Microbiome and epigenetic variation in wild fish with low genetic diversity.

Non-genetic sources of phenotypic variation, such as the epigenome and the microbiome, could be important contributors to adaptive variation for species with low genetic diversity. However, little is known about the complex interaction between these factors and the genetic diversity of the host, particularly in wild populations. Here, we examine the skin microbiome composition of two closely-related mangrove killifish species with different mating systems (self-fertilising and outcrossing) under sympatric and allopatric conditions. This allows us to partition the influence of the genotype and the environment on their microbiome and (previously described) epigenetic profiles. We find the diversity and community composition of the skin microbiome are strongly shaped by the environment and, to a lesser extent, by species-specific influences. Heterozygosity and microbiome alpha diversity, but not epigenetic variation, are associated with the fluctuating asymmetry of traits related to performance (vision) and behaviour (aggression). Our study identifies that a proportion of the epigenetic diversity and microbiome differentiation is unrelated to genetic variation, and we find evidence for an associative relationship between microbiome and epigenetic diversity in these wild populations. This suggests that both mechanisms could potentially contribute to variation in species with low genetic diversity.

Inland navigation and land use interact to impact European freshwater biodiversity.

Inland navigation in Europe is proposed to increase in the coming years, being promoted as a low-carbon form of transport. However, we currently lack knowledge on how this would impact biodiversity at large scales and interact with existing stressors. Here we addressed this knowledge gap by analysing fish and macroinvertebrate community time series across large European rivers comprising 19,592 observations from 4,049 sampling sites spanning the past 32 years. We found ship traffic to be associated with biodiversity declines, that is, loss of fish and macroinvertebrate taxonomic richness, diversity and trait richness. Ship traffic was also associated with increases in taxonomic evenness, which, in concert with richness decreases, was attributed to losses in rare taxa. Ship traffic was especially harmful for benthic taxa and those preferring slow flows. These effects often depended on local land use and riparian degradation. In fish, negative impacts of shipping were highest in urban and agricultural landscapes. Regarding navigation infrastructure, the negative impact of channelization on macroinvertebrates was evident only when riparian degradation was also high. Our results demonstrate the risk of increasing inland navigation on freshwater biodiversity. Integrative waterway management accounting for riparian habitats and landscape characteristics could help to mitigate these impacts.

Over 200,000 kilometers of free-flowing river habitat in Europe is altered due to impoundments.

European rivers are disconnected by more than one million man-made barriers that physically limit aquatic species migration and contribute to modification of freshwater habitats. Here, a Conceptual Habitat Alteration Model for Ponding is developed to aid in evaluating the effects of impoundments on fish habitats. Fish communities present in rivers with low human impact and their broad environmental settings enable classification of European rivers into 15 macrohabitat types. These classifications, together with the estimated fish sensitivity to alteration of their habitat are used for assessing the impacts of six main barrier types (dams, weirs, sluices, culverts, fords, and ramps). Our results indicate that over 200,000 km or 10% of previously free-flowing river habitat has been altered due to impoundments. Although they appear less frequently, dams, weirs and sluices cause much more habitat alteration than the other types. Their impact is regionally diverse, which is a function of barrier height, type and density, as well as biogeographical location. This work allows us to foresee what potential environmental gain or loss can be expected with planned barrier management actions in rivers, and to prioritize management actions.

Different housing conditions for zebrafish: What are the effects?

Zebrafish is a popular experimental model in several research areas but little is known about the effects of using different strains or housing conditions. Poor control of genetic background and housing conditions could affect experimental results and data reproducibility. Here we investigated the effects of two possible sources of variation on zebrafish behaviour: fish origin and environmental parameters (light intensity, water temperature and noise). Zebrafish behaviour was then examined using the 'novel tank test', one of the most common paradigms used to assess anxiety-like behaviours in zebrafish. Our results show that an increase in light intensity alters fish behaviour, particularly freezing duration and distance from the bottom of the tank, indicating increased anxiety. Swimming activity increased at the lowest temperature (25 °C). However, different levels of background noise did not cause any significant changes in behaviour. Differences were also found between zebrafish strains and populations: while the AB strain from laboratory 1 was minimally influenced by variation in holding conditions, the AB strain from laboratory 2 was highly affected by changes in temperature, light, and background noise. Our study shows that variation in strains and holding conditions can significantly influence the results of behavioural testing and should be carefully considered in the experimental design and properly reported to improve data interpretation and reproducibility.

