Phylogeny and salt-tolerance of freshwater Nostocales strains: Contribution to their systematics and evolution
Introduction
Cyanobacteria are considered to be among the most ancient of evolutionary lineages, and they are thought to be the primary oxygenic photosynthetic organisms that originated before 3.0 Ma, prior to the increase in atmospheric oxygen levels (Schirrmeister et al., 2015). They are generally considered to be key players in past and present global biogeochemical cycles (e.g. Schirrmeister et al., 2013). Cyanobacteria are one of the most diversified prokaryotic groups, with a cosmopolitan distribution ranging from freshwater and marine aquatic ecosystems, soil, and endolithic habitats to extreme environments such as deserts, glaciers, soda-lakes, and hot springs (Whitton, 2000). This broad ecological distribution coupled with a large phylogenetic diversity (Komarek et al., 2014) involved species with the ability to tolerate and/or adapt to different kinds of environmental changes during the earth’s history. Various changing environmental factors have been identified that have promoted their growth and dispersion, such as an increase in nutrients (e.g. land-use changes) and the rising temperature (e.g. global warming) of water bodies (Paerl and Paul, 2012). Such environmental changes promote global expansion of cyanobacterial blooms whereby some species appear to produce toxins that can have both ecological and sanitary consequences (Ibelings et al., 2015).
The Nostocales include heterocytous cyanobacteria and it is the most diversified order, comprising 108 of the 300 genera listed by Komarek et al. (2014). From a phylogenetic point of view, Nostocales represent a large and monophyletic lineage on the basis of their 16S rDNA sequences (Willame et al., 2006, Komarek et al., 2014). Within Nostocales, Komarek et al. (2014) defined 12 families, including two large families, the Aphanizomenonaceae Elenkin and the Nostocaceae C.A. Agardh ex Kirchner, both of which comprise genera that have undergone extensive study in light of their abundance in freshwater reservoirs and their capacities to form blooms. Systematics of among these heterocytous genera are constantly being revised, as for planktonic Anabaena which has been newly characterized as Dolichospermum (type species D. flos-aquae, formerly Anabaena flos-aquae) / Aphanizomenon (Gugger et al., 2002, Komárek and Komárková, 2006, Komárek and Zapomělová, 2007), Cylindrospermopsis / Raphidiopsis (Gugger and Hoffmann, 2004, Li et al., 2008), Anabaena (a benthic species, type species A. oscillarioides / Nostoc/ Trichormus (Rajaniemi et al., 2005), and Anabaena bergii reclassified as Chrysosporum bergii (Zapomělová et al., 2009) (for an exhaustive review, see Komarek et al., 2014). All of these revisions are based on polyphasic approaches that include assessment of morphological, molecular, biochemical, or ecological characteristics. Additionally, complete genome and phylogenetic (phylogenomics) analyses are now also being used to better define certain genera. For example, Li et al. (2016) have provided the clearest picture of the relationships within the Dolichospermum/Anabaena/Aphanizomenon group of cyanobacteria. One of the goals of genome sequencing is to identify the genes related to the metabolic and physiological capabilities that determine the ecological niche of each isolated strain. This has allowed for detection of different evolutionary events, such as lateral transfers or the loss of genes. Such events have been shown to occur frequently among prokaryotic lineages (Zhaxybayeva et al., 2006) and they could stymie the reconstruction of their common ascendant relationships. The range of environmental conditions in which a microbial strain is able to grow has been used for a long time in bacterial taxonomy (Castenholtz and Waterbury, 1989) and it can be included to study the evolution of traits of cyanobacteria (e.g. nitrogen fixation, thermophilicity, motility, and use of sulphide as an electron donor).
In the current anthropogenic period, global climate change is likely to have induced salinity fluctuations in aquatic ecosystems due to an increase in drought frequency and duration, or in precipitation (Paerl and Paul, 2012). For microbial communities, the level of salinity is one of the most common changing environmental factors in aquatic ecosystems (Moisander et al., 2002, Morbach and Krämer, 2002) and it is the major environmental determinant of their taxonomic composition on a global scale (Lozupone and Knight, 2007, Ryan et al., 2008). Unlike many freshwater eukaryotic phytoplankton that are unable to tolerate large changes in salinity (Moisander et al., 2002), a number of freshwater cyanobacterial species are able to tolerate brackish conditions (Laamanen et al., 2001, Orr et al., 2004). For instance, some cyanobacteria belonging to Dolichospermum are major contributors to blooms observed in brackish areas of the Baltic Sea, and some species can withstand salinities of up to 15 g L−1 NaCl (Moisander et al., 2002). In terms of salt-tolerance, it was assumed that early cyanobacterial lineages were equally successful in the various aquatic habitats of early Earth (Honda et al., 1999). Recently, Sánchez-Baracaldo et al. (2005), Sánchez-Baracaldo (2015) have hypothesized that the earliest cyanobacteria probably lived in freshwater/terrestrial ecosystems, and that halophytic/marine environments were secondarily colonized by cyanobacteria several times in an independent manner.
