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The recognition that all macroorganisms live in symbiotic association with microbial communities has opened up a new field in biology. Animals, plants, and algae are now considered holobionts, complex ecosystems consisting of the host, the microbiota, and the interactions among them. Accordingly, ecological concepts can be applied to understand the host-derived and microbial processes that govern the dynamics of the interactive networks within the holobiont. In marine systems, holobionts are further integrated into larger and more complex communities and ecosystems, a concept referred to as "nested ecosystems." In this review, we discuss the concept of holobionts as dynamic ecosystems that interact at multiple scales and respond to environmental change. We focus on the symbiosis of sponges with their microbial communities—a symbiosis that has resulted in one of the most diverse and complex holobionts in the marine environment. In recent years, the field of sponge microbiology has remarkably advanced in terms of curated databases, standardized protocols, and information on the functions of the microbiota. Like a Russian doll, these microbial processes are translated into sponge holobiont functions that impact the surrounding ecosystem. For example, the sponge-associated microbial metabolisms, fueled by the high filtering capacity of the sponge host, substantially affect the biogeochemical cycling of key nutrients like carbon, nitrogen, and phosphorous. Since sponge holobionts are increasingly threatened by anthropogenic stressors that jeopardize the stability of the holobiont ecosystem, we discuss the link between environmental perturbations, dysbiosis, and sponge diseases. Experimental studies suggest that the microbial community composition is tightly linked to holobiont health, but whether dysbiosis is a cause or a consequence of holobiont collapse remains unresolved. Moreover, the potential role of the microbiome in mediating the capacity for holobionts to acclimate and adapt to environmental change is ...

Marine sponges are ancient metazoans that are populated by distinct and highly diverse microbial communities. In order to obtain deeper insights into the functional gene repertoire of the Mediterranean sponge Aplysina aerophoba, we combined Illumina short-read and PacBio long-read sequencing followed by un-targeted metagenomic binning. We identified a total of 37 high-quality bins representing 11 bacterial phyla and two candidate phyla. Statistical comparison of symbiont genomes with selected reference genomes revealed a significant enrichment of genes related to bacterial defense (restriction-modification systems, toxin-antitoxin systems) as well as genes involved in host colonization and extracellular matrix utilization in sponge symbionts. A within-symbionts genome comparison revealed a nutritional specialization of at least two symbiont guilds, where one appears to metabolize carnitine and the other sulfated polysaccharides, both of which are abundant molecules in the sponge extracellular matrix. A third guild of symbionts may be viewed as nutritional generalists that perform largely the same metabolic pathways but lack such extraordinary numbers of the relevant genes. This study characterizes the genomic repertoire of sponge symbionts at an unprecedented resolution and it provides greater insights into the molecular mechanisms underlying microbial-sponge symbiosis. ; This project was funded by Grant Number CSP1291 of the DOE Joint Genome Institute, the European Commission within its FP7 Program, under the thematic area KBBE.2012.3.2.-01 with Grant Number 311932 ('SeaBio-Tech'), and by the European Union's Horizon 2020 research and innovation program under Grant Agreement No. 679849 ('SponGES'). BMS was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg. We thank Monika Bright (University of Vienna) and the Marine Biology Station Piran for support during sampling, Tanja Woyke (DOE Walnut Creek, CA, USA) for helpful discussions on ...

ABSTRACT. Establishment of adequate conservation areas represents a challenging but crucial task in the conservation of genetic diversity and biological variability. Anthropogenic pressures on marine ecosystems and organisms are steadily increasing. Whether and to what extent these pressures influence marine genetic biodiversity is only starting to be revealed. Using 16S rRNA gene amplicon sequencing, we analysed the microbial community structure of 33 individuals of the habitat-forming glass sponge Vazella pourtalesii, as well as reference seawater, sediment, and biofilm samples. We assessed how two anthropogenic impacts, i.e. habitat destruction by trawling and artificial substrate provision (moorings made of composite plastic), correspond with in situ V. pourtalesii microbiome variability. In addition, we evaluated the role of two bottom fishery closures in preserving sponge-associated microbial diversity on the Scotian Shelf, Canada. Our results illustrate that V. pourtalesii sponges collected from protected sites within fishery closures contained distinct and taxonomically largely novel microbial communities. At the trawled site we recorded significant quantitative differences in distinct microbial phyla, such as a reduction in Nitrospinae in the four sponges from this site and the environmental references. Individuals of V. pourtalesii growing on the mooring were significantly enriched in Bacteroidetes, Verrucomicrobia and Cyanobacteria in comparison to sponge individuals growing on the natural seabed. Due to a concomitant enrichment of these taxa in the mooring biofilm, we propose that biofilms on artificial substrates may 'prime' sponge-associated microbial communities when small sponges settle on such substrates. These observations likely have relevant management implications when considering the increase of artificial substrates in the marine environment, e.g., marine litter, off-shore wind parks, and petroleum platforms. ; DATA AVAILABILITY. Sample metadata were deposited in the Pangaea database: ...

