Zoothamnium mariella sp. nov., a marine, colonial ciliate with an atypcial growth pattern, and its ectosymbiont Candidatus Fusimicrobium zoothamnicola gen. nov., sp. nov.

Ciliates are unicellular eukaryotes, regularly involved in symbiotic associations. Symbionts may colonize the inside of their cells as well as their surface as ectosymbionts. Here, we report on a new ciliate species, designated as Zoothamnium mariella sp. nov. (Peritrichia, Sessilida), discovered in the northern Adriatic Sea (Mediterranean Sea) in 2021. We found this ciliate species to be monospecifically associated with a new genus of ectosymbiotic bacteria, here proposed as Candidatus Fusimicrobium zoothamnicola gen. nov., sp. nov. To formally describe the new ciliate species, we investigated its morphology and sequenced its 18S rRNA gene. To demonstrate its association with a single species of bacterial ectosymbiont, we performed 16S rRNA gene sequencing, fluorescence in situ hybridization, and scanning electron microscopy. Additionally, we explored the two partners' cultivation requirements and ecology. Z. mariella sp. nov. was characterized by a colony length of up to 1 mm. A consistent number of either seven or eight long branches alternated on the stalk in close distance to each other. The colony developed three different types of zooids: microzooids ("trophic stage"), macrozooids ("telotroch stage"), and terminal zooids ("dividing stage"). Viewed from inside the cell, the microzooids' oral ciliature ran in 1 » turns in a clockwise direction around the peristomial disc before entering the infundibulum, where it performed another ¾ turn. Phylogenetic analyses assigned Z. mariella sp. nov. to clade II of the family Zoothamnidae. The ectosymbiont formed a monophyletic clade within the Gammaproteobacteria along with two other ectosymbionts of peritrichous ciliates and a free-living vent bacterium. It colonized the entire surface of its ciliate host, except for the most basal stalk of large colonies, and exhibited a single, spindle-shaped morphotype. Furthermore, the two partners together appear to be generalists of temperate, oxic, marine shallow-water environments and were collectively cultivable in steady flow-through systems.

Comparative genomics of a vertically transmitted thiotrophic bacterial ectosymbiont and its close free-living relative.

Thiotrophic symbioses between sulphur-oxidizing bacteria and various unicellular and metazoan eukaryotes are widespread in reducing marine environments. The giant colonial ciliate Zoothamnium niveum, however, is the only host of thioautotrophic symbionts that has been cultivated along with its symbiont, the vertically transmitted ectosymbiont Candidatus Thiobius zoothamnicola (short Thiobius). Because theoretical predictions posit a smaller genome in vertically transmitted endosymbionts compared to free-living relatives, we investigated whether this is true also for an ectosymbiont. We used metagenomics to recover the high-quality draft genome of this bacterial symbiont. For comparison we have also sequenced a closely related free-living cultured but not formally described strain Milos ODIII6 (short ODIII6). We then performed comparative genomics to assess the functional capabilities at gene, metabolic pathway and trait level. 16S rRNA gene trees and average amino acid identity confirmed the close phylogenetic relationship of both bacteria. Indeed, Thiobius has about a third smaller genome than its free-living relative ODIII6, with reduced metabolic capabilities and fewer functional traits. The functional capabilities of Thiobius were a subset of those of the more versatile ODIII6, which possessed additional genes for oxygen, sulphur and hydrogen utilization and for the acquisition of phosphorus illustrating features that may be adaptive for the unstable environmental conditions at hydrothermal vents. In contrast, Thiobius possesses genes potentially enabling it to utilize lactate and acetate heterotrophically, compounds that may be provided as byproducts by the host. The present study illustrates the effect of strict host-dependence of a bacterial ectosymbiont on genome evolution and host adaptation.

The complete and closed genome of the facultative generalist Candidatus Endoriftia persephone from deep-sea hydrothermal vents.

