Molecular detection of a novel perkinsid associated with the deep-sea clam Phreagena okutanii.

Based on environmental DNA surveys, it is widely held that phylogenetically diverse protists exist in chemosynthetic ecosystems. However, knowledge regarding the protists associated with the endemic animals inhabiting these environments is still very limited. In the present study, utilizing polymerase chain reaction (PCR) techniques, we detected fragments of the small subunit ribosomal RNA (SSU rRNA) gene and the internal transcribed spacer (ITS) region of the ribosomal RNA genes from a particular protist in the gills of the vesicomyid clam Phreagena okutanii (formerly described as Calyptogena okutanii), a representative animal in chemosynthetic ecosystems. Based on the phylogeny of the SSU rRNA gene, the organism in question belongs to the genus Perkinsus, which is exclusively composed of protistan parasites infecting mollusks. Intriguingly, based on the ITS phylogeny, this protist was not related to any known Perkinsus species and was deeply branched within the radiation of this genus, thus represents an undescribed species. In addition, the protist detected by PCR was localized to the intercellular spaces in the gills of the host clam with fluorescence in situ hybridization. Although the ecological significance of this novel deep-sea perkinsid remains unclear, our present findings may provide important insights into the diversity of the genus Perkinsus.

Microbial Eukaryotes that Lack Sterols.

It is widely held that sterols are key cyclic triterpenoid lipids in eukaryotic cell membranes and are synthesized through oxygen-dependent multienzyme pathways. However, there are known exceptions-ciliated protozoans, such as Tetrahymena, along with diverse low-oxygen-adapted eukaryotes produce, instead of sterols, the cyclic triterpenoid lipid tetrahymanol that does not require molecular oxygen for its biosynthesis. Here, we report that a number of anaerobic microbial eukaryotes (protists) utilize neither sterols nor tetrahymanol in their membranes. The lack of detectable sterol-like compounds in their membranes may provide an opportunity to reconsider the physiological function of sterols and sterol-like lipids in eukaryotes.

Complex evolution of two types of cardiolipin synthase in the eukaryotic lineage stramenopiles.

The phospholipid cardiolipin is indispensable for eukaryotes to activate mitochondria, and it was previously reported that two phylogenetically distinct types of enzyme synthesizing cardiolipin, one with two phospholipase D domains (CLS_pld) and the other with a CDP-alcohol phosphatidyltransferase domain (CLS_cap), are patchily and complementarily distributed at higher taxonomic (e.g., supergroup) levels of eukaryotes. Stramenopiles, one of the major eukaryotic clades, were considered to exclusively possess CLS_cap. However, through our present surveys with genome or transcriptome data from a broad range of stramenopile taxa, species with both CLS_cap and CLS_pld and species with only CLS_pld or CLS_cap were discovered among this group. Because these homologues of CLS_cap and CLS_pld retrieved from stramenopiles were likely inherited from the last eukaryotic common ancestor, it is reasonable to assume that a common ancestor of all stramenopiles harbored both CLS_cap and CLS_pld. Furthermore, based on the robust organismal phylogeny of stramenopiles unveiled with large-scale phylogenetic analyses, the earliest diverging lineage of stramenopiles (including bicosoecids, placidids, etc.) was found to comprise species with both CLS_cap and CLS_pld along with species with only either CLS_cap or CLS_pld. These findings suggest that a common ancestor of the most basal stramenopile lineage retained these two vertically inherited enzymes and that differential losses of either CLS_cap or CLS_pld occurred in this lineage. On the other hand, in the other stramenopile lineage composed of Ochrophyta, Pseudofungi, and Labyrinthulomycetes (to the exclusion of the most basal lineage), only CLS_cap was found, and therefore a common ancestor of these three groups likely lost CLS_pld. Based on our findings, the evolution of CLS_cap/CLS_pld in stramenopiles appears to be more complex than previously thought.

