Ancestors' Gift: Parental Early Exposure to the Environmentally Realistic Pesticide Mixture Drives Offspring Phenotype in a Larger Extent Than Direct Exposure in the Pacific Oyster, Crassostrea gigas.

Marine organisms are threatened by the presence of pesticides in coastal waters. Among them, the Pacific oyster is one of the most studied invertebrates in marine ecotoxicology where numerous studies highlighted the multiscale impacts of pesticides. In the past few years, a growing body of literature has reported the epigenetic outcomes of xenobiotics. Because DNA methylation is an epigenetic mark implicated in organism development and is meiotically heritable, it raises the question of the multigenerational implications of xenobiotic-induced epigenetic alterations. Therefore, we performed a multigenerational exposure to an environmentally relevant mixture of 18 pesticides (nominal sum concentration: 2.85 µg·L-1) during embryo-larval stages (0-48 hpf) of a second generation (F1) for which parents where already exposed or not in F0. Gene expression, DNA methylation, and physiological end points were assessed throughout the life cycle of individuals. Overall, the multigenerational effect has a greater influence on the phenotype than the exposure itself. Thus, multigenerational phenotypic effects were observed: individuals descending from exposed parents exhibited lower epinephrine-induced metamorphosis and field survival rates. At the molecular level, RNA-seq and Methyl-seq data analyses performed in gastrula embryos and metamorphosis-competent pediveliger (MCP) larvae revealed a clear F0 treatment-dependent discrimination. Some genes implicated into shell secretion and immunity exhibited F1:F0 treatment interaction patterns (e.g., Calm and Myd88). Those results suggest that low chronic environmental pesticide contamination can alter organisms beyond the individual scale level and have long-term adaptive implications.

A comparison of Oxford nanopore library strategies for bacterial genomics.

BACKGROUND: Oxford nanopore Technologies (ONT) provides three main library preparation strategies to sequence bacterial genomes. These include tagmentation (TAG), ligation (LIG) and amplification (PCR). Despite ONT's recommendations, making an informed decision for preparation choice remains difficult without a side-by-side comparison. Here, we sequenced 12 bacterial strains to examine the overall output of these strategies, including sequencing noise, barcoding efficiency and assembly quality based on mapping to curated genomes established herein. RESULTS: Average read length ranged closely for TAG and LIG (> 5,000 bp), while being drastically smaller for PCR (< 1,100 bp). LIG produced the largest output with 33.62 Gbp vs. 11.72 Gbp for TAG and 4.79 Gbp for PCR. PCR produced the most sequencing noise with only 22.7% of reads mappable to the curated genomes, vs. 92.9% for LIG and 87.3% for TAG. Output per channel was most homogenous in LIG and most variable in PCR, while intermediate in TAG. Artifactual tandem content was most abundant in PCR (22.5%) and least in LIG and TAG (0.9% and 2.2%). Basecalling and demultiplexing of barcoded libraries resulted in ~ 20% data loss as unclassified reads and 1.5% read leakage. CONCLUSION: The output of LIG was best (low noise, high read numbers of long lengths), intermediate in TAG (some noise, moderate read numbers of long lengths) and less desirable in PCR (high noise, high read numbers of short lengths). Overall, users should not accept assembly results at face value without careful replicon verification, including the detection of plasmids assembled from leaked reads.

The extensive transgenerational transcriptomic effects of ocean acidification on the olfactory epithelium of a marine fish are associated with a better viral resistance.

