Aerophytic phototrophs, colonizers of different substrates exposed to air, among which are rock substrates in cave habitats, are in general not sufficiently investigated and studied. From the ecological point of view, seasonal/temporal variations and parameters that influence aerophytic phototrophic communities through seasons still remain largely unknown. Seasonal analysis of phototrophic microorganisms from biofilms developed at entrances of Samar and Jezava caves in Eastern Serbia was performed four times a year. All identified phototrophic representatives were assigned to a certain division or group. The relationship between documented groups and different measured, determined and observed ecological parameters was illustrated using principal component analysis (PCA). A high diversity of cyanobacteria and algae was observed as a result of seasonal sampling: 141 taxa in Samar and 61 taxa in Jezava Cave. Cyanobacteria were dominant in both caves (coccoid forms prevailing) and were followed by Bacillariophyta. Bacillariophyta were positively correlated with light intensity and sites where seeping water was present, and cyanobacteria with water content in biofilm, Chl a and relative air humidity. Higher diversity of phototrophs was documented in December and March, and lower in May and August in both caves with Chl a values having the same trend as the diversity of phototrophic microorganisms. This was especially the case with biofilms in which diversity was more prone to seasonal changes and where Cyanobacteria dominated. Only 24 taxa were documented during all four seasons in Samar Cave (Cyanobacteria, Bacillariophyta and Chlorophyta representatives) and three in Jezava Cave (Cyanobacteria only).

Charophyte species delineation is regularly based on a set of thallus morphological characteristics, but considering pronounced phenotypic plasticity, difficulties and doubts commonly occur in Chara species determination. DNA barcoding may contribute to solving these challenges. Here we characterize Chara contraria with an unusual set of morphological characteristics, and specimens morphologically resembling Chara connivens collected in Serbia, by describing their morphological traits and analysing matK barcoding results. Our results indicated that dioecious Chara specimens, tentatively determined as Chara “connivens” based on morphological traits, were genetically more closely related to C. globularis. These Chara “connivens” specimens formed a sister group to a monophyletic C. globularis cluster, suggesting that it may be neither C. connivens nor C. globularis. We strongly encourage further barcoding of C. “connivens” samples from freshwater, in order to find out if there are consistent genetic differences between the dioecious freshwater C. “connivens” and monoecious C. globularis. Barcoding of matK placed the monoecious Chara specimens, which based on morphological characteristics initially were determined as C. virgata, into the C. contraria group. This indicates that the microscopic traits which commonly are used for Chara species determination sometimes are misleading. In general, our study challenges the commonly used phenetic species concept in Charophyte taxonomy and illustrates the importance of molecular approaches to evaluate the validity of morphological characteristics of the plant thallus in species delineation.

Due to life in extreme environments, cyanobacteria and algae from cave biofilms that form at the entrances or deep inside the cave around artificial lights are of increasing interest to many scientists. It is well-known that many phototrophic microorganisms are first to colonize exposed substrata and produce the organic matter on which other biofilm constituents relly. Many studies dealing with phototrophic microorganisms from biofilms focus on the diversity and community composition of cyanobacteria and algae, while quantitative assessments are rarely implemented. Biofilm sampling was conducted in Degurić and Vernjikica Cave located in Western and Eastern Serbia, respectively. Ecological parameters (temperature, relative humidity, light intensity) and distance from the entrance were measured. Additionally, chlorophyll content, as well as biofilm parameters (water content, organic and inorganic matter) were determined. The abundance of phototrophic microorganisms was assessed on microscope slides which contained 1 mg of biofilm that was dehydrated for a short period of time and homogenized prior to slide preparation, and then rehydratated. The biomass of recorded cyanobacterial and algal taxa was calculated by applying geometric approximations and standard mathematical formulas. In Degurić Cave, at the sampling site where the highest biomass was documented, the higher diversity, water content and chlorophyll values were also recorded, while in Vernjikica Cave a high content of organic matter was documented. According to the multivariate analyses performed, the biomass of simple trichal Cyanobacteria, Bacillariophyta, and Xanthophyta was positively correlated with the content of organic matter in biofilm and light intensity, while coccoid and heterocytous Cyanobacteria and Chlorophyta showed a positive correlation with water content in the biofilm, relative humidity and distance from the entrance. The total biomass was positively correlated with the chlorophyll content, organic matter and light intensity, and negatively with the distance from the entrance.

Substrate specificity of the periphyton community is usually underestimated in both periphyton ecology and biomonitoring studies, thus different kinds of both natural and artificial substrates (of organic and inorganic origin) are employed. Periphyton colonization and successional trajectories are particularly debatable processes when different kinds of substrates are considered. In our field experiment, we deployed four kinds of artificial substrates (two inorganic ones – glass and ceramic, and two organic ones – willow and yew wooden tiles) for the development of periphyton in an urban reservoir referred to as Lake Savsko (Belgrade, Serbia). We comparatively investigated the structure, colonization process, diversity and successional trajectories of periphyton. We also assessed the relationship between the dynamics of algae growth forms (ecological groups) on different substrates and selected abiotic limnological factors. Our objective was to determine whether the type of substrate affects periphyton characteristics at various levels and whether an artificial substrate potentially affects the bioindication capacity of ecological groups. We concluded that all substrates behave similarly at the level of structure and colonization phases, but when considering diversity and successional trajectories, the substrate specificity was demonstrated. Our results suggest that communities developed on inert substrates (glass and ceramic) could provide more realistic insight into complex environmental changes.