Connecting the morphological and molecular species concepts to facilitate species identification within the genus Fragilaria (Bacillariophyta).

This paper explores the diversity and taxonomy of species within Fragilaria sensu stricto, an abundant and ecologically important diatom genus, taking advantage of cultured and DNA-barcoded material. The goal is to facilitate the identification of European taxa within this complex, providing a unified view on morphological and molecular diversity. There is a general agreement that the separation of species within the group of Fragilaria is difficult because morphological descriptions of species are not consistent between authorities, ongoing taxonomic revisions have resulted in species described with standards of the late 20th and 21st centuries alongside descriptions based on 19th century (light microscopical) criteria, and because not all diagnostic characters can be seen in all specimens encountered in routine analyses. Consequent confusion could blur potentially important ecological distinctions between species. Our study demonstrated that some species defined on morphological criteria could be confirmed using the rbcL chloroplast gene as a genetic marker, for example, Fragilaria gracilis, Fragilaria tenera, Fragilaria perminuta, and Fragilaria subconstricta. However, even for those species, preliminary identifications based on morphology often differed from identifications based on phylogenetic clustering combined with detailed morphological study. Clades were well-defined by rbcL, but based on morphology, the terminal taxa of these clades did not match the currently described Fragilaria species. To clarify recognition of these taxa, we describe three new species: Fragilaria agnesiae, Fragilaria heatherae, and Fragilaria joachimii.

Declining glacier cover threatens the biodiversity of alpine river diatom assemblages.

Climate change poses a considerable threat to the biodiversity of high altitude ecosystems worldwide, including cold-water river systems that are responding rapidly to a shrinking cryosphere. Most recent research has demonstrated the severe vulnerability of river invertebrates to glacier retreat but effects upon other aquatic groups remain poorly quantified. Using new data sets from the European Alps, we show significant responses to declining glacier cover for diatoms, which play a critical functional role as freshwater primary producers. Specifically, diatom α-diversity and density in rivers presently fed by glaciers will increase with future deglaciation, yet ß-diversity within and between sites will reduce because declining glacier influence will lower the spatiotemporal variability of glacier cover and its associated habitat heterogeneity. Changes in diatom assemblage composition as glacier cover declined were associated strongly with increasing riverbed stability and water temperature. At the species level, diatoms showed a gradation of responses; for example, Eunotia trinacria, found exclusively at river sites with high (≥52%) catchment glacier cover, may be affected negatively by ice loss. Conversely, seven taxa confined to sites with no glacier cover, including Gomphonema calcareum, stand to benefit. Nineteen (22%) taxa were noted as threatened, endangered, rare or decreasing on the Red List of Algae for Germany, with most at sites ≤26% glacier cover, meaning further ice loss may benefit these diatoms. However, six taxa found only in rivers ≥28% glacier cover may require reclassification of their Red List conservation status, as this habitat is threatened by deglaciation. Our identification of clear links between decreasing glacier cover and river diatom biodiversity suggests there could be significant reorganization of river ecosystems with deglaciation, for example, through alterations to primary production, biogeochemical cycles, and the shifting resource base of alpine freshwater food webs which lack significant allochthonous energy inputs.

Macrophyte assessment in European lakes: Diverse approaches but convergent views of 'good' ecological status.

The European Water Framework Directive has been adopted by Member States to assess and manage the ecological integrity of surface waters. Specific challenges include harmonizing diverse assessment systems across Europe, linking ecological assessment to restoration measures and reaching a common view on 'good' ecological status. In this study, nine national macrophyte-based approaches for assessing ecological status were compared and harmonized, using a large dataset of 539 European lakes. A macrophyte common metric, representing the average standardized view of each lake by all countries, was used to compare national methods. This was also shown to reflect the total phosphorus (r2 = 0.32), total nitrogen (r2 = 0.22) as well as chlorophyll-a (r2 = 0.35-0.38) gradients, providing a link between ecological data, stressors and management decisions. Despite differing assessment approaches and initial differences in classification, a consensus was reached on how type-specific macrophyte assemblages change across the ecological status gradient and where ecological status boundaries should lie. A marked decline in submerged vegetation, especially Charophyta (characterizing 'good' status), and an increase in abundance of free-floating plants (characterizing 'less than good' status) were the most significant changes along the ecological status gradient. Macrophyte communities of 'good' status lakes were diverse with many charophytes and several Potamogeton species. A large number of taxa occurred across the entire gradient, but only a minority dominated at 'less than good' status, including filamentous algae, lemnids, nymphaeids, and several elodeids (e.g., Zannichellia palustris and Elodea nuttallii). Our findings establish a 'guiding image' of the macrophyte community at 'good' ecological status in hard-water lakes of the Central-Baltic region of Europe.

