related to each other in complex ways. Inbred strains of mice independently derived from natural populations have the potential to increase power in genetic studies with the addition of novel genetic variation. Here, we perform exome-enrichment and high-throughput sequencing (~8× coverage) of 26 wild-derived strains known in the mouse research community as the "Montpellier strains." We identified 1.46 million SNPs in our dataset, approximately 19% of which have not been detected from other inbred strains. This novel genetic variation is expected to contribute to phenotypic variation, as they include 18,496 nonsynonymous variants and 262 early stop codons. Simulations demonstrate that the higher density of genetic variation in the Montpellier strains provides increased power for quantitative genetic studies. Inasmuch as the power to connect genotype to phenotype depends on genetic variation, it is important to incorporate these additional genetic strains into future research programs.

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum.

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

DNA Methylation Divergence and Tissue Specialization in the Developing Mouse Placenta.

The placental epigenome plays a vital role in regulating mammalian growth and development. Aberrations in placental DNA methylation are linked to several disease states, including intrauterine growth restriction and preeclampsia. Studying the evolution and development of the placental epigenome is critical to understanding the origin and progression of such diseases. Although high-resolution studies have found substantial variation between placental methylomes of different species, the nature of methylome variation has yet to be characterized within any individual species. We conducted a study of placental DNA methylation at high resolution in multiple strains and closely related species of house mice (Mus musculus musculus, Mus m. domesticus, and M. spretus), across developmental timepoints (embryonic days 15-18), and between two distinct layers (labyrinthine transport and junctional endocrine). We observed substantial genome-wide methylation heterogeneity in mouse placenta compared with other differentiated tissues. Species-specific methylation profiles were concentrated in retrotransposon subfamilies, specifically RLTR10 and RLTR20 subfamilies. Regulatory regions such as gene promoters and CpG islands displayed cross-species conservation, but showed strong differences between layers and developmental timepoints. Partially methylated domains exist in the mouse placenta and widen during development. Taken together, our results characterize the mouse placental methylome as a highly heterogeneous and deregulated landscape globally, intermixed with actively regulated promoter and retrotransposon sequences.

Phylogenomic Insights into Mouse Evolution Using a Pseudoreference Approach.

Comparative genomic studies are now possible across a broad range of evolutionary timescales, but the generation and analysis of genomic data across many different species still present a number of challenges. The most sophisticated genotyping and down-stream analytical frameworks are still predominantly based on comparisons to high-quality reference genomes. However, established genomic resources are often limited within a given group of species, necessitating comparisons to divergent reference genomes that could restrict or bias comparisons across a phylogenetic sample. Here, we develop a scalable pseudoreference approach to iteratively incorporate sample-specific variation into a genome reference and reduce the effects of systematic mapping bias in downstream analyses. To characterize this framework, we used targeted capture to sequence whole exomes (∼54 Mbp) in 12 lineages (ten species) of mice spanning the Mus radiation. We generated whole exome pseudoreferences for all species and show that this iterative reference-based approach improved basic genomic analyses that depend on mapping accuracy while preserving the associated annotations of the mouse reference genome. We then use these pseudoreferences to resolve evolutionary relationships among these lineages while accounting for phylogenetic discordance across the genome, contributing an important resource for comparative studies in the mouse system. We also describe patterns of genomic introgression among lineages and compare our results to previous studies. Our general approach can be applied to whole or partitioned genomic data and is easily portable to any system with sufficient genomic resources, providing a useful framework for phylogenomic studies in mice and other taxa.

The Composite Regulatory Basis of the Large X-Effect in Mouse Speciation.

The disruption of meiotic sex chromosome inactivation (MSCI) has been proposed to be a major developmental mechanism underlying the rapid evolution of hybrid male sterility. We tested this idea by analyzing cell-specific gene expression across spermatogenesis in two lineages of house mice and their sterile and fertile reciprocal hybrids. We found pervasive disruption of sex chromosome gene expression in sterile hybrids at every stage of spermatogenesis. Failure of MSCI was developmentally preceded by increased silencing of autosomal genes, supporting the hypothesis that divergence at the hybrid incompatibility gene, Prdm9, results in increased rates of autosomal asynapsis which in turn triggers widespread silencing of unsynapsed chromatin. We also detected opposite patterns of postmeiotic overexpression or hyper-repression of the sex chromosomes in reciprocal hybrids, supporting the hypothesis that genomic conflict has driven functional divergence that leads to deleterious X-Y dosage imbalances in hybrids. Our developmental timeline also exposed more subtle patterns of mitotic misregulation on the X chromosome, a previously undocumented stage of spermatogenic disruption in this cross. These results indicate that multiple hybrid incompatibilities have converged on a common regulatory phenotype, the disrupted expression of the sex chromosomes during spermatogenesis. Collectively, these data reveal a composite regulatory basis to hybrid male sterility in mice that helps resolve the mechanistic underpinnings of the well-documented large X-effect in mice speciation. We propose that the inherent sensitivity of spermatogenesis to X-linked regulatory disruption has the potential to be a major driver of reproductive isolation in species with chromosomal sex determination.