BACKGROUND: Progressive CO2-induced ocean acidification (OA) impacts marine life in ways that are difficult to predict but are likely to become exacerbated over generations. Although marine fishes can balance acid-base homeostasis efficiently, indirect ionic regulation that alter neurosensory systems can result in behavioural abnormalities. In marine invertebrates, OA can also affect immune system function, but whether this is the case in marine fishes is not fully understood. Farmed fish are highly susceptible to disease outbreak, yet strategies for overcoming such threats in the wake of OA are wanting. Here, we exposed two generations of the European sea bass (Dicentrarchus labrax) to end-of-century predicted pH levels (IPCC RCP8.5), with parents (F1) being exposed for four years and their offspring (F2) for 18 months. Our design included a transcriptomic analysis of the olfactory rosette (collected from the F2) and a viral challenge (exposing F2 to betanodavirus) where we assessed survival rates. RESULTS: We discovered transcriptomic trade-offs in both sensory and immune systems after long-term transgenerational exposure to OA. Specifically, RNA-Seq analysis of the olfactory rosette, the peripheral olfactory organ, from 18-months-old F2 revealed extensive regulation in genes involved in ion transport and neuronal signalling, including GABAergic signalling. We also detected OA-induced up-regulation of genes associated with odour transduction, synaptic plasticity, neuron excitability and wiring and down-regulation of genes involved in energy metabolism. Furthermore, OA-exposure induced up-regulation of genes involved in innate antiviral immunity (pathogen recognition receptors and interferon-stimulated genes) in combination with down-regulation of the protein biosynthetic machinery. Consistently, OA-exposed F2 challenged with betanodavirus, which causes damage to the nervous system of marine fish, had acquired improved resistance. CONCLUSION: F2 exposed to long-term transgenerational OA acclimation showed superior viral resistance, though as their metabolic and odour transduction programs were altered, odour-mediated behaviours might be consequently impacted. Although it is difficult to unveil how long-term OA impacts propagated between generations, our results reveal that, across generations, trade-offs in plastic responses is a core feature of the olfactory epithelium transcriptome in OA-exposed F2 offspring, and will have important consequences for how cultured and wild fish interacts with its environment.

Genomic Diversity of Campylobacter lari Group Isolates from Europe and Australia in a One Health Context.

Members of the Campylobacter lari group are causative agents of human gastroenteritis and are frequently found in shellfish, marine waters, shorebirds, and marine mammals. Within a One Health context, we used comparative genomics to characterize isolates from a diverse range of sources and geographical locations within Europe and Australia and assess possible transmission of food, animal, and environmental isolates to the human host. A total of 158 C. lari isolates from Australia, Denmark, France, and Germany, which included 82 isolates from human stool and blood, 12 from food, 14 from domestic animal, 19 from waterbirds, and 31 from the environment were analyzed. Genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance (AMR) traits was carried-out. Most of the isolates belonged to C. lari subsp. lari (Cll; 98, 62.0%), while C. lari subsp. concheus and C. lari urease-positive thermotolerant Campylobacter (UPTC) were represented by 12 (7.6%) and 15 (9.5%) isolates, respectively. Furthermore, 33 (20.9%) isolates were not assigned a subspecies and were thus attributed to distant Campylobacter spp. clades. Whole-genome sequence-derived multilocus sequence typing (MLST) and core-genome MLST (cgMLST) analyses revealed a high genetic diversity with 97 sequence types (STs), including 60 novel STs and 14 cgMLST clusters (≤10 allele differences), respectively. The most prevalent STs were ST-21, ST-70, ST-24, and ST-58 (accounting for 13.3%, 4.4%, 3.8%, and 3.2% of isolates, respectively). A high prevalence of the 125 examined virulence-related loci (from 76.8 to 98.4% per isolate) was observed, especially in Cll isolates, suggesting a probable human pathogenicity of these strains. IMPORTANCE Currently, relatedness between bacterial isolates impacting human health is easily monitored by molecular typing methods. These approaches rely on discrete loci or whole-genome sequence (WGS) analyses. Campylobacter lari is an emergent human pathogen isolated from diverse ecological niches, including fecal material from humans and animals, aquatic environments, and seafood. The presence of C. lari in such diverse sources underlines the importance of adopting an integrated One Health approach in studying C. lari population structure for conducting epidemiological risk assessment. This retrospective study presents a comparative genomics analysis of C. lari isolates retrieved from two different continents (Europe and Australia) and from different sources (human, domestic animals, waterbirds, food, and environment). It was designed to improve knowledge regarding C. lari ecology and pathogenicity, important for developing effective surveillance and disease prevention strategies.

