Biology subjects

Smith, L. M.

Publications and source records attributed to Smith, L. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Anti-amyloid immunotherapy drives APOE4 specific increases in glial reactivity, perivascular immune activation, and ARIA-like events

Anti-amyloid antibodies represent the first disease modifying therapeutics for Alzheimers disease (AD). Adoption of these novel treatments has been slowed by the occurrence of amyloid related imaging abnormalities (ARIA) - treatment-associated edema (ARIA-E) or microhemorrhages (ARIA-H) that disproportionately affect carriers of the E4 allele of apolipoprotein E (APOE). With E4 carriers comprising nearly 70% of the AD population, there is a critical need to understand the unique vulnerability of E4 carriers to these events. To address this gap, we utilized the EFAD mouse model - which expresses human APOE isoforms on the 5xFAD background of amyloidosis - to directly compare the effects of anti-amyloid therapy across APOE genotypes. 9-month-old E2, E3, and E4FAD mice received weekly injections of chimeric Aducanumab (chAdu) or IgG control for 12 weeks, to assess APOE isoform-specific effects on amyloid dynamics, ARIA-H-like microhemorrhages, and underlying cellular and transcriptomic responses. E4FAD mice demonstrated plaque reductions with accompanying increases in microhemorrhages (measured on both MRI and histology), and increases in microglial and astrocyte reactivity - especially in the perivascular compartment. Additionally, vascular branching analysis and parallel single cell and spatial transcriptomics revealed a loss of vascular plasticity and increased inflammatory and immune signaling in the neurovascular units of E4FAD mice. Together, these findings suggest the cerebrovasculature of E4s is uniquely susceptible to antibody mediated vascular damage and provide immunological targets for the assessment or mitigation of ARIA risk in this highest need population

neuroscience↗

bioRxiv2026

Rapid Peptide Mapping of Monoclonal Antibodies with Direct Infusion Mass Spectrometry

Peptide mapping is a critical tool for characterizing biotherapeutic proteins and is essential for the development of monoclonal antibody drugs. Here we describe a new direct infusion technology that streamlines peptide mapping data collection and analysis, accelerating the method by up to 100-fold. This method, which we term RaPiD-mAb-MS, combines high-throughput plate-based sample preparation with direct infusion mass spectrometry analysis. RaPiD-mAb-MS allows analysis of 96 samples within [~] 1.5 to 2 hours, routinely achieves >95% sequence coverage, and has been successfully applied to 28 unique antibodies and over 2,000 samples. Here we demonstrate that RaPiD-mAb-MS detects and quantifies oxidation, deamidation, isomerization, glycosylation, and sequence variants with results comparable to conventional LC-MS based methods in a fraction of the time. Further, by eliminating chromatography, data analysis is greatly streamlined and simplified. By allowing for the collection of [~] 1,000 peptide maps per day, RaPiD-mAb-MS is positioned to accelerate all phases of antibody-based drug discovery & development and sets the stage for collection of massive datasets that would allow artificial intelligent prediction of optimal antibody variants and formulations.

biochemistry↗

bioRxiv2026

Orai1 is required for Ca2+-dependent plasma membrane repair and mechanoadaptation

Ca2+-dependent repair of plasma membrane breaches is essential for animal cell viability. An initial passive influx of extracellular Ca2+ triggers the formation of a protein plug that rapidly seals breaches. However, the mechanism of extracellular Ca2+ requirement for subsequent repair remains undefined. EHD2 protein stabilizes the plasma membrane caveolae, which sustain membrane repair, and maintains high surface levels of the caveolae-resident Ca2+ channel Orai1. We establish the requirement of both Orai1 and EHD2 for repair of plasma membrane lesions induced by mechanical injury or by a model bacterial pore-forming toxin. We demonstrate rapid EHD2 recruitment and Orai1-mediated Ca2+ entry at plasma membrane sites of localized mechanical stimulus, the latter requiring EHD2 and CAV1. EHD2 and Orai1 are necessary for mechanosensitive YAP/TAZ-TEAD activation and positive feedback for CAV1 expression that promotes membrane repair. Our studies establish EHD2 and Orai1 as novel components of mammalian plasma membrane repair and mechanoadaptation.