Morphological Identities of Two Different Marine Stramenopile Environmental Sequence Clades: Bicosoeca kenaiensis (Hilliard, 1971) and Cantina marsupialis (Larsen and Patterson, 1990) gen. nov., comb. nov.

Although environmental DNA surveys improve our understanding of biodiversity, interpretation of unidentified lineages is limited by the absence of associated morphological traits and living cultures. Unidentified lineages of marine stramenopiles are called "MAST clades". Twenty-five MAST clades have been recognized: MAST-1 through MAST-25; seven of these have been subsequently discarded because the sequences representing those clades were found to either (1) be chimeric or (2) affiliate within previously described taxonomic groups. Eighteen MAST clades remain without a cellular identity. Moreover, the discarded "MAST-13" has been used in different studies to refer to two different environmental sequence clades. After establishing four cultures representing two different species of heterotrophic stramenopiles and then characterizing their morphology and molecular phylogenetic positions, we determined that the two different species represented the two different MAST-13 clades: (1) a lorica-bearing Bicosoeca kenaiensis and (2) a microaerophilic flagellate previously named "Cafeteria marsupialis". Both species were previously described with only light microscopy; no cultures, ultrastructural data or DNA sequences were available from these species prior to this study. The molecular phylogenetic position of three different "C. marsupialis" isolates was not closely related to the type species of Cafeteria; therefore, we established a new genus for these isolates, Cantina gen. nov.

Uncovering sibling species in Radiolaria: evidence for ecological partitioning in a marine planktonic protist.

Phylogeography of unicellular plankton, as representative pelagic organisms, is fundamental to understanding their evolution in the ocean. Historically, these microplankton were believed to have cosmopolitan distributions achieved through passive transport and little potential for speciation because of a lack of geographic barriers in the oceans. Recent phylogeographic studies of these microplankton, however, have often revealed high diversity and fine-scale geographic distributions. These apparent contradictions may result from poor knowledge of the spatial distributions of pelagic microplankton in the water column. More information about both geographic and vertical distributions of pelagic populations could reveal the dispersal pathways, gene flow, and resulting diversifications in the open ocean. Here we demonstrate that two genetic types of the radiolarian morphospecies Spongotrochus glacialis with morphological differences are vertically segregated into the upper and lower surface waters within the pycnocline of the North Pacific Subtropical Water. This vertically separated distribution of two sister species is associated with distinct ecological partitioning. These two species could survive on different food resources from their respective environments: one in oligotrophic surface waters by using nutrients from symbionts, and the other at greater depths by depending on both heterotrophic and symbiotic nutrition. Moreover, molecular divergence-time estimates suggest that the two species diverged during the period of oligotrophic surface-water development in the Pacific Ocean. Our findings suggest that genetic isolation in the vertical dimension occurs through ecological partitioning even in the absence of physical barriers in the pelagic oceans.

A novel alveolate in bivalves with chemosynthetic bacteria inhabiting deep-sea methane seeps.

It has recently been unveiled that a wide variety of microbial eukaryotes (protists) occur in chemosynthetic ecosystems, such as hydrothermal vents and methane seeps. However, there is little knowledge regarding protists associated with endemic animals inhabiting these environments. In the present study, utilizing PCR techniques, we detected fragments of the small subunit ribosomal RNA gene (SSU rRNA gene) from a particular protist from gill tissues of a significant fraction of the vesicomyid clams Calyptogena soyoae and C. okutanii complex and of the mussel Bathymodiolus platifrons and B. japonicus, all of which harbor chemosynthetic endosymbiont bacteria and dominate methane seeps in Sagami Bay, Japan. Based on the phylogeny of SSU rRNA gene, the organism in question was shown to belong to Alveolata. It is noteworthy that this protist did not affiliate with any known alveolate group, although being deeply branched within the lineage of Syndiniales, for which the monophyly was constantly recovered, but not robustly supported. In addition, the protist detected using PCR followed by sequencing was localized within gill epithelial cells of B. platifrons with whole-mount fluorescence in situ hybridization. This protist may be an endoparasite or an endocommensal of Calyptogena spp. and Bathymodiolus spp., and possibly have physiological and ecological impacts on these bivalves.

mTORC1 regulates phagosome digestion of symbiotic bacteria for intracellular nutritional symbiosis in a deep-sea mussel.