The mutualistic interactions between Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone (short Endoriftia) have been extensively researched. However, the closed Endoriftia genome is still lacking. Here, by employing single-molecule real-time sequencing we present the closed chromosomal sequence of Endoriftia. In contrast to theoretical predictions of enlarged and mobile genetic element-rich genomes related to facultative endosymbionts, the closed Endoriftia genome is streamlined with fewer than expected coding sequence regions, insertion-, prophage-sequences and transposase-coding sequences. Automated and manually curated functional analyses indicated that Endoriftia is more versatile regarding sulphur metabolism than previously reported. We identified the presence of two identical rRNA operons and two long CRISPR regions in the closed genome. Additionally, pangenome analyses revealed the presence of three types of secretion systems (II, IV and VI) in the different Endoriftia populations indicating lineage-specific adaptations. The in depth mobilome characterization identified the presence of shared genomic islands in the different Endoriftia drafts and in the closed genome, suggesting that the acquisition of foreign DNA predates the geographical dispersal of the different endosymbiont populations. Finally, we found no evidence of epigenetic regulation in Endoriftia, as revealed by gene screenings and absence of methylated modified base motifs in the genome. As a matter of fact, the restriction-modification system seems to be dysfunctional in Endoriftia, pointing to a higher importance of molecular memory-based immunity against phages via spacer incorporation into CRISPR system. The Endoriftia genome is the first closed tubeworm endosymbiont to date and will be valuable for future gene oriented and evolutionary comparative studies.

Host-symbiont stress response to lack-of-sulfide in the giant ciliate mutualism.

The mutualism between the thioautotrophic bacterial ectosymbiont Candidatus Thiobius zoothamnicola and the giant ciliate Zoothamnium niveum thrives in a variety of shallow-water marine environments with highly fluctuating sulfide emissions. To persist over time, both partners must reproduce and ensure the transmission of symbionts before the sulfide stops, which enables carbon fixation of the symbiont and nourishment of the host. We experimentally investigated the response of this mutualism to depletion of sulfide. We found that colonies released some initially present but also newly produced macrozooids until death, but in fewer numbers than when exposed to sulfide. The symbionts on the colonies proliferated less without sulfide, and became larger and more rod-shaped than symbionts from freshly collected colonies that were exposed to sulfide and oxygen. The symbiotic monolayer was severely disturbed by growth of other microbes and loss of symbionts. We conclude that the response of both partners to the termination of sulfide emission was remarkably quick. The development and the release of swarmers continued until host died and thus this behavior contributed to the continuation of the association.

Novel Insights on Obligate Symbiont Lifestyle and Adaptation to Chemosynthetic Environment as Revealed by the Giant Tubeworm Genome.

The mutualism between the giant tubeworm Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone has been extensively researched over the past 40 years. However, the lack of the host whole-genome information has impeded the full comprehension of the genotype/phenotype interface in Riftia. Here, we described the high-quality draft genome of Riftia, its complete mitogenome, and tissue-specific transcriptomic data. The Riftia genome presents signs of reductive evolution, with gene family contractions exceeding expansions. Expanded gene families are related to sulfur metabolism, detoxification, antioxidative stress, oxygen transport, immune system, and lysosomal digestion, reflecting evolutionary adaptations to the vent environment and endosymbiosis. Despite the derived body plan, the developmental gene repertoire in the gutless tubeworm is extremely conserved with the presence of a near intact and complete Hox cluster. Gene expression analyses establish that the trophosome is a multifunctional organ marked by intracellular digestion of endosymbionts, storage of excretory products, and hematopoietic functions. Overall, the plume and gonad tissues both in contact to the environment harbor highly expressed genes involved with cell cycle, programed cell death, and immunity indicating a high cell turnover and defense mechanisms against pathogens. We posit that the innate immune system plays a more prominent role into the establishment of the symbiosis during the infection in the larval stage, rather than maintaining the symbiostasis in the trophosome. This genome bridges four decades of physiological research in Riftia, whereas it simultaneously provides new insights into the development, whole organism functions, and evolution in the giant tubeworm.

Thiotrophic bacterial symbiont induces polyphenism in giant ciliate host Zoothamnium niveum.

Evolutionary theory predicts potential shifts between cooperative and uncooperative behaviour under fluctuating environmental conditions. This leads to unstable benefits to the partners and restricts the evolution of dependence. High dependence is usually found in those hosts in which vertically transmitted symbionts provide nutrients reliably. Here we study host dependence in the marine, giant colonial ciliate Zoothamnium niveum and its vertically transmitted, nutritional, thiotrophic symbiont from an unstable environment of degrading wood. Previously, we have shown that sulphidic conditions lead to high host fitness and oxic conditions to low fitness, but the fate of the symbiont has not been studied. We combine several experimental approaches to provide evidence for a sulphide-tolerant host with striking polyphenism involving two discrete morphs, a symbiotic and an aposymbiotic one. The two differ significantly in colony growth form and fitness. This polyphenism is triggered by chemical conditions and elicited by the symbiont's presence on the dispersing swarmer. We provide evidence of a single aposymbiotic morph found in nature. We propose that despite a high fitness loss when aposymbiotic, the ciliate has retained a facultative life style and may use the option to live without its symbiont to overcome spatial and temporal shortage of sulphide in nature.

