A novel target-enriched multilocus assay for sponges (Porifera): Red Sea Haplosclerida (Demospongiae) as a test case.

With declining biodiversity worldwide, a better understanding of species diversity and their relationships is imperative for conservation and management efforts. Marine sponges are species-rich ecological key players on coral reefs, but their species diversity is still poorly understood. This is particularly true for the demosponge order Haplosclerida, whose systematic relationships are contentious due to the incongruencies between morphological and molecular phylogenetic hypotheses. The single gene markers applied in previous studies did not resolve these discrepancies. Hence, there is a high need for a genome-wide approach to derive a phylogenetically robust classification and understand this group's evolutionary relationships. To this end, we developed a target enrichment-based multilocus probe assay for the order Haplosclerida using transcriptomic data. This probe assay consists of 20,000 enrichment probes targeting 2956 ultraconserved elements in coding (i.e. exon) regions across the genome and was tested on 26 haplosclerid specimens from the Red Sea. Our target-enrichment approach correctly placed our samples in a well-supported phylogeny, in agreement with previous haplosclerid molecular phylogenies. Our results demonstrate the applicability of high-resolution genomic methods in a systematically complex marine invertebrate group and provide a promising approach for robust phylogenies of Haplosclerida. Subsequently, this will lead to biologically unambiguous taxonomic revisions, better interpretations of biological and ecological observations and new avenues for applied research, conservation and managing declining marine diversity.

Influence of CNT Length on Dispersion, Localization, and Electrical Percolation in a Styrene-Butadiene-Based Star Block Copolymer.

This study followed the approach of dispersing and localizing carbon nanotubes (CNTs) in nanostructured domains of block copolymers (BCPs) by shortening the CNTs via ball milling. The aim was to selectively tune the electrical and mechanical properties of the resulting nanocomposites, e.g., for use as sensor materials. Multiwalled carbon nanotubes (MWCNTs) were ground into different size fractions. The MWCNT length distribution was evaluated via transmission electron microscopy and dynamic light scattering. The nanostructure of the BCPs and the glass transition temperatures of the PB-rich and PS phases were not strongly affected by the addition of CNTs up to 2 wt%. However, AFM and TEM investigations indicated a partial localization of the shortened CNTs in the soft PB-rich phase or at the interface of the PB-rich and PS phase, respectively. The stress-strain behavior of the solution-mixed composites differed little from the mechanical property profile of the neat BCP and was largely independent of CNT amount and CNT size fraction. Significant changes could only be observed for Young's modulus and strain at break and may be attributed to CNT localization and small changes in morphology. For nanocomposites with unmilled CNTs, the electrical percolation threshold was less than 0.1 wt%. As the CNTs were shortened, the resistivity increased and the percolation threshold shifted to higher CNT contents. Composites with CNTs ground for 7.5 h and 13.5 h showed no bulk conductivity but significantly decreased surface resistivity on the bottom side of the films, which could be attributed to a sedimentation process of the grind and thereby highly compressed CNT agglomerates during evaporation.

Carbonic Anhydrases: An Ancient Tool in Calcareous Sponge Biomineralization.

Enzymes of the α-carbonic anhydrase gene family (CAs) are essential for the deposition of calcium carbonate biominerals. In calcareous sponges (phylum Porifera, class Calcarea), specific CAs are involved in the formation of calcite spicules, a unique trait and synapomorphy of this class. However, detailed studies on the CA repertoire of calcareous sponges exist for only two species of one of the two Calcarea subclasses, the Calcaronea. The CA repertoire of the second subclass, the Calcinea, has not been investigated so far, leaving a considerable gap in our knowledge about this gene family in Calcarea. Here, using transcriptomic analysis, phylogenetics, and in situ hybridization, we study the CA repertoire of four additional species of calcareous sponges, including three from the previously unsampled subclass Calcinea. Our data indicate that the last common ancestor of Calcarea had four ancestral CAs with defined subcellular localizations and functions (mitochondrial/cytosolic, membrane-bound, and secreted non-catalytic). The evolution of membrane-bound and secreted CAs involved gene duplications and losses, whereas mitochondrial/cytosolic and non-catalytic CAs are evidently orthologous genes. Mitochondrial/cytosolic CAs are biomineralization-specific genes recruited for biomineralization in the last common ancestor of calcareous sponges. The spatial-temporal expression of these CAs differs between species, which may reflect differences between subclasses or be related to the secondary thickening of spicules during biomineralization that does not occur in all species. With this study, we extend the understanding of the role and the evolution of a key biomineralization gene in calcareous sponges.

