The effect of anaerobic digestate as an organic soil fertilizer on the diversity and structure of the indigenous soil microbial and nematode communities.

Anaerobic digestate is a popular soil additive which can promote sustainability and transition toward a circular economy. This study addresses how anaerobic digestate modifies soil health when combined with a common chemical fertilizer. Attention was given to soil microbes and, a neglected but of paramount importance soil taxonomic group, soil nematodes. A mesocosm experiment was set up in order to assess the soil's microbial and nematode community. The results demonstrated that the microbial diversity was not affected by the different fertilization regimes, although species richness increased after digestate and mixed fertilization. The composition and abundance of nematode community did not respond to any treatment. Mixed fertilization notably increased potassium (K) and boron (B) levels, while nitrate (NO3-) levels were uniformly elevated across fertilized soils, despite variations in nitrogen input. Network analysis revealed that chemical fertilization led to a densely interconnected network with mainly mutualistic relationships which could cause ecosystem disruption, while digestate application formed a more complex community based on bacterial interactions. However, the combination of both orchestrated a more balanced and less complex community structure, which is more resilient to random disturbances, but on the downside, it is more likely to collapse under targeted perturbations.

Investigating the role of genetic variation in vgll3 and six6 in the domestication of gilthead seabream (Sparus aurata Linnaeus) and European seabass (Dicentrarchus labrax Linnaeus).

Gene function conservation is crucial in molecular ecology, especially for key traits like growth and maturation in teleost fish. The vgll3 and six6 genes are known to influence age-at-maturity in Atlantic salmon, but their impact on other fish species is poorly understood. Here, we investigated the association of vgll3 and six6 in the domestication of gilthead seabream and European seabass, both undergoing selective breeding for growth-related traits in the Mediterranean. We analysed two different sets of samples using two different genotyping approaches. The first dataset comprised farmed and wild populations from Greece, genotyped for SNPs within the two genes ('gene-level genotyping'). The second dataset examined 300-600 k SNPs located in the chromosomes of the two genes, derived from a meta-analysis of a Pool-Seq experiment involving farmed and wild populations distributed widely across the Mediterranean ('chromosome-level genotyping'). The gene-level analysis revealed a statistically significant allele frequency differences between farmed and wild populations on both genes in each species. This finding was partially supported by the chromosome-level analysis, identifying highly differentiated regions may be involved in the domestication process at varying distances from the candidate genes. Noteworthy genomic features were found, such as a CpG island in gilthead seabream and novel candidate genes in European seabass, warranting further investigation. These findings support a putative role of vgll3 and six6 in the maturation and growth of gilthead seabream and European seabass, emphasizing the need for further research on their conserved function.

Prevalence of SOD1 allele associated with degenerative myelopathy in canine population in Greece.

Canine degenerative myelopathy (CDM) is a late-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (c.118G > A). The purpose of this study was to determine the genotype and allele frequencies of this mutation in 108 dogs, mainly in Belgian Malinois and German Shepherd dogs with (CDM-affected group) and without CDM clinical symptoms (control group) in Greece. Genotyping of the c.118G > A mutation was possible by Sanger sequencing and PCR-RFLP. The observed genotype frequencies for the control group were 89.4% for the homozygous (G/G), 9.6% for the heterozygous (A/G), and 0.96% for the homozygous mutant (A/A) allele. The mutant allele was not common in the Belgian Malinois dogs (allele frequency = 0.029), but quite common in the German Shepherd dogs (allele frequency = 0.138). In the CDM affected group, all 4 dogs were homozygous for the mutant allele. These frequencies were close to those expected, indicating no significant departure from Hardy-Weinberg equilibrium. A strong but not statistically significant association between the mutant allele and CDM was observed. A previously identified deletion upstream of the mutation of interest was found at a high frequency (0.361) in the population.

Fire and Rhizosphere Effects on Bacterial Co-Occurrence Patterns.

