Temporal and spatial variability in population traits of an intertidal fucoid reveals local-scale climatic refugia.

Global change is imposing significant losses in the functional traits of marine organisms. Although areas of climatic refugia ameliorate local conditions and help them to persist, the extent to which mesoscale effects contribute for intraregional variability on population traits and conservation is uncertain. Here we assess patterns of conservation status of Fucus guiryi, the main intertidal habitat-forming seaweed in the Strait of Gibraltar (southern Spain and northern Morocco). We investigated the demography, reproductive phenology, and morphology at northern and southern side populations. Population traits were compared seasonally within populations from each side, and at spatial scale in early summer 2019. In the last decade three populations became extinct; two marginal populations had dispersed individuals with a narrower fertility season and miniaturized individuals below 3 cm; and five populations showed variable density and cover with more than 20% of reproductive individuals over the seasons. Highest density, cover, morphology, and reproductive potential was detected at one population from each side, suggesting local-scale climatic refugia in upwelling areas located inside marine protected areas. Southern recruits were more warm-tolerant but grew less at colder conditions than northern ones, revealing a mesoscale heterogeneity in thermal affinities. This study evidenced functional losses and distinct reproductive strategies experienced by F. guiryi at peripheral locations and urges to prioritize its conservation and restoration at contemporary climatic refugia.

Changes in the growth rate of Chlamydomonas reinhardtii under long-term selection by temperature and salinity: Acclimation vs. evolution.

We investigated the roles of acclimation and different components involved in evolution (adaptation, chance and history) on the changes in the growth rate of the model freshwater microalga Chlamydomonas reinhardtii P. A. Dang. exposed to selective temperature and salinity. Three C. reinhardtii strains previously grown during one year in freshwater medium and 20 °C were exposed to 5 °C temperature increase and a salinity of 5 g L-1 NaCl. Cultures under each selective scenario and in combination (increase of salinity and temperature), were propagated until growth rate achieved an invariant mean value for 6 months (100-350 generations, varying as a function of scenario and strain). The changes of the growth rate under increased temperature were due to both adaptation and acclimation, as well as history. However, acclimation was the only mechanism detected under salinity increase as well as in the selective scenario of both temperature and salinity, suggesting that genetic variability would not allow survival at salinity higher than that to which experimental populations were exposed. Therefore, it could be hypothesized that under a global change scenario an increase in salinity would be a greater challenge than warming for some freshwater phytoplankton.

Unexpected resilience in relict Abies pinsapo Boiss forests to dieback and mortality induced by climate change.

Acute and early symptoms of forest dieback linked to climate warming and drought episodes have been reported for relict Abies pinsapo Boiss. fir forests from Southern Spain, particularly at their lower ecotone. Satellite, orthoimages, and field data were used to assess forest decline, tree mortality, and gap formation and recolonization in the lower half of the altitudinal range of A. pinsapo forests (850-1550 m) for the last 36 years (1985-2020). Field surveys were carried out in 2003 and in 2020 to characterize changes in stand canopy structure and mortality rates across the altitudinal range. Time series of the Normalized Difference Vegetation Index (NDVI) at the end of the dry season (derived from Landsat 5 and 7 imagery) were used for a Dynamic Factor Analysis to detect common trends across altitudinal bands and topographic solar incidence gradients (SI). Historical canopy cover changes were analyzed through aerial orthoimages classification. Here we show that extensive decline and mortality contrast to the almost steady alive basal area for 17 years, as well as the rising photosynthetic activity derived from NDVI since the mid-2000s and an increase in the forest canopy cover in the late years at mid and high altitudes. We hypothesized that these results suggest an unexpected resilience in A. pinsapo forests to climate change-induced dieback, that might be promoted by compensation mechanisms such as (i) recruitment of new A. pinsapo individuals; (ii) facilitative effects on such recruitment mediated by revegetation with other species; and (iii) a 'release effect' in which surviving trees can thrive with fewer resource competition. Future research is needed to understand these compensation mechanisms and their scope in future climate change scenarios.

