MOLECULAR

Vol.

1, No.

PHYLOGENETICS

3, September, pp.

Evolutionary

AND

EVOLUTION

231-241,

1992

Relationships within the Fungi: Analyses of Nuclear Small Subunit rRNA Sequences

THOMAS D. BRUNS,* ROTAS V1LGALys.t SUSAN M. BARNS,* DOLORES GoNzALEz,t DAVID S. HiesErr,? DAVID J. LANE,~ Luc SIMON,T SHAWN STICKEL,~~TIMOTHY M. SZARO,* WILLIAM G. WEISBURG,~ AND MITCHELL L. SOGIN~~ *Department of Plant Pathology, University of California Berkeley, California 94720; tDepartment of Botany, Duke University, Durham, North Carolina 27706; #Biology Department, 738 Jordan Hall, Indiana University, Bloomington, Indiana 47405; SGENE-TRAK Systems, 3 7 New York Avenue, Framingham, Massachusetts 01707; TCentre de Recherche en Biologie Forest&e, Facult& de Foresterie et de Geomatique, Universit@ Laval, Sainte-Foy Quebec, Canada, GIK 7P4; and “Center for Molecular Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Received

June

11, 1992;

revised

September

21,

1992

ceteae, or the Eumycota (Cavalier-Smith, 1986; Shaffer, 1975; Tehler, 1988). Evidence from the small subunit rRNA sequences has substantiated the view that the Fungi, as thus defined, are monophyletic (Bowman et al., 1992a; Bruns et al., 1991; Gunderson et al., 1987; Schlegel, 1991). Molecular studies have also revealed that the closest relatives of the Fungi are the multicellular animals and the green plant lineages, although the exact relationship among these three major groups remains unresolved (Gouy and Li, 1989). In this paper we have combined 14 18s rRNA sequences derived from independent work in four laboratories with previously published sequences (Table 1) to address the question of relationships within the Fungi. Representatives of the Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota are included and provide a framework for the phylogeny of the Fungi.

Nucleotide sequences of the small subunit ribosomal RNA (l&S) gene were used to investigate evolutionary relationships within the Fungi. The inferred tree topologies are in general agreement with traditional classifications in the following ways: (1) the Chytridiomycota and Zygomycota appear to be basal groups within the Fungi. (2) The Ascomycota and Basidiomycota are a derived monophyletic group. (3) Relationships within the Ascomycota are concordant with traditional orders and divide the hemi- and euascomycetes into distinct lineages. (4) The Basidiomycota is divided between the holobasidiomycetes and phragmobasidiomycetes. Conflicts with traditional classification were limited to weakly supported branches of the tree. Strongly supported relationships were robust to minor changes in alignment, method of analysis, and various weighting schemes. Weighting, either of transversions or by site, did not convincingly improve the status of poorly supported portions of the tree. The rate of variation at particular sites does not appear to be independent of lineage, suggesting that covariation of sites may be an important phenomenon in these genes. o 1992Academic

METHODS

Sequence Determination Sequences of small subunit rDNA coding regions from Thanatephorus, Coprinus, Mucor, Schizosaccharomyces, and Schizophyllum were determined from cloned rDNA repeats using double-stranded sequencing methods described by Elwood et al. (1985). Small subunit rDNA coding regions from Aspergillus, Blastomyces, Coccidioides, Filobasidiella, and Penicillium were amplified using the polymerase chain reaction (PCR) protocol (Mullis and Faloona, 1987; Saiki et al. 1988) as modified in Medlin et al. (1988). The population of PCR products tias sampled by preparing singlestranded sequencing templates from a pool of as many as 15 independent recombinant Ml3 clones. Sequence heterogeneity, representing microheterogeneity in the different rDNA copies from a given organism or from potential PCR artifacts appears as multiple bands at

Press, Inc.

INTRODUCTION

Many diverse eukaryotic lineages are fungal-like in their heterotrophic lifestyles and filamentous thalli. Four divisions: Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota share chitinous cell walls end unique hioaynthetic pathway for l$aine (CavalierSmith, 1986). These organisms have long been thought to constitute a monophyletic group distinct from the other fungal-like groups such as the Oomycota and the slime molds (Barr, 1983). These “true” fungi have been referred to as the Kingdom Fungi, the Kingdom Eumy231

1055-7903192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

232

BRUNS

TABLE Organisms

and Sequences Used in Study” GenBank accession numbers Fungi

10 20 13 16 9 I 21 14 11 31 4 25 2’7 3 1 2 23 26 8 29 17 5 12 19 15 22 24 18 26 30 32 6

Aspergillus fumigatus Athelia bombacinn Aureobasidium pulluhns Blastocladiella emersonii Blastomyces dermatitidis Boletus satanns Candida albicans Colletotrichum gloeosporioides Coccidioides immitis Coprinus cinereus Cronurtium ribicola Filobasidiella (Cryptococcus) Chytridium confervae En&gone pisiformis Gigaspora margarita Glomus intmradix Kluyveromyces 1acti.s Leucosporidium scotti Mucor racemosus Neocallimastix sp Neurospora crassa Peridermium harknessii Penicillium notatum Pneumaystis carinii Podospom anserina Saccharomyces cerevisiae Schizophyllum commune Schizosacchuromyces pombe Spizellomyces acuminatus Spongipellis unicolor Thunatephorus pmticola Xerocomus chrysenteron Other Achlya bisexualis Anemonia sulcata Chlamydomonas reinhardtii Ochromonus danica Prorocentrum micans Stylonychia pustulata Zea mays Zamia pumila

