Cyanobacteria are a highly diverse group of gram-negative prokaryotes that have colonized a wide range of environments, from desert crusts to fresh and marine waters, and from the tropics to the poles (Whitton and Potts, 2002). They have played a crucial role in modifying the Earth’s atmosphere through the process of oxygenic photosynthesis, which enabled the evolution of life in more complex forms (Fischer et al., 2016b). Cyanobacteria have been studied for decades, and their diversity has been described both morphologically (Rippka et al., 1979; Komárek et al., 2014) and genetically (Shih et al., 2013). Cyanobacteria are often considered a nuisance in aquatic ecosystems as they can form large blooms and decrease water quality, ecosystem functions and ecosystem services (Rigosi et al., 2014).

With the development of molecular techniques allowing the investigation of non-cultivable organisms, an unexpected diversity of cyanobacteria was unveiled in many ecosystems (Sogin et al., 2006; Di Rienzi et al., 2013; Soo et al., 2014; Meola et al., 2015). Genome sequencing recently revealed important information about a clade of non-photosynthetic prokaryotes closely related to the Cyanobacteria. The name Melainabacteria was proposed (Di Rienzi et al., 2013), because several representatives of this group have been found in aphotic environments. They were first thought to constitute a sister-phylum of the Cyanobacteria (Di Rienzi et al., 2013), but additional genomic information confirmed the position of Melainabacteria as a sister-clade of photosynthetic cyanobacteria and as part of the same phylum (Soo et al., 2014). Based on genomic evidence, a re-classification has been proposed for the phylum Cyanobacteria, with the class-level lineages Oxyphotobacteria (all cyanobacteria capable of photosynthesis) and Melainabacteria, as well as a third class called ML635J-21 (Soo et al., 2014), for which the name Sericytochromatia was recently proposed (Soo et al., 2017). Sericytochromatia is the most basal lineage and forms a paraphyletic group that is the ancestor of both Melainabacteria and photosynthetic cyanobacteria (Fischer et al., 2016a; Soo et al., 2017). After diverging from Melainabacteria, the Oxyphotobacteria developed oxygenic photosynthesis around 2.4–2.35 billion years ago based on molecular clock estimates (see Shih et al., 2016; Soo et al., 2017) and geological data (Fischer et al., 2016a).

For ease of discussion, we hereafter refer to class Oxyphotobacteria as photosynthetic cyanobacteria, and we use the nomenclature proposed by (Soo et al., 2014, 2017) for the class Melainabacteria, wherein the following orders are included: Gastranaerophilales (YS2), Obscuribacterales (mle1-12), Caenarcaniphilales (ACD20), and Vampirovibrionales (SM1D11). Because there is only a hand-full of Melainabacteria and Sericytochromatia sequenced genomes (Soo et al., 2017), the metabolism, functions, and ecological role of these organisms are not fully known. Representatives of Melainabacteria and Sericytochromatia have been found both in photic and aphotic environments, such as subsurface ground water (Di Rienzi et al., 2013), lake water and algal biofilms (Soo et al., 2017; Salmaso et al., 2018), marine and lacustrine sediment (Ley et al., 2005), animal and human feces (Soo et al., 2014) and guts (Ley et al., 2005). A list of non-photosynthetic cyanobacteria (NCY) representatives with their associated environments is presented in Table 1.

All sequenced genomes of Melainabacteria confirm that they completely lack the photosynthesis apparatus, supporting the hypothesis that the acquisition of the photosystem in photosynthetic cyanobacteria happened after divergence from the non-photosynthetic Melainabacteria (Soo et al., 2014, 2017). All representatives of Melainabacteria are thought to be able to perform fermentation and some are thought to be capable of anaerobic and aerobic respiration (Di Rienzi et al., 2013; Soo et al., 2014, 2017). Some Melainabacteria in the Caenarcaniphilales, Vampirovibrionales, and Obscuribacterales orders are also capable of aerobic respiration under both high and low oxygen conditions (Soo et al., 2017). This ability has been acquired after the evolution of oxygenic photosynthesis in the sister-clade photosynthetic cyanobacteria (Shih et al., 2016; Soo et al., 2017). The Gastranaerophilales are found in human and other animal guts and although their exact role is unknown, they are thought to have a beneficial effect for their hosts by aiding digestion and as a source of vitamins B and K (Di Rienzi et al., 2013). Some Gastranaerophilales are flagellated, but other representatives appear to have only a subset of the necessary genes for encoding a functional flagellum (Soo et al., 2014). The Gastranaerophilales and Caenarcaniphilales seem to lack the genes for anaerobic and aerobic respiration, suggesting that they support their metabolism by fermentation only (Soo et al., 2014). Vampirovibrio chlorellavorus (previously assigned to genus Bdellovibrio) is a predatory bacterium that was originally described in 1972, and which was first suggested being a member of the Deltaproteobacteria (Gromov and Mamkaeva, 1972). This bacterium was recently isolated from a 36-year old co-culture sample of Chlorella vulgaris lyophilised cells (NCBI 11383) (Coder and Starr, 1978) and whole genome sequencing confirmed the position of Vampirovibrio in the class Melainabacteria (order Vampirovibrionales) part of phylum Cyanobacteria (Soo et al., 2015). V. chlorellavorus predates on Chlorella cells and, consistently with other classes of Melainabacteria, it lacks the genes for carbon fixation and photosynthesis (Soo et al., 2014).