Impacts of existing and planned hydropower dams on river fragmentation in the Balkan Region.

The Balkan region has some of the best conserved rivers in Europe, but is also the location of ~3000 planned hydropower dams that are expected to help decarbonise energy production. A conflict between policies that promote renewable hydropower and those that prioritise river conservation has ensued, which can only be resolved with the help of reliable information. Using ground-truthed barrier data, we analysed the extent of current longitudinal river fragmentation in the Balkan region and simulated nine dam construction scenarios that varied depending on the number, location and size of the planned dams. Balkan rivers are currently fragmented by 83,017 barriers and have an average barrier density of 0.33 barriers/km after correcting for barrier underreporting; this is 2.2 times lower than the mean barrier density found across Europe and serves to highlight the relatively unfragmented nature of these rivers. However, our analysis shows that all simulated dam construction scenarios would result in a significant loss of connectivity compared to existing conditions. The largest loss of connectivity (-47 %), measured as reduction in barrier-free length, would occur if all planned dams were built, 20 % of which would impact on protected areas. The smallest loss of connectivity (-8 %) would result if only large dams (>10 MW) were built. In contrast, building only small dams (<10 MW) would cause a 45 % loss of connectivity while only contributing 32 % to future hydropower capacity. Hence, the construction of many small hydropower plants will cause a disproportionately large increase in fragmentation that will not be accompanied by a corresponding increase in hydropower. At present, hydropower development in the Balkan rivers does not require Strategic Environmental Assessment, and does not consider cumulative impacts. We encourage planners and policy makers to explicitly consider trade-offs between gains in hydropower and losses in river connectivity at the river basin scale.

Additive and non-additive epigenetic signatures of natural hybridization between fish species with different mating systems.

Hybridization is a major source of evolutionary innovation. In plants, epigenetic mechanisms can help to stabilize hybrid genomes and contribute to reproductive isolation, but the relationship between genetic and epigenetic changes in animal hybrids is unclear. We analysed the relationship between genetic background and methylation patterns in natural hybrids of two genetically divergent fish species with different mating systems, Kryptolebias hermaphroditus (self-fertilizing) and K. ocellatus (outcrossing). Co-existing parental species displayed highly distinct genetic (SNPs) and methylation patterns (37,000 differentially methylated cytosines). Hybrids had predominantly intermediate methylation patterns (88.5% of the sites) suggesting additive effects, as expected from hybridization between genetically distant species. The large number of differentially methylated cytosines between hybrids and parental species (n = 5,800) suggests that hybridization may play a role in increasing genetic and epigenetic variation. Although most of the observed epigenetic variation was additive and had a strong genetic component, we also found a small percentage of non-additive, potentially stochastic, methylation differences that might act as an evolutionary bet-hedging strategy and increase fitness under environmental instability.

Phylogenomics reveals extensive introgression and a case of mito-nuclear discordance in the killifish genus Kryptolebias.

