Terrestrial-type nitrogen-fixing symbiosis between seagrass and a marine bacterium

Symbiotic N2-fixing microorganisms have a crucial role in the assimilation of nitrogen by eukaryotes in nitrogen-limited environments1–3. Particularly among land plants, N2-fixing symbionts occur in a variety of distantly related plant lineages and often involve an intimate association between host and symbiont2,4. Descriptions of such intimate symbioses are lacking for seagrasses, which evolved around 100 million years ago from terrestrial flowering plants that migrated back to the sea5. Here we describe an N2-fixing symbiont, ‘Candidatus Celerinatantimonas neptuna’, that lives inside seagrass root tissue, where it provides ammonia and amino acids to its host in exchange for sugars. As such, this symbiosis is reminiscent of terrestrial N2-fixing plant symbioses. The symbiosis between Ca. C. neptuna and its host Posidonia oceanica enables highly productive seagrass meadows to thrive in the nitrogen-limited Mediterranean Sea. Relatives of Ca. C. neptuna occur worldwide in coastal ecosystems, in which they may form similar symbioses with other seagrasses and saltmarsh plants. Just like N2-fixing microorganisms might have aided the colonization of nitrogen-poor soils by early land plants6, the ancestors of Ca. C. neptuna and its relatives probably enabled flowering plants to invade nitrogen-poor marine habitats, where they formed extremely efficient blue carbon ecosystems7.


Statistics
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Software and code
Policy information about availability of computer code Data collection Zeiss ZEN 3.2 blue edition, xT microscope control software v6.2.6.For the acquisition of raw mass spectrometric and raw sequencing data, instrument-supplied software was utilized.
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Raw reads of the 16S rRNA gene amplicon sequencing, the MAGs of C. neptuna and C. diazotrophica (DSM18577), and the mapped reads of the transcriptomes are available under Bioproject number PRJEB37438 at the European Nucleotide Archive (ENA).Sequences that were included in the phylogenetic tree are available in Supplementary File 1 (with accession numbers and references) and as a tree file (Supplementary File 2).The comparison of 34 genomes for presence/absence of specific genes and/or pathways is available in Supplementary File 3 including their accession numbers.The PhyloFlash results (as presented in Extended Data Fig. 2) are available in Supplementary File 4. Publicly available sequences used for phylogenetic tree construction and genome comparison can be found under their respective accession numbers at NCBI (https://www.ncbi.nlm.nih.gov/) or ENA (https://www.ebi.ac.uk/ena/browser/home).Ribosomal subunit databases used for taxonomic classification can be found at the SILVA rRNA database (https://www.arb-silva.de/).Source data are provided with this paper.

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Study description
We carried out several field sampling campaigns to Posidonia oceanica meadows in the Mediterranean Sea.Sampling and experiments were set up to i) measure in situ rates of primary production and O2 penetration into the sediment, ii) measure N2 fixation rates associated with the roots of the seagrass and the subsequent transfer of freshly fixed N, iii) to subsample these rate incubations to visualize N2-fixing microorganisms, and iv) to study their potential metabolism via sequencing of metagenomes and metatranscriptomes.This study was designed to obtain a mechanistic insight into the interactions of N2-fixing microorganisms with the P. oceanica plant.

Research sample
The seagrass Posidonia oceanica is one of the most prolific seagrasses, producing large amounts of biomass.Its growth in a nutrientpoor environment indicates that microbial N2 fixation is important for this ecosystem.Our prior work showed that N2 fixation was mostly associated with the roots of the seagrass.We therefore focused on studying the root-associated N2 fixation activity as well as the transfer of the newly fixed N to the leaves.Sediment, pore water and the overlying water column were also sampled to obtain environmental data relevant to our study.Fetovaia Bay (Elba, Italy) was chosen as a study site due to its pristine nature and the oligotrophic conditions representative of Posidonia meadows.Further, the study site is near a field station with sampling and laboratory infrastructure, which allowed repeated sampling and the completion of experiments.

