Cave Thiovulum (Candidatus Thiovulum stygium) differs metabolically and genomically from marine species.

Thiovulum spp. (Campylobacterota) are large sulfur bacteria that form veil-like structures in aquatic environments. The sulfidic Movile Cave (Romania), sealed from the atmosphere for ~5 million years, has several aqueous chambers, some with low atmospheric O2 (~7%). The cave's surface-water microbial community is dominated by bacteria we identified as Thiovulum. We show that this strain, and others from subsurface environments, are phylogenetically distinct from marine Thiovulum. We assembled a closed genome of the Movile strain and confirmed its metabolism using RNAseq. We compared the genome of this strain and one we assembled from public data from the sulfidic Frasassi caves to four marine genomes, including Candidatus Thiovulum karukerense and Ca. T. imperiosus, whose genomes we sequenced. Despite great spatial and temporal separation, the genomes of the Movile and Frasassi Thiovulum were highly similar, differing greatly from the very diverse marine strains. We concluded that cave Thiovulum represent a new species, named here Candidatus Thiovulum stygium. Based on their genomes, cave Thiovulum can switch between aerobic and anaerobic sulfide oxidation using O2 and NO3- as electron acceptors, the latter likely via dissimilatory nitrate reduction to ammonia. Thus, Thiovulum is likely important to both S and N cycles in sulfidic caves. Electron microscopy analysis suggests that at least some of the short peritrichous structures typical of Thiovulum are type IV pili, for which genes were found in all strains. These pili may play a role in veil formation, by connecting adjacent cells, and in the motility of these exceptionally fast swimmers.

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

Chromosomal assembly of the flat oyster (Ostrea edulis L.) genome as a new genetic resource for aquaculture.

The European flat oyster (Ostrea edulis L.) is a native bivalve of the European coasts. Harvest of this species has declined during the last decades because of the appearance of two parasites that have led to the collapse of the stocks and the loss of the natural oyster beds. O. edulis has been the subject of numerous studies in population genetics and on the detection of the parasites Bonamia ostreae and Marteilia refringens. These studies investigated immune responses to these parasites at the molecular and cellular levels. Several genetic improvement programs have been initiated especially for parasite resistance. Within the framework of a European project (PERLE 2) that aims to produce genetic lines of O. edulis with hardiness traits (growth, survival, resistance) for the purpose of repopulating natural oyster beds in Brittany and reviving the culture of this species in the foreshore, obtaining a reference genome becomes essential as done recently in many bivalve species of aquaculture interest. Here, we present a chromosome-level genome assembly and annotation for the European flat oyster, generated by combining PacBio, Illumina, 10X linked, and Hi-C sequencing. The finished assembly is 887.2 Mb with a scaffold-N50 of 97.1 Mb scaffolded on the expected 10 pseudochromosomes. Annotation of the genome revealed the presence of 35,962 protein-coding genes. We analyzed in detail the transposable element (TE) diversity in the flat oyster genome, highlighted some specificities in tRNA and miRNA composition, and provided the first insight into the molecular response of O. edulis to M. refringens. This genome provides a reference for genomic studies on O. edulis to better understand its basic physiology and as a useful resource for genetic breeding in support of aquaculture and natural reef restoration.

Comparative genome analysis of Vagococcus fluvialis reveals abundance of mobile genetic elements in sponge-isolated strains.

BACKGROUND: Vagococcus fluvialis is a species of lactic acid bacteria found both free-living in river and seawater and associated to hosts, such as marine sponges. This species has been greatly understudied, with no complete genome assembly available to date, which is essential for the characterisation of the mobilome. RESULTS: We sequenced and assembled de novo the complete genome sequences of five V. fluvialis isolates recovered from marine sponges. Pangenome analysis of the V. fluvialis species (total of 17 genomes) showed a high intraspecific diversity, with 45.5% of orthologous genes found to be strain specific. Despite this diversity, analyses of gene functions clustered all V. fluvialis species together and separated them from other sequenced Vagococcus species. V. fluvialis strains from different habitats were highly similar in terms of functional diversity but the sponge-isolated strains were enriched in several functions related to the marine environment. Furthermore, sponge-isolated strains carried a significantly higher number of mobile genetic elements (MGEs) compared to previously sequenced V. fluvialis strains from other environments. Sponge-isolated strains carried up to 4 circular plasmids each, including a 48-kb conjugative plasmid. Three of the five strains carried an additional circular extrachromosomal sequence, assumed to be an excised prophage as it contained mainly viral genes and lacked plasmid replication genes. Insertion sequences (ISs) were up to five times more abundant in the genomes of sponge-isolated strains compared to the others, including several IS families found exclusively in these genomes. CONCLUSIONS: Our findings highlight the dynamics and plasticity of the V. fluvialis genome. The abundance of mobile genetic elements in the genomes of sponge-isolated V. fluvialis strains suggests that the mobilome might be key to understanding the genomic signatures of symbiosis in bacteria.

