Diversity and ecology of green microalgae in marine systems: an overview based on 18S rRNA gen

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The present state of Chlorophyta classification

The first description of tiny green cells growing in aquatic environments and the first ideas about the classification of microalgae occurred in the middle of the 19th century (Nägeli, 1849). This was followed by a large number of descriptions of green microalgae, leading scientists to reflect on the ecological significance of these organisms. Gaarder (1933) discovered the importance of green microalgae in the food web by looking at the source of oyster food in Norway. Twenty years later, the first marine picoeukaryotic phytoplankter to be described (Chromulina pusilla, later renamed Micromonas pusilla) was a tiny green alga (Butcher, 1952).
In the 1960s and early 1970s, Round (Round, 1963, 1971), reviewing available morphological information, divided the green algae into four divisions: Euglenophyta, Charophyta, Chlorophyta and Prasinophyta. While Round classified the Prasinophyta in a separate phylum, other authors (Bourrelly, 1966; Klein and Cronquist, 1967) included them in the order Volvocales within the Chlorophyta. The division Chlorophyta was reorganized by Mattox and Stewart (1975) mainly based on ultrastructural characteristics such as the type of mitosis (Sluiman et al., 1989), presence/absence of an interzonal spindle, the structure of the flagellar apparatus (O’Kelly and Floyd, 1983), and the presence of extracellular features such as scales and thecae. They proposed the division of Chlorophyta into four major groups: the Prasinophyceae, Charophyceae, Ulvophyceae and Chlorophyceae (Stewart and Mattox, 1978). This has been partly confirmed by molecular phylogenetic analyses over the years (Chapman et al., 1998), although it was recognized from the beginning (Christensen, 1962) that prasinophytes constitute a polyphyletic assemblage (i.e. phylogenetic branches without a common ancestor). Therefore the class name Prasinophyceae is no longer used and the generic term prasinophytes, that has no phylogenetic meaning, has replaced it (Leliaert et al., 2012). At present, the Chlorophyta is viewed as composed of two major groups: the prasinophytes and the “core” chlorophytes (Leliaert et al., 2012; Fučíková et al., 2014). The prasinophytes currently consist of nine major lineages of microalgae corresponding to different taxonomic levels (order, class, undescribed clades) that will probably all be raised to the class level in the future (Leliaert et al., 2012). These lineages share ancestral features such as flagella and organic scales. The number of prasinophyte lineages has been increasing following the availability of novel environmental sequences. Ten years ago, prasinophyte clade VII was introduced using sequences from cultured strains and environmental clone libraries (Guillou et al., 2004). Four years later, two additional clades, VIII and IX, were reported (Viprey et al., 2008) that are only known so far from environmental sequences. Prasinophytes may be divided into three informal groups (Marin and Melkonian, 2010): a group of “basal” lineages (Prasinococcales, Pyramimonadales, Mamiellophyceae), a group of “intermediate” lineages (Pseudoscourfieldiales, clade VII, Nephroselmidophyceae) and a group of “late” diverging lineages (Pedinophyceae and Chlorodendrophyceae). Recently, the “late” diverging lineages have been merged with the Ulvophyceae-Trebouxiophyceae-Chlorophyceae (UTC) clade into the “core” chlorophytes (Fučíková et al., 2014), the Chlorodendrophyceae based on common features, in particular a mode of cell division mediated by a phycoplast (Mattox and Stewart, 1984; Leliaert et al., 2012), and the Pedinophyceae based on strong phylogenetic support (Marin, 2012; Fučíková et al., 2014).