’ Number

before

fungal

M55626 M55638 M55639 M54937 M55624 M94337 x53497 M55640 M55627 M92911 M94338 M55625 M59758 X58724 X58726 X58725 X51830 x53499 X54863 M59761 x04971 M94339 M55628 X12708 X54864 M27607 X54865 X54866 M59759 M59760 M92990 M94340

neoformans

Distance Analysis

organisms

sequences

M32705 X53498 M32703 M32704 Ml4694 Ml4600 K02202 M20017 indicates

order

of taxa

in Fig.

1.

corresponding positions in sequencing gel lanes (Illingworth et al., 1991). However, such heterogeneities were not observed in any of the rRNA coding regions included in this analysis. Sequences of Cronurtium, Peridermium, Boletus, and Xerocomus were determined by direct sequencing of PCR products using the -methods and primers described by White et al. (19901, along with additional internal primers described by Gargas and Taylor (1992). Se@.&n~e Alignment

The sequences were aligned using the Editor-GJO computer program (Olsen et al., 1991). This program

AL.

is an assisted interactive editor that considers the conservation of both primary and secondary structural features. The simultaneous alignment of poorly conserved elements was facilitated by color coding different nucleotide bases in the computer display of aligned sequences. Alignments within regions that exhibit length variations were refined by juxtaposing sequences that define phylogenetically conserved higher order structures. Only those positions which are in obvious alignment (using the criteria of primary and secondary structure conservation) were used in the distance and parsimony analysis. When only higher fungi were considered, analyses included as many as 1780 aligned sites. Comparisons of fungal, animal, and plant sequences were restricted to 1740 sites that were considered to be in alignment. The slightly different treatment of gaps in distance and parsimony analyses is discussed below.

1

Organism

ET

Similarity values, defined as the number of matches divided by the sum of matched plus nonmatching nucleotides plus one-half the number of alignment gaps (Elwood et al., 19851, were computed for all possible pairwise comparisons of aligned nucleotide positions. In the case of gaps, only the first five positions were used for the calculations since large insertions or deletions probably reflect single rare events. Similarity values were converted to distance values using the formula of Jukes and Cantor (1969). This formula compensates for the probability of multiple substitutions at the same positions by assuming that all sites change at the same rate and transitions and transversions are equally probable. Fitch and Margoliash (1967) matrix methods were used to infer phylogenetic trees. Branch strengths in the distance trees were assessed by 100 bootstrap resamplings as described by Felsenstein (1985). The replicate data sets were produced with the Editor-GJO program (Olsen et al., 1991) and individual Fitch-Margoliash trees were constructed as described by Elwood et al. (1985). When analyzing only the fungi, all taxa were used, but when outgroups were included, 24 taxa were selected for bootstrap analysis due to computer time constraints. The taxa chosen included representatives of plants, animals, and the major lineages of Fungi. Parsimony

Analysis

All parsimony analyses were conducted using PAUP 3.0 (Swofford, 1990). Before using parsimony analysis the alignment was carefully examined and portions of it were recoded from the DNA format to the symbols format in order to use selected gaps as characters. Only areas with length mutations were affected by recoding. The product of the recoding was a*,Qg.bt,.of length inutations that were treated as a fifth character state, “X,” and a subset of nucleotide positions bordering length

FUNGAL A 1 2 3 4 5 6 7 8 9 :: 12 13 14 15 :t 18 19 if 22 i: 25 26 27 ii 30 31 32

CAGGWAAAC CAW3JGAAAC CUGUGAAAC CAGUGAAAC CAGUGAAAC CUGUGAAAC CUGUGAAAC WGUGAAAC CGG-WAMC CGG-IJGMAC WG-UGAMC CUGWAAAC CGG-WMAC CUGWAAAC CAGCGAAAC CAAGUGAAAC CCGCGAAAC CUGUGAAAC CAGUGAAAC CUGUGAAAC CAGUGAAAC CAGUGAAAC CAGUGAAAC CUGUGAAAC CUGUGAAAC CXG-UGAAAC CUGUGAAAC CUGUGAAAC CUGUGAAAC CUGWAAAC CUGUGAAAC CUGUGAAAC

w

* CAGGUGAAAC CAWUGAAAC CUGXUGAAAC CAGXUGAAAC CAGXUGAAAC cuGxuGAAAc CUGXUGAAAC UUGXUGAAAC CGGXUGAMC CGGXUGAAAC CGGXUGAAAC “,$gg’g CUGXUGAAAC CAGXCGAAAC CAAGUGAMC ccGxcGAAAc CUGXUGMAC CAGXUGAAAC CUGXUGAAAC CAGXUGAAAC CAGXUGAAAC CAGXWAAAC CUGXUGAAAC CUGXUGAAAC CGGXUGAAAC CUGXUGAAAC CUGXUGAAAC CUGXUGAAAC CUGXUGAAAC cuGxuGAAAc CUGXUGAAAC