Phagocytosis is one of the methods used to acquire symbiotic bacteria to establish intracellular symbiosis. A deep-sea mussel, Bathymodiolus japonicus, acquires its symbiont from the environment by phagocytosis of gill epithelial cells and receives nutrients from them. However, the manner by which mussels retain the symbiont without phagosome digestion remains unknown. Here, we show that controlling the mechanistic target of rapamycin complex 1 (mTORC1) in mussels leads to retaining symbionts in gill cells. The symbiont is essential for the host mussel nutrition; however, depleting the symbiont's energy source triggers the phagosome digestion of symbionts. Meanwhile, the inhibition of mTORC1 by rapamycin prevented the digestion of the resident symbionts and of the engulfed exogenous dead symbionts in gill cells. This indicates that mTORC1 promotes phagosome digestion of symbionts under reduced nutrient supply from the symbiont. The regulation mechanism of phagosome digestion by mTORC1 through nutrient signaling with symbionts is key for maintaining animal-microbe intracellular nutritional symbiosis.

Evolution of elongation factor-like (EFL) protein in Rhizaria is revised by radiolarian EFL gene sequences.

Elongation factor 1α (EF-1α) and elongation factor-like (EFL) proteins are considered to carry out equivalent functions in translation in eukaryotic cells. Elongation factor 1α and EFL genes are patchily distributed in the global eukaryotic tree, suggesting that the evolution of these elongation factors cannot be reconciled without multiple lateral gene transfer and/or ancestral co-occurrence followed by differential loss of either of the two factors. Our current understanding of the EF-1α/EFL evolution in the eukaryotic group Rhizaria, composed of Foraminifera, Radiolaria, Filosa, and Endomyxa, remains insufficient, as no information on EF-1α/EFL gene is available for any members of Radiolaria. In this study, EFL genes were experimentally isolated from four polycystine radiolarians (i.e. Dictyocoryne, Eucyrtidium, Collozoum, and Sphaerozoum), as well as retrieved from publicly accessible expressed sequence tag data of two acantharean radiolarians (i.e. Astrolonche and Phyllostaurus) and the endomyxan Gromia. The EFL homologs from radiolarians, foraminiferans, and Gromia formed a robust clade in both maximum-likelihood and Bayesian phylogenetic analyses, suggesting that EFL genes were vertically inherited from their common ancestor. We propose an updated model for EF-1α/EFL evolution in Rhizaria by incorporating new EFL data obtained in this study.

Opsins in the Cephalic and Extracephalic Photoreceptors in the Marine Gastropod Onchidium verruculatum.

AbstractThe marine gastropod Onchidium verruculatum has a pair of ocular photoreceptors, the stalk eyes, on the tip of its stalk near the head, as well as several extracephalic photosensory organs. The retinas of the stalk eye consist of two morphologically distinct visual cells, namely, the type I cells equipped with well-developed microvilli and the type II cells with less developed microvilli. The extracephalic photosensors comprise the dorsal eye, dermal photoreceptor, and brain photosensitive neurons. The characteristics of these cephalic and extracephalic photosensory organs have been studied from morphological and electrophysiological perspectives. However, little is known about the visual pigment molecules responsible for light detection in these organs. In the present study, we searched for opsin molecules that are expressed in the neural tissues of Onchidium and identified six putative signaling-competent opsin species, including Xenopsin1, Xenopsin2, Gq-coupled rhodopsin1, Gq-coupled rhodopsin2, Opsin-5B, and Gq-coupled rhodopsin-like. Immunohistochemical staining of four of the six opsins revealed that Xenopsin1, Gq-coupled rhodopsin1, and Gq-coupled rhodopsin2 are expressed in the rhabdomere of the stalk eye and in the dermal photoreceptor. Xenopsin2 was expressed in the type II photoreceptors of the stalk eye and in the ciliary photoreceptors of the dorsal eye. These immunohistochemical data were consistent with the results of the expression analysis, revealed by quantitative reverse transcription polymerase chain reaction. This study clarified the identities of opsins expressed in the extracephalic photosensory organs of Onchidium and the distinct molecular compositions among the photoreceptors.

