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Sri Lanka is a biodiversity hotspot and one of the richest geographical locations of marine sponges in the Indian ocean. However, the most extensive taxonomical study on Sri Lankan sponge biodiversity dates back similar to 100 years and only a limited number of studies have been conducted on sponge natural products. In the current study, 35 marine sponge specimens (collected from 16 sponge habitats around Sri Lanka) were identified, microfractionated and evaluated for antibacterial and anticancer assays. In total, 30 species were characterized, of which 19 species gave extracts with antibacterial and/or cytotoxic activities. Microfractionated organic extract of Aciculites orientalis gave the most potent antibacterial activity against Staphylococcus aureus and strongest lymphoma cell toxicity was exhibited by the organic extract of Acanthella sp. Guided by the molecular ion peaks in the bioactive fractions, large-scale extraction of Stylissa massa led to the isolation of three bromopyrrole alkaloids, sceptrin, hymenin and manzacidin A/C. Of these, sceptrin exhibited broad spectrum antibacterial activity against both Escherichia coli and S. aureus (MIC of 62.5 mu M against both species). Based on natural product literature, seven promising species were identified as understudied. Their further exploration may lead to the discovery of structurally novel compounds.

Sponges contain an astounding diversity of lipids that serve in several biological functions, including yolk formation in their oocytes and embryos. The study of lipid metabolism during reproduction can provide information on food-web dynamics and energetic needs of the populations in their habitats, however, there are no studies focusing on the lipid metabolism of sponges during their seasonal reproduction. In this study, we used histology, lipidome profiling (UHPLC-MS), and transcriptomic analysis (RNA-seq) on the deep-sea sponge Phakellia ventilabrum (Demospongiae, Bubarida), a key species of North-Atlantic sponge grounds, with the goal to (i) assess the reproductive strategy and seasonality of this species, (ii) examine the relative changes in the lipidome signal and the gene expression patterns of the enzymes participating in lipid metabolism during oogenesis. Phakellia ventilabrum is an oviparous and most certainly gonochoristic species, reproducing in May and September in the different studied areas. Half of the specimens were reproducing, generating two to five oocytes per mm(2). Oocytes accumulated lipid droplets and as oogenesis progressed, the signal of most of the unsaturated and monounsaturated triacylglycerides increased, as well as of a few other phospholipids. In parallel, we detected upregulation of genes in female tissues related to triacylglyceride biosynthesis and others related to fatty acid beta-oxidation. Triacylglycerides are likely the main type of lipid forming the yolk in P. ventilabrum since this lipid category has the most marked changes. In parallel, other lipid categories were engaged in fatty acid beta-oxidation to cover the energy requirements of female individuals during oogenesis. In this study, the reproductive activity of the sponge P. ventilabrum was studied for the first time uncovering their seasonality and revealing 759 lipids, including 155 triacylglycerides. Our study has ecological and evolutionary implications providing e

Macro and megafauna were studied in the Avile acute accent s Canyon System (ACS), southern Bay of Biscay (Cantabrian Sea), during several oceanographic cruises carried out from 2009 to 2017. The biodiversity of ACS is summarized and its description is herein updated after sampling surveys of several programmes (ECOMARG, INDEMARES, SponGES, INTEMARES) conducted by the Spanish Institute of Oceanography (IEO).This study has updated previous knowledge in the canyon area from past national and international projects, their reports and publications as well as data collected in the context of regional projects designed to gain new insight into the diversity of marine invertebrates and fishes from the ACS. Samples were taken using a range of sampling gears (Rock dredge, Beam trawl, Trawl gear GOC-73, Suprabenthic sledge, Box corer and Remoted operated vehicle), from 55 to 2291 m in depth. A total of 1015 species were identified at the ACS: 98 Porifera, 153 Cnidaria, 14 Brachiopoda, 22 Bryozoa, 97 Mollusca, 151 Annelida, 315 Arthropoda, 74 Echinodermata and 91 Chordata. New records for the Bay of Biscay fauna include 13 Porifera species, 17 Cnidaria, 7 Mollusca, Arthopoda, 3 Echinodermata and 4 Chordata. Also the bathymetric range of some species has been extended. As a result of the research projects carried out in the area in the last fifteen years, important information is now available which suggests that the ACS houses a large number of species with a high ecological value, that represents a biodiversity hotspot in terms of the presence of sponge aggregations and coral reefs in certain regions, and that it sustains important fisheries due to the abundance of comercial species. Given the relevance of the species and habitats occurring in the ACS, there is a need to implement a conservation and management plan of the area in order to maintain habitats in good state of preservation.