Setting nutrient boundaries to protect aquatic communities: The importance of comparing observed and predicted classifications using measures derived from a confusion matrix.

Defining nutrient thresholds that protect and support the ecological integrity of aquatic ecosystems is a fundamental step in maintaining their natural biodiversity and preserving their resilience. With increasing catchment pressures and climate change, it is more important than ever to develop clear methods to establish thresholds for status classification and management of waters. This must often be achieved using complex data and should be robust to interference from additional pressures as well as ameliorating or confounding conditions. We use both artificial and real data to examine challenges in setting nutrient thresholds in unbalanced and skewed data. We found significant advantages to using binary logistic regression over other techniques. However, one of the key challenges is objectively selecting a probability from which to derive the nutrient threshold. For this purpose, the examination of the proportions of matching and mismatching status classifications of nutrients and a biological quality element using a confusion matrix is a key step that should be more widely adopted in threshold selection. We examined a large array of statistical measures of classification accuracy and their performance over combinations of skewness and imbalance in the data. The most appropriate threshold probability is a compromise between maximising overall classification accuracy and reducing mismatches expressed as commission (false positives) without excessive omission (false negatives). An application to a lake type indicated total phosphorus thresholds that would be around 50 µg l-1 lower than the threshold achieved by an 'unguided' approach, indicating that this approach is a very significant development meriting attention from national authorities responsible for water management.

Maximising environmental pressure-response relationship signals from diatom-based metabarcoding in rivers.

DNA metabarcoding has been performed on a large number of river phytobenthos samples collected from the UK, using rbcL primers optimised for diatoms. Within this dataset the composition of non-diatom sequence reads was studied and the effect of including these in models for evaluating the nutrient gradient was assessed. Whilst many non-diatom taxonomic groups were detected, few contained the full diversity expected in riverine environments. This may be due to the performance of the current primers in characterising the wider phytobenthic community and influenced by the sampling method employed, as both were developed specifically for diatoms. Nevertheless, the study identified considerable diversity in some groups, e.g. Eustigmatophyceae and a wider distribution than previously thought for freshwater Phaeophyceae. These results offer a strong case for the benefits of metabarcoding for expanding knowledge of aquatic biodiversity in the UK and elsewhere. Many of the ASVs associated with non-diatoms showed significant pressure responses; however, models that included non-diatoms had similar predictive strength to those based on diatoms alone. Whilst limitations of the primers for assessing non-diatoms may play a role in explaining these results, the diatoms provide a strong signal along the nutrient gradient and other algae, therefore, add little unique information. We recommend that future developments should use ASVs to calculate metrics, with links to reference databases made as a final step to generate lists of taxa to support interpretation. Any further exploration of the potential of non-diatoms would benefit from access to a well-curated reference database, similar to diat.barcode. Such a database does not yet exist, and we caution against the indiscriminate use of NCBI GenBank as a taxonomic resource as many rbcL sequences deposited have not been curated.

Establishing ecologically-relevant nutrient thresholds: A tool-kit with guidance on its use.

One key component of any eutrophication management strategy is establishment of realistic thresholds above which negative impacts become significant and provision of ecosystem services is threatened. This paper introduces a toolkit of statistical approaches with which such thresholds can be set, explaining their rationale and situations under which each is effective. All methods assume a causal relationship between nutrients and biota, but we also recognise that nutrients rarely act in isolation. Many of the simpler methods have limited applicability when other stressors are present. Where relationships between nutrients and biota are strong, regression is recommended. Regression relationships can be extended to include additional stressors or variables responsible for variation between water bodies. However, when the relationship between nutrients and biota is weaker, categorical approaches are recommended. Of these, binomial regression and an approach based on classification mismatch are most effective although both will underestimate threshold concentrations if a second stressor is present. Whilst approaches such as changepoint analysis are not particularly useful for meeting the specific needs of EU legislation, other multivariate approaches (e.g. decision trees) may have a role to play. When other stressors are present quantile regression allows thresholds to be established which set limits above which nutrients are likely to influence the biota, irrespective of other pressures. The statistical methods in the toolkit may be useful as part of a management strategy, but more sophisticated approaches, often generating thresholds appropriate to individual water bodies rather than to broadly defined "types", are likely to be necessary too. The importance of understanding underlying ecological processes as well as correct selection and application of methods is emphasised, along with the need to consider local regulatory and decision-making systems, and the ease with which outcomes can be communicated to non-technical audiences.

