The genome of the reef-building glass sponge Aphrocallistes vastus provides insights into silica biomineralization.

Well-annotated and contiguous genomes are an indispensable resource for understanding the evolution, development, and metabolic capacities of organisms. Sponges, an ecologically important non-bilaterian group of primarily filter-feeding sessile aquatic organisms, are underrepresented with respect to available genomic resources. Here we provide a high-quality and well-annotated genome of Aphrocallistes vastus, a glass sponge (Porifera: Hexactinellida) that forms large reef structures off the coast of British Columbia (Canada). We show that its genome is approximately 80 Mb, small compared to most other metazoans, and contains nearly 2500 nested genes, more than other genomes. Hexactinellida is characterized by a unique skeletal architecture made of amorphous silicon dioxide (SiO2), and we identified 419 differentially expressed genes between the osculum, i.e. the vertical growth zone of the sponge, and the main body. Among the upregulated ones, mineralization-related genes such as glassin, as well as collagens and actins, dominate the expression profile during growth. Silicateins, suggested being involved in silica mineralization, especially in demosponges, were not found at all in the A. vastus genome and suggests that the underlying mechanisms of SiO2 deposition in the Silicea sensu stricto (Hexactinellida + Demospongiae) may not be homologous.

Metagenomic data for Halichondria panicea from Illumina and nanopore sequencing and preliminary genome assemblies for the sponge and two microbial symbionts.

OBJECTIVES: These data were collected to generate a novel reference metagenome for the sponge Halichondria panicea and its microbiome for subsequent differential expression analyses. DATA DESCRIPTION: These data include raw sequences from four separate sequencing runs of the metagenome of a single individual of Halichondria panicea-one Illumina MiSeq (2 × 300 bp, paired-end) run and three Oxford Nanopore Technologies (ONT) long-read sequencing runs, generating 53.8 and 7.42 Gbp respectively. Comparing assemblies of Illumina, ONT and an Illumina-ONT hybrid revealed the hybrid to be the 'best' assembly, comprising 163 Mbp in 63,555 scaffolds (N50: 3084). This assembly, however, was still highly fragmented and only contained 52% of core metazoan genes (with 77.9% partial genes), so it was also not complete. However, this sponge is an emerging model species for field and laboratory work, and there is considerable interest in genomic sequencing of this species. Although the resultant assemblies from the data presented here are suboptimal, this data note can inform future studies by providing an estimated genome size and coverage requirements for future sequencing, sharing additional data to potentially improve other suboptimal assemblies of this species, and outlining potential limitations and pitfalls of the combined Illumina and ONT approach to novel genome sequencing.

Profiling cellular diversity in sponges informs animal cell type and nervous system evolution.

The evolutionary origin of metazoan cell types such as neurons and muscles is not known. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identified 18 distinct cell types. These include nitric oxide­sensitive contractile pinacocytes, amoeboid phagocytes, and secretory neuroid cells that reside in close contact with digestive choanocytes that express scaffolding and receptor proteins. Visualizing neuroid cells by correlative x-ray and electron microscopy revealed secretory vesicles and cellular projections enwrapping choanocyte microvilli and cilia. Our data show a communication system that is organized around sponge digestive chambers, using conserved modules that became incorporated into the pre- and postsynapse in the nervous systems of other animals.

A chromosome-scale genome assembly and karyotype of the ctenophore Hormiphora californensis.

Here, we present a karyotype, a chromosome-scale genome assembly, and a genome annotation from the ctenophore Hormiphora californensis (Ctenophora: Cydippida: Pleurobrachiidae). The assembly spans 110 Mb in 44 scaffolds and 99.47% of the bases are contained in 13 scaffolds. Chromosome micrographs and Hi-C heatmaps support a karyotype of 13 diploid chromosomes. Hi-C data reveal three large heterozygous inversions on chromosome 1, and one heterozygous inversion shares the same gene order found in the genome of the ctenophore Pleurobrachia bachei. We find evidence that H. californensis and P. bachei share thirteen homologous chromosomes, and the same karyotype of 1n = 13. The manually curated PacBio Iso-Seq-based genome annotation reveals complex gene structures, including nested genes and trans-spliced leader sequences. This chromosome-scale assembly is a useful resource for ctenophore biology and will aid future studies of metazoan evolution and phylogenetics.