SMALL B

co-MAMUC cu-AAAACCU cu-MAAAUC CUGAAAAGa CUGMAAGCC CAAUCAAGCC CAAtl-c CMAAAAACC c-UAAAAACC c-UAAAAACC c-uAAAAAcc c-UAAAAACC c-UAAAAACC c-UAAMACC CA-AAAAAUC CACUCAAGUC c-UAAAAACC c-uAAAAAuc CA-AAAAAUC CAWAAAGCC cuuAAAAucc cuuAAAAucu cuuAAAAucu CAAUCAAGCC CUGAAAAGCC cuGMAAAuc CAAUAAAAGG co-AAMAUC CG-UAAAAAC CAAUCAAGCC CAAUCdAGCC CAUCAAAGCC

SUBUNIT l

C

t

cNNMAAAuc CNNMAACCU CNNAAAAAUC CWAAAAGCC CWAAAAGCC CAAWAAGCC cAAwAAGcc CAMAAAACC

-

233

EVOLUTION

CNNAAAAACC CNNAAAAACC cNmAAAAcc :EE”c CNNAAAAACC CNNAAAAAUC CACUCAAGUC cNNAAAAAcc CNNAAAAAUC CNNAAAAAUC CAWAAAGCC CWAAAAUCC CWAAAAUCU CWAAAAUCU CAAWAAGCC CUGAAAAGCC cuGAAAAAuc CAAUAMAGG CNNAAAAAUC cNNmAAAAc cAAwAAGcc cAAucAAGcc CAUCAAAGCC

CUUCG---GGU---uuccu GGGCAA--ccGA--uucccu GGGCAA--WAC---UCAUC UWCG---GGUC---W-CU UWCG---GGUC---UC-Cc0 GCUCG--CCGC---UCGW GCUCG---CCGC--0cGw GGGUAAAACCAGWUCCCU CUUCG---GGGC--UC-CO CUUCG---GGGC--UC-CO CUUCG---GGGC---UC-UU CUUCG---GGGC---UC-CO CWCG---GGGC---UC-CD CWCG---GGGC---UC-AC CUCCG---GGGC---UC-AC GGGCAA-CCGGGWUUCUGU CWCG---GGGC---UA-AC -WCG---GGC--UUUWU -W~----~---UUAJU GCUCG---CCGC---UCCNU -W~---~--UC-UU -wcG----GAc---UC-uu -uucG----GAc---UC-cu GCUCG---CCGC---UCACU UWCG---GCCC---UC-UA CUUCG---GGUC---CC-UA G--AA---ACGG---UUCUU GGCAA---CCGc+--UUWU GGCAA---CCAG--UUWU GCUCG---CCGC--UCCAU GCUCG---CCGC--UCCCU GWCG---CCGC---UCCUU

******it************* CUUCGX--GGu---wcccu WGCAA--ccGA--wcccu GGGCAA--ccAc--xucAuc wUcGx--GGuC--XWXCU UUUCGX--GGUC--XUCXCU GCUCGX--CCGC--XUCGUU GCUCGX--CCGC--XUCGUU GGGUA~NNUCCCU CUUCGX--Gwc--xucxcu CUUCGX--GGGc--xucxcu CUUCGX--GGGc--xucxuu CUUCGX--GGGC--XUCXCU CWCGX--GGGc--xucxcu CWCGX--GGGC--XUCXAC cuccGx--GGGc--xucxAc GGGCAA-NCGGGNNNNNNNN cuucGx--GGGc--xuAxAc XUUCGX--XGGC--UWWU XUUCGX--xGGc--xwxuu GcucGx--ccGc--xuccNu XUUCGX--xGGc--xucxw XUUCGX--xGAc--xucxuu XUUCGX--XGAC--XUCXCU GCUCGX--CCGC--XUCACU UUUCGX--GCCC--XUCXUA CUUCGX--GGUC--XCCXUA NNNNNNNNNNNN--XWCUU GGCAAX--CCGG--XWWU GGCAAX--CCAG-XWUUU GCUCGX--CCGC--XUCCAU GCUCGX-CCG’S-XUCCCU GUUCGX--CCGC--XUCCUU

