Protist, Vol. 155, 143–156, June 2004 http://www.elsevier.de/protist Published online 14 June 2004 Protist ORIGINAL PAPER Phylogenetic Position of the Parasitoid Nanoflagellate Pirsonia inferred from Nuclear-Encoded Small Subunit Ribosomal DNA and a Description of Pseudopirsonia n. gen. and Pseudopirsonia mucosa (Drebes) comb. nov. Stefanie Kühn a, Linda Medlin b, and Gundula Eller c,1 a Department of Marine Botany (FB2), University of Bremen, Leobener Str./NW2, D-28359 Bremen b Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven c Department of Physiological Ecology, Max Planck Institute for Limnology, August-Thienemann-Strasse 2, D-24306 Plön Submitted June 10, 2003; Accepted February 1, 2004 Monitoring Editor:Mitchell L. Sogin Sequences of the nuclear encoded small subunit (SSU) rRNA were determined for Pirsonia diadema, P. guinardiae, P. punctigerae, P. verrucosa, P. mucosa and three newly isolated strains 99-1, 99-2, 99- S. Based on phylogenetic analysis all Pirsonia strains, except P. mucosa, clustered together in one clade, most closely related to Hyphochytrium catenoides within the group of stramenopiles. How- ever, P. mucosa was most closely related to Cercomonas sp. SIC 7235 and Heteromita globosa and belongs to the heterogenic group of Cercozoa. In addition to the SSU rDNA sequences, P. mucosa differs from the stramenopile Pirsonia species in some characteristics and was therefore redescribed in this paper as Pseudopirsonia mucosa . The three newly isolated strains 99-1, 99-2, and 99-S dif- fered by 28 bp in their SSU rDNA sequences from their closest neighbour P. diadema and only 1 to 3 bp among themselves. These base differences and a host range similar to P. formosa were sufficient to assign them as new strains of P. formosa. Introduction Parasitoid protists are small unicellular eukaryotic groups, such as euglenozoa, dinoflagellates, cer- heterotrophs that infect, consume and thereby in- comonads, plasmodiophorids, oomycetes and evitably kill their phytoplankton hosts. Most of the chytrids (fungi), and species of unknown affiliation planktonic diatoms in the North Sea (53 species ob- (e.g. Bulman et al. 2001; Drebes 1966; Drebes and served, unpublished) are susceptible to infections. Schnepf 1988, 1998; Kühn et al. 2000; Schnepf These parasitoids comprise diverse taxonomic 1994; Schweikert and Schnepf 1996). Although host-specific parasitoids can decimate a diatom population by more than 90 per cent (Grahame 1 Corresponding author; 1976; Tillmann et al. 1999), their role in the marine fax 49-4522763310 planktonic food web is still poorly understood. Only e-mail [email protected] recently, a novel flagellate, Parvilucifera infectans , 1434-4610/04/155/02-143 $ 30.00/0 144 S. Kühn et al. was described that kills toxic dinoflagellates (Norén phagocytosis begins the diatom protoplast struc- et al. 1999), indicating the possible importance of ture becomes notably affected (Fig. 2A). Non-partic- parasitoids for the control of harmful algal blooms. ulate nutrients are transported into the main body of Additionally, parasitoid nanoflagellates (PNF) often the former flagellate, which remains outside the have small and flexible cells, possibly enabling them frustule and becomes the auxosome (Fig. 2B, 2C). to pass through filter membranes with a poresize Trophosomes of adjacent auxosomes frequently below the 3 µm pore that is normally used by fuse. As long as nutrients are transported into the oceanographers to fractionate water samples. This auxosomes these continue to grow and divide. The might lead to their detection in the so-called pi- offspring eventually grow flagella and become infec- coplanktonic fraction of the phytoplankton. PNF tive flagellates. may therefore play an important role in the hitherto Five Pirsonia species were described in detail by undefined heterotrophic fraction of the picoplank- Kühn et al. (1996). In this study, we included three tonic community. newly isolated strains Pirsonia 99-1, 99-2 and 99-S, Pirsonia species are PNF that infect planktonic di- which were assigned to P. formosa as described atoms. Their feeding mode is unique: flagellates at- below. Pirsonia species differ only slightly in their tach to the diatom frustule and squeeze a pseudo- morphological characteristics (Table 1). The flagel- pod into the cell, either between the girdle bands or late of P. verrucosa is, for example, approximately 8 through the rimoportulae or labiate processes, µm long and 3 µm wide, and thereby slightly smaller which are tubular openings that penetrate the valve than the flagellate of the new Pirsonia formosa 99-S wall. The pseudopod then becomes the tropho- with 9-13 6 µm. The anterior flagellum measures in some, which phagocytises and digests the diatom all species 15 to 25 µm, but the length of the poste- protoplast. Digested material is transported into the rior flagellum is variable with 15 µm in P. verrucosa auxosome, the part of the body that remains on the and 45 µm in P. eucampiae . Additional morphologi- outside of the frustule. The auxosome then