Molecular phylogeny suggests polyphyly of both the turban shells (family Turbinidae) and the superfamily Trochoidea (Mollusca: Vetigastropoda)

Bayesian and parsimony phylogenetic analyses of sequence from two nuclear and two mitochondrial genes suggest that neither the molluscan superfamily Trochoidea, nor the family Turbinidae are monophyletic. The family Turbinidae s.l. divides into two main groups. The first group includes taxa previously referred to the five subfamilies Angariinae, Colloniinae, Phasianellinae, Tricoliinae, Gabrieloninae, and the liotiine genus Cinysca; these subfamilies are here recognized as Angariidae, Colloniidae, and Phasianellidae (with subfamilies Phasianellinae, Tricoliinae, and Gabrieloninae). The second group, which corresponds to Turbinidae sensu stricto, includes Prisogasterinae, Turbininae, and the liotiine genus Liotina, all of which are more closely related to trochids than they are to the first group. Several morphological studies have suggested previously that the family Phasianellidae is distinct from Turbinidae. However, this is the first study to suggest that Phasianellidae forms a group with some taxa previously thought of as turbinids, but excluding the nominotypical genus Turbo and its allies. The family Turbinidae has traditionally been described as the only family in the Vetigastropoda group that has a calcified operculum. The non-monophyly of Turbinidae suggests that calcareous opercula may have arisen independently more than once within the Vetigastropoda.     

Description of the early ontogenic part of the Tentaculitids, with implications for classification

Ontogenic development and classification of tentaculitids at high systematic levels are reevaluated in the light of new findings on shell structure and morphology of larval parts, and these features are here regarded as being of primary importance for taxonomy. Class Tentaculita Bouček, 1964 is subdivided into two subclasses, of which subclass Chionioconarida Farsan, 1994 is distinguished by a tubular larval process closed at the apex and covered with microrings. The process is differentiated into a prolarval, metalarval and epilarval part, of which the latter coincides with metamorphosis. Morphology of the larval parts suggests that metamorphosis proceeds in two different manners, giving rise to superorders within this subclass. Within superorder Trompetoconarida Farsan, 1994 a bilaterally symmetrical larval cone develops with an aperture oblique to the long axis of the conch; following metamorphosis the conch becomes radially symmetrical and the aperture perpendicular to the axis; secondary shell, septa and pseudopunctae develop in the adult phase, and the structure of the shell is lamellar. In contrast, within the second superorder, Lirioconarida Farsan, 1994 the epilarval tube develops into a larval bulb with no changes in symmetry and position of the aperture; secondary shell, septa and pseudopuncta are absent. The microstructure of the shell is lamellar in the larval part whereas in postlarval parts it is either sigmoidal or lamellar. The subclass Dacryoconarida Fisher, 1962 possesses a subspherical, tear- or drop-like embryonic chamber which may have a caudal process. The microstructure of the embryonic chamber is variable within this group, being lamellar in some taxa whereas in others, a single layer of shell is present. The postembryonic parts of the lamellar forms possess nacreous or sigmoidal structures.     

Microboring in a Freshwater Fluvial Unionid Bivalve Substrate

Samples of the unionid bivalve Elliptio complanata were collected from the channel of the freshwater Saint John River, from Fredericton, New Brunswick, Canada. Scanning electron microscopy imaging of prepared shell samples revealed an assemblage of microborings. No borings are noted on the periostracum or prismatic shell layers. Boring structures are instead confined to the underlying nacreous aragonitic shell material, together with its associated organic conchiolin layers. Three main styles of boring are encountered, encompassing both predominantly surficial structures and penetrative tubular borings. Surficial structures are represented by a polygonal network on an exposed conchiolin shell layer. The penetrative borings take two forms, one being simple unbranched tubes, steeply aligned (perpendicular to the shell surface) and traversing the full thickness of the nacreous shell layer. The other penetrative boring style, again occurring within the nacreous layer, comprises a complex irregular network of randomly oriented rarely branching tubular borings. Borings generally display diameters of micron scale. Biofilm and extracellular polymeric substances, with bacterial, diatomaceous and filamentous components are also observed, often displaying a close association with both the borings and the conchiolin layers within the shell. The formation of the borings may be attributed to cyanobacteria, cyanophyte or fungal progenitors.     