FIG. 1. Examples of recoding for parsimony. (A) A single base gap surrounded by conserved sequences is scored as an informative gap (X). (B) A single base gap that could be shifted to several places is coded as missing data (N) along with adjacent nucleotides of uncertain alignment, thus avoiding the problem of generating “informative sites” by arbitrary alignment decisions. (0. A region where some sequences are easily aligned-and others are not is coded with a combination of informative gaps and missing data and is then included in the “questionable sites” exclusion set enabling the eff&ctsof these alignment decisionstobetested (see text). Note that all gaps that are ceded: with a hvnhen are equivalent to missing data, “N.” Preliminary alignments were made as described in text. *Position affected by recoding. Numbe&*refer to taxa in Table 1. -

mutations that were treated as missing data. These changes were made for two reasons: (1) Information is likely to exist in parts of the molecule that cannot be aligned in all taxa. We refer to such regions as partially alignable, because the alignment is unambiguous for a subset of taxa, while the exact position of a gap or insertion is uncertain in others. Recoding these ambiguous portions of some taxa as “missing data” (i.e., N) allows information to be extracted from such regions for those taxa whose sequences can be aligned, while avoiding arbitrary decisions where alignments are uncertain. This method results in some known nucleotides being coded as Ns (Fig. 1). As pointed out previously this method of coding does result in distortion of branch lengths (Bruns and Szaro, 19921, but we do not view this as a major problem because our main concern with parsimony analysis is topology. Furthermore, as parsimony branch lengths suffer from other distortions caused by branching density (Fitch and Bruschi, 19871, their lengths should always be viewed with some skepticism. (2) Some length mutations are likely to provide useful phylogenetic information, but by default all gaps are treated as missing data by PAUP if a “DNA format” is specified. Alternatively, PAUP allows all gaps to be defined as a fifth character state, but this latter approach is unacceptable for the 18s data since many multibase gaps probably reflect single insertion or deletion events. Our solution was to limit “fifth state gaps” to single characters and limit

their use to gaps that were bordered by well-aligned areas, while all other gaps were treated as missing data. Both simple and complex examples of the recoding method are shown (Fig. 11, and the full parsimony data set is available for detailed inspection from the first and second authors. We also defined several exclusion sets for regions in which the coding changes could have been done in more than one way. Collectively, we will refer to these sets as the “questionable sites.” Their effect on the analysis was tested and the results are discussed below. The robustness of branches was assessed by bootstrap and decay analysis. Bootstrapping was conducted using the heuristic search option of PAUP on the full dataset and on subsets of taxa. One-hundred bootstrap replicates were performed. The decay index is a measure of the number of additional steps necessary for a particular clade to collapse as parsimony is relaxed and it is correlated with bootstrapping (Brent Mishler, personal communication). In some cases, however, the decay index may actually be a more conservative estimate of support for branching than the bootstrap (Hibbett and Vilgalys, unpublished results; Brent Mishler, personal communication). The decay index of each clade was determined by obtaining the semistrict consensus of trees which are one or more steps longer than the most parsimonious tree. For relatively small sets of taxa, decay indices may be determined by screening

BRUNS ET AL.

234

longer than the most parsimonious resulted in computer crashes after saving 20,000 trees. Therefore the decay analysis of the 32-taxon tree was restricted to 20,000 trees.

all possible topologies using the tree filtering option in PAUP 3.0 (Swofford, 1990). For larger sets of taxa, as in this case, the population of longer trees was determined in two steps. First, the heuristic search option was used to find the most parsimonious trees. The search was then repeated, with the specification that PAUP save all trees less than or equal to 20 steps longer than the most parsimonious tree. The resulting trees were then screened using the filter trees option in PAUP to obtain successively shorter sets of trees which were then used to compute a set of strict consensus trees for each different tree length. When the analysis was restricted to ascomycetes and basidiomycetes, it was possible to find and save all trees (20,000) within 20 steps of the most parsimonious tree using the heuristic search algorithm on a Macintosh IIfx (equipped with 8 megabytes RAM). For the 324axon analysis of all Fungi, repeated attempts to save all trees 10 steps A loo

Peridermium ~Cmnartium l Leuwsporidium

1

I

RESULTS

The Fungi in Relation to Other Eukaryotes The initial analysis, based on pairwise distances (Fig. 2a), was rooted with selected plants, animals, and protists. It shows the Fungi as a monophyletic group with Blastocladiella as the earliest branch within the Fungi. Although this rooting is not strongly supported by our bootstrap analysis, it is in agreement with the cladistic analysis of Tehler (1988), which is based primarily on morphology. Confidence in the branch uniting the Fungi was B l

F 2

.02

AND DISCUSSION

9

s

63/3 loo/>2

A

55

‘Owl7

Blastomycws Ceaidbldes

100/19

“Z?ZZrn

1 83

58/l

Mucor + Chytridium l Newattimastix l Spkelbmyces l Mastodadelia Anemonia l Chtamydomonas t zea*

-

Pneumocystis

t

Endopne

zamia

hfuwf

6lastoctadietla

*

Ctybnychia

’