Photosynthetic performance in cyanobacteria with increased sulphide tolerance: an analysis comparing wild-type and experimentally derived strains.

Sulphide is proposed to have influenced the evolution of primary stages of oxygenic photosynthesis in cyanobacteria. However, sulphide is toxic to most of the species of this phylum, except for some sulphide-tolerant species showing various sulphide-resistance mechanisms. In a previous study, we found that this tolerance can be induced by environmental sulphidic conditions, in which two experimentally derived strains with an enhanced tolerance to sulphide were obtained from Microcystis aeruginosa, a sensitive species, and Oscillatoria, a sulphide-tolerant genus. We have now analysed the photosynthetic performance of the wild-type and derived strains in the presence of sulphide to shed light on the characteristics underlying the increased tolerance. We checked whether the sulphide tolerance was a result of higher PSII sulphide resistance and/or the induction of sulphide-dependent anoxygenic photosynthesis. We observed that growth, maximum quantum yield, maximum electron transport rate and photosynthetic efficiency in the presence of sulphide were less affected in the derived strains compared to their wild-type counterparts. Nevertheless, in 14C photoincoporation assays, neither Oscillatoria nor M. aeruginosa exhibited anoxygenic photosynthesis using sulphide as an electron donor. On the other hand, the content of photosynthetic pigments in the derived strains was different to that observed in the wild-type strains. Thus, an enhanced PSII sulphide resistance appears to be behind the increased sulphide tolerance displayed by the experimentally derived strains, as observed in most natural sulphide-tolerant cyanobacterial strains. However, other changes in the photosynthetic machinery cannot be excluded.

Detection of the maximum resistance to the herbicides diuron and glyphosate, and evaluation of its phenotypic cost, in freshwater phytoplankton.

One of the most important anthropogenic impacts on freshwater aquatic ecosystems close to intensive agriculture areas is the cumulative increase in herbicide concentrations. The threat is especially relevant for phytoplankton organisms because they have the same physiological targets as the plants for which herbicides have been designed. This led us to explore the evolutionary response of three phytoplanktonic species to increasing concentrations of two herbicides and its consequences in terms of growth and photosynthesis performance. Specifically, we used an experimental ratchet protocol to investigate the differential evolution and the limit of resistance of a cyanobacterium (Microcystis aeruginosa) and two chlorophyceans (Chlamydomonas reinhardtii and Dictyosphaerium chlorelloides) to two herbicides in worldwide use: glyphosate and diuron. Initially, the growth rate of M. aeruginosa and D. chlorelloides was completely inhibited when they were exposed to a dose of 0.23 ppm diuron or 40 ppm glyphosate, whereas a higher concentration of both herbicides (0.46 ppm diuron or 90 ppm glyphosate) was necessary to abolish C. reinhardtii growth. However, after running a ratchet protocol, the resistance of the three species to both herbicides increased by an adaptation process. M. aeruginosa and D. chlorelloides were able to grow at 1.84 ppm diuron and 80 ppm glyphosate and C. reinhardtii proliferated at twice these concentrations. Herbicide-resistant strains showed lower growth rates than their wild-type counterparts in the absence of herbicides, as well as changes on morphology and differences on photosynthetic pigment content. Besides, herbicide-resistant cells generally showed a lower photosynthetic performance than wild-type strains in the three species. These results indicate that the introduction of both herbicides in freshwater ecosystems could produce a diminution of primary production due to the selection of herbicide-resistant mutants, that would exhibit lower photosynthetic performance than wild-type populations.

The role of changes in environmental quality in multitrait plastic responses to environmental and social change in the model microalga Chlamydomonas reinhardtii.