Polyclonal symbiont populations in hydrothermal vent tubeworms and the environment.

Horizontally transmitted symbioses usually house multiple and variable symbiont genotypes that are acquired from a much more diverse environmental pool via partner choice mechanisms. However, in the deep-sea hydrothermal vent tubeworm Riftia pachyptila (Vestimentifera, Siboglinidae), it has been suggested that the Candidatus Endoriftia persephone symbiont is monoclonal. Here, we show with high-coverage metagenomics that adult R. pachyptila house a polyclonal symbiont population consisting of one dominant and several low-frequency variants. This dominance of one genotype is confirmed by multilocus gene sequencing of amplified housekeeping genes in a broad range of host individuals where three out of four loci ( atpA, uvrD and recA) revealed no genomic differences, while one locus ( gyrB) was more diverse in adults than in juveniles. We also analysed a metagenome of free-living Endoriftia and found that the free-living population showed greater sequence variability than the host-associated population. Most juveniles and adults shared a specific dominant genotype, while other genotypes can dominate in few individuals. We suggest that although generally permissive, partner choice is selective enough to restrict uptake of some genotypes present in the environment.

NanoSIMS and tissue autoradiography reveal symbiont carbon fixation and organic carbon transfer to giant ciliate host.

The giant colonial ciliate Zoothamnium niveum harbors a monolayer of the gammaproteobacteria Cand. Thiobios zoothamnicoli on its outer surface. Cultivation experiments revealed maximal growth and survival under steady flow of high oxygen and low sulfide concentrations. We aimed at directly demonstrating the sulfur-oxidizing, chemoautotrophic nature of the symbionts and at investigating putative carbon transfer from the symbiont to the ciliate host. We performed pulse-chase incubations with 14C- and 13C-labeled bicarbonate under varying environmental conditions. A combination of tissue autoradiography and nanoscale secondary ion mass spectrometry coupled with transmission electron microscopy was used to follow the fate of the radioactive and stable isotopes of carbon, respectively. We show that symbiont cells fix substantial amounts of inorganic carbon in the presence of sulfide, but also (to a lesser degree) in the absence of sulfide by utilizing internally stored sulfur. Isotope labeling patterns point to translocation of organic carbon to the host through both release of these compounds and digestion of symbiont cells. The latter mechanism is also supported by ultracytochemical detection of acid phosphatase in lysosomes and in food vacuoles of ciliate cells. Fluorescence in situ hybridization of freshly collected ciliates revealed that the vast majority of ingested microbial cells were ectosymbionts.

'Candidatus Cochliophilus cryoturris' (Coxiellaceae), a symbiont of the testate amoeba Cochliopodium minus.

Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as 'Candidatus Cochliophilus cryoturris'. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.

A Novel Colonial Ciliate Zoothamnium ignavum sp. nov. (Ciliophora, Oligohymenophorea) and Its Ectosymbiont Candidatus Navis piranensis gen. nov., sp. nov. from Shallow-Water Wood Falls.

Symbioses between ciliate hosts and prokaryote or unicellular eukaryote symbionts are widespread. Here, we report on a novel ciliate species within the genus Zoothamnium Bory de St. Vincent, 1824, isolated from shallow-water sunken wood in the North Adriatic Sea (Mediterranean Sea), proposed as Zoothamnium ignavum sp. nov. We found this ciliate species to be associated with a novel genus of bacteria, here proposed as "Candidatus Navis piranensis" gen. nov., sp. nov. The descriptions of host and symbiont species are based on morphological and ultrastructural studies, the SSU rRNA sequences, and in situ hybridization with symbiont-specific probes. The host is characterized by alternate microzooids on alternate branches arising from a long, common stalk with an adhesive disc. Three different types of zooids are present: microzooids with a bulgy oral side, roundish to ellipsoid macrozooids, and terminal zooids ellipsoid when dividing or bulgy when undividing. The oral ciliature of the microzooids runs 1» turns in a clockwise direction around the peristomial disc when viewed from inside the cell and runs into the infundibulum, where it makes another ¾ turn. The ciliature consists of a paroral membrane (haplokinety), three adoral membranelles (polykineties), and one stomatogenic kinety (germinal kinety). One circular row of barren kinetosomes is present aborally (trochal band). Phylogenetic analyses placed Z. ignavum sp. nov. within the clade II of the polyphyletic family Zoothamniidae (Oligohymenophorea). The ectosymbiont was found to occur in two different morphotypes, as rods with pointed ends and coccoid rods. It forms a monophyletic group with two uncultured Gammaproteobacteria within an unclassified group of Gammaproteobacteria, and is only distantly related to the ectosymbiont of the closely related peritrich Z. niveum (Hemprich and Ehrenberg, 1831) Ehrenberg, 1838.