An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola.

Microcolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast-like growth and constitutive melanin formation. They dominate a range of hostile natural and man-made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi's phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple genes with or without resistance cassettes were developed. This state-of-the-art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi.

NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog.

Macroautophagy/autophagy is a conserved degradation process in eukaryotic cells involving the sequestration of proteins and organelles within double-membrane vesicles termed autophagosomes. In filamentous fungi, its main purposes are the regulation of starvation adaptation and developmental processes. In contrast to nonselective bulk autophagy, selective autophagy is characterized by cargo receptors, which bind specific cargos such as superfluous organelles, damaged or harmful proteins, or microbes, and target them for autophagic degradation. Herein, using the core autophagy protein ATG8 as bait, GFP-Trap analysis followed by liquid chromatography mass spectrometry (LC/MS) identified a putative homolog of the human autophagy cargo receptor NBR1 (NBR1, autophagy cargo receptor) in the filamentous ascomycete Sordaria macrospora (Sm). Fluorescence microscopy revealed that SmNBR1 colocalizes with SmATG8 at autophagosome-like structures and in the lumen of vacuoles. Delivery of SmNBR1 to the vacuoles requires SmATG8. Both proteins interact in an LC3 interacting region (LIR)-dependent manner. Deletion of Smnbr1 leads to impaired vegetative growth under starvation conditions and reduced sexual spore production under non-starvation conditions. The human NBR1 homolog partially rescues the phenotypic defects of the fungal Smnbr1 deletion mutant. The Smnbr1 mutant can neither use fatty acids as a sole carbon source nor form fruiting bodies under oxidative stress conditions. Fluorescence microscopy revealed that degradation of a peroxisomal reporter protein is impaired in the Smnbr1 deletion mutant. Thus, SmNBR1 is a cargo receptor for pexophagy in filamentous ascomycetes.

A new species of the calcareous sponge genus Leuclathrina (Calcarea: Calcinea: Clathrinida) from the Maldives.

The diversity and phylogenetic relationships of calcareous sponges are still not completely understood. Recent integrative approaches combined analyses of DNA and morphological observations. Such studies resulted in severe taxonomic revisions within the subclass Calcinea and provided the foundation for a phylogenetically meaningful classification. However, several genera are missing from DNA phylogenies and their relationship to other Calcinea remain uncertain. One of these genera is Leuclathrina (family Leucaltidae). We here describe a new species from the Maldives, Leuclathrina translucida sp. nov., which is only the second species of the genus. Like the type species Leuclathrina asconoides, the new species has a leuconoid aquiferous system and lacks a specialized choanoskeleton. Phylogenetic analyses of the partial 28S ribosomal RNA gene revealed that L. translucida sp. nov. is most closely related to a clade containing the exclusively asconoid genera Ascandra, Levinella and Soleneiscus, and to a clade of the likewise asconoid genus Ernstia. No close relationship exists to other members of the polyphyletic family Leucaltidae, or to any other leuconoid Calcinea. Our results suggest that the leuconoid aquiferous system of Leuclathrina evolved independently from that of other calcineans and that the family assignment of the genus has to be reconsidered. Because the latter requires a more comprehensive family level revision among many genera of Calcinea, we propose to formally retain the genus in Leucaltidae for the time being.