Intraspecific variation plays a key role in species' responses to environmental change; however, little is known about the role of changes in environmental quality (the population growth rate an environment supports) on intraspecific trait variation. Here, we hypothesize that intraspecific trait variation will be higher in ameliorated environments than in degraded ones. We first measure the range of multitrait phenotypes over a range of environmental qualities for three strains and two evolutionary histories of Chlamydomonas reinhardtii in laboratory conditions. We then explore how environmental quality and trait variation affect the predictability of lineage frequencies when lineage pairs are grown in indirect co-culture. Our results show that environmental quality has the potential to affect intraspecific variability both in terms of the variation in expressed trait values, and in terms of the genotype composition of rapidly growing populations. We found low phenotypic variability in degraded or same-quality environments and high phenotypic variability in ameliorated conditions. This variation can affect population composition, as monoculture growth rate is a less reliable predictor of lineage frequencies in ameliorated environments. Our study highlights that understanding whether populations experience environmental change as an increase or a decrease in quality relative to their recent history affects the changes in trait variation during plastic responses, including growth responses to the presence of conspecifics. This points toward a fundamental role for changes in overall environmental quality in driving phenotypic variation within closely related populations, with implications for microevolution.

Using ForeStereo and LIDAR data to assess fire and canopy structure-related risks in relict Abies pinsapo Boiss. forests.

In this study we combine information from aerial LIDAR and hemispherical images taken in the field with ForeStereo-a forest inventory device-to assess the vulnerability and to design conservation strategies for endangered Mediterranean fir forests based on the mapping of fire risk and canopy structure spatial variability. We focused on the largest continuous remnant population of the endangered tree species Abies pinsapo Boiss. spanning 252 ha in Sierra de las Nieves National Park (South Andalusia, Spain). We established 49 sampling plots over the study area. Stand structure variables were derived from ForeStereo device, a proximal sensing technology for tree diameter, height and crown dimensions and stand crown cover and basal area retrieval from stereoscopic hemispherical images photogrammetry. With this information, we developed regression models with airborne LIDAR data (spatial resolution of 0.5 points∙m-2). Thereafter, six fuel models were fitted to the plots according to the UCO40 classification criteria, and then the entire area was classified using the Nearest Neighbor algorithm on Sentinel imagery (overall accuracy of 0.56 and a KIA-Kappa Coefficient of 0.46). FlamMap software was used for fire simulation scenarios based on fuel models, stand structure, and terrain data. Besides the fire simulation, we analyzed canopy structure to assess the status and vulnerability of this fir population. The assessment shows a secondary growth forest that has an increasing presence of fuel models with the potential for high fire spread rate fire and burn probability. Our methodological approach has the potential to be integrated as a support tool for the adaptive management and conservation of A. pinsapo across its whole distribution area (<4,000 ha), as well as for other endangered circum-Mediterranean fir forests, as A. numidica de Lannoy and A. pinsapo marocana Trab. in North Africa.

The limit of resistance to salinity in the freshwater cyanobacterium Microcystis aeruginosa is modulated by the rate of salinity increase.

The overall mean levels of different environmental variables are changing rapidly in the present Anthropocene, in some cases creating lethal conditions for organisms. Under this new scenario, it is crucial to know whether the adaptive potential of organisms allows their survival under different rates of environmental change. Here, we used an eco-evolutionary approach, based on a ratchet protocol, to investigate the effect of environmental change rate on the limit of resistance to salinity of three strains of the toxic cyanobacterium Microcystis aeruginosa. Specifically, we performed two ratchet experiments in order to simulate two scenarios of environmental change. In the first scenario, the salinity increase rate was slow (1.5-fold increase), while in the second scenario, the rate was faster (threefold increase). Salinity concentrations ranging 7-10 gL-1 NaCl (depending on the strain) inhibited growth completely. However, when performing the ratchet experiment, an increase in salinity resistance (9.1-13.6 gL-1 NaCl) was observed in certain populations. The results showed that the limit of resistance to salinity that M. aeruginosa strains were able to reach depended on the strain and on the rate of environmental change. In particular, a higher number of populations were able to grow under their initial lethal salinity levels when the rate of salinity increment was slow. In future scenarios of increased salinity in natural freshwater bodies, this could have toxicological implications due to the production of microcystin by this species.

