Evolution of Epidermal Growth Factor (EGF)-like and Zona Pellucida Domains Containing Shell Matrix Proteins in Mollusks.

Several types of shell matrix proteins (SMPs) have been identified in molluskan shells. Their diversity is the consequence of various molecular processes, including domain shuffling and gene duplication. However, the evolutionary origin of most SMPs remains unclear. In this study, we investigated the evolutionary process EGF-like and zona pellucida (ZP) domains containing SMPs. Two types of the proteins (EGF-like protein (EGFL) and EGF-like and ZP domains containing protein (EGFZP)) were found in the pearl oyster, Pinctada fucata. In contrast, only EGFZP was identified in the gastropods. Phylogenetic analysis and genomic arrangement studies showed that EGFL and EGFZP formed a clade in bivalves, and their encoding genes were localized in tandem repeats on the same scaffold. In P. fucata, EGFL genes were expressed in the outer part of mantle epithelial cells are related to the calcitic shell formation. However, in both P. fucata and the limpet Nipponacmea fuscoviridis, EGFZP genes were expressed in the inner part of the mantle epithelial cells are related to aragonitic shell formation. Furthermore, our analysis showed that in P. fucata, the ZP domain interacts with eight SMPs that have various functions in the nacreous shell mineralization. The data suggest that the ZP domain can interact with other SMPs, and EGFL evolution in pterimorph bivalves represents an example of neo-functionalization that involves the acquisition of a novel protein through gene duplication.

Stasis and diversity in living fossils: Species delimitation and evolution of lingulid brachiopods.

The Lingulidae are often considered living fossils, because they have shown little morphological change since the Paleozoic. Limited morphological variation has also made the taxonomic study of living lingulids challenging. We investigated species diversity and phylogenetic relationships of extant lingulids and show that they are substantially more diverse than realized, demonstrating that morphological stasis was commonly accompanied by speciation. Species delimitation based on cytochrome c oxidase subunit I (COI) gene sequences from 194 specimens sampled from East Asia, Australia, Oceania, and the Americas suggested 14-22 species in the lingulids (9-17 species in Lingula and 4-5 species in Glottidia), in contrast to the 11-12 species currently recognized globally in the family. Four-gene phylogenetic analyses supported the sister relationship between Lingula and Glottidia. Within Lingula, L. adamsi, which possesses large, brownish shells, was recovered as sister to all remaining Lingula species, which have more or less greenish shells. Within the greenish Lingula clade, the 'L. anatina' complex was sister to the clade that includes the 'L. reevei' complex. The 'L. anatina' complex was further separated into two major clades with partly separate ranges centered on (i) temperate East Asia, and (ii) the tropical west-central Pacific. Within Glottidia, Pacific species were nested within Atlantic species. Time-calibrated phylogenetic analyses suggested that Lingula likely originated in the early Cretaceous contrary to a previously proposed hypothesis advocating a Cenozoic origin. The separation of Lingula and Glottidia appears to date from the Mesozoic, not from the Carboniferous, contrary to a previous hypothesis. Overall, our results uncovered substantial cryptic diversity in lingulids, which will form the basis for conservation and further taxonomic revision.

Dual Gene Repertoires for Larval and Adult Shells Reveal Molecules Essential for Molluscan Shell Formation.

Molluscan shells, mainly composed of calcium carbonate, also contain organic components such as proteins and polysaccharides. Shell organic matrices construct frameworks of shell structures and regulate crystallization processes during shell formation. To date, a number of shell matrix proteins (SMPs) have been identified, and their functions in shell formation have been studied. However, previous studies focused only on SMPs extracted from adult shells, secreted after metamorphosis. Using proteomic analyses combined with genomic and transcriptomic analyses, we have identified 31 SMPs from larval shells of the pearl oyster, Pinctada fucata, and 111 from the Pacific oyster, Crassostrea gigas. Larval SMPs are almost entirely different from those of adults in both species. RNA-seq data also confirm that gene expression profiles for larval and adult shell formation are nearly completely different. Therefore, bivalves have two repertoires of SMP genes to construct larval and adult shells. Despite considerable differences in larval and adult SMPs, some functional domains are shared by both SMP repertoires. Conserved domains include von Willebrand factor type A (VWA), chitin-binding (CB), carbonic anhydrase (CA), and acidic domains. These conserved domains are thought to play crucial roles in shell formation. Furthermore, a comprehensive survey of animal genomes revealed that the CA and VWA-CB domain-containing protein families expanded in molluscs after their separation from other Lophotrochozoan linages such as the Brachiopoda. After gene expansion, some family members were co-opted for molluscan SMPs that may have triggered to develop mineralized shells from ancestral, nonmineralized chitinous exoskeletons.