Estimating nutrient thresholds for eutrophication management: Novel insights from understudied lake types.

Nutrient targets based on pressure-response models are essential for defining ambitions and managing eutrophication. However, the scale of biogeographical variation in these pressure-response relationships is poorly understood, which may hinder eutrophication management in regions where lake ecology is less intensively studied. In this study, we derive ecology-based nutrient targets for five major ecoregions of Europe: Northern, Central-Baltic, Alpine, Mediterranean and Eastern Continental. As a first step, we developed regressions between nutrient concentrations and ecological quality ratios (EQR) based on phytoplankton and macrophyte communities. Significant relationships were established for 13 major lake types; in most cases, these relationships were stronger for phosphorus than for nitrogen, and stronger for phytoplankton than for macrophytes. Using these regressions, we estimated the total phosphorus (TP) and total nitrogen (TN) concentrations at which lakes of different types are likely to achieve good ecological status. However, in the very shallow lakes of the Eastern Continental region, relations between nutrient and biological communities were weak or non-significant. This can be attributed to high nutrient concentrations (in the asymptotic zone of phosphorus-phytoplankton models) suggesting other factors (light, grazing) limit primary production. However, we also show that fish stocking is a major pressure on Eastern Continental lakes, negatively affecting ecological status: lakes with low fish stocking show low chlorophyll-a concentrations and good ecological status despite high nutrient levels, while the lakes with high fish stocking show high chlorophyll-a and low ecological status. This study highlights the need to better understand lakes in biogeographic regions that have been, for historical reasons, less studied. This, in turn, helps reveal factors that challenge the dominant paradigms of lake assessment and management.

Executing multi-taxa eDNA ecological assessment via traditional metrics and interactive networks.

Current approaches to ecological assessment are limited by the traditional morpho-taxonomic methods presently employed and the inability to meet increasing demands for rapid assessments. Advancements in high throughput sequencing now enable rapid high-resolution ecological assessment using environmental DNA (eDNA). Here we test the ability of using eDNA-based ecological assessment methods against traditional assessment of two key indicator groups (diatoms and macroinvertebrates) and show how eDNA across multiple gene regions (COI, rbcL, 12S and 18S) can be used to infer interactive networks that link to ecological assessment criteria. We compared results between taxonomic and eDNA based assessments and found significant positive associations between macroinvertebrate (p < 0.001 R2 = 0.645) and diatom (p = 0.015, R2 = 0.222) assessment metrics. We further assessed the ability of eDNA based assessment to identify environmentally sensitive genera and found an order of magnitude greater potential for 18S, versus COI or rbcL, to determine environmental filtering of ecologically assessed communities. Lastly, we compared the ability of traditional metrics against co-occurrence network properties of our combined 18S, COI and rbcL indicator genera to infer habitat quality measures currently used by managers. We found that transitivity (network connectivity), linkage density and cohesion were significantly associated with habitat modification scores (HMS), whereas network properties were inconsistent with linking to the habitat quality score (HQS) metric. The incorporation of multi-marker eDNA network assessment opens up a means for finer scale ecological assessment, currently limited using traditional methods. While utilization of eDNA-based assessment is recommended, direct comparisons with traditional approaches are difficult as the methods are intrinsically different and should be treated as such with regards to future research. Overall, our findings show that eDNA can be used for effective ecological assessment while offering a wider range of scope and application compared to traditional assessment methods.

European aquatic ecological assessment methods: A critical review of their sensitivity to key pressures.