Adaptation dynamics and evolutionary rescue under sulfide selection in cyanobacteria: a comparative study between Microcystis aeruginosa and Oscillatoria sp. (cyanobacteria).

Experimental evolution studies using cyanobacteria as model organisms are scarce despite the role of cyanobacteria in the evolution of photosynthesis. Three different experimental evolution approaches have been applied to shed light on the sulfide adaptation process, which played a key role in the evolution of this group. We used a Microcystis aeruginosa sulfide-sensitive strain, unable to grow above ~0.1 mM, and an Oscillatoria sp. strain, isolated from a sulfureous spa (~0.2 mM total sulfide). First, performing a fluctuation analysis design using the spa waters as selective agent, we proved that M. aeruginosa was able to adapt to this sulfide level by rare spontaneous mutations. Second, applying a ratchet protocol, we tested if the limit of adaptation to sulfide of the two taxa was dependent on their initial sulfide tolerance, finding that M. aeruginosa adapted to 0.4 mM sulfide, and Oscillatoria sp. to ~2 mM sulfide, twice it highest tolerance level. Third, using an evolutionary rescue approach, we observed that both speed of exposure to increasing sulfide concentrations (deterioration rate) and populations' genetic variation determined the survival of M. aeruginosa at lethal sulfide levels, with a higher dependence on genetic diversity. In conclusion, sulfide adaptation of sensitive cyanobacterial strains is possible by rare spontaneous mutations and the adaptation limits depend on the sulfide level present in strain's original habitat. The high genetic diversity of a sulfide-sensitive strain, even at fast environmental deterioration rates, could increase its possibility of survival even to a severe sulfide stress.

North Atlantic Oscillation drives the annual occurrence of an isolated, peripheral population of the brown seaweed Fucus guiryi in the Western Mediterranean Sea.

The canopy-forming, intertidal brown (Phaeophyceae) seaweed Fucus guiryi is distributed along the cold-temperate and warm-temperate coasts of Europe and North Africa. Curiously, an isolated population develops at Punta Calaburras (Alboran Sea, Western Mediterranean) but thalli are not present in midsummer every year, unlike the closest (ca. 80 km), perennial populations at the Strait of Gibraltar. The persistence of the alga at Punta Calaburras could be due to the growth of resilient, microscopic stages as well as the arrival of few-celled stages originating from neighbouring localities, and transported by the permanent Atlantic Jet flowing from the Atlantic Ocean into the Mediterranean. A twenty-six year time series (from 1990 to 2015) of midsummer occurrence of F. guiryi thalli at Punta Calaburras has been analysed by correlating with oceanographic (sea surface temperature, an estimator of the Atlantic Jet power) and climatic factors (air temperature, rainfall, and North Atlantic Oscillation -NAO-, and Arctic Oscillation -AO- indexes). The midsummer occurrence of thalli clustered from 1990-1994 and 1999-2004, with sporadic occurrences in 2006 and 2011. Binary logistic regression showed that the occurrence of thalli at Punta Calaburras in midsummer is favoured under positive NAO index from April to June. It has been hypothesized that isolated population of F. guiryi should show greater stress than their congeners of permanent populations, and to this end, two approaches were used to evaluate stress: one based on the integrated response during ontogeny (developmental instability, based on measurements of the fractal branching pattern of algal thalli) and another based on the photosynthetic response. Although significant differences were detected in photosynthetic quantum yield and water loss under emersion conditions, with thalli from Punta Calaburras being more affected by emersion than those from Tarifa, the developmental instability showed that the population from Tarifa suffers higher stress during ontogeny than that from Punta Calaburras. In conclusion, this study demonstrates the teleconnection between atmospheric oscillations and survival and proliferation of marine macroalgae.

