The compact genome of the sponge Oopsacas minuta (Hexactinellida) is lacking key metazoan core genes.

BACKGROUND: Explaining the emergence of the hallmarks of bilaterians is a central focus of evolutionary developmental biology-evodevo-and evolutionary genomics. For this purpose, we must both expand and also refine our knowledge of non-bilaterian genomes, especially by studying early branching animals, in particular those in the metazoan phylum Porifera. RESULTS: We present a comprehensive analysis of the first whole genome of a glass sponge, Oopsacas minuta, a member of the Hexactinellida. Studying this class of sponge is evolutionary relevant because it differs from the three other Porifera classes in terms of development, tissue organization, ecology, and physiology. Although O. minuta does not exhibit drastic body simplifications, its genome is among the smallest of animal genomes sequenced so far, and surprisingly lacks several metazoan core genes (including Wnt and several key transcription factors). Our study also provides the complete genome of a symbiotic Archaea dominating the associated microbial community: a new Thaumarchaeota species. CONCLUSIONS: The genome of the glass sponge O. minuta differs from all other available sponge genomes by its compactness and smaller number of encoded proteins. The unexpected loss of numerous genes previously considered ancestral and pivotal for metazoan morphogenetic processes most likely reflects the peculiar syncytial tissue organization in this group. Our work further documents the importance of convergence during animal evolution, with multiple convergent evolution of septate-like junctions, electrical-signaling and multiciliated cells in metazoans.

Evolution, Expression Patterns, and Distribution of Novel Ribbon Worm Predatory and Defensive Toxins.

Ribbon worms are active predators that use an eversible proboscis to inject venom into their prey and defend themselves with toxic epidermal secretions. Previous work on nemertean venom has largely focused on just a few species and has not investigated the different predatory and defensive secretions in detail. Consequently, our understanding of the composition and evolution of ribbon worm venoms is still very limited. Here, we present a comparative study of nemertean venom combining RNA-seq differential gene expression analyses of venom-producing tissues, tandem mass spectrometry-based proteomics of toxic secretions, and mass spectrometry imaging of proboscis sections, to shed light onto the composition and evolution of predatory and defensive toxic secretions in Antarctonemertes valida. Our analyses reveal a wide diversity of putative defensive and predatory toxins with tissue-specific gene expression patterns and restricted distributions to the mucus and proboscis proteomes respectively, suggesting that ribbon worms produce distinct toxin cocktails for predation and defense. Our results also highlight the presence of numerous lineage-specific toxins, indicating that venom evolution is highly divergent across nemerteans, producing toxin cocktails that might be finely tuned to subdue different prey. Our data also suggest that the hoplonemertean proboscis is a highly specialized predatory organ that seems to be involved in a variety of biological functions besides predation, including secretion and sensory perception. Overall, our results advance our knowledge into the diversity and evolution of nemertean venoms and highlight the importance of combining different types of data to characterize toxin composition in understudied venomous organisms.

Trapped DNA fragments in marine sponge specimens unveil North Atlantic deep-sea fish diversity.

Sponges pump water to filter feed and for diffusive oxygen uptake. In doing so, trace DNA fragments from a multitude of organisms living around them are trapped in their tissues. Here we show that the environmental DNA retrieved from archived marine sponge specimens can reconstruct the fish communities at the place of sampling and discriminate North Atlantic assemblages according to biogeographic region (from Western Greenland to Svalbard), depth habitat (80-1600 m), and even the level of protection in place. Given the cost associated with ocean biodiversity surveys, we argue that targeted and opportunistic sponge samples - as well as the specimens already stored in museums and other research collections - represent an invaluable trove of biodiversity information that can significantly extend the reach of ocean monitoring.

Mismatches between phenotype and environment shape fitness at hyperlocal scales.

