Axial growth of hexactinellid spicules: formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis

The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3 m and diameters of 8.5 mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size tendency. The apical elongation of the spicule proceeds by piling up cone-like structural units formed from silica. As a support of the assumption that in the extracellular space silicatein(-like) molecules exist that associate with the external surface of the respective spicule immunogold electron microscopic analyses were performed. With the primmorph system from Suberites domuncula we show that silicatein(-like) molecules assemble as string- and net-like arrangements around the spicules. At their tips the silicatein(-like) molecules are initially stacked and at a later stay also organized into net-like structures. Silicatein(-like) molecules have been extracted from the giant basal spicule of Monorhaphis. Applying the SDS–PAGE technique it could be shown that silicatein molecules associate to dimers and trimers. Higher complexes (filaments) are formed from silicatein(-like) molecules, as can be visualized by electron microscopy (SEM). In the presence of ortho-silicate these filaments become covered with 30–60 nm long small rod-like/cuboid particles of silica. From these data we conclude that the apical elongation of the spicules of Monorhaphis proceeds by piling up cone-like silica structural units, whose synthesis is mediated by silicatein(-like) molecules.     

Silicatein expression in the hexactinellid Crateromorpha meyeri: the lead marker gene restricted to siliceous sponges

The siliceous spicules of sponges (Porifera) are synthesized by the enzyme silicatein. This protein and its gene have been identified so far in the Demospongiae, e.g., Tethya aurantium and Suberites domuncula. In the Hexactinellida, the second class of siliceous sponges, the mechanism of synthesis of the largest bio-silica structures on Earth remains obscure. Here, we describe the morphology of the spicules (diactines and stauractines) of the hexactinellid Crateromorpha meyeri. These spicules are composed of silica lamellae concentrically arranged around a central axial canal and contain proteinaceous sheaths (within the siliceous mantel) and proteinaceous axial filaments (within the axial canal). The major protein in the spicules is a 24-kDa protein that strongly reacts with anti-silicatein antibodies in Western blots. Its cDNA has been successfully cloned; the deduced hexactinellid silicatein comprises, in addition to the characteristic catalytic triad amino acids Ser-His-Asn and the "conventional" serine cluster, a "hexactinellid C. meyeri-specific" Ser cluster. We show that anti-silicatein antibodies react specifically with the proteinaceous matrix of the C. meyeri spicules. The characterization of silicatein at the genetic level should contribute to an understanding of the molecular/biochemical mechanism of spiculogenesis in Hexactinellida. These data also indicate that silicatein is an autapomorphic molecule common to both classes of siliceous sponges.     

Formation of giant spicules in the deep-sea hexactinellid <Emphasis Type="Italic">Monorhaphis chuni</Emphasis> (Schulze 1904): electron-microscopic and biochemical studies

The siliceous sponge Monorhaphis chuni (Hexactinellida) synthesizes the largest biosilica structures on earth (3 m). Scanning electron microscopy has shown that these spicules are regularly composed of concentrically arranged lamellae (width: 3–10 μm). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter: ~2 μm) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100–150 μm) of electron-dense homogeneous silica. During dissolution of the spicules with hydrofluoric acid, the axial filament is first released followed by the release of a proteinaceous tubule. Two major proteins (150 kDa and 35 kDa) have been visualized, together with a 24-kDa protein that cross-reacts with antibodies against silicatein. The spicules are surrounded by a collagen net, and the existence of a hexactinellidan collagen gene has been demonstrated by cloning it from Aphrocallistes vastus. During the axial growth of the spicules, silicatein or the silicatein-related protein is proposed to become associated with the surface of the spicules and to be finally internalized through the apical opening to associate with the axial filament. Based on the data gathered here, we suggest that, in the Hexactinellida, the growth of the spicules is mediated by silicatein or by a silicatein-related protein, with the orientation of biosilica deposition being controlled by lectin and collagen. Carsten Eckert was previously with the Museum für Naturkunde, Invalidenstrasse 43, 10115 Berlin, Germany. The collagen sequence from Aphrocallistes vastus reported here, viz., [COL_APHRO] APHVACOL (accession number AM411124), has been deposited in the EMBL/GenBank data base. This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin), the National Natural Science Foundation of China (grant no. 50402023), and the International Human Frontier Science Program.     