Introgression is a widespread evolutionary process leading to phylogenetic inconsistencies among distinct parts of the genomes, particularly between mitochondrial and nuclear-based phylogenetic reconstructions (e.g., mito-nuclear discordances). Here, we used mtDNA and genome-wide nuclear sites to provide the first phylogenomic-based hypothesis on the evolutionary relationships within the killifish genus Kryptolebias. In addition, we tested for evidence of past introgression in the genus given the multiple reports of undergoing hybridization between its members. Our mtDNA phylogeny generally agreed with the relationships previously proposed for the genus. However, our reconstruction based on nuclear DNA revealed an unknown lineage - Kryptolebias sp. 'ESP' - as the sister group of the self-fertilizing mangrove killifishes, K. marmoratus and K. hermaphroditus. All individuals sequenced of Kryptolebias sp. 'ESP' had the same mtDNA haplotype commonly observed in K. hermaphroditus, demonstrating a clear case of mito-nuclear discordance. Our analysis further confirmed extensive history of introgression between Kryptolebias sp. 'ESP' and K. hermaphroditus. Population genomics analyses indicate no current gene flow between the two lineages, despite their current sympatry and history of introgression. We also confirmed introgression between other species pairs in the genus that have been recently reported to form hybrid zones. Overall, our study provides a phylogenomic reconstruction covering most of the Kryptolebias species, reveals a new lineage hidden in a case of mito-nuclear discordance, and provides evidence of multiple events of ancestral introgression in the genus. These findings underscore the importance of investigating different genomic information in a phylogenetic framework, particularly in taxa where introgression is common as in the sexually diverse mangrove killifishes.

Operational methods for prioritizing the removal of river barriers: Synthesis and guidance.

Barrier removal can be an efficient method to restore river continuity but resources available for defragmenting rivers are limited and a prioritization strategy is needed. We review methods for prioritizing barriers for removal and report on a survey asking practitioners which barrier prioritization methods they use. Opportunities for barrier removal depend to a large extent on barrier typology, as this dictates where barriers are normally located, their size, age, condition, and likely impacts. Crucially, river fragmentation depends chiefly on the number and location of barriers, not on barrier size, while the costs of barrier removal typically increase with barrier height. Acting on many small barriers will often be more cost-efficient than acting on fewer larger structures. Barriers are not randomly distributed and a small proportion of barriers have a disproportionately high impact on fragmentation, therefore targeting these 'fragmentizers' can result in substantial gains in connectivity. Barrier prioritization methods can be grouped into six main types depending on whether they are reactive or proactive, whether they are applied at local or larger spatial scales, and whether they employ an informal or a formal approach. While mathematical optimization sets the gold standard for barrier prioritization, a hybrid approach that explicitly considers uncertainties and opportunities is likely to be the most effective. The effectiveness of barrier removal can be compromised by inaccurate stream networks, erroneous barrier coordinates, and underestimation of barrier numbers. Such uncertainties can be overcome by ground truthing via river walkovers and predictive modelling, but the cost of collecting additional information must be weighed against the cost of inaction. To increase the success of barrier removal projects, we recommend that barriers considered for removal fulfill four conditions: (1) their removal will bring about a meaningful gain in connectivity; (2) they are cost-effective to remove; (3) they will not cause significant or lasting environmental damage, and (4) they are obsolete structures. Mapping barrier removal projects according to the three axes of opportunities, costs, and gains can help locate any 'low hanging fruit.'

MHC class I-α population differentiation in a commercial fish, the European sea bass (Dicentrarchus labrax).

Identifying population structuring in highly fecund marine species with high dispersal rates is challenging, but critical for conservation and stock delimitation for fisheries management. European sea bass (Dicentrarchus labrax) is a commercial species of fisheries and aquaculture relevance whose stocks are declining in the North Atlantic, despite management measures to protect them and identifying their fine population structure is needed for managing their exploitation. As for other marine fishes, neutral genetic markers indicate that eastern Atlantic sea bass form a panmictic population and is currently managed as arbitrarily divided stocks. The genes of the major histocompatibility complex (MHC) are key components of the adaptive immune system and ideal candidates to assess fine structuring arising from local selective pressures. We used Illumina sequencing to characterise allelic composition and signatures of selection at the MHC class I-α region of six D. labrax populations across the Atlantic range. We found high allelic diversity driven by positive selection, corresponding to moderate supertype diversity, with 131 alleles clustering into four to eight supertypes, depending on the Bayesian information criterion threshold applied, and a mean number of 13 alleles per individual. Alleles could not be assigned to particular loci, but private alleles allowed us to detect regional genetic structuring not found previously using neutral markers. Our results suggest that MHC markers can be used to detect cryptic population structuring in marine species where neutral markers fail to identify differentiation. This is particularly critical for fisheries management, and of potential use for selective breeding or identifying escapes from sea farms.