In the era of human-driven climate change, understanding whether behavioural buffering of temperature change is linked with organismal fitness is essential. According to the 'cost-benefit' model of thermoregulation, animals that live in environments with high frequencies of favourable thermal microclimates should incur lower thermoregulatory costs, thermoregulate more efficiently and shunt the associated savings in time and energy towards other vital tasks such as feeding, territory defence and mate acquisition, increasing fitness. Here, we explore how thermal landscapes at the scale of individual territories, physiological performance and behaviour interact and shape fitness in the southern rock agama lizard (Agama atra). We integrated laboratory assays of whole organism performance with behavioural observations in the field, fine-scale estimates of environmental temperature, and paternity assignment of offspring to test whether fitness is predicted by territory thermal quality (i.e. the number of hours that operative temperatures in a territory fall within an individual's performance breadth). Male lizards that occupied territories of low thermal quality spent more time behaviourally compensating for sub-optimal temperatures and displayed less. Further, display rate was positively associated with lizard fitness, suggesting that there is an opportunity cost to engaging in thermoregulatory behaviour that will change as climate change progresses.

Meta-transcriptomic comparison of two sponge holobionts feeding on coral- and macroalgal-dissolved organic matter.

BACKGROUND: Sponge holobionts (i.e., the host and its associated microbiota) play a key role in the cycling of dissolved organic matter (DOM) in marine ecosystems. On coral reefs, an ecological shift from coral-dominated to algal-dominated ecosystems is currently occurring. Given that benthic corals and macroalgae release different types of DOM, in different abundances and with different bioavailability to sponge holobionts, it is important to understand how the metabolic activity of the host and associated microbiota change in response to the exposure to both DOM sources. Here, we look at the differential gene expression of two sponge holobionts 6 hours after feeding on naturally sourced coral- and macroalgal-DOM using RNA sequencing and meta-transcriptomic analysis. RESULTS: We found a slight, but significant differential gene expression in the comparison between the coral- and macroalgal-DOM treatments in both the high microbial abundance sponge Plakortis angulospiculatus and the low microbial abundance sponge Haliclona vansoesti. In the hosts, processes that regulate immune response, signal transduction, and metabolic pathways related to cell proliferation were elicited. In the associated microbiota carbohydrate metabolism was upregulated in both treatments, but coral-DOM induced further lipid and amino acids biosynthesis, while macroalgal-DOM caused a stress response. These differences could be driven by the presence of distinct organic macronutrients in the two DOM sources and of small pathogens or bacterial virulence factors in the macroalgal-DOM. CONCLUSIONS: This work provides two new sponge meta-transcriptomes and a database of putative genes and genetic pathways that are involved in the differential processing of coral- versus macroalgal-DOM as food source to sponges with high and low abundances of associated microbes. These pathways include carbohydrate metabolism, signaling pathways, and immune responses. However, the differences in the meta-transcriptomic responses of the sponge holobionts after 6 hours of feeding on the two DOM sources were small. Longer-term responses to both DOM sources should be assessed to evaluate how the metabolism and the ecological function of sponges will be affected when reefs shift from coral towards algal dominance.

Molecular machineries of ciliogenesis, cell survival, and vasculogenesis are differentially expressed during regeneration in explants of the demosponge Halichondria panicea.

Sponges are interesting animal models for regeneration studies, since even from dissociated cells, they are able to regenerate completely. In particular, explants are model systems that can be applied to many sponge species, since small fragments of sponges can regenerate all elements of the adult, including the oscula and the ability to pump water. The morphological aspects of regeneration in sponges are relatively well known, but the molecular machinery is only now starting to be elucidated for some sponge species. Here, we have used an explant system of the demosponge Halichondria panicea to understand the molecular machinery deployed during regeneration of the aquiferous system. We sequenced the transcriptomes of four replicates of the 5-day explant without an osculum (NOE), four replicates of the 17-18-day explant with a single osculum and pumping activity (PE) and also four replicates of field-collected individuals with regular pumping activity (PA), and performed differential gene expression analysis. We also described the morphology of NOE and PE samples using light and electron microscopy. Our results showed a highly disorganised mesohyl and disarranged aquiferous system in NOE that is coupled with upregulated pathways of ciliogenesis, organisation of the ECM, and cell proliferation and survival. Once the osculum is formed, genes involved in "response to stimulus in other organisms" were upregulated. Interestingly, the main molecular machinery of vasculogenesis described in vertebrates was activated during the regeneration of the aquiferous system. Notably, vasculogenesis markers were upregulated when the tissue was disorganised and about to start forming canals (NOE) and angiogenic stimulators and ECM remodelling machineries were differentially expressed once the aquiferous system was in place (PE and PA). Our results are fundamental to better understanding the molecular mechanisms involved in the formation of the aquiferous system in sponges, and its similarities with the early onset of blood-vessel formation in animal evolution.

