The transcriptome assembly of the European freshwater mussel Unio elongatulus C. Pfeiffer, 1825.

Freshwater mussels of the order Unionida are a global conservation concern. Species of this group are strictly freshwater, sessile, slow-growing animals and, extremely sensitive to environmental changes. Human-mediated changes in freshwater habitats are imposing enormous pressure on the survival of freshwater mussels. Although a few flagship species are protected in Europe, other highly imperilled species receive much less attention. Moreover, knowledge about biology, ecology, and evolution and proper conservation assessments of many European species are still sparse. This knowledge gap is further aggravated by the lack of genomic resources available, which are key tools for conservation. Here we present the transcriptome assembly of Unio elongatulus C. Pfeiffer, 1825, one of the least studied European freshwater mussels. Using the individual sequencing outputs from eight physiologically representative mussel tissues, we provide an annotated panel of tissue-specific Relative Gene Expression profiles. These resources are pivotal to studying the species' biological and ecological features, as well as helping to understand its vulnerability to current and future threats.

An historical "wreck": A transcriptome assembly of the naval shipworm, Teredo navalis Linnaeus, 1978.

Historically famous for their negative impact on human-built marine wood structures, mollusc shipworms play a central ecological role in marine ecosystems. Their association with bacterial symbionts, providing cellulolytic and nitrogen-fixing activities, underscores their exceptional wood-eating and wood-boring behaviours, improving energy transfer and the recycling of essential nutrients locked in the wood cellulose. Importantly, from a molecular standpoint, a minute of omic resources are available from this lineage of Bivalvia. Here, we produced and assembled a transcriptome from the globally distributed naval shipworm, Teredo navalis (family Teredinidae). The transcriptome was obtained by sequencing the total RNA from five equidistant segments of the whole body of a T. navalis specimen. The quality of the produced assembly was accessed with several statistics, revealing a highly contiguous (1194 N50) and complete (over 90% BUSCO scores for Eukaryote and Metazoan databases) transcriptome, with nearly 38,000 predicted ORF, more than half being functionally annotated. Our findings pave the way to investigate the unique evolutionary biology of these highly modified bivalves and lay the foundation for an adequate gene annotation of a full genome sequence of the 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.

Dataset of the complete mitogenome of the deep-sea sailfin roughshark, Oxynotus paradoxus Frade, 1929.

Chondrichthyans comprise a diverse group of vertebrate species with extraordinary ecological relevance. Yet, multiple members of this evolutionary lineage are associated with significant extinction risk. The sailfin roughshark Oxynotus paradoxus is a deep-water benthic shark currently listed as vulnerable due to population declines in parts of its range. Here we provide the first complete mitochondrial genome of O. paradoxus, comprising also the first record for the genus and family Oxynotidae. These data can facilitate future monitoring of the genetic diversity in this and related species. Genomic DNA was extracted from O. paradoxus collected in the eastern North Atlantic off western Portugal (37.59°N, 9.51°W) and sent for Illumina Paired-End (2 × 150 bp) library construction and whole genome sequencing on a Novaseq6000 platform. Trimmomatic (version 0.38) was used to remove adapters and MitoZ (version 3.4) to assemble and annotate the mitogenome. This mitogenome with 17 100 bp has a total of 38 genes, 13 of which are protein-coding genes, 23 transfer RNA genes, and 2 ribosomal RNA genes. Eight transfer RNAs and 1 protein-coding gene (NADH dehydrogenase subunit 6, NAD6) are in the complementary strand. In the provided phylogenetic inference, with all available and verified Squalomorphii mitogenomes, the four orders are well separated, and as expected, O. paradoxus is placed in the Squaliformes order. This data reinforces the need for more genomic resources for the Oxynotidae family.

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.

PacBio Hi-Fi genome assembly of the Iberian dolphin freshwater mussel Unio delphinus Spengler, 1793.

