Lake Bacterial Communities in North Patagonian Andes: The Effect of the Nothofagus pumilio Treeline.

One of the most noticeable environmental discontinuities in mountains is the transition that exists in vegetation below and above the treeline. In the North Patagonian Andean lakes (between 900 and 1950 m a.s.l.), we analyzed the bacterial community composition of lakes in relation to surrounding vegetation (erected trees, krummholz belt, and bare rocks), dissolved organic carbon (DOC), and total dissolved nutrients (nitrogen, TDN and phosphorus, TDP). We observed a decrease in DOC, TDP, and TDN concentrations with altitude, reflecting shifts in the source inputs entering the lakes by runoff. Cluster analysis based on bacterial community composition showed a segregation of the lakes below treeline, from those located above. This first cluster was characterized by the cyanobacteria Cyanobium PCC-6307, while in the krummholz belt and bare rocks, bacterial communities were dominated by Actinobacteria hgcl-clade and Proteobacteria (Sandarakinorhabdus and Rhodovarius), with the presence of pigments such as actinorhodopsin, carotenoids, and bacteriochlorophyll a. The net relatedness index (NRI), which considers the community phylogenetic dispersion, showed that lakes located on bare rocks were structured by environmental filtering, while communities of lakes below treeline were structured by species interactions such as competition. Beta-diversity was higher among lakes below than among lakes located above the treeline. The contribution of species turnover was more important than nestedness. Our study brings light on how bacterial communities may respond to changes in the surrounding vegetation, highlighting the importance of evaluating different aspects of community structure to understand metacommunity organization.

Glacial Influence Affects Modularity in Bacterial Community Structure in Three Deep Andean North-Patagonian Lakes.

We analyze the bacteria community composition and the ecological processes structuring these communities in three deep lakes that receive meltwater from the glaciers of Mount Tronador (North-Patagonia, Argentina). Lakes differ in their glacial connectivity and in their turbidity due to glacial particles. Lake Ventisquero Negro is a recently formed proglacial lake and it is still in contact with the glacier. Lakes Mascardi and Frías lost their glacial connectivity during the Pleistocene-Holocene transition. Total dissolved solid concentration has a significant contribution to the environmental gradient determining the segregation of the three lakes. The newly formed lake Ventisquero Negro conformed a particular bacterial community that seemed to be more related to the microorganisms coming from glacier melting than to the other lakes of the basin. The net relatedness index (NRI) showed that the bacterial community of lake Ventisquero Negro is determined by environmental filtering, while in the other lakes, species interaction would be a more important driver. The co-occurrence network analysis showed an increase in modularity and in the number of modules when comparing Lake Ventisquero Negro with the two large glacier-fed lakes suggesting an increase in heterogeneity. At the same time, the presence of modules with phototrophic bacteria (Cyanobium strains) in lakes Frías and Mascardi would reflect the increase of this functional photosynthetic association. Overall, our results showed that the reduction in ice masses in Patagonia will affect downstream large deep Piedmont lakes losing the glacial influence in their bacterial communities.

A georeferenced rRNA amplicon database of aquatic microbiomes from South America.

The biogeography of bacterial communities is a key topic in Microbial Ecology. Regarding continental water, most studies are carried out in the northern hemisphere, leaving a gap on microorganism's diversity patterns on a global scale. South America harbours approximately one third of the world's total freshwater resources, and is one of these understudied regions. To fill this gap, we compiled 16S rRNA amplicon sequencing data of microbial communities across South America continental water ecosystems, presenting the first database µSudAqua[db]. The database contains over 866 georeferenced samples from 9 different ecoregions with contextual environmental information. For its integration and validation we constructed a curated database (µSudAqua[db.sp]) using samples sequenced by Illumina MiSeq platform with commonly used prokaryote universal primers. This comprised ~60% of the total georeferenced samples of the µSudAqua[db]. This compilation was carried out in the scope of the µSudAqua collaborative network and represents one of the most complete databases of continental water microbial communities from South America.