A modern approach to demineralization of spicules in glass sponges (Porifera: Hexactinellida) for the purpose of extraction and examination of the protein matrix

Glass sponges of the class Hexactinellida are a group of the most ancient multicellular animals, whose fossil remnants from the early Proterozoic have been registered. In order to demineralize the skeletal structures of the glass sponge Hyalonema sieboldi, we have used for the first time a strategy of slow leaching of the silicon-bearing component, based on the usage of alkaline solutions of sodium hydroxide, sodium dodecyl sulfate, and an anionic biosurfactant of a rhamnolipid nature. The obtained data unequivocally corroborate the presence of a fibrillar protein matrix functioning as a basis for silicon biomineralization in the basal spicules of H. sieboldi. Also, it has been found for the first time that the protein matrix is constructed of a collagenous protein. The technical approach proposed here might appear important for the study of the structural organization of skeletons in other silicon-bearing animals and, in an applied aspect, to work out new biomaterials for implantology and biocomposites, in order to use the latter as bioactive additives.     

Apposition of silica lamellae during growth of spicules in the demosponge Suberites domuncula: biological/biochemical studies and chemical/biomimetical confirmation

Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; their surfaces are formed by silicatein. The extracellularly located spicules are surrounded by sclerocytes which are filled with both electron-dense and electron-poor vesicles; energy dispersive X-ray analysis/scanning electron microscopical studies revealed that the electron-dense vesicles are filled of silicon/silica and therefore termed silicasomes. The release of the content of the silicasomes into the hollow cylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicatein molecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silicatein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]-poly(acetoxime methacrylate) [reactive ester polymer]-N(epsilon)-benzyloxycarbonyl L-lysine tert-butyl ester-Ni(II); finally His-tagged silicatein is immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for a technical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis of nanostructured tubes.     

Phylogenetic affinities and taphonomy of Brooksella from the Cambrian of Georgia and Alabama, USA

Siliceous “star cobbles”, referred to the enigmatic genus Brooksella, are abundant in the Conasauga Formation of the Coosa River Valley of Alabama and Georgia, USA. Explaining the phylogenetic affinities and taphonomic history of Brooksella has been difficult and contentious. Brooksella has, at times, been referred to: 1, the cnidarian order Scyphomedusae; 2, the cnidarian class Protomedusae (order Brooksellida); 3, as algae; 4, as a trace fossil; and 5, as a feature of inorganic origin.Macroscopic, microscopic, and computer-assisted tomographic analysis of Brooksella from the Conasauga Formation suggests that the “star cobbles” represent exceptionally preserved body fossils of simple construction. Morphology of star cobbles is most consistent with a siliceous (hexactinellid) sponge interpretation. Specimens show wide morphologic variation, including gradational patterns, suggesting that a single species name (Brooksella alternata) should be used to embrace all forms described from the Coosa Valley. B. alternata includes specimens having a variable number of radially disposed lobes divided by radial grooves, and often a central opening inferred to be an osculum on one side. Lobes in many specimens terminate in small openings. Small craterlike structures, inferred to be ostia, are present on the external surface. Radial internal cavities occupy the lobes. Specimens from the Conasauga Formation have siliceous spicules preserved surficially and internally.The three-dimensional nature of most “star cobbles” suggests rapid fossil diagenesis, perhaps mediated by the activities of microbial consortia that quickly formed biofilms around the dead hosts.     