Spicule formation in calcareous sponges: Coordinated expression of biomineralization genes and spicule-type specific genes.

The ability to form mineral structures under biological control is widespread among animals. In several species, specific proteins have been shown to be involved in biomineralization, but it is uncertain how they influence the shape of the growing biomineral and the resulting skeleton. Calcareous sponges are the only sponges that form calcitic spicules, which, based on the number of rays (actines) are distinguished in diactines, triactines and tetractines. Each actine is formed by only two cells, called sclerocytes. Little is known about biomineralization proteins in calcareous sponges, other than that specific carbonic anhydrases (CAs) have been identified, and that uncharacterized Asx-rich proteins have been isolated from calcitic spicules. By RNA-Seq and RNA in situ hybridization (ISH), we identified five additional biomineralization genes in Sycon ciliatum: two bicarbonate transporters (BCTs) and three Asx-rich extracellular matrix proteins (ARPs). We show that these biomineralization genes are expressed in a coordinated pattern during spicule formation. Furthermore, two of the ARPs are spicule-type specific for triactines and tetractines (ARP1 or SciTriactinin) or diactines (ARP2 or SciDiactinin). Our results suggest that spicule formation is controlled by defined temporal and spatial expression of spicule-type specific sets of biomineralization genes.

Molecular biodiversity of Red Sea demosponges.

Sponges are important constituents of coral reef ecosystems, including those around the Arabian Peninsula. Despite their importance, our knowledge on demosponge diversity in this area is insufficient to recognize, for example, faunal changes caused by anthropogenic disturbances. We here report the first assessment of demosponge molecular biodiversity from Arabia, with focus on the Saudi Arabian Red Sea, based on mitochondrial and nuclear ribosomal molecular markers gathered in the framework of the Sponge Barcoding Project. We use a rapid molecular screening approach on Arabian demosponge collections and analyze results in comparison against published material in terms of biodiversity. We use a variable region of 28S rDNA, applied for the first time in the assessment of demosponge molecular diversity. Our data constitutes a solid foundation for a future more comprehensive understanding of sponge biodiversity of the Red Sea and adjacent waters.

Calcareous sponge genomes reveal complex evolution of α-carbonic anhydrases and two key biomineralization enzymes.

BACKGROUND: Calcium carbonate biominerals form often complex and beautiful skeletal elements, including coral exoskeletons and mollusc shells. Although the ability to generate these carbonate structures was apparently gained independently during animal evolution, it sometimes involves the same gene families. One of the best-studied of these gene families comprises the α- carbonic anhydrases (CAs), which catalyse the reversible transformation of CO2 to HCO3 - and fulfill many physiological functions. Among Porifera -the oldest animal phylum with the ability to produce skeletal elements- only the class of calcareous sponges can build calcitic spicules, which are the extracellular products of specialized cells, the sclerocytes. Little is known about the molecular mechanisms of their synthesis, but inhibition studies suggest an essential role of CAs. In order to gain insight into the evolution and function of CAs in biomineralization of a basal metazoan species, we determined the diversity and expression of CAs in the calcareous sponges Sycon ciliatum and Leucosolenia complicata by means of genomic screening, RNA-Seq and RNA in situ hybridization expression analysis. Active biomineralization was located with calcein-staining. RESULTS: We found that the CA repertoires of two calcareous sponge species are strikingly more complex than those of other sponges. By characterizing their expression patterns, we could link two CAs (one intracellular and one extracellular) to the process of calcite spicule formation in both studied species. The extracellular biomineralizing CAs seem to be of paralogous origin, a finding that advises caution against assuming functional conservation of biomineralizing genes based upon orthology assessment alone. Additionally, calcareous sponges possess acatalytic CAs related to human CAs X and XI, suggesting an ancient origin of these proteins. Phylogenetic analyses including CAs from genomes of all non-bilaterian phyla suggest multiple gene losses and duplications and presence of several CAs in the last common ancestor of metazoans. CONCLUSIONS: We identified two key biomineralization enzymes from the CA-family in calcareous sponges and propose their possible interaction in spicule formation. The complex evolutionary history of the CA family is driven by frequent gene diversification and losses. These evolutionary patterns likely facilitated the numerous events of independent recruitment of CAs into biomineralization within Metazoa.