The Middle Jurassic Bivalve 'Cuspidaria'Ibbetsoni: A Corbulid Not A Septibranch

A distinctive shallow–burrowing bivalve with an elongate rostrum, common in the British Middle Jurassic, is frequently referred to as Cuspidaria ibbetsoni (Morris, 1853) and as such is recognised as one of the earliest members of the anomalodesmatan family Cuspidariidae. Since modern cuspidariids are highly specialised bivalves and are voracious carnivores in the deep sea, whereas C. ibbetsoni is prevalent in brackish–water facies, questions have been raised over its true affinities. We have investigated new and pre–existing material of C. ibbetsoni , and have concentrated on shell microstructure, morphology, and musculature. Our findings suggest that the inequivalve shell is composed of crossed–lamellar aragonite rather than either homogeneous or prismato–nacreous structures. It also lacks the complex musculature associated with the raptorial siphon of the predatory septibranchs. These characters suggest that C. ibbetsoni should more properly be placed within the myoid family Corbulidae and a new genus, Rostrocorbula , has been erected to accommodate it. It has also been necessary to establish a neotype. The significance of this familial redesignation to our understanding of the evolution of carnivory within the Bivalvia is discussed.     

Crystalline structure,orientation and nucleation of the nacreous tablets in the cephalopod <Emphasis Type="Italic">Nautilus</Emphasis>

The nacreous tablets in the Nautilus shell have similar crystalline structure as the tablets in the gastropod Gibbula shell. Etching with Mutvei’s solution reveals that each tablet is composed of vertical crystalline columns that are structurally similar to the acicular crystallites in the outer spherulitic-prismatic layer of the shell wall. The columns are attached to each other to form numerous vertical crystalline lamellae, oriented parallel to the longitudinal axis of the tablet. It is still unknown whether or not the orientation of the vertical lamellae corresponds to that of the crystallographic a- or b-axis. The orientation of the crystalline lamellae in the adjacent tablets is parallel in some nacreous laminae, but random in other laminae. Similar large variation was found in the nacreous tablets of the gastropod and bivalve shells. The nucleation sites of the nacreous tablets are predominantly situated on the peripheral portion of the upper surface of the preceding tablet, both in the shell wall and septa. The “aragonite-nucleating proteins” in the central portion of the crystal imprints of the organic interlamellar sheets, described by several writers, have therefore a negative correlation with the nucleation sites of the nacreous tablets.     

Predation and the geography of opercular thickness in turbinid gastropods

The heavily calcified opercula of turban snails (Turbinidae)function as passive-defensive structures against predators thatbreak the shell at the outer lip or that enter the shell byway of the aperture. Passive armour generally is more commonand much better developed in the tropics than in colder regions.In line with this expectation, the relative opercular thicknessof tropical turbinids (mean 0.358 ± 0.045, 33 species)significantly exceeds that of temperate species (mean 0.300± 0.051, 21 species). Small differences in relative opercularthickness occur among tropical biogeographic regions, but noneis significant. Patterns observed among turbinids as a wholealso appear in individual clades, indicating that the patternsare not dictated by a phylogenetic signal. (Received 5 September 2006; accepted 21 November 2006)     

Skeletal chemistry of Nautilus and its taxonomic significance

The strontium (Sr) and magnesium (Mg) chemistry of the shell wall and septum as well as the spherulitic-prismatic and nacreous layers of these structures was determined for Nautilus species: N. belauensis, N. macromphalus, N. pompilius and N. scrobiculatus. Each species of Nautilus exhibits greater variability and higher concentrations of Mg in juvenile portions of the shell than in more mature portions of the shell. This decrease in the variability and amount of Mg in the aragonite lattice suggests a physiochemical system which becomes more efficient with time relative to carbonate production. Statistically significant differences in the Sr and Mg content of spherulitic-prismatic and nacreous layers of the shell and septum indicate that these layers were formed from extracellular fluids of different compositions. Concentrations of Sr and Mg in aragonite of the shell wall are characteristic for each species and sufficiently invariant within species to allow species of Nautilus to be distinguished statistically on the basis of either the Sr or Mg content of the shell wall or the Mg content of septa.     