Species misidentification in the field of natural products is an acknowledged problem. These errors are especially widespread in sponge studies, albeit rarely assessed and documented. As a case study, we aim to revisit reports of isomalabaricane triterpenes, isolated from four demosponge genera: Jaspis, Geodia, Stelletta and Rhabdastrella. From a total of 44 articles (1981-2022), 27 unique vouchers were listed, 21 of which were accessed and re-examined here: 11 (52.4%) of these were misidentified. Overall, 65.9% of the studies published an incorrect species name: previously identified Jaspis and Stelletta species were all in fact Rhabdastrella globostellata. We conclude that isomalabaricane triterpenes were isolated from only two Rhabdastrella species and possibly one Geodia species. In addition to shedding a new light on the distribution of isomalabaricane triterpenes, this study is an opportunity to highlight the crucial importance of vouchers in natural product studies. Doing so, we discuss the impact of species misidentification and poor accessibility of vouchers in the field of sponge natural products. We advocate for stricter voucher guidelines in natural product journals and propose a common protocol of good practice, in the hope of reducing misidentifications in sponge studies, ensure reproducibility of studies, and facilitate follow-up work on the original material.

Background: Among all present demosponges, lithistids represent a polyphyletic group with exceptionally well-preserved fossils dating back to the Cambrian. Knowledge of their recent diversity, particularly in the Tropical Western Atlantic Ocean (TWA) where they are common in deep waters, is scarce making any comparison between present and past major 'lithistid' faunas difficult. In addition, the lack of sufficient molecular and morphological data hamper any predictions on phylogenetic relationships or phylodiversity from this region. The Harbor Branch Oceanographic Institute (HBOI, Fort Pierce, Florida) holds the largest collection of TWA lithistid sponges worldwide, however, the majority remain to be taxonomically identified and revised. Principal Findings: In this study we provide sequences of 249 lithistid demosponges using two independent molecular markers (28S rDNA (C1-D2) and cox1 mtDNA). In addition, a morphological documentation of 70 lithistid specimens is provided in the database of the Sponge Barcoding Project (SBP). This integrated dataset represents the largest and most comprehensive of the TWA lithistids to date. The phylogenetic diversity of 'lithistid' demosponges in the Bahamas and Jamaica are high in comparison to other TWA regions; Theonellidae and Corallistidae dominate the fauna, while Neopeltidae and Macandrewiidae are rare. A proposed tetractinellid suborder, one undescribed genus and several undescribed species are recognized and the Pacific 'lithistid' genera, Herengeria and Awhiowhio, are reported from the TWA for the first time. The higher-taxa relationships of desma-bearing tetractinellids are discussed and topics for revision suggested. Conclusion: This first integrative approach of TWA 'lithistid' demosponges contributes to a better understanding of their phylogenetic affinities, diversity and bathymetric distribution patterns within the TWA. As in the Pacific, the TWA 'lithistid' demosponges dominate deep-water habitats. Deeper taxonom