The European Union has embarked on a policy which aims to achieve good ecological status in all surface waters (i.e. rivers, lakes, transitional and coastal waters). In theory, ecological status assessment methods should address the effects of all relevant human pressures. In this study, we analyze the degree to which methods European countries use to assess ecological status tackle various pressures affecting European waters. Nutrient pollution is by far the best-covered pressure for all four water categories. Out of total of 423 assessment methods, 370 assess eutrophication and pressure-specific relationships have been demonstrated for 212 of these. "General degradation" is addressed by 238 methods, mostly validated by relationships to combined pressure indices. Other major pressures have received significantly less effort: hydromorphological degradation is assessed by 160 methods and pressure-specific relationships have been demonstrated for just 40 of these. Hydromorphological pressures are addressed (at least by one BQE) only by 25% countries for coastal waters and 70-80% for lakes and transitional waters. Specific diagnostic tools (i.e. single-pressure relationships) for hydromorphology have only been developed by a few countries: only 20% countries have such methods for lakes, coastal and transitional waters and less than half for rivers. Toxic contamination is addressed by 90 methods; however, pressure-specific relationships have been demonstrated for just eight of these. Only two countries have demonstrated pressure-specific acidification methods for rivers, and three for lakes. In summary, methods currently in use mostly address eutrophication and/or general degradation, but there is not much evidence that they reliably pick up the effects of other significant pressures such as hydromorphology or toxic contamination. Therefore, we recommend that countries re-examine: (1) those pressures which affect different water categories in the country; (2) relevant assessment methods to tackle those pressures; (3) whether pressure-response relationships have been developed for each of these.

Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization.

Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 18S-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data.

Deriving nutrient criteria to support 'good' ecological status in European lakes: An empirically based approach to linking ecology and management.

European water policy has identified eutrophication as a priority issue for water management. Substantial progress has been made in combating eutrophication but open issues remain, including setting reliable and meaningful nutrient criteria supporting 'good' ecological status of the Water Framework Directive. The paper introduces a novel methodological approach - a set of four different methods - that can be applied to different ecosystems and stressors to derive empirically-based management targets. The methods include Ranged Major Axis (RMA) regression, multivariate Ordinary Least Squares (OLS) regression, logistic regression, and minimising the mismatch of classifications. We apply these approaches to establish nutrient (nitrogen and phosphorus) criteria for the major productive shallow lake types of Europe: high alkalinity shallow (LCB1; mean depth 3-15 m) and very shallow (LCB2; mean depth < 3 m) lakes. Univariate relationships between nutrients and macrophyte assessments explained 29-46% of the variation. Multivariate models with both total phosphorus (TP) and total nitrogen (TN) as predictors had higher R2 values (0.50 for LCB1 and 0.49 for LCB2) relative to the use of TN or TP singly. We estimated nutrient concentrations at the boundary where lake vegetation changes from 'good' to 'moderate' ecological status. LCB1 lakes achieved 'good' macrophyte status at concentrations below 48-53 µg/l TP and 1.1-1.2 mg/l TN, compared to LCB2 lakes below 58-78 µg/l TP and 1.0-1.4 mg/l TN. Where strong regression relationships exist, regression approaches offer a reliable basis for deriving nutrient criteria and their uncertainty, while categorical approaches offer advantages for risk assessment and communication, or where analysis is constrained by discontinuous measures of status or short stressor gradients. We link ecological status of macrophyte communities to nutrient criteria in a user-friendly and transparent way. Such analyses underpin the practical actions and policy needed to achieve 'good' ecological status in the lakes of Europe.

Establishing nutrient thresholds in the face of uncertainty and multiple stressors: A comparison of approaches using simulated datasets.

Various methods have been proposed to identify threshold concentrations of nutrients that would support good ecological status, but the performance of these methods and the influence of other stressors on the underlying models have not been fully evaluated. We used synthetic datasets to compare the performance of ordinary least squares, logistic and quantile regression, as well as, categorical methods based on the distribution of nutrient concentrations categorised by biological status. The synthetic datasets used differed in their levels of variation between explanatory and response variables, and were centered at different positions along the stressor (nutrient) gradient. In order to evaluate the performance of methods in "multiple stressor" situations, another set of datasets with two stressors was used. Ordinary least squares and logistic regression methods were the most reliable when predicting the threshold concentration when nutrients were the sole stressor; however, both had a tendency to underestimate the threshold when a second stressor was present. In contrast, threshold concentrations produced by categorical methods were strongly influenced by the level of the stressor (nutrient enrichment, in this case) relative to the threshold they were trying to predict (good/moderate in this instance). Although all the methods tested had limitations in the presence of a second stressor, upper quantiles seemed generally appropriate to establish non-precautionary thresholds. For example, upper quantiles may be appropriate when establishing targets for restoration, but not when seeking to minimise deterioration. Selection of an appropriate threshold concentration should also attend to the regulatory regime (i.e. policy requirements and environmental management context) within which it will be used, and the ease of communicating the principles to managers and stakeholders.

