Both nuclear and cytoplasmic polymorphisms are involved in genetic conflicts over male fertility in the gynodioecious snail, Physa acuta.

Gynodioecy, the coexistence of hermaphrodites with females, often reflects conflicts between cytoplasmic male sterility (CMS) genes and nuclear genes restoring male fertility. CMS is frequent in plants and has been recently discovered in one animal: the freshwater snail, Physa acuta. In this system, CMS was linked to a single divergent mitochondrial genome (D), devoid of apparent nuclear restoration. Our study uncovers a second, novel CMS-associated mitogenome (K) in Physa acuta, demonstrating an extraordinary acceleration of molecular evolution throughout the entire K mitochondrial genome, akin to the previously observed pattern in D. This suggests a pervasive occurrence of accelerated evolution in both CMS-associated lineages. Through a 17-generation introgression experiment, we further show that nuclear polymorphisms in K-mitogenome individuals contribute to the restoration of male function in natural populations. Our results underscore shared characteristics in gynodioecy between plants and animals, emphasizing the presence of multiple CMS mitotypes and cytonuclear conflicts. This reaffirms the pivotal role of mitochondria in influencing male function and in generating genomic conflicts that impact reproductive processes in animals.

A collaborative backbone resource for comparative studies of subterranean evolution: The World Asellidae database.

Transition to novel environments, such as groundwater colonization by surface organisms, provides an excellent research ground to study phenotypic evolution. However, interspecific comparative studies on evolution to groundwater life are few because of the challenge in assembling large ecological and molecular resources for species-rich taxa comprised of surface and subterranean species. Here, we make available to the scientific community an operational set of working tools and resources for the Asellidae, a family of freshwater isopods containing hundreds of surface and subterranean species. First, we release the World Asellidae database (WAD) and its web application, a sustainable and FAIR solution to producing and sharing data and biological material. WAD provides access to thousands of species occurrences, specimens, DNA extracts and DNA sequences with rich metadata ensuring full scientific traceability. Second, we perform a large-scale dated phylogenetic reconstruction of Asellidae to support phylogenetic comparative analyses. Of 424 terminal branches, we identify 34 pairs of surface and subterranean species representing independent replicates of the transition from surface water to groundwater. Third, we exemplify the usefulness of WAD for documenting phenotypic shifts associated with colonization of subterranean habitats. We provide the first phylogenetically controlled evidence that body size of males decreases relative to that of females upon groundwater colonization, suggesting competition for rare receptive females selects for smaller, more agile males in groundwater. By making these tools and resources widely accessible, we open up new opportunities for exploring how phenotypic traits evolve in response to changes in selective pressures and trade-offs during groundwater colonization.

Extreme mitochondrial DNA divergence underlies genetic conflict over sex determination.

Cytoplasmic male sterility (CMS) is a form of genetic conflict over sex determination that results from differences in modes of inheritance between genomic compartments.1-3 Indeed, maternally transmitted (usually mitochondrial) genes sometimes enhance their transmission by suppressing the male function in a hermaphroditic organism to the detriment of biparentally inherited nuclear genes. Therefore, these hermaphrodites become functionally female and may coexist with regular hermaphrodites in so-called gynodioecious populations.3 CMS has been known in plants since Darwin's times4 but is previously unknown in the animal kingdom.5-8 We relate the first observation of CMS in animals. It occurs in a freshwater snail population, where some individuals appear unable to sire offspring in controlled crosses and show anatomical, physiological, and behavioral characters consistent with a suppression of the male function. Male sterility is associated with a mitochondrial lineage that underwent a spectacular acceleration of DNA substitution rates, affecting the entire mitochondrial genome-this acceleration concerns both synonymous and non-synonymous substitutions and therefore results from increased mitogenome mutation rates. Consequently, mitochondrial haplotype divergence within the population is exceptionally high, matching that observed between snail taxa that diverged 475 million years ago. This result is reminiscent of similar accelerations in mitogenome evolution observed in plant clades where gynodioecy is frequent,9,10 both being consistent with arms-race evolution of genome regions implicated in CMS.11,12 Our study shows that genomic conflicts can trigger independent evolution of similar sex-determination systems in plants and animals and dramatically accelerate molecular evolution.

Passive sampling of environmental DNA in aquatic environments using 3D-printed hydroxyapatite samplers.

