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It remains unclear how variation in the intensity of sperm competition shapes phenotypic and molecular evolution across clades. Mice and rats in the subfamily Murinae are a rapid radiation exhibiting incredible diversity in sperm morphology and production. We combined phenotypic and genomic data to perform phylogenetic comparisons of male reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass, presumably reflecting reduced sperm competition. Several sperm traits were associated with relative testes mass, suggesting that mating system evolution selects for convergent suites of traits related to sperm competitive ability. We predicted that sperm competition would also drive more rapid molecular divergence in species with large testes. Contrary to this, we found that many spermatogenesis genes evolved more rapidly in species with smaller relative testes mass due to relaxed purifying selection. While some reproductive genes evolved rapidly under recurrent positive selection, relaxed selection played a greater role in underlying rapid evolution in small testes species. Our work demonstrates that postcopulatory sexual selection can impose strong purifying selection shaping the evolution of male reproduction, and that broad patterns of molecular evolution may help identify genes that contribute to male fertility., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE). All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Mutations in glycosylation pathways, such as N-linked glycosylation, O-linked glycosylation, and GPI anchor synthesis, lead to Congenital Disorders of Glycosylation (CDG). CDG typically present with seizures, hypotonia, and developmental delay but display large clinical variability with symptoms affecting every system in the body. This variability suggests modifier genes might influence the phenotypes. Because of the similar physiology and clinical symptoms, there are likely common genetic modifiers between CDG. Here, we use evolution as a tool to identify common modifiers between CDG and glycosylation genes. Protein glycosylation is evolutionarily conserved from yeast to mammals. Evolutionary rate covariation (ERC) identifies proteins with similar evolutionary rates that indicate shared biological functions and pathways. Using ERC, we identified strong evolutionary rate signatures between proteins in the same and different glycosylation pathways. Genome-wide analysis of proteins showing significant ERC with GPI anchor synthesis proteins revealed strong signatures with ncRNA modification proteins and DNA repair proteins. We also identified strong patterns of ERC based on cellular sub-localization of the GPI anchor synthesis enzymes. Functional testing of the highest scoring candidates validated genetic interactions and identified novel genetic modifiers of CDG genes. ERC analysis of disease genes and biological pathways allows for rapid prioritization of potential genetic modifiers, which can provide a better understanding of disease pathophysiology and novel therapeutic targets., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Thorpe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The reported prevalence of hypermobility spectrum disorders (HSD) and hypermobile Ehlers-Danlos Syndrome (hEDS) is unclear due to complex presentations and lack of awareness amongst professionals, contributing to diagnostic and management uncertainties. Standardising an outcome measure to assess symptoms most important to patients should help determine the impact of interventions. This Delphi study aims to reach a consensus with stakeholders on the core outcome set for children and adults with HSD/hEDS. A three-round modified Delphi consensus study with a follow-up consensus meeting was used. Stakeholder groups consisted of (1) individuals with HSD/hEDS; (2) family/friends/carers and (3) healthcare professionals. Participants could belong to more than one stakeholder group. They rated 74 symptoms using a 9-point Likert scale: 1 "not important to 9 "critically important". Symptoms achieving a consensus rating of ≥ 70.0% critical importance across all groups were included. In Round 1, 766 responses were received from 600 participants, reducing to 566 responses from 438 participants by Round 3, with 53 participating in the consensus meeting. Overall, 30 symptoms met the ≥ 70.0% critically important threshold to be included in the final core outcome set. These were categorised under the specialties of musculoskeletal and orthopaedics, social, pain, gynaecology and urology, negative affect, neurological, gastrointestinal and "other". This study is the first to identify by consensus the core outcome set to be measured for patients with HSD/hEDS. The importance of these outcomes was confirmed by individuals living with the condition, their family, friends, carers and relevant healthcare professionals. Trial registration: IRAS ID: 326,855; East Midlands - Leicester South REC (reference: 23/EM/0143); Protocol registered with the COMET Initiative. Key Points • There is a lack of standardised outcome measure for HSD/hEDS research studies due to the heterogeneity of symptom presentations. • Symptoms across musculoskeletal and orthopaedics, social, pain, gynaecology and urology, negative affect, neurological, gastrointestinal and "other" specialties were identified as the core outcome set defined as ≥ 70.0% critically important to measure for individuals with HSD/hEDS. • There were a significant number of symptoms, widely recognised in the literature to be comorbid to HSD/hEDS, that reached a critical importance threshold of 50.0-69.9%, restricting the core outcome set to only those that met 70% or above may be limiting., Competing Interests: Declarations. Ethics approval: Ethical approval was obtained for this study by East Midlands — Leicester South Research Ethics Committee (REC) [REC reference: 23/EM/0143; IRAS Project ID: 326855]. All the participants provided electronic written informed consent. Consent for publication: Not applicable. Disclosures: None., (© 2024. The Author(s).)

Background: In May 2020, England moved to an opt-out organ donation system, meaning adults are presumed to be an organ donor unless within an excluded group or have opted-out. This change aims to improve organ donation rates following brain or circulatory death. Healthcare staff in the UK are supportive of organ donation, however, both healthcare staff and the public have raised concerns and ethical issues regarding the change. The #options survey was completed by NHS organisations with the aim of understanding awareness and support of the change. This paper analyses the free-text responses from the survey., Methods: The #options survey was registered as a National Institute of Health Research (NIHR) portfolio trial [IRAS 275992] 14 February 2020, and was completed between July and December 2020 across NHS organisations in the North-East and North Cumbria, and North Thames. The survey contained 16 questions of which three were free-text, covering reasons against, additional information required and family discussions. The responses to these questions were thematically analysed., Results: The #options survey received 5789 responses from NHS staff with 1404 individuals leaving 1657 free-text responses for analysis. The family discussion question elicited the largest number of responses (66%), followed by those against the legislation (19%), and those requiring more information (15%). Analysis revealed six main themes with 22 sub-themes., Conclusions: The overall #options survey indicated NHS staff are supportive of the legislative change. Analysis of the free-text responses indicates that the views of the NHS staff who are against the change reflect the reasons, misconceptions, and misunderstandings of the public. Additional concerns included the rationale for the change, informed decision making, easy access to information and information regarding organ donation processes. Educational materials and interventions need to be developed for NHS staff to address the concepts of autonomy and consent, organ donation processes, and promote family conversations. Wider public awareness campaigns should continue to promote the positives and refute the negatives thus reducing misconceptions and misunderstandings., Trial Registration: National Institute of Health Research (NIHR) [IRAS 275992]., (© 2024. The Author(s).)

Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat ( Rousettus aegyptiacus ) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.

Co-functional proteins tend to have rates of evolution that covary over time. This correlation between evolutionary rates can be measured over the branches of a phylogenetic tree through methods such as evolutionary rate covariation (ERC), and then used to construct gene networks by the identification of proteins with functional interactions. The cause of this correlation has been hypothesized to result from both compensatory coevolution at physical interfaces and nonphysical forces such as shared changes in selective pressure. This study explores whether coevolution due to compensatory mutations has a measurable effect on the ERC signal. We examined the difference in ERC signal between physically interacting protein domains within complexes compared to domains of the same proteins that do not physically interact. We found no generalizable relationship between physical interaction and high ERC, although a few complexes ranked physical interactions higher than nonphysical interactions. Therefore, we conclude that coevolution due to physical interaction is weak, but present in the signal captured by ERC, and we hypothesize that the stronger signal instead comes from selective pressures on the protein as a whole and maintenance of the general function., Competing Interests: JL, MC, NC No competing interests declared, (© 2023, Little et al.)

Background: Conditions such as hypermobility spectrum disorders (HSD) and Ehlers-Danlos syndrome (EDS) are most often diagnosed when an individual has joint flexibility beyond the normal physiological limits. Additional characteristics and symptoms include pain and fatigue with individuals also being more likely to report feelings of anxiety and depression. Due to the varied presentation of these conditions, there is a lack of understanding amongst the various healthcare professionals (HCPs) individuals present to, leading to delayed diagnoses and negative experiences for the individuals themselves. This scoping review therefore aims to map the known biopsychosocial impact of adults with HSD and EDS., Methods: The scoping review will follow the six-step framework as outlined by Arskey and O'Malley and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Reviews (PRISMA-ScR) checklist. The search will be conducted using the following databases: AMED, CINAHL, Cochrane Library, Embase, MEDLINE, PsycINFO, PubMed PEDro. Full-text published articles in the English language (excluding literature and systematic reviews) with adult samples (over the age of 18 years) and a diagnosis of a HSD or EDS, published between 2012 and 2022, will be included in the review., Discussion: This review will aim to explore the existing literature for the reported biopsychosocial impact of adults with a HSD or EDS. It will also aim to further acknowledge the gaps in understanding of the condition, how the condition and the impact of the condition is being measured and what HCPs are involved in supporting such individuals. These gaps will be used to inform a future systematic review. It is the overall goal to increase the knowledge of HCPs and the quality of life of adults living with a joint hypermobility condition., (© 2024. The Author(s).)

Objective: To use a previously validated veterinary clinical examination sheet, Liverpool Osteoarthritis in Dogs (LOAD) questionnaire, combined with kinetic and kinematic gait analysis in dogs with/without mobility problems to demonstrate the capacity of a novel clinical metrology instrument ("GenPup-M") to detect canine mobility impairments., Design: Quantitative study., Animals: 62 dogs (31 with mobility impairments and 31 without mobility impairments)., Procedure: The dogs' clinical history was obtained from owners and all dogs underwent a validated orthopaedic clinical examination. Mobility impairments were diagnosed in the mobility impaired group based on clinical history and orthopaedic examination. Owners were asked to complete GenPup-M along with a previously validated mobility questionnaire (Liverpool Osteoarthritis in Dogs (LOAD)) to identify construct validity. As a test of criterion validity, the correlation between instrument scores and the overall clinical examination scores, along with force-platform obtained peak vertical forces (PVF) were calculated. GenPup-M underwent internal consistency and factor analysis. Spatiotemporal parameters were calculated for dogs with/without mobility impairments to define the gait differences between these two groups., Results: Principal Component Analysis identified GenPup-M had two components with Eigenvalues >1 ("stiffness/ease of movement" and "willingness to be active/exercise"). Cronbach's α was used to test internal consistency of GenPup-M and was found to be "good" (0.87). There was a strong, positive correlation between GenPup-M and LOAD responses (r2 = 0.69, p<0.001) highlighting construct validity. Criterion validity was also shown when comparing GenPup-M to clinical examination scores (r2 = 0.74, p<0.001) and PVF (r2 = 0.43, p<0.001). Quantitative canine gait analysis showed that there were statistically significant differences between peak vertical forces (PVF) of mobility impaired and non-mobility impaired dogs (p<0.05). Analyses of PVF showed that non-mobility impaired dogs more evenly distributed their weight across all thoracic and pelvic limbs when compared to mobility impaired dogs. There were also consistent findings that mobility impaired dogs moved slower than non-mobility impaired dogs., Conclusion and Clinical Relevance: GenPup-M is a clinical metrology instrument (CMI) that can be completed by dog owners to detect all mobility impairments, including those that are early in onset, indicating the versatility of GenPup-M to assess dogs with and without mobility impairments. Results of the study found that GenPup-M positively correlated with all three objective measures of canine mobility and consequently showed criterion and construct validity. Owner-reported CMIs such as GenPup-M allow non-invasive scoring systems which veterinary surgeons and owners can use to allow communication and longitudinal assessment of a dog's mobility. It is anticipated that GenPup-M will be used by owners at yearly vaccinations/health checks, allowing identification of any subtle mobility changes, and enabling early intervention., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Clark et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: In England, everyone is considered an organ donor unless they have registered for opt-out donation. Research Question: This study evaluated positive statements and negative affective attitudes against anticipated organ donor status and whether the order in the attitudes and statements presented impacted organ donor intention under an opt-out system. Design: A quasi-experimental mixed between-within design was employed with participants assigned to 1 of 2 conditions. Participants in the first condition received negative affective attitude statements followed by positive statements. This was reversed in the second condition to combat ordering effects. Participants ( N  = 679) were asked about their donor status under an opt-out system. There were three groups: opt-in (actively), opt-out/not sure, and deemed consent (no objection). Organ donor intentions were measured at three intervals: baseline, post-positive statements, and post-negative attitudes. Results: Approximately 10% of participants would opt-out or were unsure of their intentions to be an organ donor under an opt-out system. Significant effects were found in both positive statements and negative affective attitudes. All groups expressed greater medical mistrust and were most influenced by cognitive attitudes. Conclusions: Under the opt-out system in England, it is anticipated that the majority would actively opt-in or have no objection to being automatically registered as an organ donor. Public health campaigns would benefit from promoting the most influential positive statements and refuting the most detrimental negative attitudes to increase intentions of those who plan to opt-out or are unsure.