The aim of this study was to contribute to the systematics of Nostocales with a polyphasic approaches and to provide insights regarding the debate over the evolution of salt-tolerant characteristics (Sánchez-Baracaldo et al., 2005; Sánchez-Baracaldo, 2015). New cyanobacterial strains were thus sampled from African freshwater ecosystems (e.g. lakes, rivers or channel and ponds with a salinity <3 g L−1) where salinity fluctuation was expected (i.e. evaporation, precipitation). The strains were isolated and maintained in freshwater-culture medium (salinity <1 g L−1 NaCl). We investigated if the freshwater-selected strains were able to survive and growth in culture conditions, under a gradient of salt (NaCl), allowing to define their salt tolerance (salinity where the growth is null). All strains belong to separate lineages within the Aphanizomenonaceae Elenkin and the Nostocaceae C.A. Agardh (ex Kirchner): Dolichospermum (formerly Anabaena), Anabaenopsis, Chrysosporum, Cylindrospermopsis, and Anabaena. Molecular phylogenies, morphological analyses and salt-tolerance data were combined so as to contribute to better understand the evolution of Nostocales.
Section snippets
Cyanobacterial isolation and culture
The strains used in this study, their geographical origins are listed in Table 1 and the salinity of each habitats in Table S7. The strains isolated from sub-Saharan Africa (Senegal and Burkina Faso) were previously described in Thomazeau et al. (2010) and Berger et al. (2005). Strains from Mayotte Island were obtained from freshwater samples collected during November 2006 in the Doujani water body (12° 47′ 18” N–45° 11′ 49” W). The isolation procedure was as described in Thomazeau et al. (2010)
Taxonomy of the new isolates
Samples of the new cyanobacterial isolates (Table 1) were obtained from diverse freshwater habitats (e.g. ponds, drinking water reservoirs, and natural lakes) of the African continent (Senegal and Burkina Faso) and from Mayotte Island (in the Indian Ocean). The 36 new Nostocaceae isolates were planktonic, filamentous, isopolar, and unbranched, with diversified cells (e.g. heterocytes, akinetes) and usually with aerotopes in the cells (Fig. 1). They belonged to Nostocales, either to the
Discussion
The systematics of cyanobacterial genera has changed greatly since the use of molecular characteristics, and there is still ample scope for improvements in systematics through use of a polyphasic approach (e.g. see Komarek et al., 2014; Komárek, 2013, Komárek, 2016). Conventional molecular phylogenies, based on 16S rDNA, several markers, or now even entire genomes, complement taxonomic evaluations based on morpho-anatomical or ultrastructure characteristics. Physiological traits have been
Conclusion
Comparison between molecular and physiological data showed that salt-tolerance conferred additional support for molecular relationships, and that it could be a useful marker for delineation of heterocytous cyanobacterial lineages. In addition, congruency between the 16S rDNA sequence phylogeny and salt-tolerance allowed its evolution to be inferred. Perhaps the most interesting finding was that most cyanobacterial strains isolated from freshwater environments were salt tolerant, and this
Author contributions
ST, CY, MT, FR, and CB designed the experiments. CD, ST, YD, AC, and FR performed the experimental procedures. CD, ST, MT, FR, and CB performed the data analysis. CY helped analyze the data. CD, ST, MB, MT, FR, and CB wrote the manuscript. CB co-led the project with FR and MT.
Acknowledgements
This work was funded by the National Museum of Natural History and the Institut de Recherche pour le Développement (IRD). The study was supported by the “Consortium National de Recherche en Génomique” and the “service de systématique moléculaire” of the Muséum National d’Histoire Naturelle (USM 2700). It is part of agreement no. 2005 / 67 between the Genoscope and the Muséum National d’Histoire Naturelle for the project “Macrophylogeny of life”. The authors would like B. Demenou, H. Shelaifa
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Authors who contributed equally to the data acquisition.