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Phylogenetic analysis of the small subunit ribosomal 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 clearly resolved. Thus, we have expanded the number of isolates and taxa of hyphochytrids compared. The phylogenetic analysis of the small subunit ribosomal RNA sequence of Rhizidiomyces apophysatus Zopf based on neighbour-joining and parsimony methods showed that Hyphochytrium catenoides Karling and R. apophysatus are monophyletic and probably the closest relatives of the oomycetes. Our data also confirmed the monophyly of the stramenopiles, which includes heterokont algae along with nonphotosynthetic fungallike organisms, namely the Oomycota, Hyphochytriomycota, and Labyrinthulomycota. Key words: Hyphochytriomycota, heterokont algae, phylogeny, small subunit ribosomal gene. Résumé : Les relations possibles des hyphochytrides uniflagellées avec les straménopiles biflagellées n’est toujours pas claire. 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’Hyphochytrium catenoides Karling et le R. apophysatus sont monophylétiques et probablement les voisins les plus apparentés des oomycètes. 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, Hyphochytriomycota et Labyrinthulomycota. Mots clés : Hyphochytriomycota, algues hétérokontes, phylogénie, gène de la petite sous-unité ribosomale. [Traduit par la Rédaction]
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Introduction Rhizidiomyces apophysatus Zopf has been isolated from soil and pine pollen but has also been found to be parasitic on the oogonia of plant-pathogenic members of the Oomycota and on other fungi, as well as some algae (Fuller 1990). As a member of the phylum Hyphochytriomycota, it is nonphotosynthetic, possesses cell walls during growth, and has an absorptive mode of nutrition, characteristics that in the past had them placed within the kingdom Fungi as mastigomycetes, along with other zoosporic fungi, namely Received July 8, 1999. G. Hausner. Department of Biological Sciences, University of Calgary, 2500 University Drive, Calgary, AB T2N 1N4, Canada. A. Belkhiri. Biotechnology Research Institute, National Research 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:
[email protected]).
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the oomycetes and chytridiomycetes (Ainsworth 1973; Müller and Loeffler 1976). However, the presence of a single anteriorly directed flagellum with tripartite flagella hairs, as well as biochemical data (LéJohn 1971; Bartnicki-Garcia 1987) and ultrastructural data on their mitochondria and zoospores, suggest that the hyphochytrids and oomycetes are more closely related to the heterokont algae than to the true fungi (reviewed in Cavalier-Smith 1998). Thus, they have been included with the stramenopiles (Patterson 1989) or the infrakingdom Heterokonta (Cavalier-Smith 1998). Analysis of rRNA gene sequences has confirmed these placements for oomycetes (Förster et al. 1990) and for Hyphochytrium catenoides Karling (Van der Auwera et al.1995). These data also suggest that hyphochytrids and oomycetes are more closely related to each other than to the heterokont algae. Because the evolutionary position of the Hyphochytriomycetes has been a long-standing issue among mycologists (reviewed in Fuller 1990; Barr 1992) and because the convergent evolution of the “chytrid thallus type” among the zoosporic fungi is a possibility (Förster et al. 1990; Bowman et al. 1992), we felt that the question should be evaluated based on the available H. catenoides sequence and one other morphologically distinct representative of this phylum, © 2000 NRC Canada
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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 stramenopiles based on SSrDNA sequences. The branch lengths are those as determined by the NJ program. The arrowheads (䉲) point at branches that unite species belonging to the true fungi and those branches that lead to fungus-like organisms. The circle () shows the branch that unites all the tested members of the stramenopiles. GenBank accession numbers are given in parentheses.