A Win-Loss Interaction on Fe0 Between Methanogens and Acetogens From a Climate Lake.

Diverse physiological groups congregate into environmental corrosive biofilms, yet the interspecies interactions between these corrosive physiological groups are seldom examined. We, therefore, explored Fe0-dependent cross-group interactions between acetogens and methanogens from lake sediments. On Fe0, acetogens were more corrosive and metabolically active when decoupled from methanogens, whereas methanogens were more metabolically active when coupled with acetogens. This suggests an opportunistic (win-loss) interaction on Fe0 between acetogens (loss) and methanogens (win). Clostridia and Methanobacterium were the major candidates doing acetogenesis and methanogenesis after four transfers (metagenome sequencing) and the only groups detected after 11 transfers (amplicon sequencing) on Fe0. Since abiotic H2 failed to explain the high metabolic rates on Fe0, we examined whether cell exudates (spent media filtrate) promoted the H2-evolving reaction on Fe0 above abiotic controls. Undeniably, spent media filtrate generated three- to four-fold more H2 than abiotic controls, which could be partly explained by thermolabile enzymes and partly by non-thermolabile constituents released by cells. Next, we examined the metagenome for candidate enzymes/shuttles that could catalyze H2 evolution from Fe0 and found candidate H2-evolving hydrogenases and an almost complete pathway for flavin biosynthesis in Clostridium. Clostridial ferredoxin-dependent [FeFe]-hydrogenases may be catalyzing the H2-evolving reaction on Fe0, explaining the significant H2 evolved by spent media exposed to Fe0. It is typical of Clostridia to secrete enzymes and other small molecules for lytic purposes. Here, they may secrete such molecules to enhance their own electron uptake from extracellular electron donors but indirectly make their H2-consuming neighbors-Methanobacterium-fare five times better in their presence. The particular enzymes and constituents promoting H2 evolution from Fe0 remain to be determined. However, we postulate that in a static environment like corrosive crust biofilms in lake sediments, less corrosive methanogens like Methanobacterium could extend corrosion long after acetogenesis ceased, by exploiting the constituents secreted by acetogens.

Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri.

The genomes of non-bilaterian metazoans are key to understanding the molecular basis of early animal evolution. However, a full comprehension of how animal-specific traits, such as nervous systems, arose is hindered by the scarcity and fragmented nature of genomes from key taxa, such as Porifera. Ephydatia muelleri is a freshwater sponge found across the northern hemisphere. Here, we present its 326 Mb genome, assembled to high contiguity (N50: 9.88 Mb) with 23 chromosomes on 24 scaffolds. Our analyses reveal a metazoan-typical genome architecture, with highly shared synteny across Metazoa, and suggest that adaptation to the extreme temperatures and conditions found in freshwater often involves gene duplication. The pancontinental distribution and ready laboratory culture of E. muelleri make this a highly practical model system which, with RNAseq, DNA methylation and bacterial amplicon data spanning its development and range, allows exploration of genomic changes both within sponges and in early animal evolution.

Very few sites can reshape the inferred phylogenetic tree.

The history of animal evolution, and the relative placement of extant animal phyla in this history is, in principle, testable from phylogenies derived from molecular sequence data. Though datasets have increased in size and quality in the past years, the contribution of individual genes (and ultimately amino acid sites) to the final phylogeny is unequal across genes. Here we demonstrate that removing a small fraction of sites strongly favoring one topology can produce a highly-supported tree of an alternate topology. We explore this approach using a dataset for animal phylogeny, and create a highly-supported tree with a monophyletic group of sponges and ctenophores, a topology not usually recovered. Because of the high sensitivity of such an analysis to gene selection, and because most gene sets are neither standardized nor representative of the entire genome, researchers should be diligent about making intermediate analyses available with their phylogenetic studies. Effort is needed to ensure these datasets are maximally informative, by ensuring all genes are systematically sampled across relevant species. From there, it could be determined whether any gene or gene sets introduce bias, and then deal with those biases appropriately.

Conserved novel ORFs in the mitochondrial genome of the ctenophore Beroe forskalii.