Water masses are bodies of water with distinctive physical and biogeochemical properties. They impart vertical structure to the deep ocean, participate in circulation, and can be traced over great distances, potentially influencing the distributions of deep-sea fauna. The classic potential temperature-salinity (theta-s) diagram was used to investigate deep-sea sponge (demosponge genus Geodia) association with water masses over the North Atlantic Ocean and Nordic Seas. A novel analysis was conducted, based on sampling the curvature of climatological mean theta-s curves at sponge locations. Sponges were particularly associated with turning points in the theta-s curves, indicative of intermediate and deep water masses. Arctic geodiid species (G. hentscheli and G. parva) were associated with Arctic intermediate and deep waters in the Nordic Seas, and with dense overflows into the northern North Atlantic. Boreal species (G. atlantica, G. barretti, G. macandrewii, and G. phlegraei) were associated with upper and intermediate water masses in the Northeast Atlantic and with upper, Atlantic-derived waters in the Nordic Seas. Taken together with distributional patterns, a link with thermohaline currents was also inferred. We conclude that water masses and major current pathways structure the distribution of a key deep-sea benthic faunal group on an ocean basin scale. This is most likely because of a combination of the physical constraints they place on the dispersal of early life-history stages, ecophysiological adaptation (evolved tolerances) to specific water masses, and the benefits to filter-feeders of certain phenomena linked to water column structure (e.g. nepheloid layers, internal waves/ tides, density-driven currents).

Host-microbe interactions play crucial roles in marine ecosystems. However, we still have very little understanding of the mechanisms that govern these relationships, the evolutionary processes that shape them, and their ecological consequences. The holobiont concept is a renewed paradigm in biology that can help to describe and understand these complex systems. It posits that a host and its associated microbiota with which it interacts, form a holobiont, and have to be studied together as a coherent biological and functional unit to understand its biology, ecology, and evolution. Here we discuss critical concepts and opportunities in marine holobiont research and identify key challenges in the field. We highlight the potential economic, sociological, and environmental impacts of the holobiont concept in marine biological, evolutionary, and environmental sciences. Given the connectivity and the unexplored biodiversity specific to marine ecosystems, a deeper understanding of such complex systems requires further technological and conceptual advances, e.g., the development of controlled experimental model systems for holobionts from all major lineages and the modeling of (info)chemical-mediated interactions between organisms. Here we propose that one significant challenge is to bridge cross-disciplinary research on tractable model systems in order to address key ecological and evolutionary questions. This first step is crucial to decipher the main drivers of the dynamics and evolution of holobionts and to account for the holobiont concept in applied areas, such as the conservation, management, and exploitation of marine ecosystems and resources, where practical solutions to predict and mitigate the impact of human activities are more important than ever.

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.

The poorly known sponge species Axinella vellerea (Topsent, 1904), Acarnus levii (Vacelet, 1960) and Haliclona poecillastroides (Vacelet, 1969) are reported from bottom-trawl samples off the Balearic Islands, Western Mediterranean. A re-description is provided for all three species and the Folmer fragment of cytochrome oxidase subunit I (COI) obtained for A. levii and H. poecillastroides. This is the second report of A. vellerea in the Mediterranean, the first time that A. levii is reported outside Corsica and the first time that H. poecillastroides is documented outside the Gulf of Lion, France. The systematic allocation of A. levii and H. poecillastroides is discussed based on a COI phylogenetic analysis and morphology. The poorly understood phylogeny of the Haplosclerida does not permit us to find a proper allocation for H. poecillastroides, although its current position in the genus Haliclona or the family Chalinidae is not defensible. On the other hand, A. levii currently fits best in the family Microcionidae, and seems related to some Clathria species with mixed features between Clathria and Acarnus. Considering that the species of the genus Acarnus shares a strong synapomorphy (the possession of Cladotylotes), it is plausible for all Acarnus species to be Microcionids. We conclude that H. poecillastroides needs to be reallocated to a new genus: Xestospongia poecillastroides comb. nov. (Petrosiidae). However, a reallocation of A. levii is not advisable for the moment, as this would imply major systematic changes such as the reallocation of the whole genus Acarnus to Microcionidae, and the redescription of Microcionidae and Acarnidae.