Re-annotation, improved large-scale assembly and establishment of a catalogue of noncoding loci for the genome of the model brown alga Ectocarpus.

The genome of the filamentous brown alga Ectocarpus was the first to be completely sequenced from within the brown algal group and has served as a key reference genome both for this lineage and for the stramenopiles. We present a complete structural and functional reannotation of the Ectocarpus genome. The large-scale assembly of the Ectocarpus genome was significantly improved and genome-wide gene re-annotation using extensive RNA-seq data improved the structure of 11 108 existing protein-coding genes and added 2030 new loci. A genome-wide analysis of splicing isoforms identified an average of 1.6 transcripts per locus. A large number of previously undescribed noncoding genes were identified and annotated, including 717 loci that produce long noncoding RNAs. Conservation of lncRNAs between Ectocarpus and another brown alga, the kelp Saccharina japonica, suggests that at least a proportion of these loci serve a function. Finally, a large collection of single nucleotide polymorphism-based markers was developed for genetic analyses. These resources are available through an updated and improved genome database. This study significantly improves the utility of the Ectocarpus genome as a high-quality reference for the study of many important aspects of brown algal biology and as a reference for genomic analyses across the stramenopiles.

microRNAs and the evolution of complex multicellularity: identification of a large, diverse complement of microRNAs in the brown alga Ectocarpus.

There is currently convincing evidence that microRNAs have evolved independently in at least six different eukaryotic lineages: animals, land plants, chlorophyte green algae, demosponges, slime molds and brown algae. MicroRNAs from different lineages are not homologous but some structural features are strongly conserved across the eukaryotic tree allowing the application of stringent criteria to identify novel microRNA loci. A large set of 63 microRNA families was identified in the brown alga Ectocarpus based on mapping of RNA-seq data and nine microRNAs were confirmed by northern blotting. The Ectocarpus microRNAs are highly diverse at the sequence level with few multi-gene families, and do not tend to occur in clusters but exhibit some highly conserved structural features such as the presence of a uracil at the first residue. No homologues of Ectocarpus microRNAs were found in other stramenopile genomes indicating that they emerged late in stramenopile evolution and are perhaps specific to the brown algae. The large number of microRNA loci in Ectocarpus is consistent with the developmental complexity of many brown algal species and supports a proposed link between the emergence and expansion of microRNA regulatory systems and the evolution of complex multicellularity.

The Pseudoautosomal Regions of the U/V Sex Chromosomes of the Brown Alga Ectocarpus Exhibit Unusual Features.

The recombining regions of sex chromosomes (pseudoautosomal regions, PARs) are predicted to exhibit unusual features due to their being genetically linked to the nonrecombining, sex-determining region. This phenomenon is expected to occur in both diploid (XY, ZW) and haploid (UV) sexual systems, with slightly different consequences for UV sexual systems because of the absence of masking during the haploid phase (when sex is expressed) and because there is no homozygous sex in these systems. Despite a considerable amount of theoretical work on PAR genetics and evolution, these genomic regions have remained poorly characterized empirically. We show here that although the PARs of the U/V sex chromosomes of the brown alga Ectocarpus recombine at a similar rate to autosomal regions of the genome, they exhibit many genomic features typical of nonrecombining regions. The PARs were enriched in clusters of genes that are preferentially, and often exclusively, expressed during the sporophyte generation of the life cycle, and many of these genes appear to have evolved since the Ectocarpales diverged from other brown algal lineages. A modeling-based approach was used to investigate possible evolutionary mechanisms underlying this enrichment in sporophyte-biased genes. Our results are consistent with the evolution of the PAR in haploid systems being influenced by differential selection pressures in males and females acting on alleles that are advantageous during the sporophyte generation of the life cycle.

Sexual dimorphism and the evolution of sex-biased gene expression in the brown alga ectocarpus.