cell biology↗

bioRxiv2025

High-throughput transposon mutagenesis defines the essential genome of diverse phages

Phages are important drivers of bacterial evolution with therapeutic potential as antimicrobials. However, gaps in our understanding of phages and our inability to rapidly engineer them with new genetic cargo hinders progress towards phage-based therapies. To address the lack of unbiased, genome-wide mutational tools for phages, we developed transposon mutagenesis employing CRISPR-anti-CRISPR (Acr)-based selection and deep-sequencing (Phage Tn-seq). Transposon mutagenesis was effective for phages with unmodified or hypermodified genomes and a jumbo phage that protects its DNA within a nucleus. Phage Tn-seq enabled phage gene essentiality assignment consistent with structural proteomics and core gene conservation. Insertion biases allowed prediction of transcriptional direction and early injected phage DNA regions. We exploited the method to rapidly deliver new cargo to phage genomes in just a few days and used an AI-designed Acr to expand the phage transposon toolbox. Phage Tn-seq is a versatile tool to advance our understanding and applications of phages.

microbiology↗

bioRxiv2025

Interactome screening implicates BAG6 as a suppressor of UBQLN2 misfolding in ALS-dementia

Ubiquilin-2 (UBQLN2) is a ubiquitin (Ub)-binding shuttle protein that is mutated in X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS/FTD-linked mutations in UBQLN2 disrupt its conformation, increasing its tendency to form cytoplasmic aggregates that may disrupt cellular regulation through loss-of-function (LOF) and gain-of-function (GOF) effects. To explore how ALS-associated mutations impact UBQLN2 function, we performed quantitative mass spectrometry (MS)-based interactome analysis using affinity-purified UBQLN2 from inducible pluripotent stem cells (iPSCs) and induced motor neurons (iMNs) expressing wild-type UBQLN2 (UBQLN2WT), a UBQLN2P497H clinical mutant, or a UBQLN24XALS allele harboring four disease mutations. Proteins showing enhanced association with ALS-mutant UBQLN2 proteins included PEG10, a known degradation target of UBQLN2, and BAG6, a chaperone involved in the triage of mislocalized proteins (MLPs). BAG6 knockdown inhibited the solubility recovery of both wild-type and ALS-mutant UBQLN2 proteins following heat stress (HS), suggesting it functions as a UBQLN2 holdase. In addition, knockdown of BAG6 or knockout of UBQLN2 led to PEG10 accumulation, implicating both in PEG10 turnover; however, neither BAG6 nor UBQLN2 was required for PEG10 degradation in response to HS. The aggregation prone UBQLN24XALS mutant showed increased PEG10 binding and modestly delayed PEG10 turnover while PEG10 degradation was not significantly different between UBQLN2WT and UBQLN2P497H iPSCs. The combined findings implicate BAG6 a UBQLN2 holdase and identify a suite of proteins whose altered binding may contribute to pathologic changes in UBQLN2-associated ALS/FTD.

molecular biology↗

bioRxiv2025

From recall to reset: the role of DNA (de)methylation in modulating plant immune memory

Immune memory enables plants to respond more effectively to recurrent pathogen infections. Epigenetic changes in DNA methylation and small RNAs have been implicated, yet how their temporal dynamics govern the establishment and erasure of immune memory remains unclear. Using a chemically inducible system in Arabidopsis to activate the DNA demethylase REPRESSOR OF SILENCING 1 (ROS1), we establish immune memory against biotrophic pathogens that persists for up to two weeks. Memory establishment coincides with reduced small RNA accumulation and DNA methylation along chromosome arms, revealing gene targets involved in salicylic acid-dependent immunity and DNA damage repair. These changes are progressively reversed during immune memory erasure. In contrast, transposon-rich pericentromeres retain elevated small RNA and DNA methylation levels, consistent with a CLASSY3-associated redistribution of RNA-directed DNA methylation. Our findings show that transient ROS1 activity drives contrasting epigenetic responses across chromosomal regions, shaping both the establishment and erasure of plant immune memory.