Comparative analysis of the Mercenaria mercenaria genome provides insights into the diversity of transposable elements and immune molecules in bivalve mollusks.

BACKGROUND: The hard clam Mercenaria mercenaria is a major marine resource along the Atlantic coasts of North America and has been introduced to other continents for resource restoration or aquaculture activities. Significant mortality events have been reported in the species throughout its native range as a result of diseases (microbial infections, leukemia) and acute environmental stress. In this context, the characterization of the hard clam genome can provide highly needed resources to enable basic (e.g., oncogenesis and cancer transmission, adaptation biology) and applied (clam stock enhancement, genomic selection) sciences. RESULTS: Using a combination of long and short-read sequencing technologies, a 1.86 Gb chromosome-level assembly of the clam genome was generated. The assembly was scaffolded into 19 chromosomes, with an N50 of 83 Mb. Genome annotation yielded 34,728 predicted protein-coding genes, markedly more than the few other members of the Venerida sequenced so far, with coding regions representing only 2% of the assembly. Indeed, more than half of the genome is composed of repeated elements, including transposable elements. Major chromosome rearrangements were detected between this assembly and another recent assembly derived from a genetically segregated clam stock. Comparative analysis of the clam genome allowed the identification of a marked diversification in immune-related proteins, particularly extensive tandem duplications and expansions in tumor necrosis factors (TNFs) and C1q domain-containing proteins, some of which were previously shown to play a role in clam interactions with infectious microbes. The study also generated a comparative repertoire highlighting the diversity and, in some instances, the specificity of LTR-retrotransposons elements, particularly Steamer elements in bivalves. CONCLUSIONS: The diversity of immune molecules in M. mercenaria may allow this species to cope with varying and complex microbial and environmental landscapes. The repertoire of transposable elements identified in this study, particularly Steamer elements, should be a prime target for the investigation of cancer cell development and transmission among bivalve mollusks.

Solving the Coral Species Delimitation Conundrum.

Distinguishing coral species is not only crucial for physiological, ecological, and evolutionary studies but also to enable effective management of threatened reef ecosystems. However, traditional hypotheses that delineate coral species based on morphological traits from the coral skeleton are frequently at odds with tree-based molecular approaches. Additionally, a dearth of species-level molecular markers has made species delimitation particularly challenging in species-rich coral genera, leading to the widespread assumption that interspecific hybridization might be responsible for this apparent conundrum. Here, we used three lines of evidence-morphology, breeding trials, and molecular approaches-to identify species boundaries in a group of ecologically important tabular Acropora corals. In contrast to previous studies, our morphological analysis yielded groups that were congruent with experimental crosses as well as with coalescent-based and allele sharing-based multilocus approaches to species delimitation. Our results suggest that species of the genus Acropora are reproductively isolated and independently evolving units that can be distinguished morphologically. These findings not only pave the way for a taxonomic revision of coral species but also outline an approach that can provide a solid basis to address species delimitation and provide conservation support to a wide variety of keystone organisms. [Acropora; coral reefs; hybridization; reproductive isolation; taxonomy.].

Molecular analyses of groundwater amphipods (Crustacea: Niphargidae) from Luxembourg: new species reveal limitations of morphology-based checklists.

Niphargus amphipods were collected from 2007 to 2018 at 98 sites comprising artificial caverns, springs and interstitial waters in the Grand Duchy of Luxembourg. Opportunistic sampling was combined with passive trapping. Specimen identification was achieved using morphological keys and molecular data. Initial morphological determination and literature data suggested five species, whereas sequencing of fragments of the mitochondrial cytochrome c oxidase subunit 1 gene and nuclear 28S rDNA marker supported the presence of seven species: Niphargus schellenbergi, Niphargus puteanus, Niphargus fontanus, one species of the Niphargus kochianus complex, and three species of the Niphargus aquilex complex. Niphargus schellenbergi was by far the most abundant and widespread species. Limited overlap was observed between literature-based records, our initial morphological determinations based on classical taxonomic characters, and genetic sequence data. In general, the combination of phenotypically variable taxa, such as N. schellenbergi, and cryptic or near-cryptic species, as in the N. aquilex complex, renders morphological identification of niphargids from Luxembourg a challenging or even impossible task. DNA taxonomy will therefore have to be used in future studies of the fauna of this region.