A common temperature dependence of nutritional demands in ectotherms.

In light of ongoing climate change, it is increasingly important to know how nutritional requirements of ectotherms are affected by changing temperatures. Here, we analyse the wide thermal response of phosphorus (P) requirements via elemental gross growth efficiencies of Carbon (C) and P, and the Threshold Elemental Ratios in different aquatic invertebrate ectotherms: the freshwater model species Daphnia magna, the marine copepod Acartia tonsa, the marine heterotrophic dinoflagellate Oxyrrhis marina, and larvae of two populations of the marine crab Carcinus maenas. We show that they all share a non-linear cubic thermal response of nutrient requirements. Phosphorus requirements decrease from low to intermediate temperatures, increase at higher temperatures and decrease again when temperature is excessive. This common thermal response of nutrient requirements is of great importance if we aim to understand or even predict how ectotherm communities will react to global warming and nutrient-driven eutrophication.

Light dependence in the phototrophy-phagotrophy balance of constitutive and non-constitutive mixotrophic protists.

Mixotrophic protists display contrasting nutritional strategies and are key groups connecting planktonic food webs. They comprise constitutive mixotrophs (CMs) that have an innate photosynthetic ability and non-constitutive mixotrophs (NCMs) that acquire it from their prey. We modelled phototrophy and phagotrophy of two mixotrophic protists as a function of irradiance and prey abundance. We hypothesised that differences in their physiology (constitutive versus non-constitutive mixotrophy) can result in different responses to light gradients. We fitted the models with primary production and bacterivory data from laboratory and field experiments with the nanoflagellate Chrysochromulina parva (CM) and the ciliate Ophrydium naumanni (NCM) from north Andean Patagonian lakes. We found a non-monotonic response of phototrophy and phagotrophy to irradiance in both mixotrophs, which was successfully represented by our models. Maximum values for phototrophy and phagotrophy were found at intermediate irradiance coinciding with the light at the deep chlorophyll maxima in these lakes. At lower and higher irradiances, we found a decoupling between phototrophy and phagotrophy in the NCM while these functions were more coupled in the CM. Our modelling approach revealed the difference between both mixotrophic functional types on the balance between their nutritional strategies under different light scenarios. Thus, our proposed models can be applied to account how changing environmental conditions affect both primary and secondary production within the planktonic microbial food web.

Short term fluctuating temperature alleviates Daphnia stoichiometric constraints.

In this study, we analysed how short term temperature fluctuation interacts with nutrient limitation in the vertical migrating Daphnia commutata. We hypothesize that short term (daily) temperature fluctuation will alleviate nutrient limitation. We carried out experiments analysing growth rates, phosphorus and RNA content of D. commutate grown under four different temperature regimes and two P-limited conditions. Our experiments showed that individuals grown under fluctuating temperature grew more than at the mean temperature. We estimated the expected sizes for the 15 °C treatment based on the Q10 and for the fluctuating temperature treatment. These expected sizes for both treatments resulted well below the observed ones. The P and RNA content of individuals grown at 10 °C were significantly higher than those at 20 °C, and when individuals grown at 10 °C were translocated to 20 °C they exerted an increased growth rate. Our results suggest that, under a regime of diel vertical migration, the temperature alternation would allow migrating organisms to alleviate the effect of severe nutrient limitation maintaining population growth. Under a scenario of global warming, where epilimnetic temperatures will increase, lake temperature will interact with nutrient limitation for consumers, but, organisms may be able to face these changes if they can still regularly move from a cold hypolimnion to a warmer epilimnion.

Nutritional stress by means of high C:N ratios in the diet and starvation affects nitrogen isotope ratios and trophic fractionation of omnivorous copepods.