plant biology↗

bioRxiv2025

The mutational dynamics of the Arabidopsis centromeres

Centromeres are specialized chromosomal regions essential for sister chromatid cohesion and spindle attachment during cell division. Although many centromeres consist of highly variable tandem repeat arrays, the mutational processes driving this variability remain poorly understood. Here, using replicated genome assemblies of Arabidopsis thaliana mutation accumulation (MA) lines, we define the centromeric mutation spectrum. We find that point mutations occur at an almost tenfold higher rates than in chromosome arms, largely driven by non-allelic gene conversion between closely linked repeat units. Large kilobase-scale indels are also frequent and consistently preserve the tandem repeat array by adding or removing only complete repeat units. Analysis of MA lines deficient in the helicase RTEL1 supports the involvement of homology-directed DNA repair in these mutational processes. Moreover, simulations of sequence turnover using the centromere-specific mutation spectrum recapitulate the emergence of homogenized repeat blocks characteristic of natural centromeres. Together, our results show that centromere evolution is driven by a distinct mutational spectrum shaped by homology-directed DNA repair, providing a quantitative framework for how local mutational processes generate and maintain the large-scale architecture of centromeric DNA.

genomics↗

bioRxiv2025

Chemoselective Characterization of New Extracellular Matrix Deposition in Bioengineered Tumor Tissues

The extracellular matrix (ECM), present in nearly all tissues, provides extensive support to resident cells through structural, biomechanical, and biochemical means, and in return the ECM undergoes constant remodeling from interacting cells to adapt to the evolving tissue states. Bioengineered 3D tissues, commonly known as cell-ECM composites, are robust model systems to recapitulate and investigate native pathophysiology. Key to this engineered morphogenesis process are the intricate cell-ECM interactions reflected by how cells respond to and thereby modulate their surrounding microenvironments through their ongoing ECM secretome. However, investigating ECM-regulated new ECM production has been challenging due to the proteomic background from the pre-existing biomaterial ECM. To address this hindrance, here we present a chemoselective strategy to label, enrich, and characterize newly synthesized ECM (newsECM) proteins produced by resident cells, allowing distinction from the pre-existing ECM background. Applying our analytical pipeline to bioengineered tumor tissues, either built upon decellularized ECM (dECM-tumors) or as ECM-free tumor spheroids (tumoroids), we observed distinct ECM synthesis patterns that were linked to their extracellular environments. Tumor cells responded to the dECM presence with elevated ECM remodeling activities, mediated by augmented digestion of pre-existing ECM coupled with upregulated synthesis of tumor-associated ECM. Our findings highlight the sensitivity of newsECM profiling to capture remodeling events that are otherwise under-represented by bulk proteomics and underscore the significance of dECM support for enabling native-like tumor cell behaviors. We anticipate the described newsECM analytical pipeline to be broadly applicable to other tissue-engineered systems to probe ECM-regulated ECM synthesis and remodeling, both fundamental aspects of cell-ECM crosstalk in engineered tissue morphogenesis.

bioengineering↗

bioRxiv2025

Changes in free-roaming dog population demographics and health associated with a catch-neuter-vaccinate-release program in Jamshedpur, India

Indias large free-roaming dog populations contribute to significant human health, environmental, and social challenges. Population management strategies, such as capture-neuter-vaccinate-return (CNVR), aim to reduce dog numbers, improve their welfare, and reduce human-animal conflict. The Humane Society International, in partnership with the Animal Health Foundation, implemented a CNVR program in Jamshedpur, neutering and vaccinating over 20,000 dogs. This study evaluates the impact of this program on dog health, population structure and size. The study areas encompassed 10 sites within the Jamshedpur Metropolitan Region, including both intervention sites where CNVR was directly applied and sites without direct intervention. Data was collected from May 2014 until December 2018, including bi-annual street surveys, as well as clinical data from the dogs captured and treated. We fit logistic regression, negative binomial, and binomial mixed effects models to assess changes in dog population characteristics, health, and reproductive conditions over time in relation to the CNVR intervention. We found that, over time, the CNVR program significantly reduced the probability of dogs entering the clinic with mange, transmissible venereal tumours, and pregnant. Street surveys showed an increase in sterilised dogs, with higher proportions observed in CNVR-treated sites, although the counts of dogs observed increased overall. The age-structure of free-roaming dogs remained stable over time. In CNVR-treated areas, the probability of observing lactating female dogs decreased, whereas it increased in untreated sites. This work contributes to the growing body of knowledge investigating the impact of dog population management interventions. Continued monitoring and evaluation of CNVR programs are required to identify optimal coverage required to reduce population size effectively.