Exposure to ultraviolet radiation (UVR) is harmful to living cells, leading organisms to evolve protective mechanisms against UVR-induced cellular damage and stress. 1 , 2 UVR, particularly UVB (280-320 nm), can damage proteins and DNA, leading to errors during DNA repair and replication. Excessive UVR can induce cellular death. Aquatic organisms face risk of UV exposure as biologically harmful levels of UVB can penetrate >10 m in clear water. 3 While melanin is the only known sunscreen in vertebrates, it often emerges late in embryonic development, rendering embryos of many species vulnerable during the earlier stages. Algae and microbes produce a class of sunscreening compounds known as mycosporine-like amino acids (MAAs). 4 Fish eggs contain a similar compound called gadusol, whose role as a sunscreen has yet to be tested despite its discovery over 40 years ago. 5 The recent finding that many vertebrate genomes contain a biosynthetic pathway for gadusol suggests that many fish may produce and use this molecule as a sunscreen. 6 We generated a gadusol-deficient mutant zebrafish to investigate the role of gadusol in protecting fish embryos and larvae from UVR. Our results demonstrate that maternally provided gadusol is the primary sunscreen in embryonic and larval development, while melanin provides modest secondary protection. The gadusol biosynthetic pathway is retained in the vast majority of teleost genomes but is repeatedly lost in species whose young are no longer exposed to UVR. Our data demonstrate that gadusol is a maternally provided sunscreen that is critical for early-life survival in the most species-rich branch of the vertebrate phylogeny., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Joint hypermobility affects approximately 30% of the United Kingdom (UK) population, characterised by the ability to move joints beyond the physiological limits. Associated conditions include Ehlers-Danlos syndrome and hypermobility spectrum disorders, affecting individuals across physical, psychological and social levels detrimentally impacting their health and wellbeing. The scoping review aims to describe the known biopsychosocial impact of joint hypermobility conditions in adults over the last decade. Additional objectives include to (1) identify the types of studies that address these factors, (2) to understand how the impact of the condition is measured and managed and (3) what healthcare professionals (HCPs) are involved. The scoping review was conducted using the five-stage framework by Arksey and O'Malley. The search strategy related to two main keywords, "hypermobility" and, "biopsychosocial" across a number of electronic databases. A pilot search was conducted to determine the suitability of the databases and terms. Following the search, the data was extracted and charted, summarised and narratively reported. 32 studies met the inclusion criteria. The majority were conducted in either the UK or United States of America and case-control in design. The biopsychosocial impact was wide-ranging including, but not limited to, musculoskeletal system and dermatology, gastroenterology, mood and anxiety disorders, education and employments. This review is the first of its kind to summarise all reported symptoms and impact of joint hypermobility conditions in adults, highlighting a clear need to promote a multidisciplinary and holistic approach in raising awareness of these conditions and improving their management., (© 2023. The Author(s).)

Convergent adaptation to the same environment by multiple lineages frequently involves rapid evolutionary change at the same genes, implicating these genes as important for environmental adaptation. Such adaptive molecular changes may yield either change or loss of protein function; loss of function can eliminate newly deleterious proteins or reduce energy necessary for protein production. We previously found a striking case of recurrent pseudogenization of the Paraoxonase 1 (Pon1) gene among aquatic mammal lineages-Pon1 became a pseudogene with genetic lesions, such as stop codons and frameshifts, at least four times independently in aquatic and semiaquatic mammals. Here, we assess the landscape and pace of pseudogenization by studying Pon1 sequences, expression levels, and enzymatic activity across four aquatic and semiaquatic mammal lineages: pinnipeds, cetaceans, otters, and beavers. We observe in beavers and pinnipeds an unexpected reduction in expression of Pon3, a paralog with similar expression patterns but different substrate preferences. Ultimately, in all lineages with aquatic/semiaquatic members, we find that preceding any coding-level pseudogenization events in Pon1, there is a drastic decrease in expression, followed by relaxed selection, thus allowing accumulation of disrupting mutations. The recurrent loss of Pon1 function in aquatic/semiaquatic lineages is consistent with a benefit to Pon1 functional loss in aquatic environments. Accordingly, we examine diving and dietary traits across pinniped species as potential driving forces of Pon1 functional loss. We find that loss is best associated with diving activity and likely results from changes in selective pressures associated with hypoxia and hypoxia-induced inflammation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Change in gene family size has been shown to facilitate adaptation to different selective pressures. This includes gene duplication to increase dosage or diversification of enzymatic substrates and gene deletion due to relaxed selection. We recently found that the PON1 gene, an enzyme with arylesterase and lactonase activity, was lost repeatedly in different aquatic mammalian lineages, suggesting that the PON gene family is responsive to environmental change. We further investigated if these fluctuations in gene family size were restricted to mammals and approximately when this gene family was expanded within mammals. Using 112 metazoan protein models, we explored the evolutionary history of the PON family to characterize the dynamic evolution of this gene family. We found that there have been multiple, independent expansion events in tardigrades, cephalochordates, and echinoderms. In addition, there have been partial gene loss events in monotremes and sea cucumbers and what appears to be complete loss in arthropods, urochordates, platyhelminths, ctenophores, and placozoans. In addition, we show the mammalian expansion to three PON paralogs occurred in the ancestor of all mammals after the divergence of sauropsida but before the divergence of monotremes from therians. We also provide evidence of a novel PON expansion within the brushtail possum. In the face of repeated expansions and deletions in the context of changing environments, we suggest a range of selective pressures, including pathogen infection and mitigation of oxidative damage, are likely influencing the diversification of this dynamic gene family across metazoa., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion., Competing Interests: CP, GA, MG, MD, EK, KG, SM, JS, VF, CT, CS, NW, HJ, NC, RF, JT, AL, MB, PC, MS, RW No competing interests declared, (© 2022, Pereira et al.)