Siliceous spicules in demosponges exist in a variety of shapes, some of which look like minute spheres of glass. They are called “sterrasters” when they belong to the Geodiidae family (Tetractinellida order) and “selenasters” when they belong to the Placospongiidae family (Clionaida order). Today, the Geodiidae represent a highly diverse sponge family with more than 340 species, occurring in shallow to deep waters worldwide, except for the Antarctic. The molecular phylogeny of Geodiidae is currently difficult to interpret because we are lacking morphological characters to support most of its clades. To fill this knowledge gap, the surface microornamentations of sterrasters were compared in different genera. Observations with scanning electron microscopy revealed four types of surfaces, which remarkably matched some of the Geodiidae genera: type I characteristic of Geodia, type II characteristic of Pachymatisma, Caminus, and some Erylus; type III characteristic of other Erylus; type IV characteristic of Caminella. Two subtypes were identified in Geodia species: warty vs. smooth rosettes. These different microornamentations were mapped on new Geodiidae COI (Folmer fragment) and 28S (C1–D2) phylogenetic trees. The monophyly of the Geodiidae was once again challenged, thereby suggesting that sterrasters have evolved independently at least three times: in the Geodiinae, in the Erylinae and in Caminella. Surface microornamentations were used to review the fossil record of sterrasters and selenasters through the paleontology literature and examination of fossils. It was concluded that “rhaxes” in the literature may represent mixes of sterrasters and selenasters: while Rhaxella spicules may belong to the Placospongiidae, Rhaxelloides spicules belong to the Geodiidae. The putative Geodiidae fossil genera, Geoditesia, and Geodiopsis, are reallocated to Tetractinellida incertae sedis. Isolated Miocene-Pliocene fossil sterrasters Hataina (Huang, 1967), Silicosphaera (Hughes, 19

Boreo-arctic sponge grounds are essential deep-sea structural habitats that provide important services for the ecosystem. These large sponge aggregations are dominated by demosponges of the genus Geodia (order Tetractinellida, family Geodiidae). However, little is known about the basic biological features of these species, such as their life cycle and dispersal capabilities. Here, we surveyed five deep-sea species of Geodia from the North-Atlantic Ocean and studied their reproductive cycle and strategy using light and electron microscopy. The five species were oviparous and gonochoristic. Synchronous development was observed at individual and population level in most of the species. Oocytes had diameters ranging from 8 μm in previtellogenic stage to 103 μm in vitellogenic stage. At vitellogenic stages, oocytes had high content of lipid yolk entirely acquired by autosynthesis, with no participation of nurse cells. Intense vertical transmission of bacterial symbionts to the oocytes by phagocytosis through pseudopodia was observed, especially in late stages of oogenesis. The density of oocytes within the sponge tissue was on average 10 oocytes/mm2 across all species, higher than that of most temperate and tropical oviparous species studied elsewhere. Spermatic cysts were widespread over the tissue during early stages, or fused in larger cysts, around the canals in later stages, and occupying between 1.5 and 12% of the tissue in males. The reproductive season spanned similar periods for all Geodia spp.: from late spring to early autumn. During the reproductive peak of each species, between 60 and 90% of the population was engaged in reproduction for most species. Given the present hazards that threaten the boreo-arctic tetractinellid sponge grounds, it becomes crucial to understand the processes behind the maintenance and regeneration of populations of keystone deep-sea species in order to predict the magnitude of human impacts and estimate their ability to recover. The, Title in thesis list of papers: Exploring the reproductive biology of the genus Geodia from boreo-arctic North-Atlantic deep-sea sponge grounds