ecology↗

bioRxiv2024

Identification of Gene Targets for the Sprouting Inhibitor CIPC

Sprout suppressants are widely used in industry to ensure year-round availability of potato tubers, significantly decreasing wastage by repressing premature growth of buds on the tuber surface during storage. Despite its ban from 2020 in the EU, isopropyl N-(3- chlorophenyl) carbamate (also known as chlorpropham or CIPC) remains the most widely used suppressant world-wide. However, the mechanism of action of CIPC remains obscure. Here we report on a combined targeted transcriptomic and genetic approach to identify components in the tuber bud cell-division machinery that might be involved in CIPCs mode of action. This involved RNAseq analysis of dissected, staged tuber buds during in vitro sprouting with and without CIPC to identify lead genes, followed by the development and application of an Arabidopsis root assay to assess cell division response to CIPC in selected mutants. The ease of use of this model plant, coupled with its immense genetic resources, allowed us to test the functionality of lead genes encoding cell-division associated proteins in the modulation of plant growth response to CIPC. This approach led to the identification of a component of the augmin complex (a core player in mitosis) as a potential target for CIPC.

plant biology↗

bioRxiv2024

Improved detection of differentially abundant proteins through FDR-control of peptide-identity-propagation

The goal of proteomics is to identify and quantify peptides and proteins within a biological sample. Almost all algorithms for the identification of peptides in LC-MS/MS data employ two steps: peptide/spectrum matching and peptide-identity-propagation (PIP), also known as match-between-runs. PIP was originally envisioned as a backup method to overcome measurement stochasticity. However, current PIP implementations can routinely account for up to 40% of all results, with that proportion rising as high as 75% in single-cell proteomics. Unlike peptide identities derived through peptide/spectrum matches, for which error estimation has been strictly enforced for decades, peptide identities derived through PIP have not historically been subject to statistical evaluation. As an indispensable component of label free quantification, PIP needs a simple and statistically rigorous method for estimating its error rates. Although several tools claim to control the false discovery rate (FDR) of PIP, these claims cannot be validated as there is currently no accepted method to assess the accuracy of the stated FDR. We present a method for FDR control of PIP, called PIP-ECHO, and devise a rigorous protocol for evaluating FDR control of any PIP method. Using three different benchmark datasets, we evaluate PIP-ECHO alongside the PIP procedures implemented by FlashLFQ, IonQuant, and MaxQuant. These analyses show that only PIP-ECHO can accurately control the FDR of PIP at 1% across all datasets, including single cell. When analyzing spike-in datasets where different known amounts of yeast or E. coli peptides are added to a constant background of human peptides, PIP-ECHO increases both the accuracy and sensitivity of differential expression analysis, yielding 53% more differentially abundant proteins than MaxQuant and 146% more than IonQuant.

bioinformatics↗

bioRxiv2024

Alternative splicing modulates chromatin interactome and phase separation of the RIF1 C-terminal domain

How RIF1 (RAP1 interacting factor) fulfills its diverse roles in DNA double-strand break (DSB) repair, DNA replication, and nuclear organization remains elusive. Here we show that alternative splicing (AS) of a cassette exon (Ex32) encoding a Ser/Lys-rich (S/K) cassette in the RIF1 C-terminal domain (CTD) gives rise to RIF1-Long (RIF1-L) and RIF1-Short (RIF1-S) isoforms with different functional characteristics. We demonstrate that RIF1-Ex32 splice-in is mediated by an exonic splicing enhancer that is recognized by the splicing factor SRSF1 and antagonized by splicing inhibitors SRSF3 and SRSF7. Exposure to DNA damage inhibited Ex32 splice-in, potentiated the association of SRSF3 and SRSF7 with RIF1 pre-mRNA, and caused an increase in RIF1-S protein expression, which was also observed across a diverse set of primary cancers. Isoform-specific proteomic analyses revealed RIF1-L preferentially associated with mediator of DNA damage checkpoint 1 (MDC1) and sustained MDC1 focus formation to a greater extent than RIF1-S. We further show that the S/K cassette stabilized a novel phase separation activity of the RIF1 CTD and enhanced RIF1-L chromatin retention, which was reversed by CDK1-dependent phosphorylation of the RIF1 CTD in response to G2 DNA damage checkpoint inhibition. These combined findings suggest DNA damage-dependent RIF1 AS contributes to RIF1 functional diversification in genome protection.

