Harald R. Gruber-Vodicka

Many animals benefit from microbial symbionts that aid digestion, supply essential nutrients, or provide defense. When this dependence becomes obligate, as in gutless marine oligochaetes that have lost both digestive and excretory systems and rely entirely on their bacterial partners for nutrition and recycling of waste compounds, it would seem to constrain ecological and evolutionary flexibility. Yet, these worms are globally distributed and thrive across diverse environments. To investigate how stability and adaptability coexist in such obligate symbioses, we combined global sampling with high-resolution phylogenomic analyses of 246 individuals from 63 species across 17 marine sites. Ancestral reconstructions revealed that the chemoautotrophic primary symbiont “ Candidatus Thiosymbion” was acquired early and persistently maintained, whereas additional partners were repeatedly gained through convergent evolution, expanding metabolic potential. Symbiont communities were highly consistent within host species, but this stability dissolved rapidly as hosts diverged, with communities reshaping rapidly across short macroevolutionary timescales. Even the primary symbiont “ Ca . Thiosymbion” showed low fidelity and occasional displacement, a sharp contrast to other multipartite symbioses, where core partners persist unchanged for hundreds of millions of years. Thus, gutless oligochaetes illustrate how long-term stability at microevolutionary scales can coexist with rapid reshaping at macroevolutionary scales in multipartite symbioses. More broadly, this pattern demonstrates how the paradox of stability through flexibility may underpin the persistence and diversification of complex symbioses across deep time.

Lysozymes are an essential part of immunity and nutrition in metazoans, degrading bacterial cell walls via the hydrolysis of peptidoglycan. Although various lysozymes have been reported for higher animals, the origin of animal lysozymes remains elusive as they seem to be lacking in all early branching phyla. In this study, we investigated a putative goose-type lysozyme (PLys, glycoside hydrolase family 23, GH23) of the placozoan Trichoplax sp. H2. We show that PLys is highly active and primarily produced by cells of the placozoan ventral epithelium. PLys contains a non-conserved cysteine-rich domain N-terminal of the GH23 lysozyme domain, which stabilizes the protein and is truncated during maturation. Using a pH-sensitive fluorescence reporter, we show that Trichoplax sp. H2 acidifies its temporary feeding grooves pulsatively during digestive events close to the optimum pH for PLys activity. To elucidate the evolutionary origin of the metazoan GH23 lysozyme family, we applied structure-based phylogenetics to show that the metazoan g-type GH23 lysozymes originated from a horizontal gene transfer event from bacteria to an early pre-bilaterian ancestor. GH23 lysozymes have then been retained and expanded in many phyla acting as first animal lysozyme and a key component in the antibacterial arsenal since early animal evolution.

Rickettsiales are an enigmatic clade of strictly host-associated bacteria with some very common phylotypes in aquatic habitats. Despite amplicon- and metagenomic sequencing-based observations, the roles of these aquatic Rickettsiales are still unknown, as their genomes feature both beneficial and parasitic traits. In placozoans, microscopy-based observations of intracellular and Rickettsia-like-organisms point to Rickettsiales as common and abundant symbionts. We therefore systematically studied the microbiomes of placozoans to understand the eco-evolutionary dynamics of their associated Rickettsiales. Single-animal metagenomics revealed a widespread association of placozoans with Rickettsiales from three clades of Ca. Midichloriaceae, which were present in all but two placozoan lineages. A comparative phylogenetic analysis of placozoans and their Rickettsiales symbionts revealed host species-specific switches among Rickettsiales clades in several placozoan lineages. Our combination of evolutionary analyses, genome annotations, and expression data indicate different functional roles of these Rickettsiales clades, with some clearly diverging from a classical parasitic lifestyle by the loss of all ATP scavenging machinery. One of placozoan lineages that lacked Ca. Midichloriaceae was instead associated with Rickettsiaceae symbionts, while another was completely devoid of Rickettsiales symbionts. Our imaging data of fibre cells entirely free of Rickettsiales underscores previously unrecognized variation in placozoan-microbe associations. Early-branching invertebrates, including placozoans as well as many corals, are gaining increasing traction as emerging models across the life sciences. Our data and analyses show that it is important to consider the specific endosymbiont groups and their ecology in a given host, as these symbionts likely influence the hosts' cellular and organismal biology.

A bstract: In the Mediterranean Sea, the demosponge Chondrilla nucula can occur in close association with the native seagrass Posidonia oceanica . C. nucula harbors a diverse and abundant microbial community, including potential nitrifiers. Thus, the sponge may contribute to the nitrogen (N) demand of the seagrass holobiont. In this study, we investigated potential nitrification rates (PNR) and inorganic N fluxes within this association at a site where C. nucula covered 18 ± 3 % of the seagrass meadow area, during plant growth (spring) and senescence (autumn). Using incubation experiments with ¹⁵ N-labeled ammonium, we measured PNR and inorganic N of the seagrass-sponge association, and of sponge and seagrass independently, under light and dark conditions. We supplemented these experiments with 16s rRNA gene amplicon sequencing to characterize the microbial community of the sponge. PNR was exclusively measured when the sponge was present (alone or in association with the seagrass). PNR was highest in the dark and when C. nucula was associated with the seagrass, ranging from 21 ± 7 to 267 ± 33 nmol N g DW ^-1 h ^-1 in spring and autumn, respectively. Sponge-mediated PNR can support 8% of the N demand of the P. oceanica holobiont during growth and 47 % during senescence. We identified key nitrifying bacterial and archaeal groups as members of the sponge’s microbial community. While C. nucula released inorganic N, potentially sustaining the seagrass, it benefitted from dissolved organic carbon released by P. oceanica . These results suggest that the interaction between C. nucula and P. oceanica is mutually beneficial, ultimately supporting and stabilizing the seagrass ecosystem.