Bioorganic/inorganic hybrid composition of sponge spicules: matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis

The giant basal spicules of the siliceous sponges Monorhaphis chuni and Monorhaphis intermedia (Hexactinellida) represent the largest biosilica structures on earth (up to 3m long). Here we describe the construction (lamellar organization) of these spicules and of the comitalia and highlight their organic matrix in order to understand their mechanical properties. The spicules display three distinct regions built of biosilica: (i) the outer lamellar zone (radius: >300 microm), (ii) the bulky axial cylinder (radius: <75 microm), and (iii) the central axial canal (diameter: <2 microm) with its organic axial filament. The spicules are loosely covered with a collagen net which is regularly perforated by 7-10 microm large holes; the net can be silicified. The silica layers forming the lamellar zone are approximately 5 microm thick; the central axial cylinder appears to be composed of almost solid silica which becomes porous after etching with hydrofluoric acid (HF). Dissolution of a complete spicule discloses its complex structure with distinct lamellae in the outer zone (lamellar coating) and a more resistant central part (axial barrel). Rapidly after the release of the organic coating from the lamellar zone the protein layers disintegrate to form irregular clumps/aggregates. In contrast, the proteinaceous axial barrel, hidden in the siliceous axial cylinder, is set up by rope-like filaments. Biochemical analysis revealed that the (dominant) molecule of the lamellar coating is a 27-kDa protein which displays catalytic, proteolytic activity. High resolution electron microscopic analysis showed that this protein is arranged within the lamellae and stabilizes these surfaces by palisade-like pillars. The mechanical behavior of the spicules was analyzed by a 3-point bending assay, coupled with scanning electron microscopy. The load-extension curve of the spicule shows a biphasic breakage/cracking pattern. The outer lamellar zone cracks in several distinct steps showing high resistance in concert with comparably low elasticity, while the axial cylinder breaks with high elasticity and lower stiffness. The complex bioorganic/inorganic hybrid composition and structure of the Monorhaphis spicules might provide the blueprint for the synthesis of bio-inspired material, with unusual mechanical properties (strength, stiffness) without losing the exceptional properties of optical transmission.     

Analysis of the axial filament in spicules of the demosponge Geodia cydonium: different silicatein composition in microscleres (asters) and megascleres (oxeas and triaenes)

The skeleton of the siliceous sponges (Porifera: Hexactinellida and Demospongiae) is supported by spicules composed of bio-silica. In the axial canals of megascleres, harboring the axial filaments, three isoforms of the enzyme silicatein (-alpha, -beta and -gamma) have been identified until now, using the demosponges Tethya aurantium and Suberites domuncula. Here we describe the composition of the proteinaceous components of the axial filament from small spicules, the microscleres, in the demosponge Geodia cydonium that possesses megascleres and microscleres. The morphology of the different spicule types is described. Also in G. cydonium the synthesis of the spicules starts intracellularly and they are subsequently extruded to the extracellular space. In contrast to the composition of the silicateins in the megascleres (isoforms: -alpha, -beta and -gamma), the axial filaments of the microscleres contain only one form of silicatein, termed silicatein-alpha/beta, with a size of 25kDa. Silicatein-alpha/beta undergoes three phosphorylation steps. The gene encoding silicatein-alpha/beta was identified and found to comprise the same characteristic sites, described previously for silicateins-alpha or -beta. It is hypothesized, that the different composition of the axial filaments, with respect to silicateins, contributes to the morphology of the different types of spicules.     

Formation of siliceous spicules in the marine demosponge <Emphasis Type="Italic">Suberites domuncula</Emphasis>

The siliceous skeleton of demosponges is constructed of spicules. We have studied the formation of spicules in primmorphs from Suberites domuncula. Scanning electron microscopy and transmission electron-microscopical (TEM) analyses have revealed, in the center of the spicules, an axial canal that is 0.3–1.6 m wide and filled with an axial filament. This filament is composed of the enzyme silicatein, which synthesizes the spicules. TEM analysis has shown that spicule formation starts intracellularly and ends extracellularly in the mesohyl. At the initial stage, the axial canal is composed only of silicatein, whereas membranous structures and fibrils (10–15 nm in width) can later also be identified, suggesting that intracellular components protrude into the axial canal. Antibodies against silicatein have been applied for Western blotting; intracellularly, silicatein is processed to the mature form (24 kDa), whereas the pro-enzyme with the propeptide (33 kDa) is detected extracellularly. Silicatein undergoes phosphorylation at five sites. Immunohistological analysis has shown that silicatein exists in the axial canal (axial filament) and on the surface of the spicules, suggesting that they grow by apposition. Finally, we have demonstrated that the enzymic reaction of silicatein is inhibited by anti-silicatein antibodies. These data provide, for the first time, a comprehensive outline of spicule formation.This work was supported by grants from the European Commission (SILIBIOTEC), the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin) and the International Human Frontier Science Program.     