A phylogenetic mosaic plastid proteome and unusual plastid-targeting signals in the green-colored dinoflagellate Lepidodinium chlorophorum.

BACKGROUND: Plastid replacements through secondary endosymbioses include massive transfer of genes from the endosymbiont to the host nucleus and require a new targeting system to enable transport of the plastid-targeted proteins across 3-4 plastid membranes. The dinoflagellates are the only eukaryotic lineage that has been shown to have undergone several plastid replacement events, and this group is thus highly relevant for studying the processes involved in plastid evolution. In this study, we analyzed the phylogenetic origin and N-terminal extensions of plastid-targeted proteins from Lepidodinium chlorophorum, a member of the only dinoflagellate genus that harbors a green secondary plastid rather than the red algal-derived, peridinin-containing plastid usually found in photosynthetic dinoflagellates. RESULTS: We sequenced 4,746 randomly picked clones from a L. chlorophorum cDNA library. 22 of the assembled genes were identified as genes encoding proteins functioning in plastids. Some of these were of green algal origin. This confirms that genes have been transferred from the plastid to the host nucleus of L. chlorophorum and indicates that the plastid is fully integrated as an organelle in the host. Other nuclear-encoded plastid-targeted protein genes, however, are clearly not of green algal origin, but have been derived from a number of different algal groups, including dinoflagellates, streptophytes, heterokonts, and red algae. The characteristics of N-terminal plastid-targeting peptides of all of these genes are substantially different from those found in peridinin-containing dinoflagellates and green algae. CONCLUSIONS: L. chlorophorum expresses plastid-targeted proteins with a range of different origins, which probably arose through endosymbiotic gene transfer (EGT) and horizontal gene transfer (HGT). The N-terminal extension of the genes is different from the extensions found in green alga and other dinoflagellates (peridinin- and haptophyte plastids). These modifications have likely enabled the mosaic proteome of L. chlorophorum.

Reduced genome of the thioautotrophic intracellular symbiont in a deep-sea clam, Calyptogena okutanii.

Although dense animal communities at hydrothermal vents and cold seeps rely on symbioses with chemoautotrophic bacteria [1, 2], knowledge of the mechanisms underlying these chemosynthetic symbioses is still fragmentary because of the difficulty in culturing the symbionts and the hosts in the laboratory. Deep-sea Calyptogena clams harbor thioautotrophic bacterial symbionts in their gill epithelial cells [1, 2]. They have vestigial digestive tracts and nutritionally depend on their symbionts [3], which are vertically transmitted via eggs [4]. To clarify the symbionts' metabolic roles in the symbiosis and adaptations to intracellular conditions, we present the complete genome sequence of the symbiont of Calyptogena okutanii. The genome is a circular chromosome of 1,022,154 bp with 31.6% guanine + cytosine (G + C) content, and is the smallest reported genome in autotrophic bacteria. It encodes 939 protein-coding genes, including those for thioautotrophy and for the syntheses of almost all amino acids and various cofactors. However, transporters for these substances to the host cell are apparently absent. Genes that are unnecessary for an intracellular lifestyle, as well as some essential genes (e.g., ftsZ for cytokinesis), appear to have been lost from the symbiont genome. Reductive evolution of the genome might be ongoing in the vertically transmitted Calyptogena symbionts.

A wide diversity of previously undetected free-living relatives of diplomonads isolated from marine/saline habitats.

