**3. Methylation: a primordial resemblance**

High-resolution mass spectrometry revealed the occurrence of small satellite peaks for MMXF3 and MGnXF3 /GnMXF3 in mass spectra of *P. patens* N-glycans, primarily when N-glycans were extracted from whole tissue (**Figure 2**) [10]. The mass increment of about 14 Da could also arise from the oxidation of a sugar moiety to glucuronic acid. The exact mass increment, however, and subsequent chemical analysis (linkage analysis via gas chromatography-mass spectrometry) clearly identified it as O-methylation of a mannose residue—more exactly, as 2,6-di-O-methylation. Collision-induced fragmentation in negative mode furthermore established the α1,6 linked mannose as being methylated (**Figure 2**).

Methylation in various ways was encountered in a previous study of *Chlorella*clade microalgae [11–14]. Although methyl-mannoses were also found in these algae, none of the well over a dozen glycol-types contained complex-type/paucimannosidic structures and none of them contained 2,6-O-methylated mannose. As a recent unpublished survey of several *Scenedesmaceae* strains revealed likewise varied glycosylation (Mocsai R and Altmann F, unpublished results). We, thus, cannot exclude that other algae strains share with mosses this type of methylation and may thus be considered ancestors of mosses—at least glycosylation-wise.