Chromosome-level genome assembly reveals homologous chromosomes and recombination in asexual rotifer Adineta vaga.

Bdelloid rotifers are notorious as a speciose ancient clade comprising only asexual lineages. Thanks to their ability to repair highly fragmented DNA, most bdelloid species also withstand complete desiccation and ionizing radiation. Producing a well-assembled reference genome is a critical step to developing an understanding of the effects of long-term asexuality and DNA breakage on genome evolution. To this end, we present the first high-quality chromosome-level genome assemblies for the bdelloid Adineta vaga, composed of six pairs of homologous (diploid) chromosomes with a footprint of paleotetraploidy. The observed large-scale losses of heterozygosity are signatures of recombination between homologous chromosomes, either during mitotic DNA double-strand break repair or when resolving programmed DNA breaks during a modified meiosis. Dynamic subtelomeric regions harbor more structural diversity (e.g., chromosome rearrangements, transposable elements, and haplotypic divergence). Our results trigger the reappraisal of potential meiotic processes in bdelloid rotifers and help unravel the factors underlying their long-term asexual evolutionary success.

Overcoming uncollapsed haplotypes in long-read assemblies of non-model organisms.

BACKGROUND: Long-read sequencing is revolutionizing genome assembly: as PacBio and Nanopore technologies become more accessible in technicity and in cost, long-read assemblers flourish and are starting to deliver chromosome-level assemblies. However, these long reads are usually error-prone, making the generation of a haploid reference out of a diploid genome a difficult enterprise. Failure to properly collapse haplotypes results in fragmented and structurally incorrect assemblies and wreaks havoc on orthology inference pipelines, yet this serious issue is rarely acknowledged and dealt with in genomic projects, and an independent, comparative benchmark of the capacity of assemblers and post-processing tools to properly collapse or purge haplotypes is still lacking. RESULTS: We tested different assembly strategies on the genome of the rotifer Adineta vaga, a non-model organism for which high coverages of both PacBio and Nanopore reads were available. The assemblers we tested (Canu, Flye, NextDenovo, Ra, Raven, Shasta and wtdbg2) exhibited strikingly different behaviors when dealing with highly heterozygous regions, resulting in variable amounts of uncollapsed haplotypes. Filtering reads generally improved haploid assemblies, and we also benchmarked three post-processing tools aimed at detecting and purging uncollapsed haplotypes in long-read assemblies: HaploMerger2, purge_haplotigs and purge_dups. CONCLUSIONS: We provide a thorough evaluation of popular assemblers on a non-model eukaryote genome with variable levels of heterozygosity. Our study highlights several strategies using pre and post-processing approaches to generate haploid assemblies with high continuity and completeness. This benchmark will help users to improve haploid assemblies of non-model organisms, and evaluate the quality of their own assemblies.

Genome Assembly of the Cold-Tolerant Leaf Beetle Gonioctena quinquepunctata, an Important Resource for Studying Its Evolution and Reproductive Barriers between Species.

Coleoptera is the most species-rich insect order, yet is currently underrepresented in genomic databases. An assembly was generated for ca. 1.7-Gb genome of the leaf beetle Gonioctena quinquepunctata by first assembling long-sequence reads (Oxford Nanopore; ± 27-fold coverage) and subsequently polishing the resulting assembly with short sequence reads (Illumina; ± 85-fold coverage). The unusually large size (most Coleoptera species are associated with a reported size below 1 Gb) was at least partially attributed to the presence of a large fraction of repeated elements (73.8%). The final assembly was characterized by an N50 length of 432 kb and a BUSCO score of 95.5%. The heterozygosity rate was ±0.6%. Automated genome annotation informed by RNA-Seq resulted in 40,568 predicted proteins, which is much larger than the typical range 17,000-23,000 predicted for other Coleoptera. However, no evidence of a genome duplication was detected. This new reference genome will contribute to our understanding of genetic variation in the Coleoptera. Among others, it will also allow exploring reproductive barriers between species, investigating introgression in the nuclear genome, and identifying genes involved in resistance to extreme climate conditions.

An Indo-Pacific coral spawning database.

The discovery of multi-species synchronous spawning of scleractinian corals on the Great Barrier Reef in the 1980s stimulated an extraordinary effort to document spawning times in other parts of the globe. Unfortunately, most of these data remain unpublished which limits our understanding of regional and global reproductive patterns. The Coral Spawning Database (CSD) collates much of these disparate data into a single place. The CSD includes 6178 observations (3085 of which were unpublished) of the time or day of spawning for over 300 scleractinian species in 61 genera from 101 sites in the Indo-Pacific. The goal of the CSD is to provide open access to coral spawning data to accelerate our understanding of coral reproductive biology and to provide a baseline against which to evaluate any future changes in reproductive phenology.

Population genetics of the brooding coral Seriatopora hystrix reveals patterns of strong genetic differentiation in the Western Indian Ocean.

Coral reefs provide essential goods and services but are degrading at an alarming rate due to local and global anthropogenic stressors. The main limitation that prevents the implementation of adequate conservation measures is that connectivity and genetic structure of populations are poorly known. Here, the genetic diversity and connectivity of the brooding scleractinian coral Seriatopora hystrix were assessed at two scales by genotyping ten microsatellite markers for 356 individual colonies. S. hystrix showed high differentiation, both at large scale between the Red Sea and the Western Indian Ocean (WIO), and at smaller scale along the coast of East Africa. As such high levels of differentiation might indicate the presence of more than one species, a haploweb analysis was conducted with the nuclear marker ITS2, confirming that the Red Sea populations are genetically distinct from the WIO ones. Based on microsatellite analyses three groups could be distinguished within the WIO: (1) northern Madagascar, (2) south-west Madagascar together with one site in northern Mozambique (Nacala) and (3) all other sites in northern Mozambique, Tanzania and Kenya. These patterns of restricted connectivity could be explained by the short pelagic larval duration of S. hystrix, and/or by oceanographic factors, such as eddies in the Mozambique Channel (causing larval retention in northern Madagascar but facilitating dispersal from northern Mozambique towards south-west Madagascar). This study provides an additional line of evidence supporting the conservation priority status of the Northern Mozambique Channel and should inform coral reef management decisions in the region.

Toward perfect reads: self-correction of short reads via mapping on de Bruijn graphs.

MOTIVATION: Short-read accuracy is important for downstream analyses such as genome assembly and hybrid long-read correction. Despite much work on short-read correction, present-day correctors either do not scale well on large datasets or consider reads as mere suites of k-mers, without taking into account their full-length sequence information. RESULTS: We propose a new method to correct short reads using de Bruijn graphs and implement it as a tool called Bcool. As a first step, Bcool constructs a compacted de Bruijn graph from the reads. This graph is filtered on the basis of k-mer abundance then of unitig abundance, thereby removing most sequencing errors. The cleaned graph is then used as a reference on which the reads are mapped to correct them. We show that this approach yields more accurate reads than k-mer-spectrum correctors while being scalable to human-size genomic datasets and beyond. AVAILABILITY AND IMPLEMENTATION: The implementation is open source, available at http://github.com/Malfoy/BCOOL under the Affero GPL license and as a Bioconda package. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Computational Characterization of the mtORF of Pocilloporid Corals: Insights into Protein Structure and Function in Stylophora Lineages from Contrasting Environments.

More than a decade ago, a new mitochondrial Open Reading Frame (mtORF) was discovered in corals of the family Pocilloporidae and has been used since then as an effective barcode for these corals. Recently, mtORF sequencing revealed the existence of two differentiated Stylophora lineages occurring in sympatry along the environmental gradient of the Red Sea (18.5°C to 33.9°C). In the endemic Red Sea lineage RS_LinB, the mtORF and the heat shock protein gene hsp70 uncovered similar phylogeographic patterns strongly correlated with environmental variations. This suggests that the mtORF too might be involved in thermal adaptation. Here, we used computational analyses to explore the features and putative function of this mtORF. In particular, we tested the likelihood that this gene encodes a functional protein and whether it may play a role in adaptation. Analyses of full mitogenomes showed that the mtORF originated in the common ancestor of Madracis and other pocilloporids, and that it encodes a transmembrane protein differing in length and domain architecture among genera. Homology-based annotation and the relative conservation of metal-binding sites revealed traces of an ancient hydrolase catalytic activity. Furthermore, signals of pervasive purifying selection, lack of stop codons in 1830 sequences analyzed, and a codon-usage bias similar to that of other mitochondrial genes indicate that the protein is functional, i.e., not a pseudogene. Other features, such as intrinsically disordered regions, tandem repeats, and signals of positive selection particularly in StylophoraRS_LinB populations, are consistent with a role of the mtORF in adaptive responses to environmental changes.

The hitchhiker's guide to single-locus species delimitation.