Possible co-option of engrailed during brachiopod and mollusc shell development.

In molluscs, two homeobox genes, engrailed (en) and distal-less (dlx), are transcription factors that are expressed in correlation with shell development. They are expressed in the regions between shell-forming and non-shell-forming cells, likely defining the boundaries of shell-forming fields. Here we investigate the expression of two transcription factors in the brachiopod Lingula anatina We find that en is expressed in larval mantle lobes, whereas dlx is expressed in larval tentacles. We also demonstrate that the embryonic shell marker mantle peroxidase (mpox) is specifically expressed in mantle lobes. Our results suggest that en and mpox are possibly involved in brachiopod embryonic shell development. We discuss the evolutionary developmental origin of lophotrochozoan biomineralization through independent gene co-option.

Proteome analysis of shell matrix proteins in the brachiopod Laqueus rubellus.

BACKGROUND: The calcitic brachipod shells contain proteins that play pivotal roles in shell formation and are important in understanding the evolution of biomineralization. Here, we performed a large-scale exploration of shell matrix proteins in the brachiopod Laqueus rubellus. RESULTS: A total of 40 proteins from the shell were identified. Apart from five proteins, i.e., ICP-1, MSP130, a cysteine protease, a superoxide dismutase, and actin, all other proteins identified had no homologues in public databases. Among these unknown proteins, one shell matrix protein was identified with a domain architecture that includes a NAD(P) binding domain, an ABC-type transport system, a transmembrane region, and an aspartic acid rich region, which has not been detected in other biominerals. We also identified pectin lyase-like, trypsin inhibitor, and saposin B functional domains in the amino acid sequences of the shell matrix proteins. The repertoire of brachiopod shell matrix proteins also contains two basic amino acid-rich proteins and proteins that have a variety of repeat sequences. CONCLUSIONS: Our study suggests an independent origin and unique mechanisms for brachiopod shell formation.

Metagenomic Insights into Ecophysiology of Zetaproteobacteria and Gammaproteobacteria in Shallow Zones within Deep-sea Massive Sulfide Deposits.

Deep-sea massive sulfide deposits serve as energy sources for chemosynthetic ecosystems in dark, cold environments even after hydrothermal activity ceases. However, the vertical distribution of microbial communities within sulfide deposits along their depth from the seafloor as well as their ecological roles remain unclear. We herein conducted a culture-independent metagenomic ana-lysis of a core sample of massive sulfide deposits collected in a hydrothermally inactive field of the Southern Mariana Trough, Western Pacific, by drilling (sample depth: 0.52| |m below the seafloor). Based on the gene context of the metagenome-assembled genomes (MAGs) obtained, we showed the metabolic potential of as-yet-uncultivated microorganisms, particularly those unique to the shallow zone rich in iron hydroxides. Some members of Gammaproteobacteria have potential for the oxidation of reduced sulfur species (such as sulfide and thiosulfate) to sulfate coupled to nitrate reduction to ammonia and carbon fixation via the Calvin-Benson-Bassham (CBB) cycle, as the primary producers. The Zetaproteobacteria member has potential for iron oxidation coupled with microaerobic respiration. A comparative ana-lysis with previously reported metagenomes from deeper zones (~2| |m below the seafloor) of massive sulfide deposits revealed a difference in the relative abundance of each putative primary producer between the shallow and deep zones. Our results expand knowledge on the ecological potential of uncultivated microorganisms in deep-sea massive sulfide deposits and provide insights into the vertical distribution patterns of chemosynthetic ecosystems.

Annotation of the pearl oyster genome.

The initial, manual annotation analysis of the pearl oyster genome is reported in the present issue of Zoological Science. Contributors represent a wide array of research fields, including bioinformatics, molecular and cellular biology, fisheries science, biochemistry, biomineralogy, molluscan biology, evolutionary and developmental biology, and paleobiology, reflecting the pearl oyster's broad biological and economic importance. The annotated pearl oyster genome paves the way for future studies in diverse areas including pearl aquaculture, biomineralization, and lophotrochozoan biology.

Initiating the mollusk genomics annotation community: toward creating the complete curated gene-set of the Japanese Pearl Oyster, Pinctada fucata.

