Contrasting morphological and DNA barcode-suggested species boundaries among shallow-water amphipod fauna from the southern European Atlantic coast.

In this study we compared DNA barcode-suggested species boundaries with morphology-based species identifications in the amphipod fauna of the southern European Atlantic coast. DNA sequences of the cytochrome c oxidase subunit I barcode region (COI-5P) were generated for 43 morphospecies (178 specimens) collected along the Portuguese coast which, together with publicly available COI-5P sequences, produced a final dataset comprising 68 morphospecies and 295 sequences. Seventy-five BINs (Barcode Index Numbers) were assigned to these morphospecies, of which 48 were concordant (i.e., 1 BIN = 1 species), 8 were taxonomically discordant, and 19 were singletons. Twelve species had matching sequences (<2% distance) with conspecifics from distant locations (e.g., North Sea). Seven morphospecies were assigned to multiple, and highly divergent, BINs, including specimens of Corophium multisetosum (18% divergence) and Dexamine spiniventris (16% divergence), which originated from sampling locations on the west coast of Portugal (only about 36 and 250 km apart, respectively). We also found deep divergence (4%-22%) among specimens of seven species from Portugal compared to those from the North Sea and Italy. The detection of evolutionarily meaningful divergence among populations of several amphipod species from southern Europe reinforces the need for a comprehensive re-assessment of the diversity of this faunal group.

A complex picture of associations between two host mussels and symbiotic bacteria in the Northeast Atlantic.

Among chemosymbiotic metazoans found at deep-sea hydrothermal vents, cold seeps and organic falls, members of the mussel clade Bathymodiolinae (Bivalvia: Mytilidae) have evolved interactions with a higher diversity of bacterial lineages than other bivalve groups. Here, we characterized the bacteria associated with "Bathymodiolus" mauritanicus and Idas-like specimens from three sites in the Northeast Atlantic (two mud volcanoes in the Gulf of Cadiz and one seamount of the Gorringe Bank). Phylogenetic analysis of bacterial 16S rRNA-encoding gene sequences demonstrated that "B". mauritanicus has a dual symbiosis dominated by two phylotypes of methane-oxidising bacteria and a less abundant phylotype of a sulphur-oxidising bacterium. The latter was the dominant phylotype in a sympatric population of Idas-like mussels at the Darwin mud volcano. These results are the first report of a bacterial phylotype shared between two deep-sea mussels from divergent clades. This sulphur-oxidising bacterium was absent from Idas-like specimens from the other two sites (Gorringe Bank and Meknès mud volcano), in which bacterial clone libraries were dominated by other Gammaproteobacteria related to symbionts previously identified in Idas modiolaeformis from the Eastern Mediterranean. All Idas-like specimens studied herein are closely related and also related to I. modiolaeformis. However, they probably display different associations with bacteria, with the possible absence of both methane- and sulphur-oxidising symbionts at the Gorringe Bank. These results draw a very complex picture of associations between mussels and bacteria in the Northeast Atlantic, which could be highly variable depending on locale characteristics of the habitats.

Microbial diversity in Frenulata (Siboglinidae, Polychaeta) species from mud volcanoes in the Gulf of Cadiz (NE Atlantic).

Frenulates are a group of gutless marine annelids belonging to the Siboglinidae that are nutritionally dependent upon endosymbiotic bacteria. We have characterized the bacteria associated with several frenulate species from mud volcanoes in the Gulf of Cadiz by PCR-DGGE of bacterial 16S rRNA genes, coupled with analysis of 16S rRNA gene libraries. In addition to the primary symbiont, bacterial consortia (microflora) were found in all species analysed. Phylogenetic analyses indicate that the primary symbiont in most cases belongs to the Gammaproteobacteria and were related to thiotrophic and methanotrophic symbionts from other marine invertebrates, whereas members of the microflora were related to multiple bacterial phyla. This is the first molecular evidence of methanotrophic bacteria in at least one frenulate species. In addition, the occurrence of the same bacterial phylotype in different Frenulata species, from different depths and mud volcanoes suggests that there is no selection for specific symbionts and corroborates environmental acquisition as previously proposed for this group of siboglinids.

Additions to the Tanaidomorpha (Crustacea: Tanaidacea) from mud volcanoes and coral mounds of the Gulf of Cadiz and Horseshoe Continental Rise.