To date, five ctenophore species' mitochondrial genomes have been sequenced, and each contains open reading frames (ORFs) that if translated have no identifiable orthologs. ORFs with no identifiable orthologs are called unidentified reading frames (URFs). If truly protein-coding, ctenophore mitochondrial URFs represent a little understood path in early-diverging metazoan mitochondrial evolution and metabolism. We sequenced and annotated the mitochondrial genomes of three individuals of the beroid ctenophore Beroe forskalii and found that in addition to sharing the same canonical mitochondrial genes as other ctenophores, the B. forskalii mitochondrial genome contains two URFs. These URFs are conserved among the three individuals but not found in other sequenced species. We developed computational tools called pauvre and cuttlery to determine the likelihood that URFs are protein coding. There is evidence that the two URFs are under negative selection, and a novel Bayesian hypothesis test of trinucleotide frequency shows that the URFs are more similar to known coding genes than noncoding intergenic sequence. Protein structure and function prediction of all ctenophore URFs suggests that they all code for transmembrane transport proteins. These findings, along with the presence of URFs in other sequenced ctenophore mitochondrial genomes, suggest that ctenophores may have uncharacterized transmembrane proteins present in their mitochondria.

A hybrid de novo assembly of the sea pansy (Renilla muelleri) genome.

BACKGROUND: More than 3,000 species of octocorals (Cnidaria, Anthozoa) inhabit an expansive range of environments, from shallow tropical seas to the deep-ocean floor. They are important foundation species that create coral "forests," which provide unique niches and 3-dimensional living space for other organisms. The octocoral genus Renilla inhabits sandy, continental shelves in the subtropical and tropical Atlantic and eastern Pacific Oceans. Renilla is especially interesting because it produces secondary metabolites for defense, exhibits bioluminescence, and produces a luciferase that is widely used in dual-reporter assays in molecular biology. Although several anthozoan genomes are currently available, the majority of these are hexacorals. Here, we present a de novo assembly of an azooxanthellate shallow-water octocoral, Renilla muelleri. FINDINGS: We generated a hybrid de novo assembly using MaSuRCA v.3.2.6. The final assembly included 4,825 scaffolds and a haploid genome size of 172 megabases (Mb). A BUSCO assessment found 88% of metazoan orthologs present in the genome. An Augustus ab initio gene prediction found 23,660 genes, of which 66% (15,635) had detectable similarity to annotated genes from the starlet sea anemone, Nematostella vectensis, or to the Uniprot database. Although the R. muelleri genome may be smaller (172 Mb minimum size) than other publicly available coral genomes (256-448 Mb), the R. muelleri genome is similar to other coral genomes in terms of the number of complete metazoan BUSCOs and predicted gene models. CONCLUSIONS: The R. muelleri hybrid genome provides a novel resource for researchers to investigate the evolution of genes and gene families within Octocorallia and more widely across Anthozoa. It will be a key resource for future comparative genomics with other corals and for understanding the genomic basis of coral diversity.

The Role of Homology and Orthology in the Phylogenomic Analysis of Metazoan Gene Content.

Resolving the relationships of animals (Metazoa) is crucial to our understanding of the origin of key traits such as muscles, guts, and nerves. However, a broadly accepted metazoan consensus phylogeny has yet to emerge. In part, this is because the genomes of deeply diverging and fast-evolving lineages may undergo significant gene turnover, reducing the number of orthologs shared with related phyla. This can limit the usefulness of traditional phylogenetic methods that rely on alignments of orthologous sequences. Phylogenetic analysis of gene content has the potential to circumvent this orthology requirement, with binary presence/absence of homologous gene families representing a source of phylogenetically informative characters. Applying binary substitution models to the gene content of 26 complete animal genomes, we demonstrate that patterns of gene conservation differ markedly depending on whether gene families are defined by orthology or homology, that is, whether paralogs are excluded or included. We conclude that the placement of some deeply diverging lineages may exceed the limit of resolution afforded by the current methods based on comparisons of orthologous protein sequences, and novel approaches are required to fully capture the evolutionary signal from genes within genomes.

Correction: Comparative genomics and the nature of placozoan species.

[This corrects the article DOI: 10.1371/journal.pbio.2005359.].

Integrating Embryonic Development and Evolutionary History to Characterize Tentacle-Specific Cell Types in a Ctenophore.

