Mónica Rouco

Adaptation of the chlorophycean Dictyosphaerium chlorelloides to stressful acidic, mine metal-rich waters as result of pre-selective mutations

Several species of microalgae, closely related to mesophilic lineages, inhabit the extreme environment (pH 2.5, high levels of metals) of the Spains Aguas Agrias Stream water (AASW). Consequently, AASW constitutes an interesting natural laboratory for analysis of adaptation by microalgae to extremely stressful conditions. To distinguish between the pre-selective or post-selective origin of adaptation processes allowing the existence of microalgae in AASW, a Luria-Delbrück fluctuation analysis was performed with the chlorophycean Dictyosphaerium chlorelloides isolated from non-acidic waters. In the analysis, AASW was used as selective factor. Preselective, resistant D. chlorelloides cells appeared with a frequency of 1.1 x 10(-6) per cell per generation. AASW-resistant mutants, with a diminished Malthusian fitness, are maintained in non-extreme waters as the result of a balance between new AASW-resistant cells arising by mutation and AASW-resistant mutants eliminated by natural selection (equilibrium at c. 12 AASW-resistants per 10(7) wild-type cells). We propose that the microalgae inhabiting this stressful environment could be the descendents of chance mutants that arrived in the past or are even arriving at the present.

Adaptation of green microalgae to the herbicides simazine and diquat as result of pre-selective mutations.

Aquatic ecosystems located close to agricultural areas are increasingly polluted by herbicides. We evaluated the capacity for adaptation of green microalgae to lethal concentrations of the herbicide simazine in one strain of Dictyosphaerium chlorelloides and two strains of Scenedesmus intermedius, as well as adaptation to the herbicide diquat in one of the strains of S. intermedius. A Luria-Delbrück fluctuation analysis was carried out in order to distinguish between resistant cells arising from physiological adaptation (acclimatization) or post-adaptive mutation (both events occurring after the exposure to the herbicides), and adaptation due to mutations before the exposure to the herbicides. Simazine-resistant cells arose by rare spontaneous mutations before the exposure to simazine, with a rate of 3.0 x 10(-6) mutants per cell per generation in both strains of S. intermedius, and of 9.2 x 10(-6) mutants per cell per generation in D. chlorelloides. Diquat-resistant cells in S. intermedius arose by pre-selective mutations with a rate of 17.9 x 10(-6) per cell per generation. Rare, pre-selective mutations may allow the survival of green microalgae in simazine- or diquat-polluted waters, via herbicide-resistant selection. Therefore, human-synthesized pollutants, such as the herbicides simazine and diquat, could cause the emergence of evolutionary novelties in aquatic environments.

Estimating the capability of different phytoplankton groups to adapt to contamination: herbicides will affect phytoplankton species differently

• Investigating the differential capacity of the response of phytoplankton to human-induced environmental forcing has become a key issue to understanding further the future repercussions on the functioning of aquatic ecosystems. • The initial tolerance to the widely dispersed herbicide simazine was measured in diverse phytoplankton species. An experimental ratchet system maintaining large populations of dividing cells (which ensures the occurrence of rare spontaneous mutations that confer adaptation) and a strong selection pressure (which ensures the preservation of such mutations within the population) was later applied to estimate the capability of different groups of phytoplankton to adapt to simazine. • Initially, simazine doses between 0.05 and 0.15 ppm were able to inhibit 100% growth in all the species tested. However, a significant increase in simazine resistance was achieved in all derived populations during the ratchet experiment. The differential capacity for simazine adaptation was observed among the different species. • The capacity of different species to adapt to simazine can be explained in relation to taxonomic group, ploidy, growth rate and habitat preference. Haploid populations of continental Chlorophyta showed the greatest capacity to adapt to simazine. By contrast, populations of Haptophyta of open ocean regions were the group least capable of adapting to the herbicide.

