Institute of Aquatic Ecology

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

Freshwater mussels of the Order Unionida provide important ecosystem functions and services, yet many of their populations are in decline. We comprehensively review the status of the 16 currently recognized species in Europe, collating for the first time their life-history traits, distribution, conservation status, habitat preferences, and main threats in order to suggest future management actions. In northern, central, and eastern Europe, a relatively homogeneous species composition is found in most basins. In southern Europe, despite the lower species richness, spatially restricted species make these basins a high conservation priority. Information on freshwater mussels in Europe is unevenly distributed with considerable differences in data quality and quantity among countries and species. To make conservation more effective in the future, we suggest greater international cooperation using standardized protocols and methods to monitor and manage European freshwater mussel diversity. Such an approach will not only help conserve this vulnerable group but also, through the protection of these important organisms, will offer wider benefits to freshwater ecosystems.

In ASDEX Upgrade, experimental efforts aim to establish pace making and mitigation of type-I edge localized modes (ELMs) in high confinement mode (H-mode) discharges. Injection of small size cryogenic deuterium pellets (∼(1.4 mm)2 × 0.2 mm ≈ 2.5 × 1019 D) at rates up to 83 Hz imposed persisting ELM control without significant fuelling, enabling for investigations well inside the type-I ELM regime. The approach turned out to meet all required operational features. ELM pace making was realized with the driving frequency ranging from 1 to 2.8 times the intrinsic ELM frequency, the upper boundary set by hardware limits. ELM frequency enhancement by pellet pace making causes much less confinement reduction than by engineering means like heating, gas bleeding or plasma shaping. Confinement reduction is observed in contrast to the typical for engineering parameters. Matched discharges showed triggered ELMs ameliorated with respect to intrinsic counterparts while their frequency was increased. No significant differences were found in the ELM dynamics with the available spatial and temporal resolution. By breaking the close correlation of ELM frequency and plasma parameters, pace making allows the establishment of fELM as a free parameter giving enhanced operational headroom for tailoring H-mode scenarios with acceptable ELMs. Use was made of the pellet pace making tool in several successful applications in different scenarios. It seems that further reduction of the pellet mass could be possible, eventually resulting in less confinement reduction as well.

The protection, preservation and restoration of aquatic ecosystems and their functions are of global importance. For European states it became legally binding mainly through the EUWater Framework Directive (WFD). In order to assess the ecological status of a given water body, aquatic biodiversity data are obtained and compared to a reference water body. The quantified mismatch obtained determines the extent of potential management actions. The current approach to biodiversity assessment is based on morpho-taxonomy. This approach has many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to the varying individual taxonomic expertise of the analysts. Novel genomic tools can overcome many of the aforementioned problems and could complement or even replace traditional bioassessment. Yet, a plethora of approaches are independently developed in different institutions, thereby hampering any concerted routine application. The goal of this Action is to nucleate a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel ecogenomic indices for routine application in biodiversity assessments of European fresh- and marine water bodies. Furthermore, DNAqua-Net will provide a platform for training of the next generation of European researchers preparing them for the new technologies. Jointly with water managers, politicians, and other stakeholders, the group will develop a DNAqua-Net: Developing new genetic tools for bioassessment and monitoring ... 3 conceptual framework for the standard application of eco-genomic tools as part of legally binding assessments.

Despite the large number of measurement tools developed to assess problematic Internet use, numerous studies use measures with only modest investigation into their psychometric properties. The goal of the present study was to validate the short (6-item) version of the Problematic Internet Use Questionnaire (PIUQ) on a nationally representative adolescent sample (n = 5,005; mean age 16.4 years, SD = 0.87) and to determine a statistically established cut-off value. Data were collected within the framework of the European School Survey Project on Alcohol and Other Drugs project. Results showed an acceptable fit of the original three-factor structure to the data. In addition, a MIMIC model was carried out to justify the need for three distinct factors. The sample was divided into users at-risk of problematic Internet use and those with no-risk using a latent profile analysis. Two latent classes were obtained with 14.4% of adolescents belonging to the at-risk group. Concurrent and convergent validity were tested by comparing the two groups across a number of variables (i.e., time spent online, academic achievement, self-esteem, depressive symptoms, and preferred online activities). Using the at-risk latent profile analysis class as the gold standard, a cut-off value of 15 (out of 30) was suggested based on sensitivity and specificity analyses. In conclusion, the brief version of the (6-item) PIUQ also appears to be an appropriate measure to differentiate between Internet users at risk of developing problematic Internet use and those not at risk. Furthermore, due to its brevity, the shortened PIUQ is advantageous to utilize within large-scale surveys assessing many different behaviors and/or constructs by reducing the overall number of survey questions, and as a consequence, likely increasing completion rates.

