The sponge class Demospongiae is the most speciose and morphologically diverse in the phylum Porifera, and the species within it are vital components of a range of ecosystems worldwide. Despite their ubiquity, a number of recalcitrant problems still remain to be solved regarding their phylogenetic inter-relationships, the timing of their appearance, and their mitochondrial biology, the latter of which is only beginning to be investigated. Here we generated 14 new demosponge mitochondrial genomes which, alongside previously published mitochondrial resources, were used to address these issues. In addition to phylogenomic analysis, we have used syntenic data and analysis of coding regions to forge a framework for understanding the inter-relationships between Demospongiae sub-classes and orders. We have also leveraged our new resources to study the mitochondrial biology of these clades in terms of codon usage, optimisation and gene expression, to understand how these vital cellular components may have contributed to the success of the Porifera. Our results strongly support a sister relationship between Keratosa and (Verongimorpha + Heteroscleromorpha), contradicting previous studies using nuclear markers. Our study includes one species of Clionaida, and show for the first time support for a grouping of Suberitida+(Clionaida+(Tethyida + Poecilosclerida). The findings of our phylogenetic analyses are supported by in-depth examination of structural and coding-level evidence from our mitochondrial data. A time-calibrated phylogeny estimated the origin of Demospongiae in the Cambrian (similar to 529 Mya), and suggests that most demosponge order crown-groups emerged in the Mesozoic. This work therefore provides a robust basis for considering demosponge phylogenetic relationships, as well as essential mitochondrial data for understanding the biological basis for their success and diversity.

Mitochondrial resources are of known utility to many fields of phylogenetic, population and molecular biology. Their combination of faster and slower-evolving regions and high copy number enables them to be used in many situations where other loci are unsuitable, with degraded samples and after recent speciation events. The advent of next-generation sequencing technologies (and notably the Illumina platform) has led to an explosion in the number of samples that can be studied at transcriptomic level, at relatively low cost. Here we describe a robust pipeline for the recovery of mitochondrial genomes from these RNA-seq resources. This pipeline can be used on sequencing of a variety of depths, and reliably recovers the protein coding and ribosomal gene complements of mitochondria from almost any transcriptomic sequencing experiment. The complete sequence of the mitochondrial genome can also be recovered when sequencing is performed in sufficient depth. We evidence the efficacy of our pipeline using data from a number of non-model invertebrates of four disparate phyla, namely Porifera, Nemertea, Mollusca and Annelida. Interestingly, among our poriferan data, where microbiological symbionts are known empirically to make mitochondrial assembly difficult, this pipeline proved especially useful. Our pipeline will allow the recovery of mitochondrial data from a variety of previously-sequenced samples, and add an additional angle of enquiry to future RNA-seq efforts, simplifying the process of mitochondrial genome assembly for even the most recalcitrant clades and adding this data to the scientific record for a range of future uses.

Freshwater sponges (Spongillida) are a unique lineage of demosponges that secondarily colonized lakes and rivers and are now found ubiquitously in these ecosystems. They developed specific adaptations to freshwater systems, including the ability to survive extreme thermal ranges, long-lasting dessication, anoxia, and resistance to a variety of pollutants. Although spongillids have colonized all freshwater systems, the family Lubomirskiidae is endemic to Lake Baikal and plays a range of key roles in this ecosystem. Our work compares the genomic content and microbiome of individuals of three species of the Lubomirskiidae, providing hypotheses for how molecular evolution has allowed them to adapt to their unique environments. We have sequenced deep (>92% of the metazoan "Benchmarking Universal Single-Copy Orthologs" [BUSCO] set) transcriptomes from three species of Lubomirskiidae and a draft genome resource for Lubomirskia baikalensis. We note Baikal sponges contain unicellular algal and bacterial symbionts, as well as the dinoflagellate Gyrodinium. We investigated molecular evolution, gene duplication, and novelty in freshwater sponges compared with marine lineages. Sixty one orthogroups have consilient evidence of positive selection. Transporters (e.g., zinc transporter-2), transcription factors (aristaless-related homeobox), and structural proteins (e.g. actin-3), alongside other genes, are under strong evolutionary pressure in freshwater, with duplication driving novelty across the Spongillida, but especially in the Lubomirskiidae. This addition to knowledge of freshwater sponge genetics provides a range of tools for understanding the molecular biology and, in the future, the ecology (e.g., colonization and migration patterns) of these key species.

The Lake Baikal ecosystem is unique. The largest, oldest and deepest lake in the world presents a variety of rare evolutionary opportunities and ecological niches to the species that inhabit it, and as a result the lake is a biodiversity hotspot. More than 80% of the animals found there are endemic, and they often exhibit unusual traits. The freshwater sponge Lubomirskia baicalensis and its relatives are good examples of these idiosyncratic organisms. Lake Baikal sponges are unusually large for freshwater sponges, and possess particular symbiotic microorganisms which aid their survival and growth. As sponges are the most abundant benthic organisms in Lake Baikal, with 13 extant species (and 2 subspecies), they play a variety of key roles in the maintenance of this unusual ecosystem. In recent years the flora and fauna of Lake Baikal have been challenged by human activity in the region. Pollution, invasive species, temperature increases, eutrophication and falling water levels have all had deleterious effects. Sponges have suffered in particular from bleaching events, where their symbiotes are no longer retained, with knock-on effects to their survival and that of the ecosystem as a whole. To better understand the unique biology of these sponges we have established here a variety of resources for understanding these sponges at a molecular level. We have sequenced transcriptomes from three species of Lubomirskiidae. We have also assembled a draft genome resource for L. baicalensis. With these resources in place, a sizable addition to our knowledge of freshwater sponge genetics, we have investigated the adaptations of these and other freshwater sponge species to their environment, investigated their symbioses, and established a range of molecular tools for understanding the molecular and population-level biology of these key species across their habitat in Lake Baikal.

ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and that controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP-ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesize and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor This protein is a member of an uncharacterized subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyze PARP-dependent protein ADP-ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism.

Dinaric limestone cave systems, recognized as a hotspot of subterranean biodiversity, inhabit composite microbial communities whose structure, function and importance to ecosystems was poorly considered until the last few years. Filamentous microbial biofilms from three caves in Dinaric karst were assessed using 16S rRNA-based phylogenetic approach combined with universally protein coding genes/proteins. Studied clone libraries shared divisions but phylogenetic distribution of the obtained phylotypes differed: in Veternica and Vjetrenica clone libraries, Nitrospirae prevailed with 36% and 60% respectively, while in Izvor Bistrac the most abundant were Alphaproteobacteria (41%) followed by Firmicutes (32%). Moreover, three phylotypes were associated with novel uncultured candidate divisions OP3, WS5 and OD1 revealing the diversity and uniqueness of the microbial world in caves. Deeply understanding subterranean habitats could elucidate many new aspects in phylogeny and evolution of microorganisms as well as animal taxa, adjacent to their energy suppliers in microbial communities and biofilms.

In this study we analyze the calcarean sponge diversity of the Adriatic Sea, the type locality of some of the first described species of calcarean sponges. Morphological and molecular approaches are combined for the taxonomic identification. Our results reveal six species new to science and provisionally endemic to the Adriatic Sea (Ascandra spalatensis sp. nov., Borojevia croatica sp. nov., Leucandra falakra sp. nov., L. spinifera sp. nov., Paraleucilla dalmatica sp. nov., and Sycon ancora sp. nov.), one species previously known only from the Southwestern Atlantic (Clathrina conifera), and three already known from the Adriatic Sea (Ascaltis reticulum, Borojevia cerebrum, and Clathrina primordialis). We confirm the presence of the alien species Paraleucilla magna in the Adriatic and again record Clathrina blanca, C. clathrus, and C. rubra. We emend the description of the genus Ascaltis, propose a lectotype for Borojevia cerebrum and synonymise B. decipiens with B. cerebrum. A checklist of all calcarean species previously and currently known from the Adriatic Sea (39 species) is given. The Central Adriatic is indicated as the richest calcarean sponge fauna sector; however, the biodiversity of this class is underestimated in the whole Adriatic Sea and new systematic surveys are desirable.

Hymenoporus paradoxus, a new marasmioid fungal species belonging to the new genus is described from southern China. It differs from all other marasmioid species by a true tubular hymenophore, a character previously unknown in marasmioid fungi, and from all other tubular agarics by a hymenophore attached to a free collarium, and central, tough, dark and filiform stipe. The additional diagnostic characters are small basidiocarps, mostly convex pileus, smooth, hyaline, non-amyloid spores, densely packed dendriform cheilocystidia, and pileipellis composed of repent hyphae densely covered with simple to coralloid excrescences, intermixed with dendriform cells. As peculiar morphological characters had indicated, molecular phylogenetic analyses based on the 28S rDNA sequences confirm phylogenetic position of the new species in Omphalotaceae and a need to establish a new genus. Color photographs of macro- and micromorphological characters, SEM microphotographs, and a phylogenetic tree based on the partial 28S rDNA gene are provided.

The taxonomy of the class Calcarea has long relied on histological and morphological characters, with a few molecular studies done. In character-poor genera, such as Clathrina, an integrative taxonomy, associating morphological and molecular tools, greatly aids in the species identification. In this study, we describe four Clathrina species from the northern Adriatic Sea using morphology and DNA sequences together with observations on their ecology. Clathrina clathrus and Clathrina blanca have been previously reported for the Adriatic Sea; however, it is the first time that a DNA sequence of C. blanca has been obtained. Clathrina cf. hondurensis, a Caribbean species, is being reported in the Adriatic Sea for the first time, and we present the first description and DNA sequence of Clathrina rubra, a red species originally mentioned from Naples.

In order to determine an unknown fish population from the Vrana Lake, mitochondrial cytochrome b gene and non-coding nuclear region Cyfun P were investigated. Stabile population of Bulldog rudd, Scardinius dergle Heckel & Kner, the endemic Croatian freshwater fish in the Krka River, was genetically characterized with the same markers in order to compare it with the material from the Vrana Lake. Genetic markers were sequenced and aligned with the similar ones obtained from the GenBank in order to determine taxonomic and phylogenetic position of these two species. A significant discrepancy between nuclear genetic markers of our specimens and the sequence from the GenBank was found. Phylogenetic analysis suggested that the specimens from the Vrana Lake belong to the species S. hesperidicus. Morphometric characteristics, the maximum length and body mass showed new maximum values for both S. dergle and S. hesperidicus.

It is well known today that opine production anaerobic pathways are analogs to the classical glycolytic pathway (lactate production pathway). These pathways, catalyzed by a group of enzymes called opine dehydrogenases (OpDHs), ensure continuous flux of glycolysis and a constant supply of ATP by maintaining the NADH/NAD+ ratio during exercise and hypoxia, thus regulating the cytosolic redox balance in glycolysis under anoxia. OpDHs are distributed in a wide range of marine invertebrate phyla, including sponges (Porifera). Phylogenetic analyses supported with enzymatic assays strongly indicate that sponge OpDHs constitute an enzyme class unrelated to other OpDHs. Therefore, OpDHs in marine invertebrates are divided into two groups, a mollusk/annelid type and a sponge type, which belongs to the OCD/mu-crystallin family.

The paper presents the first record of invasive calcareous sponge Paraleucilla magna Klautau, Monteiro & Borojevic, 2004 in the area of the eastern Adriatic coast. The species was observed in the central Adriatic Sea in the area of Ploce harbour (international maritime transport) in 2011. It was present at depth from 0.5 m to 7 m in fouling community of photophilic algae and filter feeders. Maximal recorded abundance of this sponge was 4-5 N m-2 in the autumn. Collected data are the base for future monitoring of sponge’s spreading in the area of the eastern Adriatic coast.

Poriferan mitochondrial DNA (mtDNA), especially large intergenic regions, is a target for the insertion of repetitive hairpin-forming elements. These elements are responsible for the large mt genome size differences observed even among closely related sponge taxa. In this study, we present the new, nearly complete, mt genome sequence of Ephydatia fluviatilis and compare it with previously published mt genomes of freshwater sponges. Special emphasis was placed on comparison with the closely related species Ephydatia muelleri, thereby comparing the only two species of the genus Ephydatia on the western Balkan Peninsula. In particular, we analyzed repetitive palindromic elements within the mitochondrial intergenic regions. The genomic distribution of these repetitive elements was analyzed and their potential role in the evolution of mt genomes discussed. We show here that palindromic elements are widespread through the whole mt genome, including the protein coding genes, thus introducing genetic variability into mt genomes.

Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with "higher" metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales.

The complete mitochondrial DNA (mtDNA) genome of the Eunapius subterraneu s (Porifera, Demospongiae), a unique stygobitic sponge, was analyzed and compared with previously published mitochondrial genomes from this group. The 24,850 bp long mtDNA genome is circular with the same gene composition as found in other metazoans. Intergenic regions (IGRs) comprise 24.7% of mtDNA and are abundant with direct and inverted repeats and palindromic elements as well as with open reading fames (ORFs) whose distribution and homology was compared with other available mt genomes with a special focus on freshwater sponges. Phylogenetic analyses based on concatenated amino acid sequences from 12 mt protein genes placed E. subterraneus in a well-supported monophyletic clade with the freshwater sponges, Ephydatia muelleri and Lubomirskia baicalensis . Our study showed high homology of mtDNA genomes among freshwater sponges, implying their recent split.

SUMMARY The metabolic energy state of sponge tissue in vivo is largely unknown. Quantitative bioluminescence-based imaging was used to analyze the ATP distribution of Suberites domuncula (Olivi 1792) tissue, in relation to differences between the cortex and the medulla. This method provides a quantitative picture of the ATP distribution closely reflecting the in vivo situation. The obtained data suggest that the highest ATP content occurs around channels in the sponge medulla. HPLC reverse-phase C-18, used for measurement of ATP content, established a value of 1.62 μmol ATP g–1 dry mass in sponge medulla, as opposed to 0.04 μmol ATP g–1 dry mass in the cortex, thus indicating a specific and defined energy distribution. These results correlate with the mitochondria localization, determined using primary antibodies against cytochrome oxidase c subunit 1 (COX1) (immunostaining), as well as with the distribution of arginine kinase (AK), essential for cellular energy metabolism (in situ hybridization with AK from S. domuncula; SDAK), in sponge sections. The highest energy consumption seemed to occur in choanocytes, the cells that drive the water through the channel system of the sponge body. Taken together, these results showed that the majority of energetic metabolism in S. domuncula occurs in the medulla, in the proximity of aqueous channels.