In this study, we used a dataset consisting of 16S rDNA sequences obtained from high-throughput amplicon sequencing of samples collected in sediment cores from ten lakes, spanning ∼100 years (from ∼1900 to present), to study the diversity and distribution of NCY over the Anthropocene in the European peri-Alpine region. Lake sediments are well known to constitute rich archives of information about freshwater pelagic communities, in terms of both species resting stages and biomolecules (Gregory-Eaves and Beisner, 2011). Sedimentary DNA (sedDNA) in particular has been instrumental in reconstructing past community composition of various plankton forms, such as bacteria, phyto- and zooplankton, as well as microbial eukaryotes (Domaizon et al., 2017). Cyanobacteria were found to be well preserved in deep hard-water lakes (Domaizon et al., 2013; Monchamp et al., 2016). We have demonstrated previously that the reconstruction of cyanobacterial community structure and phylogenetic diversity using sedDNA, amplification, and sequencing relates almost 1:1 to pelagic microscopic counts from lakes over the past 40 years (Monchamp et al., 2016). Here, we used 16S rDNA sequences from sedimentary archives to investigate changes in NCY prevalence and community composition over time across our panel of ten lakes. Since we were only interested in the diversity of environmentally associated NCY, we present the composition of all groups of NCY, but exclude the gut-associated Gastranaerophilales order from the diversity analyses. Additionally, we tested for changes in community similarity across lakes and geographic distances to infer patterns of NCY dispersal at the regional scale. Results are discussed in comparison with previously observed community changes in photosynthetic cyanobacterial assemblages in the same lake samples (Monchamp et al., 2018a).

Little is known about the recently discovered clades of NCY in nature. The class Sericytochromatia is the least understood since only three genomes have been sequenced (Soo et al., 2017). The ecological function and the niche occupied by NCY are currently unknown. As reviewed in the introduction, it appears that NCY might be present in both photic and aphotic habitats, with no clear associations between clades and particular environments. Both Melainabacteria and Sericytochromatia have been found in surface waters, including lakes, and algal biofilms (Quast et al., 2013; Gies et al., 2014; Soo et al., 2017; Salmaso et al., 2018). It appears plausible that, like photosynthetic cyanobacteria, Melainabacteria and Sericytochromatia are aquatic life forms. In this study, we found members of NCY at every depth of the sediment cores and in all the peri-Alpine lakes investigated. It could be hypothesized that these organisms are also able to thrive in sediment layers, given their ability to grow under anoxic conditions (Soo et al., 2017). We, however, consider the possibility that this organisms’ habitat is actually the sediment quite unlikely, given the documented presence of NCY in aquatic environments (Table 1). For example, recent evidence suggests that NCY tend to be more prevalent during summer and autumn in the deep, peri-Alpine, and meso-oligotrophic lake Garda (Salmaso et al., 2018). More explorative studies and genome sequencing are needed to shed light on the actual ecology of these bacteria. Here, we consider sediment layers as archives for bacteria living in the lake water and we discuss our findings in terms of changes in NCY assemblages over time.

The topology of our reconstructed phylogenetic tree (Figure 1) reflected the relationships among taxa that were expected from previous work (Di Rienzi et al., 2013; Soo et al., 2014, 2017; Fischer et al., 2016a): we found a clear separation between the Sericytochromatia, Melainabacteria, and photosynthetic cyanobacteria. The number of NCY OTUs recovered from the sedimentary archive of peri-Alpine lakes was much lower in comparison to the richness of photosynthetic cyanobacteria determined previously in the same lakes (Monchamp et al., 2018a). Most of the OTUs were assigned to the deepest-branching paraphyletic group, the Sericytochromatia, and to the Melainabacteria order Gastranaerophilales. As mentioned earlier, the latter OTUs were not retained for further analysis because of their association mostly with guts and feces samples (Di Rienzi et al., 2013; Quast et al., 2013). Gastranaerophilales diversity and distribution in our samples also appears to be purely random, suggesting that their presence in lakes might be the result of release from local point sources, for example via run-off from land, wastewater, or excretions by wild animals and livestock in the lake watershed.