Abstract Aim To assess the future climatic suitability of European catchments for freshwater species and the future utility of the current network of protected areas. Location Europe. Methods Using recently updated catchment‐scale species data and climate projections from multiple climate models, we assessed the climate change threat by the 2050s for 1648 European freshwater plants, fishes, molluscs, odonates, amphibians, crayfish and turtles for two dispersal scenarios and identified hotspots of change at three spatial scales: major river basins, countries and freshwater ecoregions. We considered both common species and the often overlooked rare species. To set our findings within the context of current and future conservation networks, we evaluated the coverage of freshwater biodiversity by Europe's protected area network. Results Six per cent of common and 77% of rare species are predicted to lose more than 90% of their current range. Eight fish species and nine mollusc species are predicted to experience 100% range loss under climate change. As the most species‐rich group, molluscs are particularly vulnerable due to the high proportion of rare species and their relatively limited ability to disperse. Furthermore, around 50% of molluscs and fish species will have no protected area coverage given their projected distributions. Main conclusions We identified the species most at threat due to projected changes in both catchment suitability and representation within the European protected area network. Our findings suggest an urgent need for freshwater management plans to facilitate adaptation to climate change.

Abstract Quantifying the relative importance of how local (environmental or niche‐based) and regional (dispersal‐related or spatial) processes regulate the assembly of communities has become one of the main research avenues of community ecology. It has been shown that the degree of isolation of local habitats in the landscape may substantially influence the relative role of environmental filtering and dispersal‐related processes in metacommunities. Dendritic stream networks are unique habitats in the landscape, where more isolated upstream sites have been predicted to be primarily structured by environmental variables, while more central mainstem rivers by both environmental and spatial variables (hereafter the network position hypothesis, NPH ). However, the NPH has almost exclusively been tested for stream macroinvertebrates, and therefore its predictions warrant confirmation from multiple taxa. We examined the validity of the NPH for benthic diatoms, macrophytes, macroinvertebrates and fish in the Pannon Ecoregion, Hungary. Following the NPH we predicted a clear dominance of environmental over spatial variables in headwaters, and a larger effect of spatial variables in rivers compared to headwaters. We tested these predictions using variance partitioning analyses separately for the different taxa in headwater and in riverine habitats. We found large differences in the explained community variance when the impact of environmental (physical and chemical) and spatial (overland and watercourse distance) variables for various taxa was studied. In general, total explained variance was lower for the more passively dispersing plant taxa than for animal taxa with more active dispersal in both streams and rivers. However, similar to other studies, the total explained variance was low for both headwater streams and rivers. Community structure of diatoms could be best explained by both environmental and spatial variables in streams, whereas their community structure could not be explained by either variable group in rivers. The significance of environmental and spatial variables depended on the distance measure (overland versus watercourse) in the case of macrophytes. Community structure of macroinvertebrates could be explained by environmental variables in streams and by both environmental and spatial variables in rivers. Moreover, variation was explained by different predictors when macroinvertebrate taxa were divided into flying and non‐flying groups, suggesting the importance of dispersal mode in explaining community variation. Finally, community structure of fishes could be explained by both environmental and spatial variables in streams and only by environmental variables in rivers. In conclusion, we found no clear evidence of the NPH in our multi‐taxa comparison. For example, while patterns in macroinvertebrate communities seem to support the NPH , those in fish communities run counter with the predictions of the NPH . This study thus shows that different taxa may behave differently to isolation effects in stream networks. We discuss alternatives in the interpretation of dispersal (or spatial) effects which may partly explain differences in the observed patterns from the NPH , and emphasise the need for further studies in unravelling the importance of isolation in stream metacommunity structuring.