Autophagic kinases SmVPS34 and SmVPS15 are required for viability in the filamentous ascomycete Sordaria macrospora.

Autophagy is a tightly controlled degradation process of all eukaryotes. It includes the sequestration of cytoplasmic contents and organelles within a double-membraned autophagosome. Autophagy involves core autophagy related (atg) genes as well as genes regulating vesicle trafficking. Previously, we analyzed the impact of proteins of the core autophagic machinery SmATG7, SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. While deletion of Smatg8 and Smatg4 abolished fruiting-body formation and impaired vegetative growth, Smatg7 is required for viability. In yeast, the phosphatidylinositol 3-kinase vacuolar protein sorting 34 (Vps34) and its myristoylated membrane targeting unit, the protein kinase Vps15 have been shown to be important regulators of autophagy and vacuolar protein sorting. However, their exact role in filamentous ascomycetes remains elusive. To determine the function of Smvps34 and Smvps15 we isolated genes with high sequence similarity to Saccharomyces cerevisiae VPS34 and VPS15. For both genes we were not able to generate a homokaryotic knockout mutant in S. macrospora, suggesting that Smvps34 and Smvps15 are required for viability. Furthermore, we analyzed the repertoire of vps genes encoded by S. macrospora and could identify putative homologs of nearly all of the 61 VPS genes of S. cerevisiae.

bZIP transcription factor SmJLB1 regulates autophagy-related genes Smatg8 and Smatg4 and is required for fruiting-body development and vegetative growth in Sordaria macrospora.

Autophagy is a precisely controlled degradation process in eukaryotic cells, during which the bulk of the cytoplasm is engulfed by a double membrane vesicle, the autophagosome. Fusion of the autophagosome with the vacuole leads to breakdown of its contents, such as proteins and organelles, and the recycling of nutrients. Earlier studies of autophagic genes of the core autophagic machinery in the filamentous ascomycete Sordaria macrospora elucidated the impact of autophagy on fungal viability, vegetative growth and fruiting-body development. To gain further knowledge about the regulation of autophagy in S. macrospora, we analyzed the function of the bZIP transcription factor SmJLB1, a homolog of the Podospora anserina basic zipper-type transcription factor induced during incompatibility 4 (IDI-4) and the Aspergillus nidulans transcription factor jun-like bZIP A (JlbA). Generation of the homokaryotic deletion mutant demonstrated S. macrospora Smjlb1 is associated with autophagy-dependent processes. Deletion of Smjlb1 abolished fruiting-body formation and impaired vegetative growth. SmJLB1 is localized to the cytoplasm and to nuclei. Quantitative real-time PCR experiments revealed an upregulated expression of autophagy-related genes Smatg8 and Smatg4 in the Smjlb1 deletion mutant, suggesting a transcriptional repression function of SmJLB1.

Self-eating to grow and kill: autophagy in filamentous ascomycetes.

Autophagy is a tightly controlled degradation process in which eukaryotic cells digest their own cytoplasm containing protein complexes and organelles in the vacuole or lysosome. Two types of autophagy have been described: macroautophagy and microautophagy. Both types can be further divided into nonselective and selective processes. Molecular analysis of autophagy over the last two decades has mostly used the unicellular ascomycetes Saccharomyces cerevisiae and Pichia pastoris. Genetic analysis in these yeasts has identified 36 autophagy-related (atg) genes; many are conserved in all eukaryotes, including filamentous ascomycetes. However, the autophagic machinery also evolved significant differences in fungi, as a consequence of adaptation to diverse fungal lifestyles. Intensive studies on autophagy in the last few years have shown that autophagy in filamentous fungi is not only involved in nutrient homeostasis but in other cellular processes such as cell differentiation, pathogenicity and secondary metabolite production. This mini-review focuses on the specific roles of autophagy in filamentous fungi.