The Tanaidacea collection from various research cruises carried out in the Gulf of Cadiz and Horseshoe Continental Rise between 2004 and 2012 yielded four species new to science that are described herein. Two belong to genera recorded for the first time since the original descriptions of their type species: Cetiopyge, described from the Gulf of Mexico and Gamboa from shallow waters of Macaronesia. The other two belong to the genera Collettea and Paragathotanais, both with a worldwide distribution. Additionally, specimens of Tumidochelia uncinata are described and illustrated to complete previous descriptions. Identification keys to all known genera of Nototanaidae, and the Eastern Atlantic species of Paragathotanais and Collettea are provided. This works raises the number of tanaidacean species known from the deep-sea habitats in the study region to a total of 22.

Phytoplankton dynamics in relation to seasonal variability and upwelling and relaxation patterns at the mouth of Ria de Aveiro (West Iberian Margin) over a four-year period.

From June 2004 to December 2007, samples were weekly collected at a fixed station located at the mouth of Ria de Aveiro (West Iberian Margin). We examined the seasonal and inter-annual fluctuations in composition and community structure of the phytoplankton in relation to the main environmental drivers and assessed the influence of the oceanographic regime, namely changes in frequency and intensity of upwelling events, over the dynamics of the phytoplankton assemblage. The samples were consistently handled and a final subset of 136 OTUs (taxa with relative abundance > 0.01%) was subsequently submitted to various multivariate analyses. The phytoplankton assemblage showed significant changes at all temporal scales but with an overriding importance of seasonality over longer- (inter-annual) or shorter-term fluctuations (upwelling-related). Sea-surface temperature, salinity and maximum upwelling index were retrieved as the main driver of seasonal change. Seasonal signal was most evident in the fluctuations of chlorophyll a concentration and in the high turnover from the winter to spring phytoplankton assemblage. The seasonal cycle of production and succession was disturbed by upwelling events known to disrupt thermal stratification and induce changes in the phytoplankton assemblage. Our results indicate that both the frequency and intensity of physical forcing were important drivers of such variability, but the outcome in terms of species composition was highly dependent on the available local pool of species and the timing of those events in relation to the seasonal cycle. We conclude that duration, frequency and intensity of upwelling events, which vary seasonally and inter-annually, are paramount for maintaining long-term phytoplankton diversity likely by allowing unstable coexistence and incorporating species turnover at different scales. Our results contribute to the understanding of the complex mechanisms of coastal phytoplankton dynamics in relation to changing physical forcing which is fundamental to improve predictability of future prospects under climate change.

Deep-sea crustacean trawling fisheries in Portugal: quantification of effort and assessment of landings per unit effort using a Vessel Monitoring System (VMS).

Mapping and quantifying bottom trawling fishing pressure on the seafloor is pivotal to understand its effects on deep-sea benthic habitats. Using data from the Vessel Monitoring System of crustacean trawlers along the Portuguese margin, we have identified the most exploited areas and characterized the most targeted habitats and water depths. We estimated a total trawling effort of 69596, 66766, and 63427 h y-1 for the years 2012, 2013, and 2014 respectively which, considering the total landings estimated for this gear, yield values of 20.76, 21.06, and 19.11 kg of landed fish per trawled hour. The main trawling pressure is exerted in the South and Southwest Portuguese margins, on muddy and muddy-sand bottoms between 200 and 700 m water depths, while in the North and Central-West coasts a minor effort, at shallower waters and across a wider range of habitats, is also applied. The most landed species are crustaceans such as rose shrimp and Norway lobster, although this varies importantly between the different regions of Portugal, being fish and cephalopods the main captures in the Northern ports. We discuss the consequences of trawling for the impacted communities as well as the characteristics of the commercialization of these captures in Portugal.

Two new species of scale worms (Polychaeta: Aphroditiformia) from deep-sea habitats in the Gulf of Cadiz (NE Atlantic).

Two new species of scale worms are described from the Gulf of Cadiz (NE Atlantic), at depths between 1100 and 2230 m. Australaugeneria iberica sp. nov. (Polynoidae) was obtained from an alcyonarian colony collected at the flank of Carlos Ribeiro mud volcano; it is characterized by the presence of neuropodial hooks only on segment two and by having the first parapodia not enlarged. This is the first report of the genus for the deep sea. The diagnosis of Australaugeneria is emended and a table comparing all species of the genus is provided. Pholoe petersenae sp. nov. (Pholoidae) was collected from the crater of three mud volcanoes (Darwin, Captain Arutyunov and Carlos Ribeiro) in areas of active seepage. This species is characterized by the presence of prostomial peaks and parapodia stylodes and the absence of eyes.

