Nova Hedwigia Stuttgart, February 2005

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1 Nova Hedwigia Stuttgart, February 2005 Phylogenetic position of Phlyctochytrium planicorne (Chytridiales, Chytridiomycota) based on zoospore ultrastructure and partial nuclear LSU rrna gene sequence analysis by Peter M. Letcher* and Martha J. Powell Department of Biological Sciences, The University of Alabama Tuscaloosa, AL USA With 10 figures and 1 table Letcher, P.M. & J.P. Powell (2005): Phylogenetic position of Phlyctochytrium planicorne (Chytridiales, Chytridiomycota) based on zoospore ultrastructure and partial nuclear LSU rrna gene sequence analysis. - Nova Hedwigia 80: Abstract: The genus Phlyctochytrium (Chytridiales, Chytridiomycota) is an assemblage of approximately 70 described taxa that exhibit morphological, ultrastructural, and molecular diversity. Although the type species Phlyctochytrium hydrodictyi is not available for study, a similar taxon, Phlyctochytrium planicorne, has been brought into pure culture. Like the type, P. planicorne is an apophysate algal parasite. In this study zoospore ultrastructure and nuclear large subunit ribosomal RNA gene sequences are used to infer the phylogenetic position of P. planicorne among the Chytridiales. This is the first use of a Phlyctochytrium in a molecular phylogeny and the first Phlyctochytrium to exhibit the zoospore type herein described. The chytridialean zoospore of P. planicorne contains a rumposome, a bundle of 5-7 microtubules in a microtubular root connecting the rumposome to the kinetosome, an electron-opaque saddle over the kinetosome, an electronopaque plug in the base of the flagellar axoneme, and a paracrystalline inclusion in the peripheral cytoplasm; in these features it is similar to the zoospore described for Chytridium lagenaria. In a parsimony analysis, Phlyctochytrium planicorne is nested as a lone taxon in a polytomy including the Lacustromyces, Nowakowskiella, and Rhizophydium clades. Because there are so many species of Phlyctochytrium for which the ultrastructure and molecular characters are unknown, further studies are necessary to determine whether the genus as described is monophyletic. Key words: chytrid, large subunit rrna gene, phylogeny, zoospore Introduction The genus Phlyctochytrium Schroeter is one of the larger and more confusing genera of the order Chytridiales (Chytridiomycota), with approximately 70 described taxa *corresponding author, letch006@bama.ua.edu DOI: / /2005/ /05/ $ J. Cramer in der Gebrüder Borntraeger Verlagsbuchhandlung, D Berlin D Stuttgart 135

2 and a number of unidentified isolates (Sparrow 1960, Karling 1977, Longcore 1996). The initial taxa in this genus were originally classified in Chytridium, Rhizidium, and Rhizophydium before being segregated to Phlyctochytrium. Confusion regarding the generic concept arises because several of the characters used to delineate the genus (sensu Sparrow 1960) are variable and convergent. Sparrow s concept included monocentric, eucarpic chytrids with an epibiotic, inoperculate, uniporous or multiporous sporangium, an epibiotic resting spore, and an endobiotic absorptive system composed of an apophysis with a branched rhizoidal system. However, the primary discriminating character of the genus, the apophysis, is neither a stable nor a constant feature, and in some taxa may be only a slight swelling of the germ tube or rhizoidal axis, or may be lacking altogether. Alternatively, in certain taxa of Phlyctochytrium (most notably the type species P. hydrodictyi (A. Braun) J. Schröt.), an apophysis is the prominent feature, while rhizoids are absent. Compounding the confusion, several species in the other inoperculate genera Rhizophydium and Rhizidium occasionally appear apophysate. Taxonomic ambiguities arising from morphological plasticity have been addressed by assessing more conserved and phylogenetically informative ultrastructural features of the uniflagellate chytrid zoospore (Barr 1980, 2001). Barr (1980) segregated the order Spizellomycetales from the Chytridiales (sensu Sparrow 1960) because of fundamental differences in zoospore ultrastructure among taxa. The zoospore of the Chytridiales ( chytridialean zoospore, Barr 1980) generally has the following features: 1) ribosomes aggregated in the core of the zoospore and enveloped by a double membrane system; 2) a single, laterally-placed lipid globule; 3) a rumposome; 4) a microtubule root extending from the kinetosome to the rumposome; 5) one or more mitochondria associated with the microbody-lipid globule complex (Powell 1978); 6) a nucleus not intimately associated with the kinetosome; and 7) a nonflagellated centriole parallel to the kinetosome, the two structures being connected by a fibrillar bridge. Many soil-inhabiting taxa of Phlyctochytrium produced zoospores with features distinct from the chytridialean zoospore, and those taxa were transferred from the Chytridiales (sensu Sparrow 1960) to the new order Spizellomycetales (Barr 1980). However, other taxa assigned to Phlyctochytrium, such as P. irregulare W.J. Koch, exhibit a chytridialean type of zoospore (McNitt 1974). These results demonstrate that the type of thallus characteristic of Phlyctochytrium is convergent, having arisen in two different orders of the Chytridiomycota. The morphological and ultrastructural variation exhibited by taxa relegated to Phlyctochytrium raises the question of what exactly constitutes a member of the genus, and which members belong to the Chytridiales or Spizellomycetales. The type species, P. hydrodictyi, is an algal parasite that has not been isolated and thus is not available for study. Alternatively, a chytrid with similarities to P. hydrodictyi, Phlyctochytrium planicorne G.F. Atk., has been isolated and brought into pure culture. Phlyctochytrium planicorne is apophysate with branched rhizoids, and is found in aquatic habitats as a parasite on various algae, including species of Spirogyra, Rhizoclonium, Cladophora, and Oedogonium, as well as a saprotroph on pollen and cellulose. The objective of this paper is to examine the zoospore ultrastructure of P. planicorne, and its placement in a nuclear large subunit ribosomal RNA (LSU rrna) gene phylogeny of members of the Chytridiales (sensu Barr 1980). 136