molecular biology↗

bioRxiv2024

Discovery of dehydroamino acids and their crosslinks in Tau and other aggregating proteins of Alzheimers disease

BackgroundAlzheimers disease (AD) is characterized by accumulation of two types of protein aggregates, senile plaques and neurofibrillary tangles, which strongly contribute to the pathogenesis of the disease. Senile plaques consist primarily of aggregated amyloid-{beta}, while neurofibrillary tangles form via aggregation of the protein Tau, as well as other microtubule-associated proteins such as CRMP2. Posttranslational modifications have been hypothesized to contribute to the initial aggregation events that lead to SPs and NFTs. Dehydroamino acids (DHAAs) are posttranslational modifications rarely observed in humans and have not previously been reported in AD. DHAAs arise from the eliminylation of serine, threonine, or cysteine, yielding a double bond with distinct molecular geometry and reactivity. Their geometry can produce secondary structure rearrangements, such as those seen in senile plaque and neurofibrillary tangle formation, while their reactivity can cause intramolecular or intermolecular (protein-protein) crosslinking. We hypothesized that this modification might be present in protein aggregation-associated neurodegenerative disorders like AD. MethodsWe performed mass spectrometry-based bottom-up proteomics on the sarkosyl-insoluble (protein aggregate-enriched) material from ten AD brains and three age-matched controls. Identifications of DHAA-mediated crosslinked peptides were validated using both an isotopic labeling strategy and spike-in experiments employing synthetic crosslinked peptide standards. Similar findings were obtained in searches of publicly available proteomic datasets from AD and control brains. ResultsWe identified 412 sites of DHAA modification in 184 proteins, with the highest prevalence in the neurofibrillary tangle-forming proteins Tau and CRMP2. Comparison with results of previous protein aggregate interactomics studies show that proteins containing the DHAA modification are more highly associated with protein aggregates than are proteins containing any other individual posttranslational modification. We further observed 11 protein crosslinks arising from DHAAs, including three from the Tau protein. Label free quantification showed that Tau crosslinks are an order of magnitude more prevalent in AD samples than in age-matched controls. ConclusionsDehydroamino acids and their derivatives are prevalent modifications in the Alzheimers disease brain proteome. These modifications give rise to protein crosslinks which may contribute to protein aggregation processes.

biochemistry↗

bioRxiv2024

Multi-generational biotic stress increases the rate of spontaneous epimutations in a ROS1-dependent manner

Mistakes in the maintenance of CG methylation are a source of spontaneous epimutations in plants that can be inherited across generations. The extent to which these stochastic events are affected by prolonged exposure to biotic and abiotic stress remains poorly characterized. Here, we grew Arabidopsis Mutation Accumulation (MA) lines for 12 generations in the presence of two biotic stressors: Pseudomonas syringae (Pst) and salicylic acid (SA). We found that multi-generational exposure to Pst and SA led to an 18%-32% and 23%-61% increase in the genome-wide epimutation rate, respectively. These rate increases were mainly targeted to subsets of genes characterized by low steady-state methylation (LM), on average, and a lack of transcriptional responsiveness to stress. We show that these effects are mediated by the DNA demethylase Repressor of Silencing 1 (ROS1). Loss of ROS1 not only buffers transcriptional responses to biotic stress, but also stabilizes epimutation rates, particularly in LM genes, rendering them insensitive to environmental perturbations. Taken together, our data demonstrates that stress can induce heritable epimutations and highlights a ROS1-mediated link between transcriptional plasticity and DNA methylation maintenance fidelity over generations.

