Lake Balaton, in Hungary, is the largest shallow lake in Central Europe. This fresh water ecosystem has been well studied, including investigations of its photoautotrophic picoplankton (PPP). Previous studies revealed that picoeukaryotes could reach an extremely high abundance in winter, while picocyanobacteria are the predominant picoplankters in warmer periods, as in other lakes in the temperate zone. In addition to epifluorescence microscopy -which allows discrimination only between phycocyanin-rich picocyanobacteria, phycoerythrin-rich picocyanobacteria and picoeukaryotic algae -we used PCR-based molecular methods to reveal the detailed genetic diversity and seasonal dynamics of the PPP in Lake Balaton for the first time. Our results show that a single integrated pelagic sample, taken vertically from the whole water column, may harbor a large number of picocyanobacterial genotypes including previously unidentified groups. Based on length polymorphism analysis of the phycocyanin operon (which contains a non-coding region of variable size), the composition of picocyanobacterial communities showed significant seasonal changes and spatial variation. The relative importance of some of the operational taxonomic units (OTUs) we detected was correlated with environmental factors, such as temperature and the concentration of available nitrogen forms. The picoeukaryotic algal community of winter PPP was dominated by chlorophytes related to the group Trebouxiophyceae. The results of this study highlight the fine internal structure and dynamics of the PPP community in freshwater ecosystems -a view that is usually blurred by the lower resolution of commonly used methods, such as epifluorescence microscopy and flow cytometry.

The spatial response of epiphytic diatom communities to environmental stress was studied in a moderately saline wetland area located in the plain of Danube-Tisza Interfluve, Hungary. The area is characterised by World War II bomb crater ponds and can be regarded as an excellent ecological model system where the dispersion of species is slightly limited by distance. To study the effect of environmental variables on the communities, canonical correspondence analysis was applied. Salinity, pH, total suspended solids, total phosphorous and depth proved to be significant environmental drivers in this analysis. The ecological status of the ponds was assessed with Ziemann’s halobity index, as the trophity-depending metric cannot be applied to these habitats (due to the naturally high phosphorus content). Ponds in “good” ecological status significantly differed from those appertaining to water quality category of “not-good” ecological status considering characteristic of natural astatic soda pans (e.g. salinity, pH, ammonium, total phosphorous concentration, nitrogen:phosphorous ratio and turbidity). The differences between epiphytic diatom communities inhabiting the ponds were detected using non-parametric multidimensional scaling. The samples formed three groups according to the types of ponds (“transparent”, “transitional” and “turbid”) based on the width of the macrophyte belt around them. Indicator species related to the ecological status of the ponds and diatom communities contributing to the separation of groups of ponds were identified. One of the indicator species differed from species already described. Light and scanning electron microscopy features and phylogenetic analyses based on three genes (18S and 28S rRNA genes, rbcL) proved that it was a new species of Nitzschia genus, closely related to Nitzschia frustulum and Nitzschia inconspicua. Therefore, description of a new species, Nitzschia reskoi Ács, Duleba, C.E.Wetzel & Ector is proposed. We concluded that the increasing abundance of Nitzschia reskoi was a signal of the degradation of the intermittent saline wetlands.

Lake snow, caused by the freshwater centric diatom Lindavia intermedia, has become problematic in several large, oligotrophic New Zealand lakes over the past decade. Macroaggregates produced by L. intermedia foul fishing lines, intake screens, and water filters, and have a negative impact on recreational values. It was confirmed that the fibers constituting lake snow are composed of chitin, two chitin synthase genes (chs1 and 2) from L. intermedia were characterized, new qPCR-based tools to quantify the abundance of the species and measure expression of chs2 relative to the reference gene act1 (the product of which has cytoskeletal functions) were developed. The strong heterogeneity and mucilaginous nature of lake snow samples create particular difficulties for calibrations of gene or transcript copy numbers with cell densities and obtaining high yields of mRNA. However, data collected from four lakes during November 2018 and February and May 2019 show that abundance of L. intermedia is always high when lake snow is also abundant, but that a full range of L. intermedia abundance can occur when lake snow is absent, suggesting that chitin production is not obligate in L. intermedia. This result is consistent with the available data for chs2 expression, which suggest higher transcription when lake snow is abundant. Lake snow production by L. intermedia therefore requires an as yet undetermined stimulus independent of cell abundance.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 IntroductionThe classic taxonomy of diatoms is morphology-based using features of their characteristic silica cell wall called frustule (e.g. Ettl et al. 1986). Medlin et al. (1988) introduced molecular methods into diatom taxonomy. Based on molecular, morphological and cytological results, Medlin and Kaczmarska (2004) proposed major clades of diatoms including Mediophyceae for bipolar centrics and the Thalassiosirales. Phylogenetic relationships of this latter order were reconstructed by Alverson et al. (2007).Recently, morphological studies are supplemented by molecular investigations on various Skeletonema species (Alverson and Kolnick 2005; Kooistra et al. 2008; Sarno et al. 2005 Sarno et al. , 2007. Medlin et al. (1988) determined the phylogenetic status of the marine diatom Skeletonema costatum among the eukaryotes based on 16S-like (=18S) rDNA. Molecular and/or morphological studies investigated S. costatum or S. costatum-like diatoms and revealed new species, such as S. pseudocostatum (Medlin et al. 1991), S. grevillei (Zingone et al. 2005), S. dohrnii, S. grethae, S. japonicum, S. marinoi (Sarno et al. 2005 and S. ardens (Sarno et al. 2007). Skeletonema was found to be monophyletic and ancestrally a marine genus (Alverson et al. 2007). There are only two non-marine species in this genus: S. subsalsum occurring mainly in saline and brackish waters and occasionally in freshwater habitats ( Aké Castillo et al. 1995; Gibson et al. 1993; Hasle Evensen 1975; Hustedt 1957) and S. potamos that is recorded from freshwater and slightly brackish habitats (Kiss et al. 2012). This latter species was not involved in molecular investigations previously.Microsiphonia potamos C.I. Weber was first collected in 1966 from the Little Miami River, Cincinatti, Ohio and described by Weber (1970). New material from the Little Miami River, 25 May 1973, was observed by Hasle and Evensen (1976) and as they found this species highly similar to species of the genus Skeletonema, suggested its transfer to this genus Page 4 of 42 A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 as Skeletonema potamos (C.I. Weber) Hasle in Hasle & Evensen; they found the same taxon in the liquid-preserved sample from the Jensensee, Plön (Germany), 19 August 1922, Hustedt Collection E 4555, which Hustedt (1928 used when preparing the description of the diatom to which he applied erroneously the name Stephanodiscus subsalsus (Cleve-Euler) Hustedt.Skeletonema potamos is a broadly distributed species (Supplementary Material Table S1) and is considered inv...