20 substitutbnr

l

Pmmcentnim Ocdwomonas Ad&a

I]

g *

FIG. 2. Phylogenetic analyses of the fungal 18s sequences. Tree A is a Fitch-Margoliash tree based on pair-wise distances. Confidence in selected branches was estimated with a bootstrap sample of a 100 trees using the indicated 24 taxa t*). Values that were greater than 50% are shown. Arrow indicates root of the fungal tree. Tree B is an unrooted parsimony analysis of the fungal 18s sequences. The strict consensus of the three shortest trees is shown. Bootstrap values from a sample of 100 replicates are shown adjacent to each internal branch and the decay values are shown below. Topologies that are supported by more than 95% of the bootstrap trees depicted in bold. Traditional divisions of fungi are indicated. For taxa lacking sexual states their placement within the Ascomycota and Basidiomycota are shown when traditional views and 18s data are congruent, although according to the rules of botanical nomenclature they are classified as Deuteromycetes. The lack of a traditional classification for Pneumocystis is indicated.

FUNGAL

SMALL

SUBUNIT

fairly high, though below the 95% level based on bootstrap analysis (Fig. 2a). The shared AAA lysine pathway (Vogel 1965) and the presence of chitin in the cell walls (Bartnicki-Garcia, 1970) provide additional strong support for a monophyletic fungal kingdom (Cavalier-Smith, 1986), which has been recognized without molecular data for nearly twenty years (Vogel, 1965; Bartnicki-Garcia, 1970; Shaffer, 1975). Gunderson et al. (1987) showed that the 18s data supported exclusion of the oomycetes from the true fungi. This result hasbeen expanded by additional data(Eorster et al., 1990) and discussed elsewhere (Bruns et al., 1991; Schlegel, 1991); thus, we have not specifically tested that branch in this analysis. Likewise, we have not addressed the branching order among plants, animals, and fungi since this issue has been analyzed in detail by others without resolution (Gouy and Li 1989). The basal placement of the Chytridiomycota and Zygomycota and even their paraphyletic relationship relative to the Ascomycota and Basidiomycota is a very traditional idea, but the mixed groupings of the former two divisions which was found in both the distance and parsimony analyses (Fig. 2) are not. Specific conflicts are the identification of Mucor and Blastocladiella as a monophyletic or paraphyletic group by the parsimony analysis and the grouping of Engogone with three of the four members of the Chytridiomycota in both parsimony and distance analyses. Both conflicts would necessitate the convergent gain or loss of flagella. Confidence in these branches, however, is very low based on bootstrap estimates. The only exception is the Glomu+Gigasporu clade (Fig. 2), and it is not a controversial grouping. Decay analysis appears to provide some support for the branches involving Chytridium, Neocallimastix, and Spizellomyces (Fig. 2b). The depiction of these three fungi as a monophyletic group is consistent with the original analysis of Bowman et al. (1992a). However, the grouping of Neocallimastix with Chytridium rather than with Spizellomyces in the parsimony analysis differs from their results, but neither our groupings nor theirs are strongly supported by bootstrap analysis. Interestingly, the selection of taxa included in the analysis affects the branching order of these three taxa. When we analyze these three taxa with only BZustocZudieZZa, we obtain exactly the same result as Bowman et al. (1992a): Neocullimustix and Spizellomyces form a monophyletic group that is supported at a high (85%), though not significant, level by bootstrap analysis. If any of the Zygomycota are added to the analysis, then Neocallimustix groups with Chytridium with a low level of bootstrap confidence. The distance analysis, however, shows the same branching pattern as Bowman et al. (1992a) but still without strong bootstrap support. By the criteria of Hillis’ (1991) tree length distribution analysis (Fig. 3) it appears that there is some phy-

235

EVOLUTION Distribution of tree lengths for all Dossible trees

ReCA

Tree

B

Tree length w/o “questionable site”

345

356 (+l 1)

Tree length with questionable sites

879

894 [+15)

likelihoods

-6790.7

1

-6801.39

FIG. 3. Phylogenetic analysis of the basal branches of the fungal tree. The shortest unrooted parsimony tree (A) is shown along with the shortest tree that depicts the Zygomycota and Chytridiomycota as potentially monophyletic groups (B). The distribution of lengths for all possible trees is shown for the analysis lacking the questionable sites (graph), and the locations of trees A and B within this distribution are indicated. The analysis conducted with the questionable sites resulted in a similar distribution (not shown) with a slightly greater left skewing (Gi = - 0.86190 vs - 0.51’7). Both Gi values are significantly more skewed than random data (P < 0.01 and P < 0.05 level, respectively) (Hillis, 1991). The likelihood values of trees A and B were calculated with DNAML (Felsenstein, 1991). A transitionkransversion ratio of 2 and empirical base frequencies were used. Although tree A has a higher likelihood the difference is less than the standard error (13.93) and thus is not significant (Kishino and Hasegawa, 1989).

logenetic signal in the 18s sequences in these basal branches (Fig. 3). However, if either Glomus or Gigusporu is eliminated from the tree length distribution analysis, then the length distributions of the resulting seven-taxon trees are no longer significantly skewed. This latter result is the same for the data sets with and without the questionable sites. Thus, within the basal branches, the phylogenetic signal present in the 18s gene appears to be essentially restricted to the Glomus-Giguspora branch. This conclusion agrees nicely with the bootstrap analysis (Fig. 2). The low confidence levels in the basal branches do

236

BRUNS ET AL.

