J. Phycol. 49, 115–129 (2013) © 2012 Phycological Society of America DOI: 10.1111/jpy.12021 MORPHOLOGY AND PHYLOGENETIC POSITION OF THE FRESHWATER GREEN MICROALGAE CHLOROCHYTRIUM (CHLOROPHYCEAE) AND SCOTINOSPHAERA (SCOTINOSPHAERALES, ORD. NOV., ULVOPHYCEAE)1 2 Pavel Skaloud, Tomas Kalina, Katarına Nemjova Charles University in Prague, Faculty of Science, Department of Botany, Benatska 2, 128 01, Prague 2, Czech Republic Olivier De Clerck, and Frederik Leliaert Phycology Research Group, Biology Department, Ghent University, Krijgslaan 281 S8, 9000, Ghent, Belgium The green algal family Chlorochytriaceae comprises plementary DNA; DAPI, 4′,6-diamidino-2-phenylin- relatively large coccoid algae with secondarily dole; EMBL, European Molecular Biology thickened cell walls. Despite its morphological Laboratory; ML, maximum likelihood; PP, posterior distinctness, the family remained molecularly probability; rbcL, ribulose-bisphosphate carboxylase uncharacterized. In this study, we investigated the morphology and phylogenetic position of 16 strains determined as members of two Chlorochytriaceae genera, Chlorochytrium and Scotinosphaera.The phylogenetic reconstructions were based on the Diversity of eukaryotic microorganisms is gener- analyses of two data sets, including a broad, ally poorly known and likely underestimated, espe- concatenated alignment of small subunit rDNA and cially when compared to animals and land plants. In rbcL sequences, and a 10-gene alignment of 32 selected the past two decades, the use of molecular tools has taxa. All analyses revealed the distant relation of the revolutionized microbial diversity research, includ- two genera, segregated in two different classes: ing the discovery of numerous deeply branching Chlorophyceae and Ulvophyceae. Chlorochytrium phylogenetic lineages (Edgcomb et al. 2002, Kaw- strains were inferred in two distinct clades of the achi et al. 2002, Moriya et al. 2002, Kawai et al. Stephanosphaerinia clade within the Chlorophyceae. 2003, Stoeck et al. 2006, Kai et al. 2008, Lopez- Whereas clade A morphologically fits the description Garcıa and Moreira 2008, Zhao et al. 2012). of Chlorochytrium, the strains of clade B coincide with Green algae are no exception. Despite their long the circumscription of the genus Neospongiococcum.The taxonomic history, new lineages are frequently Scotinosphaera strains formed a distinct and highly being identified and described as higher taxa (Rindi divergent clade within the Ulvophyceae, warranting et al. 2006, Zhang et al. 2008, Leliaert et al. 2009, the recognition of a new order, Scotinosphaerales. Neustupa et al. 2009, 2011, Elias et al. 2010, Aboal Morphologically, the order is characterized by and Werner 2011, Carlile et al. 2011, Nemcov a et al. large cells bearing local cell wall thickenings, pyrenoid 2011, Somogyi et al. 2011). Many of these new matrix dissected by numerous anastomosing higher taxa are in fact based on molecular analysis cytoplasmatic channels, sporogenesis comprising the of described species. For example, molecular and accumulation of secondary carotenoids in the cell ultrastructural data have shown that the Prasinophy- periphery and almost simultaneous cytokinesis. The ceae, traditionally comprising a diverse array of close relationship of the Scotinosphaerales with other flagellates with organic body scales, comprise a para- early diverging ulvophycean orders enforces the notion phyletic assemblage of early diverging lineages, that nonmotile unicellular freshwater organisms have which are now being defined as new orders or clas- played an important role in the early diversification of ses (Marin and Melkonian 2010, Leliaert et al. the Ulvophyceae. 2012). Similarly, the identification of an unrecog- nized deeply branching clade of green algae, the Key index words: Chlorochytrium; Chlorophyceae; chlo- Palmophyllales (Zechman et al. 2010) was based on roplast; Kentrosphaera; Phylogeny; Scotinosphaera; a genus that had been known for over a century taxonomy; ultrastructure; Ulvophyceae (Kutzing€ 1847). Other examples include the endo- Abbreviations: ACOI, Coimbra Collection of Algae; phytic marine green alga Blastophysa rhizopus Reinke, BBM, Bold’s basal medium; BI, Bayesian inference; the marine quadriflagellate Oltmannsiellopsis viridis BV, bootstrap value; CAUP, Culture Collection of (Hargraves & Steele) Chihara & Inouye, the subaer- Algae of Charles University in Prague; cDNA, com- ial, coccoid Ignatius tetrasporus Bold & MacEntee, and the epizoic, filamentous Trichophilus welckeri 1Received 26 June 2012. Accepted 22 August 2012. Weber-van Bosse, which have been recovered as 2Author for correspondence: e-mail [email protected]. distinct lineages of Ulvophyceae (Iima and Tatewaki 115 116 PAVEL SKALOUD ET AL. 1987, Nakayama et al. 1996, Friedl and O’Kelly almost simultaneous cytokinesis (Klebs 1881c, 2002, Watanabe and Nakayama 2007, Cocquyt et al. Puncocharova 1992). 