Integrating environmental DNA monitoring to inform eel (Anguilla anguilla) status in freshwaters at their easternmost range-A case study in Cyprus.

Despite significant population declines and targeted European Union regulations aimed at Anguilla anguilla conservation, little attention has been given to their status at their easternmost range. This study applies wide-scale integrated monitoring to uncover the present-day eel distribution in Cyprus' inland freshwaters. These are subject to increasing pressures from water supply requirements and dam construction, as seen throughout the Mediterranean. We applied environmental DNA metabarcoding of water samples to determine A. anguilla distribution in key freshwater catchments. In addition, we present this alongside 10 years of electrofishing/netting data. Refuge traps were also deployed to establish the timing of glass eel recruitment. These outputs are used together, alongside knowledge of the overall fish community and barriers to connectivity, to provide eel conservation and policy insights. This study confirm the presence of A. anguilla in Cyprus' inland freshwaters, with recruitment occurring in March. Eel distribution is restricted to lower elevation areas, and is negatively associated with distance from coast and barriers to connectivity. Many barriers to connectivity are identified, though eels were detected in two reservoirs upstream of dams. The overall fish community varies between freshwater habitat types. Eels are much more widespread in Cyprus than previously thought, yet mostly restricted to lowland intermittent systems. These findings make a case to reconsider the requirement for eel management plans. Environmental DNA-based data collected in 2020 indicate that "present-day" eel distribution is representative of 10-year survey trends. Suggesting that inland freshwaters may act as an unrealized refuge at A. anguilla's easternmost range. Conservation efforts in Mediterranean freshwaters should focus on improving connectivity, therefore enabling eels to access inland perennial refugia. Thus, mitigating the impact of climate change and the growing number of fragmented artificially intermittent river systems.

Using invertebrate functional traits to improve flow variability assessment within European rivers.

Rivers are among the most threatened ecosystems worldwide and are experiencing rapid biodiversity loss. Flow alteration due to climate change, water abstraction and augmentation is a severe stressor on many aquatic communities. Macroinvertebrates are widely used for biomonitoring river ecosystems although current taxonomic approaches used to characterise ecological responses to flow have limitations in terms of generalisation across biogeographical regions. A new macroinvertebrate trait-based index, Flow-T, derived from ecological functional information (flow velocity preferences) currently available for almost 500 invertebrate taxa at the European scale is presented. The index was tested using data from rivers spanning different biogeographic and hydro-climatic regions from the UK, Cyprus and Italy. The performance of Flow-T at different spatial scales and its relationship with an established UK flow assessment tool, the Lotic-invertebrate Index for Flow Evaluation (LIFE), was assessed to determine the transferability of the approach internationally. Flow-T was strongly correlated with the LIFE index using both presence-absence and abundance weighted data from all study areas (r varying from 0.46 to 0.96). When applied at the river reach scale, Flow-T was effective in identifying communities associated with distinct mesohabitats characterised by their hydraulic characteristics (e.g., pools, riffles, glides). Flow-T can be derived using both presence/absence and abundance data and can be easily adapted to varying taxonomic resolutions. The trait-based approach facilitates research using the entire European invertebrate fauna and can potentially be applied in regions where information on taxa-specific flow velocity preferences is not currently available. The inter-regional and continental scale transferability of Flow-T may help water resource managers gauge the effects of changes in flow regime on instream communities at varying spatial scales.

Cyprus' diatom diversity and the association of environmental and anthropogenic influences for ecological assessment of rivers using DNA metabarcoding.

Human activities are the leading cause of environmental impairments. Appropriate biomonitoring of ecosystems is needed to assess these activities effectively. In freshwater ecosystems, periphytic and epilithic biofilms have diatom assemblages. These assemblages respond rapidly to environmental changes, making diatoms valuable bioindicators. For this reason, freshwater biomonitoring programs are currently using diatoms (e.g., Water Framework Directive). In the past ten years, DNA metabarcoding coupled with next-generation sequencing and bioinformatics represents a complementary approach for diatom biomonitoring. In this study, this approach is used for the first time in Cyprus by considering the association of environmental and anthropogenic pressures to diatom assemblages. Statistical analysis was then applied to identify the environmental (i.e., river types, geo-morphological) and anthropogenic (i.e., physicochemical, human land-use pressures) variables' role in the observed diatom diversity. Results indicate differences in diatom assemblages between intermittent and perennial rivers. Achnanthidium minutissimum was more abundant in intermittent rivers; whereas Amphora pediculus and Planothidium caputium in perennial ones. Additionally, we could demonstrate the correlation between nutrients (e.g., nitrogen, phosphorus), stations' local characteristics (e.g., elevation), and land use activities on the observed differences in diatom diversity. Finally, we conclude that multi-stressors and anthropogenic pressures together as multiple stressors have a significant statistical relationship to the observed diatom diversity and play a pivotal role in determining Cyprus' rivers' ecological status.

Biomonitoring of intermittent rivers and ephemeral streams in Europe: Current practice and priorities to enhance ecological status assessments.

Intermittent rivers and ephemeral streams (IRES) are common across Europe and dominate some Mediterranean river networks. In all climate zones, IRES support high biodiversity and provide ecosystem services. As dynamic ecosystems that transition between flowing, pool, and dry states, IRES are typically poorly represented in biomonitoring programmes implemented to characterize EU Water Framework Directive ecological status. We report the results of a survey completed by representatives from 20 European countries to identify current challenges to IRES status assessment, examples of best practice, and priorities for future research. We identify five major barriers to effective ecological status classification in IRES: 1. the exclusion of IRES from Water Framework Directive biomonitoring based on their small catchment size; 2. the lack of river typologies that distinguish between contrasting IRES; 3. difficulties in defining the 'reference conditions' that represent unimpacted dynamic ecosystems; 4. classification of IRES ecological status based on lotic communities sampled using methods developed for perennial rivers; and 5. a reliance on taxonomic characterization of local communities. Despite these challenges, we recognize examples of innovative practice that can inform modification of current biomonitoring activity to promote effective IRES status classification. Priorities for future research include reconceptualization of the reference condition approach to accommodate spatiotemporal fluctuations in community composition, and modification of indices of ecosystem health to recognize both taxon-specific sensitivities to intermittence and dispersal abilities, within a landscape context.

Are saturating pulses indeed saturating? Evidence for considerable PSII yield underestimation in leaves adapted to high levels of natural light.

The "saturating pulse" method of in vivo Chl fluorescence measurement has been widely used by physiologists and especially ecophysiologists, as it allows a simple, rapid and non-invasive assessment of PSII function and the allocation of absorbed energy into photochemical and non-photochemical processes. It is based on the accurate determination of the so-called Fm('), i.e. the fluorescence signal emitted when a "saturating" light pulse closes all PSII centers. In this methodological investigation, we examined whether the saturating pulse intensities required to obtain maximal fluorescence yields differ between leaves of various species receiving varying actinic light irradiances. It was shown that, in leaves adapted to comparatively high (yet realistic) levels of natural irradiances, the saturating pulses usually applied are not able to close all PSII reaction centers. As a result, there is a high risk of considerable Fm(') underestimation. Accordingly, the derived values of effective PSII yields and linear electron transport rates (ETR) are also underestimated, even at the highest saturation pulse levels afforded by commercial instruments. Since the extent of underestimation increases with actinic irradiance, the ETR versus light curves are considerably distorted. The possible reasons for the apparent inability of "saturating" pulses to close all PSII centers at high actinic light and the practical implications, especially in field work, are discussed.