Vertebrates evolved mechanisms for sodium conservation and gas exchange in conjunction with migration from aquatic to terrestrial habitats. Epithelial Na + channel (ENaC) function is critical to systems responsible for extracellular fluid homeostasis and gas exchange. ENaC is activated by cleavage at multiple specific extracellular polybasic sites, releasing inhibitory tracts from the channel's α and γ subunits. We found that proximal and distal polybasic tracts in ENaC subunits coevolved, consistent with the dual cleavage requirement for activation observed in mammals. Polybasic tract pairs evolved with the terrestrial migration and the appearance of lungs, coincident with the ENaC activator aldosterone, and appeared independently in the α and γ subunits. In summary, sites within ENaC for protease activation developed in vertebrates when renal Na + conservation and alveolar gas exchange were required for terrestrial survival., Competing Interests: XW, DB, CG, NC, OK No competing interests declared, (© 2022, Wang et al.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Baker's yeast contains a large number of duplicated genes; some function redundantly, whereas others have more specialized roles. We used the MLH family of DNA mismatch repair (MMR) proteins as a model to better understand the steps that lead to gene specialization following a gene duplication event. We focused on two highly conserved yeast MLH proteins, Pms1 and Mlh3, with Pms1 having a major role in the repair of misincorporation events during DNA replication and Mlh3 acting to resolve recombination intermediates in meiosis to form crossovers. The baker's yeast Mlh3 and Pms1 proteins are significantly diverged (19% overall identity), suggesting that an extensive number of evolutionary steps, some major, others involving subtle refinements, took place to diversify the MLH proteins. Using phylogenetic and molecular approaches, we provide evidence that all three domains (N-terminal ATP binding, linker, C-terminal endonuclease/MLH interaction) in the MLH protein family are critical for conferring pathway specificity. Importantly, mlh3 alleles in the ATP binding and endonuclease domains improved MMR functions in strains lacking the Pms1 protein and did not disrupt Mlh3 meiotic functions. This ability for mlh3 alleles to complement the loss of Pms1 suggests that an ancestral Pms1/Mlh3 protein was capable of performing both MMR and crossover functions. Our strategy for analyzing MLH pathway specificity provides an approach to understand how paralogs have evolved to support distinct cellular processes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0 to 100% lethality. Association analysis on the lethality phenotype, as well as an evolutionary rate covariation analysis, generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2 ), a conserved ion transporter. Analyses in NGLY1 -/- mouse cells demonstrated that NKCC1 has an altered average molecular weight and reduced function. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function in NGLY1 deficiency patients., Competing Interests: DT, KO, EC, GM, JP, RP, KH, AZ, NC, CL, AR, CC No competing interests declared, (© 2020, Talsness et al.)

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.

Although lifespan in mammals varies over 100-fold, the precise evolutionary mechanisms underlying variation in longevity remain unknown. Species-specific genetic changes have been observed in long-lived species including the naked mole-rat, bats, and the bowhead whale, but these adaptations do not generalize to other mammals. We present a novel method to identify associations between rates of protein evolution and continuous phenotypes across the entire mammalian phylogeny. Unlike previous analyses that focused on individual species, we treat absolute and relative longevity as quantitative traits and demonstrate that these lifespan traits affect the evolutionary constraint on hundreds of genes. Specifically, we find that genes related to cell cycle, DNA repair, cell death, the IGF1 pathway, and immunity are under increased evolutionary constraint in large and long-lived mammals. For mammals exceptionally long-lived for their body size, we find increased constraint in inflammation, DNA repair, and NFKB-related pathways. Strikingly, these pathways have considerable overlap with those that have been previously reported to have potentially adaptive changes in single-species studies, and thus would be expected to show decreased constraint in our analysis. This unexpected finding of increased constraint in many longevity-associated pathways underscores the power of our quantitative approach to detect patterns that generalize across the mammalian phylogeny., Competing Interests: AK, RP, NC, MC No competing interests declared, (© 2020, Kowalczyk et al.)

Motivation: When different lineages of organisms independently adapt to similar environments, selection often acts repeatedly upon the same genes, leading to signatures of convergent evolutionary rate shifts at these genes. With the increasing availability of genome sequences for organisms displaying a variety of convergent traits, the ability to identify genes with such convergent rate signatures would enable new insights into the molecular basis of these traits., Results: Here we present the R package RERconverge, which tests for association between relative evolutionary rates of genes and the evolution of traits across a phylogeny. RERconverge can perform associations with binary and continuous traits, and it contains tools for visualization and enrichment analyses of association results., Availability and Implementation: RERconverge source code, documentation and a detailed usage walk-through are freely available at https://github.com/nclark-lab/RERconverge. Datasets for mammals, Drosophila and yeast are available at https://bit.ly/2J2QBnj., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Molecules that mediate reproductive interactions are some of the most rapidly evolving traits. Researchers have often suggested that this is due to coevolution at key physiological interfaces. However, very few of these interfaces are well understood at the functional level. One such interface is the digestion of the spermatophore in Lepidoptera. Female Lepidoptera have a specialized reproductive organ called the bursa copulatrix that receives and processes the male spermatophore, a complex proteinaceous ejaculate. In the cabbage white butterfly, Pieris rapae , the bursa secretes a mixture of proteases hypothesized to digest the spermatophore. However, these proteases remain biochemically uncharacterized. Using a zymogram approach, we identified six proteases in bursal extracts at sufficiently high concentrations to characterize their in vitro activity. We assessed the modes of action of these bursal enzymes by quantifying their activity following exposure to diagnostic protease inhibitors. A serine protease-specific inhibitor failed to reduce bursal protease digestion of casein. However, a cysteine protease-specific inhibitor did decrease the activity of some proteases. To explore the possible molecular mechanisms responsible for these responses, we created protease homology models. The models mirrored the results of our in vitro experiments, indicating that protease homology models may offer insight into underlying functional mechanisms. Whether the observed bursal protease resistance to known inhibitors is important in the context of spermatophore digestion remains to be tested. However, our results suggest the exciting possibility that bursal protease specificity may have evolved in response to interactions with various proteins and inhibitors present within the female tract during the reproductive process.