A

100 100

L I

Cronartium PefMmium Leucosponilium

Xemcomus

100199 51Aa

+&etus -

Cqptinus Thanataphows AQwtlw~~

-TE

I

24Es E

Leucospcddium Xerocomus BoletlS spo%7W~

Schkq3hytlum

SChiZlXWChWnpS

Schizosaccharomyces

3

Pneumocystts I

>

0

0.02

0.04

0.06

0.06

2owbsullHon*

FIG. 4. Unrooted analysis of the Ascomycota and Basidiomycota. Numbers of bootstrap replicates supporting each branch from a sample size of 100 are indicated for the distance tree (A) and the shortest parsimony tree (B). The parsimony analysis was conducted with and without the “questionable sites.” Bootstrap values including these sites are given above and those excluding them below. Bold numbers highlight differences between the two analyses where one or both are greater than 95%. Bold lines indicate topologies that are supported by >95% of the trees in the distance analyses (A) or in the parsimony analysis (B) using the questionable sites. Plectomycetes (Plecto) and Pyrenomycetes (Pyreno) are indicated. The lack of a traditional classification for Pneumocystis is shown, I‘?“.

not directly address the question of whether our best estimate is significantly better than one that would define the Chytridiomycota and the Zygomycota as mono- or at least paraphyletic groups. To answer this question we examined just the eight taxa involved and selected the shortest tree that would make both divisions monophyletic relative to each other (Fig. 3). We found this tree by looking at successively longer trees for one that met this stated criterion (Fig. 3b). We calculated likelihoods for both trees and compared the difference between their likelihoods to their standard errors (Kishino and Hasegawa, 1989). Based on this comparison we conclude that the two trees are not significantly different. Thus, the conflict with traditional classification is not a strongly supported result. Ascomycota

and Basidiomycota

Most of the major branches in the Ascomycota are well resolved and are compatible with very traditional classifications (Figs. 2 and 4). Strong support for the Plectomycetes and Pyrenomycetes as monophyletic groups has been previously demonstrated by Berbee

and Taylor (1992), and is also supported in our analysis with a slightly different but less diverse selection of species. The position of Aureobasidium within the euascomycetes is clear, in that it falls between the members of these two major classes. What is unclear is which class it is closer to relative to the root of the euascomycetes. The position of the human pathogens Blastomyces and Coccidioides is in agreement with Bowman et al. (1992b). The exact relationship of Schizosaccharomyces and Pneumocystis remains uncertain. This uncertainty is based on the equivocal position of the root within the Ascomycota. The rooting in the distance tree depicts Pneumocystis as the most basal branch within the Ascomycota (Fig. 2A), but with low confidence. With parsimony, both analyses that include the Ascomycota show Schizosaccharomyces and Pneumocystis as a monophyletic group (Fig. 2B and 4B), but again only with low confidence. On the parsimony tree with all fungi (Fig. 2B) this clade can be split by making Schizosaccharomyces the sister group to all ascomycetes with only one additional step.

FUNGAL

SMALL

SUBUNIT

While the relationship of Schizosuccharomyces and Pneumocystis remains equivocal, the consistent placement of Pneumocystis near the base of the Ascomycota in all of the analyses is interesting because Pneumocystis lacks a sexual state and has only recently been recognized as a fungus (Edman et al., 1988). At the time the 18s sequence of Pneumocystis was published few other fungal sequences were available for comparison. Now with additional sequences available a placement within the Ascomycota seems plausible, although it is not strongly demonstrated by our analyses. The basidiomycetous portion of the tree shows two well-defined lineages: (1) the phragmobasidiomycetes consisting of the Uredinales or rusts, Cronurtium and Peridermium, plus the smut-like Leucosporidium and (2) seven of the eight holobasidiomycetes, Boletus, Xerocomus, Spongipelis, Athelia, Schizophyllum, Coprinus, and Thunatephorus. Relationships within the holobasidiomycetes are not well defined by these data with the exception of the clade containing Xerocomus and Boletus, whose sequences are nearly identical. The position of Filobasidiella clearly lies between these two major groups, but its position relative to the root of the Basidiomycota is tenuous in the parsimony analysis. It can be shifted to the phragmobasidiomycete side of the tree with only two additional steps. The position of Filobasidiella is strongly supported by the distance analysis and is congruent with the best parsimony tree. This placement also makes sense when morphological characters are considered because Filobasidiella has a holobasidium. The position of Leucosporidium relative to the rusts is strongly supported in all analyses. This placement is surprising, however, because the behavior of its nuclear membrane during mitosis and its globular spindle pole bodies (McCully and Robinow, 1972) are more similar to those found within holobasidiomycetes than within the rusts. In terms of these ultrastructural features, the rusts are strikingly similar to the ascomycetes (Boehm and McLaughlin, 1989). The rusts are also ascomycete-like in terms of their multiple asexual spore states (Savile, 1955) and septal pore structure (Kahn and Kimbrough, 1982). With the possible exception of septal pore structure, all of these other similarities between the rusts and ascomycetes now appear to have been caused by parallelism either between the ascomycetes and rusts or between other independent lineages in the basidiomycetes. The morphological evidence mentioned above has led some to a rather different placement of the smuts relative to the rusts and holobasidiomycetes (CavalierSmith, 1986), and has also led to the speculation that the rusts may have been independently derived from the Ascomycota (Petersen, 1974). Both ideas seem very unlikely given the strength of the branches uniting all basidiomycetes and the branches uniting Leucosporid-

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ium with the rusts, but because our analyses of the Basidiomycota exclude many key orders, a clear understanding of relationships within the division awaits a better sample. According to Savile (1955) and more recently Tehler (19881, the basidiomycetes are derived from hemiascomycetes, and thus the ascomycetes constitute a paraphyletic group. In this light it is interesting to note that yeasts occur near the basal branches of both the Ascomycota and the Basidiomycota. Our best estimates (Figs. 2 and 4), however, suggest that the Ascomycota and Basidiomycota are monophyletic sister groups. This result appears to contradict the ideas of Savile (1955) and Tehler (1988) as well as the elaborate theories of Cavalier-Smith (1986) in which the evolution of the Ascomycota and Basidiomycota is attributed to three independent radiations from the entomophoralian Zygomycota. Unfortunately, confidence in the crucial branches is not high enough to strongly reject alternative rootings that would be compatible with the views of Savile or Tehler. Unsampled taxa present an additional problem, since the theories of Savile (1955) and Cavalier-Smith (1986) as well as the structure of Tehler’s (1988) tree are strongly influenced by taxa for which sequence data are not yet available. We examined the effect of taxonomic sampling by reanalyzing Tehler’s morphological data with parsimony. When we limited the analysis of Tehler’s data to taxa represented in the present study, we found nine equally parsimonious trees. Two of these trees were consistent with a sister group relationship between the Ascomycota and Basidiomycota. By adding the deleted taxa into the analysis one at a time we found that any one of the following five would restore the paraphyletic relationship of the Ascomycota: Dipodascopsis, Dipodascaceae, Endomycetaceae, Taphrinaceae, and Exobasidiales. This result argues for the need to obtain sequence data for these unsampled taxa. Effect of Minor Alignment Changes The effect of our recoding method for partially alignable regions can be compared with the alternative of eliminating these regions entirely by viewing the results of our analyses with and without these “questionable regions” (Figs. 3 and 4B). Three lines of evidence suggest that inclusion of these sites did not adversely affect the parsimony analyses and may have improved the estimates of phylogenetic relationships in several cases. (11 In all cases, topology of the best trees was identical in all branches that were strongly supported by bootstrap analysis conducted with or without the sites. Thus, inclusion of these sites did not drastically affect the results. (2) Bootstrap confidence levels for several well-resolved branches in the Ascomycota-Basidiomycota increased with inclusion of

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FIG. 5. Site distributions and level of noise in parsimony analyses. A, distribution of number of changes/site (top) for one of the component trees of Fig. 2B and the contribution to tree length for each class of site (bottom). The distributions for 95% and 99% of the sites are indicated (top). B, same distributions for just the ascomycete portion of the tree (including Pneunwcystis).

these sites (Fig. 4B, highlighted values). This result is not surprising since several of the regions involved align well within this subset of fungi, but it demonstrates that these sites are probably not exceptionally noisy relative to other sites. (3) When only the basal branches of the tree were analyzed, the length distribution of all possible trees is more strongly skewed to the left if the questionable sites are included (Fig. 3); this result suggests that these sites are adding signal to the analysis (Hillis, 1991). This conclusion, however, is weakened by the knowledge that most of the signal in these basal branches appears to be limited to the Glomus-Gigusporu branch (see above). Thus, the phylogenetic signal added by the questionable sites is likely to be attributable only to the sequence alignment of these two taxa. The branches that were strongly supported by bootstrap analysis appear to be fairly robust to minor differences in alignment. Slightly different alignments were used in the two different tree building methods. The greatest difference was that the distance analysis did not employ the recoding method used with the parsimony analyses. Yet in spite of these differences, all branches that were strongly supported by bootstrap with either distance or parsimony were congruent with the other method. Most of the weakly supported branches were also shared (Figs. 2,4)

