Phylogeny and systematics of demospongiae in light of new small-subunit ribosomal DNA (18S) sequences.

The most diverse and species-rich class of the phylum Porifera is Demospongiae. In recent years, the systematics of this clade, which contains more than 7000 species, has developed rapidly in light of new studies combining molecular and morphological observations. We add more than 500 new, nearly complete 18S sequences (an increase of more than 200%) in an attempt to further enhance understanding of the phylogeny of Demospongiae. Our study specifically targets representation of type species and genera that have never been sampled for any molecular data in an effort to accelerate progress in classifying this diverse lineage. Our analyses recover four highly supported subclasses of Demospongiae: Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha. Within Keratosa, neither Dendroceratida, nor its two families, Darwinellidae and Dictyodendrillidae, are monophyletic and Dictyoceratida is divided into two lineages, one predominantly composed of Dysideidae and the second containing the remaining families (Irciniidae, Spongiidae, Thorectidae, and Verticillitidae). Within Myxospongiae, we find Chondrosida to be paraphyletic with respect to the Verongida. We amend the latter to include species of the genus Chondrosia and erect a new order Chondrillida to contain remaining taxa from Chondrosida, which we now discard. Even with increased taxon sampling of Haploscleromorpha, our analyses are consistent with previous studies; however, Haliclona species are interspersed in even more clades. Haploscleromorpha contains five highly supported clades, each more diverse than previously recognized, and current families are mostly polyphyletic. In addition, we reassign Janulum spinispiculum to Haploscleromorpha and resurrect Reniera filholi as Janulum filholi comb. nov. Within the large clade Heteroscleromorpha, we confirmed 12 recently identified clades based on alternative data, as well as a sister-group relationship between the freshwater Spongillida and the family Vetulinidae. We transfer Stylissa flabelliformis to the genus Scopalina within the family Scopalinidae, which is of uncertain position. Our analyses uncover a large, strongly supported clade containing all heteroscleromorphs other than Spongillida, Vetulinidae, and Scopalinidae. Within this clade, there is a major division separating Axinellidae, Biemnida, Tetractinellida, Bubaridae, Stelligeridae, Raspailiidae, and some species of Petromica, Topsentia, and Axinyssa from Agelasida, Polymastiidae, Placospongiidae, Clionaidae, Spirastrellidae, Tethyidae, Poecilosclerida, Halichondriidae, Suberitidae, and Trachycladus. Among numerous results: (1) Spirophorina and its family Tetillidae are paraphyletic with respect to a strongly supported Astrophorina within Tetractinellida; (2) Agelasida is the earliest diverging lineage within the second clade listed above; and (3) Merlia and Desmacella appear to be the earliest diverging lineages of Poecilosclerida.

Sponge systematics facing new challenges.

Systematics is nowadays facing new challenges with the introduction of new concepts and new techniques. Compared to most other phyla, phylogenetic relationships among sponges are still largely unresolved. In the past 10 years, the classical taxonomy has been completely overturned and a review of the state of the art appears necessary. The field of taxonomy remains a prominent discipline of sponge research and studies related to sponge systematics were in greater number in the Eighth World Sponge Conference (Girona, Spain, September 2010) than in any previous world sponge conferences. To understand the state of this rapidly growing field, this chapter proposes to review studies, mainly from the past decade, in sponge taxonomy, nomenclature and phylogeny. In a first part, we analyse the reasons of the current success of this field. In a second part, we establish the current sponge systematics theoretical framework, with the use of (1) cladistics, (2) different codes of nomenclature (PhyloCode vs. Linnaean system) and (3) integrative taxonomy. Sponges are infamous for their lack of characters. However, by listing and discussing in a third part all characters available to taxonomists, we show how diverse characters are and that new ones are being used and tested, while old ones should be revisited. We then review the systematics of the four main classes of sponges (Hexactinellida, Calcispongiae, Homoscleromorpha and Demospongiae), each time focusing on current issues and case studies. We present a review of the taxonomic changes since the publication of the Systema Porifera (2002), and point to problems a sponge taxonomist is still faced with nowadays. To conclude, we make a series of proposals for the future of sponge systematics. In the light of recent studies, we establish a series of taxonomic changes that the sponge community may be ready to accept. We also propose a series of sponge new names and definitions following the PhyloCode. The issue of phantom species (potential new species revealed by molecular studies) is raised, and we show how they could be dealt with. Finally, we present a general strategy to help us succeed in building a Porifera tree along with the corresponding revised Porifera classification.

