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Aquatic macroinvertebrates are often identified, based on morphology, but molecular approaches like DNA barcoding, metabarcoding and metagenomics are increasingly used for species identification. These approaches require the availability of DNA references deposited in public databases. Here we report the mitochondrial genomes of 11 aquatic macroinvertebrate species from Cyprus, a European Union island country in the Mediterranean. Only three species could be provisionally assigned to a binomial species name, highlighting the current lack of molecular references for aquatic macroinvertebrates from Cyprus. Graphical Abstract

Aquatic macroinvertebrates are often identified, based on morphology, but molecular approaches like DNA barcoding, metabarcoding and metagenomics are increasingly used for species identification. These approaches require the availability of DNA references deposited in public databases. Here we report the mitochondrial genomes of 11 aquatic macroinvertebrate species from Cyprus, a European Union island country in the Mediterranean. Only three species could be provisionally assigned to a binomial species name, highlighting the current lack of molecular references for aquatic macroinvertebrates from Cyprus.

The European eel (Anguilla anguilla) is a catadromous fish species, with population trends presenting significant declines over the last four decades. These declines throughout their range have resulted in their classification as 'critically endangered' by the IUCN. In addition, the European Union has implemented specific legislation surrounding A. anguilla, requiring member states to develop eel management plans [The EC Eel Regulation (1100/2007)]. Aimed to facilitate increased recruitment, these regulations state >40% of historic eel biomass should be allowed safe passage between inland waters and the sea. Cyprus however, applied and were granted an exemption from this, on the basis that there are no rivers on the island of suitable habitat and flow regimes which naturally host A. anguilla (2009/310/EC). Following this decision, recent findings have suggested that historically eels were more widespread in Cyprus than previously recognised. Indeed, a study by Zogaris et al. (2012) indicated that eels are likely the island's most widespread native species. Cyprus' freshwater lotic systems are dominated by intermittent rivers and streams, however the natural state and fish populations of these systems are poorly understood. The freshwaters of the island are now heavily fragmented, and with an estimated 108 dams retaining water are host to one of the highest densities of dam reservoirs in Europe. These interruptions to longitudinal connectivity lead to degraded freshwater systems in the lowlands, but despite this the island does have perennial freshwaters, particularly in the western side of the island and at higher elevations. If A. anguilla are indeed present in inland Cyprus, one key deterministic factor on their survival could be access to perennial summer refugia. Here, multiple monitoring methods were applied to build knowledge on present day eel distribution in Cyprus. By increasing knowledge regarding distribution, we can re-evaluate whether conservation initiatives are in fact justified and worthwhile. ...

The European eel (Anguilla anguilla) is a catadromous fish species, with population trends presenting significant declines over the last four decades. These declines throughout their range have resulted in their classification as 'critically endangered' by the IUCN. In addition, the European Union has implemented specific legislation surrounding A. anguilla, requiring member states to develop eel management plans [The EC Eel Regulation (1100/2007)]. Aimed to facilitate increased recruitment, these regulations state >40% of historic eel biomass should be allowed safe passage between inland waters and the sea. Cyprus however, applied and were granted an exemption from this, on the basis that there are no rivers on the island of suitable habitat and flow regimes which naturally host A. anguilla (2009/310/EC). Following this decision, recent findings have suggested that historically eels were more widespread in Cyprus than previously recognised. Indeed, a study by Zogaris et al. (2012) indicated that eels are likely the island's most widespread native species. Cyprus' freshwater lotic systems are dominated by intermittent rivers and streams, however the natural state and fish populations of these systems are poorly understood. The freshwaters of the island are now heavily fragmented, and with an estimated 108 dams retaining water are host to one of the highest densities of dam reservoirs in Europe. These interruptions to longitudinal connectivity lead to degraded freshwater systems in the lowlands, but despite this the island does have perennial freshwaters, particularly in the western side of the island and at higher elevations. If A. anguilla are indeed present in inland Cyprus, one key deterministic factor on their survival could be access to perennial summer refugia. Here, multiple monitoring methods were applied to build knowledge on present day eel distribution in Cyprus. By increasing knowledge regarding distribution, we can re-evaluate whether conservation initiatives are in fact justified and worthwhile. In 2020 environmental DNA metabarcoding was applied, 130 samples were taken across 26 freshwater sites to provide an up-to-date snapshot of eel distribution. In addition to this, temporal trends were considered based on an island wide fish monitoring programme spanning 2009 - 2019 which predominantly used electric fishing. Overall the results suggest that A. anguilla is widespread in western lowland Cyprus; 11/26 study sites (31/130 samples) tested positive for eel using eDNA metabarcoding, while eels were captured in 61/299 surveys (355 individuals) over the 10 year fish monitoring programme. The trends in eel distribution are highly concordant across methods, although not all sites were monitored with both methods (Fig. 1). These data indicate widespread eel recruitment in lowland freshwaters, but a lack of eels at higher elevation perennial areas. Lowland perennial areas are few and far between, however results here suggest they are abundant in A. anguilla. While higher elevation areas had higher overall freshwater fish species richness suggesting good habitat quality, the lack of the migratory eel in upper reaches indicates that barriers including dam structures may be preventing access for such migratory fish species. Environmental DNA detected eels in intermittent outlet flows of dams, but only in 2/9 reservoirs surveyed. We provide evidence for present day widespread eel recruitment in Cyprus' inland freshwaters, however highlight the need to increase connectivity to inland perennial systems in order to enhance survival of this critically endangered species at its eastern most range.