3 Materials and methods Isolation and culture Phlyctochytrium planicorne (isolate J.E.L. 47, culture collection of Dr J. E. Longcore, University of Maine) was isolated using standard techniques (Barr 1987) from Oedogonium sp. from a small pond, Hampden, Maine, USA, 15 June Stock cultures were maintained at 5 C on PmTG agar (Barr 1987) slants in screw-capped culture tubes and transferred at 3-month intervals. Chytrid isolates used in the phylogenetic analysis are listed in Table I. Ichthyophonus hoferi and Monosiga brevicollis were chosen as outgroup taxa (Chambers 2003). Ingroup isolates were in the Order Chytridiales (sensu Barr 1980), and representative isolates from all clades (Chytriomyces [=Chytridium], Rhizophydium, Nowakowskiella, and Lacustromyces, James et al. 2000) were included in the analyses. Cultures were obtained from the chytrid collections at the University of Maine, The University of Alabama, Canadian Collection of Fungal Cultures, and Carolina Biological Supply. Cultures were grown on the following media: PmTG, mpmtg, ½ CM+, and ½ YPSs (Barr 1987, Longcore 1995). Sample preparation for electron microscopy and molecular analysis, and molecular data analysis methods as in Letcher & Powell (2004). Results Morphology The morphology of P. planicorne (Fig. 1) conformed to previously published accounts (Atkinson 1909, Sparrow 1932, 1938, Umphlett & Holland 1960). On pollen the sporangia were sessile or occasionally stalked on a short interbiotic rhizoidal axis, broadly ellipsoidal in shape, 6-24 µm high, 6-17 µm diam., with a thin hyaline wall. The sporangium was ornamented with a distinct apical crown of four slightly inwardcurving teeth approximately 2-4 µm high. The rhizoidal system consisted of a narrow rhizoidal axis that penetrated the substrate wall, and sparse to extensively branched rhizoids arising from a spherical, endobiotic subsporangial apophysis up to 12 µm in diameter. Zoospores were spherical, 3-6 µm diam., with a prominent single lipid globule. Resting spores were not observed. Development on PmTG agar Following encystment, the germling produced a slender germ tube that penetrated the agar. Coincident with the development of the incipient sporangium, from the germ tube an apophysis formed below the sporangium as a main rhizoid began to elongate and branch. Subsequent to these events the sporangium elongated slightly and the apical ornamentation developed. At maturity, generally a large oil globule remained in the apophysis. Zoospores were discharged singly, or in an evanescent vesicle, through a single apical pore. Zoospore ultrastructure The zoospore was circular to broadly elliptical in longitudinal section (Fig. 2) and circular in transverse section (Fig. 3). A cell coat (Fig. 8), external to the plasma 137