2010, Suutari et al. 2010). In other cases, molecular Chlorochytrium and Scotinosphaera have an intricate data have resulted in unexpected taxonomic trans- taxonomic history, particularly because of close mor- fers. For example, the invertebrate pathogen Helicos- phological and ecological similarities of both genera poridium Keilin, considered to be either a fungus or with Kentrosphaera (Borzi 1883), Endosphaera (Klebs a protozoan of uncertain affinity, was phylogeneti- 1881b), and Stomatochytrium (Cunningham 1887). cally inferred among the green algal class Trebouxi- West (1904, 1916) reduced Stomatochytrium and Scot- ophyceae (Tartar et al. 2002). Similarly, the inosphaera to synonyms of Chlorochytrium, based on uniflagellate genus Pedinomonas Korshikov, tradition- morphological similarities and endophytic habit. ally affiliated with either Prasinophyceae (Moestrup Later, Bristol (1920) also included Kentrosphaera as a 1991) or Ulvophyceae (Melkonian 1990), has been synonym of Chlorochytrium, based on similarities in recovered as a distinct clade, sister to the core chlo- chloroplast structure. However, some of the later rophytes (Marin 2012). investigators did not accept Bristol’s proposal. Smith Although the molecular diversity of green algae (1933) retained the genera Chlorochytrium and has been relatively well studied (reviewed in Lewis Kentrosphaera based on their different habitat and and McCourt 2004, Leliaert et al. 2012), DNA reproductive characteristics. He characterized sequence data are still lacking for many genera and Chlorochytrium as an endophytic genus with sexual families. In particular, molecular investigation of reproduction, and Kentrosphaera as free-living, repro- morphologically distinct genera could improve our ducing only by asexual zoospores. Komarek and Fott understanding of green algal evolution (Zechman (1983) followed Smith’s recognition of Chlorochytri- et al. 2010). One such group of morphologically um and Kentrosphaera as separate genera, and listed remarkable and molecularly uncharacterized green Chlorocystis Reinhard, Stomatochytrium, Scotinosphaera algae is the Chlorochytriaceae as circumscribed by and Endosphaera as synonyms of the former genus. Komarek and Fott (1983). The family comprises rel- Puncocharova (1992) regarded Scotinosphaera as an atively large (up to 400 lm), spherical to irregularly invalid name due to the absence of either descrip- shaped coccoid algae with secondarily thickened, tion or illustration of the structure of its vegetative stratified cell walls. Of seven genera recognized by cells. However, Wujek and Thompson (2005) recti- Komarek and Fott (1983), Chlorochytrium Cohn and fied this by recognizing Scotinosphaera as a validly Scotinosphaera Klebs have been deposited in public described genus having priority over Kentrosphaera. culture collections, enabling a detailed morphologi- On the basis of detailed morphological observa- cal and ultrastructural investigation, and molecular tions, Wujek and Thompson (2005) classified five characterization. species into Scotinosphaera, and considered Chlorochy- Chlorochytrium grows endophytically in intercellu- trium as a monotypic genus. lar spaces of various freshwater, aquatic plants In addition to ambiguous generic circumscrip- (Cohn 1872, Klebs 1881a, West 1916, Lewin 1984) tions, the taxonomic affinity of Chlorochytrium and or marine macro-algae (West et al. 1988). The Scotinosphaera has long been questioned. Since their spheroid, ovoid, or slightly irregular cells contain a description, both genera were considered closely single parietal chloroplast with one to several pyre- related. The genera were originally assigned to the noids. The life history of Chlorochytrium involves green algal order Chlorococcales, and placed into biflagellate isogametes that leave sporangial wall in various families, including the Endosphaeraceae common gelatinous vesicle and fuse into quadrifla- (Smith 1950), Chlorococcaceae (Bourrelly 1966), gellate planozygotes that settle on host after short and Chlorochytriaceae (Komarek and Fott 1983). motile period. The settled planozygotes develop Ultrastructural investigations, however, shed doubt into vegetative cells, which enter the intercellular on the close relation between the two genera spaces of the host plant by the formation of tubular (Watanabe and Floyd 1994). Based on different protrusions. Both gametes and zoospores arise from arrangement of kinetosomes and flagellar roots, successive bipartition of the protoplasm. During a Chlorochytrium was classified in the Chlorophyceae complex pattern of cell division, protoplasm pieces and