In freshwater ecosystems, periphytic biofilms include diatom assemblages that depend on environmental conditions (e.g., nutrient concentrations, salinity, temperature etc.). These assemblages respond rapidly to environmental changes, which makes diatoms valuable bioindicators. For this reason, they are currently used in freshwater biomonitoring programs (e.g., EU Water Framework Directive - WFD) (Foster et al., 2000). To date, diatom taxonomic identification is based on morphological criteria, which requires high taxonomic expertise to identify them to the species level needed for biomonitoring. Having this in mind, new strategies have been examined for the development of high-throughput, non-biased identification approaches. Human activities are the leading cause of environmental impairments and appropriate biomonitoring of ecosystems is needed to effectively assess the impact of their activities. In the last ten years, DNA metabarcoding combined with next-generation sequencing and bioinformatics, have been proposed as a complementary approach to morphological identification. In the past ten years, DNA metabarcoding coupled with next-generation sequencing and bioinformatics represents a complementary approach for diatom biomonitoring (Vasselon et al., 2019). In this study, this approach was used for the first time in Cyprus considering the association of environmental and anthropogenic pressures to diatom assemblages using the rbcL 312 bp barcode, next-generation sequencing (MiSeq Illumina), and bioinformatic evaluation (Mothur Software). Statistical analysis was then applied to identify the environmental (i.e., river types, geo-morphological) and anthropogenic (i.e., physical, chemical, human land-use pressures) variables' role in the observed diatom diversity. The Indice de Polluosensibilité Spécifique (IPS) index was used as it was shown to better respond to pressures that affect water quality in Cyprus rivers (WDD, 2014). Results indicate differences in diatom assemblages between intermittent and perennial rivers. Achnanthidium minutissimum was more abundant in intermittent rivers; whereas Amphora pediculus and Planothidium victorii (P. caputium) in perennial ones. Furthermore, we could demonstrate the correlation between nutrients (e.g., nitrogen, phosphorus), characteristics of the individual sampling sites (e.g., elevation), and land use activities on the observed differences in diatom diversity (Pissaridou, 2021). Additionally, results were compared to the morphotaxonomy-based approach which was conducted microscopically. Results show a positive correlation between morphological and molecular IPS scores. Points deviating from the norm are influenced by the limitations of both techniques. Fistulifera saprophila had a key role in this observation, as it negatively influences IPS scores. All in all, we conclude that DNA metabarcoding complements the morphological methodology for the ecological quality assessment of freshwaters in Cyprus. Multi-stressors and anthropogenic pressures have a significant statistical relationship to the observed diatom diversity and play a pivotal role in determining Cyprus' rivers' ecological status (Fig. 1).Foster, D., Wood, A., Griffiths, M., 2000. The Water Framework Directive (2000/60/EC) – An introduction Dave Foster – Policy Advisor (Europe), Aram Wood EP Scientist (Water), Dr Martin Griffiths – Head of Water Quality, Environment Agency, Head Office, Rio House, Waterside Drive, Aztec West, Almon 7–9.Pissaridou, P., Vasselon V., Christou A., Chonova T., Lacroix S., Papatheodoulou A., Drakou K., Tziortzis I., Dörflinger G., Rimet F., Bouchez A. and Vasquez MI. 2021 Deciphering Cyprus' diatom diversity and the effects of environmental and anthropogenic influences for ecological assessment of rivers using DNA metabarcoding.Chemosphere (In Press)Vasselon, V., Frédéric, R., Isabelle, D., Olivier, M., Yorick, R., Agnès, B., 2019. Assessing pollution of aquatic environments with diatoms' DNA metabarcoding: Experience and developments from France Water Framework Directive networks. Metabarcoding and Metagenomics 3, 101–115. https://doi.org/10.3897/mbmg.3.39646WDD, 2014. Review and update of article 5 of Directive 2000/60/EC (Water reservoirs) & Classification of water status (Rivers, natural lakes and water reservoirs), That will establish baseline information and data for the 2nd cyprus river basin management plan.