Fires are common in Mediterranean soils and constitute an important driver of their evolution. Although fire effects on vegetation dynamics are widely studied, their influence on the assembly rules of soil prokaryotes in a small-scale environment has attracted limited attention. In the present study, we reanalyzed the data from Aponte et al. (2022) to test whether the direct and/or indirect effects of fire are reflected in the network of relationships among soil prokaryotes in a Chilean sclerophyllous ecosystem. We focused on bacterial (genus and species level) co-occurrence patterns in the rhizospheres and bulk soils in burned and unburned plots. Four soils were considered: bulk-burnt (BB), bulk-unburnt (BU), rhizosphere-burnt (RB), and rhizosphere-unburnt (RU). The largest differences in network parameters were recorded between RU and BB soils, while RB and BU networks exhibited similar values. The network in the BB soil was the most compact and centralized, while the RU network was the least connected, with no central nodes. The robustness of bacterial communities was enhanced in burnt soils, but this was more pronounced in BB soil. The mechanisms mainly responsible for bacterial community structure were stochastic in all soils, whether burnt or unburnt; however, communities in RB were much more stochastic than in RU.

An experimental test of the growth rate hypothesis as a predictive framework for microevolutionary adaptation.

The growth rate hypothesis (GRH) posits that the relative body phosphorus content of an organism is positively related to somatic growth rate, as protein synthesis, which is necessary for growth, requires P-rich rRNA. This hypothesis has strong support at the interspecific level. Here, we explore the use of the GRH to predict microevolutionary responses in consumer body stoichiometry. For this, we subjected populations of the rotifer Brachionus calyciflorus to selection for fast population growth rate (PGR) in P-rich (HPF) and P-poor (LPF) food environments. With common garden transplant experiments, we demonstrate that in HP populations evolution toward increased PGR was concomitant with an increase in relative phosphorus content. In contrast, LP populations evolved higher PGR without an increase in relative phosphorus content. We conclude that the GRH has the potential to predict microevolutionary change, but that its application is contingent on the environmental context. Our results highlight the potential of cryptic evolution in determining the performance response of populations to elemental limitation of their food resources.

Cytosine methylation patterns suggest a role of methylation in plastic and adaptive responses to temperature in European grayling (Thymallus thymallus) populations.

Temperature is a key environmental parameter affecting both the phenotypes and distributions of organisms, particularly ectotherms. Rapid organismal responses to thermal environmental changes have been described for several ectotherms; however, the underlying molecular mechanisms often remain unclear. Here, we studied whole genome cytosine methylation patterns of European grayling (Thymallus thymallus) embryos from five populations with contemporary adaptations of early life history traits at either 'colder' or 'warmer' spawning grounds. We reared fish embryos in a common garden experiment using two temperatures that resembled the 'colder' and 'warmer' conditions of the natal natural environments. Genome-wide methylation patterns were similar in populations originating from colder thermal origin subpopulations, whereas single nucleotide polymorphisms uncovered from the same data identified strong population structure among isolated populations, but limited structure among interconnected populations. This was surprising because the previously studied gene expression response among populations was mostly plastic, and mainly influenced by the developmental temperature. These findings support the hypothesis of the magnified role of epigenetic mechanisms in modulating plasticity. The abundance of consistently changing methylation loci between two warmer-to-colder thermal origin population pairs suggests that local adaptation has shaped the observed methylation patterns. The dynamic nature of the methylomes was further highlighted by genome-wide and site-specific plastic responses. Our findings support both the presence of a plastic response in a subset of CpG loci, and the evolutionary role of methylation divergence between populations adapting to contrasting thermal environments.

Rotifers from inland water bodies of continental Ecuador and Galápagos Islands: An updated checklist.

We present an updated checklist of the rotifer fauna from inland aquatic habitats in continental Ecuador and the Galápagos islands based on published rotifer records found in the literature. The checklist summarizes the status of the current taxonomic and faunistic knowledge on rotifers in Ecuador, updates the nomenclature, and reports the regions where each species has been found in the country. A total of 287 valid species (269 monogononts and 18 bdelloids was found. The Ecuadorian region with the highest number of records was Amazonia (228) followed by the Coastal region (139) and the Andes (121), whereas in the Galápagos Archipelago only 40 species have been recorded. Studies of the rotifer fauna of the areas are scarce and quite recent, pointing to important gaps in our knowledge on taxonomy and biogeography of Ecuadorian rotifers.

