The Ocean Sampling Day (OSD) project provided metabarcoding data for the V4 hyper-variable regions of the 18S rRNA gene from 157 samples collected at 143 mostly coastal stations. In this paper we focus on the class Mamiellophyceae, which was found at nearly all OSD stations and represented 55 % of the green microalgae (Chlorophyta) reads in the 2014 OSD dataset. We performed phylogenetic analyses of unique OSD metabarcodes (ASV, amplicon single variants) and reference GenBank sequences from cultures and from the environment, focusing on the four most represented genera: Ostreococcus (45 % of the Mamiellophyceae reads), Micromonas (34 %), Bathycoccus (10 %) and Mantoniella (8.7 %).These analyses uncovered novel diversity within each genus except Bathycoccus. In Ostreococcus, a new clade (E) with 2 very clear base pair differences compared to the oceanic clade B in the V4 region was the second most represented clade after the coastal Ostreococcus "lucimarinus". Within Micromonas, ten clades were found exceeding the 4 species and 2 candidate species already described. Finally, we found 2 new environmental clades of Mantoniella. Each Mamiellophyceae clade had a specific distribution in the OSD dataset suggesting that they are adapted to different ecological niches. Monomastigales 10 . Monomastigales are confined to freshwater environments while Dolichomasti-20 gales, although quite diversified in marine waters 12, 13 , are a minor component of Mamiel-21 lophyceae in coastal waters. In contrast, Mamiellales and Bathycoccales host some of the most 22 common Chlorophyta microalgae such as the ubiquitous Micromonas, the smallest known eu-23 karyote Ostreococcus or the coccoid Bathycoccus 10 . Within Mamiellales, Micromonas pusilla 14 24 was recently split into four species: Micromonas bravo (previously clade B.E.3), Micromonas com-25 moda (previous clade A.ABC.1-2), Micromonas polaris (previously clade B arctic), Micromonas 26 pusilla (previously clade C.D.5) and two clades mentioned as candidate species 1 (clade B. .4) 27 2/34 and candidate species 2 (clade B warm) 15 . Within the genus Mantoniella, only two species 28 have been described: the ubiquitous Mantoniella squamata 3, 16 , first described as Micromonas 29 squamata 17 , and Mantoniella antarctica 18 . Within Bathycoccales, four Ostreococcus clades have 30 been delineated 19 : Ostreococcus tauri 20 , Ostreococcus mediterraneus 21 , both of which have 31 been formerly described, Ostreococcus "lucimarinus" (clade A) and clade B, which both lack 32 formal taxonomic description. Analyses of pigment content and response to light levels allowed 33 to distinguish two broad ecotypes: strains adapted to high (O. tauri,O. mediterraneus, O. "luci-34 marinus" ) and low (Ostreococcus clade B) light 22 . The second genus within Bathycoccales hosts 35 a single species, Bathycoccus prasinos 23 . No clades can be delineated inside this species based 36 on 18S rRNA gene sequences from cultures and the environment. However, divergence in ITS 37 sequences suggest that Bathycoccus p...