Trophosome of the Deep-Sea Tubeworm Riftia pachyptila Inhibits Bacterial Growth.

The giant tubeworm Riftia pachyptila lives in symbiosis with the chemoautotrophic gammaproteobacterium Cand. Endoriftia persephone. Symbionts are released back into the environment upon host death in high-pressure experiments, while microbial fouling is not involved in trophosome degradation. Therefore, we examined the antimicrobial effect of the tubeworm's trophosome and skin. The growth of all four tested Gram-positive, but only of one of the tested Gram-negative bacterial strains was inhibited by freshly fixed and degrading trophosome (incubated up to ten days at either warm or cold temperature), while no effect on Saccharomyces cerevisiae was observed. The skin did not show antimicrobial effects. A liquid chromatography-mass spectrometric analysis of the ethanol supernatant of fixed trophosomes lead to the tentative identification of the phospholipids 1-palmitoleyl-2-lyso-phosphatidylethanolamine, 2-palmitoleyl-1-lyso-phosphatidylethanolamine and the free fatty acids palmitoleic, palmitic and oleic acid, which are known to have an antimicrobial effect. As a result of tissue autolysis, the abundance of the free fatty acids increased with longer incubation time of trophosome samples. This correlated with an increasing growth inhibition of Bacillus subtilis and Listeria welshimeri, but not of the other bacterial strains. Therefore, the free fatty acids produced upon host degradation could be the cause of inhibition of at least these two bacterial strains.

Endosymbionts escape dead hydrothermal vent tubeworms to enrich the free-living population.

Theory predicts that horizontal acquisition of symbionts by plants and animals must be coupled to release and limited dispersal of symbionts for intergenerational persistence of mutualisms. For deep-sea hydrothermal vent tubeworms (Vestimentifera, Siboglinidae), it has been demonstrated that a few symbiotic bacteria infect aposymbiotic host larvae and grow in a newly formed organ, the trophosome. However, whether viable symbionts can be released to augment environmental populations has been doubtful, because (i) the adult worms lack obvious openings and (ii) the vast majority of symbionts has been regarded as terminally differentiated. Here we show experimentally that symbionts rapidly escape their hosts upon death and recruit to surfaces where they proliferate. Estimating symbiont release from our experiments taken together with well-known tubeworm density ranges, we suggest a few million to 1.5 billion symbionts seeding the environment upon death of a tubeworm clump. In situ observations show that such clumps have rapid turnover, suggesting that release of large numbers of symbionts may ensure effective dispersal to new sites followed by active larval colonization. Moreover, release of symbionts might enable adaptations that evolve within host individuals to spread within host populations and possibly to new environments.

Size matters at deep-sea hydrothermal vents: different diversity and habitat fidelity patterns of meio- and macrofauna.

