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According to the Water Framework Directive (WFD), biological quality elements constitute the basis for assessing the ecological status of surface waters. Biological communities are good indicators of water quality because they reflect environmental conditions over time and do not require frequent sampling. The main aims of this study were to assess the trophic and ecological status of the Lake Visovac (Krka River hydrosystem, South Croatia) using phytoplankton together with supportive physico-chemical parameters, eutrophication status and impact indicators. We also tested the viability of chlorophyll-a (Chl-a) and Hungarian Lake Phytoplankton Index (HLPI) as proposed metrics in the standard regulatory monitoring procedure. The phytoplankton samples were taken and Chl-a and physical- chemical parameters measured on a monthly basis from April to September in 2016 and 2019. In 2016, the dominant species was Pantocsekiella ocellata (Pantocsek) K.T.Kiss & Ács, while in 2019 it was accompanied by Ceratium hirundinella (O.F.Müller) Dujardin. According to Chl-a, Lake Visovac was within the limits of oligo- to mesotrophic status. Based on the HLPI index, the Lake ecological status was assessed as Good. Chl-a showed a statistically significant positive correlation with temperature, while HLPI was positively correlated with oxygen and Secchi depth. We propose the use of Chl-a for rapid bioassessment on a weekly basis, whilst the more complex HLPI index should be applied monthly. Further improvement of the confidence level of the metrics used to assess the ecological status and a comprehensive revision of boundaries for included indicators is of fundamental importance.

In lake ecosystems, changes in eukaryotic and prokaryotic microbes and the concentration and availability of dissolved organic matter (DOM) produced within or supplied to the system by allochthonous sources are components that characterize complex processes in the microbial loop. We address seasonal changes of microbial communities and DOM in the largest Croatian lake, Vrana. This shallow lake is connected to the Adriatic Sea and is impacted by agricultural activity. Microbial community and DOM structure were driven by several environmental stressors, including drought, seawater intrusion, and heavy precipitation events. Bacterial composition of different lifestyles (free-living and particle- associated) differed and only a part of the particle-associated bacteria correlated with microbial eukaryotes. Oscillations of cyanobacterial relative abundance along with chlorophyll a revealed a high primary production season characterized by increased levels of autochthonous DOM that promoted bacterial processes of organic matter degradation. From our results, we infer that in coastal freshwater lakes dependent on precipitation-evaporation balance, prolonged dry season coupled with heavy irrigation impact microbial communities at different trophic levels even if salinity increases only slightly and allochthonous DOM inputs decrease. These pressures, if applied more frequently or at higher concentrations, could have the potential to overturn the trophic state of the lake. This article is protected by copyright. All rights reserved.

The Krka River is located at Balkan Peninsula in the Dinaric region of Croatia and it is a typical groundwater-fed karst river characterized by an unusual phenomenon-tufa. Tufa designates porous CaCO3 deposits forming under specific physical and chemical conditions and hosting very diverse freshwater biota. Karst springs are considered one of the most diverse habitats for diatoms and are usually inhabited by specific diatom assemblages, which include the Krka spring zone. However, such habitats are particularly impacted by human activities, so previous studies have recently been conducted to raise awareness of the importance of karstic spring zones, especially because they are inhabited by specific and rare species. During the research at Krka River, samples were collected in the spring zone in May 2017, and the observations revealed the existence of a one Cymbopleura species characterized by slightly convex to almost linear valves, which are slightly protracted, broadly rounded and weakly dorsally bent at the apices. Based on detailed morphological analyses on light and scanning electron microscopy it appears that this population resembles two previously described taxa: C. rupicola var. minor Krammer and C. rupicola var. korana Krammer. Here we provide a more detailed description of the mentioned population under the new name Cymbopleura amicula stat. nov. et nom. nov. due to the significant differences from C. rupicola, together with a comparison with similar taxa in Cymbopleura.

