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.

SUMMARY The cyanobacterium Microcystis aeruginosa causes most of the harmful toxic blooms in freshwater ecosystems. Some strains of M. aeruginosa tolerate low‐medium levels of salinity, and because salinization of freshwater aquatic systems is increasing worldwide it is relevant to know what adaptive mechanisms allow tolerance to salinity. The mechanisms involved in the adaptation of M. aeruginosa to salinity (acclimation vs. genetic adaptation) were tested by a fluctuation analysis design, and then the maximum capacity of adaptation to salinity was studied by a ratchet protocol experiment. Whereas a dose of 10 g NaCl L−1 completely inhibited the growth of M. aeruginosa, salinity‐resistant genetic variants, capable of tolerating up to 14 g NaCl L−1, were isolated in the fluctuation analysis experiment. The salinity‐resistant cells arose by spontaneous mutations at a rate of 7.3 × 10−7 mutants per cell division. We observed with the ratchet protocol that three independent culture populations of M. aeruginosa were able to adapt to up to 15.1 g L−1 of NaCl, suggesting that successive mutation‐selection processes can enhance the highest salinity level to which M. aeruginosa cells can initially adapt. We propose that increasing salinity in water reservoirs could lead to the selection of salinity‐resistant mutants of M. aeruginosa.

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.

A seasonal cycle of sulfide, nitrate, phosphate, ammonium, chlorophyll a (Chl a) and Oscillatoria sp. abundance (<100 μm), as well as the relative contribution of taxonomic phytoplanktonic groups (cyanobacteria, green algae, cryptomonads, diatoms and dinoflagellates) to total Chl a were measured by fluorometric measurements at La Hedionda sulfide-rich spa (southern Spain). Fluorometry determined that cyanobacteria Chl a concentration correlated positively with the abundance of Oscillatoria sp. Aggregates at 45–100 μm equivalent spherical diameter (ESD) and was used as an indicator of Oscillatoria sp. Abundance, including for aggregates <45 and >100μm (ESD). In addition, air temperature, radiation and precipitation were downloaded from meteorological databases. In agreement with the meteorological annual cycle observed in air temperature, radiation and precipitation, sulfide concentration at La Hedionda Spa shows an annual cycle with concentrations around 40 μM in winter and up to 200 μM in the dry summer period. Phytoplankton composition was dominated by cyanobacteria (mainly Oscillatoria sp.), but other groups were also represented (green algae, cryptomonads, diatoms and dinoflagellates), although they remained constant throughout the year (median Chl a < 0.2 μg L−1). Cyanobacteria, in contrast, showed an annual cycle with a significantly higher median in summer (Chl a = 1.6 μg L−1) than in winter (Chl a = 0.4 μg L−1). No linear relationship between nutrients and cyanobacteria concentration was observed, but an optimum curve of cyanobacteria concentration to sulfide concentration was fitted through a general additive model (GAM). The four-fold increase of cyanobacteria concentration under exposition of an elevated sulfide concentration can be due to higher growth rates at elevated sulfide concentrations reported for an Oscillatoria sp. strain isolated during the same annual cycle at La Hedionda and we suggest that the selective agent, sulfide, positively triggers Oscillatoria sp. proliferation in summer. According to our findings, the Oscillatoria sp. population of La Hedionda not only is sulfide-resistant, but requires sulfide in its optimal niche.

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.