Drastic Environmental Changes in the Lake Dziani Dzaha Pelagic Ecosystem
Lake Dziani Dzaha is a tropical thalassohaline crater lake, with unique specific physico-chemical characteristics. The water of the Dziani Dzaha Lake probably originates from the nearby Indian Ocean with further modifications of its physico-chemical characteristics by precipitations, hydrothermal activity, precipitation of dissolved elements, and biogeochemical activity. This marine origin is original compared with that of most known other alkaline and saline lakes that are, for the great majority, inland water bodies whose salinity is due to dissolved continental minerals .
The hydrological characteristics of Lake Dziani Dzaha are mostly dependent on the seasonal climatology, which is based on alternating dry and rainy seasons. Low surface salinity and a halocline at a depth of 2.2 m can be attributed to the heavy rainfalls occurring during the rainy season in Mayotte Islands. During dry season, evaporation progressively increased the salinity of the surface layer, easing the mixing of water column and disappearance of the halocline. Therefore, the lake can be considered a monomictic lake (mixing once a year during the southern hemisphere winter) with special features in terms of oxygen content, i.e., permanent anoxia at a depth of approximately 2 m even in the absence of halocline. In summary, the water column in the deepest part of the lake undergoes important changes in its physicochemical characteristics, thus, defining several layers along the water column during both the seasons. These contrast layers, based on the physico-chemical characteristics, were observed between seasons from 2010 to 2015  and this study. They define several environmental niches, which might have strong effects on phytoplanktonic diversity and distribution, along the water column.
The estimated phytoplanktonic biomass, expressed as mean chlorophyll a concentrations, was very high in 2014 (566 μg Chla L−1) and 2015 (692 μg Chla L−1), and in the same range than the ones already observed in 2010 (685 μg Chla L−1) and 2011 (702 μg Chla L−1) .
Diversity and Composition of Phytoplanktonic Community
In the present study, the richness of phytoplanktonic community in the lake was low, with only 15 taxa being detected, eight of them belonging to cyanobacteria and seven to photosynthetic eukaryotes. To the best of our best knowledge, no study on the phytoplanktonic diversity of thalassohaline lake have been carried out with metabarcoding and metagenomic approaches. The richness of phytoplanktonic communities of soda lakes located in the Kenyan and Ethiopian Rift Valleys, which are the most studied aquatic continental ecosystems close to Lake Dziani Dzaha, have been mainly evaluated through microscopic identification [5, 51,52,53]. The few studies based on next-generation sequencing (NGS) on the microbial communities of these lakes have focused on prokaryotic communities [12, 54]. The only NGS-based study of both prokaryotic and eukaryotic organisms in several Ethiopian Lakes was by Lanzén et al. . However, the number of phytoplanktonic taxa that can be retrieved from their database was considerably lower than that of other studies on the same lakes but based on microscopy [5, 55].
Considering the microscopy-based richness estimation of phytoplanktonic communities of Kenyan and Ethiopian lakes, which range between 72 (Koka Lake; ) and seven (Abijata Lake; ), the richness of phytoplanktonic community in Lake Dziani Dzaha was in the lower range and it is among the lowest recorded in the saline-alkaline lakes in this part of the world. Among the crater lakes located in the same geographical and climatic area, a single study in Lake Oloidien (Kenya) reported the characterization of phytoplanktonic communities using NGS . These authors observed up to 30 cyanobacteria and more than 15 eukaryotic taxa. However, Lake Oloidien differs greatly from Lake Dziani Dzaha in terms of salinity, which remains quite lower and more variable (< 4 ppt), the presence of zooplankton and the non-permanent flamingo populations, along with permanent human populations that rely on the lake water for different uses.
Among the 15 phytoplanktonic OTUs, the two significantly dominant ones were A. fusiformis and P. salinarum; both the species being commonly described as dominant phytoplanktonic taxa in equatorial soda lakes [54, 56,57,58]. Among the rare cyanobacteria OTUs, one corresponds to a new species (S. komarekii), which has been recently described in strains isolated from stromatolites, and it might be considered endemic to Lake Dziani Dzaha . The other rare cyanobacterial OTUs (Leptolyngbya spp. 1–4, Synechococcus sp., and Xenococcus sp.) have also been reported with relatively high frequency in the stromatolites of the lake . The fact that all these cyanobacterial species were detected with a very low frequency in the water column and a high frequency in stromatolites, implies that the preferential habitat of these species is probably the stromatolites, where Cyanobacteria have been shown to influence their shape and mineralogy .