Integrated analysis of bacterial and microeukaryotic communities from differentially active mud volcanoes in the Gulf of Cadiz.

The present study assesses the diversity and composition of sediment bacterial and microeukaryotic communities from deep-sea mud volcanoes (MVs) associated with strike-slip faults in the South-West Iberian Margin (SWIM). We used a 16S/18S rRNA gene based pyrosequencing approach to characterize and correlate the sediment bacterial and microeukaryotic communities from MVs with differing gas seep regimes and from an additional site with no apparent seeping activity. In general, our results showed significant compositional changes of bacterial and microeukaryotic communities in sampling sites with different seepage regimes. Sediment bacterial communities were enriched with Methylococcales (putative methanotrophs) but had lower abundances of Rhodospirillales, Nitrospirales and SAR202 in the more active MVs. Within microeukaryotic communities, members of the Lobosa (lobose amoebae) were enriched in more active MVs. We also showed a strong correlation between Methylococcales populations and lobose amoeba in active MVs. This study provides baseline information on the diversity and composition of bacterial and microeukaryotic communities in deep-sea MVs associated with strike-slip faults.

New Prionospio and Aurospio Species from the Deep Sea (Annelida: Polychaeta).

The number of records of the genus Prionospio Malmgren, 1867, from the deep sea (>2000 m) are relatively few and do not reflect the actual occurrence of species nor their potential ecological importance. In this paper we describe five new species of this genus (Prionospio amarsupiata sp. nov., P. vallensis sp. nov., P. branchilucida sp. nov., P. hermesia sp. nov. and P. kaplani sp. nov.) all of which are abundant members of the deep-sea community. We also describe two new species of the genus Aurospio Maciolek, 1981 (Aurospio abranchiata sp. nov. and A. tribranchiata sp. nov.) again common elements of the abyssal fauna. Two of the new species have characters which question the generic distinctiveness of Prionospio and Aurospio. The problems in differentiating these two genera are discussed.

Effects of sample storage and shell orientation on LA-ICPMS trace element measurements on deep-sea mussels.

Geochemical markers are being increasingly applied to fundamental questions in population and community ecology in marine habitats because they allow inferences on individuals dispersal, but vital effects, small sample size and instrumental limitation are still challenging particularly in deep-sea studies. Here we use shells of the deep-sea bivalve Idas modiolaeformis to assess potential effects of sample storage, mineralogy, and valve orientation on LA-ICPMS measurements. Trace element concentrations of (24)Mg, (43)Ca, (88)Sr, (137)Ba, (208)Pb, and (238)U are not affected by the two most commonly used storage methods of biologic deep-sea samples (frozen at -20°C and fixed in 95% ethanol); thus combined analysis of differently preserved specimens is possible when the number of individuals is insufficient and distinct sample fixation is needed for multiple purposes. Valve orientation had a strong impact on quantification of trace elements in the calcitic but not in the aragonitic layer of adult shells. Hence, to enable comparisons between adult shells and entirely aragonitic embryonic shells, a reference map of site-specific signatures can potentially be generated using the aragonitic layer of the adult shells. Understanding ontogenetic changes and environmental effects in trace element incorporation is critical before geochemical fingerprinting can be used as a tool for larval dispersal studies in the deep-sea.

Adapted to change: The rapid development of symbiosis in newly settled, fast-maturing chemosymbiotic mussels in the deep sea.

Symbioses between microbiota and marine metazoa occur globally at chemosynthetic habitats facing imminent threat from anthropogenic disturbance, yet little is known concerning the role of symbiosis during early development in chemosymbiotic metazoans: a critical period in any benthic species' lifecycle. The emerging symbiosis of Idas (sensu lato) simpsoni mussels undergoing development is assessed over a post-larval-to-adult size spectrum using histology and fluorescence in situ hybridisation (FISH). Post-larval development shows similarities to that of both heterotrophic and chemosymbiotic mussels. Data from newly settled specimens confirm aposymbiotic, planktotrophic larval development. Sulphur-oxidising (SOX) symbionts subsequently colonise multiple exposed, non-ciliated epithelia shortly after metamorphosis, but only become abundant on gills as these expand with greater host size. This wide-spread bathymodiolin recorded from sulphidic wood, bone and cold-seep habitats, displays a suite of adaptive traits that could buffer against anthropogenic disturbance.

