Botanical Journal of the Linnean Socie£r 1989),99,' 3-10. 615

IDlpact of cODlparisons on systeDlatic Dlycology

C. P. KURTZMAN

]oiorthem Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604, U.S.A.

KURTZMAN, C. P., 1989. Im.pact of nucleic acid comparisons on systematic mycology. The use of molecular techniques for taxonomic purposes is reviewed and their importance pointed out. The result will be a more realistic classification system and a more thorough understanding of genetic mechanisms that will impact the control ofplant and animal diseases and the development of more efficient industrial fermentations.

ADDITIONAL KEY WORDS:-Taxonomy - evolution - yeasts.

CONTENTS

Introduction . 3 :\lechanisms ofspecies formation 3 Nuclear DNA comparisons. 5 Mitochondrial DNA relatedness 7 Ribosomal RNA relatedness 7 Conclusions 8 References. 9

INTRODUCTION Molecular biology has progressed rapidly and a variety of techniques have resulted that can now be used by mycologists to understand the phylogenetic relationships of species, genera and higher taxonomic groupings. This is an unprecedented opportunity, because our present system of classification is strongly based on phenotypic characters, such as physiological reactions and morphology. These are characters that generally have an unknown genetic basis, and frequently the mycologist must rely on intuition for their interpretation. The intent ofthis briefrevie\\' is to discuss the types ofmolecular comparisons presently in use and to assess the degree of genetic resolution that each can provide.

MECHANISMS OF SPECIES FORMATION 'What is a species? That the answer to this question has been debated so long, and still continues to provoke serious argument, attests to the difficulty ofdefining

The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned. 3 002+-407+/89/010003+07503.00/0 4 C. P. KURTZMAN a species. Dobzhansky (1976) has championed the concept that species can be defined in terms of genetics, and if this is true, the species, the primary unit of taxonomy, becomes a product of nature rather than the creation of the taxonomist. Dobzhansky (1976) states that among sexually reproducing and outbreeding organisms, species can be defined as Mendelian populations or arrays ofpopulations that are reproductively isolated from other population arrays. This definition particularly applied to higher animals where reproduction is exclusively heterosexual. In contrast, certain 100ver animals, plants, and microorganisms may escape the confines of this definition through non­ heterosexual reproduction, most notably by vegetative among microorganisms. The process of evolution, and hence of species formation, is dependent on genetic changes, and an understanding of these processes is critical to an interpretation of DNA relatedness data. Genetic changes may be brought about by point , which result from the change of one or a few in the DNA, or through chromosomal mutations, which either reflect an increase or decrease in number, or in a rearrangement of portions in one or more . Chromosomal mutations are manifested as deletions, duplications, inversions, translocations, fusions or fissions. The impact that these mutational changes have on vegetative reproduction, sexual fertility and overall competitiveness can significantly affect the speciation process. Whether the impact of these changes applies equally to both outbreeding and inbreeding organisms is an important point when attempting to define a taxonomic system based on DNA base sequence divergence as a measure of species formation. Another matter of cardinal importance is the rate of DNA change among different taxonomic groups. vVilson and co-workers (vVilson, Maxson & Sarich, 1974; Wilson, Carlson & White, 1977; Helm-Bychowski & Wilson, 1986) have argued that changes in noncoding DNA sequences proceed with time at a roughly constant rate among all organisms, thus serving as a molecular clock. Sibley & Ahlquist (1984) present evidence that the molecular clock is constant if one uses the average divergence of all genomic DNA. Britten (1986), on the other hand, has argued that rates of molecular change vary among groups of organisms. Regardless of which argument proves correct, it is apparent that divergence is occurring over long periods of time and that the rates are probably constant among phylogenetically similar organisms. Studies of other organisms' provide interesting examples that need to be considered as we interpret data derived from comparisons among fungi. Two areas of particular interest concern anatomical diversity and reproductive isolation. For example, placental mammals, which show many anatomical differences, are believed to have diverged over a period of 75 million years. However, frog species look rather similar but have a 150 million-year history (Wilson, :Maxson & Sarich, 1974; and references therein). Rates of macromolecular evolution are similar for the two groups but are certainly not predictive of anatomical changes, something worth noting as we compare morphological differences among the fungi. Extent of DNA divergence as a measure of reproductive isolation is similarly variable among different groups of organisms. It is generally accepted that man and chimpanzee differ by only about 2% in the complementarity of their primary base sequences (Sibley & Ahlquist, 1984). In contrast to this, the frog species Xenopus laevis and X. borealis show DNA MOLECULAR BIOLOGY AND SYSTEMATIC MYCOLOGY 5 relatedness that is little above background, yet certain pairings give a few viable progeny (Galau et al.) 1976; Wilson, 1976). This latter example is not so different from standards suggested for microorganisms in which some workers have proposed that microbial strains must show at least 30% DNA divergence before they are considered separate species (Brenner, 1973;Johnson, 1973; Kurtzman & Phaff, 1987). Why this disparity and how does it affect our interpretation of fungal speciation? The reproductive isolation ofhuman and chimpanzee has been attributed to a combination of factors that include a difference in chromosome number and temporal differences in gene regulation (Yunis & Prakash, 1982; \Vilson, 1976). Are these factors under less rigid control for simpler organisms? Regardless of the answer, these findings bring us to the conclusion that guidelines predicting fertility from extent of DNA relatedness cannot apply equally to all organisms and must be developed for each particular group.

