Amoebidium Parasiticum Is a Protozoan, Not a Trichomycete

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Amoebidium parasiticum is a protozoan, not a Trichomycete

Gerald L. Benny cete order Amoebidiales (Zygomycota, Fungi) (Licht Department ofPlant Pathology, 1453 Fifield Hall, wardt 1986). With 4 and 7 species, respectively, these University ofFlorida, Gainesville, Florida, USA arthropodophilous symbionts are typically found in 32611-0680 fresh water in many parts of the world on Crustacea Kerry O'DonnelP and Insecta (Lichtwardt 1986, 1997, Lichtwardt et al J.'vlicrobial Properties Research Unit, National Center 1999), bloodworms (Licht:wardt and Williams 1992) for Agricultural Utilization Research, United States and Daphnia spp. (Lichtwardt 1986), or in the hind Department ofAgriculture, Agricultural Research gut or larval gills of invertebrates (Lichtwardt 1986, Service, 1815 North University Street, Peoria, Illinois, USA 61604-3999 Lichtwardt and Arenas 1996, Lichtwardt and Williams 1992, Williams and Lichtwardt 1990). Species of Amoebidiwll produce unicellular thalli that are typi Abstract: Classification ofthe Amoebidiales (Tricho cally attached externally to a host by an acellular mycetes, Zygomycota) 'within the Fungi is problem holdfast, whereas thalli of Paramoebidiwll are at atical because their cell walls apparently lack chitin tached to the cuticle of the hindgut, or to other Tri and they produce amoeboid cells during their life chomycetes inhabiting the host hindgut. cycle, A nearly full length fragment of the nuclear Study of the Amoebidiales has been advanced sig small subunit (SSU) rRt'\JA ofAmoebidiwll parasiticum nificantly by the axenic culture of Amoebidium par was amplified by the polymerase chain reaction asiticum (Whisler 1962), which enabled vVhistler (PCR) and sequenced to examine its phylogenetic (1963) and Trotter and vVhisler (1965) to determine relationships. Results of a Blasu'\J search of GenBank that the cell walls were not composed of cellulose or revealed that the A. parasiticum SSU rRNA sequence chitin. Culturing also has facilitated sequencing the was most closely related to that of Ichthyophonus hof mitochondrial genome, which at ca 300 kbp, is sig eri, an ichthyosporean in the Protozoa near the ani nificantly larger than that reported for any fun mal-fungal divergence. Maximum parsimony analysis gus (Lang BF, Burger G. http://megasum.bch. of ichthyosporean and fungal SSU sequences, using umontreal.ca/ogmp/projects/apara/gen.html). In sequences of choanoflagellates to root the 18S rDNA addition, V\71J.isler (1968) was able to induce the gene trees, resolved A. parasiticum as a strongly sup amoeba-cyst phase of the life cycle. These findings ported sister of 1. hofeli within the Ichthyophonida indicate the Amoebidiales are unique among organ clade of the protozoan class Ichthyosporea. In con isms classified as Fungi in the autapomorphic pro trast to other members of this class, which are mostly duction of amoeboid cells. Amoebae encyst to form obligate or facultative parasites ofvarious animals, A. (cysto)spores which develop into new thalli. In con parasiticum and other members of the Amoebidiales trast to Paramoebidium, species of Amoebidium also are only known to be arthropodophilous symbionts. form sporangiospores. The results also provide the first evidence that mito The rationale for classifying Amoebidiales as Tri chondrial cristae types exhibit homoplastic distribu chomycetes has been based on putative similarity in tions within the Ichthyosporea. thallus morphology and a shared symbiotic associa