Cryptic diversity in the Western Balkan endemic copepod: Four species in one?

We use mitochondrial (mtCOI) and nuclear (nH3) sequence data to investigate differentiation of Eudiaptomus hadzici, a freshwater copepod endemic to the Western Balkans. E. hadzici has a disjunct distribution and morphological differences were observed at regional scale. In the current study 6 out of 7 known populations are included. We applied several species delimiting approaches, distance based methods (K2P p-distance and Automatic Barcode Gap Discovery, ABGD) using the mtCOI, Bayesian phylogeny and the Bayesian method implemented in bPTP and BPP programs using the concatenated sequences of both genes. Phylogenetic and species delimitation analyses all suggest that the nominal species E. hadzici consists of four isolated, cryptic evolutionary lineages in the Western Balkans. Each of the four lineages inhabits a single lake or a group of lakes in close proximity. They exhibit major differences in secondary sexual characters, e.g. right antennule in males. Denticulation of spine on 13th segment is substantially distinct among the four lineages, having different number and shape of tooth-like protrusions. Gene flow and dispersal are restricted to very small spatial scale, but with local differences, implying that diverse historical and contemporary processes are operating at small spatial scales in E. hadzici. In order to further examine spatial and temporal diversification patterns, we constructed a dated species tree analysis using (*)BEAST. Due to lack of reliable calibration points and taxa specific evolutionary rates, two evolutionary rates were applied and the faster one (2.6% myr) seems more plausible considering the geological history of the region. The divergence of E. hadzici lineages is dated from Early Miocene onwards with geographically close lineages diverging more recently, Late Miocene to Pleistocene and Pleistocene, respectively. Overall, our findings shed light on cryptic genetic complexity of endemics in one of European biodiversity hotspots. Moreover, this study represents one further example of integrative taxonomy, linking DNA methodology and classical taxonomy based on morphology. Therefore, it lays groundwork for future taxonomy and biogeography of freshwater microcrustaceans in the region.

Redundancy in the ecological assessment of lakes: Are phytoplankton, macrophytes and phytobenthos all necessary?

Although the Water Framework Directive specifies that macrophytes and phytobenthos should be used for the ecological assessment of lakes and rivers, practice varies widely throughout the EU. Most countries have separate methods for macrophytes and phytobenthos in rivers; however, the situation is very different for lakes. Here, 16 countries do not have dedicated phytobenthos methods, some include filamentous algae within macrophyte survey methods whilst others use diatoms as proxies for phytobenthos. The most widely-cited justification for not having a dedicated phytobenthos method is redundancy, i.e. that macrophyte and phytoplankton assessments alone are sufficient to detect nutrient impacts. Evidence from those European Union Member States that have dedicated phytobenthos methods supports this for high level overviews of lake condition and classification; however, there are a number of situations where phytobenthos may contribute valuable information for the management of lakes.

Benthic macroinvertebrates in lake ecological assessment: A review of methods, intercalibration and practical recommendations.

Legislation in Europe has been adopted to determine and improve the ecological integrity of inland and coastal waters. Assessment is based on four biotic groups, including benthic macroinvertebrate communities. For lakes, benthic invertebrates have been recognized as one of the most difficult organism groups to use in ecological assessment, and hitherto their use in ecological assessment has been limited. In this study, we review and intercalibrate 13 benthic invertebrate-based tools across Europe. These assessment tools address different human impacts: acidification (3 methods), eutrophication (3 methods), morphological alterations (2 methods), and a combination of the last two (5 methods). For intercalibration, the methods were grouped into four intercalibration groups, according to the habitat sampled and putative pressure. Boundaries of the 'good ecological status' were compared and harmonized using direct or indirect comparison approaches. To enable indirect comparison of the methods, three common pressure indices and two common biological multimetric indices were developed for larger geographical areas. Additionally, we identified the best-performing methods based on their responsiveness to different human impacts. Based on these experiences, we provide practical recommendations for the development and harmonization of benthic invertebrate assessment methods in lakes and similar habitats.

Water quality assessment of rivers using diatom metrics across Mediterranean Europe: a methods intercalibration exercise.

The European Water Framework Directive establishes a framework for the protection of water resources. However, common water management tools demand common understanding of assessment methods, so quality goals are equally met. Intercalibration of methods ensures the comparability of biological elements across similar geographical areas. Many aspects can influence the outcome of intercalibration: data sampling, treatment methods, taxonomic reliability of databases, choice of metrics for ecological quality status classification, and criteria for selecting reference sites. This study describes the potentials and constraints of the intercalibration of indices using diatoms for assessment of Mediterranean rivers. Harmonisation of diatom taxonomy and nomenclature was based on a previous ring test which took place at the European level. Four diatom indices (Indice de Polluosensibilité Spécifique-IPS, Indice Biologique Diatomées-IBD 2007, Intercalibration Common Metric Italy-ICMi and Slovenian Ecological Status assessment system) were intercalibrated using data from six European Mediterranean countries (Cyprus, France, Italy, Portugal, Slovenia and Spain). Boundaries between High/Good and Good/Moderate quality classes were harmonised by means of the Intercalibration Common Metric (ICM). Comparability between countries was assured through boundary bias and class agreement. The national boundaries were adjusted when they deviated more than a quarter of a class equivalent (0.25) from the global mean. All national methods correlated well with the ICM, which was sensitive to water quality (negatively correlated to nutrients). Achnanthidium minutissimum sensu lato was the most discriminative species of Good ecological status class. Planothidium frequentissimum, Gomphonema parvulum and Nitzschia palea were the most contributive to Moderate ecological status class. Some taxa were discriminative for both Good and Moderate ecological status classes due to low indication and ecological discriminative power but also due to differences in taxonomy between countries. This intercalibration exercise allowed establishment of common water quality goals across Mediterranean Europe, which is substantiated with the ICM.

Defining chlorophyll-a reference conditions in European lakes.

The concept of "reference conditions" describes the benchmark against which current conditions are compared when assessing the status of water bodies. In this paper we focus on the establishment of reference conditions for European lakes according to a phytoplankton biomass indicator--the concentration of chlorophyll-a. A mostly spatial approach (selection of existing lakes with no or minor human impact) was used to set the reference conditions for chlorophyll-a values, supplemented by historical data, paleolimnological investigations and modelling. The work resulted in definition of reference conditions and the boundary between "high" and "good" status for 15 main lake types and five ecoregions of Europe: Alpine, Atlantic, Central/Baltic, Mediterranean, and Northern. Additionally, empirical models were developed for estimating site-specific reference chlorophyll-a concentrations from a set of potential predictor variables. The results were recently formulated into the EU legislation, marking the first attempt in international water policy to move from chemical quality standards to ecological quality targets.

Effect of fixed-fraction subsampling on macroinvertebrate bioassessment of rivers.

We investigated the effect of different subsample fractions on the variability of benthic invertebrate metrics. The results of six fractions 1/12, 1/6, 1/4, 1/3, 5/12 and 1/2 were compared to the results of the whole samples. Over 120 metrics were tested using five datasets: ecoregion Alps and four river types. In general, variability of metrics decreased with increasing subsample size, but variability varied greatly with the selected metric group and river type. Independent of river type, the highest variation was observed for the composition/abundance group metrics and the richness metrics, whereas it was low for the diversity indices and for the metrics of the sensitivity/tolerance group and intermediate for the functional metric group. For all metric groups independent of river type, the main decrease in variability occurs up to 1/4 subsample. We suggest that the effect of subsample size on variability of metrics should be tested prior to selecting potential assessment metrics.