Lysozymes are an essential part of immunity and nutrition in metazoans, degrading bacterial cell walls via the hydrolysis of peptidoglycan. Although various lysozymes have been reported for higher animals, the origin of animal lysozymes remains elusive as they seem to be lacking in all early branching phyla. In this study, we investigated a putative goose-type lysozyme (PLys, glycoside hydrolase family 23, GH23) of the placozoan Trichoplax sp. H2. We show that PLys is highly active and produced in gland cells of the ventral epithelium. PLys contains a protective and non-conserved cysteine-rich domain N-terminal of the conserved GH23 lysozyme domain. A truncation of this N-terminal domain in the maturation process of PLys leads to a drastic increase in enzymatic activity at the cost of stability. As the lysozyme is most active under acidic conditions, we investigated the pH trajectories during extracellular digestion in situ . Using a pH-senstive fluorescence reporter, we show that Trichoplax sp. H2 acidifies its temporary feeding grooves pulsatively during digestive events close to the optimum pH for PLys activity. To elucidate the evolutionary origin of the metazoan GH23 lysozyme family, we applied a structure-based phylogenetics approach to show that the metazoan g-type GH23 lysozymes originated from a horizontal gene transfer event from bacteria to an early pre-bilaterian ancestor. GH23 lysozymes have then been retained and expanded in many phyla, including Porifera, Cnidaria, Placozoa and chordates, acting as first animal lysozyme and a key component in the antibacterial arsenal since early animal evolution.

Nematodes are one of the most widespread and abundant animal taxa across aquatic and terrestrial environments. In marine shallow-water, porous sediments, members of the subfamily Stilbonematinae may be found in high numbers. Stilbonematinae are characterized by their coat of symbiotic bacteria, which give the nematodes a white appearance, while the nematodes themselves are usually colourless. We identified several species of the genus Robbea (Desmodoridae: Stilbonematinae) in which live specimens had a conspicuous dark purple coloration of the glandular sense organs (GSOs), highly specialized epidermal glands that are prominent in the Stilbonematinae. The dark inclusions in the GSOs of Robbea contained high concentrations of iron and bromide, regardless of their habitat. Morphological and phylogenetic analyses show that the genus Robbea is paraphyletic and we define the new genus Cyathorobbea gen. nov. for species with sucker-shaped post-pharyngeal supplements in males. Furthermore, we describe five new Robbea species from around the world. Some Robbea species are morphologically very similar and required the use of principal component analysis of morphometric data to untangle the diversity in this taxon. Our work emphasizes the value of live animal observations that enable the identification of important characters and highlights the overlooked diversity within the Stilbonematinae.

Marine invertebrates with limited dispersal abilities exhibit high levels of genetic divergence among populations. However, the spatial extent of genetic differentiation in these species remains poorly understood because identifying natural barriers to gene flow can be challenging in the marine environment. In this study, we investigated the population genetic structure of the interstitial annelid Olavius algarvensis , a species that lays eggs in its immediate surroundings and does not have an active dispersal phase. We analyzed the mitochondrial and nuclear genome sequences of hundreds to thousands of individuals from eleven sites in the Mediterranean, spanning microgeographic scales of < 5 km to macrogeographic scales of 800 km. Comparisons of single nucleotide polymorphisms (SNPs) in mitochondrial genomes revealed a complex history of introgression events, with as many as six mitochondrial lineages co-occurring in individuals from the same site. In contrast, SNP analyses of nuclear genomes revealed clear genetic differentiation at micro- and macrographic scales, characterised by a significant isolation by distance pattern (IBD). IBD patterns further indicated the presence of a historical physical barrier to gene flow on the east coast of the island of Elba corresponding to the historical shoreline around Elba during the Last Glacial Maximum in the Late Pleistocene, and highlighting the influence of geological forces in shaping population genetic structuring in the species today. Overall, our results provide strong empirical evidence for the high genomic diversification across spatial scales in marine interstitial fauna.

Endozoicomonadaceae bacteria are widespread in many marine animals, and generally considered beneficial. Members of one clade, however, Candidatus Endonucleobacter, infect the nuclei of deep-sea mussels, where they replicate to ≥ 80,000 bacteria per nucleus and cause the nuclei to swell to 50 times their original size. How these parasites are able to persist in host nuclei without the cell undergoing apoptosis is not known. We show here that Ca. Endonucleobacter encodes and expresses 7-15 inhibitors of apoptosis (IAPs), proteins previously only known from animals and viruses. Dual RNA-seq transcriptomes of infected nuclei revealed parallel upregulation of Ca. Endonucleobacter IAPs and host caspases, suggesting an arms race between the parasite and host for control of apoptosis. Comparative phylogenetic analyses revealed that Ca. Endonucleobacter acquired IAPs repeatedly through horizontal gene transfer from their hosts in convergent acquisition, possibly mediated by herpes viruses that may infect both the parasite and the host.

Microbes perform critical functions in corals yet most knowledge is derived from the photic zone. Here, we discovered two mollicutes that dominate the microbiome of the deep-sea octocoral, Callogorgia delta, and reside in the mesoglea. These symbionts were abundant across the host’s range, absent in the water, and rare in sediments. The symbionts lack all known fermentative capabilities including glycolysis and can only generate energy from arginine provided by the coral host. Their genomes feature extensive mechanisms to interact with foreign DNA which may be indicative of their role in symbiosis. We erect the novel family Oceanoplasmataceae which includes these symbionts and others associated with four marine invertebrate phyla. Its exceptionally broad host range suggests that the diversity of this enigmatic family remains largely undiscovered. Oceanoplasmataceae genomes are the most highly reduced among mollicutes providing new insight into their reductive evolution and the roles of coral symbionts.