Key Words: Amoebidiales, 18S rRt'\JA, Ichthyo tion 'with arthropods. V\71J.istler (1963) and Lichtwardt sporea, mitochondrial cristae, phylogeny, Zygomycota (1986), however, have theorized that this order may represent a nonfungal evolutionary lineage derived from a protozoan ancestor. This conclusion was INTRODUCTION based on the premise that similarities between the Amoebidiales and other Trichomycetes may be due Amoebidium Cienkowski and Paramoebidium Leger & to convergent evolution. Efforts to examine evolu Duboscq are genera of unusual fungal-like microor tionary relationships of the Amoebidiales have in ganisms traditionally classified 'within the Trichomy- cluded phylogenetic analysis of 5S ribosomal RNA Accepted for publication June 13, 2000, (Walker 1984), serological analysis (Sanger et al I Corresponding author; email: kodonnell@sunca,ncaur,usda,gov 1972) and comparison of ribosomal Rt'\JA molecular

1133 1134 MVCOLOGIA masses (Porter and Smiley 1979). Collectively, results authentic Perkinsus spp. are nested \vithin the alvoelates), of these studies, together with those of Trotter and Psorospermllm haeckelii Hilgendorf U33180, Rhinosporidium "Vhistler (1965), suggest that the Amoebidiales are seeberi Wernicke AF118851, rosette agent L29455, Smittium not closely related to other Zygomycota; however, culisetae Lichtlv. AF007540.1, Sphaerosomo arcticum Yl6260.2 (cited in GenBank asJoestensenJP,Johansen S, Sperstad S, they do not resolve the phyletic affinities ofthe order. Landfald B. Sphaerosoma arcticmn, a new member of a clade Because phylogenetic analyses of nuclear small sub of protists near the animal-fungal divergence: systematic po unit (SSU) 18S rRNAs have helped resolve evolution sition, in vitro growth characteristics, and gross biochemical ary relationships among fungi (Bruns et al 1992) and composition), Spizellomyces ocuminatus (DJ.S. Barr) DJ.S. pseudofungi (Gunderson et al 1987), we generated Barr M59759.1, Unknown ichthyosporean AJ130859, Usti a nearly full length SSU sequence for A. pamsiticum lago moydis (DC.) Corda X62396.1. of 1720 bp to which we added sequences from 19 Sequences were aligned using ClustalX (Thompson et al taxa obtained from GenBank based on the results of 1997) and then by eye using SemWare Editor Professionall a BlastJ.~ search (Benson et al 1999). 32 vel's. 2.80b (SemWare Corporation, Marietta, Georgia). The dataset was analyzed by equally weighted maximum parsimony using PAUP*4.0b2 (Swofford 1999). Phylogenet ivLUERIALS A>m METHODS ic analysis employed a heuristic search, with gaps treated as missing data, 1000 random addition sequences with MUL jV1aterial examined.-Amoebidium parasiticum (FRA-I-14 = PARS on and TBR branch swapping. Clade stability was as NRRL 20524 = ATCC 32708) was grown in a shallow layer sessed by 1000 parsimony bootstrap replications, using 10 of distilled water covering one-tenth strength brain heart random addition sequences per replicate, and Bremer sup infusion agar in 60 X 15 mm plastic petri dishes at room port (= BS, Bremer 1988) using TreeRot (Sorenson 1996). temperature (ca 25 C) as recommended by Lichtwardt The PAUP* file has been deposited in TreeBASE as S491. (1986). Mter 1 wk cells were harvested and lyophilized over night. RESULTS jV10lecular biology.