The study of environmental DNA (eDNA) released by aquatic organisms in their habitat offers a fast, noninvasive and sensitive approach to monitor their presence. Common eDNA sampling methods such as water filtration and DNA precipitation are time-consuming, require difficult-to-handle equipment and partially integrate eDNA signals. To overcome these limitations, we created the first proof of concept of a passive, 3D-printed and easy-to-use eDNA sampler. We designed the samplers from hydroxyapatite (HAp samplers), a natural mineral with a high DNA adsorption capacity. The porous structure and shape of the samplers were designed to optimize DNA adsorption and facilitate their handling in the laboratory and in the field. Here we show that HAp samplers can efficiently collect genomic DNA in controlled set-ups, but can also collect animal eDNA under controlled and natural conditions with yields similar to conventional methods. However, we also observed large variations in the amount of DNA collected even under controlled conditions. A better understanding of the DNA-hydroxyapatite interactions on the surface of the samplers is now necessary to optimize eDNA adsorption and to allow the development of a reliable, easy-to-use and reusable eDNA sampling tool.

Transcriptome-wide deregulation of gene expression by artificial light at night in tadpoles of common toads.

Artificial light at night (ALAN) affects numerous physiological and behavioural mechanisms in various species by potentially disturbing circadian timekeeping systems and modifying melatonin levels. However, given the multiple direct and indirect effects of ALAN on organisms, large-scale transcriptomic approaches are essential to assess the global effect of ALAN on biological processes. Moreover, although studies have focused mainly on variations in gene expression during the night in the presence of ALAN, it is necessary to investigate the effect of ALAN on gene expression during the day. In this study, we combined de novo transcriptome sequencing and assembly, and a controlled laboratory experiment to evaluate the transcriptome-wide gene expression response using high-throughput (RNA-seq) in Bufo bufo tadpoles exposed to ecologically relevant light levels. Here, we demonstrated for the first time that ALAN affected gene expression at night (3.5% and 11% of differentially expressed genes when exposed to 0.1 and 5 lx compared to controls, respectively), but also during the day (11.2% of differentially expressed genes when exposed to 5 lx compared to controls) with a dose-dependent effect. ALAN globally induced a downregulation of genes (during the night, 58% and 62% of the genes were downregulated when exposed to 0.1 and 5 lx compared to controls, respectively, and during the day, 61.2% of the genes were downregulated when exposed to 5 lx compared to controls). ALAN effects were detected at very low levels of illuminance (0.1 lx) and affected mainly genes related to the innate immune system and, to a lesser extend to lipid metabolism. These results provide new insights into understanding the effects of ALAN on organism. ALAN impacted the expression of genes linked to a broad range of physiological pathways at very low levels of ALAN during night-time and during daytime, potentially resulting in reduced immune capacity under environmental immune challenges.

Bedrock radioactivity influences the rate and spectrum of mutation.

All organisms on Earth are exposed to low doses of natural radioactivity but some habitats are more radioactive than others. Yet, documenting the influence of natural radioactivity on the evolution of biodiversity is challenging. Here, we addressed whether organisms living in naturally more radioactive habitats accumulate more mutations across generations using 14 species of waterlice living in subterranean habitats with contrasted levels of radioactivity. We found that the mitochondrial and nuclear mutation rates across a waterlouse species' genome increased on average by 60% and 30%, respectively, when radioactivity increased by a factor of three. We also found a positive correlation between the level of radioactivity and the probability of G to T (and complementary C to A) mutations, a hallmark of oxidative stress. We conclude that even low doses of natural bedrock radioactivity influence the mutation rate possibly through the accumulation of oxidative damage, in particular in the mitochondrial genome.

Enhancing DNA metabarcoding performance and applicability with bait capture enrichment and DNA from conservative ethanol.

Metabarcoding is often presented as an alternative identification tool to compensate for coarse taxonomic resolution and misidentification encountered with traditional morphological approaches. However, metabarcoding comes with two major impediments which slow down its adoption. First, the picking and destruction of organisms for DNA extraction are time and cost consuming and do not allow organism conservation for further evaluations. Second, current metabarcoding protocols include a PCR enrichment step which induces errors in the estimation of species diversity and relative biomasses. In this study, we first evaluated the capacity of capture enrichment to replace PCR enrichment using controlled freshwater macrozoobenthos mock communities. Then, we tested if DNA extracted from the fixative ethanol (etDNA) of the same mock communities can be used as an alternative to DNA extracted from pools of whole organisms (bulk DNA). We show that capture enrichment provides more reliable and accurate representation of species occurrences and relative biomasses in comparison with PCR enrichment for bulk DNA. While etDNA does not permit to estimate relative biomasses, etDNA and bulk DNA provide equivalent species detection rates. Thanks to its robustness to mismatches, capture enrichment is already an efficient alternative to PCR enrichment for metabarcoding and, if coupled to etDNA, is a time-saver option in studies where presence information only is sufficient.