Species with markedly different sizes interact when sharing the same habitat. Unravelling mechanisms that control diversity thus requires consideration of a range of size classes. We compared patterns of diversity and community structure for meio- and macrofaunal communities sampled along a gradient of environmental stress at deep-sea hydrothermal vents on the East Pacific Rise (9° 50' N) and neighboring basalt habitats. Both meio- and macrofaunal species richnesses were lowest in the high-stress vent habitat, but macrofaunal richness was highest among intermediate-stress vent habitats. Meiofaunal species richness was negatively correlated with stress, and highest on the basalt. In these deep-sea basalt habitats surrounding hydrothermal vents, meiofaunal species richness was consistently higher than that of macrofauna. Consideration of the physiological capabilities and life history traits of different-sized animals suggests that different patterns of diversity may be caused by different capabilities to deal with environmental stress in the 2 size classes. In contrast to meiofauna, adaptations of macrofauna may have evolved to allow them to maintain their physiological homeostasis in a variety of hydrothermal vent habitats and exploit this food-rich deep-sea environment in high abundances. The habitat fidelity patterns also differed: macrofaunal species occurred primarily at vents and were generally restricted to this habitat, but meiofaunal species were distributed more evenly across proximate and distant basalt habitats and were thus not restricted to vent habitats. Over evolutionary time scales these contrasting patterns are likely driven by distinct reproduction strategies and food demands inherent to fauna of different sizes.

Conserved features and major differences in the outer membrane protein composition of chlamydiae.

Chlamydiae are a highly successful group of obligate intracellular bacteria infecting a variety of eukaryotic hosts. Outer membrane proteins involved in attachment to and uptake into host cells, and cross-linking of these proteins via disulfide bonds are key features of the biphasic chlamydial developmental cycle. In this study, we used a consensus approach to predict outer membrane proteins in the genomes of members of three chlamydial families. By analysing outer membrane protein fractions of purified chlamydiae with highly sensitive mass spectrometry, we show that the protein composition differs strongly between these organisms. Large numbers of major outer membrane protein-like proteins are present at high abundance in the outer membrane of Simkania negevensis and Waddlia chondrophila, whereas yet uncharacterized putative porins dominate in Parachlamydia acanthamoebae. Simkania represents the first case of a chlamydia completely lacking stabilizing cysteine-rich proteins in its outer membrane. In agreement with this, and in contrast to Parachlamydia and Waddlia, the cellular integrity of Simkania is not impaired by conditions that reduce disulfide bonds of these proteins. The observed differences in the protein composition of the outer membrane among members of divergent chlamydial families suggest different stabilities of these organisms in the environment, probably due to adaption to different niches or transmission routes.

Symbiont-driven sulfur crystal formation in a thiotrophic symbiosis from deep-sea hydrocarbon seeps.

The siboglinid tubeworm Sclerolinum contortum symbiosis inhabits sulfidic sediments at deep-sea hydrocarbon seeps in the Gulf of Mexico. A single symbiont phylotype in the symbiont-housing organ is inferred from phylogenetic analyses of the 16S ribosomal ribonucleic acid (16S rRNA) gene and fluorescent in situ hybridization. The phylotype we studied here, and a previous study from an arctic hydrocarbon seep population, reveal identical 16S rRNA symbiont gene sequences. While sulfide is apparently the energy source for the symbionts (and ultimately the gutless host), both partners also have to cope with its toxicity. This study demonstrates abundant large sulfur crystals restricted to the trophosome area. Based on Raman microspectroscopy and energy dispersive X-ray analysis, these crystals have the same S8 sulfur configuration as the recently described small sulfur vesicles formed in the symbionts. The crystals reside adjacent to the symbionts in the trophosome. This suggests that their formation is either extra- or intracellular in symbionts. We propose that formation of these crystals provides both energy-storage compounds for the symbionts and serves the symbiosis by removing excess toxic sulfide from host tissues. This symbiont-mediated sulfide detoxification may have been crucial for the establishment of thiotrophic symbiosis and continues to remain an important function of the symbionts.

The giant ciliate Zoothamnium niveum and its thiotrophic epibiont Candidatus Thiobios zoothamnicoli: a model system to study interspecies cooperation.

Symbioses between chemoautotrophic sulfur-oxidizing (thiotrophic) bacteria and protists or animals are among the most diverse and prevalent in the ocean. They are extremely difficult to maintain in aquaria and no thiotrophic symbiosis involving an animal host has ever been successfully cultivated. In contrast, we have cultivated the giant ciliate Zoothamnium niveum and its obligate ectosymbiont Candidatus Thiobios zoothamnicoli in small flow-through aquaria. This review provides an overview of the host and the symbiont and their phylogenetic relationships. We summarize our knowledge on the ecology, geographic distribution and life cycle of the host, on the vertical transmission of the symbiont, and on the cultivation of this symbiosis. We then discuss the benefits and costs involved in this cooperation compared with other thiotrophic symbioses and outline our view on the evolution and persistence of this byproduct mutualism.