Photosynthetic use of inorganic carbon in deep-water kelps from the Strait of Gibraltar.

Mechanisms of inorganic carbon assimilation were investigated in the four deep-water kelps inhabiting sea bottoms at the Strait of Gibraltar; these species are distributed at different depths (Saccorhiza polysiches at shallower waters, followed by Laminaria ochroleuca, then Phyllariopsis brevipes and, at the deepest bottoms, Phyllariopsis purpurascens). To elucidate the capacity to use HCO3(-) as a source of inorganic carbon for photosynthesis in the kelps, different experimental approaches were used. Specifically, we measured the irradiance-saturated gross photosynthetic rate versus pH at a constant dissolved inorganic carbon (DIC) concentration of 2 mM, the irradiance-saturated apparent photosynthesis (APS) rate versus DIC, the total and the extracellular carbonic anhydrase (CAext), the observed and the theoretical photosynthetic rates supported by the spontaneous dehydration of HCO3(-) to CO2, and the δ(13)C signature in tissues of the algae. While S. polyschides and L. ochroleuca showed photosynthetic activity at pH 9.5 (around 1.0 µmol O2 m(-2) s(-1)), the activity was close to zero in both species of Phyllariopsis. The APS versus DIC was almost saturated for the DIC values of natural seawater (2 mM) in S. polyschides and L. ochroleuca, but the relationship was linear in P. brevipes and P. purpurascens. The four species showed total and CAext activities but the inhibition of the CAext originated the observed photosynthetic rates at pH 8.0 to be similar to the theoretical rates that could be supported by the spontaneous dehydration of HCO3(-). The isotopic (13)C signatures ranged from -17.40 ± 1.81 to -21.11 ± 1.73 ‰ in the four species. Additionally, the δ(13)C signature was also measured in the deep-water Laminaria rodriguezii growing at 60-80 m, showing even a more negative value of -26.49 ± 1.25 ‰. All these results suggest that the four kelps can use HCO3(-) as external carbon source for photosynthesis mainly by the action of external CAext, but they also suggest that the species inhabiting shallower waters show a higher capacity than the smaller kelps living in deeper waters. In fact, the photosynthesis in the two Phyllariopsis species could be accomplished by the spontaneous dehydration of HCO3(-) to CO2. These differences in the capacity to use HCO3(-) in photosynthesis among species could be important considering the increasing levels of atmospheric CO2 predicted for the near future.

Sulphide Resistance in the Cyanobacterium Microcystis aeruginosa: a Comparative Study of Morphology and Photosynthetic Performance Between the Sulphide-Resistant Mutant and the Wild-Type Strain.

The cyanobacterium Microcystis aeruginosa is a mesophilic freshwater organism, which cannot tolerate sulphide. However, it was possible to isolate a sulphide-resistant (S(r)) mutant strain that was able to survive in a normally lethal medium sulphide. In order to evaluate the cost of the mutation conferring sulphide resistance in the S(r) strain of M. aeruginosa, the morphology and the photosynthetic performance were compared to that found in the wild-type, sulphide-sensitive (S(s)) strain. An increase in size and a disrupted morphology was observed in S(r) cells in comparison to the S(s) counterpart. Phycoerythrin and phycocyanin levels were higher in the S(r) than in the S(s) cells, whereas a higher carotenoid content, per unit volume, was found in the S(s) strain. The irradiance-saturated photosynthetic oxygen-production rate (GPR max) and the photosynthetic efficiency (measured both by oxygen production and fluorescence, α(GPR) and α(ETR)) were lower in the S(r) strain than in the wild-type. These results appear to be the result of package effect. On the other hand, the S(r) strain showed higher quantum yield of non-photochemical quenching, especially those regulated mechanisms (estimated throughout qN and Y(NPQ)) and a significantly lower slope in the maximum quantum yield of light-adapted samples (Fv'/Fm') compared to the S(s) strain. These findings point to a change in the regulation of the quenching of the transition states (qT) in the S(r) strain which may be generated by a change in the distribution of thylakoidal membranes, which somehow could protect metalloenzymes of the electron transport chain from the lethal effect of sulphide.