Copulatory plugs inhibit the reproductive success of rival males.

Ejaculated proteins play important roles in reproductive fitness. In many species, seminal fluid coagulates and forms what has been referred to as a copulatory plug in the female's reproductive tract. In mice, previous work demonstrated that knockout males missing a key seminal fluid protein were unable to form a plug and less successful at siring litters in noncompetitive matings (one female, one male), probably the result of reduced sperm transport or insufficient stimulation of the female. Here, we extend these previous studies to competitive matings (one female, two males) and make two key insights. First, when first males were unable to form a plug, they lost almost all paternity to second males to mate. Thus, the copulatory plugs of second males could not rescue the reduced fertility of first males. Second, we showed that the copulatory plug of first males effectively blocked fertilization by second males, even if first males were vasectomized. Taken together, our experiments demonstrated that first males lost almost all paternity if they never formed a plug. We discuss our results in the context of natural populations, where in spite of the strong effects seen here, pregnant female mice regularly carry litters fertilized by more than one male.

The molecular basis and reproductive function(s) of copulatory plugs.

In many animals, male ejaculates coagulate to form what has been termed a copulatory plug, a structure that varies in size and shape but often fills and seals the female's reproductive tract. The first published observation of a copulatory plug in a mammal was made more than 160 years ago, and questions about its formation and role in reproduction continue to endear evolutionary and population geneticists, behavioral ecologists, and molecular, reproductive, and developmental biologists alike. Here, we review the current knowledge of copulatory plugs, focusing on rodents and asking two main questions: how is it formed and what does it do? An evolutionary biology perspective helps us understand the latter, potentially leading to insights into the selective regimes that have shaped the diversity of this structure. Mol. Reprod. Dev. 83: 755-767, 2016 © 2016 Wiley Periodicals, Inc.

Adaptation in isolated populations: when does it happen and when can we tell?

Isolated populations with novel phenotypes present an exciting opportunity to uncover the genetic basis of ecologically significant adaptation, and genomic scans have often, but not always, led to candidate genes directly related to an adaptive phenotype. However, in many cases these populations were established by a severe bottleneck, which can make identifying targets of selection problematic. Here, we simulate severe bottlenecks and subsequent selection on standing variation, mimicking adaptation after establishment of a new small population, such as an island or an artificial selection experiment. Using simulations of single loci under positive selection and population genetics theory, we examine how population size and age of the population isolate affect the ability of outlier scans for selection to identify adaptive alleles using both single-site measures and haplotype structure. We find and explain an optimal combination of selection strength, starting frequency, and age of the adaptive allele, which we refer to as a Goldilocks zone, where adaptation is likely to occur and yet the adaptive variants are most likely to derive from a single ancestor (a 'hard' selective sweep); in this zone, four commonly used statistics detect selection with high power. Real-world examples of both island colonization and experimental evolution studies are discussed. Our study provides concrete considerations to be made before embarking on whole-genome sequencing of differentiated populations.

Contrasting Levels of Molecular Evolution on the Mouse X Chromosome.

The mammalian X chromosome has unusual evolutionary dynamics compared to autosomes. Faster-X evolution of spermatogenic protein-coding genes is known to be most pronounced for genes expressed late in spermatogenesis, but it is unclear if these patterns extend to other forms of molecular divergence. We tested for faster-X evolution in mice spanning three different forms of molecular evolution-divergence in protein sequence, gene expression, and DNA methylation-across different developmental stages of spermatogenesis. We used FACS to isolate individual cell populations and then generated cell-specific transcriptome profiles across different stages of spermatogenesis in two subspecies of house mice (Mus musculus), thereby overcoming a fundamental limitation of previous studies on whole tissues. We found faster-X protein evolution at all stages of spermatogenesis and faster-late protein evolution for both X-linked and autosomal genes. In contrast, there was less expression divergence late in spermatogenesis (slower late) on the X chromosome and for autosomal genes expressed primarily in testis (testis-biased). We argue that slower-late expression divergence reflects strong regulatory constraints imposed during this critical stage of sperm development and that these constraints are particularly acute on the tightly regulated sex chromosomes. We also found slower-X DNA methylation divergence based on genome-wide bisulfite sequencing of sperm from two species of mice (M. musculus and M. spretus), although it is unclear whether slower-X DNA methylation reflects development constraints in sperm or other X-linked phenomena. Our study clarifies key differences in patterns of regulatory and protein evolution across spermatogenesis that are likely to have important consequences for mammalian sex chromosome evolution, male fertility, and speciation.