-Total genomic DNA was isolated from the lyophilized cellular material by the phenollchloroform The dataset consisted of 20 aligned sequences 2081 procedure described by O'Donnell et al (1997) for herbar bp in length, but 732 ambiguously aligned characters ium specimens. Polymerase chain reaction (PCR) and se were excluded from the analyses. Equally weighted quencing protocols were done according to the procedure maximum parsimony analysis of the 1349 included described by O'Donnell et al (1998), using primers de characters, 150 of which were phylogenetically infor scribed by "Vhite et al (1990), and NS21d 5'-TTGATA mative, lielded 3 most-parsimonious trees 527 steps GGGCAGAAATTTG and NS41g 5'-CCAACTGTCCCTAT in length (FIG. 1) (consistency index = 0.712, reten TAATCAT. tion index = 0.709, rescaled consistency index = Dataset construction and phylogenetic analysis.-The SSU 0.505). With this dataset, Amoebidiwn pamsiticumwas rDNA sequence of Amoebidium parasiticum was submitted deeply nested witllin the Ichthyophonida (bootstrap to a Blasu'\f search (vers. 2.0.10) of GenBank (Benson et al = 90%, BS = 7), a monophyletic sister-group of the 1999). The SSU sequence of lchthyophonus hoferi (Ichthyo Dermocystida (bootstrap = 99%, BS = 7). The latter sporea, Protozoa) yielded the highest score (Spanggaard et clade contained Rllinosporidium seeberi, the etiologi al 1996). Based on the results of this search, we downloaded cal agent of rhinosporidiosis of humans and other 19 SSU 18S rRNA from GenBank or the TreeCon website animals (Herr et al 1999). As indicated by the BlastJ.~ (http://rrna.uia.ac.ve/ssu/index.html). These included ichthyosporean sequences (Baker et al 1999, Cavalier-Smith search of GenBank, A. parasiticum was strongly sup 1998a, Herr et al 1999, Ragan et al 1996) and representa ported as a sister to Ichthyophonus hoferi (FIG. 1) tives of the major clades of fungi including the Harpellales (bootstrap = 94%, BS = 5). However, relationships (Trichomycetes). In addition, sequences of tlvo choanofla of major lineages within the Ichthyophonidia and gellates, Acanthocoepsis unguiculata and Diaphanoeca gran fungi were incompletely resolved by the SSU 18S dis (see FIG. 1), were selected for rooting the tree by the rDNA data as evidenced by one node within each of outgroup method. GenBank accession numbers for the 20 these clades with a bootstrap score of < 50%. terminals are as follows: Acanthocoepsis unguicu lata Ll0823, Amoebidium parasiticum AF274051, Anumfeca richardsi Wong & Beebee AF070445.1, Aspergillus fumigatus Fres. DISCUSSION M60300.1, Capnomyces stellatus S. W. Peterson & Lichtlv. Results of the present study confirm Whistler's AF007531.1, Chytridium confervae (Wille) Minden M59758.1, Dermocystidillm sp. U21336.1, Dermocystidium sal (1963) and Lichtwardt's (1986) hypothesis that Amoe monis U21337.1, Diaphanoeca unguiculata Ellis Ll0824, bidium pamsiticum (Amoebidiales) is a protozoan lchthyophonlls hoferi Plehn & Mulsow U25637, Neocallimas rather than a Trichomycete (Zygomycota). A BlastJ.~ tix sp. LM-2 M59761.1, ''Perkinsus'' atlanticus Azevedo search of GenBank and the present phylogenetic re AF192386.1 (the generic name is placed in quotes because construction based on parsimony analysis of SSU 18S BEN"'Y· .~"D O'DONNELL: AAIOEBIDIUM PAR4STTICUM 1135 Amoebidium parasiticum 188 rDNA Ichthyophonus hoted 527 steps Anuroteca richardsi 1of 3trees 58 AJ130859 Ichthyophonida lf CI =0.712 90 100 Ifperkinsus at/anticus Ichthyosporea RI =0.709 7 12 Sphaerosoma arcticum

1...... - Psorospermium haeckelii RC =0.505 82 2 87 Dermocystidium sp. -- 20 I 94 2Dermocystidium salmonis steps Dermocystida 99 Rhinosporidium seeberi 7 100 roseffe agent 10 97 9

100 40 fungi

I choanoftagellates ------....1 (outgroups)