Major lineages within marine Chlorophyta

We extracted a set of 132 reference sequences from the PR2 database that were used to build a phylogenetic tree of marine Chlorophyta (Fig. 1). In this tree, each triangle (except for the Mamiellophyceae for which we have represented the different families) corresponds to a “lineage” which currently corresponds to either a class, an order or a “clade” sensu Guillou et al. (2004). In this section, we review what is known about the major Chlorophyta lineages in marine waters following the order used in Guillou et al. (2004).
Pyramimonadales (prasinophyte clade I, Guillou et al. 2004) are pyramidal, oval or heart-shaped cells (10 to 400 µm long on average) with generally 4, rarely, 8 or even 16 flagella (Chadefaud, 1950; Hori et al., 1985). In Pyramimonas, cells possess three layers of different organic scales on the cell body, two layers on the flagella (Pennick, 1982, 1984) and flagellar hairs (Moestrup, 1982). Twenty-two genera have been described, with Pyramimonas, Pterosperma and Halosphaera containing most species. For the genus Pyramimonas, almost 50 species (Suda et al., 2013; Harðardottir et al., 2014; Bhuiyan et al., 2015) and six sub-genera (Hori et al., 1995) have been described, but the low number of ribosomal RNA sequences from described species in public sequence databases is an obstacle to the resolution of the phylogeny of this genus ( Table S1, Suda et al. 2013). Novel species have recently been described from isolates from the North Pacific Ocean (Fig.3A, Suda et al. 2013, Bhuiyan et al. 2015) and polar regions (Moro et al., 2002; Harðardottir et al., 2014). In Disko Bay (Greenland), Pyramimonas has been found to be important in the sea ice and in the water column and plays an important role in the spring phytoplankton bloom (Harðardottir et al., 2014). Pyramimonadales have been recorded in coastal waters as well as in confined environments such as tide pools (Chisholm and Brand, 1981; Lee, 2008). Halosphaera occurs in two forms, one flagellated and one coccoid, the latter that can be up to 800 µm in size and that may sediment quickly. In the Mediterranean Sea, high abundances of Halosphaera have been recorded at depths between 1,000 and 2,000 meters (Wiebe et al., 1974). Mamiellophyceae (clade II, Guillou et al., 2004) are characterized by a wide morphological diversity. They are split into three orders: Mamiellales, which is composed of two families (Mamiellaceae and Bathycoccaceae), Dolichomastigales and Monomastigales (Fig.1, Marin and Melkonian, 2010). The Mamiellaceae contain three genera that are ecologically important. Micromonas are ellipsoid to pyriform naked cells (1 to 3 µm) with a single emergent flagellum (Butcher, 1952). Phylogenetic and ecological studies on the micro-diversity of Micromonas suggest that this genus may consists of at least three cryptic species (Šlapeta et al., 2006; Foulon et al., 2008). Micromonas is a ubiquitous genus with cultures originating from a wide range of environments extending from the poles to the tropics, but more prevalent in coastal waters. Mamiella and Mantoniella are reniform cells (up to 10 µm) covered by two types of body scales: large, more or less square, and small, less regular (Barlow and Cattolico, 1980; Moestrup, 1984). Mamiella have two long flagella and spined flagellar scales, while Mantoniella has one long and one very short flagella with flagellar scales lacking spines (Marin and Melkonian, 1994). Environmental sequences from the latter two genera have been found in the Arctic Ocean and the Mediterranean Sea using Chlorophyta specific primers or sorted samples (Viprey et al., 2008; Balzano et al., 2012).

Environmental distribution of Chlorophyta in marine ecosystems from 18S rRNA sequences