Life History Traits Impact the Nuclear Rate of Substitution but Not the Mitochondrial Rate in Isopods.

The rate of molecular evolution varies widely among species. Life history traits (LHTs) have been proposed as a major driver of these variations. However, the relative contribution of each trait is poorly understood. Here, we test the influence of metabolic rate (MR), longevity, and generation time (GT) on the nuclear and mitochondrial synonymous substitution rates using a group of isopod species that have made multiple independent transitions to subterranean environments. Subterranean species have repeatedly evolved a lower MR, a longer lifespan and a longer GT. We assembled the nuclear transcriptomes and the mitochondrial genomes of 13 pairs of closely related isopods, each pair composed of one surface and one subterranean species. We found that subterranean species have a lower rate of nuclear synonymous substitution than surface species whereas the mitochondrial rate remained unchanged. We propose that this decoupling between nuclear and mitochondrial rates comes from different DNA replication processes in these two compartments. In isopods, the nuclear rate is probably tightly controlled by GT alone. In contrast, mitochondrial genomes appear to replicate and mutate at a rate independent of LHTs. These results are incongruent with previous studies, which were mostly devoted to vertebrates. We suggest that this incongruence can be explained by developmental differences between animal clades, with a quiescent period during female gametogenesis in mammals and birds which imposes a nuclear and mitochondrial rate coupling, as opposed to the continuous gametogenesis observed in most arthropods.

Less effective selection leads to larger genomes.

The evolutionary origin of the striking genome size variations found in eukaryotes remains enigmatic. The effective size of populations, by controlling selection efficacy, is expected to be a key parameter underlying genome size evolution. However, this hypothesis has proved difficult to investigate using empirical data sets. Here, we tested this hypothesis using 22 de novo transcriptomes and low-coverage genomes of asellid isopods, which represent 11 independent habitat shifts from surface water to resource-poor groundwater. We show that these habitat shifts are associated with higher transcriptome-wide [Formula: see text] After ruling out the role of positive selection and pseudogenization, we show that these transcriptome-wide [Formula: see text] increases are the consequence of a reduction in selection efficacy imposed by the smaller effective population size of subterranean species. This reduction is paralleled by an important increase in genome size (25% increase on average), an increase also confirmed in subterranean decapods and mollusks. We also control for an adaptive impact of genome size on life history traits but find no correlation between body size, or growth rate, and genome size. We show instead that the independent increases in genome size measured in subterranean isopods are the direct consequence of increasing invasion rates by repeat elements, which are less efficiently purged out by purifying selection. Contrary to selection efficacy, polymorphism is not correlated to genome size. We propose that recent demographic fluctuations and the difficulty of observing polymorphism variation in polymorphism-poor species can obfuscate the link between effective population size and genome size when polymorphism data are used alone.

No Evidence That Nitrogen Limitation Influences the Elemental Composition of Isopod Transcriptomes and Proteomes.

The field of stoichiogenomics aims at understanding the influence of nutrient limitations on the elemental composition of the genome, transcriptome, and proteome. The 20 amino acids and the 4 nt differ in the number of nutrients they contain, such as nitrogen (N). Thus, N limitation shall theoretically select for changes in the composition of proteins or RNAs through preferential use of N-poor amino acids or nucleotides, which will decrease the N-budget of an organism. While these N-saving mechanisms have been evidenced in microorganisms, they remain controversial in multicellular eukaryotes. In this study, we used 13 surface and subterranean isopod species pairs that face strongly contrasted N limitations, either in terms of quantity or quality. We combined in situ nutrient quantification and transcriptome sequencing to test if N limitation selected for N-savings through changes in the expression and composition of the transcriptome and proteome. No evidence of N-savings was found in the total N-budget of transcriptomes or proteomes or in the average protein N-cost. Nevertheless, subterranean species evolving in N-depleted habitats displayed lower N-usage at their third codon positions. To test if this convergent compositional change was driven by natural selection, we developed a method to detect the strand-asymmetric signature that stoichiogenomic selection should leave in the substitution pattern. No such signature was evidenced, indicating that the observed stoichiogenomic-like patterns were attributable to nonadaptive processes. The absence of stoichiogenomic signal despite strong N limitation within a powerful phylogenetic framework casts doubt on the existence of stoichiogenomic mechanisms in metazoans.