Nutritional stress, from feeding on low-quality diets or starvation, may cause changes in consumers' nitrogen isotope ratios (δ15N = 15N/14N) and trophic fractionation (∆15N = δ15Nconsumer - δ15Nfood source), however, research has shown mixed results in the magnitude and the direction of the change. This is potentially more complex for omnivores whose diets span a wide range of food resources. We conducted seasonal field samplings in Patagonian lakes and analyzed the relationship between seston (SES) quality parameters and the δ15N and ∆15N of an omnivorous copepod, Boeckella gracilipes (Bg). We also performed a 7-day laboratory starvation experiment, an extreme form of nutritional stress, to investigate if lack of food led to changes in δ15NBg values. Our field results showed that increasing values of the seston carbon to nitrogen ratio (C:NSES), chlorophyll a (Chl a), and δ15NSES were related to higher δ15NBg values. C:NSES and Chl a were also positively related to ∆15N; yet, C:NSES alone explained 70% of the variation. C:NSES values correlated with the presence of mixotrophic algae and ciliates that are key food resources for B. gracilipes. In our laboratory starvation experiment, the δ15NBg values increased significantly, pointing to use of internal N sources; yet, the change associated with starvation was less pronounced than that related to C:NSES changes in the field, suggesting depletion of the substrate pool in the former. We found that ∆15N values of omnivorous species consuming a low-quality diet would be higher than that from a conspecific with a high-quality diet; though fasting animals would show intermediate values.

Sustained effects of volcanic ash on biofilm stoichiometry, enzyme activity and community composition in North- Patagonia streams.

Volcanic eruptions are extreme perturbations that affect ecosystems. These events can also produce persistent effects in the environment for several years after the eruption, with increased concentrations of suspended particles and the introduction of elements in the water column. On 4th June 2011, the Puyehue-Cordón Caulle Volcanic Complex (40.59°S-72.11°W, 2200m.a.s.l.) erupted explosively in southern Chile. The area affected by the volcano was devastated; a thick layer of volcanic ash (up to 30cm) was deposited in areas 50 km east of the volcano towards Argentina. The aim of the present study was to evaluate the effect of volcanic ash deposits on stream ecosystems four years after the eruption, comparing biofilm stoichiometry, alkaline phosphatase activity, and primary producer's assemblage in streams which were severely affected by the volcano with unaffected streams. We confirmed in the laboratory that ash deposited in the catchment of affected streams still leach phosphorus (P) into the water four years after eruption. Results indicate that affected streams still receive volcanic particles and that these particles release P, thus stream water exhibits high P concentration. Biofilm P content was higher and the C:P ratio lower in affected streams compared to unaffected streams. As a consequence of less P in unaffected streams, the alkaline phosphatase activity was higher compared to affected streams. Cyanobacteria increased their abundances (99.9% of total algal biovolume) in the affected streams suggesting that the increase in P may positively affect this group. On the contrary, unaffected streams contained a diatom dominant biofilm. In this way, local heterogeneity was created between sub-catchments located within 30 km of each other. These types of events should be seen as opportunities to gather valuable ecological information about how severe disturbances, like volcanic eruptions, shape landscapes and lotic systems for several years after the event.

Bacterial community structure in patagonian Andean Lakes above and below timberline: from community composition to community function.

Lakes located above the timberline are remote systems with a number of extreme environmental conditions, becoming physically harsh ecosystems, and sensors of global change. We analyze bacterial community composition and community-level physiological profiles in mountain lakes located in an altitude gradient in North Patagonian Andes below and above the timberline, together with dissolved organic carbon (DOC) characterization and consumption. Our results indicated a decrease in 71 % of DOC and 65 % in total dissolved phosphorus (TDP) concentration as well as in bacteria abundances along the altitude range (1,380 to 1,950 m a.s.l.). Dissolved organic matter (DOM) fluorescence analysis revealed a low global variability composed by two humic-like components (allochthonous substances) and a single protein-like component (autochthonous substances). Lakes below the timberline showed the presence of all the three components, while lakes above the timberline the protein-like compound constituted the main DOC component. Furthermore, bacterial community composition similarity and ordination analysis showed that altitude and resource concentration (DOC and TDP) were the main variables determining the ordination of groups. Community-level physiological profiles showed a mismatch with bacteria community composition (BCC), indicating the absence of a relationship between genetic and functional diversity in the altitude gradient. However, carbon utilization efficiencies varied according to the presence of different compounds in DOM bulk. The obtained results suggest that the different bacterial communities in these mountain lakes seem to have similar metabolic pathways in order to be able to exploit the available DOC molecules.