Unveiling biogeographical patterns in the worldwide distributed Ceratitis capitata (medfly) using population genomics and microbiome composition.

Invasive species are among the most important, growing threats to food security and agricultural systems. The Mediterranean medfly, Ceratitis capitata, is one of the most damaging representatives of a group of rapidly expanding species in the family Tephritidae, due to their wide host range and high invasiveness potential. Here, we used restriction site-associated DNA sequencing (RADseq) to investigate the population genomic structure and phylogeographical history of medflies collected from six sampling sites, including Africa (South Africa), the Mediterranean (Spain, Greece), Latin America (Guatemala, Brazil) and Australia. A total of 1907 single nucleotide polymorphisms (SNPs) were used to identify two genetic clusters separating native and introduced ranges, consistent with previous findings. In the introduced range, all individuals were assigned to one genetic cluster except for those in Brazil, which showed introgression of an additional genetic cluster that also appeared in South Africa, and which could not be previously identified using microsatellite markers. Moreover, we assessed the microbial composition variations in medfly populations from selected sampling sites using amplicon sequencing of the 16S ribosomal RNA (V4 region). Microbiome composition and structure were highly similar across geographical regions and host plants, and only the Brazilian specimens showed increased diversity levels and a unique composition of its microbiome compared to other sampling sites. The unique SNP patterns and microbiome features in the Brazilian specimens could point to a direct migration route from Africa with subsequent adaptation of the microbiota to the specific conditions present in Brazil. These findings significantly improve our understanding of the evolutionary history of the global medfly invasions and their adaptation to newly colonized environments.

The Molecular Machinery of Gametogenesis in Geodia Demosponges (Porifera): Evolutionary Origins of a Conserved Toolkit across Animals.

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.

Mitochondrial evolution in the Demospongiae (Porifera): Phylogeny, divergence time, and genome biology.

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 (~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.

Barrettides: A Peptide Family Specifically Produced by the Deep-Sea Sponge Geodia barretti.

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C-G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide C (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I-IV, II-III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC50 0.64 µM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts.

Symbiosis, Selection, and Novelty: Freshwater Adaptation in the Unique Sponges of Lake Baikal.

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.

Sponge microbiome stability during environmental acquisition of highly specific photosymbionts.

In this study, we used in situ transplantations to provide the first evidence of horizontal acquisition of cyanobacterial symbionts by a marine sponge. The acquisition of the symbionts by the host sponge Petrosia ficiformis, which was observed in distinct visible patches, appeared several months after transplantation and at different times on different sponge specimens. We further used 16S rRNA gene amplicon sequencing of genomic DNA (gDNA) and complementary DNA (cDNA) and metatranscriptomics to investigate how the acquisition of the symbiotic cyanobacterium Candidatus Synechococcus feldmannii perturbed the diverse microbiota associated with the host P. ficiformis. To our surprise, the microbiota remained relatively stable during cyanobacterial symbiont acquisition at both structural (gDNA content) and activity (cDNA expression) levels. At the transcriptomic level, photosynthesis was the primary function gained following the acquisition of cyanobacteria. Genes involved in carotene production and oxidative stress tolerance were among those highly expressed by Ca. S. feldmannii, suggesting that this symbiont may protect itself and its host from damaging light radiation.

Enjoying the warming Mediterranean: Transcriptomic responses to temperature changes of a thermophilous keystone species in benthic communities.

