A somatic genetic clock for clonal species

Age and longevity are key parameters for demography and life-history evolution of organisms. In clonal species, a widespread life history among animals, plants, macroalgae and fungi, the sexually produced offspring (genet) grows indeterminately by producing iterative modules, or ramets, and so obscure their age. Here we present a novel molecular clock based on the accumulation of fixed somatic genetic variation that segregates among ramets. Using a stochastic model, we demonstrate that the accumulation of fixed somatic genetic variation will approach linearity after a lag phase, and is determined by the mitotic mutation rate, without direct dependence on asexual generation time. The lag phase decreased with lower stem cell population size, number of founder cells for the formation of new modules, and the ratio of symmetric versus asymmetric cell divisions. We calibrated the somatic genetic clock on cultivated eelgrass Zostera marina genets (4 and 17 years respectively). In a global data set of 20 eelgrass populations, genet ages were up to 1,403 years. The somatic genetic clock is applicable to any multicellular clonal species where the number of founder cells is small, opening novel research avenues to study longevity and, hence, demography and population dynamics of clonal species.


Statistics
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The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g.means) or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g.Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.Filtering of the raw data -BBDuk (https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/).Mapping of short reads against reference genome -sequence reads were mapped against the chromosome-level reference genome of Zostera marina V3.1 using BWA MEM (Burrows-Wheeler Alignment Tool v0.1.17).
Calling of somatic genetic variation (SNPs -single nucleotide polymorphisms) -Mutect2 (GATK4 v4.1.1.0)-Strelka2 (strelka-2.9.2.centos6_x86_64) Mutational Spectra analysis -germline: population-wise SNPs were extracted from 11705 core SNPs from Yu et al.Nature Plants 2023 --somatic SNPs were extracted from the 4 oldest genets detected in this data set -mutational spectra were computed using the R-package Mutational.Patterns (no version, accessed Jan 2024) Calculating the variable VRF50(X1, X2) as proxy for fixed somatic genetic variation -custom-made scripts at https://github.com/leiyu37/SomaticGeneticClock.gitcreating maps (Fig. 4; Supplementary Fig. 15): https://www.qgis.org/en/site/For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Portfolio guidelines for submitting code & software for further information.

Data collection
Collectors are mentioned in the section on sampling permits.Sample extraction was performed at GEOMAR Kiel (Diana Gill and Lei Yu).DNA samples were sent to BGI Genomics (Hong Kong) for Illumina sequencing.
Timing and spatial scale Sampling for the previous population genomics project was conducted between May 2016 and August 2017.Estonian samples were collected in August 2021.At a given site, a population was defined as continuous eelgrass meadow of at least 50 m across (parallel to shore).Samples for the 4-yr-old clones were collected from the lab In 2022.

Data exclusions
SNPs not passing the filtering criteria were excluded.

Reproducibility
SNP calling: two independent SNP calling approches were used (STRELKA2, Mutect2) calibration of the somatic genetic clock: three (4-yr) and two (17-yr) old cultivated eelgrass genets (=clones) were used to obtain a calibration curve to age eelgrass genets (=clones) at other sites identification of key covariates: an agent based model was used to examine the effects of branching rate (thus asexual generation time), number of founder cells, stem cell population size and the ratio asymmetric vs symmetric cell division.Within the parameter space of the study species eelgrass, a significant deviation of the somatic genetic clock from linearity is unlikely Field work, collection and transport

Field conditions
As our study builds upon genome polymorphism and differentiation that was emerging over hundreds to thousands of years, no environmental data were collected at the time of sampling

Location
All 20 sampling locations were geo-referenced, coordinates are listed in Supplementary Data 1 and below Access & import/export For all sites, sampling permits have been obtained by the relevant national or regional authorities where required.An e-mail string can be provided upon request between the local collaborators and the respective national authorities (NFP) with respect to an obligation or waiver of CBD or general sampling permit.
Randomization no randomization was required as the study question addressed identical clone mates Blinding no blinding was required Did the study involve field work?Yes No Croatia /CZ /Pos 44.212N 15.491E.sampling: no collection permit required, waiver to Dr. Stewart Schulz & Dr. Claudia Kruschel.CBD-"Nagoya": non-signatory Disturbance At each site, in an area of several 1000 m2, some leaf shoots of eelgrass were collected, representing <0.001% of all plants of the respective meadow.This level of disturbance is negligible compared to, for example, natural physical disturbance by storms or herbivory Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.