Phylogenetic diversity of nonmarine picocyanobacteria.

We studied the phylogenetic diversity of nonmarine picocyanobacteria broadening the sequence data set with 43 new sequences of the 16S rRNA gene. The sequences were derived from monoclonal strains isolated from four volcanic high-altitude athalassohaline lakes in Mexico, five glacial ultraoligotrophic North Patagonian lakes and six Italian lakes of glacial, volcanic and morenic origin. The new sequences fall into a number of both novel and previously described clades within the phylogenetic tree of 16S rRNA gene. The new cluster of Lake Nahuel Huapi (North Patagonia) forms a sister clade to the subalpine cluster II and the marine Synechococcus subcluster 5.2. Our finding of the novel clade of 'halotolerants' close to the marine subcluster 5.3 (Synechococcus RCC307) constitutes an important demonstration that euryhaline and marine strains affiliate closely. The intriguing results obtained shed new light on the importance of the nonmarine halotolerants in the phylogenesis of picocyanobacteria.

Glacier melting and stoichiometric implications for lake community structure: zooplankton species distributions across a natural light gradient.

Glaciers around the globe are melting rapidly, threatening the receiving environments of the world's fresh water reservoirs with significant changes. The meltwater, carried by rivers, contains large amounts of suspended sediment particles, producing longitudinal gradients in the receiving lakes. These gradients may result in changes in the light : nutrient ratio that affect grazer performance by altering elemental food quality. Thus, glacial melting may induce a shift in the phytoplankton carbon : nutrient ratio and hence influence the dominance of herbivorous zooplankton through stoichiometric mechanisms. To test this hypothesis, we combined field and experimental data, taking advantage of a natural light intensity gradient caused by glacial clay input in a deep oligotrophic Patagonian lake. Across this gradient, we evaluated the abundances of two consumer taxa with different phosphorus requirements, the copepod Boeckella gracilipes and the cladoceran Daphnia commutata, using a six-station transect along the lake. We found significant differences in light : nutrient ratio and stoichiometric food quality of the seston, together with a switch from dominance of P-rich Daphnia in low carbon : nutrient stations to dominance of low-P copepods in high carbon : nutrient stations. The laboratory experiments confirmed that the difference in the carbon : nutrient ratio across the gradient is sufficient to impair Daphnia growth. The overall patterns are consistent with our prediction that shifts in the environmental light : nutrient ratio as a result of glacial melting would contribute to shifts in the dominance of stoichiometrically contrasting taxa in consumer guilds.

Rapid enzymatic response to compensate UV radiation in copepods.

Ultraviolet radiation (UVR) causes physical damage to DNA, carboxylation of proteins and peroxidation of lipids in copepod crustaceans, ubiquitous and abundant secondary producers in most aquatic ecosystems. Copepod adaptations for long duration exposures include changes in behaviour, changes in pigmentation and ultimately changes in morphology. Adaptations to short-term exposures are little studied. Here we show that short-duration exposure to UVR causes the freshwater calanoid copepod, Eudiaptomus gracilis, to rapidly activate production of enzymes that prevent widespread collateral peroxidation (glutathione S-transferase, GST), that regulate apoptosis cell death (Caspase-3, Casp-3), and that facilitate neurotransmissions (cholinesterase-ChE). None of these enzyme systems is alone sufficient, but they act in concert to reduce the stress level of the organism. The interplay among enzymatic responses provides useful information on how organisms respond to environmental stressors acting on short time scales.