Molecular phylogeny of protobranch bivalves and systematic implications of their shell microstructure

Higher systematics and evolutionary history of Protobranchia, a subclass of Bivalvia, have long been controversial due to paucity of prominent shell characters and difficulties in collecting live material for diverse taxa. Here, we evaluate the reliability of shell microstructure for protobranch higher systematics by reconstructing a molecular phylogeny of the subclass. Relationships were assessed using the nuclear (18S rRNA, 28S rRNA and histone H3) and mitochondrial (16S rRNA and cytochrome c oxidase subunit 1) gene sequences from 89 in-group species. Maximum likelihood reconstruction with the nuclear markers recognized five superfamilies (Nuculoidea, Solemyoidea, Manzanelloidea, Nuculanoidea and Sareptoidea) as the in-group clades of the monophyletic Protobranchia. Sareptoidea is herein redefined to comprise Sarepta and Setigloma in the sole family Sareptidae, whereas Pristigloma and its monotypic Pristiglomidae are transferred from this superfamily to Nuculanoidea, both in the order Nuculanida. Mapping of shell microstructure characters on the tree confirmed their conservativeness at superfamily level when only living species were taken into account. The Nuculoidea have shells with the outer prismatic and middle/inner nacreous structures; Solemyoidea are characterized by either the radially elongate simple prismatic structure or the reticulate structure in the outer shell layer; Manzanelloidea, Nuculanoidea and Sareptoidea have shells of homogeneous, fibrous prismatic and/or fine complex crossed lamellar structures, all of which lack large structural units. Our Bayesian time calibration, on the contrary, suggested frequent loss of nacre in the Paleozoic and Mesozoic history of Protobranchia, at least once each in Nuculoidea, Manzanelloidea, Solemyoidea and Sareptoidea in the Paleozoic, and perhaps multiple times in Nuculanoidea by the Mesozoic.     

Structure and composition of the septal nacreous layer of Nautilus macromphalus L. (Mollusca, Cephalopoda)

The nacreous layer of Mollusca is the best-known aragonitic structure and is the usual model for biomineralization. However, data are based on less than 10 species. In situ observations of the septal nacreous layer of the cephalopod Nautilus shell has revealed that the tablets are composed of acicular laths. These laths are composed of round nanograins surrounded by an organic sheet. No hole has been observed in the decalcified interlamellar membranes. A set of combined analytical data shows that the organic matrices extracted from the nacreous layer are glycoproteins. In both soluble and insoluble matrices, S amino acids are rare and the soluble organic matrices have a higher sulfated sugar content than the insoluble matrices. It is possible that the observed differences in the structure and composition of the nacreous layers of the outer wall and septa of the Nautilus shell have a dual origin: evolution and functional adaptation. However, we have no appropriate data as yet to answer this question.     

The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny

A need to increase sampling of mitochondrial genomes for Vetigastropoda has been identified as an important step towards resolving relationships within the Gastropoda. We used shotgun sequencing of genomic DNA, using an Illumina MiSeq, to obtain the first mitochondrial genome for the vetigastropod family Turbinidae, doubling the number of genomes for the species-rich superfamily Trochoidea. This method avoids the necessity of finding suitable primers for long PCRs or primer-walking amplicons, resulting in a timely and cost-effective method for obtaining whole mitochondrial genomes from ethanol-preserved tissue samples. Bayesian analysis of amino acid variation for all available gastropod genomes including the new turbinid mtgenome produced a well resolved tree with high nodal support for most nodes. Major clades within Gastropoda were recovered with strong support, with the exception of Littorinimorpha, which was polyphyletic. We confirm here that mitogenomics is a useful tool for molluscan phylogenetics, especially when using powerful new models of amino acid evolution, but recognise that increased taxon sampling is still required to resolve existing differences between nuclear and mitochondrial gene trees.     