Against the Odds: Hybrid Zones between Mangrove Killifish Species with Different Mating Systems.

Different mating systems are expected to affect the extent and direction of hybridization. Due to the different levels of sexual conflict, the weak inbreeder/strong outbreeder (WISO) hypothesis predicts that gametes from self-incompatible (SI) species should outcompete gametes from self-compatible (SC) ones. However, other factors such as timing of selfing and unilateral incompatibilities may also play a role on the direction of hybridization. In addition, differential mating opportunities provided by different mating systems are also expected to affect the direction of introgression in hybrid zones involving outcrossers and selfers. Here, we explored these hypotheses with a unique case of recent hybridization between two mangrove killifish species with different mating systems, Kryptolebias ocellatus (obligately outcrossing) and K. hermaphroditus (predominantly self-fertilizing) in two hybrid zones in southeast Brazil. Hybridization rates were relatively high (~20%), representing the first example of natural hybridization between species with different mating systems in vertebrates. All F1 individuals were sired by the selfing species. Backcrossing was small, but mostly asymmetrical with the SI parental species, suggesting pattern commonly observed in plant hybrid zones with different mating systems. Our findings shed light on how contrasting mating systems may affect the direction and extent of gene flow between sympatric species, ultimately affecting the evolution and maintenance of hybrid zones.

Early life stress causes persistent impacts on the microbiome of Atlantic salmon.

Farmed fish are commonly exposed to stress in intensive aquaculture systems, often leading to immune impairment and increased susceptibility to disease. As microbial communities associated with the gut and skin are vital to host health and disease resilience, disruption of microbiome integrity could contribute to the adverse consequences of stress exposure. Little is known about how stress affects the fish microbiome, especially during sensitive early life stages when initial colonisation and proliferation of host-associated microbial communities take place. Therefore, we compared the effects of two aquaculture-relevant early-life stressors on the gut and skin microbiome of Atlantic salmon fry (four months post hatching) using 16S rRNA amplicon sequencing. Acute cold stress applied during late embryogenesis had a pronounced, lasting effect on the structure of the skin microbiome, as well as a less consistent effect on the gut microbiome. Follow-up targeted qPCR assays suggested that this is likely due to disruption of the egg shell microbial communities at the initial stages of microbiome colonisation, with persistent effects on community structure. In contrast, chronic post hatching stress altered the structure of the gut microbiome, but not that of the skin. Both types of stress promoted similar Gammaproteobacteria ASVs, particularly within the genera Acinetobacter and Aeromonas, which include several important opportunistic fish pathogens. Our results demonstrate the sensitivity of the salmon microbiome to environmental stressors during early life, with potential associated health impacts on the host. We also identified common signatures of stress in the salmon microbiome, which may represent useful microbial stress biomarkers.

SNP analyses and acoustic tagging reveal multiple origins and widespread dispersal of invasive brown trout in the Falkland Islands.

Biological invasions are important causes of biodiversity loss, particularly in remote islands. Brown trout (Salmo trutta) have been widely introduced throughout the Southern Hemisphere, impacting endangered native fauna, particularly galaxiid fishes, through predation and competition. However, due to their importance for sport fishing and aquaculture farming, attempts to curtail the impacts of invasive salmonids have generally been met with limited support and the best prospects for protecting native galaxiids is to predict where and how salmonids might disperse. We analysed 266 invasive brown trout from 14 rivers and ponds across the Falkland Islands as well as 32 trout from three potential source populations, using a panel of 592 single nucleotide polymorphisms (SNPs) and acoustic tagging, to ascertain their origins and current patterns of dispersal. We identified four genetically distinct clusters with high levels of genetic diversity and low admixture, likely reflecting the different origins of the invasive brown trout populations. Our analysis suggests that many trout populations in the Falklands may have originated from one of the donor populations analysed (River Wey). The highest genetic diversity was observed in the rivers with the greatest number of introductions and diverse origins, while the lowest diversity corresponded to a location without documented introductions, likely colonized by natural dispersal. High levels of gene flow indicated widespread migration of brown trout across the Falkland Islands, likely aided by anadromous dispersal. This is supported by data from acoustically tagged fish, three of which were detected frequently moving between two rivers ~26 km apart. Our results suggest that, without containment measures, brown trout may invade the last remaining refuges for the native endangered Aplochiton spp. We provide new insights into the origin and dispersal of invasive brown trout in the Falkland Islands that can pave the way for a targeted approach to limit their impact on native fish fauna.