The limit of the genetic adaptation to copper in freshwater phytoplankton.

Copper is one of the most frequently used algaecides to control blooms of toxic cyanobacteria in water supply reservoirs. Among the negative impacts derived from the use of this substance is the increasing resistance of cyanobacteria to copper toxicity, as well as changes in the community structure of native phytoplankton. Here, we used the ratchet protocol to investigate the differential evolution and maximum adaptation capacity of selected freshwater phytoplankton species to the exposure of increasing doses of copper. Initially, a dose of 2.5 µM CuSO4·5H2O was able to completely inhibit growth in three strains of the toxic cyanobacterium Microcystis aeruginosa, whereas growth of the chlorophyceans Dictyosphaerium chlorelloides and Desmodesmus intermedius (represented by two different strains) was completely abolished at 12 µM. A significant increase in resistance was achieved in all derived populations during the ratchet experiment. All the chlorophyceans were able to adapt to up to 270 µM of copper sulfate, but 10 µM was the highest concentration that M. aeruginosa strains were able to cope with, although one of the replicates adapted to 30 µM. The recurrent use and increasing doses of copper in water reservoirs could lead to the selection of copper-resistant mutants of both chlorophyceans and cyanobacteria. However, under high concentrations of copper, the composition of phytoplankton community could undergo a drastic change with cyanobacteria being replaced by copper-resistant chlorophyceans. This result stems from a distinct evolutionary potential of these species to adapt to this substance.

Disentangling mechanisms involved in the adaptation of photosynthetic microorganisms to the extreme sulphureous water from Los Baños de Vilo (S Spain).

Los Baños de Vilo (S Spain) is a natural spa characterized by extreme sulphureous waters; however, populations of chlorophyceans inhabit in the spa. The adaptation mechanisms allowing resistance by photosynthetic microorganisms to the extreme sulphureous waters were studied by using a modified Luria-Delbrück fluctuation analysis. For this purpose, the adaptation of the chlorophycean Dictyosphaerium chlorelloides and the cyanobacterium Microcystis aeruginosa (both isolated from non-sulphureous water) were analysed in order to distinguish between physiological adaptation (acclimation) and genetic adaptation by the selection of rare spontaneous mutations. Acclimation to the extreme water was achieved by D. chlorelloides; however, M. aeruginosa cells proliferated as a consequence of selection of favoured mutants (i.e. genetic adaptation). The resistant cells of M. aeruginosa appeared with a frequency of 7.1 × 10(-7) per cell per generation, and the frequency of the resistant allele, under non-selective conditions, was estimated to be 1.1 × 10(-6) per cells as a consequence of the balance mutation-selection. It could be hypothesized that the populations of eukaryotic algae living in the Los Baños de Vilo could be the descendants of chlorophyceans that arrived fortuitously at the spa in the past. On the other hand, cyanobacteria could quickly adapt by the selection of favoured mutants. The single mutation that allows resistance to sulphureous water from Baños de Vilo in M. aeruginosa represents a phenotypic burden impairing growth rate and photosynthetic performance. The resistant-variant cells of M. aeruginosa showed a lower acclimated growth rate and a decreased maximum quantum yield and photosynthetic efficiency, in comparison to the wild-type cells.

Photoacclimation of cultured strains of the cyanobacterium Microcystis aeruginosa to high-light and low-light conditions.