4 Table I. Taxon sampling for nuclear LSU rrna gene phylogenetic analysis of Phlyctochytrium planicorne. Taxon Culture number Accession # Origin Outgroup: Ichthyophonus hoferi AY Monosiga brevicollis AY Ingroup: Allochytridium luteum ATCC AY Canada Chytriomyces appendiculatus J.E.L. 165 AY ME, USA Chytriomyces hyalinus P.L. 13 AY AL, USA Chytriomyces hyalinus P.L. AUS 14 AY NSW, AUS a Chytriomyces sp. P.L. 06 AY TN, USA Kappamyces laurelensis P.L. 98 AY GA, USA Karlingiomyces sp. J.E.L. 93 AY ME, USA Nowakowskiella elegans M. 29 AY ME, USA Obelidium mucronatum J.E.L. 57 AY ME, USA Phlyctochytrium planicorne J.E.L. 47 AY ME, USA Polychytrium aggregatum J.E.L. 109 AY ME, USA Rhizophydium sp. P.L. AUS 7 AY NSW, AUS Rhizophydium sp. P.L. AUS 8 AY NSW, AUS Rhizophydium sp. P.L. AUS 12 AY NSW, AUS Rhizophydium sp. P.L. AUS 18 AY NSW, AUS Rhizophydium sp. P.L. 01 AY VT, USA Rhizophydium sp. P.L. 03 AY VA, USA Rhizophydium sp. P.L. 04 AY VA, USA Rhizophydium sp. P.L. 05 AY TN, USA Rhizophydium sp. P.L. 08 AY AL, USA Rhizophydium sp. P.L. 10 AY AL, USA Rhizophydium sp. P.L. 11 AY FL, USA Rhizophydium sp. P.L. 73 AY AL, USA Rhizophydium sp. P.L. 88 AY AL, USA Rhizophydium sp. P.L. 102 AY GA, USA Siphonaria petersenii J.E.L. 102 AY ME, USA a New South Wales, Australia membrane, covered the body of the zoospore but did not continue over the flagellar membrane. A central core of aggregated ribosomes was surrounded and traversed by endoplasmic reticulum (ER). A single, lobed mitochondrion resided outside the ER and the ribosomal core (Figs 2, 3). The microbody-lipid globule complex (MLC) was type 5B (Powell & Roychoudhury 1992), and consisted of a large, laterally placed lipid globule with a fenestrated cisterna (= rumposome) (Fig. 2) orientated toward the exterior of the zoospore and adjacent to the plasma membrane, and a microbody appressed to the interior face of the lipid body (Figs 2, 3). Several microbodies were concentrated in the region of the lipid body. The nucleus, in both longitudinal and transverse sections, was centrally 138

5 Fig. 1. Thallus of Phlyctochytrium planicorne, on agar. Scale bar = 10 µm. Abbreviations used in Figs 1-9. Ap, apophysis; CC, cell coat; EOS, electron-opaque cap saddle; EOP, electron-opaque plug; F, flagellum; FB, fibrillar bridge; G, Golgi complex; K, kinetosome; L, lipid globule; M, mitochondrion; Mb, microbody; Mt, microtubular root; N, nucleus; NFC, nonflagellated centriole; Nu, nucleolus; P, flagellar prop; PCI, paracrystalline inclusion; R, ribosomes; Rh, rhizoids; Ru, rumposome; SER, smooth endoplasmic reticulum; Sp, sporangium; TP, terminal plate; Vac, vacuole; Ve, electron-opaque vesicle in peripheral cytoplasm. located within the ribosomal aggregation (Fig. 3), and abutted portions of the mitochondrion and microbody. At the periphery of the nucleus, the nucleolus often appeared to be appressed to a microbody (Figs 2, 3). The core area of ribosomes, MLC, and nucleus was surrounded by cytoplasm that usually contained a single vacuole, Golgi complexes, a packet of smooth endoplasmic reticulum (Fig. 7), several distinct forms of vesicles and a laterally- or anteriorly-placed paracrystalline inclusion (Fig. 3). The kinetosome and non-flagellated centriole (nfc) were parallel to each other and were joined by a fibrillar bridge (Figs 2, 6) that connected triplets 6 and 7 of the kinetosome to the nfc. A microtubular root (Figs 2, 5) originated at triplets 1 and 2 of the kinetosome and extended to the well-developed rumposome, which had a single tier of pores. An electron-opaque saddle-like structure (Figs 4-6) was adjacent to the kinetosome. The saddle-like structure consisted of two layers of electronopaque material with an electron-transparent region between the layers. It was located over and to one side of the proximal end of the kinetosome, and extended to the nfc. A terminal plate (Fig. 4) and an electron-opaque plug (Figs 2, 4) were seen in the base of the flagellum. A schematic drawing of the zoospore (Fig. 9) illustrates the ultrastructural features. 139