The origin of novel traits can promote expansion into new niches and drive speciation. Ctenophores (comb jellies) are unified by their possession of a novel cell type: the colloblast, an adhesive cell found only in the tentacles. Although colloblast-laden tentacles are fundamental for prey capture among ctenophores, some species have tentacles lacking colloblasts and others have lost their tentacles completely. We used transcriptomes from 36 ctenophore species to identify gene losses that occurred specifically in lineages lacking colloblasts and tentacles. We cross-referenced these colloblast- and tentacle-specific candidate genes with temporal RNA-Seq during embryogenesis in Mnemiopsis leidyi and found that both sets of candidates are preferentially expressed during tentacle morphogenesis. We also demonstrate significant upregulation of candidates from both data sets in the tentacle bulb of adults. Both sets of candidates were enriched for an N-terminal signal peptide and protein domains associated with secretion; among tentacle candidates we also identified orthologs of cnidarian toxin proteins, presenting tantalizing evidence that ctenophore tentacles may secrete toxins along with their adhesive. Finally, using cell lineage tracing, we demonstrate that colloblasts and neurons share a common progenitor, suggesting the evolution of colloblasts involved co-option of a neurosecretory gene regulatory network. Together these data offer an initial glimpse into the genetic architecture underlying ctenophore cell-type diversity.

Comparative genomics and the nature of placozoan species.

Placozoans are a phylum of nonbilaterian marine animals currently represented by a single described species, Trichoplax adhaerens, Schulze 1883. Placozoans arguably show the simplest animal morphology, which is identical among isolates collected worldwide, despite an apparently sizeable genetic diversity within the phylum. Here, we use a comparative genomics approach for a deeper appreciation of the structure and causes of the deeply diverging lineages in the Placozoa. We generated a high-quality draft genome of the genetic lineage H13 isolated from Hong Kong and compared it to the distantly related T. adhaerens. We uncovered substantial structural differences between the two genomes that point to a deep genomic separation and provide support that adaptation by gene duplication is likely a crucial mechanism in placozoan speciation. We further provide genetic evidence for reproductively isolated species and suggest a genus-level difference of H13 to T. adhaerens, justifying the designation of H13 as a new species, Hoilungia hongkongensis nov. gen., nov. spec., now the second described placozoan species and the first in a new genus. Our multilevel comparative genomics approach is, therefore, likely to prove valuable for species distinctions in other cryptic microscopic animal groups that lack diagnostic morphological characters, such as some nematodes, copepods, rotifers, or mites.

Fungi in Deep Subsurface Environments.

The igneous crust of the oceans and the continents represents the major part of Earth's lithosphere and has recently been recognized as a substantial, yet underexplored, microbial habitat. While prokaryotes have been the focus of most investigations, microeukaryotes have been surprisingly neglected. However, recent work acknowledges eukaryotes, and in particular fungi, as common inhabitants of the deep biosphere, including the deep igneous provinces. The fossil record of the subseafloor igneous crust, and to some extent the continental bedrock, establishes fungi or fungus-like organisms as inhabitants of deep rock since at least the Paleoproterozoic, which challenges the present notion of early fungal evolution. Additionally, deep fungi have been shown to play an important ecological role engaging in symbiosis-like relationships with prokaryotes, decomposing organic matter, and being responsible for mineral weathering and formation, thus mediating mobilization of biogeochemically important elements. In this review, we aim at covering the abundance and diversity of fungi in the various igneous rock provinces on Earth as well as describing the ecological impact of deep fungi. We further discuss what consequences recent findings might have for the understanding of the fungal distribution in extensive anoxic environments and for early fungal evolution.

The last common ancestor of animals lacked the HIF pathway and respired in low-oxygen environments.

Animals have a carefully orchestrated relationship with oxygen. When exposed to low environmental oxygen concentrations, and during periods of increased energy expenditure, animals maintain cellular oxygen homeostasis by enhancing internal oxygen delivery, and by enabling the anaerobic production of ATP. These low-oxygen responses are thought to be controlled universally across animals by the hypoxia-inducible factor (HIF). We find, however, that sponge and ctenophore genomes lack key components of the HIF pathway. Since sponges and ctenophores are likely sister to all remaining animal phyla, the last common ancestor of extant animals likely lacked the HIF pathway as well. Laboratory experiments show that the marine sponge Tethya wilhelma maintains normal transcription under oxygen levels down to 0.25% of modern atmospheric saturation, the lowest levels we investigated, consistent with the predicted absence of HIF or any other HIF-like pathway. Thus, the last common ancestor of all living animals could have metabolized aerobically under very low environmental oxygen concentrations.

