Relating environmental pressures to littoral biological water quality indicators in Western Balkan lakes: Can we fill the largest gaps?

Along six transects in each of six lakes across the Western Balkans, we collected data for three groups of littoral biological water quality indicators: epilithic diatoms, macrophytes, and benthic invertebrates. We assessed the relationships between them and three environmental pressures: nutrient load (eutrophication), hydro-morphological alteration of the shoreline, and water level variation, separating the effect of individual lakes and continuous explanatory variables. Lake water total phosphorus concentration (TP) showed substantial variation but was not related to any of the tested biological indicators, nor to any of the tested pressures. We suggest that this may be due to feedback processes such as P removal in the lake littoral zone. Instead, we found that a gradient in surrounding land-use towards increasing urbanization, and a land-use-based estimate of P run-off, served as a better descriptor of eutrophication. Overall, eutrophication and water level fluctuation were most important for explaining variation in the assessed indicators, whereas shoreline hydro-morphological alteration was less important. Diatom indicators were most responsive to all three pressures, whereas macrophyte biomass and species number responded only to water level fluctuation. The Trophic Diatom Index for Lakes (TDIL) was negatively related to urbanization and wave exposure. This indicates that it is a suitable indicator for pressures related to urbanization, although a confounding effect of wave exposure is possible. Invertebrate abundance responded strongly to eutrophication, but the indicator based on taxonomic composition (Average Score Per Taxon) did not. Our results suggest that our metrics can be applied in Western Balkan lakes, despite the high number of endemic species present in some of these lakes. We argue that local water management should focus on abating the causes of eutrophication and water level fluctuation, whilst preserving sufficient lengths of undeveloped shoreline to ensure good water quality in the long run.

Littoral eutrophication indicators are more closely related to nearshore land use than to water nutrient concentrations: A critical evaluation of stressor-response relationships.

Biological assessment metrics and water chemistry measurements are used to quantify the link between stressors and their effects on lake ecosystems, for the Water Framework Directive. However, correlations between metrics and water chemistry are often poor. This is seen as major weaknesses of Water Framework Directive-related monitoring and assessment. We analyzed macrophytes, benthic algae, benthic macroinvertebrates, water chemistry and sediment total phosphorus content in the littoral of six lakes in the Western Balkans and used CORINE land use data to estimate nutrient enrichment via runoff from the adjacent land. Lakes with a higher estimated phosphorus runoff from the adjacent land did not have higher littoral water nutrient concentrations, but littoral diatom assemblages indicated more eutrophic conditions. These lakes also had higher abundances of littoral benthic primary producers, which in turn were associated with low concentrations of dissolved nutrients, but only in autumn, not in spring. This is consistent with primary producers taking up nutrients during the summer growth season. In lakes with high abundances of benthic primary producers, it is likely that the littoral vegetation plays a large role in the transfer of nutrients from the water to the benthos. This process impairs correlations between biological metrics and water nutrient concentrations. Our results suggest that CORINE land cover may be more useful to characterize littoral nutrient enrichment than lake water chemistry. Increased benthic primary producer biomasses and "eutrophic" diatom indices may indicate littoral nutrient enrichment even if water nutrient concentrations are low.

Simulium reptans (Linnaeus, 1758) and Simulium reptantoides Carlsson, 1962 from the Balkan Peninsula.

Simulium reptans (Linnaeus, 1758) and Simulium reptantoides Carlsson, 1962 are two species of the Simulium reptans group whose distribution is unclear because of their confusing taxonomy and systematics. Their genetic variability is well known for populations in northern and central Europe and shows that both species have two forms; however, the genetic variability of these species in southern and eastern Europe is unknown. To identify the status of these two species in southeast Europe, mtDNA was extracted from 19 individuals from 12 localities across the Balkan Peninsula. Phylogenetic analysis confirmed the existence of two species with 7.38-7.94% divergence. Each species was comprised of two clades, with 2.31% and 1.43% interclade divergence for S. reptans and S. reptantoides, respectively. This study revealed the presence of both species across the Balkans and that S. reptans occurs in this area in only one form (S. reptans B), while S. reptantoides is found in two genetic forms (A and B).

Phylogeography of Simulium Subgenus Wilhelmia (Diptera: Simuliidae)-Insights From Balkan Populations.