R. apophysatus. This would confirm the relationship between the oomycetes and the hyphochytrids and would yield new data on the monophyly of the hyphochytrids themselves. A different isolate of H. catenoides was also investigated to confirm the available GenBank sequence. There are significant morphological and molecular differences between H. catenoides and R. apophysatus. Hyphochytrium catenoides is a member of the family Hyphochytriaceae and as such is characterized by a polycentric, eucarpic thallus, a structure with several reproductive sites for zoospores development. R. apophysatus is a member of the family Rhizidiomycetaceae, characterized by having monocentric thalli, consisting of a single cell that eventually develops into a sporangium (Sparrow 1960). Also, the organization of the mitochondrial DNA of the two hyphochytrids is different; only H. catenoides has the oomycetelike inverted repeat (McNabb et al. 1988). Therefore, small subunit rRNA gene (SSrDNA) sequences were obtained for R. apophysatus and a strain of H. catenoides, and these sequences were phylogenetically analyzed within a data set of 35 SSrDNA sequences that included representatives of the chlorobionts, metazoans, true fungi, alveolates, and stramenopiles.
Materials and methods Source and growth of strains Cultures of H. catenoides and R. apophysatus were obtained from D.J.S. Barr, Ottawa, Ont. (at DOAM under BR217 and
BR296, respectively). The organisms were cultured as described previously (McNabb et al. 1988).
DNA extraction, purification, fragment amplification, and sequencing The protocol employed to extract DNA was that of Garber and Yoder (1983). The polymerase chain reaction (PCR) amplification protocol used to generate sequencing templates, the preparation of DNA sequencing templates, and the sequencing of double-stranded PCR products were as described previously (Hausner et al. 1992). The oligonucleotide primers utilized for amplification of the SSrDNA were primers J (CTG GTT GAT CCT GCC AGT AG, positions 34–53 of the SSrRNA in Dams et al. (1988)) and T (ACG CCT TGT TAC GAC T, positions 3584–3608 of the LSrRNA in Dams et al. (1988)). Initial sequences were obtained by using primers J and T; thereafter, primers were designed as needed to complete the nucleotide sequences. We have included published sequences in our phylogenetic analysis as needed to provide thorough taxon sampling (see Fig. 1). This involved the use of 33 sequences in addition to the two new sequences reported here.
DNA sequence analysis The nucleotide sequences were aligned initially using CLUSTAL_X (Thompson et al. 1997) and the alignment was then refined by eye. The SSrDNA alignment was manually adjusted to improve the alignment and to remove those sequences that could not be unambiguously aligned. Also, approximately 40 nucleotide positions were omitted from the 5′ and 3′ ends of the 16s-like sequences, because many of the sequences included have incomplete ends. The alignment is available from the corresponding author by request. © 2000 NRC Canada
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Fig. 2. Majority-rule consensus tree inferred from NJ and parsimony analysis of 1000 bootstrap replicates of the SSrDNA aligned data set consisting of 35 sequences. The triangle (䉲) shows the branch that unites all the tested members of the stramenopiles. The numbers above the branches in the consensus tree are from the NJ analysis, while values below the branches are those obtained from analysing 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).
The data were analyzed using the programs contained within PHYLIP, version 3.5c (Felsenstein 1993). The NEIGHBOR program was used to generate unrooted phylogenetic 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 two parameter model (Kimura 1980). Phylogenetic estimates were also generated with the DNAPARS program to carry out unrooted parsimony on the aligned SSrDNA data set. Bootstrap resamplings (Felsenstein 1985) were used to estimate the reliability of the inferred trees obtained from both the parsimony and the neighborjoining programs. The jumble option was used in the DNAPARS and NEIGHBOR programs to randomize the nucleotide sequence input order, and the SSrDNA data set was analyzed three times using jumble seed numbers, 7, 11, and 19.
Results and discussion Phylogenetic analysis of the SSrDNA sequences clearly demonstrated that R. apophysatus is closely related to H. catenoides (Fig. 1); therefore, the Hyphochytriomycota are a potentially monophyletic group. The monophyly was supported in both the NJ and parsimony analysis with a level of support of 100% (Fig. 2). The SSrDNA sequence of our strain of H. catenoides was very similar to that of the strain used by Van der Auwera et al. (1995). We could detect dif-
ferences in nine positions between the two sequences, but most of them occurred in regions that could be prone to sequence artifacts. In seven of the positions, however, our sequence agreed with R. apophysatus and those of the oomyctes 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 AgriFoods Canada, Ottawa, Ont.) from pine pollen in Arizona. Our isolates of R. apophysatus and H. catenoides were also collected by D.J.S. Barr and both strains were isolated from organic soil in British Columbia. Overall, the data would suggest that our H. catenoides isolate and ATCC18719 very likely represent the same or closely related taxa. The NJ and the parsimony analysis combined with bootstrap analysis generated nearly identical consensus trees (Fig. 2). The stramenopiles as described by Patterson (1989), or the infrakindom Heterokonta (Cavalier-Smith 1998) were represented in our analysis by members of the Bacillariophyta, Phaeophyceae, Xanthophyceae, Chrysophyceae, Oomycota, Hyphochytriomycota, and Labyrinthulomycota, and these form a monophyletic group (Figs. 1 and 2). The monophyly of this grouping is supported by a level of confidence of 97.1% and 98.2% based on NJ and parsimony analyses, respectively. Within the stramenopiles the following groupings appear to be monophyletic based on our phylo© 2000 NRC Canada
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genetic analysis: (i) Bacillariophyta, (ii) Phaeophyceae with one member of the Xanthophyceae, (iii) Chrysophyceae (iv) Hyphochytriomycota, (v) Oomycota, and (vi) the Labyrinthulomycota. The divergence order of these six groups could not be determined with a high level of confidence (i.e., bootstrap values >95%), with the exception of the Labyrinthulomycota (thraustochytrids and slime-nets) which appear to be the first members of the stramenopiles to have diverged prior to the separation of the remaining five groups mentioned above. The overall topology of the groupings observed within the stramenopiles is congruent with data from Leipe et al. (1994), Van der Auwera et al. (1995), and Van der Auwera and De Wachter (1997). Our results also support the view that the alveolates are the closest relatives of the stramenopiles (Van der Auwera and De Wachter 1997). The phylogenetic relationship between the Oomycota and Hyphochytriomycota was not unambiguously resolved, although both the NJ and parsimony methods suggest that these two phyla share a common ancestor. Only the parsimony analysis supported this relationship at a level of confidence >95%, whereas the NJ analysis provided only weak support for this grouping (58.3% bootstrap value). Van der Auwera et al. (1995) in their NJ analysis also obtained a low bootstrap value (65%) for the node joining H. catenoides with the oomycetes. However, based on our SSrRNA sequence analysis and those of Van der Auwera et al. (1995) and Van der Auwera and De Wachter (1997), one can conclude that, among the members of the stramenopiles characterized so far, the Hyphochytriomycota are the closest relatives of the Oomycota. So there is a strong likelihood that the oomycetes and hyphochytriomycetes share a common ancestor. This ancestor had likely already lost its plastid (Cavalier-Smith 1998) but had a heterokont zoospore (two flagella types: tinsel and whiplash). The posterior flagella, then, was lost after the divergence of the Hyphochytriomycetes. The stramenopiles represent a monophyletic evolutionary lineage that includes the autotrophic heterokont algae as well as the heterotrophic oomycetes, hyphochytrids, labyrinthulids, thraustochytids, and bicosoecids (Cavalier-Smith et al. 1994; Leipe et al. 1994; Van der Auwera and De Wachter 1997). They all have tripartite tubular hairs on one flagellum, the sole synapomorphy for the kingdom Straminipila. The oomycetes and hyphochytrids were at one time placed within the kingdom Fungi because of ecology, absorptive mode of nutrition, and convergent evolution of funguslike thallus types. However, the phylogenetic evidence is now clear that they are true stramenopiles. The consensus trees derived from parsimony and NJ analysis like those published by Baldauf and Palmer (1993), Wainright et al. (1993), and Sugiyama (1998) suggest that Fungi and Metazoa share a common ancestry. It is possible that both the Metazoa and Fungi were independently derived from choanoflagellate protozoan ancestors (Cavalier-Smith 1998).
Acknowledgements We thank S.A. McNabb for helping with DNA preparations, and D.J.S. Barr for kindly providing isolates of R. apophysatus and H. catenoides. Research support was provided
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by the Natural Sciences and Engineering Research Council of Canada.
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