Sampling strategy
The manuscript reports data from several field campaigns across different seasons and years.Sampling size was largely determined by the feasibility of experiments with triplicate incubated plants for each set of experiments and at least one experiment per sampling campaign.The three replicate plants for each experimental set is warranted by the various sampling campaigns across different seasons.

Data collection
Eddy correlation measurements were carried out in situ during 13-24 hour deployments.Diving staff of Hydra Marine Sciences, Hydra Field work and Soeren Ahmerkamp were present during the deployment and recovery of the instrumentation (high-frequency current meters combined with fast O2 microsensors; sediment microsensors).Water column and sediment O2 concentrations were taken automated during this deployment time.Plant and sediment samples were collected by staff of Hydra Marine Sciences and Hydra Field Work.Nutrient data (fluorometrically/photometrically) was collected either in the nearby laboratory on Elba Island, Italy, (collected by Nadine Lehnen, Hannah Marchant, Wiebke Mohr and/or technical support staff) or using an autoanalyzer at the Max Planck Institute Bremen (operated by technical support staff).Biomass and mass spectrometric data was collected using an elemental analyzer coupled to a continuous-flow isotope ratio mass spectrometer (equipped with an autosampler, operated by technical support staff), a nanoscale secondary ion mass spectrometer (operated by Sten Littmann and technical support staff), a gas chromatograph and a gas chromatograph coupled to an isotope ratio mass spectrometer (operated by technical support staff; Eawag).Sequencing data was collected at the Max Planck Genome Centre Cologne (individual sequencing platforms are detailed in the methods section).
Timing and spatial scale Field campaigns took place in June 2014, May 2015, April 2016, August 2016, May 2017, September 2018, June and September 2019.Different seasons were chosen to observe changes in microbial community and processes with a change from conditions where nutrients are available to nutrient-deplete conditions.The same seagrass meadow was visited during these campaigns (Fetovaia Bay, Elba), and an additional meadow at a different island (Cala della Ruta, Pianosa) was visited once in May 2017.Fetovaia Bay opens easterly/southeasterly to the Mediterranean Sea with a maximum North-South and West-East extent of ~700 m.Cala della Ruta Bay opens southerly to the Mediterranean and has a maximum North-South and West-East extent of about 400 and 700 m, respectively.

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March 2021

Data exclusions
We excluded the sequencing data of three plants from the microbial community analyses (16S rRNA gene amplicon sequencing) because they did not pass our initial quality assessment (described in methods).These three plants belonged the largest group of plants for one (non-N2-fixing) season (May).

Reproducibility
All experiments described in our study are individual environmental sampling campaigns at different times and/or different years.Within each set of experiments, triplicate incubations and multiple measurements within each triplicate (where applicable) were performed to assess variability.Variability within triplicates was substantial (see Fig. 1, 2 and Extended Data Fig. 3) reflecting biological differences between individual plants and plant pieces rather than errors in measurements.All attempts at replication were successful.

Randomization
The plants were randomly collected by divers in accessible spots and considering to minimize potential damage to the seagrass meadow.Incubation plants were randomly chosen from the pool of sampled plants making sure that each incubated plant had sufficient root, rhizome and leaf material for subsequent measurements.

Blinding
Blinding was not pertinent to our study because it did not include any animals and/or human research participants.In addition, blinding was not possible since many analyses were also carried out by the persons in charge of sampling and interpretation of the data was done by persons in charge of analyses.
Did the study involve field work?Access & import/export The Posidonia oceanica meadows were accessed using minimal-invasive zodiacs/small boats and SCUBA-diving/snorkling. Local, national and international laws were followed.Access to the protected waters of the Island of Pianosa was granted by the National Park Tuscan Archipelago, Portoferraio, Italy (permit no.2930/2017).

Disturbance
Plants were carefully separated from the meadow by hand to minimize damage/disturbance to the ecosystem.In situ measurements were minimally invasive.
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Materials
Nutrient concentrations in the water column above the seagrass meadow and in porewaters of seagrass and neighboring sediments were measured during several campaigns and are detailed in Extended Data Table1.