Our results describing the long-term composition and phylogenetic diversity of NCY assemblages in peri-Alpine lakes represent the earliest attempt to explore the distribution of these microorganisms across large spatial and temporal scales, and to investigate their change over broad environmental gradients. Our studied lakes have in fact been characterized by a well-documented history of climate warming and eutrophication, which have led to significant changes in the composition and assembly of photosynthetic cyanobacterial communities (Monchamp et al., 2018a,b). Unlike photosynthetic cyanobacteria, which have increased their diversity at the local and regional scale over the past century (Monchamp et al., 2018a), the richness of environmentally associated NCY did not significantly increase over time, with the exception of Lake Annecy. We have no information to interpret the interesting pattern detected in Lake Annecy. We have no evidence for methodological biases specific for this lake, and neither for directional environmental drivers that are unique to Lake Annecy, which was the most pristine lake in our dataset and it has been impacted by only weak anthropogenic changes over the past century (Monchamp et al., 2018a). No change in community structure, however, suggests that NCY live in a relatively stable lake environment, either in terms of species composition (if they depend on other organisms for growth) or in terms of water physics and chemistry. This is a puzzle, given the evidence for seasonality in NCY dynamics in Lake Garda (Salmaso et al., 2018), and the clear changes in trends and seasonality of lake environments over the past century (Adrian et al., 2009; Smith and Schindler, 2009; Rigosi et al., 2014; Salmaso et al., 2015; Yankova et al., 2017; Monchamp et al., 2018a).

Our results suggest that NCY communities are not shaped by the same environmental drivers as photosynthetic cyanobacteria, and that their composition is most likely lake-dependent (Table 2). We have previously observed that, in the same lakes, the richness and composition of photosynthetic cyanobacterial assemblages were significantly explained by the interaction between warming and eutrophication, with climate change being the strongest driver (Monchamp et al., 2018a,b). PERMANOVA confirmed that photosynthetic cyanobacterial phylogenetic diversity is strongly influenced by temporally changing drivers (Monchamp et al., 2018a,b), and suggest that both local (lake) and regional (north–south) environments play a significant role in shaping composition of these assemblages (Table 2). NCY communities did not show significant temporal dynamics at the decadal scale, and therefore no signs of correlation with temporally changing environmental conditions (Table 2). Similarly, we found no significant effect of regional drivers, like climatic difference between north and south of the Alps (Table 2). We found, however, a significant lake effect on the phylogenetic distance estimated between each pair of NCY communities. Since we did not detect dispersal limitation between lakes, neither for NCY (Figure 6) nor photosynthetic cyanobacteria (Supplementary Figure S2; Monchamp et al., 2018b), our results suggest that local features of lakes are important determinants of community structure in Sericytochromatia and Melainabacteria.

We can only speculate about what the above-mentioned lake features could be. If we assume that NCY thrive in the water-column, as suggested by previous work (Salmaso et al., 2018), it is possible that lake depth, volume, retention time, mixing regime, trophic state and/or catchment characteristics are important factors influencing the composition and diversity of these microorganisms’ communities. Between-lake phylogenetic similarity (based on Unifrac distances) in NCY communities was higher than the average similarity estimated between lakes for photosynthetic cyanobacterial communities (Figure 5; Monchamp et al., 2018a). This could indicate that within-lake factors have, however, a weaker effect on the community composition of NCY than they have on photosynthetic cyanobacterial community change. Understanding of environmental factors driving the diversity and distribution of NCY strongly requires follow up surveys, such as the monitoring of several lakes of different characteristics over depth profiles, and during seasonal succession (Salmaso et al., 2018). It has been suggested that the scale of sampling might be relevant to determine the relative influence of local factors versus environmental gradients (Martiny et al., 2006). Extending our survey of NCY diversity to a larger region might also reveal patterns associated with changes in climate that could not emerge from our peri-Alpine study, or of other environmental gradients that may have played a significant role in the establishment of NCY clades in different lakes and regions. Another important aspect that could not be addressed in our study is the abundance of NCY in lakes, to evaluate their importance for microbial ecosystem functioning.

Eutrophication and climate warming have been strong environmental drivers during the Anthropocene, impacting lake ecosystem processes (Adrian et al., 2009; Smith and Schindler, 2009) and cyanobacterial community assembly (O’Neil et al., 2012; Rigosi et al., 2014; Huisman et al., 2018; Monchamp et al., 2018a,b). Our study suggests that NCY communities have been unaffected by large-scale anthropogenic environmental change. The lack of decay in phylogenetic similarity over time and over geographic distance across lake communities suggests temporally stable communities with no limitation to dispersal at the regional (peri-Alpine) scale. This is consistent with most reports about microbial dispersal, which rarely showed evidence for geographic distance-decay patterns at the local (0–100 km) and regional (101–5,000 km) scales (Hanson et al., 2012). As previously reported (Monchamp et al., 2018b), the dissimilarity of communities over geographic distance was also non-significant in photosynthetic cyanobacterial assemblages (Supplementary Figure S2). Being temporally stable and lake specific, NCY assemblages could perhaps be used in future studies as an internal lake reference, to which compare patterns of other ecological communities under suspected environmental forcing.

Our study represents an initial exploratory survey of the composition and diversity of ecologically unknown clades of NCY. Our results uncover the diversity of these currently cryptic organisms, which might play a role in lake biogeochemistry. High-throughput sequencing of environmental DNA has the potential to illuminate the ecological niche of these understudied and uncultivable groups of NCY, at the local as well as over large geographic scales. In combination with genome sequencing, diversity surveys and possibly outdoors experiments will help gain understanding on the ecology, evolution and function of these microbes in their natural environment.

M-EM, FP, and PS designed the study. M-EM performed the sampling, laboratory work, and data analysis. M-EM and FP wrote the manuscript, which was reviewed and commented by PS.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.