Congruence between nuclear and mitochondrial genes in Demospongiae: a new hypothesis for relationships within the G4 clade (Porifera: Demospongiae).

The current morphological classification of the Demospongiae G4 clade was tested using large subunit ribosomal RNA (LSU rRNA) sequences from 119 taxa. Fifty-three mitochondrial cytochrome oxidase 1 (CO1) barcoding sequences were also analysed to test whether the 28S phylogeny could be recovered using an independent gene. This is the largest and most comprehensive study of the Demospongiae G4 clade. The 28S and CO1 genetrees result in congruent clades but conflict with the current morphological classification. The results confirm the polyphyly of Halichondrida, Hadromerida, Dictyonellidae, Axinellidae and Poecilosclerida and show that several of the characters used in morphological classifications are homoplasious. Robust clades are clearly shown and a new hypothesis for relationships of taxa allocated to G4 is proposed.

Embryogenesis in the glass sponge Oopsacas minuta: Formation of syncytia by fusion of blastomeres.

Sponges (Porifera) are unusual animals whose body plans make interpreting phylogenetic relationships within the group and with other basal metazoan taxa a difficult task. Although molecular approaches have offered new insights, some questions require a morphological approach using detailed ultrastructural or light microscopical studies of developing embryos and larvae. Glass sponges (Hexactinellida) have perhaps the most unusual body plan within the Metazoa because the majority of the tissue of the adult consists of a single giant multinucleated syncytium that forms the inner and outer layers of the sponge and is joined by cytoplasmic bridges to uninucleate cellular regions. Here we have used serial section transmission and high-resolution scanning electron microscopy to examine when syncytia first form in the cave-dwelling glass sponge Oopsacas minuta. We confirm that in O. minuta blastomeres are separate until the 32-cell stage; cleavage is equal but asynchronous until a hollow blastula is formed. The sixth division yields a collection of variously sized micromeres at the surface of the embryo and large yolk- and lipid-filled macromeres lining the blastocoel. Syncytia then form by the fusion of micromeres to form cytoplasmic bridges with each other and the fusion of macromeres to form the future multinucleated trabecular tissue of the larva and adult sponge. The multinucleated trabecular tissue envelops and forms cytoplasmic bridges with all uninucleate cells, covering the developing larva with a continuous syncytial epithelium. Differentiation of tissues occurs very early during embryogenesis with the separation of uninucleate and multinucleate lineages, but all cells and syncytia are joined by cytoplasmic bridges such that there is cytoplasmic continuity throughout the entire larva. Although glass sponges begin life as a cellular embryo, the unusual mechanism of syncytia formation at such an early stage in development distinguishes this group of animals from their closest multicellular relatives, the Demospongiae. Most important, however, these data lend support to the hypothesis that the original metazoans were cellular, not syncytial.

Sponge paraphyly and the origin of Metazoa.

In order to allow critical evaluation of the interrelationships between the three sponge classes, and to resolve the question of mono- or paraphyly of sponges (Porifera), we used the polymerase chain reaction (PCR) to amplify almost the entire nucleic acid sequence of the 18S rDNA from several hexactinellid, demosponge and calcareous sponge species. The amplification products were cloned, sequenced and then aligned with previously reported sequences from other sponges and nonsponge metazoans and variously distant outgroups, and trees were constructed using both neighbour-joining and maximum parsimony methods. Our results suggest that sponges are paraphyletic, the Calcarea being more related to monophyletic Eumetazoa than to the siliceous sponges (Demospongiae, Hexactinellida). These results have important implications for our understanding of metazoan origins, because they suggest that the common ancestor of Metazoa was a sponge. They also have consequences for basal metazoan classification, implying that the phylum Porifera should be abandoned. Our results support the upgrading of the calcareous sponge class to the phylum level.

Phylogenetic analysis of the Hsp70 sequences reveals the monophyly of Metazoa and specific phylogenetic relationships between animals and fungi.

To understand the early evolution of the Metazoa, it is necessary to determine the correct phylogenetic status of diploblastic animals. Despite cladistic studies of morphological characters and recent molecular phylogenetic studies, it remains uncertain whether diploblasts are monophyletic or paraphyletic, and how the phyla of diploblasts are phylogenetically related. The heat shock protein 70 (Hsp70) sequences, because of their ubiquity and high degree of conservation, could provide a useful model for phylogenetic analysis. We have sequenced almost the entire nucleic acid sequence of cytoplasmic Hsp70 from eight diploblastic species. Our data support the monophyly of diploblastic animals. However, the phylogenetic relationships of the diploblast groups were not significantly resolved. Our phylogenetic trees also support the monophyly of Metazoa with high bootstrap values, indicating that animals form an extremely robust clade.