UV radiation simultaneously affects phototrophy and phagotrophy in nanoflagellate-dominated phytoplankton from an Andean shallow lake.

Mixotrophic nanoflagellates, that combine photosynthesis and phagotrophy, are important members of planktonic food webs in many aquatic environments depending on the balance among the different carbon and energy sources. We carried out field sampling and laboratory experiments with natural nanoflagellate assemblages from an Andean North-Patagonian lake exposing them or not to UVR, and measuring photosynthetic parameters and bacterivory. The effect of different light treatments on the photosynthetic efficiency was studied by the non-invasive, pulse amplitude-modulated (PAM) fluorescence technique, and bacterivory was assessed with fluorescently labeled bacteria (FLB). Mixotrophic nanoflagellates were clearly dominant (up to 90% of total phytoplankton and 88% of total nanoflagellate abundance), and in the experiments labeled bacteria were observed in more than 75% of mixotrophic cells. These results support the idea that these phytoflagellates were never entirely photosynthetic. The high light : phosphorus ratio and the high C : N : P ratio suggest a strong nutrient limitation towards P. Our results show that both functions, photosynthesis and bacteria ingestion, were simultaneously reduced by the same level of UVR. We estimated that UVR exposure of mixotrophic nanoflagellates reduced photosystem II activity between 23% and 31% while ingestion rates were reduced between 23% and 28%. Therefore, our results suggest that the different cell functions could be concurrently impacted by UVR, implying that patterns and rates of C transfer would be substantially altered in the microbial food web.

Ultraviolet radiation induces filamentation in bacterial assemblages from North Andean Patagonian lakes.

Through laboratory experiments, we tested whether UV radiation (UVR) induces filamentation in natural bacteria assemblages from North Andean Patagonian lakes. We incubated water from three different lakes for 72 h in four separate treatments: (1) UVR + PAR (photosynthetically active radiation), (2) 50% UVR + PAR, (3) PAR and (4) 50% PAR. The irradiance levels used in the experiments were equivalent to those registered at the epilimnion of the lakes. In the UVR treatments filamentation was induced after the first 24 h and the proportion continued to increase for the next 48-72 h. A comparison of the gross composition and diversity of the entire community (cells >0.2 microm) with bacterial filaments alone (>5.0 microm) showed that UVR-induced filamentation is not a feature of any particular cluster. By sequencing part of the 16S rRNA gene of the taxonomic units obtained using denaturing gels, we observed that strains in the beta-Proteobacteria group were of relatively high importance in filament formation, followed by Cytophaga-Flavobacterium-Bacteroides, gamma-Proteobacteria and alpha-Proteobacteria, whereas Actinobacteria were almost nonexistent in the filaments. We propose that UVR doses equivalent to those of Andean lakes produce bacterial morphological changes, and that all bacterial groups except Actinobacteria can potentially form filaments.

Effect of ultraviolet radiation on acetylcholinesterase activity in freshwater copepods.

We analyzed the effects of UV radiation (UVR) effects on acetylcholinesterase (AChE) activity in two calanoid copepods, Boeckella gibbosa and Parabroteas sarsi that inhabit Patagonian shallow lakes. We studied the effect of experimental UVR (UV-B and UV-A) exposure on AChE activity in relation to basal antioxidant capacities of both copepods. Our experiments showed that UVR can effectively depress AChE activity, although with differences between species. In both copepods AChE was affected by UV-B, whereas UV-A only affected AChE in B. gibbosa. Both copepods also differed in body elemental composition (C:N:P), photoprotecting compound content (carotenoids and mycosporine-like amino acids) and enzymatic antioxidant capacity (glutathione S-transferase [GST]). Our results suggest that when exposed to UVR, AChE activity would depend more on the antioxidant capacity (GST) and P availability for enzyme synthesis than on the photoprotective compounds.