The extent of asexual reproduction in sponges of the genus Chondrilla (Demospongiae: Chondrosida) from the Caribbean and the Brazilian coasts

In this study, we estimated the extent of asexual reproduction and genotypic diversity at the intra- and interspecific levels in sponges of the genus Chondrilla in the Caribbean and along the coast of Brazil. Allozymes were used to identify the genotypes of specimens of Chondrilla in each location. The four species studied displayed a large variation in the extent of clonal reproduction and genotypic diversity, with the two species from the Bahamas having a greater proportion of asexually produced individuals than those along the coast of Brazil. Conspecific Brazilian populations of Chondrilla sp. had large differences in clonality: the population from a heterogeneous environment and under the influence of a strong upwelling had little clonality (7%), whereas the population located 350 km south along the coast, in a more homogeneous and temporally stable environment, had a five-fold larger (39%) proportion of asexually derived individuals. Finally, we were able to confirm that, besides fission, the genus Chondrilla displays a second mode of asexual reproduction, by fragmentation of teardrop shaped individuals.     

Spicule dimensions as taxonomic criteria in the identification of haplosclerid sponges from the shores of Anglesey

The lengths and widths of at least 100 spicules from each of 126 specimens, comprising at least 12 species of haplosclerid sponges, mainly from Church Island and Rhosneigr, Anglesey, North Wales have been measured. It was found that spicule dimensions by themselves would be unreliable in the identification of species. The sample means, medians and maxima, when plotted using width and length axes, form specific clusters that overlap, sometimes to a considerable extent. The correlation coefficients for width against length varied widely, even when single species were considered. The variation arises from differences in the numbers of juvenile spicules in various samples. The best correlation coefficients exceeded 0.8 and the regression constants for linear correlation in such samples could be useful in the diagnosis and taxonomy of the species. The coefficients of variation for length and width also yielded parameters for species characterization. In general the coefficient for length tended to increase as the mean length increased, whereas that for width tended to decrease with increasing mean width. Some species stood apart from the general trends, however. The coefficients varied widely from one sample to another of the same species, identified using a variety of diagnostic features. To some extent the variation was linked with the date of collection. When all the data were combined, the average coefficient for width decreased markedly in May. The same was true for two species that were separately considered. The decrease was not simply caused by a change in mean width, changes in standard deviation also being involved. There was a tendency for certain species, in particular Reniera rosea and Gellius angulatus , to produce thin spicules in the spring, which became incorporated distally in the primary spicule bundles in July-August.     

Isolation and characterization of a protease from the marine sponge Spheciospongia vesparia

A protein that showed activity against proteic (casein and hide powder azure) and synthetic (BAEE and HLPA) substrates was isolated from the marine sponge Spheciospongia vesparia. The protease was purified from an aqueous extract by ammonium sulfate precipitation, gel filtration, hydrophobic and HPLC-anion exchange chromatographies. The purified protease showed a single band in SDS-PAGE minigels and had a molecular weight of 29,600, but when submitted to isoelectric focusing it showed 2 bands with isoelectric points of 4.56 and 4.43. Its catalytic action was inhibited by EDTA and 1,10-phenanthroline, so it seemed to be a metalloprotease.     

Biosilica formation in spicules of the sponge Suberites domuncula: synchronous expression of a gene cluster

The formation of spicules is a complicated morphogenetic process in sponges (phylum Porifera). The primmorph system was used to demonstrate that in the demosponge Suberites domuncula the synthesis of the siliceous spicules starts intracellularly and is dependent on the concentration of silicic acid. To understand spicule formation, a cluster of genes was isolated. In the center of this cluster is the silicatein gene, which codes for the enzyme that synthesizes spicules. This gene is flanked by an ankyrin repeat gene at one side and by a tumor necrosis factor receptor-associated factor and a protein kinase gene at the other side. All genes are strongly expressed in primmorphs and intact animals after exposure to silicic acid, and this expression is restricted to those areas where the spicule formation starts or where spicules are maintained in the animals. Our observations suggest that in S. domuncula a coordinated expression of physically linked genes is essential for the synthesis of the major skeletal elements.     