evolutionary biology↗

bioRxiv2023

Imaging the master regulator of the antioxidant response in non-small cell lung cancer with positron emission tomography

Mutations in the NRF2-KEAP1 pathway are common in non-small cell lung cancer (NSCLC) and confer broad-spectrum therapeutic resistance, leading to poor outcomes. The cystine/glutamate antiporter, system xc-, is one of the >200 cytoprotective proteins controlled by NRF2, which can be non-invasively imaged by (S)-4-(3-18F-fluoropropyl)--glutamate ([18F]FSPG) positron emission tomography (PET). Through genetic and pharmacologic manipulation, we show that [18F]FSPG provides a sensitive and specific marker of NRF2 activation in advanced preclinical models of NSCLC. We validate imaging readouts with metabolomic measurements of system xc- activity and their coupling to intracellular glutathione concentration. A redox gene signature was measured in patients from the TRACERx 421 cohort, suggesting an opportunity for patient stratification prior to imaging. Furthermore, we reveal that system xc- is a metabolic vulnerability that can be therapeutically targeted for sustained tumour growth suppression in aggressive NSCLC. Our results establish [18F]FSPG as predictive marker of therapy resistance in NSCLC and provide the basis for the clinical evaluation of both imaging and therapeutic agents that target this important antioxidant pathway.

cancer biology↗

bioRxiv2023

The Chicken Chorioallantoic Membrane as a Low-Cost, High-Throughput Model for Cancer Imaging

PurposeMouse models are invaluable tools for radiotracer development and validation. They are, however, expensive, low throughput, and are constrained by animal welfare considerations. Here, we assessed the chicken chorioallantoic membrane (CAM) as an alternative to mice for preclinical cancer imaging studies. MethodsGrowth of NCI-H460 Fluc tumors on the CAM was optimized using a range of physical and chemical supports. Tumor-bearing eggs were imaged by dynamic 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) or (4S)-4-(3-18F-fluoropropyl)-L-glutamate (18F-FSPG) PET/CT following intravenous injection, with mice bearing subcutaneous NCI-H460 Fluc xenografts imaged with 18F-FDG for comparison. The dependence of the transporter system xc- on in ovo 18F-FSPG tumor uptake was determined through treatment with imidazole ketone erastin. Additionally, 18F-FSPG PET/CT was used to monitor treatment response in ovo 24 h following external beam radiotherapy. ResultsNCI-H460 Fluc cells grown in Matrigel formed vascularized tumors of reproducible size without compromising embryo viability. By designing a simple method for cannulation it was possible to perform dynamic PET imaging in ovo, producing high tumor-to-background signal for both 18F-FDG and 18F-FSPG. 18F-FDG tumor uptake kinetics were similar in ovo and in vivo, with 18F-FSPG providing an early marker of both treatment response and target inhibition in CAM-grown tumors. ConclusionsThe CAM provides a low-cost alternative to tumor xenograft mouse models which may broaden access to PET and SPECT imaging. Rapid tumor growth and high-quality PET images that can be obtained with this model suggest its potential use for early radiotracer screening, pharmacological studies, and monitoring response to therapy.

cancer biology↗

bioRxiv2023

Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

molecular biology↗

bioRxiv2023

An evolutionary epigenetic clock in plants

Molecular clocks are the basis for dating the divergence between lineages over macro-evolutionary timescales (~105-108 years). However, classical DNA-based clocks tick too slowly to inform us about the recent past. Here, we demonstrate that stochastic DNA methylation changes at a subset of cytosines in plant genomes possess a clock-like behavior. This epimutation-clock is orders of magnitude faster than DNA-based clocks and enables phylogenetic explorations on a scale of years to centuries. We show experimentally that epimutation-clocks recapitulate known topologies and branching times of intra-species phylogenetic trees in the selfing plant A. thaliana and the clonal seagrass Z. marina, which represent two major modes of plant reproduction. This discovery will open new possibilities for high-resolution temporal studies of plant biodiversity.

evolutionary biology↗