With respect to the rare eukaryotic plastid-related OTUs, only Kryptoperidinium sp. was detected in stromatolites with a higher frequency than that in the water column . Regarding Bacillariophyta, the assignation level of Cymbellaceae spp. clusters (Family) does not allow comparison with the composition of diatoms from other aquatic ecosystems. However, both Cymbellaceae species and Gyrosigma sp., affiliated with Gyrosigma fasciola, have already been observed in the Mozambique Channel . Gyrosigma sp. has never been reported as a typical species of soda or thalassohaline lakes. Thus, it can be hypothesized that most of the rare eukaryotic species detected in Lake Dziani Dzaha are probably of marine origin. Finally, some common thalassohaline or soda lakes taxa (e.g., Chlorophyta such as Kirchneriella, Monoraphidium, Raphidocelis, Selenastrum, and Tetranephris  were not detected in Lake Dziani Dzaha.
Overall, these results support our initial hypothesis of very low taxonomic richness of phytoplanktonic community in the Lake Dziani Dzaha pelagic ecosystem. However, the two-dominant species (A. fusiformis and P. salinarum) were the same as those reported in most saline-alkaline lakes, whereas, the very rare OTUs observed in the water column appeared to originate either from stromatolites or surrounding marine environment. Different and complementary processes can explain the low richness. First, the geographical isolation of this insular crater lake from other alkaline-saline lakes avoids or significantly limits the movement of phytoplanktonic species, which might occur in continental lakes through dispersal agents (such as water, air, animal, and human) . Second, the small size of the lake coupled with drastic environmental conditions in the water column limits the suitable niches for phytoplankton to the thin upper layer of the water column. Third, a main difference from other alkaline or thalassohaline lakes is the absence of grazers (zooplankton, fishes, and birds such as flamingos ). The absence of top-down control of phytoplanktonic populations might allow dominant species to establish and persist [63, 64]. Fourth, the large amount of phytoplanktonic biomass suggests that there is no nutrient limitation and implies a strong light attenuation, resulting in a competition for light among phytoplanktonic species leading to the dominance of the best competitors .
Only One Prokaryotic and One Eukaryotic Taxa Dominated the Phytoplanktonic Community from the Surface to Bottom Layers of the Water Column
Considering that the above-mentioned processes can explain the observed very low diversity, both competitive exclusion  and killing the winner theories [24, 67] predict that only one species can dominate and exclude the others. However, in Lake Dziani Dzaha, we observed the co-dominance of two distinct species in the same environmental niche, which, to the best of our knowledge, has not been reported in saline-alkaline lakes.
A first explanation of this unexpected result might be that co-occurrence is possible because of the distinct capacities of the two taxa to cope with light-limitation (Tables 4 and S3). Light limitation mostly refers to the quantitative aspect, but light is not a single resource, such as nutrients (e.g., NH4+ and PO43−). Light is composed of different wavelengths, and photosynthetic organisms have developed adaptations to use different regions of the photosynthetically active radiation (PAR) through a diverse range of pigments. Recently, Burson et al.  proposed an explanation for the unexpected co-occurrence of different phytoplanktonic species, where the best competitor for light is expected to dominate the phytoplanktonic community. In the two dominant taxa of Lake Dziani Dzaha, differences in their pigment composition were obvious (Table 4). For instance, if they share the ubiquitous Chla pigment with absorption peaks at 440 and 680 nm (blue and red regions of the spectrum), P. salinarum cells can exhibit higher concentration of Chla than A. fusiformis, given the potential better efficiency of P. salinarum to capture energy associated with Chla absorption wavelengths. The cyanobacteria A. fusiformis contains the accessory pigment phycocyanin, which has an absorption peak at 630 nm (orange region), whereas, P. salinarum contains Chlb with an absorption peak at 475 (blue-green region) and 650 nm (red region). Furthermore, P. salinarum also contains several other pigments, including diatoxanthin (451 and 479 nm) and monadoxanthin (448 and 475 nm), which enables them to efficiently use the blue-green part of the light spectrum.