Nutrient criteria for surface waters under the European Water Framework Directive: Current state-of-the-art, challenges and future outlook.

The aim of European water policy is to achieve good ecological status in all rivers, lakes, coastal and transitional waters by 2027. Currently, more than half of water bodies are in a degraded condition and nutrient enrichment is one of the main culprits. Therefore, there is a pressing need to establish reliable and comparable nutrient criteria that are consistent with good ecological status. This paper highlights the wide range of nutrient criteria currently in use by Member States of the European Union to support good ecological status and goes on to suggest that inappropriate criteria may be hindering the achievement of good status. Along with a comprehensive overview of nutrient criteria, we provide a critical analysis of the threshold concentrations and approaches by which these are set. We identify four essential issues: (1) Different nutrients (nitrogen and/or phosphorus) are used for different water categories in different countries. (2) The use of different nutrient fractions (total, dissolved inorganic) and statistical summary metrics (e.g., mean, percentiles, seasonal, annual) currently hampers comparability between countries, particularly for rivers, transitional and coastal waters. (3) Wide ranges in nutrient threshold values within shared water body types, in some cases showing more than a 10-fold difference in concentrations. (4) Different approaches used to set threshold nutrient concentrations to define the boundary between "good" and "moderate" ecological status. Expert judgement-based methods resulted in significantly higher (less stringent) good-moderate threshold values compared with data-driven approaches, highlighting the importance of consistent and rigorous approaches to criteria setting. We suggest that further development of nutrient criteria should be based on relationships between ecological status and nutrient concentrations, taking into account the need for comparability between different water categories, water body types within these categories, and countries.

Diat.barcode, an open-access curated barcode library for diatoms.

Diatoms (Bacillariophyta) are ubiquitous microalgae which produce a siliceous exoskeleton and which make a major contribution to the productivity of oceans and freshwaters. They display a huge diversity, which makes them excellent ecological indicators of aquatic ecosystems. Usually, diatoms are identified using characteristics of their exoskeleton morphology. DNA-barcoding is an alternative to this and the use of High-Throughput-Sequencing enables the rapid analysis of many environmental samples at a lower cost than analyses under microscope. However, to identify environmental sequences correctly, an expertly curated reference library is needed. Several curated libraries for protists exists; none, however are dedicated to diatoms. Diat.barcode is an open-access library dedicated to diatoms which has been maintained since 2012. Data come from two sources (1) the NCBI nucleotide database and (2) unpublished sequencing data of culture collections. Since 2017, several experts have collaborated to curate this library for rbcL, a chloroplast marker suitable for species-level identification of diatoms. For the latest version of the database (version 7), 605 of the 3482 taxonomical names originally assigned by the authors of the rbcL sequences were modified after curation. The database is accessible at https://www6.inra.fr/carrtel-collection_eng/Barcoding-database .

Protecting and restoring Europe's waters: An analysis of the future development needs of the Water Framework Directive.

The Water Framework Directive (WFD) is a pioneering piece of legislation that aims to protect and enhance aquatic ecosystems and promote sustainable water use across Europe. There is growing concern that the objective of good status, or higher, in all EU waters by 2027 is a long way from being achieved in many countries. Through questionnaire analysis of almost 100 experts, we provide recommendations to enhance WFD monitoring and assessment systems, improve programmes of measures and further integrate with other sectoral policies. Our analysis highlights that there is great potential to enhance assessment schemes through strategic design of monitoring networks and innovation, such as earth observation. New diagnostic tools that use existing WFD monitoring data, but incorporate novel statistical and trait-based approaches could be used more widely to diagnose the cause of deterioration under conditions of multiple pressures and deliver a hierarchy of solutions for more evidence-driven decisions in river basin management. There is also a growing recognition that measures undertaken in river basin management should deliver multiple benefits across sectors, such as reduced flood risk, and there needs to be robust demonstration studies that evaluate these. Continued efforts in 'mainstreaming' water policy into other policy sectors is clearly needed to deliver wider success with WFD goals, particularly with agricultural policy. Other key policy areas where a need for stronger integration with water policy was recognised included urban planning (waste water treatment), flooding, climate and energy (hydropower). Having a deadline for attaining the policy objective of good status is important, but even more essential is to have a permanent framework for river basin management that addresses the delays in implementation of measures. This requires a long-term perspective, far beyond the current deadline of 2027.

Implementation options for DNA-based identification into ecological status assessment under the European Water Framework Directive.

Assessment of ecological status for the European Water Framework Directive (WFD) is based on "Biological Quality Elements" (BQEs), namely phytoplankton, benthic flora, benthic invertebrates and fish. Morphological identification of these organisms is a time-consuming and expensive procedure. Here, we assess the options for complementing and, perhaps, replacing morphological identification with procedures using eDNA, metabarcoding or similar approaches. We rate the applicability of DNA-based identification for the individual BQEs and water categories (rivers, lakes, transitional and coastal waters) against eleven criteria, summarised under the headlines representativeness (for example suitability of current sampling methods for DNA-based identification, errors from DNA-based species detection), sensitivity (for example capability to detect sensitive taxa, unassigned reads), precision of DNA-based identification (knowledge about uncertainty), comparability with conventional approaches (for example sensitivity of metrics to differences in DNA-based identification), cost effectiveness and environmental impact. Overall, suitability of DNA-based identification is particularly high for fish, as eDNA is a well-suited sampling approach which can replace expensive and potentially harmful methods such as gill-netting, trawling or electrofishing. Furthermore, there are attempts to replace absolute by relative abundance in metric calculations. For invertebrates and phytobenthos, the main challenges include the modification of indices and completing barcode libraries. For phytoplankton, the barcode libraries are even more problematic, due to the high taxonomic diversity in plankton samples. If current assessment concepts are kept, DNA-based identification is least appropriate for macrophytes (rivers, lakes) and angiosperms/macroalgae (transitional and coastal waters), which are surveyed rather than sampled. We discuss general implications of implementing DNA-based identification into standard ecological assessment, in particular considering any adaptations to the WFD that may be required to facilitate the transition to molecular data.

Small Water Bodies in Great Britain and Ireland: Ecosystem function, human-generated degradation, and options for restorative action.

Small, 1st and 2nd-order, headwater streams and ponds play essential roles in providing natural flood control, trapping sediments and contaminants, retaining nutrients, and maintaining biological diversity, which extend into downstream reaches, lakes and estuaries. However, the large geographic extent and high connectivity of these small water bodies with the surrounding terrestrial ecosystem makes them particularly vulnerable to growing land-use pressures and environmental change. The greatest pressure on the physical processes in these waters has been their extension and modification for agricultural and forestry drainage, resulting in highly modified discharge and temperature regimes that have implications for flood and drought control further downstream. The extensive length of the small stream network exposes rivers to a wide range of inputs, including nutrients, pesticides, heavy metals, sediment and emerging contaminants. Small water bodies have also been affected by invasions of non-native species, which along with the physical and chemical pressures, have affected most groups of organisms with consequent implications for the wider biodiversity within the catchment. Reducing the impacts and restoring the natural ecosystem function of these water bodies requires a three-tiered approach based on: restoration of channel hydromorphological dynamics; restoration and management of the riparian zone; and management of activities in the wider catchment that have both point-source and diffuse impacts. Such activities are expensive and so emphasis must be placed on integrated programmes that provide multiple benefits. Practical options need to be promoted through legislative regulation, financial incentives, markets for resource services and voluntary codes and actions.

Benthic algal assessment of ecological status in European lakes and rivers: Challenges and opportunities.

This opinion paper introduces a special series of articles dedicated to freshwater benthic algae and their use in assessment and monitoring. This special series was inspired by talks presented at the 9th International Congress on the Use of Algae for Monitoring Rivers and Comparable Habitats (Trento, Italy, 2015), the latest of a series of meetings started in 1991. In this paper, we will first provide a brief overview of phytobenthos methods in Europe. Then, we will turn towards the 'dark side' of phytobenthos and describe four particular problems for phytobenthos assessment in the European Union: (1) over-reliance on a single group of algae (mostly diatoms) to the exclusion of other groups; (2) relatively low adoption of benthic algae for ecological assessments in lakes; (3) absence of measures of phytobenthos abundance; (4) approaches used to define boundaries between ecological classes. Following this, we evaluate the strengths and limitations of current phytobenthos assessment methods against 12 criteria for method evaluation addressing four areas: ecological rationale, performance, feasibility of implementation, and use in communication and management. Using these criteria, we identify and discuss three general challenges for those developing new methods for phytobenthos-based assessment: a weak ecological rationale and insufficient consideration of the role of phytobenthos as a diagnostic tool and for communicating ecosystem health beyond a narrow group of specialists. The papers in the special series allow a comparison with the situation and approaches in the USA, present new methods for the assessment of ecological status and acidification, provide tools for an improved management of headwaters and petrifying springs, discuss the utility of phytobenthos for lake assessments, and test the utility of functional measures (such as biofilm phosphorus uptake capacity, PUC).

RAPPER: A new method for rapid assessment of macroalgae as a complement to diatom-based assessments of ecological status.

Most methods for ecological assessment developed since the onset of the Water Framework Directive require substantial effort by skilled analysts and are therefore expensive to use. RAPPER ("Rapid Assessment of PeriPhyton Ecology in Rivers") is a high level ecological "triage" method that enables rapid screening of sites within a water body to enable managers to identify areas subject to nutrient pressures. The method involves a survey of macroscopic algae within 10m lengths of watercourses, taking samples for subsequent identification, and assessing cover. Genus-level identification is used to ensure rapid assessment and comparability, and that the method can be used by a wide range of users. Genera of alga that form conspicuous growths recognisable with the naked eye are designated as either "stress-tolerant" ("S-taxa") or "competitive" taxa ("C-taxa"), depending on their preference for locations with low or high nutrient concentrations. Genera whose representatives span a wide range of nutrient conditions, or for which few data are available, are placed in a third class, "unclassified". The presence of S-taxa and the relative cover of C-taxa are then used to determine whether a site is at risk from eutrophication. Field trials in Scotland demonstrated that the method discriminates between sites with low and high nutrient concentrations. Significant differences were also observed in values of the Trophic Diatom Index between RAPPER classification categories. RAPPER can be used alone (allowing greater spatial or temporal coverage within water bodies at lower cost than conventional assessment methods) or to increase confidence in assessments of the condition of the phytobenthos by incorporating algae other than diatoms. The outcomes also relate directly to the experiences of non-technical stakeholders, and will have benefits for communicating ecosystem health concepts to the wider public, for example through "citizen science".

A Water Framework Directive-compatible metric for assessing acidification in UK and Irish rivers using diatoms.

Freshwater acidification continues to be a major problem affecting large areas of Europe, and while there is evidence for chemical recovery, similar evidence for biological recovery of freshwaters is sparse. The need for a methodology to identify waterbodies impacted acidification and to assess the extent of biological recovery is relevant to the EU Water Framework Directive, which requires methods to quantify differences in biology between impacted and unimpacted or reference sites. This study presents a new WFD-compliant metric based on diatoms (Diatom Acidification Metric: DAM) for assessing the acidification status of rivers. A database of 558 benthic diatom samples and associated water chemistry data was assembled. Diatom taxa were assigned to one of 5 indicator classes on the basis of their pH optimum, assessed using Gaussian logistic regression, and these indicator values used to calculate a DAM score for each site using weighted averaging. Reference sites were selected on the basis of their acid neutralising capacity (ANC) and calcium concentration, and a regression model developed to predict expected DAM for each site using pH and total organic carbon (TOC) concentration. Site-specific DAM scores were used to calculate ecological quality ratios ranging from ≥1, where the diatom assemblage showed no impact, to (theoretically) 0, when the diatom assemblage was indicative of major anthropogenic activities. The boundary between 'high' and 'good' status was defined as the 25th percentile of Ecological Quality Ratios (EQRs) of all reference sites. The boundary between 'good' and 'moderate' status was set at the point at which nutrient-sensitive and nutrient-tolerant taxa were present in equal relative abundance. The methodology was evaluated using long-term data from 11 sites from the UK Uplands Waters Monitoring Network and is shown to perform well in discriminating naturally acid from acidified sites.