Males and females often have marked phenotypic differences, and the expression of these dissimilarities invariably involves sex differences in gene expression. Sex-biased gene expression has been well characterized in animal species, where a high proportion of the genome may be differentially regulated in males and females during development. Male-biased genes tend to evolve more rapidly than female-biased genes, implying differences in the strength of the selective forces acting on the two sexes. Analyses of sex-biased gene expression have focused on organisms that exhibit separate sexes during the diploid phase of the life cycle (diploid sexual systems), but the genetic nature of the sexual system is expected to influence the evolutionary trajectories of sex-biased genes. We analyze here the patterns of sex-biased gene expression in Ectocarpus, a brown alga with haploid sex determination (dioicy) and a low level of phenotypic sexual dimorphism. In Ectocarpus, female-biased genes were found to be evolving as rapidly as male-biased genes. Moreover, genes expressed at fertility showed faster rates of evolution than genes expressed in immature gametophytes. Both male- and female-biased genes had a greater proportion of sites experiencing positive selection, suggesting that their accelerated evolution is at least partly driven by adaptive evolution. Gene duplication appears to have played a significant role in the generation of sex-biased genes in Ectocarpus, expanding previous models that propose this mechanism for the resolution of sexual antagonism in diploid systems. The patterns of sex-biased gene expression in Ectocarpus are consistent both with predicted characteristics of UV (haploid) sexual systems and with the distinctive aspects of this organism's reproductive biology.

Molecular and phenotypic effects of early exposure to an environmentally relevant pesticide mixture in the Pacific oyster, Crassostrea gigas.

Early life stages are crucial for organism development, especially for those displaying external fertilization, whose gametes and early stages face environmental stressors such as xenobiotics. The pacific oyster, Crassostrea gigas, is considered a model species in ecotoxicology because of its ecological characteristics (benthic, sessile, filter feeding). So far studies have investigated the impact of xenobiotics at embryotoxic, genotoxic and physiological endpoints, sometimes at the multigenerational scale, highlighting the role of epigenetic mechanisms in transmitting alterations induced by exposure to single xenobiotics. However, to date, little is known about the impact of environmentally-mimicking contaminants cocktails. Thus, we examined the impact of an early exposure to environmentally relevant mixture on the Pacific oyster life history. We studied transcriptomic, epigenetic and physiological alterations induced in oysters exposed to 18 pesticides and metals at environmental concentration (nominal sum concentration: 2.85 µg.L-1, measured sum concentration: 3.74 ± 0.013 µg.L-1) during embryo-larval stage (0-48 h post fertilization, hpf). No significant differences in embryo-larval abnormalities at 24 hpf were observed during larval and spat rearing; the swimming behaviour of exposed individuals was disturbed, while they were longer and heavier at specific time points, and exhibited a lower epinephrine-induced metamorphosis rate as well as a higher survival rate in the field. In addition, RNA-seq analyses of gastrula embryos revealed the differential expression of development-related genes (e.g. Hox orthologues and cell cycle regulators) between control and exposed oysters. Whole-genome DNA methylation analyses demonstrated a significant modification of DNA methylation in exposed larvae marked by a demethylation trend. Those findings suggest that early exposure to an environmentally relevant pesticide mixture induces multi-scale latent effects possibly affecting life history traits in the Pacific oyster.

Draft Genome Sequences of Thelohania contejeani and Cucumispora dikerogammari, Pathogenic Microsporidia of Freshwater Crustaceans.

We announce the draft genome sequences of two pathogenic microsporidia of European freshwater crustaceans, Thelohania contejeani (the causative agent of porcelain disease) and Cucumispora dikerogammari Both species are implicated in mass mortalities in natural populations of their crayfish and amphipod hosts, respectively.

Comparative Analysis of Fecal Microbiomes From Wild Waterbirds to Poultry, Cattle, Pigs, and Wastewater Treatment Plants for a Microbial Source Tracking Approach.