Colonization of plant substrates at hydrothermal vents and cold seeps in the northeast Atlantic and Mediterranean and occurrence of symbiont-related bacteria.

Reducing conditions with elevated sulfide and methane concentrations in ecosystems such as hydrothermal vents, cold seeps or organic falls, are suitable for chemosynthetic primary production. Understanding processes driving bacterial diversity, colonization and dispersal is of prime importance for deep-sea microbial ecology. This study provides a detailed characterization of bacterial assemblages colonizing plant-derived substrates using a standardized approach over a geographic area spanning the North-East Atlantic and Mediterranean. Wood and alfalfa substrates in colonization devices were deployed for different periods at 8 deep-sea chemosynthesis-based sites in four distinct geographic areas. Pyrosequencing of a fragment of the 16S rRNA-encoding gene was used to describe bacterial communities. Colonization occurred within the first 14 days. The diversity was higher in samples deployed for more than 289 days. After 289 days, no relation was observed between community richness and deployment duration, suggesting that diversity may have reached saturation sometime in between. Communities in long-term deployments were different, and their composition was mainly influenced by the geographical location where devices were deployed. Numerous sequences related to horizontally-transmitted chemosynthetic symbionts of metazoans were identified. Their potential status as free-living forms of these symbionts was evaluated based on sequence similarity with demonstrated symbionts. Results suggest that some free-living forms of metazoan symbionts or their close relatives, such as Epsilonproteobacteria associated with the shrimp Rimicaris exoculata, are efficient colonizers of plant substrates at vents and seeps.

Fickle or Faithful: The Roles of Host and Environmental Context in Determining Symbiont Composition in Two Bathymodioline Mussels.

The Mediterranean Sea and adjoining East Atlantic Ocean host a diverse array of small-sized mussels that predominantly live on sunken, decomposing organic remains. At least two of these, Idas modiolaeformis and Idas simpsoni, are known to engage in gill-associated symbioses; however, the composition, diversity and variability of these symbioses with changing habitat and location is poorly defined. The current study presents bacterial symbiont assemblage data, derived from 454 pyrosequencing carried out on replicate specimens of these two host species, collected across seven sample sites found in three oceanographic regions in the Mediterranean and East Atlantic. The presence of several bacterial OTUs in both the Mediterranean Sea and eastern Atlantic suggests that similar symbiont candidates occur on both sides of the Strait of Gibraltar. The results reveal markedly different symbiotic modes in the two species. Idas modiolaeformis displays high symbiont diversity and flexibility, with strong variation in symbiont composition from the East Mediterranean to the East Atlantic. Idas simpsoni displays low symbiont diversity but high symbiont fidelity, with a single dominant OTU occurring in all specimens analysed. These differences are argued to be a function of the host species, where subtle differences in host evolution, life-history and behaviour could partially explain the observed patterns. The variability in symbiont compositions, particularly in Idas modiolaeformis, is thought to be a function of the nature, context and location of the habitat from which symbiont candidates are sourced.

Are organic falls bridging reduced environments in the deep sea? - results from colonization experiments in the Gulf of Cádiz.

Organic falls create localised patches of organic enrichment and disturbance where enhanced degradation is mediated by diversified microbial assemblages and specialized fauna. The view of organic falls as "stepping stones" for the colonization of deep-sea reducing environments has been often loosely used, but much remains to be proven concerning their capability to bridge dispersal among such environments. Aiming the clarification of this issue, we used an experimental approach to answer the following questions: Are relatively small organic falls in the deep sea capable of sustaining taxonomically and trophically diverse assemblages over demographically relevant temporal scales? Are there important depth- or site-related sources of variability for the composition and structure of these assemblages? Is the proximity of other reducing environments influential for their colonization? We analysed the taxonomical and trophic diversity patterns and partitioning (α- and ß-diversity) of the macrofaunal assemblages recruited in small colonization devices with organic and inorganic substrata after 1-2 years of deployment on mud volcanoes of the Gulf of Cádiz. Our results show that small organic falls can sustain highly diverse and trophically coherent assemblages for time periods allowing growth to reproductive maturity, and successive generations of dominant species. The composition and structure of the assemblages showed variability consistent with their biogeographic and bathymetric contexts. However, the proximity of cold seeps had limited influence on the similarity between the assemblages of these two habitats and organic falls sustained a distinctive fauna with dominant substrate-specific taxa. We conclude that it is unlikely that small organic falls may regularly ensure population connectivity among cold seeps and vents. They may be a recurrent source of evolutionary candidates for the colonization of such ecosystems. However, there may be a critical size of organic fall to create the necessary intense and persistent reducing conditions for sustaining typical chemosymbiotic vent and seep organisms.