FIG. 1. One of three most-parsimonious phylograms 527 steps in length found by PAUP*, using the heuristic search option with 1000 random addition sequences. Sequences of two choanoflagellates were used to root the tree. Bootstrap intervals 2:50% are indicated above nodes; numbers below nodes represent Bremer support calculated with TreeRot (So renson 1996). rDNA sequence data identified the ichthyosporean posed of protozoan parasites of crustaceans and fish intracellular fish parasite Ichthyophonus hoferi as its (Ragan et al 1996). Subsequently, five additional closest knmvn relative. The present molecular phy members of this clade were discovered using phylo logeny indicates that both taxa are nested within the genetics of ISS rRJ.'JA sequences: Anurofeca richardsi order Ichthyophonida of the class Ichthyosporea from tadpole larvae (Baker et al 1999), Sphaerosoma (Cavalier-Smith 1995a). This finding is consistent arcticum from a marine arctic invertebrate (B. Land with preliminary phylogenetic analysis of mitochon fald pers comm), "Perkinsus" atlanticus from a ma drial DNA that places A. pamsiticum at the root of rine clam, a cloned sequence from an experimental the animal-fungal clade as a sister taxon to choano microbial community (van Hannen et al 1999), and flagellates (Lang BF, Burger G. http://megasum. Rhinosporidium seebm? from humans with rhinospo bch.umontreal.ca/ogmp/projects/apara/gen.html). ridiosis (Herr et al 1999). Like Amoebidium pamsiti Phylogenetic analysis of a more inclusive SSU ISS cum, R seberi and 1. hofm? were thought to be fungi rDNA data set (data not shown) that included se because their thalli and/or sporangia are fungal-like quences of Ginkgo biloba and Zamia pumila as an out (reviewed in Herr et al 1999, Rand 1994); however, group did not resolve a possible relationship of the the latter two taxa were treated as incertae sedis by Ichthyosporea with the animals, fungi or choanofla Hawks-worth et al (1995). gellates (Wainright et al 1993). The Ichthyosporea Although all Ichthyosporea were thought to pos was initially identified near the animal-fungal diver sess parasitic stages (Cavalier-Smith 1995a), there is gence and informally named the DRIP clade com- no evidence that either Amoebidium pamsiticum or 1136 MYCOLOGL\

Anumfeca richardsi (Baker et al 1999) are paraSItic. use of the name by USDA implies no approval of the prod Furthermore, in addition to Amoebidium parasiticum uct to the exclusion of others that may also be suitable. (vVhistler 1962), pure cultures have been established for Anurafeca richardsi (cited in van Hannen et al 1999),1. hoferi (Spanggaard et al 1995), "Perkinsus" LITERATURE CITED atlanticus, and S. arcticum (B. Landfald pers comm) , Baker GC, Beebee TJC, Ragan MA. 1999. Prototheca richard indicating that these taxa are not obligate parasites. si, a pathogen of anuran larvae, is related to a clade of Interestingly, all five ichthyosporeans that have been protistan parasites near the animal-fungal divergence. cultured axenically are members of the Ichthyophon Microbiology 145:1777-1784. ida clade. Furthermore, the experimental conditions Benson DA, Boguski MS, Lipman DJ, Ostell J, Ouellette established in the microbial ecology study by van BFS, Rapp BA, v\'heeler DL. 1999. GenBank. Nucl Ac Hannen et al (1999) apparently supported growth of ids Res 27:12-17. a free living ichthyosporean from which the SSU Bremer K. 1988. The limits of amino acid sequence data in rDNA sequence i\J130859 was cloned. Nevertheless, angiosperm phylogenetic reconstruction. Evolution 42: because knowledge of their life cycles is incomplete, 795-803. Bruns TD, Vilgalys R, Barns SM, Gonzalez D, Hibbett DS, we cannot rule out the possibility that parasitic stages Lane DJ, Simon L, Stickel S, Szaro TM, Weisburg WG, are present in all of the ichthyosporeans. Sogin ML. 1992. Evolutionary relationships within the Results of the present molecular phylogeny sup Fungi: analyses of nuclear small subunit rRJ.'