Over the last 15 years classical culturing and environmental PCR techniques have revealed a modest number of genuinely new major lineages of protists; however, some new groups have greatly influenced our understanding of eukaryote evolution. We used culturing techniques to examine the diversity of free-living protists that are relatives of diplomonads and retortamonads, a group of evolutionary and parasitological importance. Until recently, a single organism, Carpediemonas membranifera, was the only representative of this region of the tree. We report 18 new isolates of Carpediemonas-like organisms (CLOs) from anoxic marine sediments. Only one is a previously cultured species. Eleven isolates are conspecific and were classified within a new genus, Kipferlia n. gen. The remaining isolates include representatives of three other lineages that likely represent additional undescribed genera (at least). Small-subunit ribosomal RNA gene phylogenies show that CLOs form a cloud of six major clades basal to the diplomonad-retortamonad grouping (i.e. each of the six CLO clades is potentially as phylogenetically distinct as diplomonads and retortamonads). CLOs will be valuable for tracing the evolution of diplomonad cellular features, for example, their extremely reduced mitochondrial organelles. It is striking that the majority of CLO diversity was undetected by previous light microscopy surveys and environmental PCR studies, even though they inhabit a commonly sampled environment. There is no reason to assume this is a unique situation - it is likely that undersampling at the level of major lineages is still widespread for protists.

Molecular evidence that phylogenetically diverged ciliates are active in microbial mats of deep-sea cold-seep sediment.

Cold seeps are areas of the seafloor where hydrogen sulfide- and methane-rich fluid seepage occurs, often sustaining chemosynthetic ecosystems. It is well known that both archaea and bacteria oxidize sulfides and methane to produce chemical energy and that several endemic animals use this energy to thrive in cold seeps. On the other hand, there is little knowledge regarding diversity and ecology of microbial eukaryotes in this ecosystem. In this study we isolated environmental RNA and DNA from microbial mats of cold-seep sediment in Sagami Bay, Japan, and retrieved eukaryotic small-subunit ribosomal RNA sequences with polymerase chain reaction methods followed by clone library construction. Most RNA-derived clones obtained were from ciliates, although DNA-derived clones were mainly from the fungus Cryptococcus curvatus, suggesting that ciliates are active in the environment. The ciliate sequences were phylogenetically diverse, and represented eight known class lineages as well as undesignated lineages. Because most ciliates are bacterivorous, it is highly likely that the ciliates for which sequences were recovered play a role in the food web of this ecosystem as grazers of microbial mats. In addition, given that the environment studied is under highly reduced (anoxic) conditions, based on the prokaryotic community structure deduced from T-RFLP profiles, the ciliates detected may be obligatory or facultative anaerobes.

Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum.

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.

Molecular identification of the ichthyosporean protist "Pseudoperkinsus tapetis" from the mytilid mussel Adipicola pacifica associated with submerged whale carcasses in Japan.

A protist tentatively designated "Pseudoperkinsus tapetis" belonging to the eukaryotic group Ichthyosporea (Mesomycetozoa) was previously isolated from carpet shell clams in Galicia (northwest Spain). In the present study, based on molecular data, a potential P. tapetis specimen was identified from the gill tissues of the mussel Adipicola pacifica associated with whale carcasses (generating chemosynthetic-based ecosystems) collected at shelf depths in the northwest Pacific (southwest Japan). Small subunit ribosomal DNA sequences (1751 sites) of the genotypes of P. tapetis from Spain and Japan were almost identical (only one substitution and one insertion/deletion difference). On the other hand, differences of 10 and 8 substitutions were found in two internal transcribed spacer regions of ribosomal DNA, ITS1 (288 sites) and ITS2 (251 sites) between these two genotypes, respectively, indicating that they are genetically different at the population level. These findings suggest that P. tapetis occurs worldwide and can associate with (and possibly infect) various types of bivalves. Further, a PCR method to specifically detect the P. tapetis cells in the host was also established.

