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.

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 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.

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.

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.

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.

Genetic variability associated with photosynthetic pigment concentration, and photochemical and nonphotochemical quenching, in strains of the cyanobacterium Microcystis aeruginosa.

Although populations of cyanobacteria are usually considered to be clonal, their capacity to survive environmental changes suggests intrapopulation genetic variation. We therefore estimated the genetic variability on the basis of two processes important for any photoautotroph - photochemical and nonphotochemical quenching - as well as photosynthetic pigment concentrations. For this purpose, two parameters related to photochemical and nonphotochemical quenching were measured using specific experimental and statistical procedures, in 25 strains of the cyanobacterium Microcystis aeruginosa, along with their contents of chlorophyll a, total carotenoids and phycocyanin. The experimental procedure allowed discrimination between genetic and nongenetic (or residual) variability among strains. The high genetic variability found in photosynthetic pigments and both photosynthetic parameters denotes large differences even among strains isolated from the same community. The high genetic diversity within a population could be important for the evolutionary success of cyanobacteria.

Adaptation of Spirogyra insignis (Chlorophyta) to an extreme natural environment (sulphureous waters) through preselective mutations.

Adaptation of Spirogyra insignis (Chlorophyceae) to growth and survival in an extreme natural environment (sulphureous waters from La Hedionda Spa, S. Spain) was analysed by using an experimental model. Photosynthesis and growth of the alga were inhibited when it was cultured in La Hedionda Spa waters (LHW), but after further incubation for several weeks, the culture survived due to the growth of a variant that was resistant to LHW. A Luria-Delbruck fluctuation analysis was carried out to distinguish between resistant filaments arising from rare spontaneous mutations and resistant filaments arising from other mechanisms of adaptation. It was demonstrated that the resistant filaments arose randomly by rare spontaneous mutations before the addition of LHW (preselective mutations). The rate of spontaneous mutation from sensitivity to resistance was 2.7 x 10(-7) mutants per cell division. Since LHW(resistant) mutants have a diminished growth rate, they are maintained in nonsulphureous natural waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, recurrence of rare spontaneous preselective mutations ensures the survival of the alga in sulphureous waters.