Phytoplankton is one of the key Biological Quality Elements within the Water Framework Directive, used to assess the ecological status of surface water bodies. Water samples for phytoplankton identification were collected from April to September at a total of eight sampling sites in all six Croatian natural karstic lakes with an area greater than 0.5 km2. The main objective was to show the comparability of environmental DNA metabarcoding (Illumina sequencing using the hypervariable V9 region of the eukaryotic SSU rRNA gene) with morphologically based assessment and its applicability in assessing the ecological status of lakes. The value of Hungarian lake phytoplankton index (HLPI) indicating the final ecological status was calculated for both datasets using biomass and composition metrics. Chlorophyll a concentration measured using Ultra-High-Performance Liquid Chromatography and Spectrophotometer giving two biomass metrics along with the functional group approach (FG) as the composition metric for the complete taxa/operational taxonomic units (OTUs) lists as well as for the taxa/OTUs that contributed more than 5% to the total biomass/number of amplicons gave four to four HLPI values per sample. HLPI values from both approaches were highly correlated (Pearson's r > 0.92) and classified into good or high ecological status, although different compositions and proportions of FGs were recorded, thus giving the important role to the equal or similar factors assignment to different FGs with similar ecological demands and favourable habitats. In 89% of the samples, HLPI values indicate an equal range of ecological status and most differences were found due to different methods of Chlorophyll a measurement. Different composition metrics between approaches showed significant differences (p < 0.05) only in lakes Prošće and Crniševo. This study showed the applicability of the V9 region of 18S rRNA in ecological status assessment for oligotrophic and mesotrophic lakes due to the comparable results between approaches, but further development and standardization of eDNA metabarcoding are needed for the implementation in routine monitoring programs.

The biodiversity of algal communities and environmental conditions were investigated in the springs of Mt. Konjuh. The assemblages of 20 springs emerging from different lithologies (limestones and ophiolites, respectively) comprised 234 algal taxa. Diatoms and cyanobacteria were the most species-rich groups. The most common alkaliphilic, circumneutral, and eutraphentic diatoms were represented by the genera Gomphonema, Nitzschia, Navicula, Cymbella, and Achnanthidium, and by the cyanobacterial genus Phormidium. Hierarchical clustering and SIMPROF analysis based on relative algal abundance clustered springs into six groups, separating them mainly according to spring type and lithology. Indicator species for groups and springs on different lithological substrata were singled out, revealing 33 taxa with preferences for ophiolites, and 20 taxa with preferences for carbonates. The values of the Shannon-Wiener diversity index were moderately high per spring location, and similar for the two groups of springs on different lithologies. A higher similarity in species composition was noted between springs on ophiolites and limestones than between springs on ophiolites and other types of siliceous substrata. The present study suggests that algal assemblages in springs emerging from ophiolites, even those made up by a preponderance of silicates, should be analyzed separately from those related to springs on other siliceous substrata. The results obtained showed that most of the springs studied are affected by anthropogenic impacts and morphological alterations leading to the dominance of highly competitive meso- and eutraphentic algal species, thus emphasizing the importance of further investigation and conservation of these habitats.

Ciliates are single celled eukaryotes recognized as key players in the microbial loop of aquatic ecosystems. The present study was carried out on the Krka River (Croatia), a karst freshwater ecosystem characterized by tufa barriers, biomineralization and highly diverse aquatic communities. The main aims of the study were to investigate ciliate community structure in the biofilm (i.e. periphyton) samples collected from light- and dark-exposed lithified tufa/stones. Furthermore, by establishing links between ciliate community patterns and environmental parameters, we aimed to assess the bioindicator potential of specific ciliate taxa for environmental monitoring of freshwater habitats. The periphyton sampling was performed at four representative sites of the river source, upstream, middle and downstream river sections. Ciliate community was investigated via traditional microscopy analyses and environmental DNA (eDNA) metabarcoding (Illumina sequencing of the hypervariable V9-region of the SSU rRNA gene). The molecular approach recorded a substantially higher number of ciliate taxa, most of which taxonomically belonging to genera typically occurring in tufa barriers. The results from microscopy analyses did not show any links between ciliate community structure and sampling location. However, eDNA approach indicated significant differences among the sampling locations regarding the ciliate community structure. Thereby, hydrological parameters and saprobiological classification of the sampling sites were the main structuring factors for ciliate community. The coupling of eDNA metabarcoding with the morphological approach provides a robust, in-depth analytical system in elucidating the bioindicator potential of ciliated protists.