Mussels of order Unionida are a group of strictly freshwater bivalves with nearly 1,000 described species widely dispersed across world freshwater ecosystems. They are highly threatened showing the highest record of extinction events within faunal taxa. Conservation is particularly concerning in species occurring in the Mediterranean biodiversity hotspot that are exposed to multiple anthropogenic threats, possibly acting in synergy. That is the case of the dolphin freshwater mussel Unio delphinus Spengler, 1793, endemic to the western Iberian Peninsula with recently strong population declines. To date, only four genome assemblies are available for the order Unionida and only one European species. We present the first genome assembly of Unio delphinus. We used the PacBio HiFi to generate a highly contiguous genome assembly. The assembly is 2.5 Gb long, possessing 1254 contigs with a contig N50 length of 10 Mbp. This is the most contiguous freshwater mussel genome assembly to date and is an essential resource for investigating the species' biology and evolutionary history that ultimately will help to support conservation strategies.

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.

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.

Identifying freshwater priority areas for cross-taxa interactions.

Information about biotic interactions (e.g. competition, predation, parasitism, diseases, mutualism, allelopathy) is fundamental to better understand species distribution and abundance, ecosystem functioning, and ultimately guide conservation efforts. However, conservation planning often overlooks these important interactions. Here, we aim to demonstrate a new framework to include biotic interactions into Marxan. For that, we use freshwater mussels and fish interaction (as mussels rely on fishes to complete their life cycle) in the Douro River basin (Iberian Peninsula) as a case study. While doing that, we also test the importance of including biotic interactions into conservation planning exercises, by running spatial prioritisation analysis considering either: 1) only the target species (freshwater mussels); 2) freshwater mussels and their obligatory hosts (freshwater fishes); 3) freshwater mussels, fishes and their interactions. With this framework we found that biotic interactions tend to be underrepresented when the data on both freshwater mussels and fishes is not simultaneously included in the spatial prioritisation. Overall, the priority areas selected across all scenarios are mostly located in the western part of the Douro River basin, where most freshwater mussels and fishes still occur. Given the low overlap of priority areas identified here and the current Natura 2000 network, our approach may be useful for establishing (or enlarging) protected areas, especially in light of the EU Biodiversity Strategy for 2030. Also, this work may provide guidance for future habitat restoration and management of main threats to freshwater biodiversity.

Evaluating a species phylogeny using ddRAD SNPs: Cyto-nuclear discordance and introgression in the salmonid genus Thymallus (Salmonidae).

Hybridization and introgression are very common among freshwater fishes due to the dynamic nature of hydrological landscapes. Cyclic patterns of allopatry and secondary contact provide numerous opportunities for interspecific gene flow, which can lead to discordant paths of evolution for mitochondrial and nuclear genomes. Here, we used double digest restriction-site associated DNA sequencing (ddRADseq) to obtain a genome-wide single nucleotide polymorphism (SNP) dataset comprehensive for allThymallus (Salmonidae)species to infer phylogenetic relationships and evaluate potential recent and historical gene flow among species. The newly obtained nuclear phylogeny was largely concordant with a previously published mitogenome-based topology but revealed a few cyto-nuclear discordances. These incongruencies primarily involved the placement of internal nodes rather than the resolution of species, except for one European species where anthropogenic stock transfers are thought to be responsible for the observed pattern. The analysis of four contact zones where multiple species are found revealed a few cases of mitochondrial capture and limited signals of nuclear introgression. Interestingly, the mechanisms restricting interspecific gene flow might be distinct; while in zones of secondary contact, small-scale physical habitat separation appeared as a limiting factor, biologically based reinforcement mechanisms are presumed to be operative in areas where species presumably evolved in sympatry. Signals of historical introgression were largely congruent with the routes of species dispersal previously inferred from mitogenome data. Overall, the ddRADseq dataset provided a robust phylogenetic reconstruction of the genus Thymallus including new insights into historical hybridization and introgression, opening up new questions concerning their evolutionary history.

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.

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.

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.

Complete mitochondrial genome of the ragworm annelid Hediste diversicolor (of Müller, 1776) (Annelida: Nereididae).

