J Mol Evol (2006) 62:388–420 DOI: 10.1007/s00239-004-0353-8 Phylogeny and Megasystematics of Phagotrophic Heterokonts (Kingdom Chromista) Thomas Cavalier-Smith, Ema E-Y. Chao Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK Received: 11 December 2004 / Accepted: 21 September 2005 [Reviewing Editor: Patrick J. Keeling] Abstract. Heterokonts are evolutionarily important gyristea cl. nov. of Ochrophyta as once thought. The as the most nutritionally diverse eukaryote supergroup zooflagellate class Bicoecea (perhaps the ancestral and the most species-rich branch of the eukaryotic phenotype of Bigyra) is unexpectedly diverse and a kingdom Chromista. Ancestrally photosynthetic/ major focus of our study. We describe four new bicil- phagotrophic algae (mixotrophs), they include several iate bicoecean genera and five new species: Nerada ecologically important purely heterotrophic lineages, mexicana, Labromonas fenchelii (=Pseudobodo all grossly understudied phylogenetically and of tremulans sensu Fenchel), Boroka karpovii (=P. uncertain relationships. We sequenced 18S rRNA tremulans sensu Karpov), Anoeca atlantica and Cafe- genes from 14 phagotrophic non-photosynthetic het- teria mylnikovii; several cultures were previously mis- erokonts and a probable Ochromonas, performed ph- identified as Pseudobodo tremulans. Nerada and the ylogenetic analysis of 210–430 Heterokonta, and uniciliate Paramonas are related to Siluania and revised higher classification of Heterokonta and its Adriamonas; this clade (Pseudodendromonadales three phyla: the predominantly photosynthetic Och- emend.) is probably sister to Bicosoeca. Genetically rophyta; the non-photosynthetic Pseudofungi; and diverse Caecitellus is probably related to Anoeca, Bigyra (now comprising subphyla Opalozoa, Bicoecia, Symbiomonas and Cafeteria (collectively Anoecales Sagenista). The deepest heterokont divergence is emend.). Boroka is sister to Pseudodendromonadales/ apparently between Bigyra, as revised here, and Och- Bicoecales/Anoecales. Placidiales are probably diver- rophyta/Pseudofungi. We found a third universal gent bicoeceans (the GenBank Placidia sequence is a heterokont signature sequence, and deduce three basidiomycete/heterokont chimaera). Two GenBank independent losses of ciliary hairs, several of 1-2 cilia, ‘opalinid’ sequences are fungal; Pseudopirsonia is 10 of photosynthesis, but perhaps only two plastid cercozoan; two previous GenBank ‘Caecitellus’ se- losses. In Ochrophyta, heterotrophic Oikomonas is quences are Adriamonas. sister to the photosynthetic Chrysamoeba, whilst the abundant freshwater predator Spumella is biphyletic; Key words: Heterokonta — Oikomonas — Anoeca neither clade is specifically related to Paraphysomonas, — Bicoecea — Caecitellus — Paramonas — Nerada indicating four losses of photosynthesis by chry- — Opalozoa — Labromonas — Bigyra somonads. Sister to Chrysomonadea (Chrysophyceae) is Picophagea cl. nov. (Picophagus, Chlamydomyxa). The diatom-parasite Pirsonia belongs in Pseudofungi. Introduction Heliozoan-like actinophryids (e.g. Actinosphaerium) are Opalozoa, not related to pedinellids within Hypo- Heterokonts are of particular evolutionary interest as their evolutionary diversification has produced a Correspondence to: Thomas Cavalier-Smith; email: tom.cavalier- greater panoply of nutritional modes and body forms [email protected] than in any other major group; Heterokonta include 389 the multicellular brown seaweeds that can grow their secondarily non-photosynthetic descendants; longer than blue whales or Sequoia trees, and the secondly the heterotrophic phylum Bigyra, compris- usually parasitic oomycetes (e.g. Phytophthora, the ing the zooflagellate Developayella and osmotrophic pseudofungus that caused the great 1845 Irish potato Pseudofungi (Oomycetes, Hyphochytrea) and Opali- famine, for centuries confused with fungi because of nata (Opalinea, Proteromonas, Blastocystis); thirdly its filamentous body), as well as numerous protists of the also purely heterotrophic Sagenista, comprising major importance for aquatic biology e.g. the pho- the osmotrophic Labyrinthulea and phagotrophic tosynthetic diatoms (having tens of thousands of Bicoecea [for previous treatments of the heterokonts species), chrysomonads, xanthophytes and numerous see Cavalier-Smith and Chao (1996) and Cavalier- smaller groups of chlorophyll-c-containing algae, Smith 1997, 2004a]. For many years the evolutionary plus several non-photosynthetic groups that may feed arguments for the kingdom Chromista (Cavalier- phagotrophically, e.g. bicoeceans, or absorptively, Smith 1981) with a common photosynthetic ancestry e.g. labyrinthuleans, opalinates. This paper focuses and multiple losses of plastids following the single on the non-photosynthetic phagotrophic