Plant dispersal syndromes are allocated based on diaspore morphology and used to predict the dominant mechanisms of dispersal. Many authors assume that only angiosperms with endozoochory, epizoochory or anemochory syndromes have a long‐distance dispersal (LDD) mechanism. Too much faith is often placed in classical syndromes to explain historical dispersal events and to predict future ones. What is usually recorded as the ‘endozoochory syndrome' is in reality a ‘frugivory syndrome' and this has often diverted attention from endozoochory by non‐frugivores (e.g. waterbirds and large herbivores) that disperse a broad range of angiosperms, for which they likely provide the maximum dispersal distances. Neither the endozoochory nor the epizoochory syndromes provide helpful predictions of which plants non‐frugivores disperse, or by which mechanism. We combined data from previous studies to show that only 4% of European plant species dispersed by ungulate endozoochory belong to the corresponding syndrome, compared to 36% for ungulate epizoochory and 8% for endozoochory by migratory ducks. In contrast, the proportions of these species that are assigned to an ‘unassisted syndrome' are 37, 31 and 28%, respectively. Since allocated syndromes do not adequately account for zoochory, empirical studies often fail to find the expected relationship between syndromes and LDD events such as those underlying the colonization of islands or latitudinal migration rates. We need full incorporation of existing zoochory data into dispersal databases, and more empirical research into the relationship between plant traits and the frequency and effectiveness of different dispersal mechanisms (paying attention to unexpected vectors). Acknowledging the broad role of non‐frugivores in facilitating LDD is crucial to improve predictions of the consequences of global change, such as how plant distributions respond to climate change, and how alien plants spread. Networks of dispersal interactions between these vertebrates and plants are a vital but understudied part of the Web of Life.

Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s.

Our understanding on phytoplankton diversity has largely been progressing since the publication of Hutchinson on the paradox of the plankton. In this paper, we summarise some major steps in phytoplankton ecology in the context of mechanisms underlying phytoplankton diversity. Here, we provide a framework for phytoplankton community assembly and an overview of measures on taxonomic and functional diversity. We show how ecological theories on species competition together with modelling approaches and laboratory experiments helped understand species coexistence and maintenance of diversity in phytoplankton. The non-equilibrium nature of phytoplankton and the role of disturbances in shaping diversity are also discussed. Furthermore, we discuss the role of water body size, productivity of habitats and temperature on phytoplankton species richness, and how diversity may affect the functioning of lake ecosystems. At last, we give an insight into molecular tools that have emerged in the last decades and argue how it has broadened our perspective on microbial diversity. Besides historical backgrounds, some critical comments have also been made.

Abstract Centric diatoms of 107 different Hungarian running waters were investigated. Among them the largest was the River Danube, from which more than one hundred plankton samples were analysed by scanning electron microscopy (SEM). Only one sample was analysed from creeks, which were the smallest running waters analysed in this study. There were also channels with slow currents flowing out of rivers or connecting different rivers. In total, 41 centric taxa belonging to 11 genera were found during this study. The average number of taxa found in a single watercourse was 7, the maximum 40 and the minimum 1. Cyclotella meneghiniana was the most frequently encountered species (present in 60% of sites). Twelve taxa were found in more than 20% of sites, 7 taxa between 5-10% and 6 taxa only in one site.

The characteristic striped appearance of zebras has provoked much speculation about its function and why the pattern has evolved, but experimental evidence is scarce. Here, we demonstrate that a zebra-striped horse model attracts far fewer horseflies (tabanids) than either homogeneous black, brown, grey or white equivalents. Such biting flies are prevalent across Africa and have considerable fitness impact on potential mammalian hosts. Besides brightness, one of the likely mechanisms underlying this protection is the polarization of reflected light from the host animal. We show that the attractiveness of striped patterns to tabanids is also reduced if only polarization modulations (parallel stripes with alternating orthogonal directions of polarization) occur in horizontal or vertical homogeneous grey surfaces. Tabanids have been shown to respond strongly to linearly polarized light, and we demonstrate here that the light and dark stripes of a zebra's coat reflect very different polarizations of light in a way that disrupts the attractiveness to tabanids. We show that the attractiveness to tabanids decreases with decreasing stripe width, and that stripes below a certain size are effective in not attracting tabanids. Further, we demonstrate that the stripe widths of zebra coats fall in a range where the striped pattern is most disruptive to tabanids. The striped coat patterns of several other large mammals may also function in reducing exposure to tabanids by similar mechanisms of differential brightness and polarization of reflected light. This work provides an experimentally supported explanation for the underlying mechanism leading to the selective advantage of a black-and-white striped coat pattern.