DNA repair is critical for genome stability and is maintained through conserved pathways. Traditional genome-wide mammalian screens are both expensive and laborious. However, computational approaches circumvent these limitations and are a powerful tool to identify new DNA repair factors. By analyzing the evolutionary relationships between genes in the major DNA repair pathways, we uncovered functional relationships between individual genes and identified partners. Here we ranked 17,487 mammalian genes for coevolution with 6 distinct DNA repair pathways. Direct comparison to genetic screens for homologous recombination or Fanconi anemia factors indicates that our evolution-based screen is comparable, if not superior, to traditional screening approaches. Demonstrating the utility of our strategy, we identify a role for the DNA damage-induced apoptosis suppressor ( DDIAS ) gene in double-strand break repair based on its coevolution with homologous recombination. DDIAS knockdown results in DNA double-strand breaks, indicated by ATM kinase activation and 53BP1 foci induction. Additionally, DDIAS-depleted cells are deficient for homologous recombination. Our results reveal that evolutionary analysis is a powerful tool to uncover novel factors and functional relationships in DNA repair., Competing Interests: The authors declare no conflict of interest.

Identifying genomic elements underlying phenotypic adaptations is an important problem in evolutionary biology. Comparative analyses learning from convergent evolution of traits are gaining momentum in accurately detecting such elements. We previously developed a method for predicting phenotypic associations of genetic elements by contrasting patterns of sequence evolution in species showing a phenotype with those that do not. Using this method, we successfully demonstrated convergent evolutionary rate shifts in genetic elements associated with two phenotypic adaptations, namely the independent subterranean and marine transitions of terrestrial mammalian lineages. Our original method calculates gene-specific rates of evolution on branches of phylogenetic trees using linear regression. These rates represent the extent of sequence divergence on a branch after removing the expected divergence on the branch due to background factors. The rates calculated using this regression analysis exhibit an important statistical limitation, namely heteroscedasticity. We observe that the rates on branches that are longer on average show higher variance, and describe how this problem adversely affects the confidence with which we can make inferences about rate shifts. Using a combination of data transformation and weighted regression, we have developed an updated method that corrects this heteroscedasticity in the rates. We additionally illustrate the improved performance offered by the updated method at robust detection of convergent rate shifts in phylogenetic trees of protein-coding genes across mammals, as well as using simulated tree data sets. Overall, we present an important extension to our evolutionary-rates-based method that performs more robustly and consistently at detecting convergent shifts in evolutionary rates., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The adherens junction couples the actin cytoskeletons of neighboring cells to provide the foundation for multicellular organization. The core of the adherens junction is the cadherin-catenin complex that arose early in the evolution of multicellularity to link actin to intercellular adhesions. Over time, evolutionary pressures have shaped the signaling and mechanical functions of the adherens junction to meet specific developmental and physiological demands. Evolutionary rate covariation (ERC) identifies proteins with correlated fluctuations in evolutionary rate that can reflect shared selective pressures and functions. Here we use ERC to identify proteins with evolutionary histories similar to the Drosophila E-cadherin (DE-cad) ortholog. Core adherens junction components α-catenin and p120-catenin displayed positive ERC correlations with DE-cad, indicating that they evolved under similar selective pressures during evolution between Drosophila species. Further analysis of the DE-cad ERC profile revealed a collection of proteins not previously associated with DE-cad function or cadherin-mediated adhesion. We then analyzed the function of a subset of ERC-identified candidates by RNAi during border cell (BC) migration and identified novel genes that function to regulate DE-cad. Among these, we found that the gene CG42684, which encodes a putative GTPase activating protein (GAP), regulates BC migration and adhesion. We named CG42684 raskol ("to split" in Russian) and show that it regulates DE-cad levels and actin protrusions in BCs. We propose that Raskol functions with DE-cad to restrict Ras/Rho signaling and help guide BC migration. Our results demonstrate that a coordinated selective pressure has shaped the adherens junction and this can be leveraged to identify novel components of the complexes and signaling pathways that regulate cadherin-mediated adhesion., Competing Interests: The authors have declared that no competing interests exist.

Interactions between herbivorous insects and their host plants are a central component of terrestrial food webs and a critical topic in agriculture, where a substantial fraction of potential crop yield is lost annually to pests. Important insights into plant-insect interactions have come from research on specific plant defences and insect detoxification mechanisms. Yet, much remains unknown about the molecular mechanisms that mediate plant-insect interactions. Here we use multiple genome-wide approaches to map the molecular basis of herbivory from both plant and insect perspectives, focusing on butterflies and their larval host plants. Parallel genome-wide association studies in the cabbage white butterfly, Pieris rapae, and its host plant, Arabidopsis thaliana, pinpointed a small number of butterfly and plant genes that influenced herbivory. These genes, along with much of the genome, were regulated in a dynamic way over the time course of the feeding interaction. Comparative analyses, including diverse butterfly/plant systems, showed a variety of genome-wide responses to herbivory, as well as a core set of highly conserved genes in butterflies as well as their host plants. These results greatly expand our understanding of the genomic causes and evolutionary consequences of ecological interactions across two of nature's most diverse taxa, butterflies and flowering plants.

Mammals diversified by colonizing drastically different environments, with each transition yielding numerous molecular changes, including losses of protein function. Though not initially deleterious, these losses could subsequently carry deleterious pleiotropic consequences. We have used phylogenetic methods to identify convergent functional losses across independent marine mammal lineages. In one extreme case, Paraoxonase 1 ( PON1 ) accrued lesions in all marine lineages, while remaining intact in all terrestrial mammals. These lesions coincide with PON1 enzymatic activity loss in marine species' blood plasma. This convergent loss is likely explained by parallel shifts in marine ancestors' lipid metabolism and/or bloodstream oxidative environment affecting PON1's role in fatty acid oxidation. PON1 loss also eliminates marine mammals' main defense against neurotoxicity from specific man-made organophosphorus compounds, implying potential risks in modern environments., (Copyright © 2018, American Association for the Advancement of Science.)

The underground environment imposes unique demands on life that have led subterranean species to evolve specialized traits, many of which evolved convergently. We studied convergence in evolutionary rate in subterranean mammals in order to associate phenotypic evolution with specific genetic regions. We identified a strong excess of vision- and skin-related genes that changed at accelerated rates in the subterranean environment due to relaxed constraint and adaptive evolution. We also demonstrate that ocular-specific transcriptional enhancers were convergently accelerated, whereas enhancers active outside the eye were not. Furthermore, several uncharacterized genes and regulatory sequences demonstrated convergence and thus constitute novel candidate sequences for congenital ocular disorders. The strong evidence of convergence in these species indicates that evolution in this environment is recurrent and predictable and can be used to gain insights into phenotype-genotype relationships.

