ABSTRACT Species differ in longevity, physiology, and social organization, and these properties expose them to distinct endogenous and environmental mutagens. Mutational spectra generated by these processes can shape downstream molecular evolution, influencing synonymous nucleotide composition, codon usage, and even amino acid composition. Tracing this signal from life history to proteome through mutagenesis could reveal how mutational pressure interacts with the fitness landscape, including the direction of molecular change and the extent to which proteins remain functional while following mutational biases. Here, building on the recently identified age-associated mitochondrial A>G mutational signature in mammals, we test the universality of this signature and its downstream effects on genome and proteome evolution by comparing long-lived termites with short-lived non-termite cockroaches. We find that termite mtDNA exhibits a stronger A>G mutational signature than that of non-termite cockroaches, accompanied by coordinated shifts in synonymous nucleotide composition, codon usage, and amino acid composition. Our results show that ecological and life-history-associated mutational pressures can be transmitted through a hierarchy from mutational spectra to nucleotide composition and ultimately to proteome evolution. Mitochondrial genomes may therefore function not only as records of ancestry but also as molecular archives of the biological conditions under which species evolve.

The mutational spectrum is an increasingly important molecular phenotype that quantitatively describes mutagenesis in a given gene and species, enabling future comparative analyses to reveal differences in underlying mutagenic processes, whether internal, such as DNA repair processes, or external, such as ecological niches and conditions. Mutation accumulation experiments, although time-consuming and costly, remain the standard approach for reconstructing bacterial neutral mutation spectra. Here, we present BacNeMu, a phylogenetically informed pipeline that reconstructs neutral mutational spectra of bacterial genomes using open databases GTDB, AnnoTree and KEGG Orthology, building on previously developed NeMu pipeline. BacNeMu reconstructs mutation spectra that closely match mutation accumulation experiments results while requiring substantially less time, enabling comparative analyses across diverse bacterial taxa. Applied to obligate aerobes and anaerobes, BacNeMu recovered the expected excess of T:A>C:G transitions, consistent with oxidative-damage-associated mutational patterns previously described in mitochondrial genomes and yeast single-strand. We further asked if any other ecologic factors influence a mutational spectrum. As a pilot we compared three species living under different temperatures: one strong thermophile - Thermotoga maritima, one psychrophile - Clostridium algidicarnis, and one with intermediate temperature tolerance - Psychrobacter sanguinis. In the thermophile, the relative frequency of T:A>C:G substitutions was higher than in the psychrophile, consistent with the hypothesis that GC-biased mutagenesis contributes to thermal adaptation, although C:G>T:A transitions predominate across all three species. BacNeMu provides a rapid, phylogenetically informed framework for generating biologically meaningful mutation spectra from open databases.

BACKGROUND:Clonal hematopoiesis (CH), defined by the expansion of hematopoietic cells with somatic mutations in leukemogenic genes (CH of indeterminate potential (CHIP)) or with mosaic chromosomal alterations (mCAs), is associated with aging and adverse health outcomes in the general population. CHIP prevalence is higher in People with HIV (PWH) than in controls. However, the full spectrum, prevalence, and clinical consequences of CH in PWH remain incompletely understood. METHODS:We assessed CHIP and mCAs in a large sample of PWH (N∼2,500) from the Swiss HIV Cohort Study. Using high-depth targeted sequencing of CHIP genes and genome-wide genotyping to call mCAs, we quantified the prevalence and clone size of both CH types and investigated an association of CH with clinical variables. RESULTS:CHIP (25% of individuals) and mCAs (16% of individuals) were common, positively correlated with age, often co-occurring (OR=1.7, p=0.02 for autosomal mCAs), and associated with various clinical outcomes, including all-cause mortality (HR=1.3, p=0.02 for CHIP) and hematologic malignancies (HR=9.4, p=0.01 for the effect of CHIP on the risk of myeloid cancer; HR>20, p<0.001 for the effect of co-occurring CHIP and mCAs on the risk of lymphoid cancer). We also observed associations of CH with several proxies of inflammatory status (CD4:CD8 ratio, HIV viral load, late initiation of antiretroviral therapy, and toxicity of antiretroviral drugs). CONCLUSION:The study provides a comprehensive assessment of the CH landscape in PWH, highlighting potential causes and consequences in this population and suggesting an interaction between CH and chronic immune activation.