The cyanobacterium Microcystis aeruginosa forms blooms that can consist of colonies. We have investigated how M. aeruginosa acclimatizes to changing light conditions such as can occur during blooms. Three different strains were exposed to two irradiance levels: lower (LL) and higher (HL) than the irradiance-onset saturation parameter. We measured the photosynthetic pigment concentrations, PSII photochemical efficiency, electron transport rate (ETR), irradiance-saturated ETR and ETR efficiency. The relationship between ETR and photosynthetic oxygen production and the excess in PSII capacity were also studied for one strain. Higher values of chlorophyll a and phycocyanin and lower values of total carotenoids were found under LL conditions in the three strains. The strains showed clear differences in the irradiance-saturated ETR and in ETR efficiency under both LL and HL treatments. No differences were found in the linear relationship between ETR and photosynthetic oxygen production under both irradiance treatments. LL-acclimated cells showed higher PSII excess capacity than HL ones, possibly because their higher pigment content could result in a higher light stress than HL cells when forming surface blooms. The fact that the genetically different strains show different photosynthetic physiologies suggests that the very dynamic light climate observed in lakes may allow their coexistence.

Effects of adaptation, chance, and history on the evolution of the toxic dinoflagellate Alexandrium minutum under selection of increased temperature and acidification.

The roles of adaptation, chance, and history on evolution of the toxic dinoflagellate Alexandrium minutum Halim, under selective conditions simulating global change, have been addressed. Two toxic strains (AL1V and AL2V), previously acclimated for two years at pH 8.0 and 20°C, were transferred to selective conditions: pH 7.5 to simulate acidification and 25°C. Cultures under selective conditions were propagated until growth rate and toxin cell quota achieved an invariant mean value at 720 days (ca. 250 and ca. 180 generations for strains AL1V and AL2V, respectively). Historical contingencies strongly constrained the evolution of growth rate and toxin cell quota, but the forces involved in the evolution were not the same for both traits. Growth rate was 1.5-1.6 times higher than the one measured in ancestral conditions. Genetic adaptation explained two-thirds of total adaptation while one-third was a consequence of physiological adaptation. On the other hand, the evolution of toxin cell quota showed a pattern attributable to neutral mutations because the final variances were significantly higher than those measured at the start of the experiment. It has been hypothesized that harmful algal blooms will increase under the future scenario of global change. Although this study might be considered an oversimplification of the reality, it can be hypothesized that toxic blooms will increase but no predictions can be advanced about toxicity.

Photosynthetic performance of the aquatic macrophyte Althenia orientalis to solar radiation along its vertical stems.

We have studied the plasticity of the photosynthetic apparatus in the endangered aquatic macrophyte Althenia orientalis to the gradient of light availability within its meadow canopy. We determined diurnal change in situ irradiance, light quality, in vivo chlorophyll a fluorescence, ex situ oxygen evolution rates, respiration rate and pigment concentration. The levels of photosynthetic photon flux density (PFD) and ultraviolet radiation (UVR) and the red/far-red ratio decreased with depth within the canopies of A. orientalis. Apical leaves had a greater decrease of the maximal quantum yield (F(v)/F(m)) in the morning and a faster recovery rate in the afternoon than those in the basal ones. The relative electron transport rate (ETRr) was not saturated at any time of the day, even in the apical leaves that received the highest light. The maximum light-saturated rate of gross photosynthesis (GP(max)) took place in apical leaves around noon. The chlorophyll a/b ratio values were higher, and the chlorophyll/carotenoid ratio values lower, in apical leaves than basal ones. The highest concentrations in total carotenoids were reached in the apical leaves around noon. A. orientalis has a high capacity to acclimatize to the changes in the light environment, both in quality and quantity, presenting sun and shade leaves in the same stem through the vertical gradient in the canopy.

Evolutionary changes in growth rate and toxin production in the cyanobacterium Microcystis aeruginosa under a scenario of eutrophication and temperature increase.