Animal origins and the Tonian Earth system.

The Neoproterozoic Era (1000-541 million years ago, Ma) was characterized by dramatic environmental and evolutionary change, including at least two episodes of extensive, low-latitude glaciation, potential changes in the redox structure of the global ocean, and the origin and diversification of animal life. How these different events related to one another remains an active area of research, particularly how these environmental changes influenced, and were influenced by, the earliest evolution of animals. Animal multicellularity is estimated to have evolved in the Tonian Period (1000-720 Ma) and represents one of at least six independent acquisitions of complex multicellularity, characterized by cellular differentiation, three-dimensional body plans, and active nutrient transport. Compared with the other instances of complex multicellularity, animals represent the only clade to have evolved from wall-less, phagotrophic flagellates, which likely placed unique cytological and trophic constraints on the evolution of animal multicellularity. Here, we compare recent molecular clock estimates with compilations of the chromium isotope, micropaleontological, and organic biomarker records, suggesting that, as of now, the origin of animals was not obviously correlated to any environmental-ecological change in the Tonian Period. This lack of correlation is consistent with the idea that the evolution of animal multicellularity was primarily dictated by internal, developmental constraints and occurred independently of the known environmental-ecological changes that characterized the Neoproterozoic Era.

Predicted microbial secretomes and their target substrates in marine sediment.

Scientific drilling has identified a biosphere in marine sediments 1 , which contain many uncultivated microbial groups known only by their DNA sequences 2-4 . Recycling of organic matter in sediments is an important component of biogeochemical cycles because marine sediments are critical for long-term carbon storage 5 . Turnover of carbon is hypothesized to be driven by the secretion of enzymes by microbial organisms 5-7 , which act to break down macromolecules into constitutive monomers that can be transported into cells. As such, the nature of the microbial secretome often influences the function of a community 6 . However, the microbial groups involved in this process and the biochemistry they encode is poorly understood. Here, we show that expressed genes from 5 to 159 meters below the seafloor 8 (mbsf) encode numerous candidate peptidases and carbohydrate-active enzymes ('CAZymes') 9 targeted for secretion. The majority (90-99%) were assigned to Bacteria, of which 12% shared the highest sequence similarity with candidate phyla 10,11 . The remaining putatively secreted proteins shared highest sequence similarity with archaeal and fungal enzymes, which peak in two redox transition zones 12 . In the shallower redox zone at 30 mbsf, 20% of the transcripts encoding putative secreted peptidases were assigned to lineages 7,13,14 of uncultivated Archaea. The target compounds of the predicted secreted proteome show a preference for necromass in the form of microbial cell envelopes as well as plankton and algal detritus. The predicted fungal secreted proteome encodes CAZymes not present in the predicted bacterial or archaeal secreted proteomes, indicating that fungi putatively play a minimal but specialized role in subseafloor carbohydrate recycling.

Symplectin evolved from multiple duplications in bioluminescent squid.

The squid Sthenoteuthis oualaniensis, formerly Symplectoteuthis oualaniensis, generates light using the luciferin coelenterazine and a unique enzyme, symplectin. Genetic information is limited for bioluminescent cephalopod species, so many proteins, including symplectin, occur in public databases only as sequence isolates with few identifiable homologs. As the distribution of the symplectin/pantetheinase protein family in Metazoa remains mostly unexplored, we have sequenced the transcriptomes of four additional luminous squid, and make use of publicly available but unanalyzed data of other cephalopods, to examine the occurrence and evolution of this protein family. While the majority of spiralians have one or two copies of this protein family, four well-supported groups of proteins are found in cephalopods, one of which corresponds to symplectin. A cysteine that is critical for symplectin functioning is conserved across essentially all members of the protein family, even those unlikely to be used for bioluminescence. Conversely, active site residues involved in pantetheinase catalysis are also conserved across essentially all of these proteins, suggesting that symplectin may have multiple functions including hydrolase activity, and that the evolution of the luminous phenotype required other changes in the protein outside of the main binding pocket.

Similar Ratios of Introns to Intergenic Sequence across Animal Genomes.

One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought.