Functional diversity enables multiple symbiont strains to coexist in deep-sea mussels

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Abstract

Genetic diversity of closely related free-living microorganisms is widespread and underpins ecosystem functioning, but most evolutionary theories predict that it destabilizes intimate mutualisms. Accordingly, strain diversity is assumed to be highly restricted in intracellular bacteria associated with animals. Here, we sequenced metagenomes and metatranscriptomes of 18 Bathymodiolus mussel individuals from four species, covering their known distribution range at deep-sea hydrothermal vents in the Atlantic. We show that as many as 16 strains of intracellular, sulfur-oxidizing symbionts coexist in individual Bathymodiolus mussels. Co-occurring symbiont strains differed extensively in key functions, such as the use of energy and nutrient sources, electron acceptors and viral defence mechanisms. Most strain-specific genes were expressed, highlighting their potential to affect fitness. We show that fine-scale diversity is pervasive in Bathymodiolus sulfur-oxidizing symbionts, and hypothesize that it may be widespread in low-cost symbioses where the environment, rather than the host, feeds the symbionts.

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Fig. 1: Overview of the workflow developed for this study.
Fig. 2: The population genetic measures π and FST show that mussels from the same site host similar symbiont populations.
Fig. 3: Gene version reconstruction reveals up to 16 co-occurring SOX symbiont strains in individual Bathymodiolus mussels.
Fig. 4: Strain number estimates from PacBio sequencing confirm the strain estimation approach from gene version reconstruction of Illumina sequences.
Fig. 5: Strain-specific genes encode potential key functions in SOX symbionts, including energy production and interactions with hosts and phages.
Fig. 6: Simultaneous FISH of key genes of the hydrogenase operon and 16S rRNA of the SOX symbiont in gill tissue of B. azoricus from Lucky Strike.

Data availability

Sequence data were deposited in the European Nucleotide Archive92 using the data brokerage service of the German Federation for Biological Data93, in compliance with the Minimal Information about any (x) Sequence standard94. All metagenomic sequencing reads and symbiont bins used in this study can be found at the European Nucleotide Archive under project accession number PRJEB32787, and all metatranscriptomic reads are under project accession number PRJEB32788. Any further data that support the findings of this study are available from the corresponding author upon request.

Code availability

Custom code and detailed information on the computing steps are available from the GitHub repository (https://github.com/rbcan/MARsym_paper). The code used to calculate the nucleotide diversity, π, and the fixation index, FST, is available from the GitHub repository at https://github.com/deropi/BathyBrooksiSymbionts/tree/master/Population_structure_analyses.

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Acknowledgements

We thank the captains, crews and remotely operated underwater vehicle teams on the cruises BioBaz (2013), ODEMAR (2014), M78-2 (2009) and Atalante Cruise Leg 2 (2008) on board the research vessels Pourquoi Pas?, FS Meteor and L’Atalante, as well as the chief scientists F. Lallier, J. Excartin and M. Andreani, and R. Seifert and C. Devey. We are grateful to M. Meyer for de novo production of the geneFISH probes, to A. Assié, C. Borowski, C. Breusing and K. van der Heijden for sample collection and fixation on board, and to M. Tietjen for the extraction of RNA from the samples of the vent fields Semenov, Clueless and Lilliput. We also thank C. Quast and H. Teeling for technical support, as well as T. Dagan for discussions and input during the project and on the written manuscript. This study was funded by the Max Planck Society, an ERC Advanced Grant (BathyBiome, 340535), a Gordon and Betty Moore Foundation Marine Microbial Initiative Investigator Award to N.D. (grant GBMF3811), the MARUM DFG Research Center/Excellence Cluster ‘The Ocean in the Earth System’ at the University of Bremen, the DFG CRC 1182 ‘Origin and Function of Metaorganisms’ and the German Research Foundation (RV Meteor M78-2 cruise).

Author information

R.A., J.P., L.S. and N.D. conceived the study. R.A. and J.P. wrote the manuscript, with support from N.D. and contributions and revisions from all other co-authors. R.A. developed the metagenomic workflow for polymorphism detection, strain reconstruction and the identification of strain-specific genes, and analysed the data with the exceptions described hereafter. S.R. conducted the core-genome calculation, read simulation analyses, provided support for the statistical analyses and drafted respective manuscript sections. L.S. extracted nucleic acids for samples from Lucky Strike, Semenov and Wideawake, and conducted and evaluated the PacBio assembly. M.A.G.P. designed the geneFISH probes, produced geneFISH images on gill tissue sections, and drafted the respective sections in the manuscript. A.K. developed and provided an R script for the calculation of π and FST. H.E.T. sequenced the metagenomes from the vent fields Clueless and Lilliput.

Correspondence to Nicole Dubilier or Jillian Petersen.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Number of SNPs in the symbiont 16S rRNA gene within individual hosts at the four vent fields.

The position on the 16S rRNA gene, SNP frequencies and nucleotide changes are indicated in light grey boxes. The following host species correspond to listed vent fields: Lucky Strike – B. azoricus, Semenov – B. puteoserpentis, Clueless – B. sp., Lilliput – B. sp.

Extended Data Fig. 2 Single nucleotide polymorphisms (SNPs) of within-host symbiont populations.

SNPs/kbp are shown in (a) core genes and (b) whole genomes including non-coding regions. Vent fields (host species) are LS: Lucky Strike (B. azoricus), Se: Semenov (B. puteoserpentis), Cl: Clueless (B. sp.), Li: Lilliput (B. sp.).

Extended Data Fig. 3 Counts and percentages of low-coverage genes in within-host symbiont populations.

Subsets represent counts that excluded all genes annotated as “hypothetical protein” and are further defined as strain- specific genes (one-copy genes with lower coverage in most or all symbiont populations from that site) and low-coverage genes with further copies in the genome. The following host species correspond to listed vent fields: Lucky Strike – B.azoricus, Semenov – B. puteoserpentis, Clueless – B. sp., Lilliput – B. sp.

Extended Data Fig. 4 Theoretical model predicting the influence of symbiont transmission on population genomic signatures (π, FST).

On the left a scenario is depicted in which the symbionts are acquired only once by juvenile mussels during a restricted time window, followed exclusively by repeated self-infection. On the right a scenario is depicted in which the symbionts are continuously released and taken up by host individuals throughout their lifetime.

Extended Data Fig. 5 π-values within symbiont populations of single host individuals and in pairwise calculation between each two host individuals from all four vent fields.

(a) Each within-host π value (white) per individual and between-host π value (colored) per pair of individuals is shown separately. NLS = 3320, NSe = 2,435, NCl = 3,023, NLi = 3,352 genes for which a π value was calculated. (b) Within-host (white) and between-host (colored) π values are grouped together, respectively. LS: Lucky Strike, Se: Semenov, Cl: Clueless, Li: Lilliput vent fields. In the box-plots, line represents mean, upper and lower hinges represent the first (25th percentile) and third (75th percentile) quartiles, whiskers represent 1.5x interquartile range, individual points are outliers. NLS = 16,600 (white) / 33,200 (colored), NSe = 7,305 (both, white and colored), NCl = 15,115 (white) / 30,230 (colored), NLi = 16,760 (white) / 33,520 (colored) genes for which a π value was calculated. Source data

Extended Data Fig. 6 Spearman correlation between the difference (a) or sum (b) in shell lengths of two compared hosts with the pairwise FST; and correlation of shell length with intra-host SNP density (c).

= spearmans correlation coefficient rho, p = p-value (two- sided); for vent field Lucky Strike white symbols = host pairs from different sites Eiffel Tower and Montsegur, red symbols = host pairs from the same site; LS: Lucky Strike, Se: Semenov, Cl: Clueless, Li: Lilliput. For a and b NLS = 10, NSe = 3, NCl = 10, NLi = 10 pairwise FST between each two per-mussel symbiont populations; for c NLS = 5, NSe = 3, NCl = 5, NLi = 5 per-mussel symbiont populations. Semenov did not have enough data to infer values for r and p. Source data

Extended Data Fig. 7 Cumulative gene counts of distinct numbers of gene versions.

Cumulative gene version count is shown for gammaproteobacterial marker genes from PhylaAmphora and the extended set of genes that had a read coverage within the coverage range of gammaproteobacterial marker genes, indicating that each strain in the population encoded these. Strain numbers were estimated for the marker gene set with 100x read coverage (a) and full read coverage (b) and for the entire gene set with full read coverage per host individual. Full read coverage was 100–120x for LS, 280–370x for Se, 150–215x for Cl and 190–218x for Li mussels. LS: Lucky Strike, Se: Semenov, Cl: Clueless, Li: Lilliput, cov = coverage. Source data

Extended Data Fig. 8 Comparison of reported SNP densities from published studies to our present dataset.

Data from Bathymodiolus SOX symbionts of this study are depicted in bold. Many of the reported values were previously summed up in Wilmes et al.95. NA = information was missing or could not be retrieved96,97,98,99,100,101,102,103,104,105.

Extended Data Fig. 9 Representation of denitrification genes among strains of the SOX symbiont.

A gene is absent (white mussel symbols), strain specific (blue mussel symbols), or present in all strains (red mussel symbols) in a single host individual. When some host individuals from the same vent site had symbiont populations where a gene is strain specific and others where the entire population encoded that gene, mussel symbol is split into red and blue color. LS: Lucky Strike (B. azoricus), Se: Semenov (B. puteoserpentis), Cl: Clueless (B. sp.), Li: Lilliput (B. sp.), NAR: respiratory nitrate reductase, NIR: nitrite reductase, NOR: nitric oxide reductase, NOS: nitrous oxide reductase, NarK: nitrate transporter, NAS A: assimilatory nitrate reductase.

Extended Data Fig. 10 Representation of CRISPR-Cas gene clusters in the SOX symbiont strains showing strain- specific genes (blue), genes present in all strains (red), and CRISPR- arrays (striped boxes).

LS: Lucky Strike, Se: Semenov, Cl: Clueless, Li: Lilliput.

Supplementary information

Supplementary Information

Supplementary Discussion, Supplementary Tables 1–5 and 7–11, and Supplementary Figs. 1–5.

Reporting Summary

Supplementary Table 6

List of strain-specific genes, excluding all hypothetical proteins.

Source data

Source Data Fig. 2

Pi values per gene used in Permanova analysis and for Fig. 2a, as well as FST values used in Fig. 2b.

Source Data Fig. 3

Source data for cumulative density plots and maximum strain numbers of 100× coverage (Fig. 3a) and source data for correlation of strain count with coverage (Fig. 3b).

Source Data Fig. 4

Cumulative density data of strain counts for PacBio and Illumina Data for vent field Wideawake.

Source Data Extended Data Fig. 5

Pi values per gene plotted in Extended Data Fig. 5.

Source Data Extended Data Fig. 6

Source Data for correlation analysis of FST and SNP counts with mussel size, size sum and size differences.

Source Data Extended Data Fig. 7

Source data for cumulative density plots and maximum strain numbers of full coverage, and phylaAmphora marker genes in full and 100× coverage.

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Ansorge, R., Romano, S., Sayavedra, L. et al. Functional diversity enables multiple symbiont strains to coexist in deep-sea mussels. Nat Microbiol (2019) doi:10.1038/s41564-019-0572-9

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