ABSTRACT Most animals, including sponges (Porifera), have species‐specific microbiomes. Which genetic or environmental factors play major roles structuring the microbial community at the intraspecific level in sponges is, however, largely unknown. In this study, we tested whether geographic location or genetic structure of conspecific sponges influences their microbial assembly. For that, we used three sponge species with different rates of gene flow, and collected samples along their entire distribution range (two from the Mediterranean and one from the Southern Ocean) yielding a total of 393 samples. These three sponge species have been previously analysed by microsatellites or single nucleotide polymorphisms, and here we investigate their microbiomes by amplicon sequencing of the microbial 16S rRNA gene. The sponge Petrosia ficiformis, with highly isolated populations (low gene flow), showed a stronger influence of the host genetic distance on the microbial composition than the spatial distance. Host‐specificity was therefore detected at the genotypic level, with individuals belonging to the same host genetic cluster harbouring more similar microbiomes than distant ones. On the contrary, the microbiome of Ircinia fasciculata and Dendrilla antarctica ‐ both with weak population structure (high gene flow) ‐ seemed influenced by location rather than by host genetic distance. Our results suggest that in sponge species with high population structure, the host genetic cluster influence the microbial community more than the geographic location. ; ACKNOWLEDGEMENTS This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie Individual Fellowships Grant Agreement No. 796011 ('DeepSym'), and Grant Agreement No. 679849 ('SponGES'). We thank the team around Andre Franke at the Institute of Clinical Molecular Biology, Kiel, for sequencing support. We also want to thank Shaun Nielsen for support and guidance through data analysis. Finally, we ...

ABSTRACT. Deep-sea sponge grounds are underexplored ecosystems that provide numerous goods and services to the functioning of the deep-sea. This study assessed the prokaryotic diversity in embryos, recruits, and adults of Craniella zetlandica and Craniella infrequens, common and abundant representatives of deep-sea sponge grounds in the North Atlantic. Our results reveal that symbiont transmission in the two Craniella sponge species likely occurs vertically, as highly similar microbial consortia have been identified in adults, embryos, and recruits. Moreover, transmission electron microscopy revealed high abundances of sponge-associated microorganisms, among which Chloroflexi (SAR202) were identified as common representatives by amplicon sequencing and fluorescence in situ hybridization (FISH). Equal diversity metrices, a similar overall prokaryotic community composition and a distinct dominance of the phylum Chloroflexi within all life stages are the key findings of our analyses. Information such as presented here provide understanding on the recruitment of deep-sea sponge holobionts which is needed to develop integrated management tools of such vulnerable marine ecosystems. ; FUNDING. This study was funded by the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 679849 (the SponGES project). This document reflects only the authors' view and the Executive Agency for Small and Medium-sized Enterprises (EASME) is not responsible for any use that may be made of the information it contains. ACKNOWLEDGEMENTS. We express our gratitude to HR, coordinator of the H2020-SponGES project on deep-sea sponge grounds of the North Atlantic, deep-sea sponge taxonomist, explorer, mentor, and friend. We thank the crews and scientific parties of RV G.O.Sars cruises "GS2017110" and "GS2018108". On these cruises the ROV AEgir-team (University of Bergen) enabled sampling of precious samples. Ina Clefsen and Andrea Hethke provided valuable support in the lab back on land. We also thank Heikki ...