\lA sequenc port the classification of Cavalier-Smith (1998a) that es. Mol Phylogenet Evol 1:231-241. recognizes the monophyletic sister clades Ichthy Cavalier-Smith T. 1998a. Neomonada and the origin of an ophonida and Dermocystida within the Ichthyospo imals and fungi. In: Coombs GH, Vickerman K, Sleigh rea. Although tubulovesiculate and flat mitochondri MA, Warren A, eds. Evolutionary relationships among al cristae were thought to characterize these respec Protozoa. London: Chapman and Hall. p 375-407. tive sister orders (Ragan et al 1996, Herr et al 1999), ---. 1998b. A revised sLx-kingdom system oflife. BioI Rev cristae of Amoebidium parasiticum mitochondria are 73:203-266. mostly flat (vVhistler and Fuller 1968), indicating that Gunderson JH, Elwood H, Ingold A, Kindle K, Sogin ML. this character is homoplasious. Ironically, in addition 1987. Phylogenetic relationships betlveen chlorophytes, chrysophytes, and oomycetes. Proc Nat! Acad Sci USA to erecting a protozoan class for the ichthyosporeans 84:5823-5827. (Cavalier-Smith 1998a), Cavalier-Smith (1998b) also Hawksworth DL, Kirk PM, Sutton BC, Pegler DN. 1995. described the fungal class Enteromycetes to accom Ainsworth & Bisby's dictionary of the fungi. 8th ed. modate the Amoebidiales and Eccrinales (Lichnvardt Wallingford, UK: CAB International. 616 p. 1986) . Herr RA, Ajello L, Taylor jW, Arseculeratne SN, Mendoza Results of the present study also highlight the need L. 1999. Phylogenetic analysis of Rhinosporidium see to further test the monophyly of the Trichomycetes. beds 18S small-subunit ribosomal DNA groups this Fortunately, Lichnvardt (1973, 1986) and Moss and pathogen among members of the protoctistan Meso Young (1978) have published explicit testable hy mycetozoa clade. J Clin Microbiol 37:2750-2754. potheses outlining putative phyletic relationships of . Lichtlvardt RW. 1973. The Trichomycetes: what are their the Trichomycetes, including the poorly known and relationships? Mycologia 65:1-20. ---. 1986. The Trichomycetes, fungal associates of ar uncultured Eccrinales and Asellariales for which no thropods. New York: Springer-Verlag. 343 p. molecular systematic data are currently available. ---. 1997. Costa Rican gut fungi (Trichomycetes) in fecting lotic insect larvae. Rev BioI Trop 45:1349-1383. ---, Arenas 1996. Trichomycetes in aquatic insects ACKc"iOWLEDGMENTS J. from southern Chile. Mycologia 88:844--857. We dedicate this study to Robert \V. Lichtlvardt, University ---, Ferrington LC, L6pez Lama C. 1999. Trichomyce of Kansas, whose scholarly studies on the Trichomycetes tes in Argentinean aquatic insect larvae. Mycologia 91: stimulated our interest in their phylogeny, for his friendly 1060-1082. encouragement in all phases of this research and for sup ---, Williams Me. 1992. Two new Australian species of plying the pure culture of Amoebidium pamsiticum. Thanks Amoebidiales associated with aquatic insect larvae, and are due Bjarne Landfald, Norwegian College of Fishery Sci comments on their biogeography. Mycologia 84:376 ence, University of Troms0 for sharing unpublished data, 383. Elizabeth Cigelnik for assistance in preparing the sequence Moss ST, Young TWK. 1978. Phyletic considerations of the data, Larry W. Tjarks for primer synthesis, and Steve Prath Harpellales and Asellariales (Trichomycetes, Zygomy er for preparing FIG. 1. Names are necessary to report fac cotina) and the Kickxellales (Zygomycetes, Zygomyco tually on available data; however, the USDA neither guar tina). Mycologia 70:944-963. antees nor warrants the standard of the product, and the O'Donnell K, Cigelnik E, Benny GL. 1998. Phylogenetic re- BEN,,;Y AJ"iD O'DONNELL: AMOEBIDIUlvf P/iR1SITICUM 1137