Fecal pollution in coastal areas is of a high concern since it affects bathing and shellfish harvesting activities. Wild waterbirds are non-negligible in the overall signal of the detectable pollution. Yet, studies on wild waterbirds' gut microbiota focus on migratory trajectories and feeding impact on their shape, rare studies address their comparison to other sources and develop quantitative PCR (qPCR)-based Microbial Source Tracking (MST) markers to detect such pollution. Thus, by using 16S rRNA amplicon high-throughput sequencing, the aims of this study were (i) to explore and compare fecal bacterial communities from wild waterbirds (i.e., six families and 15 species, n = 275 samples) to that of poultry, cattle, pigs, and influent/effluent of wastewater treatment plants (n = 150 samples) and (ii) to develop new MST markers for waterbirds. Significant differences were observed between wild waterbirds and the four other groups. We identified 7,349 Amplicon Sequence Variants (ASVs) from the hypervariable V3-V4 region. Firmicutes and Proteobacteria and, in a lesser extent, Actinobacteria and Bacteroidetes were ubiquitous while Fusobacteria and Epsilonbacteraeota were mainly present in wild waterbirds. The clustering of samples in non-metric multidimensional scaling (NMDS) ordination indicated a by-group clustering shape, with a high diversity within wild waterbirds. In addition, the structure of the bacterial communities was distinct according to bird and/or animal species and families (Adonis R 2 = 0.13, p = 10-4, Adonis R 2 = 0.11, p = 10-4, respectively). The Analysis of Composition of Microbiomes (ANCOM) showed that the wild waterbird group differed from the others by the significant presence of sequences from Fusobacteriaceae (W = 566) and Enterococcaceae (W = 565) families, corresponding to the Cetobacterium (W = 1427) and Catellicoccus (W = 1427) genera, respectively. Altogether, our results suggest that some waterbird members present distinct fecal microbiomes allowing the design of qPCR MST markers. For instance, a swan- and an oystercatcher-associated markers (named Swan_2 and Oyscab, respectively) have been developed. Moreover, bacterial genera harboring potential human pathogens associated to bird droppings were detected in our dataset, including enteric pathogens, i.e., Arcobacter, Clostridium, Helicobacter, and Campylobacter, and environmental pathogens, i.e., Burkholderia and Pseudomonas. Future studies involving other wildlife hosts may improve gut microbiome studies and MST marker development, helping mitigation of yet unknown fecal pollution sources.

Comparative Genomics of Strictly Vertically Transmitted, Feminizing Microsporidia Endosymbionts of Amphipod Crustaceans.

Microsporidia are obligate intracellular eukaryotic parasites of vertebrates and invertebrates. Microsporidia are usually pathogenic and undergo horizontal transmission or a mix of horizontal and vertical transmission. However, cases of nonpathogenic microsporidia, strictly vertically transmitted from mother to offspring, have been reported in amphipod crustaceans. Some of them further evolved the ability to feminize their nontransmitting male hosts into transmitting females. However, our understanding of the evolution of feminization in microsporidia is hindered by a lack of genomic resources. We report the sequencing and analysis of three strictly vertically transmitted microsporidia species for which feminization induction has been demonstrated (Nosema granulosis) or is strongly suspected (Dictyocoela muelleri and Dictyocoela roeselum), along with a draft genome assembly of their host Gammarus roeselii. Contrary to horizontally transmitted microsporidia that form environmental spores that can be purified, feminizing microsporidia cannot be easily isolated from their host cells. Therefore, we cosequenced symbiont and host genomic DNA and devised a computational strategy to obtain genome assemblies for the different partners. Genomic comparison with feminizing Wolbachia bacterial endosymbionts of isopod crustaceans indicated independent evolution of feminization in microsporidia and Wolbachia at the molecular genetic level. Feminization thus represents a remarkable evolutionary convergence of eukaryotic and prokaryotic microorganisms. Furthermore, a comparative genomics analysis of microsporidia allowed us to identify several candidate genes for feminization, involving functions such as DNA binding and membrane fusion. The genomic resources we generated contribute to establish Gammarus roeselii and its microsporidia symbionts as a new model to study the evolution of symbiont-mediated feminization.

