Tufa deposits in karst rivers are unique habitats created by mutual interactions between specific environmental and biotope features and inhabited by diatoms as a highly abundant and diverse algal group. This pilot study aimed to investigate the diversity of diatom communities on tufa depositing habitats and assess the Una River’s ecological status using a comparative molecular and morphological approach for diatom identification. The 312 base pairs of the rbcL gene were barcoded and analyzed using MiSeq reads and amplicon sequence variants (ASVs) obtained by the DADA2 pipeline. The reference database Diat.barcode v7 was used for taxonomic assignment. The morphological identification of the diatoms was carried out in parallel. In total, the combined dataset revealed 46 taxa identified at genus rank, 125 on the subgenus, and 145 on combined taxonomy rank. The metabarcoding approach mostly leads to a lower number of identified taxa at species rank (58 in molecular vs. 119 in optical inventory), resulting in higher values of beta diversity and heterogeneity in diatom assemblages in samples obtained by morphological approach. Despite the high percentage of taxonomically not assigned diatom ASVs to the species rank, high Shannon diversity index values and a similar number of taxa per locations compared to the morphological approach were obtained. Taxa Achnanthidium minutissimum (Kützing) Czarnecki, Achnanthidium pyrenaicum (Hustedt) H.Kobayasi, Amphora pediculus (Kützing) Grunow, Diatoma vulgaris Bory, Navicula cryptotenella Lange-Bertalot, and Navicula tripunctata (O.F.Müller) Bory were identified at all locations in both inventories. Although limited consistency in the diatom abundances between the two inventory datasets was found, a similar grouping of samples was observed connected to the river’s longitudinal gradient. The data obtained using molecular approach in most sites indicated a mostly lower ecological status (good or moderate) compared to the data obtained from the morphological approach (high, good, and moderate). The potential of environmental DNA (eDNA) diatom metabarcoding for water monitoring and diversity studies is undeniable, but to fully realize the benefits of these methods in the future, it is essential to standardize protocols and expand the reference database for species found in specific habitats, such as tufa deposits.

Nitrogen is one of the essential elements limiting growth in aquatic environments. Being primarily of anthropogenic origin, it exerts negative impacts on freshwater ecosystems. The present study was carried out at the nitrate-vulnerable zone within the alluvial aquifer of the large lowland Drava River. The main aim was to investigate the ecosystem’s functionality by characterizing the bacterial and phytoplankton diversity of a small inactive gravel pit by using interdisciplinary approaches. The phytoplankton community was investigated via traditional microscopy analyses and environmental DNA (eDNA) metabarcoding, while the bacterial community was investigated by a molecular approach (eDNA). Variations in the algal and bacterial community structure indicated a strong correlation with nitrogen compounds. Summer samples were characterized by a high abundance of bloom-forming Cyanobacteria. Following the cyanobacterial breakdown in the colder winter period, Bacillariophyceae and Actinobacteriota became dominant groups. Changes in microbial composition indicated a strong correlation between N forms and algal and bacterial communities. According to the nitrogen dynamics in the alluvial aquifer, we emphasize the importance of small water bodies as potential buffer zones to anthropogenic nitrogen pressures and sentinels of the disturbances displayed as algal blooms within larger freshwater systems.

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.

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.

Phytoplankton are considered to be one of the most sensitive indicators of the ecological status of lakes. Nowadays, it is essential to recognize the prospects of the molecular approach (eDNA metabarcoding) in phytoplankton community assessments and combine them with the existing traditional microscopy-based morphological approach before its standardization. In this study, the aim was to characterize the phytoplankton community of a natural karstic lake by combining and comparing the morphological and molecular approach to check the applicability of eDNA metabarcoding as a biomonitoring tool. A total of 51 phytoplankton taxa were found using the morphological approach, whilst the molecular approach discovered 97 ASVs that corresponded to the algal community. The comparability of both approaches in describing phytoplankton communities is evident in the designation of centric diatoms, dinoflagellates and cryptophytes as descriptive taxa. Furthermore, both approaches proved reliable in detecting functional groups (Lo, C, X2, X3) with similar ecological demands. Moreover, the results have shown that euphotic zone samples can be reliably exchanged by composite samples to provide an accurate characterization of phytoplankton communities in the euphotic zone. It was confirmed that eDNA metabarcoding is an applicable tool for biodiversity monitoring of a natural karst lake and should be used as a feasible supplement to traditional microscopy in the phytoplankton community assessments, with regards to the drawbacks of each method.