ABSTRACT Members of the widespread bacterial phylum Chloroflexi can dominate high-microbial-abundance (HMA) sponge microbiomes. In the Sponge Microbiome Project, Chloroflexi sequences amounted to 20 to 30% of the total microbiome of certain HMA sponge genera with the classes/clades SAR202, Caldilineae, and An- aerolineae being the most prominent. We performed metagenomic and single- cell genomic analyses to elucidate the functional gene repertoire of Chloroflexi symbionts of Aplysina aerophoba. Eighteen draft genomes were reconstructed and placed into phylogenetic context of which six were investigated in detail. Common genomic features of Chloroflexi sponge symbionts were related to central energy and carbon converting pathways, amino acid and fatty acid metabolism, and respiration. Clade-specific metabolic features included a massively expanded genomic repertoire for carbohydrate degradation in Anaerolineae and Caldilineae ge- nomes, but only amino acid utilization by SAR202. While Anaerolineae and Caldilin- eae import cofactors and vitamins, SAR202 genomes harbor genes encoding compo- nents involved in cofactor biosynthesis. A number of features relevant to symbiosis were further identified, including CRISPR-Cas systems, eukaryote-like repeat proteins, and secondary metabolite gene clusters. Chloroflexi symbionts were visualized in the sponge extracellular matrix at ultrastructural resolution by the fluorescence in situ hybridization-correlative light and electron microscopy (FISH-CLEM) method. Carbo- hydrate degradation potential was reported previously for "Candidatus Poribacteria" and SAUL, typical symbionts of HMA sponges, and we propose here that HMA sponge symbionts collectively engage in degradation of dissolved organic matter, both labile and recalcitrant. Thus, sponge microbes may not only provide nutrients to the sponge host, but they may also contribute to dissolved organic matter (DOM) recycling and primary productivity in reef ecosystems via a pathway termed the sponge loop. I M P O R T A N C E ...

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. ; Most animals, including sponges (Porifera), have species-specific microbiomes. Which genetic or environmental factors play major roles structuring the microbial community at the intraspecific level in sponges is, however, largely unknown. In this study, we tested whether geographic location or genetic structure of conspecific sponges influences their microbial assembly. For that, we used three sponge species with different rates of gene flow, and collected samples along their entire distribution range (two from the Mediterranean and one from the Southern Ocean) yielding a total of 393 samples. These three sponge species have been previously analysed by microsatellites or single nucleotide polymorphisms, and here we investigate their microbiomes by amplicon sequencing of the microbial 16S rRNA gene. The sponge Petrosia ficiformis, with highly isolated populations (low gene flow), showed a stronger influence of the host genetic distance on the microbial composition than the spatial distance. Host-specificity was therefore detected at the genotypic level, with individuals belonging to the same host genetic cluster harbouring more similar microbiomes than distant ones. On the contrary, the microbiome of Ircinia fasciculata and Dendrilla antarctica - both with weak population structure (high gene flow) - seemed influenced by location rather than by host genetic distance. Our results suggest that in sponge species with high population structure, the host genetic cluster influence the microbial community more than the geographic location. ; European Union's Horizon 2020 Research and Innovation Actions, Grant/Award Number: 679849; Marie Skłodowska-Curie Individual Fellowships, Grant/Award Number: 796011

ABSTRACT. Few studies have explored the microbiomes of glass sponges (Hexactinellida). The present study seeks to elucidate the composition of the microbiota associated with the glass sponge Vazella pourtalesii and the functional strategies of the main symbionts. We combined microscopic approaches with metagenome-guided microbial genome reconstruction and amplicon community profiling toward this goal. Microscopic imaging revealed that the host and microbial cells appeared within dense biomass patches that are presumably syncytial tissue aggregates. Based on abundances in amplicon libraries and metagenomic data, SAR324 bacteria, Crenarchaeota, Patescibacteria, and Nanoarchaeota were identified as abundant members of the V. pourtalesii microbiome; thus, their genomic potentials were analyzed in detail. A general pattern emerged in that the V. pourtalesii symbionts had very small genome sizes, in the range of 0.5 to 2.2 Mb, and low GC contents, even below those of seawater relatives. Based on functional analyses of metagenome-assembled genomes (MAGs), we propose two major microbial strategies: the "givers", namely, Crenarchaeota and SAR324, heterotrophs and facultative anaerobes, produce and partly secrete all required amino acids and vitamins. The "takers," Nanoarchaeota and Patescibacteria, are anaerobes with reduced genomes that tap into the microbial community for resources, e.g., lipids and DNA, likely using pilus-like structures. We posit that the existence of microbial cells in sponge syncytia together with the low-oxygen conditions in the seawater environment are factors that shape the unique compositional and functional properties of the microbial community associated with V. pourtalesii. IMPORTANCE. We investigated the microbial community of V. pourtalesii that forms globally unique, monospecific sponge grounds under low-oxygen conditions on the Scotian Shelf, where it plays a key role in its vulnerable ecosystem. The microbial community was found to be concentrated within biomass patches and is dominated ...