Many morphologically similar species of the simuliid (Diptera: Simuliidae) subgenus Wilhelmia, Enderlein are difficult to distinguish. Thus, the revision of the subgenus using various morphological, cytogenetic, and genetic analyses has been attempted. Neglected until now, the Balkan Peninsula, a crossroad between Europe and Anatolia, provides insight which could resolve problematic interrelationships of the taxa within this subgenus. To uncover the status and relations within the subgenus Wilhelmia, mtDNA was extracted from 47 individuals of six morphospecies: Simulium balcanicum (Enderlein, 1924), Simulium turgaicum Rubtsov, 1940, Simulium lineatum (Meigen, 1804), Simulium pseudequinum Séguy, 1921, Simulium equinum (Linnaeus, 1758), and Simulium paraequinum Puri, 1933 from 21 sites throughout the Balkan Peninsula. Phylogenetic analysis of the Wilhelmia species using mitochondrial DNA barcoding (COI) gene showed two major branches, the lineatum branch, which includes the lineages sergenti, paraequinum, and lineatum, and the equinum branch. In the equinum branch, the mtDNA sequences formed six clades, with high genetic distances, suggesting the existence of different species. Historically, the clades of the equinum branch appeared at numerous islands, perhaps as a result of allopatric speciation. The paraequinum lineage (lineatum branch) is composed of two species. However, six clades of the lineatum lineage overlapped with intra- and interspecific genetic distances. Our results revealed that the species S. balcanicum, S. pseudequinum B, and S. equinum were omnipresent in the Balkans. The results point to not only the fair diversity of Wilhelmia species in the Balkans, but also indicate that most Wilhelmia species live in sympatry.

The response of chironomid taxonomy- and functional trait-based metrics to fish farm effluent pollution in lotic systems.

The lotic habitats affected by trout farm waste are colonized with a particular invertebrate community of which chironomids are the most abundant group. However, there is little information available regarding how chironomid community structures respond to this type of pollution at the highest taxonomic resolution. Eight fish farms, together with their lotic systems as recipients, were used to test the variability of the chironomid community and its surrogates (taxonomic and functional metrics) across spatially arranged sampling sites to form a gradual decrease in the trout farm influence. The self organizing map (SOM) classified six different types of chironomid communities which were characteristic for both the control and affected habitats. The species indicator analyses listed 32 taxa as positive indicators of water pollution. The SOM and Kruskal-Wallis test revealed that the pattern of chironomid community structure obtained was mainly driven by six environmental parameters (Altitude, conductivity, distance from the outlet, hardness, HN4-N, NO3-N). Categorical principal components analysis (CATPCA) derived three models for each type of biotic metric, in which for diversity-, taxonomy- and functional feeding group-based metrics, the first two dimensions explained 55.2%, 58.3% and 55.4%, of the total variance respectively for 315 sampling sites. According to this analysis, the total number of taxa (S), abundance and the Shannon-Wiener index (H') (as a diversity metric), as well as the proportion of Tanypodinae (as taxonomic group) and grazers/scraper (GRA) and gatherer collector (GAT)(as FFG metrics), were related to the outlet distance gradient, thus showing great potential to be used in the multimetric approach in bioassessment.

Evaluation of oxidative stress biomarkers in the freshwater gammarid Gammarus dulensis exposed to trout farm outputs.

The current study reports a combined seasonal monitoring of the Crnica River watercourse using physico-chemical parameters and biomarkers measured in Gammarus dulensis with the aim of correlating the effects of pollution with the level of oxidative stress biomarkers in this amphipod. The pollution source is identified as a trout farm in the upper part of the Crnica River (Eastern Serbia). Physico-chemical water parameters and sediment trace element concentrations were measured in the Crnica River during three seasons. Three localities downstream [CR2, CR3, and CR4 (20 m, 400 m, and 1.3 km from the trout farm discharge, respectively)] were assessed in comparison with a reference location (CR1), 250 m upstream from the trout aquaculture in the Crnica River. Antioxidant biomarkers including the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione-S-transferase (GST) were determined in pooled samples of G. dulensis. Our findings showed statistically different antioxidant enzyme levels in gammarids from the first downstream locality (CR2) compared to those from other three localities. The results indicate that oxidative stress occurred to some extent despite the assumed antioxidant induction as a result of exposure to elevated levels of some elements and changed water parameters, the effects seeming in part to be mediated by suppression of SOD and CAT (during summer and autumn); and by suppression of GPX and GR (during autumn) at the CR2 locality. The activity of GST in all seasons was higher at the CR2 locality than at the other localities, suggesting the presence of some permanent xenobiotic next to the aquaculture output. Seasonal difference in the physical and chemical water parameters and sediment trace element concentrations were observed. Overall, our results indicate a locality- and season-dependent antioxidant enzymes response in G. dulensis, pointing at progressively reduced deleterious effect of trout farm outputs with movement further downstream.

Integrative taxonomy by molecular species delimitation: multi-locus data corroborate a new species of Balkan Drusinae micro-endemics.