Information about the genomic processes underlying responses to temperature changes is still limited in non-model marine invertebrates. In this sense, transcriptomic analyses can help to identify genes potentially related to thermal responses. We here investigated, via RNA-seq, whole-transcriptomic responses to increased and decreased temperatures in a thermophilous keystone sea urchin, Arbacia lixula, whose populations are increasing in the Mediterranean. This species is a key driver of benthic communities' structure due to its grazing activity. We found a strong response to experimentally induced cold temperature (7°C), with 1,181 differentially expressed transcripts relative to the control condition (13°C), compared to only 179 in the warm (22°C) treatment. A total of 84 (cold treatment) and three (warm treatment) gene ontology terms were linked to the differentially expressed transcripts. At 7°C the expression of genes encoding different heat shock proteins (HSPs) was upregulated, together with apoptotic suppressor genes (e.g., Bcl2), genes involved in the infection response and/or pathogen-recognition (e.g., echinoidin) and ATP-associated genes, while protein biosynthesis and DNA replication pathways were downregulated. At 22°C neither HSPs induction nor activation of the previously mentioned pathways were detected, with the exception of some apoptotic-related activities that were upregulated. Our results suggest a strong transcriptional response associated with low temperatures, and support the idea of low water temperature being a major limitation for A. lixula expansion across deep Mediterranean and northern Atlantic waters.

On the way to specificity - Microbiome reflects sponge genetic cluster primarily in highly structured populations.

Most animals, including sponges (Porifera), have species-specific microbiomes. Which genetic or environmental factors play major roles structuring the microbial community at the intraspecific level in sponges is, however, largely unknown. In this study, we tested whether geographic location or genetic structure of conspecific sponges influences their microbial assembly. For that, we used three sponge species with different rates of gene flow, and collected samples along their entire distribution range (two from the Mediterranean and one from the Southern Ocean) yielding a total of 393 samples. These three sponge species have been previously analysed by microsatellites or single nucleotide polymorphisms, and here we investigate their microbiomes by amplicon sequencing of the microbial 16S rRNA gene. The sponge Petrosia ficiformis, with highly isolated populations (low gene flow), showed a stronger influence of the host genetic distance on the microbial composition than the spatial distance. Host-specificity was therefore detected at the genotypic level, with individuals belonging to the same host genetic cluster harbouring more similar microbiomes than distant ones. On the contrary, the microbiome of Ircinia fasciculata and Dendrilla antarctica - both with weak population structure (high gene flow) - seemed influenced by location rather than by host genetic distance. Our results suggest that in sponge species with high population structure, the host genetic cluster influence the microbial community more than the geographic location.

Genetic diversity, connectivity and gene flow along the distribution of the emblematic Atlanto-Mediterranean sponge Petrosia ficiformis (Haplosclerida, Demospongiae).

BACKGROUND: Knowledge about the distribution of the genetic variation of marine species is fundamental to address species conservation and management strategies, especially in scenarios with mass mortalities. In the Mediterranean Sea, Petrosia ficiformis is one of the species most affected by temperature-related diseases. Our study aimed to assess its genetic structure, connectivity, and bottleneck signatures to understand its evolutionary history and to provide information to help design conservation strategies of sessile marine invertebrates. RESULTS: We genotyped 280 individuals from 19 locations across the entire distribution range of P. ficiformis in the Atlanto-Mediterranean region at 10 microsatellite loci. High levels of inbreeding were detected in most locations (especially in the Macaronesia and the Western Mediterranean) and bottleneck signatures were only detected in Mediterranean populations, although not coinciding entirely with those with reported die-offs. We detected strong significant population differentiation, with the Atlantic populations being the most genetically isolated, and show that six clusters explained the genetic structure along the distribution range of this sponge. Although we detected a pattern of isolation by distance in P. ficiformis when all locations were analyzed together, stratified Mantel tests revealed that other factors could be playing a more prominent role than isolation by distance. Indeed, we detected a strong effect of oceanographic barriers impeding the gene flow among certain areas, the strongest one being the Almeria-Oran front, hampering gene flow between the Atlantic Ocean and the Mediterranean Sea. Finally, migration and genetic diversity distribution analyses suggest a Mediterranean origin for the species. CONCLUSIONS: In our study Petrosia ficiformis showed extreme levels of inbreeding and population differentiation, which could all be linked to the poor swimming abilities of the larva. However, the observed moderate migration patterns are highly difficult to reconcile with such poor larval dispersal, and suggest that, although unlikely, dispersal may also be achieved in the gamete phase. Overall, because of the high genetic diversity in the Eastern Mediterranean and frequent mass mortalities in the Western Mediterranean, we suggest that conservation efforts should be carried out specifically in those areas of the Mediterranean to safeguard the genetic diversity of the species.