ABSTRACT. Seamounts represent ideal systems to study the influence and interdependency of environmental gradients at a single geographic location. These topographic features represent a prominent habitat for various forms of life, including microbiota and macrobiota, spanning benthic as well as pelagic organisms. While it is known that seamounts are globally abundant structures, it still remains unclear how and to which extent the complexity of the sea floor is intertwined with the local oceanographic mosaic, biogeochemistry, and microbiology of a seamount ecosystem. Along these lines, the present study aimed to explore whether and to what extent seamounts can have an imprint on the microbial community composition of seawater and of sessile benthic invertebrates, sponges. For our high-resolution sampling approach of microbial diversity (16S rRNA gene amplicon sequencing) along with measurements of inorganic nutrients and other biogeochemical parameters, we focused on the Schulz Bank seamount ecosystem, a sponge ground ecosystem which is located on the Arctic Mid-Ocean Ridge. Seawater samples were collected at two sampling depths (mid-water, MW, and near-bed water, BW) from a total of 19 sampling sites. With a clustering approach we defined microbial microhabitats within the pelagic realm at Schulz Bank, which were mapped onto the seamount's topography and related to various environmental parameters (such as suspended particulate matter, SPM; dissolved inorganic carbon, DIC; silicate, SiO−4; phosphate, PO3−4; ammonia, NH+4; nitrate, NO2−3; nitrite, NO−2; depth; and dissolved oxygen, O2). The results of our study reveal a "seamount effect" (sensu stricto) on the microbial mid-water pelagic community at least 200 m above the sea floor. Further, we observed a strong spatial heterogeneity in the pelagic microbial landscape across the seamount, with planktonic microbial communities reflecting oscillatory and circulatory water movements, as well as processes of bentho-pelagic coupling. Depth, NO2−3, SiO−4, and O2 ...

11 pages, 5 figures, 1 table, supplementary information https://doi.org/10.1038/s41396-021-00900-6 ; Bacteriophages (phages) are ubiquitous elements in nature, but their ecology and role in animals remains little understood. Sponges represent the oldest known extant animal-microbe symbiosis and are associated with dense and diverse microbial consortia. Here we investigate the tripartite interaction between phages, bacterial symbionts, and the sponge host. We combined imaging and bioinformatics to tackle important questions on who the phage hosts are and what the replication mode and spatial distribution within the animal is. This approach led to the discovery of distinct phage-microbe infection networks in sponge versus seawater microbiomes. A new correlative in situ imaging approach ('PhageFISH-CLEM') localised phages within bacterial symbiont cells, but also within phagocytotically active sponge cells. We postulate that the phagocytosis of free virions by sponge cells modulates phage-bacteria ratios and ultimately controls infection dynamics. Prediction of phage replication strategies indicated a distinct pattern, where lysogeny dominates the sponge microbiome, likely fostered by sponge host-mediated virion clearance, while lysis dominates in seawater. Collectively, this work provides new insights into phage ecology within sponges, highlighting the importance of tripartite animal-phage-bacterium interplay in holobiont functioning. We anticipate that our imaging approach will be instrumental to further understanding of viral distribution and cellular association in animal hosts ; We acknowledge funding by the DFG CRC1182 to UH (TPC4.3), TL (TPC4.2). MTJ was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Wuerzburg, and a Young Investigator Award of the CRC1182. SMM was supported by the Studienstiftung des Deutschen Volkes (German Academic Scholarship Foundation). CS was supported by the grants DFG STI700/1-1 and GRK2581 (P6). MR was supported by the Spanish Government grant (RTI2018-094187-B100) and 'Generalitat de Catalunya' research group grant (2017SGR1011). BED was supported by the Netherlands Organisation for Scientific Research (NWO) Vidi grant 864.14.004 and by the European Research Council (ERC) Consolidator grant 865694: DiversiPHI. /./ Open Access funding enabled and organized by Projekt DEA ; With the funding support of the 'Severo Ochoa Centre of Excellence' accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI) ; Peer reviewed