The defensive role of scutes in juvenile fluted giant clams (Tridacna squamosa)

This study tests the hypothesis that the scaly projections (scutes) on the shells of juvenile giant fluted clams, Tridacna squamosa, are an adaptation against crushing predators such as crabs. The forces required to crush scutes and clams were measured with a universal testing machine whereas crab chela strength was measured with a digital force gauge connected to a set of lever arms. Results for shell properties and chela strength are used to create two, non-mutually exclusive, predator–defense models. In Model 1, scutes increase the overall shell size, consequently reducing the number of crab predators with chelae that are large enough to seize and crush the prey. In Model 2, the chela has to open more to grasp a prey with these projecting structures which leads to a loss of claw-closing force such that crabs fail to crush the scutes, and consequently the clam. Clam scutes may also deter crab predators by increasing the risk of claw damage and/or handling time.     

Formation of cytoplasts from giant protoplasts in culture

Summary Protoplasts isolated from calli ofNicotiana plumbaginifolia can dramatically increase in volume without showing indications of cell wall synthesis. After reaching a critical size, the plasma-rich giant protoplasts show multiple formation of cytoplasts, which are released from the mother cells. The anucleate cytoplasts display the same increase in size as the nucleate protoplasts. Both cell types retain a spherical shape for several months, indicating that no major synthesis of cell wall occurred.     

Polarity factor 'Frizzled' in the demosponge Suberites domuncula: identification, expression and localization of the receptor in the epithelium/pinacoderm(1)

Until recently, it was assumed that polarity and axis formation have evolved only in metazoan phyla higher than Cnidaria. One key molecule involved in the signal transduction causing tissue polarity is Frizzled, a seven-transmembrane receptor that is activated by the Wnt family of secreted proteins. We report the isolation and characterization of a Frizzled gene from the demosponge Suberites domuncula (Sd-Fz). The deduced polypeptide comprises all characteristic domains known from Frizzled receptors of higher metazoans. In situ hybridization studies show that Sd-Fz is expressed in cells close to the surface of the sponges and in the pinacocytes of some canals. Northern blot analysis demonstrates its upregulation during the formation of three-dimensional sponge cell aggregates in culture. These data provide for the first time experimental evidence that already in the lowest metazoan phylum (Porifera) genes are present which are very likely involved in tissue polarity.     

Tube formation by complex cellular processes in Ciona intestinalis notochord

In the course of embryogenesis multicellular structures and organs are assembled from constituent cells. One structural component common to many organs is the tube, which consists most simply of a luminal space surrounded by a single layer of epithelial cells. The notochord of ascidian Ciona forms a tube consisting of only 40 cells, and serves as a hydrostatic “skeleton” essential for swimming. While the early processes of convergent extension in ascidian notochord development have been extensively studied, the later phases of development, which include lumen formation, have not been well characterized. Here we used molecular markers and confocal imaging to describe tubulogenesis in the developing Ciona notochord. We found that during tubulogenesis each notochord cell established de novo apical domains, and underwent a mesenchymal–epithelial transition to become an unusual epithelial cell with two opposing apical domains. Concomitantly, extracellular luminal matrix was produced and deposited between notochord cells. Subsequently, each notochord cell simultaneously executed two types of crawling movements bi-directionally along the anterior/posterior axis on the inner surface of notochordal sheath. Lamellipodia-like protrusions resulted in cell lengthening along the anterior/posterior axis, while the retraction of trailing edges of the same cell led to the merging of the two apical domains. As a result, the notochord cells acquired endothelial-like shape and formed the wall of the central lumen. Inhibition of actin polymerization prevented the cell movement and tube formation. Ciona notochord tube formation utilized an assortment of common and fundamental cellular processes including cell shape change, apical membrane biogenesis, cell/cell adhesion remodeling, dynamic cell crawling, and lumen matrix secretion.     