All animals are capable of undergoing gametogenesis. The ability of forming haploid cells from diploid cells through meiosis and recombination appeared early in eukaryotes, whereas further gamete differentiation is mostly a metazoan signature. Morphologically, the gametogenic process presents many similarities across animal taxa, but little is known about its conservation at the molecular level. Porifera are the earliest divergent animals and therefore are an ideal phylum to understand evolution of the gametogenic toolkits. Although sponge gametogenesis is well known at the histological level, the molecular toolkits for gamete production are largely unknown. Our goal was to identify the genes and their expression levels which regulate oogenesis and spermatogenesis in five gonochoristic and oviparous species of the genus Geodia, using both RNAseq and proteomic analyses. In the early stages of both female and male gametogenesis, genes involved in germ cell fate and cell-renewal were upregulated. Then, molecular signals involved in retinoic acid pathway could trigger the meiotic processes. During later stages of oogenesis, female sponges expressed genes involved in cell growth, vitellogenesis, and extracellular matrix reassembly, which are conserved elements of oocyte maturation in Metazoa. Likewise, in spermatogenesis, genes regulating the whole meiotic cycle, chromatin compaction, and flagellum axoneme formation, that are common across Metazoa were overexpressed in the sponges. Finally, molecular signals possibly related to sperm capacitation were identified during late stages of spermatogenesis for the first time in Porifera. In conclusion, the activated molecular toolkit during gametogenesis in sponges was remarkably similar to that deployed during gametogenesis in vertebrates.

Background: Lithistid demosponges, also known as rock sponges, are a polyphyletic group of sponges which are widely distributed. In the Northeast Atlantic (NEA), 17 species are known and the current knowledge on their distribution is mainly restricted to the Macaronesian islands. In the Mediterranean Sea, 14 species are recorded and generally found in marine caves. Methods: Lithistids were sampled in nine NEA seamounts during the scientific expeditions Seamount 1 (1987) and Seamount 2 (1993) organized by the MNHN of Paris. Collected specimens were identified through the analyses of external and internal morphological characters using light and scanning electron microscopy, and compared with material from various museum collections as well as literature records. Results: A total of 68 specimens were analysed and attributed to 17 species across two orders, seven families, and seven genera, representing new records of distribution. Ten of these species are new to science, viz. Neoschrammeniella inaequalis sp. nov., N. piserai sp. nov., N. pomponiae sp. nov., Discodermia arbor sp. nov., D. kellyae sp. nov., Macandrewia schusterae sp. nov., M. minima sp. nov., Exsuperantia levii sp. nov., Leiodermatium tuba sp. nov. and Siphonidium elongatus sp. nov., and are here described and illustrated. New bathymetric records were also found for D. ramifera, D. verrucosa and M. robusta. The Meteor seamount group has a higher species richness (15 species) compared to the Lusitanian seamount group (six species). The majority of the species had their distribution restricted to one seamount, and ten are only known from a single locality, but this can be a result of sample bias. Discussion: The number of species shared between the seamounts and the Macaronesian islands is very reduced. The same pattern repeats between the NEA and Mediterranean Sea. This study demonstrates that NEA seamounts are ecosystems with a higher diversity of lithistids than previously thought, increasing the numbe

Response to B. J. Nettersheim et al. Nature Ecology & Evolution https://doi.org/10.1038/s41559-019-0806-5 (2019)

The Rosemary Bank Seamount in the NE Atlantic was designated a Marine Protected Area in 2014 by the Scottish Government. Visual and trawl surveys of the seamount have been undertaken since 2007. Here these data are compiled and analysed to provide an assessment of the communities of demersal fish and benthic invertebrates found there. The fish and benthic invertebrate communities changed markedly with depth. Cluster analysis revealed at least four distinct communities of fish: those on the summit, the mid slope, the lower slope and the deep moat at the base of the seamount. The invertebrate community changed at a depth of 1100 m, where mixed-species sponge aggregations dominated to depths of 1500 m. The seamount is an important site for vulnerable marine ecosystems, most notably the extensive and unusually diverse deep-sea sponge grounds on the lower slope. Other prioritised conservation species and habitats recorded included cold water corals, orange roughy, blue ling, leafscale gulper shark and the Portuguese dogfish. Due to sampling constraints some areas of the seamount still remain unknown. A precautionary approach to protecting the entire seamount would achieve multiple conservation objectives. The data presented here serve as a base-line to assess the impact of management intervention in the future.