The genome sequence of the Japanese pearl oyster, the first draft genome from a mollusk, was published in February 2012. In order to curate the draft genome assemblies and annotate the predicted gene models, two annotation Jamborees were held in Okinawa and Tokyo. To date, 761 genes have been surveyed and curated. A preparatory meeting and a debriefing were held at the Misaki Marine Biological Station before and after the Jamborees. These four events, in conjunction with the sequence-decoding project, have facilitated the first series of gene annotations. Genome annotators among the Jamboree participants added 22 functional categories to the annotation system to date. Of these, 17 are included in Generic Gene Ontology. The other five categories are specific to molluskan biology, such as "Byssus Formation" and "Shell Formation", including Biomineralization and Acidic Proteins. A total of 731 genes from our latest version of gene models are annotated and classified into these 22 categories. The resulting data will serve as a useful reference for future genomic analyses of this species as well as comparative analyses among mollusks.

An in-silico genomic survey to annotate genes coding for early development-relevant signaling molecules in the pearl oyster, Pinctada fucata.

The pearl oyster Pinctada fucata has great potential as a model system for lophotrochozoan developmental biology research. Pinctada fucata is an important commercial resource, and a significant body of primary research on this species has emphasized its basic aquaculture biology such as larval biology and growth, aquaculture, pearl formation and quality improvement, shell formation, and biomineralization. Recently, a draft genome sequence of this species was published, and many experimental resources are currently being developed, such as bioinformatics tools, embryo and larva manipulation methods, gene knockdown technique, etc. In this paper, we report the results from our genomic survey pertaining to gene families that encode developmental signaling ligands (Fgf, Hedgehog, PDGF/VEGF, TGFß, and Wnt families). We found most of the representative genes of major signaling pathways involved in axial patterning, as well as copies of the signaling molecule paralogs. Phylogenetic character mapping was used to infer a possible evolutionary scenario of the signaling molecules in the protostomes, and to reconstruct possible copy numbers of signaling molecule-coding genes for the ancestral protostome. Our reconstruction suggests that P. fucata retains the ancestral protostome gene complement, providing further justifications for the use of this taxon as a model organism for developmental genomics research.

The diversity of shell matrix proteins: genome-wide investigation of the pearl oyster, Pinctada fucata.

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/pinctada_fucata ). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

A comparative study of the shell matrix protein aspein in pterioid bivalves.

Aspein is one of the unusually acidic shell matrix proteins originally identified from the pearl oyster Pinctada fucata. Aspein is thought to play important roles in the shell formation, especially in calcite precipitation in the prismatic layer. In this study, we identified Aspein homologs from three closely related pterioid species: Pinctada maxima, Isognomon perna, and Pteria penguin. Our immunoassays showed that they are present in the calcitic prismatic layer but not in the aragonitic nacreous layer of the shells. Sequence comparison showed that the Ser-Glu-Pro and the Asp-Ala repeat motifs are conserved among these Aspein homologs, indicating that they are functionally important. All Aspein homologs examined share the Asp-rich D-domain, suggesting that this domain might have a very important function in calcium carbonate formation. However, sequence analyses showed a significantly high level of variation in the arrangement of Asp in the D-domain even among very closely related species. This observation suggests that specific arrangements of Asp are not required for the functions of the D-domain.

Possible functions of Dpp in gastropod shell formation and shell coiling.

We examined dpp expression patterns in the pulmonate snail Lymnaea stagnalis and analyzed the functions of dpp using the Dpp signal inhibitor dorsomorphin in order to understand developmental mechanisms and evolution of shell formation in gastropods. The dpp gene is expressed in the right half of the circular area around the shell gland at the trochophore stage and at the right-hand side of the mantle at the veliger stage in the dextral snails. Two types of shell malformations were observed when the Dpp signals were inhibited by dorsomorphin. When the embryos were treated with dorsomorphin at the 2-cell and blastula stages before the shell gland is formed, the juvenile shells grew imperfectly and were not mineralized. On the other hand, when treated at the trochophore and veliger stage after the shell gland formation, juvenile shells grew to show a cone-like form rather than a normal coiled form. These results indicated that dpp plays important roles in the formation and coiling of the shell in this gastropod species.

Hydrophilic Shell Matrix Proteins of Nautilus pompilius and the Identification of a Core Set of Conchiferan Domains.