Adaptation of microalgae to lindane: A new approach for bioremediation

Lindane is especially worrisome because its persistence in aquatic ecosystems, tendency to bioaccumulation and toxicity. We studied the adaptation of freshwater cyanobacteria and microalgae to resist lindane using an experimental model to distinguish if lindane-resistant cells had their origin in random spontaneous pre-selective mutations (which occur prior to the lindane exposure), or if lindane-resistant cells arose by a mechanism of physiological acclimation during the exposure to the selective agent. Although further research is needed to determine the different mechanisms contributing to the bio-elimination of lindane, this study, however, provides an approach to the bioremediation abilities of the lindane-resistant cells. Wild type strains of the experimental organisms were exposed to increasing lindane levels to estimate lethal concentrations. Growth of wild-type cells was completely inhibited at 5mg/L concentration of lindane. However, after further incubation in lindane for several weeks, occasionally the growth of rare lindane-resistant cells was found. A fluctuation analysis demonstrated that lindane-resistant cells arise only by rare spontaneous mutations that occur randomly prior to exposure to lindane (lindane-resistance did not occur as a result of physiological mechanisms). The rate of mutation from lindane sensitivity to resistance was between 1.48 × 10(-5) and 2.35 × 10(-7) mutations per cell per generation. Lindane-resistant mutants exhibited a diminished fitness in the absence of lindane, but only these variants were able to grow at lindane concentrations higher than 5mg/L (until concentrations as high as 40 mg/L). Lindane-resistant mutants may be maintained in uncontaminated waters as the result of a balance between new resistant mutants arising from spontaneous mutation and resistant cells eliminated by natural selection waters via clone selection. The lindane-resistant cells were also used to test the potential of microalgae to remove lindane. Three concentrations (4, 15 and 40 mg/L) were chosen as a model. In these exposures the lindane-resistant cells showed a great capacity to remove lindane (until 99% lindane was eliminated). Apparently, bioremediation based on lindane-resistant cells could be a great opportunity for cleaning up of lindane- and other chlorinated organics-polluted habitats.

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.

Toxicity and adaptation of Dictyosphaerium chlorelloides to extreme chromium contamination

Metals are often spilled by industries into inland water environments, with adverse consequences. Numerous papers have reported that heavy metals produce massive destruction of algae. Nevertheless, algal populations seem to become tolerant when they have had previous exposures to heavy metals. Because the mechanisms allowing heavy metal tolerance of algae are not yet known, the present study analyzed the effect of hexavalent chromium on growth and photosynthetic performance of Dictyosphaerium chlorelloides, stressing on the adaptation mechanisms to chromium contamination. Growth and photosynthetic performance of algal cells were inhibited by Cr(VI) at 10 mg/L, and the 72-h median inhibition concentration was established as 1.64 and 1.54 mg/L, respectively. However, after further incubation for a three month period in an environment with 25 mg/L of chromium, some rare, chromium-resistant cells occasionally were found. A Luria-Delbrück fluctuation analysis was performed to distinguish between resistant algae arising from rare, spontaneous mutations and resistant algae arising from physiological adaptation and other adaptive mechanisms. Resistant cells arose only by spontaneous mutations before the addition of chromium, with a rate of 1.77 x 10(-6) mutants per cell division. From a practical point of view, the use of both chromium-sensitive and chromium-resistant genotypes could make possible a specific algal biosensor for chromium.

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.

Living in Vulcan's forge: Algal adaptation to stressful geothermal ponds on Vulcano Island (southern Italy) as a result of pre‐selective mutations

Four species of eukaryotic algae proliferate in the sulfureous, acidic (pH 3.1) water of the largest geothermal pond on Vulcano Island (southern Italy). Consequently, this pond constitutes a natural laboratory for analysis of adaptation by phytoplankters to extremely stressful conditions. To distinguish between the pre‐selective or post‐selective origin of adaptation processes allowing the existence of phytoplankters in the pond, a Luria‐Delbrück fluctuation test was carried out with the chlorophycean Dictyosphaerium chlorelloides and the cyanobacterium Microcystis aeruginosa, both isolated from non‐extreme waters; natural water from the Vulcano Island pond was used as selective factor. Preselective, resistant D. chlorelloides cells appeared with a frequency of 4.7 × 10−7 per cell per generation. We propose that the micro‐algae inhabiting this stressful pond could be the descendents of chance mutants that arrived in the past or are even arriving at the present. The genetic adaptation of D. chlorelloides to Vulcano waters could help to explain the survival of photosynthesizers in very stressful geothermal waters during the Neoproterozoic ‘snowball Earth’, a period when primary production collapsed in the biosphere. On the other hand, adaptation to these conditions was not observed in M. aeruginosa, suggesting that cyanobacteria may not be able to develop any kind of adaptation to Vulcano pond water.

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.