Organotin levels in Nazaré canyon (west Iberian Margin, NE Atlantic) and adjacent coastal area.

Organotin compounds (OTs) are ubiquitous in the marine environment and high concentrations (µg g(-1) range) in sediments from different coastal areas around the world have been reported. However, few reports have described the OTs contamination status in the offshore and deep sea environment. This work investigated organotin levels in Nazaré canyon for the first time. Levels of monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), diphenyltin (DPT), triphenyltin (TPT), dioctyltin (DOT) and trioctyltin (TOT) were quantified in sediment samples from the upper flanks of the canyon and from the adjacent coastal area. TBT levels detected in the canyon flanks are about two to three orders of magnitude lower than those found in the coastal area. Nevertheless, when quantifiable, TBT levels in the canyon samples were higher than the Environmental Assessment Criteria set for TBT in sediments by the OSPAR Commission indicating that at those locations negative ecological impacts are likely to occur.

Chemosymbiotic bivalves from the mud volcanoes of the Gulf of Cadiz, NE Atlantic, with descriptions of new species of Solemyidae, Lucinidae and Vesicomyidae.

The chemosymbiotic bivalves collected from the mud volcanoes of the Gulf of Cadiz are reviewed. Of the thirteen species closely associated with chemosynthetic settings two Solemyidae, Solemya (Petrasma) elarraichensissp. n. and Acharax gadiraesp. n., one Lucinidae, Lucinoma asapheussp. n., and one Vesicomyidae, Isorropodon megadesmussp. n. are described and compared to close relatives of their respective families. The biodiversity and distribution of the chemosymbiotic bivalves in the Gulf of Cadiz are discussed and compared to the available information from other cold seeps in the Eastern Atlantic and Mediterranean. Although there is considerable similarity at the genus level between seep/mud volcano fields in the Eastern Atlantic and Mediterranean, there is little overlap at the species level. This indicates a high degree of endemism within chemosymbiotic bivalve assemblages.

Systematic and evolutionary insights derived from mtDNA COI barcode diversity in the Decapoda (Crustacea: Malacostraca).

BACKGROUND: Decapods are the most recognizable of all crustaceans and comprise a dominant group of benthic invertebrates of the continental shelf and slope, including many species of economic importance. Of the 17635 morphologically described Decapoda species, only 5.4% are represented by COI barcode region sequences. It therefore remains a challenge to compile regional databases that identify and analyse the extent and patterns of decapod diversity throughout the world. METHODOLOGY/PRINCIPAL FINDINGS: We contributed 101 decapod species from the North East Atlantic, the Gulf of Cadiz and the Mediterranean Sea, of which 81 species represent novel COI records. Within the newly-generated dataset, 3.6% of the species barcodes conflicted with the assigned morphological taxonomic identification, highlighting both the apparent taxonomic ambiguity among certain groups, and the need for an accelerated and independent taxonomic approach. Using the combined COI barcode projects from the Barcode of Life Database, we provide the most comprehensive COI data set so far examined for the Order (1572 sequences of 528 species, 213 genera, and 67 families). Patterns within families show a general predicted molecular hierarchy, but the scale of divergence at each taxonomic level appears to vary extensively between families. The range values of mean K2P distance observed were: within species 0.285% to 1.375%, within genus 6.376% to 20.924% and within family 11.392% to 25.617%. Nucleotide composition varied greatly across decapods, ranging from 30.8 % to 49.4 % GC content. CONCLUSIONS/SIGNIFICANCE: Decapod biological diversity was quantified by identifying putative cryptic species allowing a rapid assessment of taxon diversity in groups that have until now received limited morphological and systematic examination. We highlight taxonomic groups or species with unusual nucleotide composition or evolutionary rates. Such data are relevant to strategies for conservation of existing decapod biodiversity, as well as elucidating the mechanisms and constraints shaping the patterns observed.

Multigene molecular systematics confirm species status of morphologically convergent Pagurus hermit crabs.