ABSTRACT Marine sponges are early-branching, filter-feeding metazoans that usually host complex microbiomes comprised of several, currently uncultivatable symbiotic lineages. Here, we use a low-carbon based strategy to cultivate low-abundance bacteria from Spongia officinalis. This approach favoured the growth of Alphaproteobacteria strains in the genera Anderseniella, Erythrobacter, Labrenzia, Loktanella, Ruegeria, Sphingorhabdus, Tateyamaria and Pseudovibrio, besides two likely new genera in the Rhodobacteraceae family. Mapping of complete genomes against the metagenomes of S. officinalis, seawater, and sediments confirmed the rare status of all the above-mentioned lineages in the marine realm. Remarkably, this community of low-abundance Alphaproteobacteria possesses several genomic attributes common to dominant, presently uncultivatable sponge symbionts, potentially contributing to host fitness through detoxification mechanisms (e.g. heavy metal and metabolic waste removal, degradation of aromatic compounds), provision of essential vitamins (e.g. B6 and B12 biosynthesis), nutritional exchange (especially regarding the processing of organic sulphur and nitrogen) and chemical defence (e.g. polyketide and terpenoid biosynthesis). None of the studied taxa displayed signs of genome reduction, indicative of obligate mutualism. Instead, versatile nutrient metabolisms along with motility, chemotaxis, and tight-adherence capacities - also known to confer environmental hardiness – were inferred, underlying dual host-associated and free-living life strategies adopted by these diverse sponge-associated Alphaproteobacteria. ; ACKNOWLEDGEMENTS The authors would like to thank Telma Franco for her help during sample processing and bacterial DNA extractions, Marta Valente for Sanger-sequencing of16S rRNA genes, and Martin T. Jahn for providing insights into data analysis. We thank Jochen Blom for his assistance with the software package EDGAR. This work was supported by the Portuguese Foundation for Science and Technology ...

Estee artículo contiene 14 páginas, 6 figuras. ; Sponges are the oldest known extant animal-microbe symbiosis. These ubiquitous benthic animals play an important role in marine ecosystems in the cycling of dissolved organic matter (DOM), the largest source of organic matter on Earth. The conventional view on DOM cycling through microbial processing has been challenged by the interaction between this efficient filter-feeding host and its diverse and abundant microbiome. Here we quantify, for the first time, the role of host cells and microbial symbionts in sponge heterotrophy. We combined stable isotope probing and nanoscale secondary ion mass spectrometry to compare the processing of different sources of DOM (glucose, amino acids, algal-produced) and particulate organic matter (POM) by a high-microbial abundance (HMA) and low-microbial abundance (LMA) sponge with single-cell resolution. Contrary to common notion, we found that both microbial symbionts and host choanocyte (i.e. filter) cells and were active in DOM uptake. Although all DOM sources were assimilated by both sponges, higher microbial biomass in the HMA sponge corresponded to an increased capacity to process a greater variety of dissolved compounds. Nevertheless, in situ feeding data demonstrated that DOM was the primary carbon source for both the LMA and HMA sponge, accounting for ~90% of their heterotrophic diets. Microbes accounted for the majority (65–87%) of DOM assimilated by the HMA sponge (and ~60% of its total heterotrophic diet) but <5% in the LMA sponge. We propose that the evolutionary success of sponges is due to their different strategies to exploit the vast reservoir of DOM in the ocean. ; This work was funded by the DFG CRC1182-TP B1 "Origin and Function of Metaorganisms" to UH, the Spanish Government grant (RTI2018–094187-B100) and "Generalitat de Catalunya" research group grant (2017SGR1011) to MR and RC, the Netherlands Organization for Scientific Research (NWO-VIDI 864.13.007) to DvO, the European Research Council under the European Union's Horizon 2020 research and innovation programme (Starting Grant 715513) to JMG, and the Swiss Science Foundation Grant (200021_179092) to AM. ; Peer reviewed

ABSTRACT Connectivity is a fundamental process driving the persistence of marine populations and their adaptation potential in response to environmental change. In this study, we analysed the population genetics of two morphologically highly similar deep-sea sponge clades (Phakellia hirondellei and the 'Topsentia-and-Petromica' clade, (hereafter referred to as 'TaP clade')) at three locations in the Cantabrian Sea and simultaneously assessed the corresponding host microbiome by 16S rRNA gene sequencing. A virtual particle tracking approach (Lagrangian modelling) was applied to assess oceanographic connectivity in the study area. We observed overall genetic uniformity for both sponge clades. Notably, subtle genetic differences were observed for sponges of the TaP clade and also their microbiomes between a canyon and bank location, < 100 km apart and with the same depth range. The Lagrangian model output suggests a strong retention of larvae in the study area with variable inter-annual connectivity via currents between the three sampling regions. We conclude that geologic features (canyons) and the prevailing ocean currents may dictate sponge holobiont connectivity and that differentiation can emerge even on small spatial scales. ; ABBREVIATIONS TaP cladeTopsentia-and-Petromica clade ACKNOWLEDGEMENTS We dedicate this study to Hans Tore Rapp, friend, mentor and esteemed colleague whose outstanding expertise in deep-sea sponge taxonomy will be dearly missed. We greatly acknowledge the crew and scientific party of RV Ángeles Alvariño cruise SponGES0617 for their valuable support at sea. As well as members of the IEO Gijón for logistical support during a guest stay of KB before the cruise. We appreciate Andrea Hethke's and Ina Clefsen's, as well as Thomas Hansen's assistance in the laboratory after the cruise while generating the microbial amplicon and flow cytometry data. We thank Willi Rath for support in technical issues with the modelling part, and Lara Schmittmann and Ina Clefsen for technical support with 18S ...