zooflagel- enslavement of a red alga (Cavalier-Smith 1986a, late heterokonts, which have been much less studied 1989,1992,1995a) were widely ignored — initially than the heterokont algae and the absorptive het- because of a mistaken view that symbiogenetic gains erotrophs, but are of considerable ecological and of chloroplasts are easier than losses (Margulis 1970) evolutionary importance. Some heterotrophic het- and later because single-gene sequence trees seldom erokonts (notably paraphysomoands and pedinellids) cluster all three chromist groups (Heterokonta, are known to have retained colourless plastids when Haptophyta, Cryptista) together (Bhattacharya et al. they lost photosynthesis, but most are assumed to 1995; Delwiche 1999; Medlin et al. 1997). Multiple have lost both photosynthesis and plastids at an early chloroplast gene trees recently confirmed that all stage in evolution. Two major questions in hetero- chromist chloroplasts are monophyletic and that kont evolution are inter-related: (1) how many times those of heterokonts and haptophytes are sisters did plastid loss occur (if at all; many of the hetero- (Yoon et al. 2002), as long argued (Cavalier-Smith trophs have been insufficiently studied to exclude the 1986a, 1994, 2000a) and as effected taxonomically by possibility of relict leucoplasts remaining); and (2) the grouping of heterokonts and haptophytes to- what is the basal branching order for the group? gether as the chromist subkingdom Chromobiota The unique cell structure of heterokonts (Manton (Cavalier-Smith 1989). The name Chromobiota re- and Clarke 1950; Gibbs 1962; Hibberd 1971) presents placed Chromophyta sensu Cavalier-Smith (1981), many intriguing problems in molecular and organel- which was not an ideal name for a group embracing lar evolution that cannot be studied in more familiar former fungi and protozoa as well as algae; ‘strame- organisms (Cavalier-Smith 2004a). Heterokonta was nopiles’ (Patterson 1989) is an unwarranted junior formally established as a phylum/division by Cava- synonym for heterokonts (see Cavalier-Smith lier-Smith (1986a) for all eukaryotes with motile bi- 1993a)—not for chromists, as often incorrectly as- ciliate cells having an anterior cilium with tripartite serted, though Stramenipila of Dick (2001) confus- rigid tubular mastigonemes and a posterior hairless ingly is a similarly unnecessary recent synonym for (smooth) cilium, plus all their descendants that have Chromista of Cavalier-Smith (1981). secondarily lost one or both cilia (e.g. diatoms, hyp- The argument that all heterotrophic heterokonts hochytrids). Heterokontae originally embraced only and other chromists evolved from algal ancestors by xanthophyte and raphidophyte algae (Luther 1899), multiple losses of photosynthesis and/or plastids but the informal term heterokonts was extended to all (Cavalier-Smith 1986a) was extended further by biciliate heterokonts by Leedale (1974) and is now Cavalier-Smith (1999), who argued that chromists are applied to all Heterokonta irrespective of whether sisters of and share a photosynthetic common they have one, two, several, or no cilia and whether ancestry with alveolate protozoa, which include di- they are algae, or purely heterotrophic like oomycetes noflagellates, apicomplexans (both often with plast- and bicoeceans. Molecular sequence trees strongly ids), and the purely phagotrophic ciliates and support the holophyly of heterokonts, as do two suctorians. On this chromalveolate hypothesis chro- unique sequence signatures in 18S rRNA (Cavalier- mists and alveolates are a major clade that originated Smith et al. 1994), but the basal branching order by the unique enslavement of a red alga by a bikont within the supergroup has remained controversial, common ancestor; ciliates, like heterotrophic and needs to be clarified if their evolution is to be heterokonts, evolved from chromophyte algae (those properly understood. having chlorophylls a and c). Thus chromophyte al- Heterokonta was elevated to an infrakingdom gae are not polyphyletic but paraphyletic, chlorophyll within the kingdom Chromista (Cavalier-Smith c having originated in their common ancestor as did a 1995a,b) to allow its subdivision into three phyla: novel protein-targeting machinery for the import via Ochrophyta comprising all heterokont algae and the ER lumen of nuclear-coded chloroplast proteins 390 bearing bipartite N-terminal topogenic sequences. revealed to be a Caecitellus) were included in an The monophyly of chromalveolate protein-targeting earlier tree simply to illustrate the probable mono- mechanisms is strongly supported by all we have phyly of Bicoecea (Cavalier-Smith 2000a). Our pres- learned about these targeting mechanisms (Cavalier- ent analysis including 28 bicoecean