Reassessment of homology of morphological characters in tetractinellid sponges based on molecular data.

In sponges, as in other taxa with simple organization, the evaluation and use of morphological characters is difficult. Phylogenetic analysis of the first 850 nucleotides from the 5' end of the 28S rRNA gene is used here to assess the homology of spicules used in the classification of the subclass Tetractinellida. A single well-supported MP tree was obtained. The monophyly of the nine Tetractinellida species studied confirms the tetraxon megasclere as a morphological synapomorphy for the Tetractinellida. Two species are reallocated, Penares helleri as a Geodiidae, now thought to have lost sterraster microscleres, and Stryphnus mucronatus to the Streptosclerophorida. SEM micrographs of Stryphnus microscleres show that the morphology of the sanidasters is compatible with the hypothesis that they are homologous with streptoscleres and confirm this reallocation. Two other synapomorphies are confirmed within the tetractinellid clade, the simultaneous presence of tetraxon megasclere and aster-type microsclere (Astrophorida) and the loss of the streptosclere and persistence of the euaster s.s. microscleres (Euastrophorida) evidenced by the reallocation of Stryphnus mucronatus. The streptosclere microscleres cannot be evaluated in terms of homology because Streptosclerophorida may be paraphyletic (although these nodes are not supported by reliable bootstrap proportions) contrary to the currently accepted classification.

Polyphyly of "sclerosponges" (Porifera, Demospongiae) supported by 28S ribosomal sequences.

To test the competing hypotheses of polyphyly and monophyly of "sclerosponges," sequences from the 5' end of 28S ribosomal RNA were obtained for Astrosclera willeyana, Acanthochaetetes wellsi, and six other demosponge species. Phylogenetic relationships deduced from parsimony and neighbor-joining analyses suggest that these sclerosponges belong to two different orders of Demospongiae: Astrosclera willeyana, being closely related to the Agelasidae, belongs to the Agelasida, Acanthochaetetes wellsi, being closely related to the Spirastrellidae, belongs to the Hadromerida. These results contradict the hypothesis that sclerosponges are monophyletic and imply that a massive calcareous skeleton has evolved independently in several lineages of sponges.

Type IV collagen in sponges, the missing link in basement membrane ubiquity.

Basement membrane structures, or their main component, type IV collagen, have been detected in all multicellular animal species, except sponges. We cancel this exception by the demonstration of type IV collagenous sequences in a new marine sponge species by cDNA and genomic DNA studies. One of these sequences is long enough to demonstrate the specific characteristics of type IV collagen chains. The 12 cysteines are at conserved positions in the carboxyl-terminal non-helical NCl domain, as are the interruptions in the carboxyl-terminal end of the triple helical domain. The gene organization of the region coding for the NCl domain is similar to that of the human genes COL4A2, COL4A4 and COL4A6. An additional, shorter sequence suggests the presence of a second chain. The expected tissue localization of this collagen has been confirmed using polyclonal antibodies raised against a sponge recombinant protein. These results demonstrate that type IV collagen is representated in all animal phyla. It is actually the only known ubiquitous collagen and it has at least two different alpha chains in all the species where it has been characterized.

An analysis of partial 28S ribosomal RNA sequences suggests early radiations of sponges.

Sequences from the 5' end terminal part of 28S ribosomal RNA were obtained and compared for 22 animals belonging to all diploblastic phyla and for a large number of representatives of triploblastic Metazoa and protists. Phylogenetic analyses undertaken using different methods showed deep radiations of phyla such as Ctenophora, Cnidaria and Placozoa but also for groups of Porifera of low taxonomic rank. Short internodes between these radiations suggested an early rapid diversification of diploblasts. A long internal branch preceding the diversification of all triploblasts analyzed could be explained either by a long period with a single ancestor or by the extinction of the earliest triploblastic radiations. Finally some unexpected relationships were revealed among Porifera.

A cell type in sponges involved in the metabolism of glycogen. The gray cells.

The gray cells of four orders of demosponges contain basophilic inclusions and glycogen. They are capable of synthesis and accumulation of glycogen and responsible for its transfer to sites of more intense metabolism (growth, bud, blastema). They do not occur in larvae; but all the phases of their differentiation from the flagellar cells of the larva have been demonstrated.