Autophagy genes Smatg8 and Smatg4 are required for fruiting-body development, vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora.

Autophagy is a tightly controlled degradation process involved in various developmental aspects of eukaryotes. However, its involvement in developmental processes of multicellular filamentous ascomycetes is largely unknown. Here, we analyzed the impact of the autophagic proteins SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. A Saccharomyces cerevisiae complementation assay demonstrated that the S. macrospora Smatg8 and Smatg4 genes can functionally replace the yeast homologs. By generating homokaryotic deletion mutants, we showed that the S. macrospora SmATG8 and SmATG4 orthologs were associated with autophagy-dependent processes. Smatg8 and Smatg4 deletions abolished fruiting-body formation and impaired vegetative growth and ascospore germination, but not hyphal fusion. We demonstrated that SmATG4 was capable of processing the SmATG8 precursor. SmATG8 was localized to autophagosomes, whereas SmATG4 was distributed throughout the cytoplasm of S. macrospora. Furthermore, we could show that Smatg8 and Smatg4 are not only required for nonselective macroautophagy, but for selective macropexophagy as well. Taken together, our results suggest that in S. macrospora, autophagy seems to be an essential and constitutively active process to sustain high energy levels for filamentous growth and multicellular development even under nonstarvation conditions.

Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes, fragmented rRNAs, tRNA editing, and a novel genetic code.

Sponges (phylum Porifera) are a large and ancient group of morphologically simple but ecologically important aquatic animals. Although their body plan and lifestyle are relatively uniform, sponges show extensive molecular and genetic diversity. In particular, mitochondrial genomes from three of the four previously studied classes of Porifera (Demospongiae, Hexactinellida, and Homoscleromorpha) have distinct gene contents, genome organizations, and evolutionary rates. Here, we report the mitochondrial genome of Clathrina clathrus (Calcinea, Clathrinidae), a representative of the fourth poriferan class, the Calcarea, which proves to be the most unusual. Clathrina clathrus mitochondrial DNA (mtDNA) consists of six linear chromosomes 7.6-9.4 kb in size and encodes at least 37 genes: 13 protein codings, 2 ribosomal RNAs (rRNAs), and 24 transfer RNAs (tRNAs). Protein genes include atp9, which has now been found in all major sponge lineages, but no atp8. Our analyses further reveal the presence of a novel genetic code that involves unique reassignments of the UAG codons from termination to tyrosine and of the CGN codons from arginine to glycine. Clathrina clathrus mitochondrial rRNAs are encoded in three (srRNA) and ≥6 (lrRNA) fragments distributed out of order and on several chromosomes. The encoded tRNAs contain multiple mismatches in the aminoacyl acceptor stems that are repaired posttranscriptionally by 3'-end RNA editing. Although our analysis does not resolve the phylogenetic position of calcareous sponges, likely due to their high rates of mitochondrial sequence evolution, it confirms mtDNA as a promising marker for population studies in this group. The combination of unusual mitochondrial features in C. clathrus redefines the extremes of mtDNA evolution in animals and further argues against the idea of a "typical animal mtDNA."

Molecular phylogenetic evaluation of classification and scenarios of character evolution in calcareous sponges (Porifera, Class Calcarea).