Transgenerational regulation of cbln11 gene expression in the olfactory rosette of the European sea bass (Dicentrarchus labrax) exposed to ocean acidification.

Elevated amounts of atmospheric CO2 are causing ocean acidification (OA) that may affect marine organisms including fish species. While several studies carried out in fish revealed that OA induces short term dysfunction in sensory systems including regulation of neurons activity in olfactory epithelium, information on the effects of OA on other physiological processes and actors is scarcer. In the present study we focused our attention on a European sea bass (Dicentrarchus labrax) sghC1q gene, a member of the C1q-domain-containing (C1qDC) protein family. In vertebrates, C1qDC family includes actors involved in different physiological processes including immune response and synaptic organization. Our microsynteny analysis revealed that this sghC1q gene is the orthologous gene in European sea bass to zebrafish (Danio rerio) cbln11 gene. We cloned the full length cbln11 mRNA and identified the different domains (the signal peptide, the coiled coil region and the globular C1q domain) of the deduced protein sequence. Investigation of mRNA expression by qPCR and in situ hybridization revealed that cbln11gene is especially expressed in the non-sensory epithelium of the olfactory rosette at larval and adult stages. The expression of cbln11 mRNA was analysed by qPCR in the first generation (F0) of European sea bass broodstock exposed since larval stages to water pH of 8.0 (control) or 7.6 (predicted for year 2100) and in their offspring (F1) maintained in the environmental conditions of their parents. Our results showed that cbln11 mRNA expression level was lower in larvae exposed to OA then up-regulated at adult stage in the olfactory rosette of F0 and that this up-regulation is maintained under OA at larval and juvenile stages in F1. Overall, this work provides evidence of a transgenerational inheritance of OA-induced up-regulation of cbln11 gene expression in European sea bass. Further studies will investigate the potential immune function of cbln11 gene and the consequences of these regulations, as well as the possible implications in terms of fitness and adaptation to OA in European sea bass.

instaGRAAL: chromosome-level quality scaffolding of genomes using a proximity ligation-based scaffolder.

Hi-C exploits contact frequencies between pairs of loci to bridge and order contigs during genome assembly, resulting in chromosome-level assemblies. Because few robust programs are available for this type of data, we developed instaGRAAL, a complete overhaul of the GRAAL program, which has adapted the latter to allow efficient assembly of large genomes. instaGRAAL features a number of improvements over GRAAL, including a modular correction approach that optionally integrates independent data. We validate the program using data for two brown algae, and human, to generate near-complete assemblies with minimal human intervention.

The genome of Ectocarpus subulatus - A highly stress-tolerant brown alga.

Brown algae are multicellular photosynthetic stramenopiles that colonize marine rocky shores worldwide. Ectocarpus sp. Ec32 has been established as a genomic model for brown algae. Here we present the genome and metabolic network of the closely related species, Ectocarpus subulatus Kützing, which is characterized by high abiotic stress tolerance. Since their separation, both strains show new traces of viral sequences and the activity of large retrotransposons, which may also be related to the expansion of a family of chlorophyll-binding proteins. Further features suspected to contribute to stress tolerance include an expanded family of heat shock proteins, the reduction of genes involved in the production of halogenated defence compounds, and the presence of fewer cell wall polysaccharide-modifying enzymes. Overall, E. subulatus has mainly lost members of gene families down-regulated in low salinities, and conserved those that were up-regulated in the same condition. However, 96% of genes that differed between the two examined Ectocarpus species, as well as all genes under positive selection, were found to encode proteins of unknown function. This underlines the uniqueness of brown algal stress tolerance mechanisms as well as the significance of establishing E. subulatus as a comparative model for future functional studies.

Correction to: Rapid turnover of life-cycle-related genes in the brown algae.

Following publication of the original article [1], it was noticed that the author names were published with initials instead of full names. The article [1] has been updated.

Rapid turnover of life-cycle-related genes in the brown algae.