Dual symbiosis with co-occurring sulfur-oxidizing symbionts in vestimentiferan tubeworms from a Mediterranean hydrothermal vent.

Vestimentiferan Tws colonize hydrothermal vents and cold seeps worldwide. They lack a digestive system and gain nutrition from endosymbiotic sulfur-oxidizing bacteria. It is currently assumed that vestimentiferan Tws harbour only a single endosymbiont type. A few studies found indications for additional symbionts, but conclusive evidence for a multiple symbiosis is still missing. We investigated Tws from Marsili Seamount, a hydrothermal vent in the Mediterranean Sea. Molecular and morphological analyses identified the Tws as Lamellibrachia anaximandri. 16S ribosomal RNA clone libraries revealed two distinct gammaproteobacterial phylotypes that were closely related to sequences from other Lamellibrachia symbionts. Catalysed reporter deposition fluorescence in situ hybridization with specific probes showed that these sequences are from two distinct symbionts. We also found two variants of key genes for sulfur oxidation and carbon fixation, suggesting that both symbiont types are autotrophic sulfur oxidizers. Our results therefore show that vestimentiferans can host multiple co-occurring symbiont types. Statistical analyses of vestimentiferan symbiont diversity revealed that host genus, habitat type, water depth and geographic region together accounted for 27% of genetic diversity, but only water depth had a significant effect on its own. Phylogenetic analyses showed a clear grouping of sequences according to depth, thus confirming the important role water depth played in shaping vestimentiferan symbiont diversity.

Morphology, microanatomy and sequence data of Sclerolinum contortum (Siboglindae, Annelida) of the Gulf of Mexico.

Sclerolinum is a small genus of Siboglinidae (Annelida) living in an obligate mutualistic association with thiotrophic bacteria as adults. Its taxonomic position, based on morphology, has been controversial; however, molecular data point to a sister taxa relationship with vestimentiferans. 16S rRNA gene sequencing and comparative morphology revealed that the studied population from deep-sea hydrocarbon seeps of the Gulf of Mexico belongs to Sclerolinum contortum known from the Arctic Sea. Since no anatomical and microanatomical studies have been published yet, we conducted such a study on S. contortum using serial sectioning and light and transmission electron microscopy. We show that the Sclerolinum body, divided into a head, trunk, and opisthosoma, is very similar to that of the vestimentiferans, and therefore we propose that the body regions are homologous in both taxa.

The metatrochophore of a deep-sea hydrothermal vent vestimentiferan (Polychaeta: Siboglinidae).

Vestimentiferans (Siboglinidae, Polychaeta) live as juveniles and adults in an obligate mutualistic association with thiotrophic bacteria. Since their development is aposymbiotic, metatrochophores of vestimentiferans from the East Pacific Rise colonizing deep-sea hydrothermal vents are infected with the specific symbiont, develop the trophosome, and reduce their digestive system. To gain insight into the anatomy and ultrastructure and to compare this stage with metatrochophores from other siboglinids, we serial sectioned and reconstructed three specimens using light and transmission electron microscopy. The metatrochophore was composed of a prostomium, a small peristomium, two chaetigers (or two chaetigers and one additional segment without chaetae), and a minute pygidium. A digestive system and an intraepidermal nervous system were developed. Larval organs such as the prototroch, the neurotroch, and an apical organ were present, along with juvenile/adult organs such as tentacles, uncini, pyriform glands, and the anlage of the nephridial organ. We propose that in vestimentiferans, the vestimentum is the head arising from the prostomium, peristomium, and the anterior part of the first chaetiger. In frenulates, in contrast, the head is composed on the one hand of the cephalic lobe arising from the prostomium and on the other of the forepart developing from the peristomium and the anterior part of the first chaetiger. In frenulates the muscular septum between the forepart and trunk develops later than the first two chaetigers. Since this septum has no counterpart in vestimentiferans, the forepart-trunk border of frenulates is not considered homologous with the vestimentum-trunk border in vestimentiferans. The obturacular region in vestimentiferans does not appear to be a body region but rather the head appendages arising from the first chaetiger. In contrast, the tentacles in frenulates are prostomial head appendages. In both taxa, the trunk is the posterior part of the first chaetiger, and the opisthosoma is the following chaetigers and the pygidium. Comparisons with other polychaetes suggest that two larval segments are autapomorphic for the monophyletic Siboglinidae.