A second explanation of the non-competitive access to light resources by these two taxa in the thin surface layer of the water column might be the significant difference in their size. To prevent sinking, the large filaments of A. fusiformis benefit from intracellular aerotopes, which regulate their buoyancy . Due to their very small size, P. salinarum cells have a very low intrinsic sinking rate  that can be lowered by the viscosity of lake water where salinity is higher than that of seawater. Thus, these two taxa exhibited distinct but non-competitive approach to remain in the upper part of the euphotic layer, where they attained the maximum abundance.
Coupled with the low sinking property they confer to the phytoplanktonic cells , high surface/volume ratio (Table S3 and Fig. S3), as in picophytoplanktonic cells, compared with that of nano- or microphytoplanktonic cells, has also been proposed as a way to cope with light limitation . In the present study, these two co-dominant taxa were also observed and enumerated in all the samples by both microscopy and flow cytometry. The abundance of these two-species showed that they mostly co-occur and exhibit a significant decrease with depth, especially when the water column is stratified. However, if we hypothesize that most, if not all, the cells of both taxa were produced in the top layer of the lake and then sank, it is remarkable that during non-stratified periods, approximately 25% of Picocystis cells were able to survive in the extreme hostile physico-chemical environments that prevail in the bottom layers of the water column. Contrarily, and irrespective of the water column structure, only a small percent of abundance of Arthrospira cells in the top layer was observed in these bottom layers. To the best of our knowledge, such differential behavior has not been reported by the studies on soda lakes (Table S3).
Factors Regulating the Dynamics of the Two Dominant Phytoplanktonic Taxa
As mentioned previously, it is probable that high PAR in the upper layer allowed active growth of the two photosynthetic taxa, but they also seem to maintain minimum growth when the PAR decreased. The ability to grow in low-light environments has been demonstrated for P. salinarum, with a positive growth at very low irradiance (0.6 μM photons m−2 s−1), showing its high potential for photoacclimatation and shade-adapted photosynthesis . The high Chla pigment concentration, as observed in the isolated strains of the present study, might also help this species to optimize its ability to cope with very low light intensity . Similarly, specific adaptive mechanisms to very low irradiance have been developed by Arthrospira, which can increase the cellular pigment concentrations .
From the upper layer to the bottom of the water column, the results of statistical modeling showed that the abundance of the two over-dominant taxa were significantly correlated with all the tested environmental factors. The abundance of P. salinarum and A. fusiformis positively correlated with the temperature. Temperatures of near or over 30 °C has been shown to potentially favor the growth of the two taxa [73,74,75,76]. The negative relationships between the abundance of the two taxa versus salinity, H2S, and NH4+ indicated that below the upper layer, they were both subjected to adverse conditions.
The negative relationship with H2S can result from the effect of anoxic conditions and/or the direct toxic effect of H2S. These two taxa might have different ways to cope with high levels of H2S, but the underlying mechanisms remain to be elucidated.
Growth and photosynthetic activity of P. salinarum strains were observed under anoxic conditions and after treatment with 100 μM Na2S with comparable rates than those measured under oxic conditions . The results suggested that the ability to maintain oxygenic photosynthesis even under low light condition might allow the proximal environment of P. salinarum cells to be suboxic, thus, reducing the redox stress. However, below the surface layer of the lake, no light was available for photosynthesis and during the stratified season the H2S concentration was significantly above 1000 μM. The significant abundance of P. salinarum in the bottom layers of the lake suggests that they are highly efficient, but unknown processes must operate to maintain the cell integrity.
Different types of adaptations to H2S have been defined among cyanobacteria based on the differential sensitivity of photosystems II and I to sulfide, and the capacity to carry out anoxygenic photosynthesis [76, 77]. However, the described processes can only function if a minimum intensity of light is available . In dark and in anoxic conditions, cyanobacteria have the capacity to perform fermentative metabolism coupled with sulfur reduction . Dark and anaerobic conditions have been shown to enhance the survival of Oscillatoria terebreformis compared with that under aerobic conditions .
The negative relationships between the abundance of the two taxa and NH4+ content probably originated from the significant increase in its concentration in the bottom waters. The observed values were among the highest recorded in aquatic ecosystems , and therefore, might be toxic or sub-toxic for both the taxa. Additionally, under high temperature and pH conditions observed in the lake, the ratio between toxic free ammonia (NH3) versus ammonium (NH4+) might increase, further affecting cell growth and viability .