Genome Content and Phylogenomics Reveal both Ancestral and Lateral Evolutionary Pathways in Plant-Pathogenic Streptomyces Species.

Streptomyces spp. are highly differentiated actinomycetes with large, linear chromosomes that encode an arsenal of biologically active molecules and catabolic enzymes. Members of this genus are well equipped for life in nutrient-limited environments and are common soil saprophytes. Out of the hundreds of species in the genus Streptomyces, a small group has evolved the ability to infect plants. The recent availability of Streptomyces genome sequences, including four genomes of pathogenic species, provided an opportunity to characterize the gene content specific to these pathogens and to study phylogenetic relationships among them. Genome sequencing, comparative genomics, and phylogenetic analysis enabled us to discriminate pathogenic from saprophytic Streptomyces strains; moreover, we calculated that the pathogen-specific genome contains 4,662 orthologs. Phylogenetic reconstruction suggested that Streptomyces scabies and S. ipomoeae share an ancestor but that their biosynthetic clusters encoding the required virulence factor thaxtomin have diverged. In contrast, S. turgidiscabies and S. acidiscabies, two relatively unrelated pathogens, possess highly similar thaxtomin biosynthesis clusters, which suggests that the acquisition of these genes was through lateral gene transfer.

Genome based phylogeny and comparative genomic analysis of intra-mammary pathogenic Escherichia coli.

Escherichia coli is an important cause of bovine mastitis and can cause both severe inflammation with a short-term transient infection, as well as less severe, but more chronic inflammation and infection persistence. E. coli is a highly diverse organism that has been classified into a number of different pathotypes or pathovars, and mammary pathogenic E. coli (MPEC) has been proposed as a new such pathotype. The purpose of this study was to use genome sequence data derived from both transient and persistent MPEC isolates (two isolates of each phenotype) to construct a genome-based phylogeny that places MPEC in its phylogenetic context with other E. coli pathovars. A subsidiary goal was to conduct comparative genomic analyses of these MPEC isolates with other E. coli pathovars to provide a preliminary perspective on loci that might be correlated with the MPEC phenotype. Both concatenated and consensus tree phylogenies did not support MPEC monophyly or the monophyly of either transient or persistent phenotypes. Three of the MPEC isolates (ECA-727, ECC-Z, and ECA-O157) originated from within the predominately commensal clade of E. coli, referred to as phylogroup A. The fourth MPEC isolate, of the persistent phenotype (ECC-1470), was sister group to an isolate of ETEC, falling within the E. coli B1 clade. This suggests that the MPEC phenotype has arisen on numerous independent occasions and that this has often, although not invariably, occurred from commensal ancestry. Examination of the genes present in the MPEC strains relative to the commensal strains identified a consistent presence of the type VI secretion system (T6SS) in the MPEC strains, with only occasional representation in commensal strains, suggesting that T6SS may be associated with MPEC pathogenesis and/or as an inter-bacterial competitive attribute and therefore could represent a useful target to explore for the development of MPEC specific inhibitors.

Inflammation-associated adherent-invasive Escherichia coli are enriched in pathways for use of propanediol and iron and M-cell translocation.

BACKGROUND: Perturbations of the intestinal microbiome, termed dysbiosis, are linked to intestinal inflammation. Isolation of adherent-invasive Escherichia coli (AIEC) from intestines of patients with Crohn's disease (CD), dogs with granulomatous colitis, and mice with acute ileitis suggests these bacteria share pathoadaptive virulence factors that promote inflammation. METHODS: To identify genes associated with AIEC, we sequenced the genomes of phylogenetically diverse AIEC strains isolated from people with CD (4), dogs with granulomatous colitis (2), and mice with ileitis (2) and 1 non-AIEC strain from CD ileum and compared them with 38 genome sequences of E. coli and Shigella. We then determined the prevalence of AIEC-associated genes in 49 E. coli strains from patients with CD and controls and correlated genotype with invasion of intestinal epithelial cells, persistence within macrophages, AIEC pathotype, and growth in standardized conditions. RESULTS: Genes encoding propanediol utilization (pdu operon) and iron acquisition (yersiniabactin, chu operon) were overrepresented in AIEC relative to nonpathogenic E. coli. PduC (propanediol dehydratase) was enriched in CD-derived AIEC, correlated with increased cellular invasion, and persistence in vitro and was increasingly expressed in fucose-containing media. Growth of AIEC required iron, and the presence of chuA (heme acquisition) correlated with persistence in macrophages. CD-associated AIEC with lpfA 154 (long polar fimbriae) demonstrated increased invasion of epithelial cells and translocation across M cells. CONCLUSIONS: Our findings provide novel insights into the genetic basis of the AIEC pathotype, supporting the concept that AIEC are equipped to exploit and promote intestinal inflammation and reveal potential targets for intervention against AIEC and inflammation-associated dysbiosis.