Male ejaculates are often structurally complex, and this complexity is likely to influence key reproductive interactions between males and females. However, despite its potential evolutionary significance, the molecular underpinnings of ejaculate structural complexity have received little empirical attention. To address this knowledge gap, we sought to understand the biochemical and functional properties of the structurally complex ejaculates of Pieris rapae butterflies. Males in this species produce large ejaculates called spermatophores composed of an outer envelope, an inner matrix, and a bolus of sperm. Females are thought to benefit from the nutrition contained in the soluble inner matrix through increases in longevity and fecundity. However, the indigestible outer envelope of the spermatophore delays female remating, allowing males to monopolize paternity for longer. Here, we show that these two nonsperm-containing spermatophore regions, the inner matrix and the outer envelope, differ in their protein composition and functional properties. We also reveal how these divergent protein mixtures are separately stored in the male reproductive tract and sequentially transferred to the female reproductive tract during spermatophore assembly. Intriguingly, we discovered large quantities of female-derived proteases in both spermatophore regions shortly after mating, which may contribute to spermatophore digestion and hence, female control over remating rate. Finally, we report evidence of past selection on these spermatophore proteins and female proteases, indicating a complex evolutionary history. Our findings illustrate how structural complexity of ejaculates may allow functionally and/or spatially associated suites of proteins to respond rapidly to divergent selective pressures, such as sexual conflict or reproductive cooperation., Competing Interests: The authors declare no conflict of interest.

Mammal species have made the transition to the marine environment several times, and their lineages represent one of the classical examples of convergent evolution in morphological and physiological traits. Nevertheless, the genetic mechanisms of their phenotypic transition are poorly understood, and investigations into convergence at the molecular level have been inconclusive. While past studies have searched for convergent changes at specific amino acid sites, we propose an alternative strategy to identify those genes that experienced convergent changes in their selective pressures, visible as changes in evolutionary rate specifically in the marine lineages. We present evidence of widespread convergence at the gene level by identifying parallel shifts in evolutionary rate during three independent episodes of mammalian adaptation to the marine environment. Hundreds of genes accelerated their evolutionary rates in all three marine mammal lineages during their transition to aquatic life. These marine-accelerated genes are highly enriched for pathways that control recognized functional adaptations in marine mammals, including muscle physiology, lipid-metabolism, sensory systems, and skin and connective tissue. The accelerations resulted from both adaptive evolution as seen in skin and lung genes, and loss of function as in gustatory and olfactory genes. In regard to sensory systems, this finding provides further evidence that reduced senses of taste and smell are ubiquitous in marine mammals. Our analysis demonstrates the feasibility of identifying genes underlying convergent organism-level characteristics on a genome-wide scale and without prior knowledge of adaptations, and provides a powerful approach for investigating the physiological functions of mammalian genes., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We used ribonucleic acid sequencing to profile Candida albicans transcription within biliary fluid from a patient with cholangitis; samples were collected before and after treatment with fluconazole and drainage. Candida albicans transcriptomes at the infection site distinguished treated from untreated cholangitis. After treatment, 1131 C. albicans genes were differentially expressed in biliary fluid. Up-regulated genes were enriched in hyphal growth, cell wall organization, adhesion, oxidation reduction, biofilm, and fatty acid and ergosterol biosynthesis. This is the first study to define Candida global gene expression during deep-seated human infection. Successful treatment of cholangitis induced C. albicans genes involved in fluconazole responses and pathogenesis.

DNA damage must be repaired in an accurate and timely fashion to preserve genome stability. Cellular mechanisms preventing genome instability are crucial to human health because genome instability is considered a hallmark of cancer. Collectively referred to as the DNA damage response, conserved pathways ensure proper DNA damage recognition and repair. The function of numerous DNA damage response components is fine-tuned by posttranslational modifications, including ubiquitination. This not only involves the enzyme cascade responsible for conjugating ubiquitin to substrates but also requires enzymes that mediate directed removal of ubiquitin. Deubiquitinases remove ubiquitin from substrates to prevent degradation or to mediate signaling functions. The Saccharomyces cerevisiae deubiquitinase Ubp7 has been characterized previously as an endocytic factor. However, here we identify Ubp7 as a novel factor affecting S phase progression after hydroxyurea treatment and demonstrate an evolutionary and genetic interaction of Ubp7 with DNA damage repair pathways of homologous recombination and nucleotide excision repair. We find that deletion of UBP7 sensitizes cells to hydroxyurea and cisplatin and demonstrate that factors that stabilize replication forks are critical under these conditions. Furthermore, ubp7Δ cells exhibit an S phase progression defect upon checkpoint activation by hydroxyurea treatment. ubp7Δ mutants are epistatic to factors involved in histone maintenance and modification, and we find that a subset of Ubp7 is chromatin-associated. In summary, our results suggest that Ubp7 contributes to S phase progression by affecting the chromatin state at replication forks, and we propose histone H2B ubiquitination as a potential substrate of Ubp7., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Changes in synaptic physiology underlie neuronal network plasticity and behavioral phenomena, which are adjusted during development. The Drosophila larval glutamatergic neuromuscular junction (NMJ) represents a powerful synaptic model to investigate factors impacting these processes. Amino acids such as glutamate have been shown to regulate Drosophila NMJ physiology by modulating the clustering of postsynaptic glutamate receptors and thereby regulating the strength of signal transmission from the motor neuron to the muscle cell. To identify amino acid transporters impacting glutmatergic signal transmission, we used Evolutionary Rate Covariation (ERC), a recently developed bioinformatic tool. Our screen identified ten proteins co-evolving with NMJ glutamate receptors. We selected one candidate transporter, the SLC7 (Solute Carrier) transporter family member JhI-21 (Juvenile hormone Inducible-21), which is expressed in Drosophila larval motor neurons. We show that JhI-21 suppresses postsynaptic muscle glutamate receptor abundance, and that JhI-21 expression in motor neurons regulates larval crawling behavior in a developmental stage-specific manner.

Unlabelled: The recent explosion of comparative genomics data presents an unprecedented opportunity to construct gene networks via the evolutionary rate covariation (ERC) signature. ERC is used to identify genes that experienced similar evolutionary histories, and thereby draws functional associations between them. The ERC Analysis website allows researchers to exploit genome-wide datasets to infer novel genes in any biological function and to explore deep evolutionary connections between distinct pathways and complexes. The website provides five analytical methods, graphical output, statistical support and access to an increasing number of taxonomic groups., Availability and Implementation: Analyses and data at http://csb.pitt.edu/erc&#95;analysis/, Contact: nclark@pitt.edu., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Across a diversity of animals, male seminal fluid coagulates upon ejaculation to form a hardened structure known as a copulatory plug. Previous studies suggest that copulatory plugs evolved as a mechanism for males to impede remating by females, but detailed investigations into the time course over which plugs survive in the female's reproductive tract are lacking. Here, we cross males from eight inbred strains to females from two inbred strains of house mice (Mus musculus domesticus). Plug survival was significantly affected by male genotype. Against intuition, plug survival time was negatively correlated with plug size: long-lasting plugs were small and relatively more susceptible to proteolysis. Plug size was associated with divergence in major protein composition of seminal vesicle fluid, suggesting that changes in gene expression may play an important role in plug dynamics. In contrast, we found no correlation to genetic variation in the protein-coding regions of five genes thought to be important in copulatory plug formation (Tgm4, Svs1, Svs2, Svs4 and Svs5). Our study demonstrates a complex relationship between copulatory plug characteristics and survival. We discuss several models to explain unexpected variation in plug phenotypes.

Persistent adaptive challenges are often met with the evolution of novel physiological traits. Although there are specific examples of single genes providing new physiological functions, studies on the origin of complex organ functions are lacking. One such derived set of complex functions is found in the Lepidopteran bursa copulatrix, an organ within the female reproductive tract that digests nutrients from the male ejaculate or spermatophore. Here, we characterized bursa physiology and the evolutionary mechanisms by which it was equipped with digestive and absorptive functionality. By studying the transcriptome of the bursa and eight other tissues, we revealed a suite of highly expressed and secreted gene products providing the bursa with a combination of stomach-like traits for mechanical and enzymatic digestion of the male spermatophore. By subsequently placing these bursa genes in an evolutionary framework, we found that the vast majority of their novel digestive functions were co-opted by borrowing genes that continue to be expressed in nonreproductive tissues. However, a number of bursa-specific genes have also arisen, some of which represent unique gene families restricted to Lepidoptera and may provide novel bursa-specific functions. This pattern of promiscuous gene borrowing and relatively infrequent evolution of tissue-specific duplicates stands in contrast to studies of the evolution of novelty via single gene co-option. Our results suggest that the evolution of complex organ-level phenotypes may often be enabled (and subsequently constrained) by changes in tissue specificity that allow expression of existing genes in novel contexts, such as reproduction. The extent to which the selective pressures encountered in these novel roles require resolution via duplication and sub/neofunctionalization is likely to be determined by the need for specialized reproductive functionality. Thus, complex physiological phenotypes such as that found in the bursa offer important opportunities for understanding the relative role of pleiotropy and specialization in adaptive evolution., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Reproductive traits experience high levels of selection because of their direct ties to fitness, often resulting in rapid adaptive evolution. Much of the work in this area has focused on male reproductive traits. However, a more comprehensive understanding of female reproductive adaptations and their relationship to male characters is crucial to uncover the relative roles of sexual cooperation and conflict in driving co-evolutionary dynamics between the sexes. We focus on the physiology of a complex female reproductive adaptation in butterflies and moths: a stomach-like organ in the female reproductive tract called the bursa copulatrix that digests the male ejaculate (spermatophore). Little is known about how the bursa digests the spermatophore. We characterized bursa proteolytic capacity in relation to female state in the polyandrous butterfly Pieris rapae. We found that the virgin bursa exhibits extremely high levels of proteolytic activity. Furthermore, in virgin females, bursal proteolytic capacity increases with time since eclosion and ambient temperature, but is not sensitive to the pre-mating social environment. Post copulation, bursal proteolytic activity decreases rapidly before rebounding toward the end of a mating cycle, suggesting active female regulation of proteolysis and/or potential quenching of proteolysis by male ejaculate constituents. Using transcriptomic and proteomic approaches, we report identities for nine proteases actively transcribed by bursal tissue and/or expressed in the bursal lumen that may contribute to observed bursal proteolysis. We discuss how these dynamic physiological characteristics may function as female adaptations resulting from sexual conflict over female remating rate in this polyandrous butterfly., (© 2015. Published by The Company of Biologists Ltd.)

A diverse subset of pattern recognition receptors (PRRs) detects pathogen-associated nucleic acids to initiate crucial innate immune responses in host organisms. Reflecting their importance for host defense, pathogens encode various countermeasures to evade or inhibit these immune effectors. PRRs directly engaged by pathogen inhibitors often evolve under recurrent bouts of positive selection that have been described as molecular 'arms races.' Cyclic GMP-AMP synthase (cGAS) was recently identified as a key PRR. Upon binding cytoplasmic double-stranded DNA (dsDNA) from various viruses, cGAS generates the small nucleotide secondary messenger cGAMP to signal activation of innate defenses. Here we report an evolutionary history of cGAS with recurrent positive selection in the primate lineage. Recent studies indicate a high degree of structural similarity between cGAS and 2'-5'-oligoadenylate synthase 1 (OAS1), a PRR that detects double-stranded RNA (dsRNA), despite low sequence identity between the respective genes. We present comprehensive comparative evolutionary analysis of cGAS and OAS1 primate sequences and observe positive selection at nucleic acid binding interfaces and distributed throughout both genes. Our data revealed homologous regions with strong signatures of positive selection, suggesting common mechanisms employed by unknown pathogen encoded inhibitors and similar modes of evasion from antagonism. Our analysis of cGAS diversification also identified alternately spliced forms missing multiple sites under positive selection. Further analysis of selection on the OAS family in primates, which comprises OAS1, OAS2, OAS3 and OASL, suggests a hypothesis where gene duplications and domain fusion events result in paralogs that provide another means of escaping pathogen inhibitors. Together our comparative evolutionary analysis of cGAS and OAS provides new insights into distinct mechanisms by which key molecular sentinels of the innate immune system have adapted to circumvent viral-encoded inhibitors.