Impacts of large and small barriers on fish assemblage composition assessed using environmental DNA metabarcoding.

River fragmentation caused by instream barriers is a leading cause of biodiversity loss, particularly for freshwater migratory fish, the vertebrate group that has suffered the steepest decline. However, most studies have tended to focus on the impacts of large dams on only a few taxa. We estimated the cumulative impact of both large and small barriers on fish species richness and relative abundance along an altitudinal gradient in the main stem of the River Allier (France). Using eDNA metabarcoding, we identified 24 fish zero-radius operational taxonomic units (zOTUs), corresponding to 26 species distributed along the main stem of the river. Elevation explained the greatest amount of variation in fish distribution, together with average flow, barrier density and its interaction with cumulative barrier height. Based on eDNA, the largest discontinuity in species richness was not related to the location of Poutès, the largest dam in the system, but located downstream from it. Our results indicate that, in addition to the more obvious effects of large dams on migratory fish such as the Atlantic salmon, the cumulative effects of small barriers can have widespread impacts on fish species richness and relative abundance, which should not be overlooked. We suggest that, as for other fragmented rivers, acting on numerous small barriers might bring about greater benefits in fish species richness than focusing only on the largest dams.

Quantitative assessment of fish larvae community composition in spawning areas using metabarcoding of bulk samples.

Accurate assessment of larval community composition in spawning areas is essential for fisheries management and conservation but is often hampered by the cryptic nature of many larvae, which renders them difficult to identify morphologically. Metabarcoding is a rapid and cost-effective method to monitor early life stages for management and environmental impact assessment purposes but its quantitative capability is under discussion. We compared metabarcoding with traditional morphological identification to evaluate taxonomic precision and reliability of abundance estimates, using 332 fish larvae from multinet hauls (0-50 m depth) collected at 14 offshore sampling sites in the Irish and Celtic seas. To improve quantification accuracy (relative abundance estimates), the amount of tissue for each specimen was standardized and mitochondrial primers (12S gene) with conserved binding sites were used. Relative family abundance estimated from metabarcoding reads and morphological assessment were positively correlated, as well as taxon richness (RS  = 0.81, P = 0.007) and diversity (RS  = 0.90, P = 0.002). Spatial patterns of community composition did not differ significantly between metabarcoding and morphological assessments. Our results show that DNA metabarcoding of bulk tissue samples can be used to monitor changes in fish larvae abundance and community composition. This represents a feasible, efficient, and faster alternative to morphological methods that can be applied to terrestrial and aquatic habitats.

More than one million barriers fragment Europe's rivers.

Rivers support some of Earth's richest biodiversity1 and provide essential ecosystem services to society2, but they are often fragmented by barriers to free flow3. In Europe, attempts to quantify river connectivity have been hampered by the absence of a harmonized barrier database. Here we show that there are at least 1.2 million instream barriers in 36 European countries (with a mean density of 0.74 barriers per kilometre), 68 per cent of which are structures less than two metres in height that are often overlooked. Standardized walkover surveys along 2,715 kilometres of stream length for 147 rivers indicate that existing records underestimate barrier numbers by about 61 per cent. The highest barrier densities occur in the heavily modified rivers of central Europe and the lowest barrier densities occur in the most remote, sparsely populated alpine areas. Across Europe, the main predictors of barrier density are agricultural pressure, density of river-road crossings, extent of surface water and elevation. Relatively unfragmented rivers are still found in the Balkans, the Baltic states and parts of Scandinavia and southern Europe, but these require urgent protection from proposed dam developments. Our findings could inform the implementation of the EU Biodiversity Strategy, which aims to reconnect 25,000 kilometres of Europe's rivers by 2030, but achieving this will require a paradigm shift in river restoration that recognizes the widespread impacts caused by small barriers.