BACKGROUND: Sexual life cycles in eukaryotes involve a cyclic alternation between haploid and diploid phases. While most animals possess a diploid life cycle, many plants and algae alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. In many algae, gametophytes and sporophytes are independent and free-living and may present dramatic phenotypic differences. The same shared genome can therefore be subject to different, even conflicting, selection pressures during each of the life cycle generations. Here, we analyze the nature and extent of genome-wide, generation-biased gene expression in four species of brown algae with contrasting levels of dimorphism between life cycle generations. RESULTS: We show that the proportion of the transcriptome that is generation-specific is broadly associated with the level of phenotypic dimorphism between the life cycle stages. Importantly, our data reveals a remarkably high turnover rate for life-cycle-related gene sets across the brown algae and highlights the importance not only of co-option of regulatory programs from one generation to the other but also of a role for newly emerged, lineage-specific gene expression patterns in the evolution of the gametophyte and sporophyte developmental programs in this major eukaryotic group. Moreover, we show that generation-biased genes display distinct evolutionary modes, with gametophyte-biased genes evolving rapidly at the coding sequence level whereas sporophyte-biased genes tend to exhibit changes in their patterns of expression. CONCLUSION: Our analysis uncovers the characteristics, expression patterns, and evolution of generation-biased genes and underlines the selective forces that shape this previously underappreciated source of phenotypic diversity.

Untangling Heteroplasmy, Structure, and Evolution of an Atypical Mitochondrial Genome by PacBio Sequencing.

The highly compact mitochondrial (mt) genome of terrestrial isopods (Oniscidae) presents two unusual features. First, several loci can individually encode two tRNAs, thanks to single nucleotide polymorphisms at anticodon sites. Within-individual variation (heteroplasmy) at these loci is thought to have been maintained for millions of years because individuals that do not carry all tRNA genes die, resulting in strong balancing selection. Second, the oniscid mtDNA genome comes in two conformations: a ∼14 kb linear monomer and a ∼28 kb circular dimer comprising two monomer units fused in palindrome. We hypothesized that heteroplasmy actually results from two genome units of the same dimeric molecule carrying different tRNA genes at mirrored loci. This hypothesis, however, contradicts the earlier proposition that dimeric molecules result from the replication of linear monomers-a process that should yield totally identical genome units within a dimer. To solve this contradiction, we used the SMRT (PacBio) technology to sequence mirrored tRNA loci in single dimeric molecules. We show that dimers do present different tRNA genes at mirrored loci; thus covalent linkage, rather than balancing selection, maintains vital variation at anticodons. We also leveraged unique features of the SMRT technology to detect linear monomers closed by hairpins and carrying noncomplementary bases at anticodons. These molecules contain the necessary information to encode two tRNAs at the same locus, and suggest new mechanisms of transition between linear and circular mtDNA. Overall, our analyses clarify the evolution of an atypical mt genome where dimerization counterintuitively enabled further mtDNA compaction.

High-density genetic map and identification of QTLs for responses to temperature and salinity stresses in the model brown alga Ectocarpus.

Deciphering the genetic architecture of adaptation of brown algae to environmental stresses such as temperature and salinity is of evolutionary as well as of practical interest. The filamentous brown alga Ectocarpus sp. is a model for the brown algae and its genome has been sequenced. As sessile organisms, brown algae need to be capable of resisting the various abiotic stressors that act in the intertidal zone (e.g. osmotic pressure, temperature, salinity, UV radiation) and previous studies have shown that an important proportion of the expressed genes is regulated in response to hyposaline, hypersaline or oxidative stress conditions. Using the double digest RAD sequencing method, we constructed a dense genetic map with 3,588 SNP markers and identified 39 QTLs for growth-related traits and their plasticity under different temperature and salinity conditions (tolerance to high temperature and low salinity). GO enrichment tests within QTL intervals highlighted membrane transport processes such as ion transporters. Our study represents a significant step towards deciphering the genetic basis of adaptation of Ectocarpus sp. to stress conditions and provides a substantial resource to the increasing list of tools generated for the species.