A multi-tissue de novo transcriptome assembly and relative gene expression of the vulnerable freshwater salmonid Thymallus ligericus.

Freshwater ecosystems are among the most endangered ecosystems worldwide. While numerous taxa are on the verge of extinction as a result of global changes and direct or indirect anthropogenic activity, genomic and transcriptomic resources represent a key tool for comprehending species' adaptability and serve as the foundation for conservation initiatives. The Loire grayling, Thymallus ligericus, is a freshwater European salmonid endemic to the upper Loire River basin. The species is comprised of fragmented populations that are dispersed over a small area and it has been identified as a vulnerable species. Here, we provide a multi-tissue de novo transcriptome assembly of T. ligericus. The completeness and integrity of the transcriptome were assessed before and after redundancy removal with lineage-specific libraries from Eukaryota, Metazoa, Vertebrata, and Actinopterygii. Relative gene expression was assessed for each of the analyzed tissues, using the de novo assembled transcriptome and a genome-based analysis using the available T. thymallus genome as a reference. The final assembly, with a contig N50 of 1221 and Benchmarking Universal Single-Copy Orthologs (BUSCO) scores above 94%, is made accessible along with structural and functional annotations and relative gene expression of the five tissues (NCBI SRA and FigShare databases). This is the first transcriptomic resource for this species, which provides a foundation for future research on this and other salmonid species that are increasingly exposed to environmental stressors.

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.

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.

Complete Inactivation of Sebum-Producing Genes Parallels the Loss of Sebaceous Glands in Cetacea.

Genomes are dynamic biological units, with processes of gene duplication and loss triggering evolutionary novelty. The mammalian skin provides a remarkable case study on the occurrence of adaptive morphological innovations. Skin sebaceous glands (SGs), for instance, emerged in the ancestor of mammals serving pivotal roles, such as lubrication, waterproofing, immunity, and thermoregulation, through the secretion of sebum, a complex mixture of various neutral lipids such as triacylglycerol, free fatty acids, wax esters, cholesterol, and squalene. Remarkably, SGs are absent in a few mammalian lineages, including the iconic Cetacea. We investigated the evolution of the key molecular components responsible for skin sebum production: Dgat2l6, Awat1, Awat2, Elovl3, Mogat3, and Fabp9. We show that all analyzed genes have been rendered nonfunctional in Cetacea species (toothed and baleen whales). Transcriptomic analysis, including a novel skin transcriptome from blue whale, supports gene inactivation. The conserved mutational pattern found in most analyzed genes, indicates that pseudogenization events took place prior to the diversification of modern Cetacea lineages. Genome and skin transcriptome analysis of the common hippopotamus highlighted the convergent loss of a subset of sebum-producing genes, notably Awat1 and Mogat3. Partial loss profiles were also detected in non-Cetacea aquatic mammals, such as the Florida manatee, and in terrestrial mammals displaying specialized skin phenotypes such as the African elephant, white rhinoceros and pig. Our findings reveal a unique landscape of "gene vestiges" in the Cetacea sebum-producing compartment, with limited gene loss observed in other mammalian lineages: suggestive of specific adaptations or specializations of skin lipids.

Cartilaginous fishes offer unique insights into the evolution of the nuclear receptor gene repertoire in gnathostomes.

Nuclear receptors (NRs) are key transcription factors that originated in the common ancestor of metazoans. The vast majority of NRs are triggered by binding to either endogenous (e.g. retinoic acid) or exogenous (e.g. xenobiotics) ligands, and their evolution and expansion is tightly linked to the function of endocrine systems. Importantly, they represent classic targets of physiological exploitation by endocrine disrupting chemicals. The NR gene repertoire in different lineages has been shaped by gene loss, duplication and mutation, denoting a dynamic evolutionary route. As the earliest diverging class of gnathostomes (jawed vertebrates), cartilaginous fishes offer an exceptional opportunity to address the early diversification of NR gene families and the evolution of the endocrine system in jawed vertebrates. Here we provide an exhaustive analysis into the NR gene composition in five elasmobranch (sharks and rays) and two holocephalan (chimaeras) species. For this purpose, we generated also a low coverage draft genome assembly of the chimaera small-eyed rabbitfish, Hydrolagus affinis. We show that cartilaginous fish retain an archetypal NR gene repertoire, similar to that of mammals and coincident with the two rounds of whole genome duplication that occurred in the gnathostome ancestor. Furthermore, novel gene members of the non-canonical NR0B receptors were found in the genomes of this lineage. Our findings provide an essential view into the early diversification of NRs in gnathostomes, paving the way for functional studies.

The Quantitative Proteome of the Cement and Adhesive Gland of the Pedunculate Barnacle, Pollicipes pollicipes.