lationships among the Harpellales and Kichellales. gins DG. 1997. The ClustaLX windows interface: flexi Mycologia 90:624-639. ble strategies for multiple sequence alignment aided by ---,---, Weber NS, Trappe JS. 1997. Phylogenetic quality analysis tools. Nucl Acids Res 25:4876-4882. relationships among ascomycetous truffles and the true Trotter MJ, \Nhistler He. 1965. Chemical composition of and false morels inferred from 18S and 28S ribosomal the cell wall of Amoebidium parasiticlim. Can J Bot 43: DNA sequence analysis. Mycologia 89:48-65. 869-876. Porter D, Smiley R. 1979. Ribosomal RL'lA molecular van Hannen EJ, Mooij W, van Agterveld MP, Gons HJ, Laan weights of Trichomycetes and Zygomycetes. Exp Mycol broek HJ. 1999. Detritus-dependent development of 3:188-193. the microbial community in an experimental system: Ragan NIA, Goggin CL, Cawthorn RJ, Cerenius L,Jamieson qualitative analysis by denaturing gradient gel electro AVC, Plourde SM, Rand TG, Soderhall K, Gutell RR. phoresis. Appl Environ Microbiol 65:2478-2484. 1996. A novel clade of protistan parasites near the an Wainright PO, Hinkle G, Sogin ML, Stickel SK. 1993. Mono imal-fungal divergence. Proc Natl Acad Sci USA 93: phyletic origins of the Metazoa: an evolutionary link 11907-11912. with Fungi. Science 260:340-342. Rand ,TG. 1994. An unusual form of Ichthyaphanus hofm Walker \NF. 1984. 5S ribosomal RL'lA sequences from Zyg (Ichthyophonales; Ichthyophonaceae) from yellowtail omycotina and evolutionary implications. Syst Appl Mi flounder Limanda ferruginea from the Nova Scotia crobiol 5:448-456. v\lhistler HC. 1962. Culture and nutrition of Amoebidium shelf. Dis Aquatic Organisms 18:21-28. parasiticum. AmJ Bot 49:193-199. Sanger VK, Lichtwardt RW, Kirsch JAW, Lester RL'l. 1972. ---. 1963. Observations on some new and unusual en, Immunological studies on the fungal genus Smittium terophilous Phycomycetes. CanJ Bot 41:887-900. (Trichomycetes). Mycologia 64:342-358. ---. 1968. Developmental control of Amoebidium par Sorenson MD. 1996. TreeRot. Computer program and doc, asiticlim. Dev BioI 17: 562-570. umentation. Ann Arbor: University of Michigan. ---, Fuller MS. 1968. Preliminary observations on the Spanggaard B, Huss HH, Bresciani J. 1995. Morphology of holdfast of Amoebidium parasiticlim. Mycologia 60: Ichthyaphonus hoferi assessed by light and scanning elec 1068-1079. tron microscopy. J Fish Dis 18:567-577. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and ---, Skouboe P, Rossen L, TaylorJW. 1996. Phylogenetic direct sequencing of fungal ribosomal RL'lA genes for relationships of the intercellular fish pathogen Ichthy phylogenetics. In: Innis MA, Gelfand DH, Sninsky lJ, ophonlls hoferi and fungi, choanoflagellates and the ro White TJ, eds. PCR protocols: a guide to methods and sette agent. Marine BioI 126:109-115. applications. San Diego: Academic Press. p 315-322. Swofford DL. 1999. PAUP*4.0: Phylogenetic Analysis Using Williams MC, Licht\vardt RW 1990. Trichomycete gut fungi Parsimony. Sunderland, Massachusetts: Sinauer. in New Zealand aquatic larvae. Can J Bot 68:1045 Thompson JD, Gibson TJ, Ple\vuiak F, Jeanmougin F, Hig- 1056.

Su~pfied by U.S. Dept of Agriculture NatIonal Center for Agricultural ! I:,:,,,,.l.; R h . . ""/Hl ,esearc J Peona, "'mois