In recent years, decline of freshwater resources has been recognized as one of the main environmental problems on global level. In addition to the increasing extent of primary salinization due to climate change, secondary salinization caused by human interventions is also a significantly increasing problem, therefore, the development of various chemical-free, biological desalination and removal procedures will become increasingly important. In the present study, the salinity tolerance, salinity, and nutrient reducing ability of nine common freshwater microalgae species from the genera Chlorella, Chlorococcum, Desmodesmus, Scenedesmus, and Monoraphidium were investigated. Our results proved that the studied green microalgae species are halotolerant ones, which are able to proliferate in environments with high salt concentrations. Furthermore, most of the species were able to reduce conductivity and remove significant amounts of chloride (up to 39%) and nutrients (more than 90% nitrate). The results proved that nitrate removal of the studied species was not influenced by salt concentration, only indirectly via growth inhibition. However, the results also highlighted that N:P ratio of the medium has primarily importance in satisfactory phosphorous removal. It can be concluded that assemblages of the studied microalgae species could be able to adapt to changing conditions even of salt-rich wastewaters and improve water quality during bioremediation processes.

Abstract Long‐term dynamics of phytoplankton have been addressed in marine and lake systems, but rarely in rivers. Large rivers, however, are highly human‐impacted, whereas global warming may further affect the functioning of phytoplankton at long‐term scale. In the middle section of the large European Danube River, long‐term decrease in phytoplankton biomass (Chl‐ a ) and increase in species diversity have formerly been revealed. The functional community composition that relates to ecosystem functioning directly has not been addressed previously. We analyse a 34‐year‐long phytoplankton data set from the middle river section at Göd (N‐Budapest), Hungary. We focus on gradual changes in the functional composition and functional diversity components based on the functional trait and functional group approaches. We hypothesised that long‐term gradual changes in major environmental constraints should be followed by gradual shifts in dominance relationships among functional traits and functional groups of phytoplankton. We further hypothesised that functional shifts were highlighted by gradual changes in functional diversity components: evenness, divergence and dispersion. Water discharge of the middle Danube shifted towards the more frequent occurrence of lower values. On the other hand, high floods (>3,000 m 3 /s) increased significantly with shortening tendency in duration and altered seasonality. The concentration of N and P forms, as well as total suspended solids decreased significantly. Water temperature increased significantly, especially in summer. In the phytoplankton, single‐celled eutrophic centric diatoms decreased in relative abundance, but flagellated, elongated and filamentous forms increased. A clear functional shift was the dominance decrease in planktonic taxa and the relative abundance increase in benthic diatoms. All functional diversity components increased significantly in the entire data set, except functional evenness (FEVE) based on the functional group approach. At seasonal scale, all significant trends showed increases, except the FEVE components of the functional group approach, which decreased in winter and spring significantly. Long‐term increase in functional diversity components alone could indicate enhanced ecosystem functioning of phytoplankton in the middle section of the Danube. However, we argue that the observed increase in functional diversity may be related to a gradual shift from high‐biomass communities with the dominance of eutrophic centric diatoms towards the relative increase in several, but low‐biomass elements. These include a few planktonic algae well adapted to the altered conditions, diatoms with benthic origin and dispersed limnophilic taxa. Our results provide the first evidence for a long‐term phytoplankton functional regime shift in a European large river. Global warming, human impacts and oligotrophication might potentially increase the functional diversity of large river phytoplankton, but the origin and functional role of taxa should carefully be considered. The observed functional shift in phytoplankton might also be indicative for alterations in the food‐web structure of the middle section of the Danube River at long‐term scale.