INTRODUCTION: In spite of contemporary morphological taxonomy appraisals, apparent high morphological similarity raises uncertainty about the species status of certain Pagurus hermit crabs. This is exemplified between two European species, Pagurus excavatus (Herbst, 1791) and Pagurus alatus (Fabricius 1775), whose species status is still difficult to resolve using morphological criteria alone. METHODOLOGY/PRINCIPAL FINDINGS: To address such ambiguities, we used combinations of Maximum Likelihood (ML) and Bayesian Inference (BI) methods to delineate species boundaries of P. alatus and P. excavatus and formulate an intermediate Pagurus phylogenetic hypothesis, based upon single and concatenated mitochondrial (cytochrome oxidase I [COI]) and nuclear (16S and 28s ribosomal RNA) gene partitions. The molecular data supported the species status of P. excavatus and P. alatus and also clearly resolved two divergent clades within hermit crabs from the Northeast Atlantic Ocean and the Mediterranean Sea. CONCLUSIONS/SIGNIFICANCE: Despite the abundance and prominent ecological role of hermit crabs, Pagurus, in North East Atlantic Ocean and Mediterranean Sea ecosystems, many important aspects of their taxonomy, biology, systematics and evolution remain poorly explored. The topologies presented here should be regarded as hypotheses that can be incorporated into the robust and integrated understanding of the systematic relationships within and between species of the genus Pagurus inhabiting the Northeast Atlantic Ocean and the Mediterranean Sea.

Chemosynthetic bacteria found in bivalve species from mud volcanoes of the Gulf of Cadiz.

As in other cold seeps, the dominant bivalves in mud volcanoes (MV) from the Gulf of Cadiz are macrofauna belonging to the families Solemyidae (Acharax sp., Petrasma sp.), Lucinidae (Lucinoma sp.), Thyasiridae (Thyasira vulcolutre) and Mytilidae (Bathymodiolus mauritanicus). The delta(13)C values measured in solemyid, lucinid and thyasirid specimens support the hypothesis of thiotrophic nutrition, whereas isotopic signatures of B. mauritanicus suggest methanotrophic nutrition. The indication by stable isotope analysis that chemosynthetic bacteria make a substantial contribution to the nutrition of the bivalves led us to investigate their associated bacteria and their phylogenetic relationships based on comparative 16S rRNA gene sequence analysis. PCR-denaturing gradient gel electrophoresis analysis and cloning of bacterial 16S rRNA-encoding genes confirmed the presence of sulfide-oxidizing symbionts within gill tissues of many of the studied specimens. Phylogenetic analysis of bacterial 16S rRNA gene sequences demonstrated that most bacteria were related to known sulfide-oxidizing endosymbionts found in other deep-sea chemosynthetic environments, with the co-occurrence of methane-oxidizing symbionts in Bathymodiolus specimens. This study confirms the presence of several chemosynthetic bivalves in the Gulf of Cadiz and further highlights the importance of sulfide- and methane-oxidizing symbionts in the trophic ecology of macrobenthic communities in MV.

Comparative myoanatomy of cycliophoran life cycle stages.

The metazoan phylum Cycliophora includes small cryptic epibionts that live attached to the mouthparts of clawed lobsters. The life cycle is complex, with alternating sexual and asexual generations, and involves several sessile and free-living stages. So far, the morphological and genetic characterization of cycliophorans has been unable to clarify the phylogenetic position of the phylum. In this study, we add new details on the muscular anatomy of the feeding stage, the attached Prometheus larva, the dwarf male, and the female of one of the two hitherto described species, Symbion pandora. The musculature of the feeding stage is composed of myofibers that run longitudinally in the buccal funnel (two fibers) and in the trunk (variable number of fibers). The mouth opening is lined by a myoepithelial ring musculature. A complex myoepithelial sphincter is situated proximal to the anus. In the attached Prometheus larva, three longitudinal sets of myofilaments run dorsally, laterally, and ventrally along the entire anterior-posterior body axis. The muscular architecture of the dwarf male is complex, especially close to the penis, in the posterior part of the body. An X-shaped muscle structure is found on the dorsal side, whereas on the ventral side, longitudinal muscles and a V-shaped muscle structure are present. These muscles are complemented by additional dorsoventral muscles. The mesodermal muscle fibers attach to the cuticle via the epidermis in all life cycle stages studied herein. The musculature of the female is similar to that of the Pandora larva of Symbion americanus and includes dorsoventral muscles and longitudinal muscles that run in the dorsal and ventral body region. Overall, our results reveal striking similarities in the muscular arrangement of the life cycle stages of both Symbion species.