(1) Urbanization significantly influences the ecosystems of rivers in various ways, including the so-called loading effect of wastewater production. Benthic diatoms are used in ecological status assessments of waters. Beside species composition, traits can be used as indicators. We aimed to evaluate how the loading of the large city of Budapest manifests in the physico-chemical variables of the River Danube and what species composition and trait response this loading results in for the benthic diatom communities. (2) Weekly samplings were performed at points upstream and downstream of Budapest on both riverbanks. Samples were compared, based on general physical-chemical variables and the concentration of thirty-four elements, as well as species composition and seven traits of species of diatom communities. Ecological status was assessed using the Specific Pollution Sensitivity Index (IPS). (3) Only a few measured environmental variables showed differences between the sampling points, suggesting that the nutrient loading has significantly decreased due to the installation of several efficiently working wastewater treatment plants since the introduction of the European Union Water Framework Directive. In contrast, the species composition and traits of species showed the effect of land use. Benthic diatoms indicate the environmental changes caused by land use in the longer-term, while chemical measurements reflect instantaneous status.

Centric diatom taxa in the Cyclotella ocellata and C. comensis complexes show high morphological variability and often apparently continuous morphological transitions. In this study we investigated natural assemblages of the C. ocellata/C. comensis complex from Hungarian and Croatian lakes and from Turkish streams using morphological and molecular methods. The studied assemblages contained cells with morphologies resembling C. ocellata as well as other, closely related, species: C. comensis, C. pseudocomensis, C. costei, and C. trichnoidea. The goal of our paper was to assess whether the observed morphological differences were due to intraspecific variability or suggest the existence of several, putatively distinct species.Ten morphometric characters were measured, which, either individually, or in pairs, did not differentiate the nominal taxa in our assemblages. However, multivariate discriminant analysis has revealed a group including C. ocellata and C. trichonidea morphologies could be separated from another containing C. comensis, C. pseudocomensis and C. costei.A nuclear (18S rDNA) and a chloroplast (rbcL) gene were amplified and partially sequenced from environmental DNA or from isolated cells. The sequences showed little variability among the assemblages and nominal species. Although general congruence of molecular and morphometric separation supports the species level separation of C. ocellata/trichonidea from the probably conspecific C. comensis/pseudocomensis/costei, sequence divergences between the groups are in the same range as within them, so that a onspecificity of all four taxa cannot be unequivocally excluded.

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Diatoms are valuable bioindicators and their traditional classification and identification are mainly based on the morphological characteristics of their frustules. However, in recent years, DNA-based methods have been proposed and are rapidly growing in the scientific literature as a complementary tool to assess the ecological status of freshwaters. Diatom-based ecological status assessment uses indices calculated from sensitivity and tolerance values as well as relative abundance of species. Correct assessment requires an accurate identification of species. In the present study, diatom assemblages of an oxbow lake were investigated using light and scanning electron microscopy as well as metabarcoding using rbcL marker, and the identification results were compared, intending to match barcode sequences of species that are currently missing in the diatom reference database. The investigated oxbow is an important wetland for bird conservation, although it is impacted by land use. Taxon lists based on morphology and metabarcoding considerably differed when bioinformatics analysis involved DADA2 pipeline with Diat.barcode database. Previously unknown sequence variants of four pennate species were found with additional BLAST search. Using phylogeny and p-distance calculations sequences could be matched to three small-celled naviculoid species that were found under a microscope. One of them was found to be a new species of the genus Mayamaea and was described as a new species, Mayamaea ectorii. Additionally, spatial distribution maps for several small-celled naviculoid species are provided for the Hungarian territory.