NUCLEAR DNA COMPARISONS The first of the molecular comparisons employed by taxonomists was determination of the -plus- (G + C) content of nuclear DNA. Although the G+ C content of fungi ranges from about 28-70%, overlap of values for unrelated species is inevitable. Consequently, the value of G + C content is exclusionary. For example, we would expect strains differing by more than 1-2% in G+ C content (depending on the analytical method used) to be separate species, but no such prediction can be made when G + C contents are within this range (Price, Fuson & Phaff, 1978). More definitive separations are realized when DNA reassociation techniques are employed because these data, expressed as percent relatedness, give an overall view of similarity. However, use of such an assessment depends on equating percent DNA relatedness with what we perceive to be actual biological relatedness. If we assume, as discussed earlier, that species represent reproductively isolated populations, then we have a means for calibrating the extent ofDNA relatedness with fertility and for defining species on the basis ofthe extent of DNA relatedness. Comparisons of DNA complementarity and fertility among nearly a dozen heterothallic yeasts (Kurtzman, Smiley & Johnson, 1980; Kurtzman & Phaff, 1987) have provided considerable insight into the meaning of percent DNA relatedness. The mating reaction exhibited between compatible mating types usually ceases just as DNA complementarity approaches background levels, that is, 0-15%, the values found between unrelated species. Generally, as DNA relatedness decreases, the mating response becomes w'eaker, but the pairs Issatchenkia scutulata var. scutulata/1. scutulata var. exigua and Pichia amylophila/ P. mississippiensis show a strong mating reaction though DNA relatedness between each pair is only 25%. A more definitive measure of biological relatedness is whether fertile progeny are obtained from mated pairs. Fertility should be tested through at least the F2 generation to ensure that chromosome pairs are homologous. In the case of our earlier examples, crosses between P. am]lophila and P. mississippiensis gave only poorly formed ascospores that are not viable. By contrast, the pairing ofI. scutulata varieties scutulata and exigua produced 5% viable ascospores, and the F2 generation yielded 17% viability. These varieties may be exceptional because no 6 c. P. KURTZMAN other pamng exhibiting such low DNA relatedness has produced viable ascospores. In general, fertility seems to decrease significantly when DNA relatedness is less than 65-70S~, and in all but one case, is lacking at 25% or less DNA complementarity. Does the trend that we have seen for heterothallic yeasts also hold for homothallic species? This question was addressed through comparisons ofspecies in the homothallic genus Williopsis. An interesting aspect of most homothallic yeast species is that they show some conjugation between independent cells before ascosporulation. Wickerham (1969) and Naumov (1987) used strain-specific nutritional markers to follow the course of mating and subsequent segregation of meiotic products. From this, they found which 'species' could interbreed and they were then able to provide a preliminary definition ofbiological species. Kurtzman et al. (1981) showed that DNA relatedness paralleled fertility in much the same manner as observed among heterothallic yeasts. Most of the examples used so far have suggested that species formation progresses at a relatively constant rate over time and results only from base substitutions in the nuclear DNA. Large-scale chromosomal changes also playa major role in species formation in other groups of , and we might expect this to occur among the fungi. Such an event could result in the genetic isolation ofgroups that would still show relatively high DNA relatedness. Several examples were recently found in which this is the situation. Saccharomyces pastorianus (synonym S. carlsbergensis) shows substantial DNA homology with both S. cerevisiae and S. bayanus (58S~ and 70%, respectively) even though these latter two species demonstrate little relatedness between themselves (Vaughan Martini & Kurtzman, 1985). A comparison of genome sizes suggests S. pastorianus to be a partial amphidiploid which arose as a natural hybrid ofS. cerevisiae and S. bayanus. In this case, we would predict S. pastorianus to be infertile with the proposed parents despite relatively high DNA relatedness. Another example ofapparent differences influencing speciation is to be found in comparisons of Saccharom)'ces exiguus and its anamorph Candida holmii (Vaughan Martini & Kurtzman, unpublished data). The type strains ofS. exiguus and C. holmii exhibit 76% DNA relatedness and are of the same genome size indicating a teleomorphjanamorph relationship, albeit somewhat divergent. Other strains of each of the two morphs show a somewhat smaller genome size despite relatively high relatedness with their respective type strains. This suggests that they have different chromosome numbers and may even represent separate biological species. The conclusions that we can draw from use ofDNA relatedness as an indicator ofbiological species boundaries are several. In most cases, DNA relatedness of65­ 70% or greater suggests strains to be conspecific. DNA complementarity between 65-70% and background indicates the speciation process is under way and that we must consider strains showing this diminished relatedness as either varieties or separate species, depending on what we perceive of their chromosomal makeup and other genetic factors. Exceptions to those general trends are found in the comparison of S. bayanus and S. pastorianus where two apparently infertile taxa show high DNA complementarity but also a large difference in genome size. Such exceptions, most ofwhich are probably due to chromosomal changes, prevent us from using percent DNA relatedness as an infallible indicator of which strains belong to the same biological species. Hovvever, by considering percent MOLECULAR BIOLOGY AND SYSTEMATIC MYCOLOGY relatedness as a predictor with perhaps 90-95% reliability for discriminating among closely related taxa, we, nonetheless, have a remarkably good method for determining which strains are conspecific. Extent of relatedness beyond the sibling species level cannot be resolved by using reassociation of whole genomic nuclear DNA. More distant relationships need to be detected by other techniques.