ABSTRACT In the present study, we profiled bacterial and archaeal communities from 13 phylogenetically diverse deep-sea sponge species (Demospongiae and Hexactinellida) from the South Pacific by 16S rRNA-gene amplicon sequencing. Additionally, the associated bacteria and archaea were quantified by real-time qPCR. Our results show that bacterial communities from the deep-sea sponges are mostly host-species specific similar to what has been observed for shallow-water demosponges. The archaeal deep-sea sponge community structures are different from the bacterial community structures in that they are almost completely dominated by a single family, which are the ammonia-oxidizing genera within the Nitrosopumilaceae. Remarkably, the archaeal communities are mostly specific to individual sponges (rather than sponge-species), and this observation applies to both hexactinellids and demosponges. Finally, archaeal 16s gene numbers, as detected by quantitative real-time PCR, were up to three orders of magnitude higher than in shallow-water sponges, highlighting the importance of the archaea for deep-sea sponges in general. ; ACKNOWLEDGMENTS This work is dedicated to Hans Tore Rapp, coordinator of the H2020-SponGES project, mentor and friend. We thank Andrea Hethke, Ina Clefsen, and the CRC1182 Z3 team (Katja Cloppenborg-Schmidt, Malte Rühlemann, John Baines) for assistance with the amplicon pipeline. We greatly acknowledge the crew and scientific party of RV Sonne cruise SO254, as well as the ROV Kiel 6000 team for their valuable support at sea. We also thank Sven Rohde, Tessa Clemens and the entire benthic invertebrate team of the RV Sonne Cruise SO254 for their assistance in sample collection, processing and cataloging. We thank Henry Reiswig for advice on identification of hexactinellid samples. Sample collection was carried out under the "Application for consent to conduct marine scientific research in areas under national jurisdiction of New Zealand (dated 7.6.2016)." This is publication 68 of Senckenberg am Meer ...

Sponges host a remarkable diversity of microbial symbionts, however, the benefit their microbes provide is rarely understood. Here, we describe two new sponge species from deep-sea asphalt seeps and show that they live in a nutritional symbiosis with methane-oxidizing (MOX) bacteria. Metagenomics and imaging analyses revealed unusually high amounts of MOX symbionts in hosts from a group previously assumed to have low microbial abundances. These symbionts belonged to the Marine Methylotrophic Group 2 clade. They are host-specific and likely vertically transmitted, based on their presence in sponge embryos and streamlined genomes, which lacked genes typical of related free-living MOX. Moreover, genes known to play a role in host–symbiont interactions, such as those that encode eukaryote-like proteins, were abundant and expressed. Methane assimilation by the symbionts was one of the most highly expressed metabolic pathways in the sponges. Molecular and stable carbon isotope patterns of lipids confirmed that methane-derived carbon was incorporated into the hosts. Our results revealed that two species of sponges, although distantly related, independently established highly specific, nutritional symbioses with two closely related methanotrophs. This convergence in symbiont acquisition underscores the strong selective advantage for these sponges in harboring MOX bacteria in the food-limited deep sea. ; ACKNOWLEDGEMENTS The authors thank all individuals who helped during the R/V Meteor research cruise M114, including onboard technical and scientific personnel, the captain and crew, and the ROV MARUM-Quest team. We thank the Max Planck-Genome-Centre Cologne (http://mpgc.mpipz.mpg.de/home/) for generating the meta- genomic and the metatranscriptomic data used in this study, the Imaging Core Facility at the University of Würzburg, Germany for embedding of the TEM samples, the Central Microscopy unit at the University of Kiel, Germany for access to their electron microscope facilities, and Ralf Lendt (University of Hamburg) ...