The Chromosome-Level Genome Assembly of European Grayling Reveals Aspects of a Unique Genome Evolution Process Within Salmonids.

Salmonids represent an intriguing taxonomical group for investigating genome evolution in vertebrates due to their relatively recent last common whole genome duplication event, which occurred between 80 and 100 million years ago. Here, we report on the chromosome-level genome assembly of European grayling (Thymallus thymallus), which represents one of the earliest diverged salmonid subfamilies. To achieve this, we first generated relatively long genomic scaffolds by using a previously published draft genome assembly along with long-read sequencing data and a linkage map. We then merged those scaffolds by applying synteny evidence from the Atlantic salmon (Salmo salar) genome. Comparisons of the European grayling genome assembly to the genomes of Atlantic salmon and Northern pike (Esox lucius), the latter used as a nonduplicated outgroup, detailed aspects of the characteristic chromosome evolution process that has taken place in European grayling. While Atlantic salmon and other salmonid genomes are portrayed by the typical occurrence of numerous chromosomal fusions, European grayling chromosomes were confirmed to be fusion-free and were characterized by a relatively large proportion of paracentric and pericentric inversions. We further reported on transposable elements specific to either the European grayling or Atlantic salmon genome, on the male-specific sdY gene in the European grayling chromosome 11A, and on regions under residual tetrasomy in the homeologous European grayling chromosome pairs 9A-9B and 25A-25B. The same chromosome pairs have been observed under residual tetrasomy in Atlantic salmon and in other salmonids, suggesting that this feature has been conserved since the subfamily split.

Reverse taxonomy applied to the Brachionus calyciflorus cryptic species complex: Morphometric analysis confirms species delimitations revealed by molecular phylogenetic analysis and allows the (re)description of four species.

The discovery and exploration of cryptic species have been profoundly expedited thanks to developments in molecular biology and phylogenetics. In this study, we apply a reverse taxonomy approach to the Brachionus calyciflorus species complex, a commonly studied freshwater monogonont rotifer. By combining phylogenetic, morphometric and morphological analyses, we confirm the existence of four cryptic species that have been recently suggested by a molecular study. Based on these results and according to an exhaustive review of the taxonomic literature, we name each of these four species and provide their taxonomic description alongside a diagnostic key.

The genome of the freshwater monogonont rotifer Brachionus calyciflorus.

Monogononta is the most speciose class of rotifers, with more than 2,000 species. The monogonont genus Brachionus is widely distributed at a global scale, and a few of its species are commonly used as ecological and evolutionary models to address questions related to aquatic ecology, cryptic speciation, evolutionary ecology, the evolution of sex and ecotoxicology. With the importance of Brachionus species in many areas of research, it is remarkable that the genome has not been characterized. This study aims to address this lacuna by presenting, for the first time, the whole-genome assembly of the freshwater species Brachionus calyciflorus. The total length of the assembled genome was 129.6 Mb, with 1,041 scaffolds. The N50 value was 786.6 kb, and the GC content was 24%. A total of 16,114 genes were annotated with repeat sequences, accounting for 21% of the assembled genome. This assembled genome may form a basis for future studies addressing key questions on the evolution of monogonont rotifers. It will also provide the necessary molecular resources to mechanistically investigate ecophysiological and ecotoxicological responses.

Modularity Facilitates Flexible Tuning of Plastic and Evolutionary Gene Expression Responses during Early Divergence.