Intragenomic variation of the rDNA internal transcribed spacers in sponges (Phylum Porifera): implications for phylogenetic studies

The internal transcribed spacer regions (ITS1 and ITS2) of the tandemly repeated nuclear ribosomal DNA clusters are frequently used as markers for fine scale analyses in diverse animals. In certain taxa, ITS is nearly exclusively used for population level or inter-specific studies, despite the frequent presence of divergent paralogs within individual genomes that can be phylogenetically misleading. For the first time we survey diverse marine sponges to determine the extent and phylogenetic implications of intragenomic polymorphisms (IGPs) exhibited at their ITS loci. We discover that the extent of IGP varies greatly between taxa (with most taxa exhibiting very few) and cannot be predicted by taxonomy. Furthermore, we demonstrate that ITS can be phylogenetically informative between species when moderate levels of IGPs are detected, but that ITS paralogy can interfere with population level studies. We caution against the routine use of ITS in phylogenetic studies of sponges without (1) screening for IGPs in specimens from every population sampled; (2) including all divergent paralogs in phylogenetic analyses; (3) testing ITS data using other single-copy, unlinked loci (such as nuclear introns).     

Block of sodium conductance by n-octanol in crayfish giant axons

The block of the Na⁺ current by $\text{n-}\text{octanol}$ was studied in crayfish giant axons under axial wire voltage-clamp conditions. Standard kinetic analysis of the Na⁺ currents was undertaken to test the hypothesis that the $\text{n-}\text{octanol-induced}$ block of the Na⁺ current could be accounted for on the basis of changes in the voltage dependence of the kinetic parameters. Alterations in the membrane dipolar potential arising from rearrangement of membrane lipids would be the anticipated source of changes in the voltage dependence. Although some changes in voltage dependence did evolve with the block by $\text{n-}\text{octanol}$, the changes were not of sufficient magnitude to account for the block. In conclusion, although higher concentrations of $\text{n-}\text{octanol}$ produced shifts along the voltage axis of the kinetic parameters, direct blocking action of $\text{n-}\text{octanol}$ on the channel appears to be the most important mechanism of the block.     

Genetic structure of the Caribbean giant barrel sponge Xestospongia muta using the I3-M11 partition of COI

In recent years, reports of sponge bleaching, disease, and subsequent mortality have increased alarmingly. Population recovery may depend strongly on colonization capabilities of the affected species. The giant barrel sponge Xestospongia muta is a dominant reef constituent in the Caribbean. However, little is known about its population structure and gene flow. The 5′-end fragment of the mitochondrial gene cytochrome oxidase subunit I is often used to address these kinds of questions, but it presents very low intraspecific nucleotide variability in sponges. In this study, the usefulness of the I3-M11 partition of COI to determine the genetic structure of X. muta was tested for seven populations from Florida, the Bahamas and Belize. A total of 116 sequences of 544 bp were obtained for the I3-M11 partition corresponding to four haplotypes. In order to make a comparison with the 5′-end partition, 10 sequences per haplotype were analyzed for this fragment. The 40 resulting sequences were of 569 bp and corresponded to two haplotypes. The nucleotide diversity of the I3-M11 partition (π = 0.00386) was higher than that of the 5′-end partition (π = 0.00058), indicating better resolution at the intraspecific level. Sponges with the most divergent external morphologies (smooth vs. digitate surface) had different haplotypes, while those with the most common external morphology (rough surface) presented a mixture of haplotypes. Pairwise tests for genetic differentiation among geographic locations based on F _ST values showed significant genetic divergence between most populations, but this genetic differentiation was not due to isolation by distance. While limited larval dispersal may have led to differentiation among some of the populations, the patterns of genetic structure appear to be most strongly related to patterns of ocean currents. Therefore, hydrological features may play a major role in sponge colonization and need to be considered in future plans for management and conservation of these important components of coral reef ecosystems. Communicated by Biology Editor Dr Ruth Gates