Marine annelids are a globally distributed and species-rich group, performing important ecological roles in macrobenthic communities. Yet, the availability of molecular resources to study these organisms is scarcer, comparatively with other phyla. Here, we present the first complete mitogenome of the Atlantic ragworm Hediste diversicolor (OF Muller, 1776). The mitogenome (15,904 bp long) contains 13 protein-coding genes, 22 transfer RNA, and two ribosomal RNA genes, all encoded in the same strand. Gene arrangement and composition are identical to those observed in two available congeneric species, Hediste diadroma and Hediste japonica. The phylogenetic analysis using both maximum-likelihood and Bayesian inference methods reveal a well-supported monophyly of genus Hediste and the already reported paraphyletic relationships within the subfamilies Nereidinae and Gymnonereidinae. Our results highlight the relevance of increasing the molecular sampling within this diverse group of marine fauna.

Diversity, biogeography, evolutionary relationships, and conservation of Eastern Mediterranean freshwater mussels (Bivalvia: Unionidae).

Located at the junction between Europe, Africa, and Asia, with distinct evolutionary origins and varied ecological and geographical settings, together with a marked history of changes in orogeny and configuration of the main river basins, turned the Eastern Mediterranean into a region of high diversity and endemism of freshwater taxa. Freshwater mussels (Bivalvia, Unionidae) from the Western Palearctic have been widely studied in their European range, but little attention has been dedicated to these taxa in the Eastern Mediterranean region and their diversity and phylogeography are still poorly understood. The present study aims to resolve the diversity, biogeography, and evolutionary relationships of the Eastern Mediterranean freshwater mussels. To that end, we performed multiple field surveys, phylogenetic analyses, and a thorough taxonomic revaluation. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens across Turkey, Israel, and Iran, combining COI + 16S + 28S and COI phylogenies with molecular species delineation methods. Phylogeographical patterns were characterized based on published molecular data, newly sequenced specimens, and species distribution data, as well as ancestral range estimations. We reveal that Unionidae species richness in the Eastern Mediterranean is over 70% higher than previously assumed, counting 19 species within two subfamilies, the Unioninae (14) and Gonideinae (5). We propose two new species, Anodonta seddonisp. nov. and Leguminaia anatolicasp. nov. Six additional taxa, Unio delicatusstat. rev., Unio eucirrusstat. rev., Unio huetistat. rev., Unio sesirmensisstat. rev., Unio terminalisstat. rev. removed from the synonymy of Unio tigridis, as well as Unio damascensisstat. rev. removed from the synonymy of Unio crassus, are re-described. The nominal taxa Unio rothi var. komarowi O. Boettger, 1880 and Unio armeniacus Kobelt, 1911 are proposed as new synonyms of Unio bruguierianus, and Anodonta cyrea Drouët, 1881 and Anodonta cilicica Kobelt & Rolle, 1895 as new synonyms of Anodonta anatina. Also, the presence of Unio tumidus in the Maritza River is confirmed. The phylogeographic patterns described here are interpreted concerning major past geological events. Conservation needs and implications are presented, together with populations and species conservation priorities.

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.

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.

The genetic diversity and differentiation of mussels with complex life cycles and relations to host fish migratory traits and densities.

Many landscape and biotic processes shape the genetic structure of populations. The genetic structure of species with parasitic stages may also depend on the life history and ecology of their host. We investigated population genetic structure of the mussel Margaritifera margaritifera in Southern Sweden, and in relation to the population size and life history of its hosts, Salmo trutta and S. salar. Mussel populations were genetically differentiated into two clusters, further subdivided into four clusters and distinct conservation units. Regardless of host species, the genetic differentiation was lower among mussel populations sustained by sea-migrating than by resident hosts, while the genetic diversity was higher in mussel populations sustained by sea-migrating than by resident hosts. Genetic diversity of mussel populations was positively related to host abundance. Mussel population size was positively related to high genetic diversity of mussels sustained by resident hosts, while low mussel population size sustained by sea-migrating hosts had a high genetic diversity. The results of our study suggest a combined influence of mussels and host fish on genetic structure of unionoid mussels. We suggest to conserve not only mussel population sizes and host fish species, but also consider host migratory/resident behaviour and abundance when designing conservation programs.