MITOCHONDRIAL DNA RELATEDNESS Relatedness among the fungi has been examined through comparisons of fragment patterns of mitochondrial DNA (mtDNA) generated with restriction endonucleases (Grossman & Hudspeth, 1985). The small size of the mitochondrial genome makes such comparisons practical, whereas the larger nuclear genome would present more fragments than could be reasonably handled. McArthur & Clark-Walker (1983) used mtDNA restriction patterns to correlate teleomorph-anamorph relationships between the yeast genera Dekkera and Bretlanom)'ces. They found identical restriction patterns for the pair D. bruxellensisjB. lambicus, as well as for the anamorphs B. abstinenslB. custersii and B. anomalusjB. clausenii. This strongly suggests the pairs to be conspecific. Size differences in mtDNA among the other species assigned to these genera prevented an unambiguous assessment of their relationships. An analysis of mtDNA from seven species of the genus Aspergillus was undertaken by Kozlowski & Stepien (1982). Of particular interest was the inclusion of A. oT)'zae and A. tamarii, species also compared by Kurtzman et al. (1986) through reassociation of nuclear DNA. Restriction patterns of mtDNA from these two species showed considerable similarity, as would be expected from the nuclear DNA study. One surprise in this study was the great similarity of mtDNAs from A. tamarii and A. wentii. Garber & Yoder (1984) examined the restriction pattern of mtDNA from 23 isolates of Cochliobus heterostrophus that had been collected worldw·ide. They noted polymorphisms among these strains and suggested that one polymorphism ,vas so great as to suggest the presence of two genetically distinct but geographically overlapping populations. In the preceding studies, it was pointed out that one potential difficulty with restriction pattern analysis is that mtDNA polymorphisms, which arise from insertions or deletions, will give the erroneous appearance of greater sequence divergence than really exists. Because the rate of change in mtDNA of some organisms may be up to la-fold more rapid than that of nuclear DNA, the resolution afforded by mtDNA patterns may not be sufficient to recognize the more divergent strains of a species (Groot, Flavell & Sanders, 1975; Brown, George & 'Wilson, 1979). On the other hand, mtDNA restriction patterns might allow detection ofsubspecies from specialized habitats.