The number of publicly available sequences (Fig.2A, based on the PR2 database, Guillou et al. 2013) varies widely between the Chlorophyta groups from 3 for the Palmophyllales up to 560 for the sole genus Micromonas. Mamiellophyceae and in particular Micromonas, Bathycoccus and Ostreococcus are the most represented green algal taxa in public sequence databases, followed by Chlorophyceae and Trebouxiophyceae, two groups which were previously mostly seen as continental (Fig.2). The proportion between sequences from cultures and environmental samples is also highly variable (Fig.2B). Some groups are mostly represented by sequences from cultures (e.g. Nephroselmidophyceae and « core” chlorophytes) while others are predominantly or wholly uncultured (e.g. prasinophyte clade IX). The geographic distribution obtained from cultures and from environmental sequences is quite different (compare A and B in Fig.3 and S1). While Mamiellophyceae dominate environmental sequences, this is not true for culture sequences, which offer a better balance between the different Chlorophyta groups (Fig. S1). The contribution of different classes to environmental sequences differs between latitudinal bands and coastal vs. oceanic stations (Fig.4).
Polar waters, whether oceanic or coastal, are totally dominated by Mamiellophyceae (Fig.4), in particular the arctic Micromonas clade (Lovejoy et al., 2007; Balzano et al., 2012). The diversity of classes recovered is minimal (Supplementary Fig. S1), with representatives of the Pyramimonadales, Ulvophyceae and Prasinococcales in addition to the Mamiellophyceae. It is noteworthy that few sequences have been recovered from the Southern Ocean in comparison to the Arctic (Supplementary Fig. S1).
The dominance of Mamiellophyceae is less marked for temperate waters where other classes such as the Trebouxiophyceae can be important, especially away from the coast (Fig.4). Indeed, it is in temperate waters that Chlorophyta environmental sequence diversity is maximal, in particular in the North-West Atlantic and North-East Pacific Oceans with more than 10 Chlorophyta classes recovered (Supplementary Fig. S1). Chlorophyceae, Prasinococcales Pseudoscourfieldiales, and clade IX sequences have also been recovered from coastal temperate areas including the Mediterranean Sea (Fig.3B and 4). Nephroselmidophyceae have been repeatedly isolated from Japanese coastal waters (Fig.3). Chlorodendrophyceae, Trebouxiophyceae, Pyramimonadales and clade VII have been found in both coastal and oceanic temperate waters (North Pacific Ocean and Mediterranean Sea, Fig.3 and 4).
The decrease in the dominance of Mamiellophyceae is even more marked in tropical waters. While it shares dominance with prasinophyte clade VII in coastal waters, it becomes a minor component offshore where it is replaced by clade VII and the uncultured clade IX. Trebouxiophyceae, Prasinococcales, Pyramimonadales and Chlorophyceae have also been found at some locations in the subtropical Pacific and Atlantic Oceans (Fig.3B and Fig.4).
With respect to depth distribution, both Mamiellophyceae, Pyramimonadales, as well as prasinophyte clade VII and IX sequences have been found throughout the photic zone, even below 60 meters (Fig.5). Pseudoscourfieldiales, Trebouxiophyceae and Prasinococcales sequences seem to be restricted to surface waters, while Chlorodendrophyceae sequences appear to be preferentially found at the bottom of the photic zone, below 60 m (Fig.5). The deepest Mamiellophyceae sequences have been recovered from 500 m depth for Micromonas and down to 2500 m depth for Ostreococcus (Lie et al., 2014).

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Mamiellophyceae genera

Mamiellophyceae dominated at most OSD stations and were further investigated at the genus level. Nine genera of Mamiellophyceae were found in the OSD datasets, seven of which were found in both datasets, one only in V4, assigned to RCC391, and one only in V9, assigned to Monomastix. The latter is a freshwater genus and the OTUs assigned to it were badly assigned (BLAST analysis showed 100% identity with sequences of several land plants genera, see Supplementary data), while the RCC391 genus has eight references sequences for V4 against only one for V9. Micromonas and Ostreococcus were the two dominant genera, except at OSD80 in the Greenland Sea where Mantoniella was dominant and in the Adriatic Sea (OSD49, 76, 77 and 99) where Dolichomastigales and Mamiella were dominant (Fig.5A). Procrustean comparison showed that V4 and V9 provided similar Mamiellophyceae genus distribution (m²=0.075 and r=0.96). The relative contributions per station of the four major genera Micromonas, Mamiella, Ostreococcus and Bathycoccus (Fig. S10) was statistically similar in the two datasets (Table 3). Stations located in the Adriatic Sea (OSD49, 76, 77, 99) showed a different pattern in the heatmap (Fig. S9B) because V9 failed to discriminate the Dolichomastigales clades at the genus level. V9 recorded only Crustomastix contribution while V4 found 4 to 6 different clades of Crustomastigaceae and Dolichomastigaceae (Fig.5A). The relative contribution of the four Mamiellophyceae genera Micromonas, Mamiella, Ostreococcus and Bathycoccus was similar in V4 and V9 except at some stations (OSD22, 49, 132, 123) for Mamiella. Bray-Curtis distances always clustered together V4 and V9 (Fig.5B). Four groups of stations were observed depending on the Mamiellophyceae genus dominant at the station: Micromonas, Ostreococcus, Dolichomastigales or Mantoniella (Fig.5B).

