Molecular Phylogeny and Classification of The
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ARTICLE IN PRESS Protist, Vol. ], ]]]–]]], ]] ]]]] http://www.elsevier.de/protis Published online date 10 December 2009 ORIGINAL PAPER Molecular Phylogeny and Classification of the Mamiellophyceae class. nov. (Chlorophyta) based on Sequence Comparisons of the Nuclear- and Plastid-encoded rRNA Operons Birger Marin1, and Michael Melkonian Biowissenschaftliches Zentrum, Botanisches Institut, Lehrstuhl I, Universitat¨ zu Koln,¨ Otto-Fischer-Str. 6, 50674 Koln,¨ Germany Submitted August 10, 2009; Accepted October 19, 2009 Monitoring Editor: David Moreira Molecular phylogenetic analyses of the Mamiellophyceae classis nova, a ubiquitous group of largely picoplanktonic green algae comprising scaly and non-scaly prasinophyte unicells, were performed using single and concatenated gene sequence comparisons of the nuclear- and plastid-encoded rRNA operons. The study resolved all major clades within the class, identified molecular signature sequences for most clades through an exhaustive search for non-homoplasious synapomorphies [Marin et al. (2003): Protist 154: 99-145] and incorporated these signatures into the diagnoses of two novel orders, Monomastigales ord nov., Dolichomastigales ord. nov., and four novel families, Monomastigaceae fam. nov., Dolichomastigaceae fam. nov., Crustomastigaceae fam. nov., and Bathycoccaceae fam. nov., within a revised classification of the class. A database search for the presence of environmental rDNA sequences in the Monomastigales and Dolichomastigales identified an unexpectedly large genetic diversity of Monomastigales confined to freshwater, a novel clade (Dolicho_B) in the Dolichomastigaceae from deep sea sediments and a novel freshwater clade in the Crustomastigaceae. The Mamiellophyceae represent one of the ecologically most successful groups of eukaryotic, photosynthetic picoplankters in marine and likely also freshwater environments. & 2009 Elsevier GmbH. All rights reserved. Key words: prasinophytes; SSU rDNA; LSU rDNA; ITS2; systematics; freshwater; Monomastigales; Dolichomastigales; Mamiellales; Monomastigaceae; Dolichomastigaceae; Crustomastigaceae; Bathycoccaceae. Introduction The prasinophytes or scaly green flagellates have pioneer Irene Manton (Manton and Parke 1960; attracted the attention of protistologists and review: Sym and Pienaar 1993). Other small, phycologists ever since their major attribute, the essentially dorsiventral uni- or biflagellate green ‘‘garniture’’ of organic scales on cell and flagellar algae from both fresh- and seawater habitats surfaces, was discovered by electron microscopy lacking scales altogether or displaying cell surface structures not resembling prasinophyte scales (e.g. 1Corresponding author; fax: +49 221 470 5181 Monomastix, Pedinomonas tuberculata) were initi- e-mail [email protected] (B. Marin). ally combined with asymmetrical (dorsiventral) & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2009.10.002 Please cite this article as: Marin B; Melkonian M. Molecular Phylogeny and Classification of the Mamiellophyceae class. nov. (Chlorophyta) based on Sequence Comparisons of the Nuclear- and Plastid-encoded rRNA Operons, Protist (2009), doi:10.1016/j.protis.2009.10.002 ARTICLE IN PRESS 2 B. Marin and M. Melkonian scaly green flagellates within a class separate from 1998; Karol et al. 2001; Lemieux et al. 2007; Marin the prasinophytes, the Loxophyceae (Christensen and Melkonian 1999; Melkonian et al. 1995; 1962), which later was emended to include only the Rodrı´guez-Ezpeleta et al. 2007). While the three genera, Monomastix, Pedinomonas, and Loxophyceae sensu Moestrup (1982) were soon Scourfieldia (Moestrup 1982). While a consider- abandoned (Moestrup 1991; Moestrup and able body of knowledge has accumulated on the Throndsen 1988), and two of its three structure, biogenesis and chemical composition of genera, Pedinomonas and Scourfieldia distributed prasinophyte scales over time (e.g. Becker et al. into different classes (Pedinophyceae and 1994; Wustman et al. 2004), their functional and Prasinophyceae, respectively; Moestrup 1991), thus evolutionary significance has, for the most Monomastix remained in limbo. Only very recently part, eluded us. Interest in prasinophytes, never- has the phylogenetic position of Monomastix and theless, gained momentum also among plant Pedinomonas been studied by molecular meth- scientists, when Pickett-Heaps (1968) and Turner ods, and Monomastix shown to be affiliated with (1968) described scales, structurally identical to a the Mamiellales, an order of marine, mostly scale type (small, square-shaped underlayer picoplanktonic prasinophytes (Turmel et al. scales) previously found in several prasinophytes, 2009a), whereas Pedinomonas, based on a on the flagellar