Revisiting metazoan phylogeny with genomic sampling of all phyla.

Proper biological interpretation of a phylogeny can sometimes hinge on the placement of key taxa-or fail when such key taxa are not sampled. In this light, we here present the first attempt to investigate (though not conclusively resolve) animal relationships using genome-scale data from all phyla. Results from the site-heterogeneous CAT + GTR model recapitulate many established major clades, and strongly confirm some recent discoveries, such as a monophyletic Lophophorata, and a sister group relationship between Gnathifera and Chaetognatha, raising continued questions on the nature of the spiralian ancestor. We also explore matrix construction with an eye towards testing specific relationships; this approach uniquely recovers support for Panarthropoda, and shows that Lophotrochozoa (a subclade of Spiralia) can be constructed in strongly conflicting ways using different taxon- and/or orthologue sets. Dayhoff-6 recoding sacrifices information, but can also reveal surprising outcomes, e.g. full support for a clade of Lophophorata and Entoprocta + Cycliophora, a clade of Placozoa + Cnidaria, and raising support for Ctenophora as sister group to the remaining Metazoa, in a manner dependent on the gene and/or taxon sampling of the matrix in question. Future work should test the hypothesis that the few remaining uncertainties in animal phylogeny might reflect violations of the various stationarity assumptions used in contemporary inference methods.

Population substructure and signals of divergent adaptive selection despite admixture in the sponge Dendrilla antarctica from shallow waters surrounding the Antarctic Peninsula.

Antarctic shallow-water invertebrates are exceptional candidates to study population genetics and evolution, because of their peculiar evolutionary history and adaptation to extreme habitats that expand and retreat with the ice sheets. Among them, sponges are one of the major components, yet population connectivity of none of their many Antarctic species has been studied. To investigate gene flow, local adaptation and resilience to near-future changes caused by global warming, we sequenced 62 individuals of the sponge Dendrilla antarctica along the Western Antarctic Peninsula (WAP) and the South Shetlands (spanning ~900 km). We obtained information from 577 double digest restriction site-associated DNA sequencing (ddRADseq)-derived single nucleotide polymorphism (SNP), using RADseq techniques for the first time with shallow-water sponges. In contrast to other studies in sponges, our 389 neutral SNPs data set showed high levels of gene flow, with a subtle substructure driven by the circulation system of the studied area. However, the 140 outlier SNPs under positive selection showed signals of population differentiation, separating the central-southern WAP from the Bransfield Strait area, indicating a divergent selection process in the study area despite panmixia. Fourteen of these outliers were annotated, being mostly involved in immune and stress responses. We suggest that the main selective pressure on D. antarctica might be the difference in the planktonic communities present in the central-southern WAP compared to the Bransfield Strait area, ultimately depending on sea-ice control of phytoplankton blooms. Our study unveils an unexpectedly long-distance larval dispersal exceptional in Porifera, broadening the use of genome-wide markers within nonmodel Antarctic organisms.

Sterol and genomic analyses validate the sponge biomarker hypothesis.