Comparison of the photosynthetic communities assessed by the V4 vs. V9 regions

The V9 dataset provided 20% more OTUs than the V4. This difference between the number of OTUs for V4 and V9 is the same as the one unveiled in other environmental study such as the Naples times series results (Piredda et al., 2017). Piredda et al. (2017) also found 20% more OTUs built at 97% identity for V9 than for V4. This could be linked to the size difference between V4 and V9 as discussed above. Interestingly, these authors showed that the number of OTUs built at 95% identity was similar for V4 and V9, suggesting that at lower identity thresholds, the size difference has a lower impact.
The number of OTUs for the main photosynthetic phyla Ochrophyta, Chlorophyta, Haptophyta and Cryptophyta falls in the range found in European coastal waters using the V4 and 97% identity OTUs (1905, 314, 221 and 77 respectively, Massana et al., 2015) except for the Haptophyta for which three times less OTUs were found in the OSD V4 dataset. The number of OTUs of the main photosynthetic phyla in the OSD V9 dataset were considerably lower than the numbers of Tara Oceans V9 OTUs, 3900, 1420, 713 and 195 respectively (de Vargas et al., 2015). However, the depth of sequencing was much higher than in the OSD dataset (around one to two million reads per sample, i.e. 20 to 40 more than for OSD) which increases the occurrence of the rare OTUs. Mamiellophyceae dominated nutrient rich coastal waters, which is consistent with studies in European coastal waters (Massana et al., 2015) in particular in the English Channel (Not et al., 2004) and in the South East Pacific Ocean (Rii et al., 2016). The stations located in the Adriatic Sea (OSD49, 76, 77 and 99) showed a specific pattern with a high contribution of Pseudoscourfieldiales and Chlorodendrophyceae. Several studies using optical microscopy found in the Adriatic Sea a high contribution of phytoflagellates, most of which could not be identified (Revelante and Gilmartin, 1976; Cerino et al., 2012).

Table of contents :

Introduction
The planktonic compartment
Delimiting biogeographic units in the sea
Thesis questions
Investigating protist diversity in the Sea with molecular biology
Metabarcoding
Chapter 1 – Diversity and ecology of green microalgae in marine systems: an overview based on 18S rRNA gen
Present state of Chlorophyta classification
Major lineages within marine Chlorophyta
Environmental distribution of Chlorophyta in marine ecosystems from 18S rRNA sequences
Advantages and limitations of 18S rRNA as a marker gene for Chlorophyta
Conclusion and perspectives
Methodology
Lists
Chapter 2 – Comparison of coastal phytoplankton composition estimated from the V4 and V9 regions of 18S rRNA gene with a focus on photosynthetic groups and especially Chlorophyta
Introduction
Materials and Methods
Results
Discussion
Lists
Chapter 3 – Communities of green microalgae in marine coastal waters: the OSD datasets 
Introduction
Materials and Methods
Results
Discussion
Lists
Chapter 4 – Novel diversity within Micromonas and Ostreococcus (Mamiellophyceae) unveiled by metabarcoding analyses
Introduction
Methodology
Ostreococcus
Micromonas
Conclusion
Lists
Conclusions et Perspectives
List of publications

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