RIBOSOMAL RNA RELATEDNESS The rather narrow resolution afforded by whole genome DNA reassociation and mtDNA restriction analysis does not allow verification ofspecies assignments within genera or an understanding ofintergeneric relationships. The DNA coding for ribosomal RNA (rRNA) appears to be among the most highly conserved sequences known, and it offers a means for assessing affinities above the species 8 C. P. KURTZMAN level (Bicknell & Douglas, 1970; Kennell, 1971 ; Johnson, 1981; Baharaeen, Melcher & Vishniac, 1983). Bicknell & Douglas (1970) were among the first to employ rRNA comparisons to assess phylogenetic relatedness among fungi. Their study focussed on the genus Saccharom)'ces, some species of which now have been assigned to the genera :(ygosaccharom)'ces, Klzl)'veromyces and Torulaspora. In general, the more highly related species clustered into groups corresponding to present generic assignments. Similar results were obtained for this same group of species by Adoutte-Panvier et al. (1980) through electrophoretic and immunochemical comparisons of ribosomal proteins. The study of Bicknell & Douglas (1970), as well as the comparisons of other earlier workers, assessed relatedness from hybridization of rRNA to its complementary DNA. This methodology is unable to resolve the evolutionary distance between closely related species. Another approach, which provides greater resolution of both distantly related as well as closely related species, has been to compare the actual sequence ofrRNA nucleotides. Hori (1976) sequenced the 5S rRNA from a number of prokaryotes and eukaryotes including several yeasts. Using substitution as an evolutionary clock, Hori suggested that prokaryotes and eukaryotes diverged approximately 2.5 x 109 years ago. Andersen, Andresini & Delihas (1982) compared the 5S rRNA sequence of Phycomyces blakesleeanus with that of other eukaryotes and observed this fungus to show greater similarity with the protozoan Tetrah)'mena thermophila than with certain ascomycetes. Walker & Doolittle (1982, 1983) also used 5S rRNA sequences to investigate relatedness among the basidiomycetes. Their 'work identified two distinct clusters that correlated with the presence or absence ofseptal dolipores rather than with the traditional separation of these species into the classes Heterobasidiomycetae and Homobasidiomycetae. Further, the data suggest that capped dolipores evolved from capless dolipores, which may have evolved from single septal pores. Comparisons from the 5S rRNA molecule are somewhat limited because only about 120 nucleotides are available for comparison (Blanz & Gottschalk, 1986). Another aspect ofthese comparisons has been the discovery that different types of 5S rRNA can exist within single strains of certain fungi, thus making the phylogeny based on 5S rRNA sequence data uncertain (Metzenberg et al., 1985a, b). Sequences of the larger rRNA molecules, which seem not to have multiple forms, potentially offer much greater resolution than the 5S cistron, but sequencing the whole 18S or 25S rRNA molecule is at present an enormous task, especially considering the large number ofspecies that most taxonomists need to compare. Another approach is to partially sequence the larger molecules. Lane et al. (1985) have demonstrated that certain short sequences are highly conserved across evolutionary lines and may be used as primers to copy much larger stretches of rRNA, which can then be sequenced by the dideoxy method. It is frequently possible to detect 200-300 nucleotides on a single sequencing gel. Several primers may be used to compare large numbers of nucleotides.

CONCLUSIONS Comparisons of nucleic acids among fungi have only recently begun, but the impact on conventional taxonomy has already been significant. At present, one of ~IOLECULAR BIOLOGY AND SYSTEMATIC MYCOLOGY 9 the main obstacles to use of molecular techniques for taxonomic purposes is proper interpretation of the data. Comparisons among yeasts have provided insight into the correlation of DNA relatedness with the biological species concept. Many more studies are needed to determine ifthese guidelines also apply to other groups of fungi. Molecular comparisons must also be calibrated to provide us with the boundaries of genera and higher orders of classification, something not now reliably obtained. It is apparent from the literature that molecular studies offungi are increasing at a rapid rate, and from this we should expect not only a more realistic classification system but also a more thorough understanding ofgenetic mechanisms that will impact in the control ofplant and animal diseases and in the development ofmore efficient industrial fermentations.

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