Salinity has also been reported by the model as a significant explanatory variable with a potential negative effect on the abundance of Picocystis and Arthrospira. The regression coefficients were relatively high for Picocystis, suggesting the higher sensitivity of Picocystis to salinity variations in the water column. However, the field observations of different studies suggest that P. salinarum is more tolerant to high salinity values (10–300 psu) than A. fusiformis (20–70 psu) . In a laboratory experiment, Kebede  studied the growth of A. fusiformis strains subjected to a wide range of salinity and different salt compositions. They found an inverse relationship between salinity and growth, although the growth rate was still positive at a salinity of 90 psu (Table S3).
The apparent high sensitivity of P. salinarum to salinity in Lake Dziani Dzaha might be attributed to the conjunction of several adverse conditions that probably did not co-exist in the other ecosystems, where this species has been observed, and that have not been experimentally tested simultaneously.
Considering the above results, we can hypothesize that the co-existence of these two taxa in the same environmental niche is based on different adaptive features they might have to cope with light-limitation and adverse environmental conditions. To the best of our knowledge, this is the first in situ example of the role that niche differentiation in the light spectrum can play to complete the “nutrient-load hypothesis” . However, one can ask why the coexistence of these two taxa was not reported in other saline-alkaline lakes, with the exception of one crater lake investigated by Krienitz et al. . These authors provided the most probable explanation for the few reports of Picocystis as a co-occurrent species of cyanobacteria, writing that “This tiny protist is difficult to recognize and distinguish in field samples and is probably often neglected or wrongly identified.” During this study, we had the opportunity to use flow cytometry, which is currently the most efficient tool to enumerate picophytoplanktonic cells  and we also benefited from P. salinarum isolates to calibrate FCM analyses.
Regarding the ecological consequences of this co-existence, the two species might sustain two different trophic networks owing to their different morphological traits. The small size of P. salinarum might support a microbial loop  in the surface layer of the lake. Not only bacteria but also protozoans that have been detected in the lake  might benefit from the presence of these small phytoplanktonic cells either as a source of organic matter for aerobic heterotrophic bacteria or as a prey for nanoflagellates or protozoa. In contrast, due to their large size A. fusiformis filaments cannot be ingested by protozoans. By sedimentation, these filamentous organisms might act as a biological CO2 pump  and as the main source of (i) organic matter in the detrital bacterial loop of the anoxic bottom layer of the lake and (ii) long-term carbon stock stored in the sediment of the lake.
The presence of intact A. fusiformis and P. salinarum cells in the deepest layers of the lake, although representing only a small fraction of the abundance observed in the upper layer, implies that a part of the population of these two phylogenetically and morphologically distinct taxa was able to persist in extremely adverse conditions (anoxia, high H2S and NH4+ concentration, and no light). It is probable that the physiological status of the upper- and lower-layer cells is different. In the future, the survival behavior and physiological status of cells from isolated strains of these two taxa under controlled conditions should be evaluated. Furthermore, the transcriptome and metabolome of the two taxa along the depth profile should also be analyzed in order to understand the changes in certain metabolic pathways as a response to such extreme environmental conditions.
In conclusion, due to its geographical isolation, low anthropogenic pressure, thalassohaline signature associated with extreme physico-chemical conditions with a thin euphotic layer, and absence of grazers, Lake Dziani Dzaha is an excellent model to study the composition and functioning of a “simple” microbial aquatic ecosystem. By analyzing the phytoplanktonic community of this lake through high-depth sequencing approach, we verified the hypothesis that this community exhibits a very low richness and diversity like in other isolated and extreme aquatic ecosystems. Two OTUs (A. fusiformis and P. salinarum) appeared as the sole true pelagic phytoplanktonic OTUs in this lake. Furthermore, they not only appeared to form the minimal community that can be encountered in a present or “primitive” aquatic ecosystem, but also co-existed as a stable co-dominant consortium, at least according to the data collected during our four campaigns. Our findings are different from what is proposed by the classical community ecology hypotheses, according to which, in Lake Dziani Dzaha, only one species should have dominated the ecosystem. The two phylogenetically distinct OTUs (Cyanobacteria and Chlorophyta) showed distinct functional traits (pigment composition and size) that can explain their co-existence in the euphotic layer. Because of adverse environmental conditions that prevail below the surface layer, both the taxa exhibited a significant decrease in their abundances. Their behavior in the aphotic and anoxic water layers should be explored, and it constitutes one of the interesting challenges for future studies.