Algal Community Change in Mountain Lakes of the Alps Reveals Effects of Climate Warming and Shifting Treelines1.

The biological communities of mountain lakes are suspected to be highly sensitive to global warming and associated catchment changes. To identify the parameters determining algal community responses, subfossil pigments from 21 different mountain lakes in the Bavarian-Tyrolean Limestone Alps were investigated. Sediment cores were radio-isotopically dated, and their pigment preservation evaluated. General additive models (GAM) of pigment compositions were calculated with temperature as the explanatory variable and generalized linear models with several lake parameters explaining log-transformed GAM P-values. Lake depth and trophic state were identified as major control variables of the algal community and productivity changes. Shifts in a deep oligotrophic alpine lake (lg(P) = -1.04) were half as strong as in a shallow mesotrophic alpine lake (lg(P) = -1.86) with faster warming and higher productivity forcing the development of biomass. Phytoplankton and macrophyte pigments increased clearly with warming, at lower altitudes, and decreased at the treeline, so that periphytic pigments dominated alpine sediments. This pattern is probably the result of interactions of UV radiation and allochthonous inputs of DOM. Our findings suggest that (sub)alpine shallow lakes with higher nutrient levels are most vulnerable to climate change-driven changes whereas deep, nutrient-poor lakes appear more resilient.

Synechococcus diversity along a trophic gradient in the Osterseen Lake District, Bavaria.

Picocyanobacteria are important primary producers in freshwater; however, there is still a knowledge gap regarding their diversity at the strain level. For this reason, the microbial diversity of four lakes with different trophic states was investigated by sequencing of the 16S rRNA gene using universal primers. The study was performed in selected lakes of the Osterseen Lake District, Germany, from 2012 to 2014 (Lake Schiffhuettensee: eutrophic; Lake Ostersee: meso-oligotrophic; Lake Groebensee: oligotrophic; Lake Lustsee: oligotrophic). It was determined that the bacterial community of each of these lakes was characterized by one or more specific phyla. Within the autotrophic plankton, the picocyanobacterium Synechococcus sp. dominated oligotrophic habitats, whereas eukaryotic algae prevailed in eutrophic lakes. The study focused on the occurrence of cyanobacteria, specifically the genus Synechococcus. Genetic analysis of the 16S rRNA gene revealed an extendend diversity of freshwater Synechococcus. The occurrence of the identified operational taxonomic units of Synechococcus did not correlate with the trophic state of their habitat, suggesting that the current, underestimated diversity of picocyanobacteria deserves increased consideration in assessments of microbial and freshwater biodiversity.

Occurrence, Distribution and Toxins of Benthic Cyanobacteria in German Lakes.

Cyanobacteria are favored by climate change and global warming; however, to date, most research and monitoring programs have focused on planktic cyanobacteria. Benthic cyanobacteria blooms also increase and pose a risk to animal and human health; however, there is limited knowledge of their occurrence, distribution and the toxins involved, especially in relation to their planktic conspecifics. Therefore, we analyzed the benthic and planktic life forms of cyanobacterial communities in 34 lakes in Germany, including a monitoring of cyanotoxins. Community analyses were based on microscopic examination and Illumina sequencing of the 16S rRNA gene. The analyses of cyanotoxins were carried out using LC-MS/MS and ELISA. Observed benthic mats containing cyanobacteria consisted mainly of Nostocales and Oscillatoriales, being present in 35% of the lakes. Anatoxin was the most abundant cyanotoxin in the benthic samples, reaching maximum concentrations of 45,000 µg/L, whereas microcystin was the predominate cyanotoxin in the open-water samples, reaching concentrations of up to 18,000 µg/L. Based on the results, specific lakes at risk of toxic cyanobacteria could be identified. Our findings suggest that monitoring of benthic cyanobacteria and their toxins should receive greater attention, ideally complementing existing open-water sampling programs with little additional effort.

Spatio-Temporal Monitoring of Benthic Anatoxin-a-Producing Tychonema sp. in the River Lech, Germany.

Incidents with toxic benthic cyanobacteria blooms have been increasing recently. In 2019, several dogs were poisoned in the river Lech (Germany) by the benthic anatoxin-a-producing genus Tychonema. To characterize spatial and temporal distribution of potentially toxic Tychonema in this river, a systematic monitoring was carried out in 2020, focusing on the occurrence of the genus, its toxin production and habitat requirements. Tychonema and cyanobacterial community composition in benthic mats and pelagic samples were identified using a combined approach of microscopy and DNA sequencing of the 16S rRNA gene. In addition, anatoxin-a concentrations of selected samples were measured using the ELISA method. The habitat was characterized to assess the ecological requirements and growth conditions of Tychonema. Tychonema mats and anatoxin-a were detected at several sampling sites throughout the entire study period. Toxin concentrations increased with the progression of the vegetation period and with flow direction, reaching values between 0 and 220.5 µg/L. Community composition differed among pelagic and benthic samples, with life zone and substrate condition being the most important factors. The results of this study highlight the importance of monitoring and understanding the factors determining occurrence and toxin production of both pelagic and benthic cyanobacteria due to their relevance for the health of humans and aquatic ecosystems.