Gene expression changes have been recognized as important drivers of adaptation to changing environmental conditions. Little is known about the relative roles of plastic and evolutionary responses in complex gene expression networks during the early stages of divergence. Large gene expression data sets coupled with in silico methods for identifying coexpressed modules now enable systems genetics approaches also in nonmodel species for better understanding of gene expression responses during early divergence. Here, we combined gene coexpression analyses with population genetics to separate plastic and population (evolutionary) effects in expression networks using small salmonid populations as a model system. We show that plastic and population effects were highly variable among the six identified modules and that the plastic effects explained larger proportion of the total eigengene expression than population effects. A more detailed analysis of the population effects using a QST - FST comparison across 16,622 annotated transcripts revealed that gene expression followed neutral expectations within modules and at the global level. Furthermore, two modules showed enrichment for genes coding for early developmental traits that have been previously identified as important phenotypic traits in thermal responses in the same model system indicating that coexpression analysis can capture expression patterns underlying ecologically important traits. We suggest that module-specific responses may facilitate the flexible tuning of expression levels to local thermal conditions. Overall, our study indicates that plasticity and neutral evolution are the main drivers of gene expression variance in the early stages of thermal adaptation in this system.

Integrative Taxonomy Recognizes Evolutionary Units Despite Widespread Mitonuclear Discordance: Evidence from a Rotifer Cryptic Species Complex.

Mitonuclear discordance across taxa is increasingly recognized as posing a major challenge to species delimitation based on DNA sequence data. Integrative taxonomy has been proposed as a promising framework to help address this problem. However, we still lack compelling empirical evidence scrutinizing the efficacy of integrative taxonomy in relation to, for instance, complex introgression scenarios involving many species. Here, we report remarkably widespread mitonuclear discordance between about 15 mitochondrial and 4 nuclear Brachionus calyciflorus groups identified using different species delimitation approaches. Using coalescent-, Bayesian admixture-, and allele sharing-based methods with DNA sequence or microsatellite data, we provide strong evidence in support of hybridization as a driver of the observed discordance. We then describe our combined molecular, morphological, and ecological approaches to resolving phylogenetic conflict and inferring species boundaries. Species delimitations based on the ITS1 and 28S nuclear DNA markers proved a more reliable predictor of morphological variation than delimitations using the mitochondrial COI gene. A short-term competition experiment further revealed systematic differences in the competitive ability between two of the nuclear-delimited species under six different growth conditions, independent of COI delimitations; hybrids were also observed. In light of these findings, we discuss the failure of the COI marker to estimate morphological stasis and morphological plasticity in the B. calyciflorus complex. By using B. calyciflorus as a representative case, we demonstrate the potential of integrative taxonomy to guide species delimitation in the presence of mitonuclear phylogenetic conflicts.

Plastic and Evolutionary Gene Expression Responses Are Correlated in European Grayling (Thymallus thymallus) Subpopulations Adapted to Different Thermal Environments.

Understanding how populations adapt to changing environmental conditions is a long-standing theme in evolutionary biology. Gene expression changes have been recognized as an important driver of local adaptation, but relatively little is known regarding the direction of change and in particular, about the interplay between plastic and evolutionary gene expression. We have previously shown that the gene expression profiles of European grayling (Thymallus thymallus) populations inhabiting different thermal environments include both plastic and evolutionary components. However, whether the plastic and evolutionary responses were in the same direction was not investigated in detail, nor was the identity of the specific genes involved. In this study, we show that the plastic changes in protein expression in response to different temperatures are highly correlated with the evolutionary response in grayling subpopulations adapted to different thermal environments. This finding provides preliminary evidence that the plastic response most likely facilitates adaptation during the early phases of colonization of thermal environments. The proteins that showed significant changes in expression level between warm and cold temperature treatments were mostly related to muscle development, which is consistent with earlier findings demonstrating muscle mass differentiation between cold and warm grayling populations.

Rapid adaptation of herbivore consumers to nutrient limitation: eco-evolutionary feedbacks to population demography and resource control.

