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Phylogenetic Analysis of the Small Subunit Ribosomal RNA of Marteilia.refringens Validates the Existence of Phylum Paramyxea (Desportes and Perkins, 1990)1

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ABSTRACT. Marteilia refringens is recognized as one of the most significant pathogens of bivalve molluscs. The nucleotide sequence of the small subunit ribosomal RNA gene of Marteilia refringens is used to elucidate the phylogenetic position of the
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  NOTES Phylogenetic analysis of the small subunitribosomal RNA gene of the hyphochytrid  Rhizidiomyces apophysatus Georg Hausner, Abbes Belkhiri, and Glen R. Klassen Abstract : The potential relationship of the uniflagellate hyphochytrids with biflagellate stramenopiles is still not clearlyresolved. Thus, we have expanded the number of isolates and taxa of hyphochytrids compared. The phylogenetic analy-sis of the small subunit ribosomal RNA sequence of   Rhizidiomyces apophysatus  Zopf based on neighbour-joining andparsimony methods showed that  Hyphochytrium catenoides  Karling and  R. apophysatus  are monophyletic and probablythe closest relatives of the oomycetes. Our data also confirmed the monophyly of the stramenopiles, which includesheterokont algae along with nonphotosynthetic fungallike organisms, namely the Oomycota, Hyphochytriomycota, andLabyrinthulomycota. Key words : Hyphochytriomycota, heterokont algae, phylogeny, small subunit ribosomal gene. Résumé  : Les relations possibles des hyphochytrides uniflagellées avec les straménopiles biflagellées n’est toujours pasclaire. A cette fin les auteurs ont augmenté le nombre d’isolats et de taxons d’hyphochytrides soumis à la comparaison.L’analyse phylogénétique des séquences de la petite sous-unité de l’ARN ribosomal du  Rhizidiomyces apophysatus ,Zopf basée sur les méthodes des liens de voisinage (neighbour-joining) et de parsimonie, montre que l’  Hyphochytriumcatenoides  Karling et le  R. apophysatus  sont monophylétiques et probablement les voisins les plus apparentés des oo-mycètes. Les données confirment également la monophylie des straménopiles, lesquels incluent des algues hétérokontes(heterokont) ainsi que des organismes d’apparence fongique non-chlorophylliens, nommément les Oomycota, Hypho-chytriomycota et Labyrinthulomycota.  Mots clés  : Hyphochytriomycota, algues hétérokontes, phylogénie, gène de la petite sous-unité ribosomale.[Traduit par la Rédaction]  Notes 128 Introduction  Rhizidiomyces apophysatus  Zopf has been isolated fromsoil and pine pollen but has also been found to be parasiticon the oogonia of plant-pathogenic members of the Oo-mycota and on other fungi, as well as some algae (Fuller1990). As a member of the phylum Hyphochytriomycota, itis nonphotosynthetic, possesses cell walls during growth,and has an absorptive mode of nutrition, characteristics thatin the past had them placed within the kingdom Fungi asmastigomycetes, along with other zoosporic fungi, namelythe oomycetes and chytridiomycetes (Ainsworth 1973;Müller and Loeffler 1976). However, the presence of a sin-gle anteriorly directed flagellum with tripartite flagella hairs,as well as biochemical data (LéJohn 1971; Bartnicki-Garcia1987) and ultrastructural data on their mitochondria andzoospores, suggest that the hyphochytrids and oomycetes aremore closely related to the heterokont algae than to the truefungi (reviewed in Cavalier-Smith 1998). Thus, they havebeen included with the stramenopiles (Patterson 1989) or theinfrakingdom Heterokonta (Cavalier-Smith 1998). Analysisof rRNA gene sequences has confirmed these placements foroomycetes (Förster et al. 1990) and for  Hyphochytrium cat-enoides  Karling (Van der Auwera et al.1995). These dataalso suggest that hyphochytrids and oomycetes are moreclosely related to each other than to the heterokont algae.Because the evolutionary position of the Hyphochytrio-mycetes has been a long-standing issue among mycologists(reviewed in Fuller 1990; Barr 1992) and because the con-vergent evolution of the “chytrid thallus type” among thezoosporic fungi is a possibility (Förster et al. 1990; Bowmanet al. 1992), we felt that the question should be evaluatedbased on the available  H. catenoides  sequence and one othermorphologically distinct representative of this phylum, Can. J. Bot.  78 : 124–128 (2000) © 2000 NRC Canada 124 Received July 8, 1999. G. Hausner.  Department of Biological Sciences, Universityof Calgary, 2500 University Drive, Calgary, AB T2N 1N4,Canada. A. Belkhiri.  Biotechnology Research Institute, NationalResearch Council of Canada, Montréal, QC H4P 2R2,Canada. G.R. Klassen. 1 Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. 1 Author to whom all correspondence should be addressed(e-mail: Glen_Klassen@umanitoba.ca).   