Quellenin, a new anti-Saprolegnia compound isolated from the deep-sea fungus, Aspergillus sp. YK-76.

Saprolegnia parasitica, belonging to oomycetes, is one of virulent pathogen of fishes such as salmon and trout, and causes tremendous damage and losses in commercial aquacultures by saprolegniasis. Previously, malachite green, an effective medicine, had been used to control saprolegniasis. However, this drug has been banned around the world due to its mutagenicity. Therefore, novel anti-saprolegniasis compounds are urgently needed. As a new frontier to discover bioactive compounds, we focused on the deep-sea fungi for the isolation of anti-saprolegniasis compounds. In this paper, on the course of anti-saprolegniasis agents from 546 cultured broths of 91 deep-sea fungal strains, we report a new compound, named quellenin (1) together with three known compounds, diorcinol (2), violaceol-I (3) and violaceol-II (4), from deep-sea fungus Aspergillus sp. YK-76. This strain was isolated from an Osedax sp. annelid, commonly called bone-eating worm, collected at the São Paulo Ridge in off Brazil. Compounds 2, 3 and 4 showed anti-S. parasitica activity. Our results suggest that diorcinol and violaceol analogs and could be good lead candidates for the development of novel agents to prevent saprolegniasis.

Genetic Diversity of Microbial Eukaryotes in Anoxic Sediment of the Saline Meromictic Lake Namako-ike (Japan): On the Detection of Anaerobic or Anoxic-tolerant Lineages of Eukaryotes.

Available sequence data on eukaryotic small-subunit ribosomal DNA (SSU rDNA) directly retrieved from various environments have increased recently, and the diversity of microbial eukaryotes (protists) has been shown to be much greater than previously expected. However, the molecular information accumulated to date does still not thoroughly reveal ecological distribution patterns of microbial eukaryotes. In the ongoing challenge to detect anaerobic or anoxic-tolerant lineages of eukaryotes, we directly extracted DNA from the anoxic sediment of a saline meromictic lake, constructed genetic libraries of PCR-amplified SSU rDNA, and performed phylogenetic analyses with the cloned SSU rDNA sequences. Although a few sequences could not be confidently assigned to any major eukaryotic groups in the analyses and are debatable regarding their taxonomic positions, most sequences obtained have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, Stramenopiles, and Opisthokonta). Among these sequences, some branched with lineages predominantly composed of uncultured environmental clones retrieved from other anoxic environments, while others were closely related to those of eukaryotic parasites (e.g. Phytomyxea of Cercozoa, Gregarinea of Alveolata, and Ichthyosporea of Opisthokonta).

Molecular evidence suggesting interspecific hybridization in Zoanthus spp. (Anthozoa: Hexacorallia).

Interspecific hybridization has been proposed as a possible explanation for the incredible diversity seen in reef-dwelling corals, but until now little proof of such hybridization in other reef-dwelling anthozoans has been reported. Without further observation of hybridization, the question of such a phenomenon being widespread in Anthozoa remains. Here we have examined the mitochondrial cytochrome oxidase I gene (COI) and the nuclear internal transcribed spacer of ribosomal DNA (ITS-rDNA) from three species of the mass-spawning, encrusting anemone genus Zoanthus (Z. sansibaricus, Z. kuroshio, Z. gigantus) to investigate possible hybridization. The three species coexist at two of three sampling locations in southern Japan. Zoanthus spp. ITS-rDNA region spacers (ITS-1 and ITS-2) were shown to have very high rates of divergence. At locations where all three species co-existed, several of our sampled Z. sansibaricus individuals (with identical "sansi" COI sequences) possessed two very divergent (i.e., species-level difference) ITS-rDNA alleles, the expected "sansi" allele and the divergent "B" allele. Additionally, two Z. sansibaricus individuals possessed only "B" alleles despite having "sansi" COI sequences. These results indicate that Z. sansibaricus has possibly experienced interspecific hybridization at least once with a Zoanthus partner possessing the "B" allele, and that these resulting hybrids may also sexually reproduce, demonstrating potential hybridization occurring in the order Zoantharia (Hexacorallia).