Current developments in groundwater ecology--from biodiversity to ecosystem function and services.

Groundwater ecosystems constitute the largest terrestrial freshwater biome. They are dark, extremely low in energy and do not provide much space but they contain an unexpectedly high diversity of living forms showing characteristic adaptive features. The restricted accessibility along with the enormous 'invisible' heterogeneity challenged for a long time testing of scientific theories and unraveling of ecosystem functioning. Triggered by an improved interdisciplinarity, comprehensive sampling strategies and current developments in biotechnology and statistical analysis, groundwater ecology gains momentum entering a new era of research. We are only beginning to understand adaptive mechanisms, species distribution patterns and ecosystem functioning. Ninety-five percent of global liquid freshwater is stored in the terrestrial subsurface constituting a major source of water for drinking, irrigation and industrial purposes. There is an urgent need to integrate evolutionary and ecological research for developing a holistic perspective of the functional roles of biodiversity and ecosystem services and predicting global changes under alternative groundwater resource use scenarios.

Microsatellite development and first population size estimates for the groundwater isopod Proasellus walteri.

Effective population size (N e) is one of the most important parameters in, ecology, evolutionary and conservation biology; however, few studies of N e in surface freshwater organisms have been published to date. Even fewer studies have been carried out in groundwater organisms, although their evolution has long been considered to be particularly constrained by small N e. In this study, we estimated the contemporary effective population size of the obligate groundwater isopod: Proaselluswalteri (Chappuis, 1948). To this end, a genomic library was enriched for microsatellite motifs and sequenced using 454 GS-FLX technology. A total of 54,593 reads were assembled in 10,346 contigs or singlets, of which 245 contained candidate microsatellite sequences with suitable priming sites. Ninety-six loci were tested for amplification, polymorphism and multiplexing properties, of which seven were finally selected for N e estimation. Linkage disequilibrium and approximate Bayesian computation methods revealed that N e in this small interstitial groundwater isopod could reach large sizes (> 585 individuals). Our results suggest that environmental conditions in groundwater, while often referred to as extreme, are not necessarily associated with small N e.

Phenotypic heterogeneity of genomically-diverse isolates of Streptococcus mutans.

High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease.

Complete Genome Sequence of the Porcine Strain Brachyspira pilosicoli P43/6/78(T.).

Reported herein is the complete genome sequence of Brachyspira pilosicoli strain P43/6/78T, isolated from a pig with clinical disease. This sequence will aid in the study of genome-wide comparison among Brachyspira species.

Timetree of Aselloidea reveals species diversification dynamics in groundwater.

A key challenge for biologists is to document and explain global patterns of diversification in a wide range of environments. Here, we explore patterns of continental-scale diversification in a groundwater species-rich clade, the superfamily Aselloidea (Pancrustacea: Isopoda). Our analyses supported a constant diversification rate during most of the course of Aselloidea evolution, until 4-15 Ma when diversification rates started to decrease. This constant accumulation of lineages challenges the view that groundwater species diversification in temperate regions might have been primarily driven by major changes in physical environment leading to the extinction of surface populations and subsequent synchronous isolation of multiple groundwater populations. Rather than acting synchronously over broad geographic regions, factors causing extinction of surface populations and subsequent reproductive isolation of groundwater populations may act in a local and asynchronous manner, thereby resulting in a constant speciation rate over time. Our phylogeny also revealed several cases of parapatric distributions among closely related surface-water and groundwater species suggesting that species diversification could also arise from a process of disruptive selection along the surface-subterranean environmental gradient. Our results call for re-evaluating the spatial scale and timing of factors causing diversification events in groundwater.

Comparative characterization of the virulence gene clusters (lipooligosaccharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species.

Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosaccharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain-Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking.