SEQUENCES OF RIBOSOMAL GENES AND INTERNAL TRANSCRIBED

J. Gen. Appl. Microbiol., 41, 31-42 (1995)

SEQUENCES OF RIBOSOMAL GENES AND INTERNAL TRANSCRIBED SPACERS MOVE THREE PLANT

PARASITIC FUNGI, ER EMOTHECIUM ASHBYI, ASHB YA GOSSYPH, AND NEMA TOSPORA COR YLI, TOWARDS SACCHAROMYCES CEREVISIAEt

ROBERT MESSNER,* HANSJORG PRILLINGER, MARTIN IBL,'

AND GOTTFRIED HIMMLER

Institut fur Angewandte Mikrobiologie, Universitdt fur Bodenkultur, A-1190 Wien, Austria ' Codon Genetic Systems, A-1180 Wien, Austria

(Received September 6, 1994; Accepted December 6, 1994)

Phylogenetic relationships between two species from the genera Saccha-romyces (S. cerevisiae, S. kluyveri) and Kluyveromyces (K. aestuarii, K. marxianus), a plant parasitic dimorphic yeast (Nematospora coryli), and two phytopathogenic filamentous fungi (Ashbya gossypii, Eremothecium ashbyi) were investigated by comparing partial nucleotide sequences of ribosomal DNA (rDNA). The three types of sequenced regions evolve with different speed and allow the analysis at distinct taxonomic levels: conserved regions (18S rDNA, small ribosomal subunit), variable regions

(25S rDNA, large ribosomal subunit), and highly variable regions ITS1 and ITS2 (internal transcribed spacers). Segments homologous to posi-tions 619 to 1035 and 1205 to 1617 of the 18S rDNA and positions 470 to 890 and 1535 to 1984 of the 25S rDNA of S. cerevisiae were sequenced. The maximal resolving power of rDNA sequence analysis is provided by including the rapidly evolving ITS 1 and ITS2 regions. In the phylogenetic sequence analysis of all regions by the genera Kluyveromyces and Saccha-romyces cluster together with two filamentous pathogens on cotton, A.

gossypii and E. ashby, as well as the plant pathogenic yeast N. coryli. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores, the persistence of ascus walls as well as mycelium formation should not be used as differentiating characters in

yeast taxonomy, especially in family delimitation. Our sequence data support the inclusion of plant pathogenic, predominantly filamentous

t Dedicated to Prof . DI. Dr. Hermann Katinger for his efforts to establish the new IAM in

Vienna. * Address reprint requests to: Dr . Robert Messner, Institut fur Angewandte Mikrobiologie,

Universitat fur Bodenkultur, NuBdorfer Lande 11, A-1190 Wien, Austria.

31

32 MESSNER et al. VOL. 41

genera like Ashbya or Eremothecium or dimorphic genera like Nemato-spora with falcate ascospores within a new family of the Saccharomy-

cetaceae. Similarly, the saprophytic genus Kluyveromyces with reniform

ascospores and deliquescent ascus walls unequivocally belongs to the

family of Saccharomycetaceae.

As indicated by the etymology of the name Saccharomyces, Meyen acknowl-edged the fact that yeasts are true fungi (16). Yeast taxonomy, unlike the systematics of filamentous fungi, however, has a traditionally applied origin, with its roots in the fermentation industries of the late 19th and early 20th centuries

(26). Yeast and fungal systematics have developed distinctly during the last century, since they were characterized by different methods, especially physiologi-cal properties in yeast taxonomy and morphological characters for filamentous fungi. Wickerham (29, 30) concluded that the classification of yeasts will be incorrect until the related filamentous forms could be studied thoroughly by persons who knew these fungi as well as the yeasts. Although von Arx and van der Walt

(27) recently reinforced morphological characters like the shape and ornamenta-tion of ascospores, the consistency of the ascus wall, and the presence or absence of hyphae to define families of the Endomycetales, their system remains artificial when compared with classifications based on molecular characters (1, 2, 8,11,12, 21, 22).

In the present paper, we investigated the phylogenetic relationships between saprophytic yeasts of the genera Saccharomyces and Kluyveromyces and the plant

pathogenic, filamentous fungi Ashbya gossypii and Eremothecium ashbyi. Nemato-spora coryli, a dimorphic fungus, parasitic on hazel nut, soybeans and other hosts

(31) was also included in our investigations. The ascospores of Saccharomyces species are smooth and spherical; in Kluyvero-myces they are reniform. Spindle-shaped until acicular ascospores, often whip-like at one end, and occasionally septate, are known in N. coryli, A. gossypii and E. ashbyi (27).

Yamada and Nagahama (32) showed a close relationship between N. coryli and S. cerevisiae based on partial sequences of the 18S ribosomal ribonucleic acid. We used the partial sequences of ribosomal DNA to establish affinities between the above mentioned filamentous and unicellular fungi. With respect to the resolution desired, one to three appropriately chosen sections of the 18S and 25S rDNAs in a size range of 200 to 300 basepairs supports decisions in systematics (17). Two hundred bases near the 3' end of the 18S rRNA allow differentiation of the ascomycetous yeasts Holleya, Nematospora and Metschnikowia (32). Resolu-tion may be increased by sequencing the entire 18S gene (2, 8,18). Combination of two sections within the 25S rDNA together with 18S rDNA partial sequences results in a broader base for phylogenetic interpretations (11, 32). For phylogenetic analysis of closely related species, we have chosen the internal transcribed spacers ITS 1 and ITS2, which are aligned over 575 basepairs.

1995 Plant Parasitic Relatives of Saccharomyces cerevisiae 33

These highly variable sequences may show variation within a single species as shown in Fusarium sambucinum (19). To check the consistency of the relationships investigated, we sequenced four sections with an average length of 450 basepairs, selected within the 18S and 25S rDNAs, as well as the ITS 1, the ITS2 and the 5.8S rDNA.

MATERIALS AND METHODS

Cultivation of organisms. Yeasts and yeast-stages of filamentous fungi (see Table 1) were cultured in 20 ml GYP-medium in 100-ml Erlenmeyer flasks at 150 rpm and 28°C for 1 to 3 days.

DNA preparation. Culture broth (1.5 ml) was subjected to a CTAB-extraction of chromosomal DNA according to Lieckfeldt et al. (13). RNA was not removed from nucleic acid preparations. The DNA concentration was estimated by agarose gel electrophoresis of the samples and comparison of ethidium bromide-stained fluorescence with undigested lambda DNA of known concentrations. Amplification of ribosomal DNA. Sections of 1 and 1.5 kb were amplified from 18S and 25S rDNA, respectively, as described in Ref. 15), using homologous Taq-DNA polymerase (Biomedica, Wien, Austria), 4.5 mM magnesium sulfate, a volume of 50,al per reaction and 50 to 100 ng template DNA. Thirty-five cycles of the program 98 ° C/ 15 s, 55°C/60 s, and 72°C/i50 s were performed in a thermo-cycler (Trio-Thermoblock TB1, Biometra, Gottingen, Germany). Primers for the ITS 1 and ITS2 regions were synthesized corresponding to

primers ITS 1, -2, -3 and -4 (28) : The primers for amplification of the 18S and 25S rDNA parts, respectively, have been designed for optimal use in PCR and sequenc-ing by the OLIGO Primer Analysis Software (National Biosciences, MA, U.S.A.). The primer pair used to amplify 25S rDNA from base 450 to base 2016 was 5'-dACC GAT AGC GAA CAA GTA-3' and 5'-dCGA CTT CCC TTA TCT ACA TT-3'. The primer pair for 18S rDNA from base 576 to base 1628 was 5'-dGGT AAT TCC AGC TCC AAT A-3' and 5'-dGAC GGG CGG TGT GTA-3'. The numbering of bases relates to the respective sequences of Saccharomyces cerevisiae.

Table 1. List of organisms used for DNA sequencing.


The internal transcribed spacer regions including the 5.8S rDNA gene was ampli-fied with the primer pair ITS 1 and ITS4 (28). Primer synthesis was performed by Codon Genetic Systems (Vienna, Austria) using a 392 DNA synthesizer (Applied Biosystems, CA, U.S.A.). PCR reactions containing a single fragment were diluted by 3 volumes of TE buffer and precipitated by 4 volumes of PEG-buffer (13 % w/v polyethylene glycol 6000, 1.6 M NaCI) for at least 2 h on ice. After centrifugation, the pellet was washed in 70% (v/v) ethanol, dried and dissolved in 20 al water. Sequencing of PCR fragments. The fragments obtained after PEG precipita-tion and ethanol washing are free of amplification primers. After estimation of DNA concentration by agarose gel electrophoresis, the sequencing reactions were

performed by Codon Genetic Systems using a 373 A automatic DNA sequencer (Applied Biosystems). The respective primers mentioned in the PCR amplification protocol were used as sequencing primers in the fluorescent dye dideoxy nucleotide termination procedure. At least 600 bases per run were recorded, and not less than 2 independent runs on different preparations of PCR fragments were used for data collection. For the sequencing of the ITS regions on the 5.8S rDNA gene, additional runs along the reverse strand have been necessary, using primers ITS2 and ITS3. The 19 sequences are accessible from GenBank under the accession numbers U09310 to U09328 (see Table 2). Sequence analysis. Partial sequences of rDNA obtained and reference se-

quences derived from complete subunit data (GenBank) were aligned by the CLUSTAL V program (9). In the alignment options the penalty for interruption of sequences was set slightly higher or equal to the penalty for extending an existing

gap. All gap positions within the alignment exceeding a single base in length were replaced by question marks. Sequence insertions without homology to any of the other sequences were deleted in the alignment and a single base was left over causing a minimal gap. In this way gaps of all sizes were weighted equal corresponding to a single event in evolution. Thirty-four positions of the 18S rDNA alignment and about 30% of the ITS sequence alignment were omitted, since they were ambiguous. Phylogenetic computation of final alignments was done with the programs dnadist (set to Kimura "2 parameter"; (10)), fitch (6), segboot and consense in the PHYLIP package (S). Bootstrap confidence values were calculated from 1,000 samples.

RESULTS

Partial sequences of chromosomal ribosomal genes and the sequences of both internal transcribed spacers ITS 1 and ITS2 (see Table 2) were used to investigate the evolutionary distances between two genera of the classical yeasts (Kluyvero-myces and Saccharomyces), the dimorphic, plant parasitic yeast, Nematospora, and two phytopathogenic filamentous fungi (Ashbya and Eremothecium). For determining the relationships among distant taxa, two regions of the 18S

1995

Table

Plant Parasitic Relatives of Saccharomyces cerevisiae

2. Origin of rDNA sequences for phylogeny analysis: Species names

and accession numbers.

35

rDNA have been chosen: positions 619 to 1035 and 1205 to 1617 according to the

sequence of the respective gene in S. cerevisiae. Although the 18S rRNA gene

represents the most conserved ribosomal gene, considerable heterogeneity was

observed in the alignment, especially within the 150 bases at the 5' end. The

second, downstream region was found less variable, although the content of


diagnostic sites has already led to heavy use of this part of the ribosomal gene for systematic purposes (11, 32). Analysis of the combined regions of the 18S rDNA (Fig. 1) shows the clustering of the three phytopathogenic fungi A. gossypii, E, ashbyi, and N. coryli together with the members of the Saccharomycetaceae as well as with Kluyvero-myces lactis. The data obtained from the 829-basepair alignment of partial gene sequences are in good agreement with analyses of the complete 18S rDNA sequences (8,18). The Glade of Saccharomyces, Zygosaccharomyces, Torulaspora, and C. glabrata is well supported by 92% bootstrap confidence and joins the Glade of the plant

parasitic fungi Ashbya, Eremothecium, and Nematospora together with Kluyvero-myces in a node of 78% bootstrap confidence. This means a good barrier against the Glade of Pichia, Debaryomyces, and C. albicans, which represent the closest taxons not affiliated with the Saccharomycetaceae. The firmness of the tree calculated from partial sequences is demonstrated by the bootstrapping values against the outgroups. Separation as well as clustering of the "classical yeasts" in two different orders, Endomycetales and Schizosaccharomycetales, is supported at both nodes by 100% bootstrap confidence, respectively (11, 21). Outgroups sampled from the filamentous fungi were Podospora, Neurospora, Leucostoma, and Ophiostoma. They

yield a tree topology comparable to analysis from complete 18S rRNA gene sequences (8,18).

Fig. 1. Phylogenetic tree of 16 fungi and yeasts (see Table 2) derived from two

partial 18S rDNA sequences, positions 619 to 1035 and 1205 to 1617. The algorithms used for tree construction are referred to in MATERIALS AND METHODS. Bootstrapping values were calculated from 1,000 cycles.


The two regions sequenced from the 25S rDNA cover the positions 470 to 890 and 1535 to 1984, according to the nomenclature of the S, cerevisiae gene. Since these regions are longer than the partial sequences available from literature, in the respective alignment only 7 species are integrated, 4 of which derive from total 255 rDNA sequences in public databases (see Table 2). Furthermore, the number of ambiguous or misidentified bases is usually higher in small, partial sequences. Figure 2 shows the phylogenetic tree calculated from the 908-basepair alignment of the combined partial sequences. In contrast to the alignments from the 185 rDNA and the ITS regions, no ambiguously aligned positions had to be removed. Al-though the number of taxons is limited, the topology of the tree derived from 185 rDNA data (Fig. 1) is corroborated. The clustering of the three plant pathogenic fungi Ashbya, Eremothecium, and Nematospora is supported again with 100% bootstrap confidence. The affiliation of Saccharomyces to this Glade is ascertained by 64%. The attachment of C. albicans as well as the evolutionary distances

(summarized branch lengths parallel to the x-axis between two taxons) agrees with Fig. 1. Again, this cluster is well separated from the outgroup Mucor racemosus and from Schizosaccharomyces with 92% bootstrap confidence. The highest sequence variation was obtained from the rapidly evolving internal transcribed spacers, ITS 1 and ITS2. The disadvantage in the use of these regions is that the distance between organisms to be analyzed is limited. Figure 3 shows the

phylogenetic tree obtained from the alignment of both internal transcribed spacers regions, ITS 1 and ITS2. Due to the variability of these regions, the alignment

program CLUSTAL V was set to the highest window size for checking the location of matching parts. Nevertheless, correction by eye and omission of ambiguously aligned parts was necessary, since too many variable sites are concerned at once. The two members of the old family of the Saccharomycetaceae, S. cerevisiae, and S. kluyveri, represent the largest distance within a genus and is supported with 100% bootstrap confidence. The same quality is assigned to the Glade comprising the two

i/i WV V I

Fig. 2. Phylogenetic tree of 7 fungi and yeasts (see Table 2)

partial 25S rDNA sequences, positions 470 to 890 and 1535 to 1984. The algorithms used for tree construction are referred to in METHODS. Bootstrapping values were calculated from 1,000 cycles.

I W V V I I VVV WV

derived from two

MATERIALS AND


Kluyveromyces strains. By the stringent association with K. marxianus the correct

genus of the marine yeast K. aestuarii is proven. The short evolutionary distances within the Glade of Ashbya, Eremothecium, and Nematospora in relation to those

found in the good genus Saccharomyces suggest that the three plant pathogens

could meet in one genus, because the high resolution of the ITS sequence compar-

ison supplies us with the best evidence for relationships at the genus level.

DISCUSSION

The extent of divergence in partial nucleotide sequences from 18S and 25S ribosomal RNAs was used recently by Kurtzman (I1) and Kurtzman and Robnett

(12) to assess the placement of genera among families and orders of ascosporoge-nous yeasts and yeast-like fungi. The RNA comparisons showed four phylogeneti-cally distinct groups, which comprise the following taxa: Group 1. The budding

yeasts Saccharomyces, Saccharomycopsis, Debaryomyces, Metschnikowia, Saturno-spora, and Lipomyces together with the yeast-like genera Ascoidea, Cephaloascus, Dipodascus, Dipodascopsis, and Galactomyces were included in a common order Endomycetales; Group 2. Eremascus, Emericella, and Ceratocystis; this group exhibits affinities to the higher ascomycetes; Group 3. Taphrina and Protomyces

(order Taphrinales); Group 4. Schizosaccharomyces (new order Schizosaccharomyc-etales). In the present paper, we have investigated genera suspected to be closely related to the genus Saccharomyces based on the presence of dityrosine in the meiotic endospores (Breitenbach and Briza, personal communication, 22), the

qualitative and quantitative cell wall monosaccharide composition, and ubiquinone spectra (21-23). Six partial rDNA sequences were analyzed by establishment of a Kimura "2 parameter" distance matrix (10) and subsequent construction of a

Fig. 3. Phylogenetic tree of 9 fungi derived from ITS 1 and ITS2 region se-

quences.

Ambiguous parts of the alignment have been omitted before computation. The

algorithms used for tree construction are referred to in MATERIALS AND METHODS.

Bootstrapping values were calculated from 1,000 cycles.


phylogenetic tree (6) using the PHYLIP software package (5). The use of partial sequences from genes, which differ in the speed of their evolution, is superior to complete sequences of single genes, when phylogenetic relationships at different taxonomic levels are investigated.

In this study, the results of the partial 18S rDNA sequences generally meet the analysis of entire gene comparisons (8). A comparable analysis of the entire 18S rDNA sequences was performed on a wider field of fungal orders by Nishida and Sugiyama (18), using the Neighbor Joining algorithm. Although the evolutionary distances in both papers were equivalent, there is a difference in the tree topologies, as demonstrated by the respective branches leading to Mucor racemosus. The differences between the 18S rDNA trees of those two papers and Fig. 1 affect the tree topology rather than the evolutionary distances. The fine structure of the tree inferred from partial sequence data is altered in some less supported nodes when compared with that one of Hendriks and coworkers (8). In the cluster of the ascomycetous yeasts excluding Schizosaccharomyces, the positions of Pichia and C.

glabrata are moved over one node. The integration of sequences from the 25S rRNA gene is thought to add regions of higher variability, yielding a phylogeny of enlarged quality (11,12) . Still some details of analysis may be surprising, as reported by Yamada and Nagahama

(32). Partial sequencing showed that in Holleya sinecauda and Nematospora coryli the substitution rate in the 185 rDNA was 7 times higher than in the 255 rDNA.

Since the partial 255 rDNA sequences investigated in the present paper are longer than partial sequences reported in the literature, only 7 species are included in the analysis. The clustering from the 18S rDNA data are corroborated (Fig. 2). A. gossypii, E. ashbyi, and N. coryli are close to S. cerevisiae. The bootstrap confidence values for this Glade with 64% is slightly less than in Fig. 1 with 78%, but upon joining of C. albicans a bootstrap confidence of 92% against Schizosac-charomyces and the outgroup is achieved.

When the partial sequences of Metschnikowia bicuspidata (12) are joined to the appropriate parts of the sequences used for Figs. 1 and 2, the recalculation again

points to a rather distant relationship of the genus Metschnikowia to the genera Ashbya, Eremothecium, Kluyveromyces, Nematospora, and Saccharomyces. Yamada and Nagahama (32) elucidated the position of Metschnikowia biscupidata and M. reukaufii, which possesses ascospores similar to that known for Nematospora, Eremothecium and Ashbya (31). Ribosomal sequence analyses clusters the latter

group apart from Metschnikowia (11,12). Although the spores of Metschnikowia are spindle-shaped, they differ from those of Nematospora in a number of character-istics: the absence of a whip-like appendage and a homogeneous appearance when observed with a light microscope (3). A different habitat and an independence of inositol may be further characteristics which point to a not-too-close relationship between the genus Metschnikowia and Nematospora, Ashbya, and Eremothecium. Sequencing of the internal transcribed spacers ITS 1 and IT52 provides the best

reliability in detection of close phylogenetic distances. This method generally is


used for joining anamorph and teleomorph stages of a single taxon (4,14). Figure 3 demonstrates the resolving power of this approach. Three clades can be distinguished:

1. S. cerevisiae and S. kluyveri 2. K. marxianus and K, aestuarii 3. N. coryli, E. ashbyi, and A. gossypii

All clades together are strictly distinguished from C. albicans and the outgroup Fusarium solani, and are supported by sufficient bootstrapping values. It is noteworthy that the evolutionary distances within the Glade of A. gossypii, E. ashbyi and N. coryli are smaller than within the genera Saccharomyces or Kluyveromyces. This finding supports the assignment of these three plant pathogenic fungi to a single genus (Kurtzman, personal communication). On the base of molecular data, we agree with Wingard (31) that parasitic yeasts and fungi around Nematospora with its interesting relations to insects appears to be excellent material for the study of the general problems as to the origin and relationship of the entire yeast series. From the general summarization of our data (Table 3) we conclude that the family of Saccharomycetaceae should harbour the genera Saccharomyces, Torulaspora, Zygosaccharomyces, Arxiozyma, and Pachytichospora as suggested by von Arx and van der Walt (27). From the

present investigations and data from Hendriks et al. (8) the genera Kluyveromyces, Nematospora, Eremothecium, and Ashbya and species like Candida glabrata can be included. Furthermore the close relationships between Saccharomyces cerevisiae and Ashbya gossypii was found upon comparison of translation elongation factor la

(25). Nucleotide homology of 88.6% including activating elements upstream of the promoter are reported as a surprising result in the light of traditional taxonomy.

Genera from the borderline between polykaryotic sporangia and uninucleate asci (20, 21, 23) like Ashbya, Eremothecium (7), and Nematospora (3) corroborate

Table 3. Pronosal for classification of some ascosnororenous yeasts and yeast-like fungi.


the interpretation from Smith (24) and Kurtzman and Robnett (12), that the Endomycetales are indeed primitive fungi ancestral to the present day asco- and basidiomycetes and are not reduced forms of extant filamentous fungi. A final, speculative statement deals on evolution: The genera mentioned above may have developed in general as filamentous, originally "siphonal" and polykaryotic plant

pathogens (20), with yeast stages present. The lack of plant pathogens fitting to Saccharomyces and Kluyveromyces can be explained by an extinction of the respec-tive hosts. This, however, was not the fate of cotton and hazel nut and their

parasites analyzed in this paper.

We gratefully acknowledge W. Pinsker for correcting the manuscript. We thank C. P. Kurtzman, U. Stahl, and K. Scheide for providing strains and valuable literature, F. Ri ker and S. Huss for helpful comments in computing data as well as M. Breitenbach, P. Briza, E. Staudacher, K. Lopandic, and 0. Molnar for discussing unpublished results. This work was supported by the Austrian "Fonds zur Forderung der Wissenschaftlichen Forschung," project P9255.

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SEQUENCES OF RIBOSOMAL GENES AND INTERNAL TRANSCRIBED