R. apophysatus . This would confirm the relationshipbetween the oomycetes and the hyphochytrids and wouldyield new data on the monophyly of the hyphochytrids them-selves. A different isolate of   H. catenoides  was also investi-gated to confirm the available GenBank sequence.There are significant morphological and molecular differ-ences between  H. catenoides  and  R. apophysatus. Hypho-chytrium catenoides  is a member of the family Hypho-chytriaceae and as such is characterized by a polycentric,eucarpic thallus, a structure with several reproductive sitesfor zoospores development.  R. apophysatus  is a memberof the family Rhizidiomycetaceae, characterized by havingmonocentric thalli, consisting of a single cell that eventuallydevelops into a sporangium (Sparrow 1960). Also, the orga-nization of the mitochondrial DNA of the two hyphochytridsis different; only  H. catenoides  has the oomycetelike in-verted repeat (McNabb et al. 1988).Therefore, small subunit rRNA gene (SSrDNA) sequenceswere obtained for  R. apophysatus  and a strain of   H. caten-oides , and these sequences were phylogenetically analyzedwithin a data set of 35 SSrDNA sequences that included rep-resentatives of the chlorobionts, metazoans, true fungi, al-veolates, and stramenopiles. Materials and methods Source and growth of strains Cultures of   H. catenoides  and  R. apophysatus  were obtainedfrom D.J.S. Barr, Ottawa, Ont. (at DOAM under BR217 andBR296, respectively). The organisms were cultured as describedpreviously (McNabb et al. 1988). DNA extraction, purification, fragment amplification,and sequencing The protocol employed to extract DNA was that of Garber andYoder (1983). The polymerase chain reaction (PCR) amplificationprotocol used to generate sequencing templates, the preparation of DNA sequencing templates, and the sequencing of double-strandedPCR products were as described previously (Hausner et al. 1992).The oligonucleotide primers utilized for amplification of the SSr-DNA were primers J (CTG GTT GAT CCT GCC AGT AG, posi-tions 34–53 of the SSrRNA in Dams et al. (1988)) and T (ACGCCT TGT TAC GAC T, positions 3584–3608 of the LSrRNAin Dams et al. (1988)). Initial sequences were obtained by using primers J and T; thereafter, primers were designed as needed tocomplete the nucleotide sequences.We have included published sequences in our phylogenetic anal-ysis as needed to provide thorough taxon sampling (see Fig. 1).This involved the use of 33 sequences in addition to the two newsequences reported here. DNA sequence analysis The nucleotide sequences were aligned initially using CLUS-TAL_X (Thompson et al. 1997) and the alignment was then refinedby eye. The SSrDNA alignment was manually adjusted to improvethe alignment and to remove those sequences that could not be un-ambiguously aligned. Also, approximately 40 nucleotide positionswere omitted from the 5 ′  and 3 ′  ends of the 16s-like sequences, be-cause many of the sequences included have incomplete ends. Thealignment is available from the corresponding author by request. © 2000 NRC Canada Notes 125 Fig. 1.  Unrooted NJ phylogenetic network showing the potential evolutionary relationships of the hyphochytrids (  R. apophysatus  and  H. catenoides , GenBank accession Nos. AF163295 and AF163294, respectively) with the true fungi and members of the stramenopilesbased on SSrDNA sequences. The branch lengths are those as determined by the NJ program. The arrowheads (  ) point at branchesthat unite species belonging to the true fungi and those branches that lead to fungus-like organisms. The circle (  ) shows the branchthat unites all the tested members of the stramenopiles. GenBank accession numbers are given in parentheses.  The data were analyzed using the programs contained withinPHYLIP, version 3.5c (Felsenstein 1993).The NEIGHBOR program was used to generate unrooted phylo-genetic distance networks based on the neighbor-joining (NJ)method (Saitou and Nei 1987). Divergence (or distance) between two sequences was calculated by DNADIST using Kimura’s twoparameter model (Kimura 1980). Phylogenetic estimates were also generated with the DNAPARS program to carry out unrooted par-simony on the aligned SSrDNA data set. Bootstrap resamplings(Felsenstein 1985) were used to estimate the reliability of the in-ferred trees obtained from both the parsimony and the neighbor- joining programs. The jumble option was used in the DNAPARSand NEIGHBOR programs to randomize the nucleotide sequenceinput order, and the SSrDNA data set was analyzed three times us-ing jumble seed numbers, 7, 11, and 19. Results and discussion Phylogenetic analysis of the SSrDNA sequences clearlydemonstrated that  R. apophysatus  is closely related to  H. cat-enoides  (Fig. 1); therefore, the Hyphochytriomycota are apotentially monophyletic group. The monophyly was sup-ported in both the NJ and parsimony analysis with a level of support of 100% (Fig. 2). The SSrDNA sequence of ourstrain of   H. catenoides  was very similar to that of the strainused by Van der Auwera et al. (1995). We could detect dif-ferences in nine positions between the two sequences, butmost of them occurred in regions that could be prone tosequence artifacts. In seven of the positions, however, oursequence agreed with  R. apophysatus  and those of theoomyctes included in our analysis. Van der Auwera et al.(1995) used isolate ATCC18719, a strain isolated by D.J.S.Barr (Biosystematics Research Centre, Agriculture and Agri-Foods Canada, Ottawa, Ont.) from pine pollen in Arizona.Our isolates of   R. apophysatus  and  H. catenoides  were alsocollected by D.J.S. Barr and both strains were isolated fromorganic soil in British Columbia. Overall, the data wouldsuggest that our  H. catenoides  isolate and ATCC18719 verylikely represent the same or closely related taxa.The NJ and the parsimony analysis combined with boot-strap analysis generated nearly identical consensus trees(Fig. 2). The stramenopiles as described by Patterson (1989),or the infrakindom Heterokonta (Cavalier-Smith 1998) wererepresented in our analysis by members of the Bacil-lariophyta, Phaeophyceae, Xanthophyceae, Chrysophyceae,Oomycota, Hyphochytriomycota, and Labyrinthulomycota,and these form a monophyletic group (Figs. 1 and 2). Themonophyly of this grouping is supported by a level of confi-dence of 97.1% and 98.2% based on NJ and parsimony anal-yses, respectively. Within the stramenopiles the followinggroupings appear to be monophyletic based on our phylo- © 2000 NRC Canada 126 Can. J. Bot. Vol. 78, 2000 Fig. 2.  Majority-rule consensus tree inferred from NJ and parsimony analysis of 1000 bootstrap replicates of the SSrDNA aligned dataset consisting of 35 sequences. The triangle (  ) shows the branch that unites all the tested members of the stramenopiles. The num-bers above the branches in the consensus tree are from the NJ analysis, while values below the branches are those obtained from ana-lysing the bootstrap replicates by the parsimony program (DNAPARS).  Hyphochytrium catenoides  1, GenBank accession No.AF163294;  Hyphochytrium catenoides  2, GenBank accession No. X80344 (Van der Auwera et al. 1995).  genetic analysis: ( i ) Bacillariophyta, ( ii ) Phaeophyceae withone member of the Xanthophyceae, ( iii ) Chrysophyceae( iv ) Hyphochytriomycota, ( v ) Oomycota, and ( vi ) the La-byrinthulomycota. The divergence order of these six groupscould not be determined with a high level of confidence (i.e.,bootstrap values >95%), with the exception of the Labyrin-thulomycota (thraustochytrids and slime-nets) which appearto be the first members of the stramenopiles to have di-verged prior to the separation of the remaining five groupsmentioned above. The overall topology of the groupings ob-served within the stramenopiles is congruent with data fromLeipe et al. (1994), Van der Auwera et al. (1995), and Vander Auwera and De Wachter (1997). Our results also supportthe view that the alveolates are the closest relatives of thestramenopiles (Van der Auwera and De Wachter 1997).The phylogenetic relationship between the Oomycota andHyphochytriomycota was not unambiguously resolved, al-though both the NJ and parsimony methods suggest thatthese two phyla share a common ancestor. Only the parsi-mony analysis supported this relationship at a level of confi-dence >95%, whereas the NJ analysis provided only weak support for this grouping (58.3% bootstrap value). Van derAuwera et al. (1995) in their NJ analysis also obtained a lowbootstrap value (65%) for the node joining  H. catenoides with the oomycetes. However, based on our SSrRNA se-quence analysis and those of Van der Auwera et al. (1995)and Van der Auwera and De Wachter (1997), one can con-clude that, among the members of the stramenopiles char-acterized so far, the Hyphochytriomycota are the closestrelatives of the Oomycota. So there is a strong likelihoodthat the oomycetes and hyphochytriomycetes share a com-mon ancestor. This ancestor had likely already lost its plastid(Cavalier-Smith 1998) but had a heterokont zoospore (twoflagella types: tinsel and whiplash). The posterior flagella,then, was lost after the divergence of the Hyphochytrio-mycetes.The stramenopiles represent a monophyletic evolutionarylineage that includes the autotrophic heterokont algae as wellas the heterotrophic oomycetes, hyphochytrids, labyrinthu-lids, thraustochytids, and bicosoecids (Cavalier-Smith et al.1994; Leipe et al. 1994; Van der Auwera and De Wachter1997). They all have tripartite tubular hairs on one flagel-lum, the sole synapomorphy for the kingdom Straminipila.The oomycetes and hyphochytrids were at one time placedwithin the kingdom Fungi because of ecology, absorptivemode of nutrition, and convergent evolution of funguslikethallus types. However, the phylogenetic evidence is nowclear that they are true stramenopiles.The consensus trees derived from parsimony and NJ anal-ysis like those published by Baldauf and Palmer (1993),Wainright et al. (1993), and Sugiyama (1998) suggest thatFungi and Metazoa share a common ancestry. It is possiblethat both the Metazoa and Fungi were independently derivedfrom choanoflagellate protozoan ancestors (Cavalier-Smith1998).  Acknowledgements We thank S.A. McNabb for helping with DNA prepara-tions, and D.J.S. Barr for kindly providing isolates of   R. apo- physatus  and  H. catenoides . Research support was providedby the Natural Sciences and Engineering Research Councilof Canada. 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