Humans alter biogeochemical cycles of essential elements such as phosphorus (P). Prediction of ecosystem consequences of altered elemental cycles requires integration of ecology, evolutionary biology and the framework of ecological stoichiometry. We studied micro-evolutionary responses of a herbivorous rotifer to P-limited food and the potential consequences for its population demography and for ecosystem properties. We subjected field-derived, replicate rotifer populations to P-deficient and P-replete algal food, and studied adaptation in common garden transplant experiments after 103 and 209 days of selection. When fed P-limited food, populations with a P-limitation selection history suffered 37% lower mortality, reached twice the steady state biomass, and reduced algae by 40% compared to populations with a P-replete selection history. Adaptation involved no change in rotifer elemental composition but reduced investment in sex. This study demonstrates potentially strong eco-evolutionary feedbacks from shifting elemental balances to ecosystem properties, including grazing pressure and the ratio of grazer:producer biomass.

Updating the description and taxonomic status of Brachionus sessilis Varga, 1951 (Rotifera: Brachionidae) based on detailed morphological analysis and molecular data.

Brachionus sessilis Varga, 1951 is an epizoic rotifer living exclusively on cladocerans of the genus Diaphanosoma. Current taxonomic knowledge relies solely on limited morphological information, whereas there is no type material. Here, we aim to resolve issues concerning its morphology and taxonomy using both morphological and genetic characters on material sampled from Lake Balaton (Hungary), as well as Lake Doirani (Greece) that was selected for comparison purposes. Biometrical analysis was based on extensive lorica measurements. Phylogenetic reconstruction was based on DNA sequence information of the mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA gene regions as well as of the nuclear internal transcribed spacer 1 (ITS1). Well-supported evidence for substantial differentiation of B. sessilis from its closest phylogenetic relatives supports its species-rank status. Our phylogenetic analysis suggests a highly supported clade encompassing B. sessilis and another epizoic rotifer, namely B. rubens. 

Gene pleiotropy constrains gene expression changes in fish adapted to different thermal conditions.

Understanding the factors that shape the evolution of gene expression is a central goal in biology, but the molecular mechanisms behind this remain controversial. A related major goal is ascertaining how such factors may affect the adaptive potential of a species or population. Here we demonstrate that temperature-driven gene expression changes in fish adapted to differing thermal environments are constrained by the level of gene pleiotropy estimated by either the number of protein interactions or gene biological processes. Genes with low pleiotropy levels were the main drivers of both plastic and evolutionary global expression profile changes, while highly pleiotropic genes had limited expression response to temperature treatment. Our study provides critical insights into the molecular mechanisms by which natural populations can adapt to changing environments. In addition to having important implications for climate change adaptation, these results suggest that gene pleiotropy should be considered more carefully when interpreting expression profiling data.

Proteome variance differences within populations of European whitefish (Coregonus lavaretus) originating from contrasting salinity environments.

Variation in gene expression is an important component of the phenotypic differences observed in nature. Gene expression variance across biological groups and environmental conditions has been studied extensively and has revealed specific genes and molecular mechanisms of interest. However, little is known regarding the importance of within-population gene expression variation to environmental adaptation. To address this issue, we quantified the proteomes of individuals of European whitefish (Coregonus lavaretus) from populations that have previously been shown to have adapted during early development to freshwater and brackishwater salinity environments. Using MS-based label-free proteomics, we studied 955 proteins in eight hatch-stage fish embryos from each population that had been reared in either freshwater or brackishwater salinity conditions. By comparing the levels of within-population protein expression variance over individuals and per protein between populations, we found that fish embryos from the population less affected by salinity level had also markedly higher levels of expression variance. Gene Ontologies and molecular pathways associated with osmoregulation showed the most significant difference of within-population proteome variance between populations. Several new candidate genes for salinity adaptation were identified, emphasising the added value of combining assessments of within-population gene expression variation with standard gene expression analysis practices for better understanding the mechanisms of environmental adaptation. BIOLOGICAL SIGNIFICANCE: We demonstrate the benefits of studying within-population gene expression variance together with more typical methods of gene expression profiling. Proteome variance differences within European whitefish populations originating from different salinity environments allowed us to identify several new candidate genes for salinity adaptation in teleost fish and generate many further hypotheses to be tested. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

A proteomics approach reveals divergent molecular responses to salinity in populations of European whitefish (Coregonus lavaretus).