A new species of Tethya Lamarck, 1815 is described from a depth of 1000 m on the continental slope of the Great Australian Bight (GAB), southern Australia. The GAB slope was explored as part of systematic benthic surveys to understand unexplored communities in the light of current oil and gas exploration activity in the area. Tethya irisae sp. nov. was present at 1000 m in six of eight longitudinal depth surveys. Three molecular markers were obtained: COI, 28S (D3-D5) and ITS1-5.8S-ITS2. COI and 28S phylogenetic analyses show that the new species fits clearly within the genus Tethya. This is the 28th species of Tethya reported from Australia; it is unusual in that it has a stalk. The presence of a stalk as a morphological character to split genera in this family is questioned. The description of this new species is an opportunity to revisit the molecular phylogeny of the Tethyida Morrow & Cardenas, 2015 using comprehensive datasets of COI and 28S markers. As in previous analyses, four Tethya clades were retrieved; we discuss the possibility of using external colour to support some of these clades. Despite unclear phylogenetic relationships amongst Tethyidae Gray, 1848 from Australia, our results suggest that tethyid genera Tethytimea Laubenfels, 1936, Tectitethya Sara, 1994, Laxotethya Sara & Sara, 2002, Stellitethya Sara, 1994, and Xenospongia Gray, 1858 derive from species of Tethya. We show that asters have been secondarily lost at least twice in the Hemiasterellidae Lendenfeld, 1889: in Liosina Thiele, 1899 and a potential new genus from northern Australia. We formally propose the reallocation of Liosina from Dictyonellidae van Soest, Diaz & Pomponi, 1990 to Hemiasterellidae Lendenfeld, 1889.

Blending synthetic biology and synthetic chemistry represents a powerful approach to diversity complex molecules. To further enable this, compatible synthetic tools are needed. We report the first Buchwald Hartwig amination reactions with unprotected halo-tryptophans under aqueous conditions and demonstrate this methodology is applicable also to the modification of unprotected tripeptides and the natural product barettin.

This study reinforces and extends the findings of previous molecular studies showing that there is a dose relationship between species assigned to the sponge genera Halicnemia, Higginsia, Paratimea and Stelligera and that the family Heteroxyidae is polyphyletic. The present study has led to the description of one new species of Halicnemia and six new species of Paratimea, the resurrection of Halicnemia gallica and a better understanding of the characters uniting Stelligeridae. A new species of Heteroxya is also described. We demonstrate that many of the taxa assigned to Heteroxyidae are more closely related to other families, and we propose several changes to the classification of Heteroscleromorpha. Desmoxyidae is resurrected from synonymy and transferred to Poecilosclerida; Higginsia anfractuosa is transferred to Hymedesmiidae, and a new genus, Hooperia, is erected for its reception; Higginsia durissima is returned to Bubaris (Bubaridae); Higginsia fragilis is transferred to Spanioplon (Hymedesmiidae); Hemiasterella camelus is transferred to Paratimea; and Raspailia (Parasyringella) australiensis and Ceratopsion axiferum are transferred to Adreus (Hemiasterellidae).

The blending of synthetic chemistry with biosynthetic processes provides a powerful approach to synthesis. Biosynthetic halogenation and synthetic cross-coupling have great potential to be used together, for small molecule generation, access to natural product analogues and as a tool for chemical biology. However, to enable enhanced generality of this approach, further synthetic tools are needed. Though considerable research has been invested in the diversification of phenylalanine and tyrosine, functionalisation of tryptophans thorough cross-coupling has been largely neglected. Tryptophan is a key residue in many biologically active natural products and peptides; in proteins it is key to fluorescence and dominates protein folding. To this end, we have explored the Heck cross-coupling of halo-indoles and halo-tryptophans in water, showing broad reaction scope. We have demonstrated the ability to use this methodology in the functionalisation of a brominated antibiotic (bromo-pacidamycin), as well as a marine sponge metabolite, barettin.

Sponges host a remarkable diversity of microbial symbionts, however, the benefit their microbes provide is rarely understood. Here, we describe two new sponge species from deep-sea asphalt seeps and show that they live in a nutritional symbiosis with methane-oxidizing (MOX) bacteria. Metagenomics and imaging analyses revealed unusually high amounts of MOX symbionts in hosts from a group previously assumed to have low microbial abundances. These symbionts belonged to the Marine Methylotrophic Group 2 Glade. They are host-specific and likely vertically transmitted, based on their presence in sponge embryos and streamlined genomes, which lacked genes typical of related free-living MOX. Moreover, genes known to play a role in host-symbiont interactions, such as those that encode eukaryote-like proteins, were abundant and expressed. Methane assimilation by the symbionts was one of the most highly expressed metabolic pathways in the sponges. Molecular and stable carbon isotope patterns of lipids confirmed that methane-derived carbon was incorporated into the hosts. Our results revealed that two species of sponges, although distantly related, independently established highly specific, nutritional symbioses with two closely related methanotrophs. This convergence in symbiont acquisition underscores the strong selective advantage for these sponges in harboring MOX bacteria in the food-limited deep sea.

We report the first records of the sponge genus Geodia (Demospongiae, Tetractinellida, Geodiidae) from the New England Seamounts and Muir Seamount, at lower bathyal depths. Nine specimens collected between 2000 and 2005 belong to two boreal species (Geodia macandrewii and Geodia barretti) and a temperate species (Geodia megastrella). These records extend the distributions of these deep-sea amphi-Atlantic species to the west. Most of these specimens were originally fixed in formalin, which substantially degraded the DNA. We nonetheless managed to sequence two cytochrome c oxidase subunit I (COI) mini-barcodes: the universal mini-barcode at the 5′ end of the Folmer barcode (130 bp) and a newly proposed mini-barcode at the 3′ end of the Folmer barcode (296 bp). These mini-barcodes unambiguously confirmed our identifications. As an additional test, we also successfully sequenced these two mini-barcodes from the holotype of G. barretti, collected in 1855. We conclude by advocating the use of mini-barcodes on formalin-fixed or old specimens with degraded DNA.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

Sterane biomarkers preserved in ancient sedimentary rocks hold promise for tracking the diversification and ecological expansion of eukaryotes. The earliest proposed animal biomarkers from demosponges (Demospongiae) are recorded in a sequence around 100 Myr long of Neoproterozoic-Cambrian marine sedimentary strata from the Huqf Supergroup, South Oman Salt Basin. This C-30 sterane biomarker, informally known as 24-isopropylcholestane (24-ipc), possesses the same carbon skeleton as sterols found in some modern-day demosponges. However, this evidence is controversial because 24-ipc is not exclusive to demosponges since 24-ipc sterols are found in trace amounts in some pelagophyte algae. Here, we report a new fossil sterane biomarker that co-occurs with 24-ipc in a suite of late Neoproterozoic-Cambrian sedimentary rocks and oils, which possesses a rare hydrocarbon skeleton that is uniquely found within extant demosponge taxa. This sterane is informally designated as 26-methylstigmastane (26-mes), reflecting the very unusual methylation at the terminus of the steroid side chain. It is the first animal-specific sterane marker detected in the geological record that can be unambiguously linked to precursor sterols only reported from extant demosponges. These new findings strongly suggest that demosponges, and hence multicellular animals, were prominent in some late Neoproterozoic marine environments at least extending back to the Cryogenian period.

Discorhabdella pseudaster n. sp. is an incrusting sponge from the upper bathyal zone of the 'Banc du Geyser', north of Madagascar, Mozambique Channel. This new species is described only from a single specimen but it is remarkable by the presence of spicules similar to euasters, a type of microsclere unknown in Poecilosclerida. These spicules are in fact a new example of homoplasy, being derivatives of the typical Discorhabdella pseudoastrose acanthostyles, which are here reduced to the aster-like tyles. The isochelae with a large lamella on the shaft are also quite unique in Poeciloclerida.

Caminella Lendenfeld, 1894 is a poorly known Geodiidae genus with unclear phylogenetic relationships. In order to find new lines of evidence that could shed light on the evolutionary history of Caminella, we decided to revise type material and museum material, as well as examine new material from underwater caves and deep-sea ecosystems. In doing so, we formally show that Isops maculosus Vosmaer, 1894 and Caminella loricata Lendenfeld, 1894 are junior synonyms of Caminella intuta (Topsent, 1892). We discuss different spicule morphological phenotypes in C. intuta, which may be linked to silica availability. We also discovered two new species of deep-sea Caminella: 1) from Cape Verde (Caminella caboverdensis sp. nov.) and 2) from seamounts located south of the Azores archipelago and the North of Spain (Caminella pustula sp. nov.). We reveal that Caminella sterrasters have complex surface microstructures, unique amongst the Geodiidae, where actin tips are linked to each other. Molecular markers (COI, 28S (C1-D2) and 18S) sequenced for some specimens led to new phylogenetic analyses, which continue to suggest a close relationship of Caminella with the Erylinae and Calthropella; these affinities are discussed in light of morphological characters.

The Thoosidae (Porifera, Demospongiae, Tetractinellida) currently includes the genera Thoosa, Alectona, and Delectona. To this date, molecular data are only available for Alectona. In this study, the phylogenetic affinities of the genera Thoosa and Alectona have been investigated with the species T. mismalolli, T. calpulli, and T. purpurea from the Mexican Pacific using morphology and three molecular loci: the mitochondrial cytochrome oxidase subunit 1 (CO1 mtDNA), 28S rRNA (fragment D2), and 18S rRNA. Morphology and embryology showed that these genera are quite different from the rest of the tetractinellids because larvae of Alectona and Thoosa have unique features in sponges, such as the presence of monaxonic discs in Thoosa and tetraxonic discs in Alectona which disappear in the adult stages. A phylogenetic analysis using selected species from the order Tetractinellida revealed that Thoosa groups with Alectona thus confirming morphological studies. The peculiarities in spiculation and embryology of the Thoosa and Alectona larvae, which are markedly different from species belonging to the suborders Astrophorina and Spirophorina and their distant phylogenetic position (based on three molecular loci), suggest that Thoosidae could be placed in the new suborder Thoosina.

Reduced representation sequencing appraches such as ddRADseq allow to assess population connectivity and infer population summary statistics, both with and without a reference genome. However, as ddRADseq employs total DNA indiscriminate of the origin, the method warrants validation prior to application in microbial rich systems. One example of a complex system are sponges such as the North Atlantic high microbial abundance sponge Geodia barretti. This species is known to maintain large, putatively disjoint populations across the deep-sea, but its dispersal capabilities remain unclear as larvae have never been observed. To study the effect of microbial contamination on data processing and population genetic inference in ddRADseq, we produced a reference genome of G. barretti and collected 163 individuals across its habitat range and bathymetry (35–1560 m) in the North Atlantic. We processed the data with Stacks2 both with and without a reference genome (de novo and hybrid/‘reference-integrated’ approach). We found that strong population structures are recovered by both approaches and across different population genetic analyses (fastStructure, PCA, FST). Compared to previous work using microsatellites in shallow populations, we found only very weak population structure across large geographic stretches (>1000 km). However, over a third (34%) of the final loci produced by the de novo pipeline did not map to the reference genome indicating that these might be of microbial origin. For comparably complex systems this means that de novo RRS genotyping approaches may contain a considerable amount of off-target loci potentially biasing the results.