Benthic diatoms are widely used in Europe and worldwide to access ecological status of running waters. One of key goals of Water Framework Directive is to classify rivers and streams using biological quality elements and type specific reference conditions. According to system B which incorporates additional abiotic descriptors, there are 24 water types in Croatia. For biological analyses 92 rivers and streams with 140 sampling points were chosen and sampled for benthic diatoms and water chemistry simultaneously. Self organizing map (SOM) analysis was used to define biotypes from species composition and abundance of benthic diatoms. Grouping of samples in SOM resulted in 10 distinctive groups. Based on their geographical position and site characteristics, groups represent sites with similar properties (as waterbed, catchment size, altitude, size of stream) belonging to different ecoregions in Croatia. Analysis of variance revealed statistically significant differences (p

The genus Entomoneis Ehrenberg includes diatoms with structurally complex frustules having a bilobate keel elevated above the valve surface and numerous girdle bands. We describe here a new member of the genus, Entomoneis tenera sp. nov. , a minute species found in the plankton of the south-eastern Adriatic Sea. The description and proposed taxonomic affiliation are based on both morphological observations and molecular analyses obtained from three cultivated strains. The cultures were established from plankton net samples taken during BIOTA (Bio-tracing Adriatic Water Masses) cruise conducted in February–March 2015. In addition to characteristic Entomoneis features such as panduriform cells, often twisted around the apical axis and a raphe with simple endings positioned on the sigmoid keel, morphological characteristics of E. tenera are: (1) very small cells, 16–21 μm long and 5–20 μm wide (2) very lightly silicified, delicate frustules without valve striation discernible in light microscopy, (3) broad lanceolate valves with scalpeliform apices (4) a straight to slightly arcuate junction line. Phylogenetic analyses using SSU, rbc L and psb C supported the position of E . tenera within the Entomoneis genus with a clear separation from the other described species.

Crveno jezero (Red Lake) is one of the largest karstic sinkhole lakes in the Europe. Diatom flora of this lake was unknown due to difficulties in collecting samples. Recently, samples were collected by speleodivers in the frame of systematical natural sciences research, during the cave diving expedition “Crveno jezero 2017” and observations revealed the existence of a new species, Gomphosphenia plenkoviciae Gligora Udovič & Žutinić sp. nov. The species is described herein as new to science based on light and scanning electron microscopy. Gomphosphenia plenkoviciae is characterized by having narrowly clavate to linear valves, which are slightly heteropolar in larger specimens to clavate in smaller specimens, thus differing from the closest taxon by the valve outline and shape of the central area. Smaller valves of G. plenkoviciae differ from other small-sized Gomphosphenia species by valve outline, striae morphology and the shape of axial and central areas.

In four deep karstic lakes (Lake Vransko, Lake Visovacko, Kozjak and Prosce) several functionally well-adapted phytoplankton groups interchange in dominance during the seasonal cycle from April to September and likely tolerate the constraining conditions of nutrients deficiency more successfully as an aggregated group than as taxonomic units. This paper considers the functional approach by Reynolds et al. (2002), a method capturing much of the species ecology and which is successfully used for describing phytoplankton successions. Deep karstic lakes are attributed with the early spring (April) formation of stratification, low phytoplankton biomass, and with maximum biomass accumulating in July. The co-occurrence of functional groups in those lakes is evident but typically distinct according to different biomass proportions. The functional groups A, B, C, D, P, E and LO, represented by different Cyclotella, Ulnaria/Fragilaria, Dinobryon, Ceratium/Peridinium species, co-occurred in 50% and up to 90% of phytoplankton biomass in all investigated lakes. By observing the co- occurrence across the Croatian karstic region we: (i) demonstrate the regional distribution of phytoplankton assemblages in relation to low nutrient environment, and (ii) build a simple uniform pattern which successfully describes a variety of phytoplankton in deep karstic lakes and provides information of reliable indicators for those systems.