Genomic Evidence that Methanotrophic Endosymbionts Likely Provide Deep-Sea Bathymodiolus Mussels with a Sterol Intermediate in Cholesterol Biosynthesis.

Sterols are key cyclic triterpenoid lipid components of eukaryotic cellular membranes, which are synthesized through complex multi-enzyme pathways. Similar to most animals, Bathymodiolus mussels, which inhabit deep-sea chemosynthetic ecosystems and harbor methanotrophic and/or thiotrophic bacterial endosymbionts, possess cholesterol as their main sterol. Based on the stable carbon isotope analyses, it has been suggested that host Bathymodiolus mussels synthesize cholesterol using a sterol intermediate derived from the methanotrophic endosymbionts. To test this hypothesis, we sequenced the genome of the methanotrophic endosymbiont in Bathymodiolus platifrons. The genome sequence data demonstrated that the endosymbiont potentially generates up to 4,4-dimethyl-cholesta-8,14,24-trienol, a sterol intermediate in cholesterol biosynthesis, from methane. In addition, transcripts for a subset of the enzymes of the biosynthetic pathway to cholesterol downstream from a sterol intermediate derived from methanotroph endosymbionts were detected in our transcriptome data for B. platifrons. These findings suggest that this mussel can de novo synthesize cholesterol from methane in cooperation with the symbionts. By in situ hybridization analyses, we showed that genes associated with cholesterol biosynthesis from both host and endosymbionts were expressed exclusively in the gill epithelial bacteriocytes containing endosymbionts. Thus, cholesterol production is probably localized within these specialized cells of the gill. Considering that the host mussel cannot de novo synthesize cholesterol and depends largely on endosymbionts for nutrition, the capacity of endosymbionts to synthesize sterols may be important in establishing symbiont-host relationships in these chemosynthetic mussels.

Ancient Occasional Host Switching of Maternally Transmitted Bacterial Symbionts of Chemosynthetic Vesicomyid Clams.

Vesicomyid clams in deep-sea chemosynthetic ecosystems harbor sulfur-oxidizing bacteria in their gill epithelial cells. These symbionts, which are vertically transmitted, are species-specific and thought to have cospeciated with their hosts. However, recent studies indicate incongruent phylogenies between some vesicomyid clams and their symbionts, suggesting that symbionts are horizontally transmitted. To more precisely understand the evolution of vesicomyid clams and their symbionts, we compared the evolution of vesicomyid clams and their symbionts through phylogenetic analyses using multi-gene data sets. Many clades in the phylogenetic trees of 13 host species (Abyssogena mariana, Ab. phaseoliformis, Akebiconcha kawamurai, Calyptogena fausta, C. laubieri, C. magnifica, C. nautilei, C. pacifica, Isorropodon fossajaponicum, Phreagena kilmeri, Ph. okutanii, Ph. soyoae, and Pliocardia stearnsii) and their symbionts were well resolved. Six of the 13 host-symbiont pairs (C. fausta, C. magnifica, C. pacifica, Ph. kilmeri, Ph. okutanii, and Ph. soyoae, and their respective symbionts) showed topological congruence. However, the remaining seven pairs (Ak. kawamurai, Ab mariana, Ab. phaseoliformis, C. laubieri, C. nautilei, I. fossajaponicum, and Pl. stearnsii and their corresponding symbionts) showed incongruent topologies, which were supported by the approximately unbiased and Bayes factor tests. Coevolution analyses indicated that six pairs cospeciated, whereas host switching events occurred in the remaining seven pairs. Markedly, multiple host switching events may have occurred in the lineages from the common ancestral symbiont of C. pacifica and C. fausta. Our phylogenetic and coevolution analyses provide additional evidence for host switching during the evolution of vesicomyids.