Calcareous sponges (Phylum Porifera, Class Calcarea) are known to be taxonomically difficult. Previous molecular studies have revealed many discrepancies between classically recognized taxa and the observed relationships at the order, family and genus levels; these inconsistencies question underlying hypotheses regarding the evolution of certain morphological characters. Therefore, we extended the available taxa and character set by sequencing the complete small subunit (SSU) rDNA and the almost complete large subunit (LSU) rDNA of additional key species and complemented this dataset by substantially increasing the length of available LSU sequences. Phylogenetic analyses provided new hypotheses about the relationships of Calcarea and about the evolution of certain morphological characters. We tested our phylogeny against competing phylogenetic hypotheses presented by previous classification systems. Our data reject the current order-level classification by again finding non-monophyletic Leucosolenida, Clathrinida and Murrayonida. In the subclass Calcinea, we recovered a clade that includes all species with a cortex, which is largely consistent with the previously proposed order Leucettida. Other orders that had been rejected in the current system were not found, but could not be rejected in our tests either. We found several additional families and genera polyphyletic: the families Leucascidae and Leucaltidae and the genus Leucetta in Calcinea, and in Calcaronea the family Amphoriscidae and the genus Ute. Our phylogeny also provided support for the vaguely suspected close relationship of several members of Grantiidae with giantortical diactines to members of Heteropiidae. Similarly, our analyses revealed several unexpected affinities, such as a sister group relationship between Leucettusa (Leucaltidae) and Leucettidae and between Leucascandra (Jenkinidae) and Sycon carteri (Sycettidae). According to our results, the taxonomy of Calcarea is in desperate need of a thorough revision, which cannot be achieved by considering morphology alone or relying on a taxon sampling based on the current classification below the subclass level.

A homologue of the human STRIPAK complex controls sexual development in fungi.

Sexual development in fungi is a complex process involving the generation of new cell types and tissues - an essential step for all eukaryotic life. The characterization of sterile mutants in the ascomycete Sordaria macrospora has led to a number of proteins involved in sexual development, but a link between these proteins is still missing. Using a combined tandem-affinity purification/mass spectrometry approach, we showed in vivo association of developmental protein PRO22 with PRO11, homologue of mammalian striatin, and SmPP2AA, scaffolding subunit of protein phosphatase 2A. Further experiments extended the protein network to the putative kinase activator SmMOB3, known to be involved in sexual development. Extensive yeast two-hybrid studies allowed us to pinpoint functional domains involved in protein-protein interaction. We show for the first time that a number of already known factors together with new components associate in vivo to form a highly conserved multi-subunit complex. Strikingly, a similar complex has been described in humans, but the function of this so-called striatin interacting phosphatase and kinase (STRIPAK) complex is largely unknown. In S. macrospora, truncation of PRO11 and PRO22 leads to distinct defects in sexual development and cell fusion, indicating a role for the fungal STRIPAK complex in both processes.

The mitochondrial genomes of sponges provide evidence for multiple invasions by Repetitive Hairpin-forming Elements (RHE).

BACKGROUND: The mitochondrial (mt) genomes of sponges possess a variety of features, which appear to be intermediate between those of Eumetazoa and non-metazoan opisthokonts. Among these features is the presence of long intergenic regions, which are common in other eukaryotes, but generally absent in Eumetazoa. Here we analyse poriferan mitochondrial intergenic regions, paying particular attention to repetitive sequences within them. In this context we introduce the mitochondrial genome of Ircinia strobilina (Lamarck, 1816; Demospongiae: Dictyoceratida) and compare it with mtDNA of other sponges. RESULTS: Mt genomes of dictyoceratid sponges are identical in gene order and content but display major differences in size and organization of intergenic regions. An even higher degree of diversity in the structure of intergenic regions was found among different orders of demosponges. One interesting observation made from such comparisons was of what appears to be recurrent invasions of sponge mitochondrial genomes by repetitive hairpin-forming elements, which cause large genome size differences even among closely related taxa. These repetitive hairpin-forming elements are structurally and compositionally divergent and display a scattered distribution throughout various groups of demosponges. CONCLUSION: Large intergenic regions of poriferan mt genomes are targets for insertions of repetitive hairpin- forming elements, similar to the ones found in non-metazoan opisthokonts. Such elements were likely present in some lineages early in animal mitochondrial genome evolution but were subsequently lost during the reduction of intergenic regions, which occurred in the Eumetazoa lineage after the split of Porifera. Porifera acquired their elements in several independent events. Patterns of their intra-genomic dispersal can be seen in the mt genome of Vaceletia sp.