Osmoregulation is a vital physiological function for fish, as it helps maintain a stable intracellular concentration of ions in environments of variable salinities. We focused on a primarily freshwater species, the European whitefish (Coregonus lavaretus), to investigate the molecular mechanisms underlying salinity tolerance and examine whether these mechanisms differ between genetically similar populations that spawn in freshwater vs. brackishwater environments. A common garden experiment involving 27 families in two populations and five salinity treatments together with a large-scale, high-resolution mass spectrometry experiment that quantified 1500 proteins was conducted to assess phenotypic and proteomic responses during early development, from fertilization until hatching, in the studied populations. The populations displayed drastically different phenotypic and proteomic responses to salinity. Freshwater-spawning whitefish showed a significantly higher mortality rate in higher salinity treatments. Calcium, an ion involved in osmotic stress sensing, had a central role in the observed proteomic responses. Brackishwater-spawning fish were capable of viable osmoregulation, which was modulated by cortisol, an important seawater-adaptation hormone in teleost fish. Several proteins were identified to play key roles in osmoregulation, most importantly a highly conserved cytokine, tumour necrosis factor, whereas calcium receptor activities were associated with salinity adaptation. These results imply that individuals from these populations are most likely adapted to their local environments, even though the baseline level of genetic divergence between them is low (F(ST)=0.049). They also provide clues for choosing candidate loci for studying the molecular basis of salinity adaptation in other species. Further, our approach provides an example of how proteomic methods can be successfully used to obtain novel insights into the molecular mechanisms behind adaptation in non-model organism.

Is polyploidy a persevering accident or an adaptive evolutionary pattern? The case of the brine shrimp Artemia.

Asexual organisms are confronted with substantial drawbacks, both immediate and delayed, threatening their evolutionary persistence. Yet, genetic associations with asexuality may refresh the gene pool promoting adaptation of clonal lineages; polyploidy is one of them. Parthenogenesis itself and/or polyploidy are responsible for the maintenance and spread of clones in Artemia, a sexual-asexual genus of halophilic anostracans. We applied flow cytometry, microsatellite genotyping, and mtDNA sequencing to 23 asexual populations. Artemia parthenogens have evolved multiple times either through hybridization or spontaneously. Nine out of 23 populations contained clones of mixed ploidy (2n, 3n, 4n). Most clones were diploid (20/31) while two and nine clones were triploid and tetraploid, respectively. Apomictic triploids and tetraploids formed two distinct groups of low genetic diversity compared with the more divergent automictic diploids. Polyploidy is also polyphyletic in Artemia, with triploids and tetraploids having independent origins from different sexual ancestors. We discern a pattern of geographical parthenogenesis with all clonal groups being more widespread than their closest sexuals. In favour of a specialist model, asexual diploids are restricted to single locations and are strikingly segregated from generalist triploids and tetraploids occupying a variety of sites. This is a rare pattern of mixed life-history strategies within an asexual complex.

Proteomic profiling of early life stages of European grayling (Thymallus thymallus).

Salmonids are teleost fish of profound research and economic interest. Embryonic development is a key aspect of salmonid biology that can be critically affected by environmental parameters. Still, their proteome during embryogenesis remains largely unexplored. This study investigates the proteome of the eyed-egg and hatching stages of embryonic development of a salmonid species, European grayling (Thymallus thymallus), using a shotgun proteomic approach. To deal with limited grayling protein resources, the generated spectra were compared against an all-salmonid database using search and multiple protein grouping algorithms to infer identifications. Functional enrichment analysis was carried out at different levels (gene ontologies, pathways, networks) using zebrafish as a reference genome. A total of 213 and 239 proteins were confidently detected in eyed and hatching stages, respectively. Cell cycle, energy, and protein metabolism were the major processes common to both stages. Nuclear activity and brain and eye development were the predominant functions in the eyed-stage proteome, while central nervous system, skeletal muscle, and heart development prevailed in the hatching stage. Overall, this research constitutes the first effort to describe the proteome during embryogenesis in grayling or any salmonid species. It also presents a systematic approach by which existing resources can enable proteome research in salmonids.