A whole-body transcriptome assembly of the annelid worm Hediste diversicolor.

The Annelida phylum is composed of a myriad of species exhibiting key phenotypic adaptations. They occupy key ecological niches in a variety of marine, freshwater and terrestrial ecosystems. Importantly, the increment of omic resources is rapidly modifying the taxonomic landscape and knowledge of species belonging to this phylum. Here, we comprehensively characterised and annotated a transcriptome of the common ragworm, Hediste diversicolor (OF Müller). This species belongs to the family Nereididae and inhabits estuarine and lagoon areas on the Atlantic coasts of Europe and North America. Ecologically, H. diversicolor plays an important role in benthic food webs. Given its commercial value, H. diversicolor is a promising candidate for aquaculture development and production in farming facilities, under a circular economy framework. We used Illumina next-generation sequencing technology, to produce a total of 105 million (M) paired-end (PE) raw reads and generate the first whole-body transcriptome assembly of H. diversicolor species. This high-quality transcriptome contains 69,335 transcripts with an N50 transcript length of 2313 bp and achieved a BUSCO gene completeness of 97.7% and 96% in Eukaryota and Metazoa lineage-specific profile libraries. Our findings offer a valuable resource for multiple biological applications using this species.

Population Genomics Reveals the Underlying Structure of the Small Pelagic European Sardine and Suggests Low Connectivity within Macaronesia.

The European sardine (Sardina pilchardus, Walbaum 1792) is indisputably a commercially important species. Previous studies using uneven sampling or a limited number of makers have presented sometimes conflicting evidence of the genetic structure of S. pilchardus populations. Here, we show that whole genome data from 108 individuals from 16 sampling areas across 5000 km of the species' distribution range (from the Eastern Mediterranean to the archipelago of Azores) support at least three genetic clusters. One includes individuals from Azores and Madeira, with evidence of substructure separating these two archipelagos in the Atlantic. Another cluster broadly corresponds to the center of the distribution, including the sampling sites around Iberia, separated by the Almeria-Oran front from the third cluster that includes all of the Mediterranean samples, except those from the Alboran Sea. Individuals from the Canary Islands appear to belong to the Mediterranean cluster. This suggests at least two important geographical barriers to gene flow, even though these do not seem complete, with many individuals from around Iberia and the Mediterranean showing some patterns compatible with admixture with other genetic clusters. Genomic regions corresponding to the top outliers of genetic differentiation are located in areas of low recombination indicative that genetic architecture also has a role in shaping population structure. These regions include genes related to otolith formation, a calcium carbonate structure in the inner ear previously used to distinguish S. pilchardus populations. Our results provide a baseline for further characterization of physical and genetic barriers that divide European sardine populations, and information for transnational stock management of this highly exploited species towards sustainable fisheries.

Mitochondrial replication's role in vertebrate mtDNA strand asymmetry.

Mitogenomes are defined as compact and structurally stable over aeons. This perception results from a vertebrate-centric vision, where few types of mtDNA rearrangements are described. Here, we bring a new light to the involvement of mitochondrial replication in the strand asymmetry of the vertebrate mtDNA. Using several species of deep-sea hatchetfish (Sternoptychidae) displaying distinct mtDNA structural arrangements, we unravel the inversion of the coding direction of protein-coding genes (PCGs). This unexpected change is coupled with a strand asymmetry nucleotide composition reversal and is shown to be directly related to the strand location of the Control Region (CR). An analysis of the fourfold redundant sites of the PCGs (greater than 6000 vertebrates), revealed the rarity of this phenomenon, found in nine fish species (five deep-sea hatchetfish). Curiously, in Antarctic notothenioid fishes (Trematominae), where a single PCG inversion (the only other record in fish) is coupled with the inversion of the CR, the standard asymmetry is disrupted for the remaining PCGs but not yet reversed, suggesting a transitory state. Our results hint that a relaxation of the classic vertebrate mitochondrial structural stasis promotes disruption of the natural balance of asymmetry of the mtDNA. These findings support the long-lasting hypothesis that replication is the main molecular mechanism promoting the strand-specific compositional bias of this unique and indispensable molecule.

Extensive gene loss parallels kidney aglomerulism in Syngnathidae.

The eccentric seahorses, seadragons, pipehorses and pipefishes (Syngnathidae) have an aglomerular kidney1. Here, we show that nephron genes2 conserved in Bilateria are secondarily eroded/deleted in Syngnathidae genomes. A transcriptome enrichment analysis suggests the predominance of excretion processes in the Syngnathidae kidney. In a lineage where crypsis and idleness are tightly associated, we propose that aglomerulism evolved as an energy-saving strategy.

A PacBio Hi-Fi Genome Assembly of the Painter's Mussel Unio pictorum (Linnaeus, 1758).

The highly diverse group of freshwater mussels from order Unionida is found in the world's freshwater systems due to several fascinating evolutionary adaptations, including "parental care," and most notably, an obligatory parasitic phase in their early life cycle, called glochidia, which infests and uses fish for nutrition and dispersal. Freshwater mussels play essential ecological roles in freshwater habitats, including water filtration, sediment bioturbation, and nutrient cycling. However, these species are also highly threatened, being one of the faunal groups with the highest recorded extinction rate in the wild. Genomics methods have an incredible potential to promote biodiversity conservation, allowing the characterization of population health, identification of adaptive genetic elements, delineation of conservation units, and providing a framework for predictive assessments of the impact of anthropogenic threats and climate change. Unfortunately, only six freshwater mussel species have had their whole genomes sequenced to date, and only two of these are European species. Here, we present the first genome assembly of the Painter's Mussel, Unio pictorum (Linnaeus, 1758), the type species representative of the order and the most widespread species of the genus in Europe. We used long-read PacBio Hi-Fi sequencing reads to produce a highly contiguous assembly that will pave the way for the study of European freshwater mussels in the Genome Era.

Corrigendum: Uncovering a 500 million year old history and evidence of pseudogenization for TLR15.

[This corrects the article DOI: 10.3389/fimmu.2022.1020601.].

The Crown Pearl V2: an improved genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758).

Contiguous assemblies are fundamental to deciphering the composition of extant genomes. In molluscs, this is considerably challenging owing to the large size of their genomes, heterozygosity, and widespread repetitive content. Consequently, long-read sequencing technologies are fundamental for high contiguity and quality. The first genome assembly of Margaritifera margaritifera (Linnaeus, 1758) (Mollusca: Bivalvia: Unionida), a culturally relevant, widespread, and highly threatened species of freshwater mussels, was recently generated. However, the resulting genome is highly fragmented since the assembly relied on short-read approaches. Here, an improved reference genome assembly was generated using a combination of PacBio CLR long reads and Illumina paired-end short reads. This genome assembly is 2.4 Gb long, organized into 1,700 scaffolds with a contig N50 length of 3.4 Mbp. The ab initio gene prediction resulted in 48,314 protein-coding genes. Our new assembly is a substantial improvement and an essential resource for studying this species' unique biological and evolutionary features, helping promote its conservation.

A novel assembly pipeline and functional annotations for targeted sequencing: A case study on the globally threatened Margaritiferidae (Bivalvia: Unionida).

The proliferation of genomic sequencing approaches has significantly impacted the field of phylogenetics. Target capture approaches provide a cost-effective, fast and easily applied strategy for phylogenetic inference of non-model organisms. However, several existing target capture processing pipelines are incapable of incorporating whole genome sequencing (WGS). Here, we develop a new pipeline for capture and de novo assembly of the targeted regions using whole genome re-sequencing reads. This new pipeline captured targeted loci accurately, and given its unbiased nature, can be used with any target capture probe set. Moreover, due to its low computational demand, this new pipeline may be ideal for users with limited resources and when high-coverage sequencing outputs are required. We demonstrate the utility of our approach by incorporating WGS data into the first comprehensive phylogenomic reconstruction of the freshwater mussel family Margaritiferidae. We also provide a catalogue of well-curated functional annotations of these previously uncharacterized freshwater mussel-specific target regions, representing a complementary tool for scrutinizing phylogenetic inferences while expanding future applications of the probe set.

The gill transcriptome of threatened European freshwater mussels.

Genomic tools applied to non-model organisms are critical to design successful conservation strategies of particularly threatened groups. Freshwater mussels of the Unionida order are among the most vulnerable taxa and yet almost no genetic resources are available. Here, we present the gill transcriptomes of five European freshwater mussels with high conservation concern: Margaritifera margaritifera, Unio crassus, Unio pictorum, Unio mancus and Unio delphinus. The final assemblies, with N50 values ranging from 1069-1895 bp and total BUSCO scores above 90% (Eukaryote and Metazoan databases), were structurally and functionally annotated, and made available. The transcriptomes here produced represent a valuable resource for future studies on these species' biology and ultimately guide their conservation.

The repertoire of the elongation of very long-chain fatty acids (Elovl) protein family is conserved in tambaqui (Colossoma macropomum): Gene expression profiles offer insights into the sexual differentiation process.

Elongation of very long-chain fatty acids (Elovl) proteins are critical players in the regulation of the length of a fatty acid. At present, eight members of the Elovl family (Elovl1-8), displaying a characteristic fatty acid substrate specificity, have been identified in vertebrates, including teleost fish. In general, Elovl1, Elovl3, Elovl6 and Elovl7 exhibit a substrate preference for saturated and monounsaturated fatty acids, while Elovl2, Elovl4, Elovl5 and Elovl8 use polyunsaturated fatty acids (PUFA) as substrates. PUFA elongases have received considerable attention in aquatic animals due to their involvement in the conversion of C18 PUFAs to long-chain polyunsaturated fatty acids (LC-PUFA). Here, we identified the full repertoire of elovl genes in the tambaqui Colossoma macropomum genome. A detailed phylogenetic and synteny analysis suggests a conservation of these genes among teleosts. Furthermore, based on RNAseq gene expression data, we discovered a gender bias expression of elovl genes during sex differentiation of tambaqui, toward future males. Our findings suggest a role of Elovl enzymes and fatty acid metabolism in tambaqui sexual differentiation.

A mitochondrial genome assembly of the opal chimaera, Chimaera opalescens Luchetti, Iglésias et Sellos 2011, using PacBio HiFi long reads.

Chondrichthyans (sharks, rays and chimeras) are a fascinating and highly vulnerable group of early branching gnathostomes. However, they remain comparatively poorly sampled from the point of view of molecular resources, with deep water taxa being particularly data deficient. The development of long-read sequencing technologies enables the analysis of phylogenetic relationships through a precise and reliable assembly of complete mtDNA genomes. The sequencing and characterization of the complete mitogenome of the opal chimera Chimera opalescens Luchetti, Iglésias et Sellos 2011, using the long-read technique PacBio HiFi is presented. The entire mitogenome was 23,411 bp long and shows the same overall content, i.e. 13 protein-coding genes, 22 transfer RNA and 2 ribosomal RNA genes, as all other examined Chondrichthyan mitogenomes. Phylogenetic reconstructions using all available Chondrichthyan mitogenomes, including 11 Holocephali (chimeras and ratfishes), places C. opalescens within the Chimaeridae family. Furthermore, the results reinforce previous findings, showing the genus Chimera as paraphyletic and thus highlighting the need to expand molecular approaches in this group of cartilaginous fishes.

The Preservation of PPARγ Genome Duplicates in Some Teleost Lineages: Insights into Lipid Metabolism and Xenobiotic Exploitation.

Three peroxisome proliferator-activated receptor paralogues (PPARα, -ß and -γ) are currently recognized in vertebrate genomes. PPARγ is known to modulate nutrition, adipogenesis and immunity in vertebrates. Natural ligands of PPARγ have been proposed; however, the receptor also binds synthetic ligands such as endocrine disruptors. Two paralogues of PPARα and PPARß have been documented in teleost species, a consequence of the 3R WGD. Recently, two PPARγ paralogue genes were also identified in Astyanax mexicanus. We aimed to determine whether the presence of two PPARγ paralogues is prevalent in other teleost genomes, through genomic and phylogenetic analysis. Our results showed that besides Characiformes, two PPARγ paralogous genes were also identified in other teleost taxa, coinciding with the teleost-specific, whole-genome duplication and with the retention of both genes prior to the separation of the Clupeocephala. To functionally characterize these genes, we used the European sardine (Sardina pilchardus) as a model. PPARγA and PPARγB display a different tissue distribution, despite the similarity of their functional profiles: they are unresponsive to tested fatty acids and other human PPARγ ligands yet yield a transcriptional response in the presence of tributyltin (TBT). This observation puts forward the relevance of comparative analysis to decipher alternative binding architectures and broadens the disruptive potential of man-made chemicals for aquatic species.

Uncovering a 500 million year old history and evidence of pseudogenization for TLR15.

Introduction: Toll like receptors (TLRs) are at the front line of pathogen recognition and host immune response. Many TLR genes have been described to date with some being found across metazoans while others are restricted to specific lineages. A cryptic member of the TLR gene family, TLR15, has a unique phylogenetic distribution. Initially described in extant species of birds and reptiles, an ortholog has been reported for cartilaginous fish. Methods: Here, we significantly expanded the evolutionary analysis of TLR15 gene evolution, taking advantage of large genomic and transcriptomic resources available from different lineages of vertebrates. Additionally, we objectively search for TLR15 in lobe-finned and ray-finned fish, as well as in cartilaginous fish and jawless vertebrates. Results and discussion: We confirm the presence of TLR15 in early branching jawed vertebrates - the cartilaginous fish, as well as in basal Sarcopterygii - in lungfish. However, within cartilaginous fish, the gene is present in Holocephalans (all three families) but not in Elasmobranchs (its sister-lineage). Holocephalans have long TLR15 protein sequences that disrupt the typical TLR structure, and some species display a pseudogene sequence due to the presence of frameshift mutations and early stop codons. Additionally, TLR15 has low expression levels in holocephalans when compared with other TLR genes. In turn, lungfish also have long TLR15 protein sequences but the protein structure is not compromised. Finally, TLR15 presents several sites under negative selection. Overall, these results suggest that TLR15 is an ancient TLR gene and is experiencing ongoing pseudogenization in early-branching vertebrates.

A genome assembly of the Atlantic chub mackerel (Scomber colias): a valuable teleost fishing resource.

The Atlantic chub mackerel, Scomber colias (Gmelin, 1789), is a medium-sized pelagic fish with substantial importance in the fisheries of the Atlantic Ocean and the Mediterranean Sea. Over the past decade, this species has gained special relevance, being one of the main targets of pelagic fisheries in the NE Atlantic. Here, we sequenced and annotated the first high-quality draft genome assembly of S. colias, produced with PacBio HiFi long reads and Illumina paired-end short reads. The estimated genome size is 814 Mbp, distributed into 2,028 scaffolds and 2,093 contigs with an N50 length of 4.19 and 3.34 Mbp, respectively. We annotated 27,675 protein-coding genes and the BUSCO analyses indicated high completeness, with 97.3% of the single-copy orthologs in the Actinopterygii library profile. The present genome assembly represents a valuable resource to address the biology and management of this relevant fishery. Finally, this genome assembly ranks fourth in high-quality genome assemblies within the order Scombriformes and first in the genus Scomber.

A Highly Complex, MHC-Linked, 350 Million-Year-Old Shark Nonclassical Class I Lineage.

Cartilaginous fish, or Chondrichthyes, are the oldest extant vertebrates to possess the MHC and the Ig superfamily-based Ag receptors, the defining genes of the gnathostome adaptive immune system. In this work, we have identified a novel MHC lineage, UEA, a complex multigene nonclassical class I family found in sharks (division Selachii) but not detected in chimaeras (subclass Holocephali) or rays (division Batoidea). This new lineage is distantly related to the previously reported nonclassical class I lineage UCA, which appears to be present only in dogfish sharks (order Squaliformes). UEA lacks conservation of the nine invariant residues in the peptide (ligand)-binding regions (PBR) that bind to the N and C termini of bound peptide in most vertebrate classical class I proteins, which are replaced by relatively hydrophobic residues compared with the classical UAA. In fact, UEA and UCA proteins have the most hydrophobic-predicted PBR of all identified chondrichthyan class I molecules. UEA genes detected in the whale shark and bamboo shark genome projects are MHC linked. Consistent with UEA comprising a very large gene family, we detected weak expression in different tissues of the nurse shark via Northern blotting and RNA sequencing. UEA genes fall into three sublineages with unique characteristics in the PBR. UEA shares structural and genetic features with certain nonclassical class I genes in other vertebrates, such as the highly complex XNC nonclassical class I genes in Xenopus, and we anticipate that each shark gene, or at least each sublineage, will have a unique function, perhaps in bacterial defense.

A drastic shift in the energetic landscape of toothed whale sperm cells.

Mammalian spermatozoa are a notable example of metabolic compartmentalization.1 Energy in the form of ATP production, vital for motility, capacitation, and fertilization, is subcellularly separated in sperm cells. While glycolysis provides a local, rapid, and low-yielding input of ATP along the flagellum fibrous sheath, oxidative phosphorylation (OXPHOS), far more efficient over a longer time frame, is concentrated in the midpiece mitochondria.2 The relative weight of glycolysis and OXPHOS pathways in sperm function is variable among species and sensitive to oxygen and substrate availability.3-5 Besides partitioning energy production, sperm cell energetics display an additional singularity: the occurrence of sperm-specific gene duplicates and alternative spliced variants, with conserved function but structurally bound to the flagellar fibrous sheath.6,7 The wider selective forces driving the compartmentalization and adaptability of this energy system in mammalian species remain largely unknown, much like the impact of ecosystem resource availability (e.g., carbohydrates, fatty acids, and proteins) and dietary adaptations in reproductive physiology traits.8 Here, we investigated the Cetacea, an iconic group of fully aquatic and carnivorous marine mammals, evolutionarily related to extant terrestrial herbivores.9 In this lineage, episodes of profound trait remodeling have been accompanied by clear genomic signatures.10-14 We show that toothed whales exhibit impaired sperm glycolysis, due to gene and exon erosion, and demonstrate that dolphin spermatozoa motility depends on endogenous fatty acid ß-oxidation, but not carbohydrates. Such unique energetic rewiring substantiates the observation of large mitochondria in toothed whale spermatozoa and emphasizes the radical physiological reorganization imposed by the transition to a carbohydrate-depleted marine environment.

The complete mitochondrial genome of the endemic Iberian pygmy skate Neoraja iberica Stehmann, Séret, Costa, & Baro 2008 (Elasmobranchii, Rajidae).

Skates, Chondrichthyes fishes from order Rajiformes, are the most species-rich group of all Batoidea. However, their phylogenetic relationships and systematics is still a highly discussed and controversial subject. The use of complete mitogenome has shown to be a promising tool to fill this gap of knowledge. Here, the complete mitogenome of the Iberian pygmy skate Neoraja iberica (Stehmann, Séret, Costa & Baro 2008) was sequenced and assembled. The mitogenome is 16,723 bp long and its gene content (i.e. 13 protein-coding genes, 22 transfer RNA, and 2 ribosomal RNA genes) and arrangement are the expected for Batoidea. Phylogenetic reconstructions, including 89 Rajiformes and two outgroup Rhinopristiformes, recovered family Rajidae as monophyletic, and further divided in the monophyletic tribe Rajini, sister to tribes Amblyrajini and Rostrorajini. The newly sequenced N. iberica mitogenome is the first representative of the tribe Rostrorajini.

Proteogenomic Characterization of the Cement and Adhesive Gland of the Pelagic Gooseneck Barnacle Lepas anatifera.

We focus on the stalked goose barnacle L. anatifera adhesive system, an opportunistic less selective species for the substrate, found attached to a variety of floating objects at seas. Adhesion is an adaptative character in barnacles, ensuring adequate positioning in the habitat for feeding and reproduction. The protein composition of the cement multicomplex and adhesive gland was quantitatively studied using shotgun proteomic analysis. Overall, 11,795 peptide sequences were identified in the gland and 2206 in the cement, clustered in 1689 and 217 proteinGroups, respectively. Cement specific adhesive proteins (CPs), proteases, protease inhibitors, cuticular and structural proteins, chemical cues, and many unannotated proteins were found, among others. In the cement, CPs were the most abundant (80.5%), being the bulk proteins CP100k and -52k the most expressed of all, and CP43k-like the most expressed interfacial protein. Unannotated proteins comprised 4.7% of the cement proteome, ranking several of them among the most highly expressed. Eight of these proteins showed similar physicochemical properties and amino acid composition to known CPs and classified through Principal Components Analysis (PCA) as new CPs. The importance of PCA on the identification of unannotated non-conserved adhesive proteins, whose selective pressure is on their relative amino acid abundance, was demonstrated.

The Crown Pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758).

Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species' unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.

Shedding light on the Chimaeridae taxonomy: the complete mitochondrial genome of the cartilaginous fish Hydrolagus mirabilis (Collett, 1904) (Holocephali: Chimaeridae).

Cartilaginous fish are fascinating taxa, present in the folklore and art of many different cultures. Moreover, they display several unique anatomical, physiological, molecular, and behavioral characteristics making them extremely interesting from a biological perspective. Nevertheless, some crucial knowledge gaps remain, including phylogenetic relationships among extant species. Here, we produced the complete mitogenome sequence of the large-eyed rabbitfish, Hydrolagus mirabilis (Chimaeriformes). The complete mitogenome is 19,435 bp long and shows the same overall content, i.e. 13 protein-coding genes, 22 transfer RNA, and two ribosomal RNA genes, as all other examined Chondrichthyan mitogenomes. Phylogenetic reconstructions including 12 Holocephalan and three outgroup Elasmobranch mitogenomes place the H. mirabilis within the family Chimaeridae but revealed paraphyletic Hydrolagus and Chimaera, in line with a previous study, highlighting the importance for collecting additional molecular data to improve phylogenetic reconstruction in this group of vertebrates.