BACKGROUND: Taxonomy offers precise species identification and delimitation and thus provides basic information for biological research, e.g. through assessment of species richness. The importance of molecular taxonomy, i.e., the identification and delimitation of taxa based on molecular markers, has increased in the past decade. Recently developed exploratory tools now allow estimating species-level diversity in multi-locus molecular datasets. RESULTS: Here we use molecular species delimitation tools that either quantify differences in intra- and interspecific variability of loci, or divergence times within and between species, or perform coalescent species tree inference to estimate species-level entities in molecular genetic datasets. We benchmark results from these methods against 14 morphologically readily differentiable species of a well-defined subgroup of the diverse Drusinae subfamily (Trichoptera, Limnephilidae). Using a 3798 bp (6 loci) molecular data set we aim to corroborate a geographically isolated new species by integrating comparative morphological studies and molecular taxonomy. CONCLUSIONS: Our results indicate that only multi-locus species delimitation provides taxonomically relevant information. The data further corroborate the new species Drusus zivici sp. nov. We provide differential diagnostic characters and describe the male, female and larva of this new species and discuss diversity patterns of Drusinae in the Balkans. We further discuss potential and significance of molecular species delimitation. Finally we argue that enhancing collaborative integrative taxonomy will accelerate assessment of global diversity and completion of reference libraries for applied fields, e.g., conservation and biomonitoring.

The larvae of the European Helicopsyche species (Trichoptera: Helicopsychidae).

This synoptic paper gives descriptions of the hitherto unknown or poorly known larvae of Helicopsyche megalochari Malicky 1974 and H. revelieri McLachlan 1884 (Trichoptera: Helicopsychidae). We present information on the morphology of the larvae and illustrate the most important diagnostic features. This information is used for the construction of a comprehensive larval key to the five European species of family Helicopsychidae. In the context of this key, larvae can be easily diagnosed by setation patterns of the dorsal head capsule, submentum shape, and propleuron morphology. With respect to distribution, Helicopsyche megalochari has been reported from the Greek Islands of Andros, Euboea, Ikaria, and Naxos whereas H. revelieri is known from the islands of Capraia, Corsica, and Sardinia. In addition, ecological characteristics and distributions of the European species are briefly discussed.

Morphological features of larvae of Drusus plicatus Radovanovic (Insecta, Trichoptera) from the Republic of Macedonia with molecular, ecological, ethological, and distributional notes.

A description of the larva of Drusus plicatus Radovanovic is given for the first time. The most important diagnostic characters enabling separation from larvae of the other Drusinae from the southeast Europe are listed. Molecular, ecological, and ethological features and distribution patterns of the species are given. Additionally, information on the sympatric caddisfly species of the three springs where larvae and adults of Drusus plicatus were found and presented.

The trout farm effect on Dinocras megacephala (Plecoptera: Perlidae) larvae: Antioxidative defense.

Trout production represents a major agricultural activity in Serbia. Organic compounds are drained into the environment, usually without previous sedimentation, thus affecting the stream biota. Considering that biological monitoring is commonly based on aquatic macroinvertebrates as target organisms, the authors used larvae of Dinocras megacephala to estimate trout farm effects on the mass of the larvae and their antioxidative defense in pollution stress conditions. Four locations were chosen along the channel of the Raska River, 2 upstream (L1, L2) and 2 downstream (L3, L4) from the trout farm outlet. Basic physical and chemical water parameters were measured. Superoxide dismutase (SOD) and catalase (CAT) activity changes were analyzed to determine the level of oxidative stress caused by the increase of organic compounds originating from the trout farm. Dissolved oxygen concentration decreased from the upstream to downstream locations. Furthermore, the concentration of ionized ammonia was almost 10 times higher at the downstream locations than at the upstream locations. Larval mass, as well as CAT activity, was significantly higher at L3 compared with the other 3 locations. Activity of SOD was significantly higher at L3 than at L1. The results indicate that higher concentrations of organic compounds from the trout farm induce clear changes in the status of the antioxidant defense of D. megacephala larvae. Environ Toxicol Chem 2016;35:1775-1782. © 2015 SETAC.

Larval morphology and phylogenetic position of Drusus balcanicus, Drusus botosaneanui, Drusus serbicus and Drusus tenellus (Trichoptera: Limnephilidae: Drusinae).

In a recent 3-gene phylogeny of the Trichoptera subfamily Drusinae Banks, 1916 molecular data clearly correlated with the morphology and feeding ecology of larvae. The largest of three main groups, the Drusinae grazer clade, exhibits an unusual larval feeding ecology for Limnephilidae, and is the most diverse group. In this paper we describe four previously unknown Drusinae larvae from this clade: Drusus balcanicus Kumanski, 1973 (micro-endemic to Eastern Balkans); Drusus botosaneanui Kumanski, 1968 (Dinaric Western Balkans, Hellenic and Eastern Balkan, Asia Minor), Drusus serbicus Marinkovic-Gospodnetic, 1971a (micro-endemic to Dinaric Western Balkans); and Drusus tenellus (Klapálek, 1898) (Carpathians, Dinaric Eastern Balkans). Characteristically, the larvae of these species develop toothless mandibles typical for the Drusinae grazer clade. Larvae and adults were unambiguously associated by a phylogenetic approach based on two mitochondrial (mtCOI, mtLSU= 16S rDNA) and two nuclear genes (nuWG, nuCAD). In addition, information on the morphology of the larvae is given and the diagnostic features necessary for identification are illustrated.