The sterols of calcareous sponges (Calcarea, Porifera).

Sponges are sessile suspension-feeding organisms whose internal phylogenetic relationships are still the subject of intense debate. Sterols may have the potential to be used as independent markers to test phylogenetic hypotheses. Twenty representative specimens of calcareous sponges (class Calcarea, phylum Porifera) with a broad coverage within both subclasses Calcinea and Calcaronea were analysed for their sterol content. Two major pseudohomologous series were found, accompanied by some additional sterols. The first series encompassing conventional C(27) to C(29)Delta(5,7,22) sterols represented the major sterols, with ergosterol (ergosta-5,7,22-trien-3beta-ol, C(28)Delta(5,7,22)) being most prominent in many species. The second series consisted of unusual C(27) to C(29)Delta(5,7,9(11),22) sterols. Cholesterol occurred sporadically, mostly in trace amounts. The sterol patterns did not resolve intraclass phylogenetic relationships, namely the distinction between the subclasses, Calcinea and Calcaronea. This pointed towards major calcarean lipid traits being established prior to the separation of subclasses. Furthermore, calcarean sterol patterns clearly differ from those found in Hexactinellida, whereas partial overlap occurred with some Demospongiae. Hence, sterols only partly reflect the phylogenetic separation of Calcarea from both of the other poriferan classes that was proposed by recent molecular work and fatty acid analyses.

A fragmented metazoan organellar genome: the two mitochondrial chromosomes of Hydra magnipapillata.

BACKGROUND: Animal mitochondrial (mt) genomes are characteristically circular molecules of approximately 16-20 kb. Medusozoa (Cnidaria excluding Anthozoa) are exceptional in that their mt genomes are linear and sometimes subdivided into two to presumably four different molecules. In the genus Hydra, the mt genome comprises one or two mt chromosomes. Here, we present the whole mt genome sequence from the hydrozoan Hydra magnipapillata, comprising the first sequence of a fragmented metazoan mt genome encoded on two linear mt chromosomes (mt1 and mt2). RESULTS: The H. magnipapillata mt chromosomes contain the typical metazoan set of 13 genes for respiratory proteins, the two rRNA genes and two tRNA genes. All genes are unidirectionally oriented on mt1 and mt2, and several genes overlap. The gene arrangement suggests that the two mt chromosomes originated from one linear molecule that separated between nd5 and rns. Strong correlations between the AT content of rRNA genes (rns and rnl) and the AT content of protein-coding genes among 24 cnidarian genomes imply that base composition is mainly determined by mt genome-wide constraints. We show that identical inverted terminal repeats (ITR) occur on both chromosomes; these ITR contain a partial copy or part of the 3' end of cox1 (54 bp). Additionally, both mt chromosomes possess identical oriented sequences (IOS) at the 5' and 3' ends (5' and 3' IOS) adjacent to the ITR. The 5' IOS contains trnM and non-coding sequences (119 bp), whereas the 3' IOS comprises a larger part (mt2) with a larger partial copy of cox1 (243 bp). CONCLUSION: ITR are also documented in the two other available medusozoan mt genomes (Aurelia aurita and Hydra oligactis). In H. magnipapillata, the arrangement of ITR and 5' IOS and 3' IOS suggest that these regions are crucial for mt DNA replication and/or transcription initiation. An analogous organization occurs in a highly fragmented ichthyosporean mt genome. With our data, we can reject a model of mt replication that has previously been proposed for Hydra. This raises new questions regarding replication mechanisms probably employed by all medusozoans, and also has general implications for the expected organization of fragmented linear mt chromosomes of other taxa.