We examined the abundance and meso-habitat use of gobiid species during both day and night along 43 stretches (500-m long) of the littoral zone at five locations of the Middle Danube, Hungary, in spring and in summer 2004. Electro-fishing catch per unit effort sampling revealed significantly higher relative densities at night than during the day. Gobiids occupied all the available habitats encountered during sampling. Habitat-abundance relationships from night-time samples revealed that the two most abundant species, round goby Neogobius melanostomus and bighead goby Neogobius kessleri, were found in highest relative density along gravel beach and artificial rip-rap habitats; these species were in relatively low density in sandy areas. Monkey goby Neogobius fluviatilis was more abundant in natural shorelines than in rip-rap habitats and dispersed more consistently between sandy and gravel beaches. Tubenose goby Proterorhinus marmoratus demonstrated great plasticity in meso-habitat use. In conclusion, Ponto-Caspian gobies occurred rather consistently along the Hungarian section of the Danube, occupying all available habitats examined. Abundance-habitat relations suggest plasticity in meso-habitat use but partial segregation between species. Further studies are required at multiple spatial scales to assess how micro- and meso-habitat use varies with fluctuating population densities.

Surface waters are becoming increasingly contaminated by pharmaceutically active compounds (PhACs), which is a potential risk factor for drinking water quality owing to incomplete riverbank filtration. This study examined the efficiency of riverbank filtration with regard to 111 PhACs in a highly urbanized section of the river Danube. One hundred seven samples from the Danube were compared to 90 water samples from relevant drinking water abstraction wells (DWAW) during five sampling periods. The presence of 52 PhACs was detected in the Danube, the quantification of 19 agents in this section of the river was without any precedent, and 10 PhACs were present in >80% of the samples. The most frequent PhACs showed higher concentrations in winter than in summer. In the DWAWs, 32 PhACs were quantified. For the majority of PhACs, the bank filtration efficiency was >95%, and not influenced by concentrations measured in the river. For carbamazepine lidocaine, tramadol, and lamotrigine, low (<50%) filtration efficiency was observed; however, no correlations were observed between the concentrations detected in the Danube and in the wells. These frequently occurring PhACs in surface waters have a relatively even distribution, and their sporadic appearance in wells is a function of both space and time, which may be caused by the constantly changing environment and micro-biological parameters, the dynamic operating schedule of abstraction wells, and the resulting sudden changes in flow rates. Due to the changes in the efficiency of riverbank filtration in space and time, predicting the occurrence and concentrations of these four PhACs poses a further challenge to ensuring a safe drinking water supply.

Abstract Recent studies clearly support a positive biodiversity–ecosystem functioning ( BEF ) relationship in phytoplankton. As taxon richness does not quantify functional diversity, functional approaches have been developed to link community functioning to diversity. Compared to terrestrial plant communities, only a few studies have validated phytoplankton functional approaches in BEF relationships. Furthermore, the ability of functional and taxonomic richness measures in predicting ecosystem functioning of natural phytoplankton communities has not been compared yet. Here, we analysed the BEF relationship using taxonomic and functional (trait categories and response groups sensu Reynolds) approaches in a broad‐scale phytoplankton dataset from Fennoscandia. First, we analysed how taxonomic and functional compositions were related to local environmental predictors. We then compared how taxonomic and functional richness performed in predicting resource‐use efficiency (the yield in phytoplankton biomass standardised by total phosphorus) as an ecosystem functioning measure. Finally, we tested whether the relationship between ecosystem functioning and taxonomic richness is further enhanced once each of the functional richness measure is also considered. Among the approaches, phytoplankton functional trait categories as community matrix showed the best correspondence with the local environment. The richness of phytoplankton response groups predicted ecosystem functioning significantly better than the taxonomic and the functional trait category richness—both in the full dataset and in almost all Fennoscandian countries. On top of taxonomic richness, only the residual variation in response group richness predicted ecosystem functioning positively in the entire dataset and in all individual countries. Applying functional approaches, reduced complexity of data should come along with reduced ecological information. We showed, however, that both functional approaches represented some functional redundancy among taxa in a meaningful way, and enhanced our ability in predicting community composition from environmental predictors. Moreover, phytoplankton response group richness sensu Reynolds summarises information on ecosystem functioning contained in the taxonomic data in a way that represents functional diversity better than the richness of functional trait categories. Interestingly, the response group approach, which is exclusively derived from field observations rather than from quantified phytoplankton traits, outperforms taxonomic richness and trait category richness in predicting ecosystem functioning in our dataset. This may highlight that our ability to quantify phytoplankton traits is still limited. Existing phytoplankton functional approaches, however, can translate the taxonomic information into a reduced but reliable functional matrix already and predict ecosystem functioning better than taxonomic data.

