The level of integration between associated partners can range from ectosymbioses to extracellular and intracellular endosymbioses, and this range has been assumed to reflect a continuum from less intimate to evolutionarily highly stable associations. In this study, we examined the specificity and evolutionary history of marine symbioses in a group of closely related sulphur-oxidizing bacteria, called Candidatus Thiosymbion, that have established ecto-and endosymbioses with two distantly related animal phyla, Nematoda and Annelida. Intriguingly, in the ectosymbiotic associations of stilbonematine nematodes, we observed a high degree of congruence between symbiont and host phylogenies, based on their ribosomal RNA (rRNA) genes. In contrast, for the endosymbioses of gutless phallodriline annelids (oligochaetes), we found only a weak congruence between symbiont and host phylogenies, based on analyses of symbiont 16S rRNA genes and six host genetic markers. The much higher degree of congruence between nematodes and their ectosymbionts compared to those of annelids and their endosymbionts was confirmed by cophylogenetic analyses. These revealed 15 significant codivergence events between stilbonematine nematodes and their ectosymbionts, but only one event between gutless phallodrilines and their endosymbionts. Phylogenetic analyses of 16S rRNA gene sequences from 50 Cand. Thiosymbion species revealed seven well-supported clades that contained both stilbonematine ectosymbionts and phallodriline endosymbionts. This closely coupled evolutionary history of marine ecto-and endosymbionts suggests that switches between symbiotic lifestyles and between the two host phyla occurred multiple times during the evolution of the Cand. Thiosymbion clade, and highlights the remarkable flexibility of these symbiotic bacteria.

Background Many animals live in intimate associations with a species-rich microbiome. A key factor in maintaining these beneficial associations is fidelity, defined as the stability of associations between hosts and their microbiota over multiple host generations. Fidelity has been well studied in terrestrial hosts, particularly insects, over longer macroevolutionary time. In contrast, little is known about fidelity in marine animals with species-rich microbiomes at short microevolutionary time scales, that is at the level of a single host population. Given that natural selection acts most directly on local populations, studies of microevolutionary partner fidelity are important for revealing the ecological and evolutionary processes that drive intimate beneficial associations within animal species. Results In this study on the obligate symbiosis between the gutless marine annelid Olavius algarvensis and its consortium of seven co-occurring bacterial symbionts, we show that partner fidelity varies across symbiont species from strict to absent over short microevolutionary time. Using a low-coverage sequencing approach that has not yet been applied to microbial community analyses, we analysed the metagenomes of 80 O. algarvensis individuals from the Mediterranean and compared host mitochondrial and symbiont phylogenies based on single-nucleotide polymorphisms across genomes. Fidelity was highest for the two chemoautotrophic, sulphur-oxidizing symbionts that dominated the microbial consortium of all O. algarvensis individuals. In contrast, fidelity was only intermediate to absent in the sulphate-reducing and spirochaetal symbionts with lower abundance. These differences in fidelity are likely driven by both selective and stochastic forces acting on the consistency with which symbionts are vertically transmitted. Conclusions We hypothesize that variable degrees of fidelity are advantageous for O. algarvensis by allowing the faithful transmission of their nutritionally most important symbionts and flexibility in the acquisition of other symbionts that promote ecological plasticity in the acquisition of environmental resources.

In obligate symbioses, partner fidelity plays a central role in maintaining the stability of the association across multiple host generations. Fidelity has been well studied in hosts with a very restricted diversity of symbionts, but little is known about how fidelity is maintained in hosts with multiple co-occurring symbionts. The marine annelid Olavius algarvensis lives in an obligate association with at least five co-occurring bacterial symbionts that are inherited vertically. The symbionts so efficiently supply their hosts with nutrition that these worms have completely reduced their mouth and digestive tract. Here, we investigated partner fidelity in the O. algarvensis symbiosis by sequencing the metagenomes of 80 host individuals from two mitochondrial lineages and two locations in the Mediterranean. Comparative phylogenetic analyses of mitochondrial and symbiont genotypes based on single nucleotide polymorphisms revealed high fidelity for the primary symbiont that dominated the microbial consortium of all 80 O. algarvensis individuals. In contrast, the secondary symbionts of O. algarvensis, which occurred in lower abundance and were not always present in all host individuals, showed only intermediate to low fidelity. We hypothesize that harbouring symbionts with variable levels of fidelity ensures faithful transmission of the most abundant and nutritionally important symbiont, while flexibility in the acquisition of secondary symbionts enhances genetic exchange and retains ecological and evolutionary adaptability.

In addition to abundant animal communities, corals from all ocean depths support diverse microbial associates that are important to coral health. While some of these microbes have been classified taxonomically, understanding the metabolic potential of coral-associated bacteria and how they interact with their coral hosts is limited by a lack of genomic data. One example is Mycoplasma and other members of the class Mollicutes which are widespread coral associates. Here we investigated the association between two novel members of the class Mollicutes and the deep-sea octocoral Callogorgia delta. We screened C. delta, a closely related species C. americana, sediment, and water for mollicutes using 16S metabarcoding. One ASV was found in most colonies screened (99/108) and often dominated the microbiome (up to 99%). Another ASV was detected at lower abundance and prevalence in these corals. Both were absent in all water and were absent or rare in the sediment. We sequenced metagenomes and metatranscriptomes to assemble and annotate genomes and propose the names Ca. Oceanoplasma callogorgiae and Ca. Thalassoplasma callogorgiae. The genomes were small, revealed a reliance on the arginine dihydrolase pathway for ATP production, and contained CRISPR-Cas systems with extensive arrays. CARD-FISH microscopy unveiled an abundant bacterium in the mesoglea which is likely to be Ca. O. callogorgiae. These novel mollicutes cluster with others from diverse invertebrate hosts. Altogether, this work describes the association of these novel mollicutes in C. delta, provides insight into widespread coral associates, and identifies a novel clade of marine mollicutes whose diversity remains largely undiscovered.