Molecular approaches to species delimitation are increasingly used to ascertain the number of species in a sample prior to taxonomic, ecological or physiological studies. Although multilocus approaches are gaining fast in popularity, single-gene methods still predominate in the literature. However, available simulation benchmarks of these methods focus exclusively on species-poor samples and/or tree-based approaches: as a result, travellers in the land of single-locus species delimitation lack a comprehensive "hitchhiker's guide" highlighting the sweet spots and dangers on their road. To fill this gap, we compared the performances of distance-based (ABGD, "automatic barcode gap discovery"), allele sharing-based (haplowebs) and tree-based approaches (GMYC, "generalized mixed Yule-coalescent" and PTP, "Poisson tree processes") to detect interspecific boundaries in samples of 6, 60 and 120 simulated species with various speciation rates, effective population sizes, mutation rates and sampling patterns. We found that all approaches performed poorly when population sizes and speciation rates were large, with haplowebs yielding best results followed by ABGD then tree-based approaches. The latter's error type was mostly oversplitting, whereas ABGD chiefly overlumped and haplowebs leaned either way depending on simulation parameters: such widely divergent error patterns suggest that, if all three types of methods agree, then the resulting delimitation is probably correct. Perfect congruence being quite rare, travellers in search of a one-size-fit-all approach to single-locus species delimitation should forget it; however, our hitchhiker's guide raises hope that such species delimitation's Holy Grail may be found in the relatively uncharted nearby land of multilocus species delimitation.

Quantification of complex modular architecture in plants.

Morphometrics, the assignment of quantities to biological shapes, is a powerful tool to address taxonomic, evolutionary, functional and developmental questions. We propose a novel method for shape quantification of complex modular architecture in thalloid plants, whose extremely reduced morphologies, combined with the lack of a formal framework for thallus description, have long rendered taxonomic and evolutionary studies extremely challenging. Using graph theory, thalli are described as hierarchical series of nodes and edges, allowing for accurate, homologous and repeatable measurements of widths, lengths and angles. The computer program MorphoSnake was developed to extract the skeleton and contours of a thallus and automatically acquire, at each level of organization, width, length, angle and sinuosity measurements. Through the quantification of leaf architecture in Hymenophyllum ferns (Polypodiopsida) and a fully worked example of integrative taxonomy in the taxonomically challenging thalloid liverwort genus Riccardia, we show that MorphoSnake is applicable to all ramified plants. This new possibility of acquiring large numbers of quantitative traits in plants with complex modular architectures opens new perspectives of applications, from the development of rapid species identification tools to evolutionary analyses of adaptive plasticity.

Comparative analysis of the genomes of Stylophora pistillata and Acropora digitifera provides evidence for extensive differences between species of corals.

Stony corals form the foundation of coral reef ecosystems. Their phylogeny is characterized by a deep evolutionary divergence that separates corals into a robust and complex clade dating back to at least 245 mya. However, the genomic consequences and clade-specific evolution remain unexplored. In this study we have produced the genome of a robust coral, Stylophora pistillata, and compared it to the available genome of a complex coral, Acropora digitifera. We conducted a fine-scale gene-based analysis focusing on ortholog groups. Among the core set of conserved proteins, we found an emphasis on processes related to the cnidarian-dinoflagellate symbiosis. Genes associated with the algal symbiosis were also independently expanded in both species, but both corals diverged on the identity of ortholog groups expanded, and we found uneven expansions in genes associated with innate immunity and stress response. Our analyses demonstrate that coral genomes can be surprisingly disparate. Future analyses incorporating more genomic data should be able to determine whether the patterns elucidated here are not only characteristic of the differences between S. pistillata and A. digitifera but also representative of corals from the robust and complex clade at large.

A genomic glance through the fog of plasticity and diversification in Pocillopora.

Scleractinian corals of the genus Pocillopora (Lamarck, 1816) are notoriously difficult to identify morphologically with considerable debate on the degree to which phenotypic plasticity, introgressive hybridization and incomplete lineage sorting obscure well-defined taxonomic lineages. Here, we used RAD-seq to resolve the phylogenetic relationships among seven species of Pocillopora represented by 15 coral holobiont metagenomic libraries. We found strong concordance between the coral holobiont datasets, reads that mapped to the Pocillopora damicornis (Linnaeus, 1758) transcriptome, nearly complete mitochondrial genomes, 430 unlinked high-quality SNPs shared across all Pocillopora taxa, and a conspecificity matrix of the holobiont dataset. These datasets also show strong concordance with previously published clustering of the mitochondrial clades based on the mtDNA open reading frame (ORF). We resolve seven clear monophyletic groups, with no evidence for introgressive hybridization among any but the most recently derived sister species. In contrast, ribosomal and histone datasets, which are most commonly used in coral phylogenies to date, were less informative and contradictory to these other datasets. These data indicate that extant Pocillopora species diversified from a common ancestral lineage within the last ~3 million years. Key to this evolutionary success story may be the high phenotypic plasticity exhibited by Pocillopora species.