Ecological photopollution created by artificial night lighting can alter animal behavior and lead to population declines and biodiversity loss. Polarized light pollution is a second type of photopollution that triggers water-seeking insects to ovisposit on smooth and dark man-made objects, because they simulate the polarization signatures of natural water bodies. We document a case study of the interaction of these two forms of photopollution by conducting observations and experiments near a lamp-lit bridge over the river Danube that attracts mass swarms of the mayfly Ephoron virgo away from the river to oviposit on the asphalt road of the bridge. Millions of mayflies swarmed near bridge-lights for two weeks. We found these swarms to be composed of 99% adult females performing their upstream compensatory flight and were attracted upward toward unpolarized bridge-lamp light, and away from the horizontally polarized light trail of the river. Imaging polarimetry confirmed that the asphalt surface of the bridge was strongly and horizontally polarized, providing a supernormal ovipositional cue to Ephoron virgo, while other parts of the bridge were poor polarizers of lamplight. Collectively, we confirm that Ephoron virgo is independently attracted to both unpolarized and polarized light sources, that both types of photopollution are being produced at the bridge, and that spatial patterns of swarming and oviposition are consistent with evolved behaviors being triggered maladaptively by these two types of light pollution. We suggest solutions to bridge and lighting design that should prevent or mitigate the impacts of such scenarios in the future. The detrimental impacts of such scenarios may extend beyond Ephoron virgo.

The timing of early cellular evolution, from the divergence of Archaea and Bacteria to the origin of eukaryotes, is poorly constrained. The ATP synthase complex is thought to have originated prior to the Last Universal Common Ancestor (LUCA) and analyses of ATP synthase genes, together with ribosomes, have played a key role in inferring and rooting the tree of life. We reconstruct the evolutionary history of ATP synthases using an expanded taxon sampling set and develop a phylogenetic cross-bracing approach, constraining equivalent speciation nodes to be contemporaneous, based on the phylogenetic imprint of endosymbioses and ancient gene duplications. This approach results in a highly resolved, dated species tree and establishes an absolute timeline for ATP synthase evolution. Our analyses show that the divergence of ATP synthase into F- and A/V-type lineages was a very early event in cellular evolution dating back to more than 4 Ga, potentially predating the diversification of Archaea and Bacteria. Our cross-braced, dated tree of life also provides insight into more recent evolutionary transitions including eukaryogenesis, showing that the eukaryotic nuclear and mitochondrial lineages diverged from their closest archaeal (2.67-2.19 Ga) and bacterial (2.58-2.12 Ga) relatives at approximately the same time, with a slightly longer nuclear stem-lineage.

Nutrient pollution remains one of the leading causes of river degradation, making it important to set thresholds that support good ecological condition, which is the main objective of managing Europe's aquatic environment. A wide range of methods has been used by European member states to set river nutrient thresholds in the past, and these vary greatly among countries, even for similar river types. In some countries, thresholds have been set using expert judgement or the statistical distribution of nutrient concentrations. Application of such thresholds creates problems for planning strategies to achieve good ecological status and for managing transboundary river basins. An alternative approach is to examine the statistical relationship between nutrient concentration and one, or more, biological variables. Such relationships can then be used to inform decisions by water managers. We use such 'ecology-based' approaches (univariate regression and mismatch analyses) to derive nutrient thresholds for several river types in Central Europe. Our analysis focused on soluble reactive phosphorus (SRP) and total nitrogen (TN), two variables which were responsible for significant variation (40–55%) in river benthic floras. In this study, for the first time, river nutrient thresholds are estimated using both macrophytes and phytobenthos (EQRs) separately and in combination, calculated as the minimum and the average of the EQRs of the two sub-elements. The resulting thresholds supporting good ecological status range from 21 to 42 µg/L SRP and 0.9–3.5 mg/L TN for the low alkalinity lowland river type, and 32–90 µg/L SRP and 1.0–2.5 mg/L TN for the low alkalinity mid-altitude river type. These targets are compared to the values set by member states. We demonstrate that some national nutrient thresholds fall within the range of predicted values if uncertainty is taken into consideration; however, several threshold values considerably exceed this range. Adopting ecology-based nutrient targets should improve sustainable river management where nutrients are the major pressure preventing the achievement of good ecological status.