Toxic blooms of the cyanobacterium Microcystis aeruginosa affect humans and animals in inland water systems worldwide, and it has been hypothesized that the development of these blooms will increase under the future scenario of global change, considering eutrophication and temperature increase as two important consequences. The importance of genetic adaptation, chance and history on evolution of growth rate, and toxin production of M. aeruginosa was studied under these new conditions. The experiment followed the idea of "replaying life's tape" by means of the simultaneous propagation of 15 independent isolates of three M. aeruginosa strains, which were grown under doubled nutrient concentration and temperature during c. 87 generations. Adaptation by new mutations that resulted in the enhancement of growth rate arose during propagation of derived cultures under the new environmental conditions was the main component of evolution; however, chance also contributed in a lesser extension to evolution of growth rate. Mutations were selected, displacing the wild-type ancestral genotypes. In contrast, the effect of selection on mutations affecting microcystin production was neutral. Chance and history were the pacemakers in evolution of toxin production. Although this study might be considered an oversimplification of the reality, it suggest that a future scenario of global change might lead to an increase in M. aeruginosa bloom frequency, but no predictions about the frequency of toxicity can be made.

GENETIC ADAPTATION AND ACCLIMATION OF PHYTOPLANKTON ALONG A STRESS GRADIENT IN THE EXTREME WATERS OF THE AGRIO RIVER-CAVIAHUE LAKE (ARGENTINA)(1).

We tested if different adaptation strategies were linked to a stress gradient in phytoplankton cells. For this purpose, we studied the adaptation and acclimation of Dictyosphaerium chlorelloides (Naumann) Komárek et Perman (Chlorophyta) and Microcystis aeruginosa (Kütz.) Kütz. (Cyanobacteria) to different water samples (from extremely acid, metal-rich water to moderate stressful conditions) of the Agrio River-Caviahue Lake system (Neuquén, Argentina). Both experimental strains were isolated from pristine, slightly alkaline waters. To distinguish between physiological acclimation and genetic adaptation (an adaptive evolution event), a modified Luria-Delbrück fluctuation analysis was carried out with both species by using as selective agent sample waters from different points along the stress gradient. M. aeruginosa did not acclimate to any of the waters tested from different points along the stress gradient nor did D. chlorelloides to the two most acidic and metal-rich waters. However, D. chlorelloides proliferated by rapid genetic adaptation, as the consequence of a single mutation (5.4 × 10(-7) resistant mutants per cell per division) at one locus, in less extreme water and also by acclimation in the least extreme water. It is hypothesized that the stress gradient resulted in different strategies of adaptation in phytoplankton cells from nonextreme waters. Thus, very extreme conditions were lethal for both organisms, but as stressful conditions decreased, adaptation of D. chlorelloides cells was possible by the selection of resistant mutants, and in less extreme conditions, by acclimation.

Elastase Activity in Aspergillus fumigatus Can Arise by Random, Spontaneous Mutations.

Aspergillus fumigatus Fresenius has the capacity to degrade elastin (the principal protein of the lungs) and it is considered that elastase activity (EA) is among the most important pathogenicity factors of this mold. In particular, there is a strong correlation between EA in A. fumigatus and invasive aspergillosis. However, EA is not universal in this mold, and it is unknown whether the capacity to degrade elastin is the consequence of physiological mechanisms and/or genetic changes (putative adaptive mutations) induced after the exposure to this substrate or, on the contrary, it is due to random spontaneous mutations that occur under nonselective conditions. In order to discriminate between these possibilities, a Luria-Delbrück fluctuation analysis was carried out on an elastase-negative (EA(-)) A. fumigatus strain, using as selective factor a culture medium containing elastin as the sole source of nitrogen. Here we show that the EA(-) → EA(+) transformation in A. fumigatus appears by rare, random mutations before the exposure of the strain to selective conditions. This work represents the first experimental evidence of pathogenicity factor acquisition in mycelial fungi by preselective mutation.