Molecular fossils (or biomarkers) are key to unraveling the deep history of eukaryotes, especially in the absence of traditional fossils. In this regard, the sterane 24-isopropylcholestane has been proposed as a molecular fossil for sponges, and could represent the oldest evidence for animal life. The sterane is found in rocks ∼650-540 million y old, and its sterol precursor (24-isopropylcholesterol, or 24-ipc) is synthesized today by certain sea sponges. However, 24-ipc is also produced in trace amounts by distantly related pelagophyte algae, whereas only a few close relatives of sponges have been assayed for sterols. In this study, we analyzed the sterol and gene repertoires of four taxa (Salpingoeca rosetta, Capsaspora owczarzaki, Sphaeroforma arctica, and Creolimax fragrantissima), which collectively represent the major living animal outgroups. We discovered that all four taxa lack C30 sterols, including 24-ipc. By building phylogenetic trees for key enzymes in 24-ipc biosynthesis, we identified a candidate gene (carbon-24/28 sterol methyltransferase, or SMT) responsible for 24-ipc production. Our results suggest that pelagophytes and sponges independently evolved C30 sterol biosynthesis through clade-specific SMT duplications. Using a molecular clock approach, we demonstrate that the relevant sponge SMT duplication event overlapped with the appearance of 24-isopropylcholestanes in the Neoproterozoic, but that the algal SMT duplication event occurred later in the Phanerozoic. Subsequently, pelagophyte algae and their relatives are an unlikely alternative to sponges as a source of Neoproterozoic 24-isopropylcholestanes, consistent with growing evidence that sponges evolved long before the Cambrian explosion ∼542 million y ago.

Implications of population connectivity studies for the design of marine protected areas in the deep sea: An example of a demosponge from the Clarion-Clipperton Zone.

The abyssal demosponge Plenaster craigi inhabits the Clarion-Clipperton Zone (CCZ) in the northeast Pacific, a region with abundant seafloor polymetallic nodules with potential mining interest. Since P. craigi is a very abundant encrusting sponge on nodules, understanding its genetic diversity and connectivity could provide important insights into extinction risks and design of marine protected areas. Our main aim was to assess the effectiveness of the Area of Particular Environmental Interest 6 (APEI-6) as a potential genetic reservoir for three adjacent mining exploration contract areas (UK-1A, UK-1B and OMS-1A). As in many other sponges, COI showed extremely low variability even for samples ~900 km apart. Conversely, the 168 individuals of P. craigi, genotyped for 11 microsatellite markers, provided strong genetic structure at large geographical scales not explained by isolation by distance (IBD). Interestingly, we detected molecular affinities between samples from APEI-6 and UK-1A, despite being separated ~800 km. Although our migration analysis inferred very little progeny dispersal of individuals between areas, the major differentiation of OMS-1A from the other areas might be explained by the occurrence of predominantly northeasterly transport predicted by the HYCOM hydrodynamic model. Our study suggests that although APEI-6 does serve a conservation role, with species connectivity to the exploration areas, it is on its own inadequate as a propagule source for P. craigi for the entire eastern portion of the CCZ. Our new data suggest that an APEI located to the east and/or the south of the UK-1, OMS-1, BGR, TOML and NORI areas would be highly valuable.

The Syllis gracilis species complex: A molecular approach to a difficult taxonomic problem (Annelida, Syllidae).

Syllis gracilis is an emblematic member of the subfamily Syllinae (Syllidae, Annelida), which inhabits shallow, temperate coastal waters and can be found on algae, coral rubble, and sponges. Their distinctive ypsiloid chaetae, usually found in specimens from populations all around the world, led to the consideration of the species as cosmopolitan, even though four other species have similar chaetae: Syllis magellanica, S. picta, S. mayeri and S. ypsiloides. The discovery of deeply divergent lineages in the Mediterranean Sea, that were morphologically similar, questioned the cosmopolitanism of S. gracilis and suggested the possibility of it being a species complex. In order to assess the speciation patterns within the putative S. gracilis complex, we undertook species delimitation and phylogenetic analyses on 61 specimens morphologically ascribed to Syllis gracilis and closely related species using a multilocus molecular dataset (two mitochondrial and two nuclear markers). Our results suggest high levels of genetic differentiation between the S. gracilis populations analyzed, some of which have morphologically distinctive features. Five to eight distinct lineages (depending on the analysis) were identified, all with geographically restricted distributions. Although the presence of ypsiloid chaetae has been traditionally considered the main character to identify S. gracilis, we conclude that this feature is homoplastic. Instead, we propose that characters such as the degree of fusion of blades and shafts in chaetae, the morphology of the posterior chaetae or the animal color pattern should be considered to differentiate lineages within the S. gracilis species complex. Our study does not support the cosmopolitanism of S. gracilis, and instead provides morphological and molecular evidence of the existence of a complex of pseudo-cryptic species.