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the basally diverging Nautilids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautilid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we performed a multiomics study on the shell of N. pompilius, by conducting transcriptomics of its mantle tissue and proteomics of its shell matrix. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet and the Euhadra snail) revealed that three proteins and six protein domains were conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six protein domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were probably present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Even though further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be carried out in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

Functional shell matrix proteins tentatively identified by asymmetric snail shell morphology.

Molluscan shell matrix proteins (SMPs) are essential in biomineralization. Here, we identify potentially important SMPs by exploiting the asymmetric shell growth in snail, Lymnaea stagnalis. Asymmetric shells require bilaterally asymmetric expression of SMP genes. We examined expression levels of 35,951 transcripts expressed in the left and right sides of mantle tissue of the pond snail, Lymnaea stagnalis. This transcriptome dataset was used to identify 207 SMPs by LC-MS/MS. 32 of the 207 SMP genes show asymmetric expression patterns, which were further verified for 4 of the 32 SMPs using quantitative PCR analysis. Among asymmetrically expressed SMPs in dextral snails, those that are more highly expressed on the left side than the right side are 3 times more abundant than those that are more highly expressed on the right than the left, suggesting potentially inhibitory roles of SMPs in shell formation. The 32 SMPs thus identified have distinctive features, such as conserved domains and low complexity regions, which may be essential in biomineralization.

Phylogenetic comparisons reveal mosaic histories of larval and adult shell matrix protein deployment in pteriomorph bivalves.

Molluscan shells are organo-mineral composites, in which the dominant calcium carbonate is intimately associated with an organic matrix comprised mainly of proteins and polysaccharides. However, whether the various shell matrix proteins (SMPs) date to the origin of hard skeletons in the Cambrian, or whether they represent later deployment through adaptive evolution, is still debated. In order to address this issue and to better understand the origins and evolution of biomineralization, phylogenetic analyses have been performed on the three SMP families, Von Willebrand factor type A (VWA) and chitin-binding domain-containing protein (VWA-CB dcp), chitobiase, and carbonic anhydrase (CA), which exist in both larval and adult shell proteomes in the bivalves, Crassostrea gigas and Pinctada fucata. In VWA-CB dcp and chitobiase, paralogs for larval and adult SMPs evolved before the divergence of these species. CA-SMPs have been taken as evidence for ancient origins of SMPs by their presumed indispensable function in biomineralization and ubiquitous distribution in molluscs. However, our results indicate gene duplications that gave rise to separate deployments as larval and adult CA-SMPs occurred independently in each lineage after their divergence, which is considerably more recent than hitherto assumed, supporting the "recent heritage and fast evolution" scenario for SMP evolution.

Insights into the Evolution of Shells and Love Darts of Land Snails Revealed from Their Matrix Proteins.

Over the past decade, many skeletal matrix proteins that are possibly related to calcification have been reported in various calcifying animals. Molluscs are among the most diverse calcifying animals and some gastropods have adapted to terrestrial ecological niches. Although many shell matrix proteins (SMPs) have already been reported in molluscs, most reports have focused on marine molluscs, and the SMPs of terrestrial snails remain unclear. In addition, some terrestrial stylommatophoran snails have evolved an additional unique calcified character, called a "love dart," used for mating behavior. We identified 54 SMPs in the terrestrial snail Euhadra quaesita, and found that they contain specific domains that are widely conserved in molluscan SMPs. However, our results also suggest that some of them possibly have evolved independently by domain shuffling, domain recruitment, or gene co-option. We then identified four dart matrix proteins, and found that two of them are the same proteins as those identified as SMPs. Our results suggest that some dart matrix proteins possibly have evolved by independent gene co-option from SMPs during dart evolution events. These results provide a new perspective on the evolution of SMPs and "love darts" in land snails.

In vitro regulation of CaCO(3) crystal polymorphism by the highly acidic molluscan shell protein Aspein.

Biominerals, especially molluscan shells, generally contain unusually acidic proteins. These proteins are believed to function in crystal nucleation and inhibition. We previously identified an unusually acidic protein Aspein from the pearl oyster Pinctada fucata. Here we show that Aspein can control the CaCO(3) polymorph (calcite/aragonite) in vitro. While aragonite is preferentially formed in Mg(2+) -rich solutions imitating the extrapallial fluids of marine molluscs, Aspein exclusively induced calcite precipitation. Our results suggest that Aspein is involved in the specific calcite formation in the prismatic layer. Experiments using truncated Aspein demonstrated that the aspartic acid rich domain is crucial for the calcite precipitation.

Biphasic and dually coordinated expression of the genes encoding major shell matrix proteins in the pearl oyster Pinctada fucata.

Regional expression patterns of shell matrix protein genes of Pinctada fucata were investigated using real-time quantitative polymerase chain reaction (PCR) and in situ hybridization. Six shell matrix proteins examined in this study indicated a distinct biphasic pattern of expression, falling into one of the following three groups: (1) expressed only in the more dorsal region of the mantle (MSI60 and N16); (2) expressed only in the more ventral region (MSI31, Prismalin-14, and Aspein); and (3) expressed in both regions (nacrein). The ubiquity of the last protein probably reflects its general role as a carbonate-producing enzyme, while the other groups are interpreted as corresponding to the distinction between the two varieties of shell layers, the aragonitic nacreous layer and the calcitic prismatic layer. In addition, the constituent genes of each of these two groups indicated similar levels of relative expression among different sites even among different individuals, suggesting that the genes of each group share a single upstream regulatory factor, respectively, and that these genes are expressed in a dually coordinated fashion.

Molecular phylogeny of acantharian and polycystine radiolarians based on ribosomal DNA sequences, and some comparisons with data from the fossil record.

Polycystines (spumellarians, nassellarians, and collodarians), phaeodarians, and acantharians are marine planktonic protists that have been conventionally and collectively called "radiolaria". Recent molecular phylogenetic studies revealed radiolarian polyphyly with phaeodarians being a separate offshoot. Collodarians and nassellarians are also shown to form a monophyletic group, but other aspects of radiolarian phylogeny, such as interrelations among polycystines and acantharians, remained uncertain. Here, we present molecular phylogenetic analyses including new ribosomal RNA sequences from ten spumellarians and nine nassellarians, based on Bayesian and maximum-likelihood methods. Results indicate that the Polycystinea is a paraphyletic group, with Bayesian analysis suggesting that spumellarians form a clade with acantharians. The heliozoan-like protist Sticholonche appears as a sister to the spumellarian clade. The nassellarian Eucyrtidium is located outside the clade including the other nassellarians and collodarians. The mineralogy of the test of extant radiolarians and the tree topology obtained in this work suggest that acantharians and spumellarians evolved from an ancestor with a siliceous skeleton. Collodarians and nassellarians form a well-supported clade and one might infer from the fossil record that they may have diverged between the Jurassic and the Eocene.

Bivalve-specific gene expansion in the pearl oyster genome: implications of adaptation to a sessile lifestyle.

INTRODUCTION: Bivalve molluscs have flourished in marine environments, and many species constitute important aquatic resources. Recently, whole genome sequences from two bivalves, the pearl oyster, Pinctada fucata, and the Pacific oyster, Crassostrea gigas, have been decoded, making it possible to compare genomic sequences among molluscs, and to explore general and lineage-specific genetic features and trends in bivalves. In order to improve the quality of sequence data for these purposes, we have updated the entire P. fucata genome assembly. RESULTS: We present a new genome assembly of the pearl oyster, Pinctada fucata (version 2.0). To update the assembly, we conducted additional sequencing, obtaining accumulated sequence data amounting to 193× the P. fucata genome. Sequence redundancy in contigs that was caused by heterozygosity was removed in silico, which significantly improved subsequent scaffolding. Gene model version 2.0 was generated with the aid of manual gene annotations supplied by the P. fucata research community. Comparison of mollusc and other bilaterian genomes shows that gene arrangements of Hox, ParaHox, and Wnt clusters in the P. fucata genome are similar to those of other molluscs. Like the Pacific oyster, P. fucata possesses many genes involved in environmental responses and in immune defense. Phylogenetic analyses of heat shock protein70 and C1q domain-containing protein families indicate that extensive expansion of genes occurred independently in each lineage. Several gene duplication events prior to the split between the pearl oyster and the Pacific oyster are also evident. In addition, a number of tandem duplications of genes that encode shell matrix proteins are also well characterized in the P. fucata genome. CONCLUSIONS: Both the Pinctada and Crassostrea lineages have expanded specific gene families in a lineage-specific manner. Frequent duplication of genes responsible for shell formation in the P. fucata genome explains the diversity of mollusc shell structures. These duplications reveal dynamic genome evolution to forge the complex physiology that enables bivalves to employ a sessile lifestyle in the intertidal zone.