Evaluating climate change impacts on mountain lakes by applying the new silicification value to paleolimnological samples.

The evaluation of climate change impact on lakes typically relies on statistical methods like the reorganisation of organism communities (beta diversity) or transfer functions. A new method uses the silicification of diatoms that correlates with temperature and nutrients. The so-called silicification value (SiVa) overcomes problems of descriptive statistics or absent indicator species. Averaged over diatom communities, it related inversely to lake surface temperatures in mountain lakes. Hence, its change over time (δ SiVa) in a lake was hypothesised to reflect global change-driven lake warming quantitatively, which supposedly climaxes in shallow lakes. Sixteen different δ SiVa calculation approaches were tested. They (1) included or excluded planktic diatoms, (2) integrated fixed or variable time series referring to climate data or changes in diatom assemblages, (3) employed a top-bottom or regression approach and (4) expressed the δ SiVa as relative or absolute values. Subfossil diatom assemblages from 24 sediment cores from Bavarian and north Tyrolian mountain lakes served as sample set. All possible approaches were evaluated for their explanatory power for lake characteristics using GLMs. The top-bottom benthic approach with fixed climate data-based time series appeared to be the best model based on AIC and the extent of variable integration. In line with the hypothesis, the strongest decrease of δ SiVa was evident in most shallow lakes. Segmented regression further highlighted a positive correlation with depth if shallower than 10 m. By referring to the negative SiVa-summer temperature relation, δ SiVa also enabled the quantification of lake warming within the last decades, which ranged mainly between 0.1 °C and 1.1 °C per decade, consistent with existing literature. Additionally, a 100 year temperature reconstruction from a varved sediment core successfully validated the approach. Further studies may focus and extend its application to deeper lakes, but it can already serve as a powerful tool in palaeolimnological studies of shallow lakes like hard-water mountain lakes.

Mass Occurrence of Anatoxin-a- and Dihydroanatoxin-a-Producing Tychonema sp. in Mesotrophic Reservoir Mandichosee (River Lech, Germany) as a Cause of Neurotoxicosis in Dogs.

In August 2019, three dogs died after bathing in or drinking from Mandichosee, a mesotrophic reservoir of the River Lech (Germany). The dogs showed symptoms of neurotoxic poisoning and intoxication with cyanotoxins was considered. Surface blooms were not visible at the time of the incidents. Benthic Tychonema sp., a potential anatoxin-a (ATX)-producing cyanobacterium, was detected in mats growing on the banks, as biofilm on macrophytes and later as aggregations floating on the lake surface. The dogs' pathological examinations showed lung and liver lesions. ATX and dihydroanatoxin-a (dhATX) were detected by LC-MS/MS in the stomachs of two dogs and reached concentrations of 563 and 1207 µg/L, respectively. Anatoxins (sum of ATX and dhATX, ATXs) concentrations in field samples from Mandichosee ranged from 0.1 µg/L in the open water to 68,000 µg/L in samples containing a large amount of mat material. Other (neuro)toxic substances were not found. A molecular approach was used to detect toxin genes by PCR and to reveal the cyanobacterial community composition by sequencing. Upstream of Mandichosee, random samples were taken from other Lech reservoirs, uncovering Tychonema and ATXs at several sampling sites. Similar recent findings emphasize the importance of focusing on the investigation of benthic toxic cyanobacteria and applying appropriate monitoring strategies in the future.

Influence of cyanobacteria, mixotrophic flagellates, and virioplankton size fraction on transcription of microcystin synthesis genes in the toxic cyanobacterium Microcystis aeruginosa.

Toxic cyanobacteria such as Microcystis aeruginosa are a worldwide concern in freshwater reservoirs. Problems associated with their mass occurrence are predicted to increase in the future due to global warming. The hepatotoxic secondary metabolite microcystin is of particular concern in this context. This study aimed to determine whether co-occurring microorganisms influence the expression of microcystin biosynthesis genes. To this end, we performed cocultivation experiments and measured mcyB and mcyD transcripts in M. aeruginosa using RT-qPCR. We utilized representatives from three different plankton groups: the picocyanobacterium Synechococcus elongatus, the unicellular flagellate grazer Ochromonas danica, and virioplankton from two different lakes. The presence of S. elongatus significantly increased mcyB and mcyD transcription in M. aeruginosa. Cocultivation with the mixotrophic chrysophyte O. danica did not increase the transcription of mcyB and mcyD; in fact, mcyD transcripts decreased significantly. The virioplankton size fraction of environmental water samples induced a significant increase in mcyB and mcyD transcription when obtained from lakes with cyanobacterial blooms. Our results show that co-occurring microorganisms influence the expression of microcystin biosynthesis genes in M. aeruginosa.