surface of the spermatozoids of plastid-encoded multigene data set, was surpris- Chara and Nitella, i.e. in stoneworts (Charales), a ingly positioned in the Trebouxiophyceae, nested group of macroscopic, multicellular green algae, within the Chlorellales (Turmel et al. 2009b). that had been and still are, often regarded as the With the advent of molecular analyses of green likely sister group of the embryophyte land plants algal phylogeny in the early 1990s, it was soon (Lewis and McCourt 2004; McCourt et al. 2004). evident that green algae constituted two major Since that time, textbook knowledge holds that groups, previously referred to as Chlorophyta and scaly green flagellates were likely ancestral to all Streptophyta (Bremer 1985) and that the latter other Viridiplantae [small square-shaped scales included the embryophyte land plants thus corro- similar to the ones found in the Charales had also borating the conclusions from ultrastructural ana- been discovered on zoospores of the marine, lyses (for a summary of the early molecular filamentous green algae Trichosarcina and Pseu- phylogenetic data, see Melkonian and Surek dendoclonium by Mattox and Stewart (1973)]. 1995). The prasinophytes (except Mesostigma, When Manton (1965) in her review on phylogenetic see above) formed several early branching, para- aspects of flagellar structure in algae and plants phyletic lineages within the Chlorophyta concluded that ‘‘the fundamental asymmetry of (Steinkotter¨ et al. 1994; Melkonian et al. 1995). the motile cells of land plants points to an By 1998 Nakayama et al. had recognized four anisokont ancestry within the Chlorophyta, for independent lineages in the prasinophytes which which therefore Chlamydomonas is not on the they termed clades I-IV and which roughly direct line’’ (p. 21), scaly and naked asymmetric corresponded to four prasinophyte ‘orders’ infor- green flagellates soon became the focus of mally recognized earlier by Melkonian (1990) as attention for algal electron microscopists during Pyramimonadales (clade I, but excluding Mesos- the 1960s and 1970s in search of the ‘‘primitive tigma), Mamiellales (clade II), Pseudoscourfiel- flagellate’’ (Manton 1965) or the ‘‘Ancestral Green diales (clade III; Pseudoscourfieldia marina CCMP Flagellate’’ (Mattox and Stewart 1984). The search 717 was, however, misidentified and the strain struck gold with the discovery that the freshwater, refers to Nephroselmis pyriformis; Fawley et al. biflagellate, scaly green alga Mesostigma viride 1999), and Chlorodendrales (clade IV; the thecate shared several ultrastructural features (among prasinophyte genera Scherffelia and Tetraselmis). them the presence of a cruciate flagellar root In addition, the coccoid picoplanktonic system with two opposing d-roots that display Pycnococcus provasolii formed an individual proximal MLS structures, the latter hitherto known branch, later shown to form a separate clade only from reproductive cells of charophyte algae (clade V) with Pseudoscourfieldia marina K0017 and embryophyte land plants) with motile cells of (Fawley et al. 2000; in fact, P. marina and P. embryophytes (Melkonian 1983; Rogers et al. provasolii are genetically identical and presumably 1981) suggesting that Mesostigma could be the represent different life history stages of a single earliest offshoot in the evolutionary radiation taxon; unpubl. observations). A sixth clade leading to the embryophyte plants (Melkonian of entirely coccoid prasinophytes comprising 1982), a proposal later corroborated by molecular the genera Prasinococcus (Miyashita et al. 1993) phylogenetic evidence (e.g. Bhattacharya et al. and Prasinoderma (Hasegawa et al. 1996) was Please cite this article as: Marin B; Melkonian M. Molecular Phylogeny and Classification of the Mamiellophyceae class. nov. (Chlorophyta) based on Sequence Comparisons of the Nuclear- and Plastid-encoded rRNA Operons, Protist (2009), doi:10.1016/j.protis.2009.10.002 ARTICLE IN PRESS 3 Phylogeny and Classification of the Mamiellophyceae class. nov. established by Fawley et al. (2000; see also Potter lyses using these sequences demonstrate that the et al. 1997 and Courties et al. 1998 for initial Monomastigales comprise a group of freshwater studies on members of this clade); finally an Mamiellophyceae that is genetically at least as additional coccoid prasinophyte (strain CCMP1205) diverse as their marine counterpart, the Mamiel- formed another independent branch in the prasi- lales. In the Dolichomastigales, family Crustomas- nophyte radiation (Fawley et al. 2000). With the tigaceae, phylogenetic analyses of environmental addition of these novel clades, the previously well- sequences of nuclear-encoded SSU rDNA identi- supported paraphyletic branching order of prasi- fied the first freshwater lineage in this order. nophyte lineages