Adhesive secretion has a fundamental role in barnacles' survival, keeping them in an adequate position on the substrate under a variety of hydrologic regimes. It arouses special interest for industrial applications, such as antifouling strategies, underwater industrial and surgical glues, and dental composites. This study was focused on the goose barnacle Pollicipes pollicipes adhesion system, a species that lives in the Eastern Atlantic strongly exposed intertidal rocky shores and cliffs. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using a label-free LC-MS high-throughput proteomic analysis, searched against a custom transcriptome-derived database. Overall, 11,755 peptide sequences were identified in the gland while 2880 peptide sequences were detected in the cement, clustered in 1616 and 1568 protein groups, respectively. The gland proteome was dominated by proteins of the muscle, cytoskeleton, and some uncharacterized proteins, while the cement was, for the first time, reported to be composed by nearly 50% of proteins that are not canonical cement proteins, mainly unannotated proteins, chemical cues, and protease inhibitors, among others. Bulk adhesive proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Some unannotated proteins highly expressed in the proteomes, as well as at the transcriptomic level, showed similar physicochemical properties to the known surface-coupling barnacle adhesive proteins while the function of the others remains to be discovered. New quantitative and qualitative clues are provided to understand the diversity and function of proteins in the cement of stalked barnacles, contributing to the whole adhesion model in Cirripedia.

Convergent inactivation of the skin-specific C-C motif chemokine ligand 27 in mammalian evolution.

The appearance of mammalian-specific skin features was a key evolutionary event contributing for the elaboration of physiological processes such as thermoregulation, adequate hydration, locomotion, and inflammation. Skin inflammatory and autoimmune processes engage a population of skin-infiltrating T cells expressing a specific C-C chemokine receptor (CCR10) which interacts with an epidermal CC chemokine, the skin-specific C-C motif chemokine ligand 27 (CCL27). CCL27 is selectively produced in the skin by keratinocytes, particularly upon inflammation, mediating the adhesion and homing of skin-infiltrating T cells. Here, we examined the evolution and coding condition of Ccl27 in 112 placental mammalian species. Our findings reveal that a number of open reading frame inactivation events such as insertions, deletions, and start and stop codon mutations independently occurred in Cetacea, Pholidota, Sirenia, Chiroptera, and Rodentia, totalizing 18 species. The diverse habitat settings and lifestyles of Ccl27-eroded lineages probably implied distinct evolutionary triggers rendering this gene unessential. For example, in Cetacea, the rapid renewal of skin layers minimizes the need for an elaborate inflammatory mechanism, mirrored by the absence of epidermal scabs. Our findings suggest that the convergent and independent loss of Ccl27 in mammalian evolution concurred with unique adaptive roads for skin physiology.

Evolutionary Plasticity in Detoxification Gene Modules: The Preservation and Loss of the Pregnane X Receptor in Chondrichthyes Lineages.

To appraise how evolutionary processes, such as gene duplication and loss, influence an organism's xenobiotic sensitivity is a critical question in toxicology. Of particular importance are gene families involved in the mediation of detoxification responses, such as members of the nuclear receptor subfamily 1 group I (NR1I), the pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). While documented in multiple vertebrate genomes, PXR and CAR display an intriguing gene distribution. PXR is absent in birds and reptiles, while CAR shows a tetrapod-specific occurrence. More elusive is the presence of PXR and CAR gene orthologs in early branching and ecologically-important Chondrichthyes (chimaeras, sharks and rays). Therefore, we investigated various genome projects and use them to provide the first identification and functional characterization of a Chondrichthyan PXR from the chimaera elephant shark (Callorhinchus milii, Holocephali). Additionally, we substantiate the targeted PXR gene loss in Elasmobranchii (sharks and rays). Compared to other vertebrate groups, the chimaera PXR ortholog displays a diverse expression pattern (skin and gills) and a unique activation profile by classical xenobiotic ligands. Our findings provide insights into the molecular landscape of detoxification mechanisms and suggest lineage-specific adaptations in response to xenobiotics in gnathostome evolution.

Cetacea are natural knockouts for IL20.

The Cetacea infraorder comprises a very unique group within the mammalian lineage. While sharing common ancestors with terrestrial mammals, their exclusive dependence on aquatic environments makes them attractive models to explore the landscape of molecular shifts in radical habitat transitions. Among their diverse anatomical and physiological solutions, we find detectable genetic remodeling of the immune system. In agreement, here we show that the gene sequence of interleukin-20 (IL20) displays unambiguous signs of inactivation with several disruptive mutations, including stop codons, insertions, and a conserved trans-species mutation abolishing a canonical splice site, in nine analyzed cetacean genomes. Considering the suggested role of IL20 in skin immunity processes, including inflammation, epithelization, and remodeling, we propose that gene inactivation follows specific adaptations of cetacean skin to the aquatic environment, in frame with the less-is-more hypothesis.

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.

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

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

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.

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.

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.

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.

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 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.

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.

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.