Influence of temperature, mixing, and addition of microcystin-LR on microcystin gene expression in Microcystis aeruginosa.

Cyanobacteria, such as the toxin producer Microcystis aeruginosa, are predicted to be favored by global warming both directly, through elevated water temperatures, and indirectly, through factors such as prolonged stratification of waterbodies. M. aeruginosa is able to produce the hepatotoxin microcystin, which causes great concern in freshwater management worldwide. However, little is known about the expression of microcystin synthesis genes in response to climate change-related factors. In this study, a new RT-qPCR assay employing four reference genes (GAPDH, gltA, rpoC1, and rpoD) was developed to assess the expression of two target genes (the microcystin synthesis genes mcyB and mcyD). This assay was used to investigate changes in mcyB and mcyD expression in response to selected environmental factors associated with global warming. A 10°C rise in temperature significantly increased mcyB expression, but not mcyD expression. Neither mixing nor the addition of microcystin-LR (10 µg L-1 or 60 µg L-1 ) significantly altered mcyB and mcyD expression. The expression levels of mcyB and mcyD were correlated but not identical.

Temporal Dynamics of the Microbial Community Composition with a Focus on Toxic Cyanobacteria and Toxin Presence during Harmful Algal Blooms in Two South German Lakes.

Bacterioplankton plays an essential role in aquatic ecosystems, and cyanobacteria are an influential part of the microbiome in many water bodies. In freshwaters used for recreational activities or drinking water, toxic cyanobacteria cause concerns due to the risk of intoxication with cyanotoxins, such as microcystins. In this study, we aimed to unmask relationships between toxicity, cyanobacterial community composition, and environmental factors. At the same time, we assessed the correlation of a genetic marker with microcystin concentration and aimed to identify the main microcystin producer. We used Illumina MiSeq sequencing to study the bacterioplankton in two recreational lakes in South Germany. We quantified a microcystin biosynthesis gene (mcyB) using qPCR and linked this information with microcystin concentration to assess toxicity. Microcystin biosynthesis gene (mcyE)-clone libraries were used to determine the origin of microcystin biosynthesis genes. Bloom toxicity did not alter the bacterial community composition, which was highly dynamic at the lowest taxonomic level for some phyla such as Cyanobacteria. At the OTU level, we found distinctly different degrees of temporal variation between major bacteria phyla. Cyanobacteria and Bacteroidetes showed drastic temporal changes in their community compositions, while the composition of Actinobacteria remained rather stable in both lakes. The bacterial community composition of Alpha- and Beta-proteobacteria remained stable over time in Lake Klostersee, but it showed temporal variations in Lake Bergknappweiher. The presence of potential microcystin degraders and potential algicidal bacteria amongst prevalent Bacteroidetes and Alphaproteobacteria implied a role of those co-occurring heterotrophic bacteria in cyanobacterial bloom dynamics. Comparison of both lakes studied revealed a large shared microbiome, which was shaped toward the lake specific community composition by environmental factors. Microcystin variants detected were microcystin-LR, -RR, and -YR. The maximum microcystin concentrations measured was 6.7 µg/L, a value still acceptable for recreational waters but not drinking water. Microcystin concentration correlated positively with total phosphorus and mcyB copy number. We identified low abundant Microcystis sp. as the only microcystin producer in both lakes. Therefore, risk assessment efforts need to take into account the fact that non-dominant species may cause toxicity of the blooms observed.

Seasonal and spatial patterns of microbial diversity along a trophic gradient in the interconnected lakes of the Osterseen Lake District, Bavaria.

The Osterseen Lake District in Bavaria consists of 19 small interconnected lakes that exhibit a pronounced trophic gradient from eutrophic to oligotrophic. It therefore presents a unique model system to address ecological questions regarding niche adaptation and Baas Becking's long standing hypothesis of "everything is everywhere, but the environment selects." Here, we present the first assessment of the microbial diversity in these lakes. We sampled the lakes in August and December and used 454 pyrosequencing of 16S rRNA amplicons to analyze the microbial diversity. The diversity patterns between lakes and seasons were compared and the bacterial community composition was correlated with key chemical and physical parameters. Distinct patterns of bacterial diversity only emerged at the level of individual OTUs (operational taxonomic units), but not at the level of the major bacterial phyla. This emphasizes the high functional and physiological diversity among bacterial species within a phylum and calls for analysis of biodiversity at the level of OTUs in order to understand fine-scale biogeography. We were able to identify a number of cosmopolitan OTUs as well as specialist OTUs that were restricted to certain lakes or seasons, suggesting adaptation to specific ecological niches.