CONVERGENT SHELL MORPHOLOGY IN INTERTIDAL GASTROPODS

The functional morphology of shell infrastructure in 2 speciesof intertidal trochid was compared with that in 2 species ofnerite. The shell of Monodonta constrictais typical of the majorityof trochids. The shell is composed of 4 layers: a distal layer(calcite), anouter prismatic layer (aragonite), a nacreous layer(aragonite), and an oblique prismatic layer (aragonite). Monodontalabio lacks a distal layer and an oblique prismatic layer. Theoblique prismatic layer is replaced by an inner prismatic layerwhich forms an apertural ridge as a result of deposition andresorption. The shells of Nerita versicolor and N. tessellataconsistof 3 layers: an outer prismatic layer (calcite), a crossedlamellar layer (aragonite), and a complex crossed lamellar layer(aragonite). The complex crossed lamellar layer is covered withinclined platelets which superficially resemble the surfaceof the ique prismatic layer of trochids. In addition, the complexcrossed lamellar layer forms an apertural ridge which is similarin appearance to that of Monodonta labio. The outer surfaceof the mantle of Nerita versicolor and N. tessellata is throwninto a series of large folds which lie in contact with the inclinedplatelets of the omplex crossed lamellar layer. The interactionof the mantle folds with the inclined platelets is thought toserve as a rachet mechanism to aid in extension of themantle;a similar function has previously been proposed for trochids.The apertural ridges of Monodonta labio and Nerita are thoughtto prevent excessive desiccation when these gastropodsare exposedat low tide. ¹Contribution No. 56 of the Tallahassee, Sopchoppy & GulfCoast Marine Biological Association (Received 6 July 1979;     

Characteristics of shell microstructure and growth analysis of the Antarctic bivalve <Emphasis Type="Italic">Laternula elliptica</Emphasis> from Lützow-Holm Bay,Antarctica

Growth performance of the Antarctic bivalve Laternula elliptica was examined both by shell microstructural observation and by applying a fluorescent substance, tetracycline, as a shell growth marker. The shell was composed of two calcareous layers: the thick outer layer was homogeneous or granular in structure and the thin inner layer was nacreous. The architecture of Antarctic L. elliptica was different from that of temperate L. marilina, and the ratio of thickness between the outer and inner layers appeared to be different. The growth rate of the nacreous layer was analyzed to be very low. High correlations were found between the major axis of chondrophore and both shell length and shell dry weight, respectively. It is suggested that the chondrophore is an appropriate growth indicator, and combining the information of growth increments with the fluorescent method may be useful in estimating the bivalve growth performance in the Antarctic sea.     

Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the 'aragonitic line'. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P. fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the 'aragonitic line', and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.     

A Novel Acidic Matrix Protein,PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium.     

Irregular calcareous sheets preserved in a conch of the Cretaceous ammonoid Hypophylloceras from Hokkaido,Japan

The mantle tissue is essential for understanding the diverse ecology and shell morphology of ammonoid cephalopods. Here, we report on irregular calcareous sheets in a well-preserved shell of a Late Cretaceous phylloceratid ammonoid Hypophylloceras subramosum from Hokkaido, Japan, and their significance for repairing the conch through the mantle inside the body chamber. The sheets are composed of nacreous layers arranged parallel to the irregularly distorted outer whorl surface. The nacreous sheets formed earlier are unevenly distributed and attached to the outer shell wall locally, whereas the last formed sheet covers a wide area of the outer shell wall. The absence of any interruption of ribbing around the irregular area suggests that these sheets were secreted inside the body chamber from the inner mantle. Gross morphological and X-ray computed tomography observations revealed that the spacing of septal formation was not affected by this event. The complex structure of the irregular sheets suggests a highly flexible mantle inside the body chamber.     

几种生物CaCO_3霰石结晶的取向性

ＣａＣＯ３结晶广泛分布于生物界,其主要结晶形式为方解石、霰石及球霰石。用Ｘ－射线衍射法对三角帆蚌及合浦珍珠母贝的珍珠层、墨鱼骨和大黄鱼耳石的ＣａＣＯ３结晶进行测定,发现各样品均有一定取向性,以三角帆蚌和合浦珍珠母贝珍珠层的取向性为最强,墨鱼骨的取向性次之,大黄鱼耳石的取向性最小,以上材料粉末样的衍射分析表明,各样品对应ｄ值间差异极小,均为Ｘ射线衍射卡（５—０４５３）所表征的ＣａＣＯ３霰石结构。     

几种生物CaCO3霰石结晶的取向性

CaCO3结晶广泛分布于生物界,其主要结晶形式为方解石、霰石及球霰石。用X－射线衍射法对三角帆蚌及合浦珍珠母贝的珍珠层、墨鱼骨和大黄鱼耳石的CaCO3结晶进行测定,发现各样品均有一定取向性,以三角帆蚌和合浦珍珠母贝珍珠层的取向性为最强,墨鱼骨的取向性次之,大黄鱼耳石的取向性最小,以上材料粉末样的衍射分析表明,各样品对应d值间差异极小,均为X射线衍射卡（5－0453）所表征的CaCO3霰石结构。     

Geometrical and crystallographic constraints determine the self-organization of shell microstructures in Unionidae (Bivalvia: Mollusca)

Unionid shells are characterized by an outer aragonitic prismatic layer and an inner nacreous layer. The prisms of the outer shell layer are composed of single-crystal fibres radiating from spheruliths. During prism development, fibres progressively recline to the growth front. There is competition between prisms, leading to the selection of bigger, evenly sized prisms. A new model explains this competition process between prisms, using fibres as elementary units of competition. Scanning electron microscopy and X-ray texture analysis show that, during prism growth, fibres become progressively orientated with their three crystallographic axes aligned, which results from geometric constraints and space limitations. Interestingly transition to the nacreous layer does not occur until a high degree of orientation of fibres is attained. There is no selection of crystal orientation in the nacreous layer and, as a result, the preferential orientation of crystals deteriorates. Deterioration of crystal orientation is most probably due to accumulation of errors as the epitaxial growth is suppressed by thick or continuous organic coats on some nacre crystals. In conclusion, the microstructural arrangement of the unionid shell is, to a large extent, self-organized with the main constraints being crystallographic and geometrical laws.     

The mechanical behaviour of some molluscan hard tissues

Pieces of shell from 19 species of molluscs exhibiting various microstructures were tested for tensile strength, modulus of elasticity in bending and modulus of rupture. In tensile strength most shells with cross-foliated, foliated, homogeneous and crossed-lamellar structures did not exceed 60 MNm ² but prismatic and nacreous structures often exceeded this value. Nacreous structure was generally superior to all others in modulus of rupture tests; that of Turbo being about equal to bone. Values of modulus of elasticity were more uniform between structures. There is a general relation between mechanical properties, microstructure and the life style of each animal. Nacreous structure, which is very strong but not widely used, apparently evolved earlier than the less strong but widely used crossed-lamellar structure.     

Identification of chitin in the prismatic layer of the shell and a chitin synthase gene from the Japanese pearl oyster, Pinctada fucata

The shell of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the prismatic layer on the outside and the nacreous layer on the inside, both of which comprise calcium carbonate and organic matrices. Previous studies indicate that the nacreous organic matrix of the central layer of the framework surrounding the aragonite tablet is beta-chitin, but it remains unknown whether organic matrices in the prismatic layer contain chitin or not. In the present study, we identified chitin in the prismatic layer of the Japanese pearl oyster, Pinctada fucata, with a combination of Calcofluor White staining with IR and NMR spectral analyses. Furthermore, we cloned a cDNA encoding chitin synthase (PfCHS1) that produces chitin, contributing to the formation of the framework for calcification in the shell.