grows, cal features like the size and shape of the auxo- divides and reproduces, forming offspring as long somes, or whether flagellate mother cells remain as food supply continues. Up to now, seven Pirsonia connected to the diatom frustule show only minor species have been described from the North Sea differences for the hitherto identified species. Cyst (Schnepf et al. 1990; Kühn et al. 1996; Schweikert formation was only observed for P. guinardiae and P. and Schnepf 1997), and another 5 strains have been formosa 99-2. Hatching was never observed. isolated. Ultrastructural examination clearly as- signed Pirsonia to the stramenopiles (Heterokonta) (Schnepf and Schweikert 1996). Stramenopile is a term that was introduced as a rankless, informal name for eukaryotes possessing a flagellum with tri- partite, tubular hair-like projections (mastigonemes) (Patterson 1989), and comprises a huge range of taxa that include parasites, saprotrophs, het- erotrophs and autotrophs (e.g. brown algal kelps and diatoms). Here we show that, based on SSU rDNA se- quence analysis, the genus Pirsonia forms a distinct clade within the stramenopiles, with Hyphochytrium catenoides and Rhizidiomyces apophysatus as their next known relatives. Molecular analysis, however, shows that Pirsonia mucosa Drebes does not be- long to the stramenopiles but is related to the cer- comonads. Furthermore, we describe a new genus, Pseudopirsonia gen. nov., for this taxon. Figure 1. Developmental stages of Pirsonia and Pseu- dopirsonia. Motile flagellates attach and penetrate frustule with a pseudopod. The pseudopod phagocy- Results tises and digests portions of the diatom protoplast and thus differentiates into the trophosome. Nutrients are Morphology and host range transported into the body of the former flagellate, now called auxosome. The auxosome grows and divides, The life cycle of all Pirsonia species and Pseudopir- forming offspring as long as trophosomes continue to sonia is similar (Fig. 1) (Kühn 1996). As soon as phagocytise. Phylogeny of Pirsonia and Pseudopirsonia 145 A characteristic feature for the Pirsonia species is formosa 99-1 was attracted to Stephanopyxis turris their host range (Table 2). Some species, e.g. P. di- and attached to the frustule but failed to penetrate adema and P. punctigerae, are host specific and in- (not shown). Pirsoniaformosa 99-2 was clearly fect only one diatom genus whereas the host range chemosensory attracted to Thalassiosira punctig- of the newly isolated Pirsonia formosa strains 99-1, era, but did not attach to the frustule (Fig. 2D), even 99-2 and 99-S is relatively broad and similar to that though it attached to the diatom protoplast (Fig. of P. formosa. Circumstantial evidence indicates 2E). Pirsoniaformosa 99-S was strongly attracted that there might be a transition between an initial by Guinardia flaccida and attached to the frustule chemosensory attraction and possibly successful (Fig. 2F). Successful infections, however, were infection by later parasitoid generations. Pirsonia scarce. Figure 2. Pirsonia species and Pseudopirsonia mucosa. A–C. Pirsonia diadema infecting the marine diatom Cos- cinodiscus wailesii . A. Infection after approximatly 6 hours at room temperature. B. P. diadema, auxosomes and trophosomes. C. P. diadema, detail of cytoplasmatic connection between divided auxosomes. D. P. formosa 99- 2 flagellates are clearly chemosensory attracted by Thalassiosira punctigera but do not attach to the diatom frus- tule. E. P. formosa 99-2 flagellates attach to the naked protoplast of T. punctigera . F. P. formosa 99-S attach to Guinardia flaccida but only succeed occassionally to infect the diatom. G–I. Pseudopirsonia mucosa . G. Auxo- somes with mucilagenous coat and adhering bacteria and trophosomes (feeding on Rhizosolenia imbricata ). H. Flagellate, dorsal view. I. Flagellate, lateral view. J. Pirsonia diadema flagellates. (Bars = 10 µm.) 146 S. Kühn et al. Phylogenetic Analysis sonia mucosa . All methods used for phylogenetic analysis placed the Pirsonia spp. next to Hy- Full length sequences of the nuclear encoded SSU phochytrium catenoides (accession numbers rRNA gene were determined for Pirsonia diadema , P. X80344 and AF163294) and Rhizidiomyces guinardiae, P. punctigerae, P. verrucosa , Pirsonia apophysatus (accession number AF163295) within formosa strains99-1, 99-2 and 99-Sand Pseudopir- the group of stramenopiles (Fig. 3A). In contrast to Table 1. Morphological characteristics of several Pirsonia species. Pirsonia/ Size of Length of RetractionShape of Size of Cysts Pseudopirsonia flagellates anterior and of flagellaprimary primary (µm) posterior after auxosomes auxosomes flagellum (µm)infection (µm) P. diadema 8–10 3–4 16–18, 35–40 early apple-shaped 10 – P. eucampiae 7–9 4–5 15, 45 never globular 12 – completely P. formosa 7–8 5–6 18, 25–30 early globular 13 – P. formosa 99-1 9 5 18–22, 20–24 early apple-shaped 10 12 – P. formosa 99-2 6–8 3–4 12–14, 17–19 early globular 10 + P. formosa 99-S 9–13 6 16–20, 22–25 never apple-shaped 10 11 – completelyto globular P.