All multicellular organisms are genetic mosaics owing to somatic mutations. The accumulation of somatic genetic variation in clonal species undergoing asexual (or clonal) reproduction may lead to phenotypic heterogeneity among autonomous modules (termed ramets). However, the abundance and dynamics of somatic genetic variation under clonal reproduction remain poorly understood. Here we show that branching events in a seagrass (Zostera marina) clone or genet lead to population bottlenecks of tissue that result in the evolution of genetically differentiated ramets in a process of somatic genetic drift. By studying inter-ramet somatic genetic variation, we uncovered thousands of single nucleotide polymorphisms that segregated among ramets. Ultra-deep resequencing of single ramets revealed that the strength of purifying selection on mosaic genetic variation was greater within than among ramets. Our study provides evidence for multiple levels of selection during the evolution of seagrass genets. Somatic genetic drift during clonal propagation leads to the emergence of genetically unique modules that constitute an elementary level of selection and individuality in long-lived clonal species.
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The DNA sequence data are available in the NCBI short read archive, BioProject no. PRJNA557092, SRA accession no. SRR9879327- SRR9879353. The data on genes and putative gene functions and on SNP verification are included in the supplementary tables. If not included in the supplementary material, the data visualized in Figs. 3–5 are deposited in PANGAEA (ref. 72).
The custom-made computer code is available on Github at https://github.com/leiyu37/Finnish_eelgrass_milleniumClone.
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This study was supported by a four-year PhD scholarship from the China Scholarship Council to L.Y. and by a fellowship from the Åbo Akademi University Foundation to C.B. We thank J. L. Olsen and B. Werner for helpful comments on earlier versions of the manuscript and in particular I. Baums for discussing methods for sequencing-independent SNP verification via restriction enzyme digestion. We thank the Archipelago Centre Korpoström (Finland) for excellent working facilities, K. Gagnon for field assistance and S. Landis for creating some of the illustrations. Sampling permit no. MH 5448/2015 was granted through Parks and Wildlife Finland (Metsähallitus).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Somatic genetic drift in clonal organisms and possible categories of single nucleotide polymorphisms (SNPs).
Multicellular organisms originate from a single zygote and achieve growth and development via mitosis. The diagram indicates possible genotypes at a locus within the ramet (=box). Heterozygous polymorphisms present in the zygote (blue) are inherited in all offspring cells, and result in consistent heterozygous calls across all ramets. This class of SNPs needs to be excluded before making inferences on somatically derived polymorphisms (cf. Extended Data Fig. 2, ‘Remove 31,777 SNPs that are identically heterozygous among all 24 ramets’). During growth, cells acquire somatic mutations (red) owing to mitotic errors that initially emerge as genetic mosaics, that is the somatic polymorphisms are present in only a subset of cells. When clonal organisms form another iterative unit (=ramet), a subpopulation of cells in the parent ramet are progenitors for the asexual offspring. The genetic bottleneck accompanying this random sampling process determines the segregation of somatic polymorphisms into the new ramets through somatic genetic drift. This process may convert intra-ramet SNP into different fixed ramet genotypes (cf. Fig. 5d). In case 1, somatic genetic drift restores the wild-type genotype present in the zygote. In cases 2, somatic genetic drift separates the intra-ramet SNP at that locus into a fixed state. In case 3, a novel mutation emerges as an intra-ramet SNP, which would require additional rounds of somatic genetic drift to either being fixed or lost.
All 24 ramets were sequenced at an average coverage of 81x on an Illumina Hiseq 4000 platform (left, for details see Supplementary Table 3), while a subset of three ramets (R08, R10, and R12) were further sequenced at an average coverage of 1370x on Novaseq 6000 platform (right, for details see Supplementary Table 8). The filtered 81x dataset was used to (i) estimate the number of heterozygous loci in the initial zygote in a conservative way with respect to not underestimate the true nucleotide diversity, (ii) call fixed SNPs among ramets, and to (iii) identify SNPs that were genetically different between the reference genome and the initial zygote. Here, ‘informative’ SNPs with respect to a somatic generation of polymorphism refer to all SNPs but those homozygously different to the reference. Informative SNPs were used to verify the clonality of the 24 samples, and to make inferences on somatic mutations after subtracting the identical heterozygous loci shared among R01-24. The 1370x dataset was used to call intra-ramet somatic polymorphisms (composed of both, fixed heterozygous genotypes and mosaic ones with f < 0.5), which allowed us to check the proportion of mosaic intra-ramet SNPs vs. fixed heterozygous genotypes. In the 81x data set, custom filtering steps (hard filtering) using the GATK pipeline with conservative parameters intended to minimize the error of detecting false positive SNP calls. During subsequent custom filtering using the Vcftools, identical homozygous SNPs with respect to the reference were removed. In the ultra-deep sequenced data set, each of the three SNP calling approaches were technically independent and thus verified. Note that for the intra-ramet level, the analysis focused on SNPs that were co-occurring in two or three ramets.
Extended Data Fig. 3 Neighbor-joining tree using insertion/deletion (indel) based genetic differences among 24 seagrass Zostera marina ramets.
The 1,654 inter-ramet indel polymorphisms among the 24 ramets were used to quantify the pairwise genetic differences as number of different alleles. The genetic distance matrix was used to construct a neighbor-joining tree, which displays a similar topology to that revealed by inter-ramet SNPs (Fig. 2d). Scale bar = 50 differences.
We genotyped all ramets for 8 microsatellite loci (Genbank accession numbers: AJ009904.1, AJ009901.1, AJ249304.1, AJ249306.1, AJ009898.1, AJ009900.1, AJ249307.1, and AJ249305.1) and detected a total of 7 somatic polymorphisms as microsatellite alleles not present in the predominant genotype. Their presence/absence among the 24 ramets is consistent with the NJ tree based on 7,054 SNPs (Fig. 2d) and 1,652 indels (Extended Data Fig. 3). Two polymorphisms were present in more than one ramet and their appearance within one branch is indicated by the microsatellite locus designation. Small insets show two identified three-allelic, mosaic genotypes with novel microsatellite alleles that were only present in one ramet each.
Extended Data Fig. 5 Dirichlet clustering of variant read frequency of SNPs shared by 3 ramets based on 1370x resequencing.
The 2,246 SNPs shared among all three ramets were filtered by a minimum coverage of 500x after which 1,768 SNPs were retained. These were divided into clusters using a Dirichlet process. All pairwise VRF among ramets R08, R10 and R12 of the resulting six Dirichlet clusters (top number) are depicted. Each panel was divided into grids, a color scale depicts the density of points in each grid point representing SNP counts. Lines indicate expected allele frequencies for fixed heterozygote SNPs at x = 0.5 and y = 0.5, respectively, while the line x = y depicts equal allele frequencies among ramets. a, VRF in R08 vs. R10. b, VRF in R12 vs. R10. c, VRF in R08 vs. R12.
Ramets are physiologically independent and genetically unique ‘individuals’. Somatic mutations emerge as intra-ramet SNPs. Along with growth and branching of the genet, somatic genetic drift separates the intra-ramet SNP into different fixed genotypes among ramets (case 1a or 1b), while in others, they may persist as mosaic, intra-ramet polymorphism (case 2). Note that ramets quickly become physiologically independent, as the rhizome connection among them will rot away within one or two years (case 3).
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Yu, L., Boström, C., Franzenburg, S. et al. Somatic genetic drift and multilevel selection in a clonal seagrass. Nat Ecol Evol 4, 952–962 (2020). https://doi.org/10.1038/s41559-020-1196-4
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