Cortisol-Related Signatures of Stress in the Fish Microbiome.

Exposure to environmental stressors can compromise fish health and fitness. Little is known about how stress-induced microbiome disruption may contribute to these adverse health effects, including how cortisol influences fish microbial communities. We exposed juvenile Atlantic salmon to a mild confinement stressor for two weeks. We then measured cortisol in the plasma, skin-mucus, and feces, and characterized the skin and fecal microbiome. Fecal and skin cortisol concentrations increased in fish exposed to confinement stress, and were positively correlated with plasma cortisol. Elevated fecal cortisol was associated with pronounced changes in the diversity and structure of the fecal microbiome. In particular, we identified a marked decline in the lactic acid bacteria Carnobacterium sp. and an increase in the abundance of operational taxonomic units within the classes Clostridia and Gammaproteobacteria. In contrast, cortisol concentrations in skin-mucus were lower than in the feces, and were not related to any detectable changes in the skin microbiome. Our results demonstrate that stressor-induced cortisol production is associated with disruption of the gut microbiome, which may, in turn, contribute to the adverse effects of stress on fish health. They also highlight the value of using non-invasive fecal samples to monitor stress, including simultaneous determination of cortisol and stress-responsive bacteria.

More than meets the eye: syntopic and morphologically similar mangrove killifish species show different mating systems and patterns of genetic structure along the Brazilian coast.

Different mating systems can strongly affect the extent of genetic diversity and population structure among species. Given the increased effects of genetic drift on reduced population size, theory predicts that species undergoing self-fertilisation should have greater population structure than outcrossed species; however, demographic dynamics may affect this scenario. The mangrove killifish clade is composed of the two only known examples of self-fertilising species among vertebrates (Kryptolebias marmoratus and Kryptolebias hermaphroditus). A third species in this clade, Kryptolebias ocellatus, inhabits mangrove forests in southeast Brazil; however, its mating system and patterns of genetic structure have been rarely explored. Here, we examined the genetic structure and phylogeographic patterns of K. ocellatus along its distribution, using mitochondrial DNA and microsatellites to compare its patterns of genetic structure with the predominantly selfing and often-syntopic, K. hermaphroditus. Our results indicate that K. ocellatus reproduces mainly by outcrossing, with no current evidence of selfing, despite being an androdioecious species. Our results also reveal a stronger population subdivision in K. ocellatus compared to K. hermaphroditus, contrary to the theoretical predictions based on reproductive biology of the two species. Our findings indicate that, although morphologically similar, K. ocellatus and K. hermaphroditus had remarkably different evolutionary histories when colonising the same mangrove areas in southeastern Brazil, with other factors (e.g., time of colonisation, dispersal/establishment capacity) having more profound effects on the current population structuring of those species than differences in mating systems.

Environmental enrichment induces intergenerational behavioural and epigenetic effects on fish.

Parental effects influence offspring phenotypes through pre- and post-natal routes but little is known about their molecular basis, and therefore their adaptive significance. Epigenetic modifications, which control gene expression without changes in the DNA sequence and are influenced by the environment, may contribute to parental effects. We investigated the effects of environmental enrichment on the behaviour, metabolic rate and brain DNA methylation patterns of parents and offspring of the highly inbreed mangrove killifish (Kryptolebias marmoratus). Parental fish reared in enriched environments had lower cortisol levels, lower metabolic rates and were more active and neophobic than those reared in barren environments. They also differed in 1,854 methylated cytosines (DMCs). Offspring activity and neophobia were determined by the parental environment. Among the DMCs of the parents, 98 followed the same methylation patterns in the offspring, three of which were significantly influenced by parental environments irrespective of their own rearing environment. Our results suggest that parental environment influences the behaviour and, to some extent, the brain DNA methylation patterns of the offspring.