Purifying selection of mtDNA mutations is a vital process that cleanses the mitochondrial genome of detrimental variants that may endanger individuals and populations. A common measure of purifying selection is the increase of average synonymity by reduction the proportion of mostly detrimental non-synonymous mutations. The mechanisms underlying purifying selection are still debated. The Makova group has recently published high- fidelity analysis of mtDNA mutations in individual human oocytes (Arbeithuber et al., 2025). The authors observed a decrease in the proportion of potentially detrimental coding and conservative mutations at higher mutant fractions (MFs) and interpreted this as purifying selection removing detrimental mutations at higher MFs. We noted, however, that, in contrast to what would be expected under purifying selection, the synonymity of oocyte mutations was very low and decreased, rather than increased, at higher MFs. We hypothesized that this inconsistency resulted from non-synonymous mutations being prone to strong positive selection which erroneously made coding mutations appear negatively selected in comparison. In support of our hypothesis, we show that non-coding oocytes mutations indeed are under strong positive selection. To alleviate this setback, we reanalyzed the data using a new metric of intracellular clonal selection and neutral synonymous mutations as the reference. We demonstrated that coding mutations are in fact under prevailing positive selection. This is in line with previous estimates of positive selection in primordial germ cells (PGCs) and in mother-child pairs. Importantly, prevailing positive selection does not imply the absence of negative selection. We show that specific types of mutations may be under prevailing purifying selection (e.g., the Co1 gene). Of note, this prevailing positive selection pertains only to the most recent, germline mtDNA mutations which have not been yet inherited into the next generation. Purifying selection steps in as germline mutations proceed to subsequent generations. The implications of these findings and the potential benefits of positive selection of detrimental mtDNA mutations are discussed. ### Competing Interest Statement The authors have declared no competing interest.

Background: Ageing is often associated with clonal expansion of somatic mitochondrial (mtDNA) deletions, while their origin is still poorly known. Deletions are often flanked by direct nucleotide repeats, however, repeats solely do not provide an exhaustive explanation of deletion distribution. Here, we aim to decipher additional factors affecting formation of mtDNA deletions and create a score for estimation risk of deletion formation. Materials and methods: Using a collection of human mtDNA deletions and global and local mtDNA properties (distribution of repeats and other structures), we reexamine the risks of somatic mtDNA deletion. Results: Comparison of the probability of mtDNA deletions in regions flanked by different combinations of direct and inverted repeats reveals that in addition to direct repeats, which are known to influence deletions, there is a strong influence of the secondary structure of single- stranded mtDNA during replication. The secondary structure during replication is formed by inverted repeats, which can form stems and reduce the effective distance between two direct repeats, thereby increasing the probability of deletions. Conclusion: As we can see, the mitochondrial genome has a specific structure and composition of nucleotides that can influence the formation of deletions. We want to find out how the human mitochondrial anatomy predisposes to deletions. Regions of mitochondrial DNA have a different risk of deletion, and for each region a metric can be obtained to estimate the probability of deletion based on sequencing data. Nucleotide motifs such as repeats may be associated with a higher risk of deletions in the mitochondrial genome. We are going to study the various features of the global and local structure of mitochondrial genomes (direct and inverted repeats, Gibbs energies, G-quadruplexes, microhomology, rare deletions), primarily in human ones. By finding fragile places in various human haplogroups, we will be able to create a universal metric for assessing the fragility of regions of the mitochondrial genome. Acknowledgement: This work was supported by grants from the Russian Foundation for Basic Research No. 21-75-20143, No. 21-75-20145, No. 21-75-10081, EOT was supported by a PhD scholarship from the Austrian Science Foundation FWF (DOC 33-B27). Key words: mtDNA, deletions, repeats, secondary structures References: Persson et al. Copy-Choice Recombination during Mitochondrial L-Strand Synthesis Causes DNA Deletions. Nature Communications 10 (1): 1–10 (2019). Albertini et al. On the Formation of Spontaneous Deletions: The Importance of Short Sequence Homologies in the Generation of Large Deletions. Cell 29 (2): 319–28 (1982). Shamanskiy et al. Secondary structure of the human mitochondrial genome affects formation of deletions. BMC Biol 21, 103 (2023).

Osteoarthritis (OA) is one of the most common diseases of the musculoskeletal system, accomplished by a high level of disability. The leading pathogenetic factors of these age-associated diseases include the interrelated processes of “inflammatory aging” and mitochondrial dysfunction, which lead to the development of chronic inflammation and degradation of different joint tissues. The present article contains results about point mutations in mitochondrial genome of peri-articular muscular tissues in the patients with primary OA (experimental group) and post-traumatic osteoarthritis (control group). The study involved 67 volunteers over 53 years old with basic diagnosis of post-traumatic or primary gonarthrosis / coxarthrosis stage 3. Clinical diagnosis was made on the basis of medical history, complaints, clinical and X-ray examination data. The material for the study included the samples of muscles (80 to 100 mm3) obtained at knee or hip replacement surgery. Several techniques have been adapted for isolation, enrichment and purification of nucleic acids, thus allowing to obtain up to 500 ng of mitochondrial DNA (mtDNA) from the biopsies. The prepared mtDNA libraries were sequenced at NGS platform. Bioinformatic analysis was carried out using the following programs: MitoHPC (to detect rare single-nucleotide mutations of mtDNA), MitoSAlt (to detect rare deletions at the mtDNA level) and Splice-Break2 (to detect rare deletions at the level of mtDNA RNA transcripts). Common point mutations A189G (adenine to guanine at position 189) and T408A (thymine to adenine at position 408) were detected. In the control group, the A189G mutation was revealed in 7 patients and T408A mutation was found in 8 volunteers (both mutations were detected in 6 out of 9 persons). In experimental group, the A189G mutation was found in 43 of 58 patients, T408A – in 35 volunteers. In this group of volunteers, both mutations were registered in 19 subjects. The level of mutation frequency, expressed as allele frequency (VAF) in the experimental group significantly exceeded that of the control group. Moreover, in experimental group, unlike control group, a significant correlative relationship was established between the presence of an increased level of mutations in the mitochondrial genome, and a number of clinical and laboratory parameters in volunteers. The described mutations in the mitochondrial genome of periarticular muscle tissue seem to be associated both with aging process and with the direct development of age-associated pathology, i.e., osteoarthritis. The increased levels of mutations in positions 189 and 408 of the regulatory region of mitochondrial genes detected in our patients are apparently associated with both increased level of mutations typical of pathological aging and, possibly, with genotoxic effects of high-dose therapy with non-steroidal anti-inflammatory drugs on mitochondrial genome.

Mitochondrial DNA (mtDNA) mutagenesis remains poorly understood despite its crucial role in disease, aging, and evolutionary tracing. In this study, we reconstructed a comprehensive 192-component mtDNA mutational spectrum for chordates by analyzing 118,397 synonymous mutations in the CytB gene across 1,697 species and five classes. This analysis revealed three primary forces shaping mtDNA mutagenesis: (i) symmetrical, replication-driven errors by mitochondrial polymerase (POLG), resulting in C > T and A > G mutations that are highly conserved across classes; (ii) asymmetrical, damage-driven C > T mutations on the single-stranded heavy strand with clock-like dynamics; and (iii) asymmetrical A > G mutations on the heavy strand, with dynamics suggesting sensitivity to oxidative damage. The third component, sensitive to oxidative damage, positions mtDNA mutagenesis as a promising marker for metabolic and physiological processes across various classes, species, organisms, tissues, and cells. The deconvolution of the mutational spectra into mutational signatures uncovered deficiencies in both base excision repair (BER) and mismatch repair (MMR) pathways. Further analysis of mutation hotspots, abasic sites, and mutational asymmetries underscores the critical role of single-stranded DNA damage (components ii and iii), which, uncorrected due to BER and MMR deficiencies, contributes roughly as many mutations as POLG-induced errors (component i).

The extent to which somatic mitochondrial DNA (mtDNA) mutations are subject to selection is a fundamental question relevant to development, mitochondrial disease, cancer, and aging. Recently a study from the Sudmant laboratory that used an advanced, high fidelity mutational analysis reported that somatic mutations in protein-coding genes exhibit signatures of negative selection. This report came as surprise as several other studies including those that used same technology reported either lack of selection or positive (destructive) selection on somatic mutations. We hypothesized that these discrepancies may stem, in part, from the inclusion of germline mutations in addition to somatic ones, which could bias selection analyses due to the high synonymity of the latter. To test this, we reanalyzed the Sudmant dataset by separating mutations into germline (defined as shared between related animals) and somatic (not shared between tissues of an animal). We then employed a cumulative curve approach to assess selection without bias. Our analysis reveals that, indeed, an apparent purifying selection signal is driven by an admixture of synonymous germline mutations and disappears upon their removal. The remaining somatic mutations for most part show overall dynamics consistent with neutral drift. However, mutations at higher mutant fractions show positive selection trend, most compatible with a low proportion of mutations experiencing positive selection. While we do not exclude rare or context-specific selection events, our results argue against pervasive somatic selection and highlight the importance of rigorous stratification when interpreting mtDNA mutational patterns. ### Competing Interest Statement The authors have declared no competing interest.

The origin and expansion of mitochondrial somatic variants, influenced by tissue-specific mutagenesis and selection, are not well understood despite their relevance to aging and age-related diseases. Postmitotic tissues, such as skeletal muscles, are particularly underexplored, even though mtDNA variant evolution in these tissues can differ significantly from that in proliferative tissues. To address this, we analyzed mitochondrial heteroplasmy in skeletal muscle samples from an osteoarthritic cohort (N = 105). We observed that the age-related dynamics of two famous variants m.189A > G and m.408T > A in our cohort is indistinguishable from their dynamics in random control cohort, suggesting that they are not a cause of muscular problems, but rather mark the age-related processes in muscles. We also observed that when adjusted by age and gender, carriers of these variants tend to have higher BMI, body weight, and muscle strength than non-carriers. Putting together all the lines of evidence, we propose that these variants are able to rapidly expand through selfish dynamics, which is especially pronounced in hypertrophic muscle fibers of individuals with higher body weight. Further investigation is necessary to clarify this hypothesis.

Aging, characterized by a series of functional declines correlated with advancing chronological age, has a significant mitochondrial DNA (mtDNA) component, with somatic mtDNA deletions playing a central role. In post-mitotic or slow-dividing cells like neurons and skeletal muscles, selfish mtDNA deletions clonally expand within a cell, ultimately leading to the deterioration and death of host cells and appearence of age-related phenotypes. Thus reducing the burden of somatic deletions could have far-reaching systemic benefits for the entire human body. Given the crucial role of direct nucleotide repeats in the formation of mitochondrial deletions, we hypothesize that minimizing these repeats in the human mitochondrial genome could enhance healthspan by decreasing somatic deletions. To investigate this hypothesis, we focus on the "common repeat", a 13-base pair perfect direct repeat sequence (ACCTCCCTCACCA) located at positions 8470-8482 and 13447-13459, respectively. This perfect repeat: (i) is highly prevalent, with its potential deleterious consequences affecting the majority of humans; (ii) represents one of the most fragile sites, highly prone to forming deletions; (iii) when disrupted, is associated with a decreased somatic deletion load and enhanced human healthspan; (iv) is likely to experience positive selection in the present or near future due to indirect fitness effects, such as the "grandmother effect", and direct fitness effects, such as (v) a decreased mutation rate. These observations support the argument that reducing the mtDNA somatic deletion load through targeted disruption of these repeats, or by using naturally occurring polymorphisms with disrupted repeats in mitochondrial medicine, could be an effective approach to increasing human longevity. ### Competing Interest Statement The authors have declared no competing interest.

The recognized importance of mutational spectra in molecular evolution is yet to be fully exploited beyond human cancer studies and model organisms. The wealth of intraspecific polymorphism data in the GenBank repository, covering a broad spectrum of genes and species, presents an untapped opportunity for detailed mutational spectrum analysis. Existing methods fall short by ignoring intermediate substitutions on the inner branches of phylogenetic trees and lacking the capability for cross-species mutational comparisons. To address these challenges, we present the NeMu pipeline, available at https://nemu-pipeline.com, a tool grounded in phylogenetic principles designed to provide comprehensive and scalable analysis of mutational spectra. Utilizing extensive sequence data from numerous available genome projects, NeMu rapidly and accurately reconstructs the neutral mutational spectrum. This tool, facilitating the reconstruction of gene- and species-specific mutational spectra, contributes to a deeper understanding of evolutionary mechanisms across the broad spectrum of known species.

Numerous empirical studies have revealed epistatic interactions among deleterious variants. In this paper, we assume such interactions are widespread and analyze the resulting shift in mutational burden and average population fitness following the introduction of a strong and universal stress (hereafter “handicap”). We demonstrate that organisms with a low burden of slightly-deleterious variants (SDVs) are more likely to survive exposure to a handicap, whether genetic or environmental, leading to a purifying effect on the population. We further discuss the potential applications of harnessing such interactions for evolutionary and population studies as well as for population management.### Competing Interest StatementThe authors have declared no competing interest.

The primary genetic challenge encountered in artificial populations lies in the strong genetic drift, which leads to the accumulation of numerous slightly deleterious mutations across the genome. Such mutations diminish the adaptability of the entire population. The objective of this project involves the investigation and implementation of genetic selection methods within cultivated fish populations such Cyprinus carpio L . In order to maintain a high level of genome quality in productive species, we conducted a proof-of-principle experiment employing stress-induced strong purifying selection. This selection process is based on negative epistasis and effectively eliminates organisms carrying an excess of deleterious variants. The first step involves the creation of mutant and intact groups of fish. To obtain mutant groups, we treated male gametes with the ENU mutagen, which primarily induces single-nucleotide substitutions uniformly throughout the genome, thereby imitating natural mutations. This methodology is paramount for the accurate interpretation of experimental outcomes. Notably, temperature stands as a pivotal factor influencing the embryonic development of fish. Therefore, we subjected the embryos to a diverse range of temperatures and varied the duration of exposure during critical stages of embryogenesis. Through meticulous examination, we ascertained that the stage most susceptible to screening purposes is the 22-somite pair stage, occurring at a temperature of 38°C, with a 40-minute exposure period. We suppose, this comprehensive approach can be applied to improve the quality of the gene pool within domestic fish populations, ultimately enhancing the economic efficacy of fish farms. The future prospects of this method encompass its potential application to various species. ### Competing Interest Statement The authors have declared no competing interest.

In mitochondrial DNA (further mtDNA) A>G substitution in protein-coding genes on a heavy strand is the main result of chemical damage (for example, spontaneous deamination). Some vertebrates, like mammals [1] and fishes [2] show strong correlations between mtDNA intense mutagenesis and life-history traits. Here we are trying to discover these connections in birds.

Serrano et al. (Serrano et al., 2024) use a high-fidelity somatic mtDNA mutation analysis in conplastic mice in which mtDNA was replaced with exogenous mtDNA of different mouse strains. Serrano reported apparent abundant somatic reversion mutations in the exogenous mtDNA that seemed to restore the original mito-nuclear match. If real, such a phenomenon would have important implications for health and genetics. In todays highly mixed human population, the pairing of potentially mismatched nuclear and mitochondrial genomes is widespread, so the proposed reversion mutagenesis should be commonplace. We demonstrate, however, that these reversion mutations are not real but originate from cross-contamination between samples and from NUMTs, the mtDNA pseudogenes located in the nuclear genome. ### Competing Interest Statement The authors have declared no competing interest.