6 Figs 2-8. Ultrastructural features of the zoospore of Phlyctochytrium planicorne. 2. Longitudinal section. 3. Transverse section. 4. Longitudinal section through kinetosome and base of flagellum. 5, 6. Transverse serial sections through kinetosome. 7. Smooth endoplasmic reticulum in peripheral cytoplasm. 8. Cell coat on zoospore body. Scale bars = 1 µm (in Fig. 2, also for Fig. 3); 0.5 µm (Figs 4-7); 0.25 µm (Fig. 8). 140

7 Fig. 9. Schematic drawing of the zoospore of Phlyctochytrium planicorne. Features include a rumposome, a bundle of 5-7 microtubules in a microtubular root connecting the rumposome to the kinetosome, an electron-opaque saddle-like structure over the kinetosome, an electron-opaque plug in the base of the flagellum, and a paracrystalline inclusion in the peripheral cytoplasm. Molecular analysis The alignment of sequences yielded 371 parsimony-informative sites after uninformative characters were excluded. A strict consensus, most parsimonious tree (Fig. 10) was 1104 steps in length and had a consistency index (CI) = and a retention index (RI) = The strict consensus tree shows the relationship between the Chytriomyces, Nowakowskiella, Lacustromyces and Rhizophydium clades and P. planicorne. All clades were monophyletic and exhibited strong bootstrap support values. Phlyctochytrium planicorne nested as a lone taxon in a polytomy that included Lacustromyces, Nowakowskiella, and Rhizophydium clade members; the polytomy was sister to the Chytriomyces clade. G + C content for P. planicorne (874 bases) was similar to those of its neighbors Polychytrium aggregatum Ajello (861 bases) of the Lacustromyces clade, and Chytriomyces hyalinus Karling (isolate P.L. AUS 14, 856 bases) of the Chytriomyces clade (47.48%, 47.50%, and 48.13%, respectively). Percent similarity values were highest between P. planicorne and P. aggregatum 141

8 Fig. 10. Parsimony analysis of the nuclear LSU rrna gene for 26 isolates in the Chytridiales, with Monosiga brevicollis and Ichthyophonus hoferi as the outgroup taxa. Strict consensus tree (L= 1104 steps, CI = 0.565, RI = 0.661). Values are bootstrap values (1000 replicates). (72%), and between P. planicorne and C. hyalinus isolate P.L. AUS 14 (76%). In variable regions of the sequence alignment, the molecular signature of P. planicorne was more similar to members of the Chytriomyces clade than to members of the Lacustromyces or Nowakowskiella clades. Discussion The position of Phlyctochytrium planicorne in an inferred phylogeny based on LSU rrna sequences, and its zoospore ultrastructure unequivocally place this organism at a distinct position in the Chytridiales. Phylogenetic position The use of the partial LSU rrna sequence of Phlyctochytrium planicorne in this analysis gives the first indication of the phylogenetic position of this chytrid. Previous 142

9 studies of members of the Chytridiales (James et al. 2000, Chambers 2003) excluded P. planicorne from the analysis, as it had two large introns (>400 bp) in the nuclear small subunit rrna molecule that prevented contiguous sequence construction and alignment with other members of the Chytridiales. In nesting within a polytomy with Lacustromyces, Nowakowskiella, and Rhizophydium clade members, P. planicorne occupies a novel and unique position in the phylogeny. It clearly does not group with, but rather is sister to, the Chytriomyces clade, with which it has several zoospore ultrastructural features in common. Likewise, it does not group with either the Nowakowskiella or Lacustromyces clades, whose members also have zoospore ultrastructural features similar to those of P. planicorne. Molecular analyses of additional isolates of Phlyctochytrium that are parasitic on algae and exhibit a chytridialean type of zoospore may be necessary before the phylogenetic affiliations of this chytrid can be resolved. Zoospore ultrastructure The type of zoospore exhibited by Phlyctochytrium planicorne is remarkably similar to the Group-II type of zoospore based on Chytridium lagenaria A. Schenk (Barr & Hartmann 1976, Barr 1980). (Barr s isolate of C. lagenaria is no longer available). These two chytrids are the only taxa in the Chytridiales for which this type of zoospore has been observed. The differences between the zoospores of C. lagenaria and P. planicorne are: (1) P. planicorne has a cell coat external to the plasma membrane, with a structure similar to that reported for Chytriomyces hyalinus and C. aureus (Dorward & Powell 1982), but not for C. lagenaria; (2) P. planicorne has no ribosome-like bodies surrounding the microtubular root that extends from the kinetosome to the rumposome, as observed with C. lagenaria; (3) in P. planicorne, the nucleolus is often located at the periphery of the nucleus and appears to be closely associated with a microbody, a character not observed in C. lagenaria. These ultrastructural differences between the two taxa are minor when the suite of characters that defines the Group II-type of zoospore specifically is considered. As only two taxa in the Chytridiales observed thus far have exhibited this zoospore type, the range of ultrastructural variability of the Group II-type zoospore is unknown. However, the ultrastructural differences between C. lagenaria and P. planicorne may be important at the species level. Phlyctochytrium planicorne has several zoospore ultrastructural features similar to isolates of the Chytriomyces clade. In common with P. planicorne, all taxa in the Chytriomyces clade thus far examined have an electron-opaque plug (EOP) in the base of the flagellum, a rumposome, a microtubular root consisting of a bundle of 5-7 microtubules that extends from the side of the kinetosome to the rumposome, and a paracrystalline inclusion. Phlyctochytrium planicorne also has ultrastructural features in common with isolates in the Nowakowskiella clade (a rumposome and an EOP) and the Lacustromyces clade (an EOP). Zoospores of members of the Rhizophydium clade (Group III-type zoospore, Barr 1980, Letcher 2003) exhibit significant differences from the zoospore of P. planicorne. Many taxa in Phlyctochytrium and Rhizophydium often have been confused, as the morphological distinctions between the two genera are not always clear. Both genera 143

10 are large and unwieldy taxonomic entities, and consequently each genus exhibits wide morphological variation. Zoospore ultrastructure of several Phlyctochytrium taxa has been examined, but none have the Group-II type of zoospore exhibited by P. planicorne. Phlyctochytrium irregulare W.J. Koch (McNitt 1974), Rhizophydium littoreum Amon (Amon 1984, synonymous with Phlyctochytrium sp. Kazama 1972), and Phlyctochytrium aestuarii Ulken (Lange & Olson 1977) have Group III, Rhizophydium-type zoospores (Barr & Hadland-Hartmann 1978, Barr 1980), and lack distinctive features of the Group-II type of zoospore exhibited by P. planicorne. Two additional species, Phlyctochytrium powhatanense Roane (unpublished EM studies by Barr, see Barr 1980) and P. mucronatum Canter (Lange & Olson 1979) possibly exhibited a Group I-type zoospore as described for Chytriomyces confervae (Wille) Batko and Chytridium olla A. Braun. None of these isolates are presently available in culture for further ultrastructural or molecular analyses. Thus, existing evidence suggests that the genus Phlyctochytrium may be polyphyletic as now understood. For example, the allied taxa (Barr 1980) P. powhatanense, P. mucronatum Canter, and all members of the Chytriomyces clade thus far examined have Group I-type zoospores, P. planicorne has a Group II-type zoospore, P. irregulare has a Group III-type zoospore, and other taxa in Phlyctochytrium yet to be examined ultrastructurally may be properly placed in genera in the Spizellomycetales (Barr 1980), as were P. punctatum W.J. Koch (Spizellomyces punctatus [W.J. Koch] D.J.S. Barr), P. semiglobiferum Uebelm. (Gaertneriomyces semiglobiferus [Uebelm.] D.J.S. Barr), P. arcticum D.J.S. Barr (Triparticalcar arcticum [D.J.S. Barr] D.J.S. Barr), and P. dichotomum Umphlett (Kochiomyces dichotomus [Umphlett] D.J.S. Barr) (Barr 1980). Clarifying these inconsistencies will require extensive sampling for, and isolation of, described Phlyctochytrium species. A directed effort at locating and isolating both Chytridium lagenaria (from which the Group II-type zoospore, exhibited by P. planicorne, was described) and Phlyctochytrium hydrodictyi (the type of Phlyctochytrium) with subsequent ultrastructural and molecular analyses, would contribute to resolving many of the taxonomic and phylogenetic questions posed by the unavailability of these taxa. Acknowledgements This paper is based on a dissertation submitted by the first author to the Graduate School of The University of Alabama in partial fulfillment of the requirements for a doctorate degree in Biology, Completion of this study was supported by the National Science Foundation through PEET grant DEB ; The University of Alabama, Department of Biological Sciences Aquatic Ecology and Systematics Graduate Enhancement Program; and a scholarship from the Alabama Power Company. We express our appreciation to Dr Joyce E. Longcore for providing chytrid cultures, including Phlyctochytrium planicorne. We are indebted to Drs J. G. Chambers and W. E. Holznagel for invaluable assistance with the RNA protocol and sequencing. References AMON, J.P. (1984): Rhizophydium littoreum: a chytrid from siphonaceous marine algae an ultrastructural examination. - Mycologia 76: ATKINSON, G.F. (1909): Some fungus parasites of algae. - Bot. Gaz. 48:

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