The Saccharomyces cerevisiae Shu2 protein is an important regulator of Rad51, which promotes homologous recombination (HR). Shu2 functions in the Shu complex with Shu1 and the Rad51 paralogs Csm2 and Psy3. Shu2 belongs to the SWS1 protein family, which is characterized by its SWIM domain (CXC...Xn...CXH), a zinc-binding motif. In humans, SWS1 interacts with the Rad51 paralog SWSAP1. Using genetic and evolutionary analyses, we examined the role of the Shu complex in mitotic and meiotic processes across eukaryotic lineages. We provide evidence that the SWS1 protein family contains orthologous genes in early-branching eukaryote lineages (e.g., Giardia lamblia), as well as in multicellular eukaryotes including Caenorhabditis elegans and Drosophila melanogaster. Using sequence analysis, we expanded the SWIM domain to include an invariant alanine three residues after the terminal CXH motif (CXC…Xn…CXHXXA). We found that the SWIM domain is conserved in all eukaryotic orthologs, and accordingly, in vivo disruption of the invariant residues within the canonical SWIM domain inhibits DNA damage tolerance in yeast and protein-protein interactions in yeast and humans. Furthermore, using evolutionary analyses, we found that yeast and Drosophila Shu2 exhibit strong coevolutionary signatures with meiotic proteins, and in yeast, its disruption leads to decreased meiotic progeny. Together our data indicate that the SWS1 family is an ancient and highly conserved eukaryotic regulator of meiotic and mitotic HR., (Copyright © 2015 by the Genetics Society of America.)

Genes involved in the same function tend to have similar evolutionary histories, in that their rates of evolution covary over time. This coevolutionary signature, termed Evolutionary Rate Covariation (ERC), is calculated using only gene sequences from a set of closely related species and has demonstrated potential as a computational tool for inferring functional relationships between genes. To further define applications of ERC, we first established that roughly 55% of genetic diseases posses an ERC signature between their contributing genes. At a false discovery rate of 5% we report 40 such diseases including cancers, developmental disorders and mitochondrial diseases. Given these coevolutionary signatures between disease genes, we then assessed ERC's ability to prioritize known disease genes out of a list of unrelated candidates. We found that in the presence of an ERC signature, the true disease gene is effectively prioritized to the top 6% of candidates on average. We then apply this strategy to a melanoma-associated region on chromosome 1 and identify MCL1 as a potential causative gene. Furthermore, to gain global insight into disease mechanisms, we used ERC to predict molecular connections between 310 nominally distinct diseases. The resulting "disease map" network associates several diseases with related pathogenic mechanisms and unveils many novel relationships between clinically distinct diseases, such as between Hirschsprung's disease and melanoma. Taken together, these results demonstrate the utility of molecular evolution as a gene discovery platform and show that evolutionary signatures can be used to build informative gene-based networks.

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks.

Evolutionary rates of functionally related proteins tend to change in parallel over evolutionary time. Such evolutionary rate covariation (ERC) is a sequence-based signature of coevolution and a potentially useful signature to infer functional relationships between proteins. One major hypothesis to explain ERC is that fluctuations in evolutionary pressure acting on entire pathways cause parallel rate changes for functionally related proteins. To explore this hypothesis we analyzed ERC within DNA mismatch repair (MMR) and meiosis proteins over phylogenies of 18 yeast species and 22 mammalian species. We identified a strong signature of ERC between eight yeast proteins involved in meiotic crossing over, which seems to have resulted from relaxation of constraint specifically in Candida glabrata. These and other meiotic proteins in C. glabrata showed marked rate acceleration, likely due to its apparently clonal reproductive strategy and the resulting infrequent use of meiotic proteins. This correlation between change of reproductive mode and change in constraint supports an evolutionary pressure origin for ERC. Moreover, we present evidence for similar relaxations of constraint in additional pathogenic yeast species. Mammalian MMR and meiosis proteins also showed statistically significant ERC; however, there was not strong ERC between crossover proteins, as observed in yeasts. Rather, mammals exhibited ERC in different pathways, such as piRNA-mediated defense against transposable elements. Overall, if fluctuation in evolutionary pressure is responsible for ERC, it could reveal functional relationships within entire protein pathways, regardless of whether they physically interact or not, so long as there was variation in constraint on that pathway.

Evolutionary rate covariation (ERC) is a phylogenetic signature that reflects the covariation of a pair of proteins over evolutionary time. ERC is typically elevated between interacting proteins and so is a promising signature to characterize molecular and functional interactions across the genome. ERC is often assumed to result from compensatory changes at interaction interfaces (i.e., intermolecular coevolution); however, its origin is still unclear and is likely to be complex. Here, we determine the biological factors responsible for ERC in a proteome-wide data set of 4459 proteins in 18 budding yeast species. We show that direct physical interaction is not required to produce ERC, because we observe strong correlations between noninteracting but cofunctional enzymes. We also demonstrate that ERC is uniformly distributed along the protein primary sequence, suggesting that intermolecular coevolution is not generally responsible for ERC between physically interacting proteins. Using multivariate analysis, we show that a pair of proteins is likely to exhibit ERC if they share a biological function or if their expression levels coevolve between species. Thus, ERC indicates shared function and coexpression of protein pairs and not necessarily coevolution between sites, as has been assumed in previous studies. This full interpretation of ERC now provides us with a powerful tool to assign uncharacterized proteins to functional groups and to determine the interconnectedness between entire genetic pathways.

Protein components of the Drosophila male ejaculate are critical modulators of reproductive success, several of which are known to evolve rapidly. Recent evidence of adaptive evolution in female reproductive tract proteins suggests this pattern may reflect sexual selection at the molecular level. Here we explore the evolutionary dynamics of a five-paralog gene family of female reproductive proteases within geographically isolated subspecies of Drosophila mojavensis. Remarkably, four of five paralogs show exceptionally low differentiation between subspecies and unusually structured haplotypes that suggest the retention of old polymorphisms. These gene genealogies are accompanied by deviations from neutrality consistent with diversifying selection. While diversifying selection has been observed among the reproductive molecules of mammals and marine invertebrates, our study provides the first evidence of this selective regime in any Drosophila reproductive protein, male or female., (© 2011 by the Genetics Society of America)