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Branches

and Weighting

Effects

The level of homoplasy in the data is the likely cause of the low confidence in the deepest basal branches. The distribution of the number of inferred changes per site reveals that many sites change multiple times in the best tree (Fig. 5). Furthermore, with equal weighting given to all sites, the noisiest sites supply a disproportionately large contribution to branch lengths. For example, 99% of all sites change fewer than eight times, yet those that change eight times or more contribute almost twice as much length to the tree as sites that change only once (Fig. 5). Under situations such as these, where homoplastic changes are more likely than consistent changes, one might expect parsimony to fail, and the most susceptible portions of the tree should be those with long unbranched lineages (Felsenstein, 1978; Swofford and Olsen, 1990). It is probably not a coincidence that in our analyses the long basal branches of the tree and the long branches at the base of the Ascomycota are not strongly supported (Figs. 2, 4). Under such conditions, additional sequence data would not be expected to resolve these branches, unless these data are more consistent than the present data (Felsenstein, 1978). Because the problem of interpreting homoplasy is most acute with long unbranched lineages (Swofford and Olsen, 1990), sam-

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pling of additional related taxa that fall along these branches may improve the resolution. A better sampling of both the Zygomycota and the Chytridiomycota is certainly warranted, as many major groups within both divisions are completed uninvestigated. The lack of confidence within the branches of the holobasidiomycetes is harder to explain, since the branches involved are not particularly long, and the site distribution in the Basidiomycota (not shown) is virtually identical to that of the much better reLeuaxwddium solved Ascomycota (Fig. 5B). We suspect the fairly short branch lengths between the nodes relative to the terminal branch lengths may be the cause of the problem. This pattern of branch lengths may be an artifact of the taxonomic sample, but it could also be caused by I Fi/obasidie//lla' an ancient, rapid radiation of the holobasidiomycetes. Weighting of characters or of character state C changes has the potential to sort out the noise from 1 Leuwspooriium the signal. We tried two weighting schemes, one based on transversion weighting and one based on site site weighted weighing; neither had a significant impact on tree topology or branch confidence. c Transversion weighting has been suggested for FilobasidisNa I rRNA genes exhibiting the level of divergence obFIG. 6. Weighted and unweighted parsimony analyses within served in these Fungi (Mindell and Honeycutt, 1990). tha Basidiomycote Tree.-A is ‘&he shontest amrooted. tree based en The rationale for ?I& is based on thn &mat+unt&t unweighted parsimony. Tree B is based on weighting transversions transitions are more frequent than transversions and over transitions by 2: 1. Tree C is based on weighting sites by their when transversions occur, they cover up the evidence behavior in the best Ascomycota tree as described in text. Bold lines of previous transitions (DeSalle et al. 1987). A 2: 1 indicate branches that are supported by 100% of the bootstrap replicates. Other branches were supported by less than 95% of the replitransversions/transitions weighting is a conservative cates, most by less than 50%. Note that top and bottom trees are choice which is within the range observed in fungi and topologically identical, although their relative branch lengths vary. other eukaryotic nuclear 18s rRNA genes (Mindell and Honeycutt, 1990; Bruns and Szaro, 1992). Using a step matrix to apply a 2 : 1 weight, we reanato a spreadsheet program (Microsoft Excel), sorted, and lyzed portions of the tree with parsimony and found graphed. We then assigned sites that changed 0, 1, 2, that neither topology nor confidence levels were 3, and 4 or more times weights of 20, 10, 5, 2, and 1, strongly affected. Within the eight. members of the Zy- respectively. These weights were then used to analyze gomycota and Chytridiomycota, the topology was iden- two other portions of the tree. Within the eight memtical to the unweighted analyses (i.e., tree A, Fig. 3) bers of the Zygomycota and Chytridiomycota the topoland the Glomus-Gigasporu lineage remained the only ogy remained unchanged and bootstrap confidence levstrongly suported branch. Within the 11 members of els remained below the 95% level for all branches that Basidiomycota topological differences occurred (Fig. 61, were weakly supported in the unweighted analysis. but were limited to branches that were poorly sup- The same result was obtained within the Basidiomyported in both the weighted and unweighted analyses. cetes: the weighting scheme did not alter the topology Transversion weighting also had no significant effect nor make any meaningful differences in the bootstrap on bootstrap confidence levels within the Basidiomyconfidence values (Fig. 6). cota (Fig. 6). One major drawback to transversion Our site weighting was based on the assumption weighting via step matrix was that the computer time that the rate of evolution at a particular site is a propnecessary to complete an analysis increased more than erty of the site and is therefore independent of lineage. lo-fold. To examine the validity of this assumption we removed Weighting by site also had little effect, even with all sites that were variable within the ascomycetes an intentionally severe weighting scheme. To identify and then superimposed the remaining ascomycetedifferent rate classes of sites we examined the number invariant sites back onto the best parsimony tree (i.e., tree 4B). The results of this analysis (Fig. 7) showed of changes at each site within the ascomycete portion of the tree (Fig. 5B). This was done by using the “de- that sites that were completely invariant within the scribe trees” option of PAUP to output the number of ascomycetes varied dramatically in other parts of the changes at each site. The data were then transferred tree and their distribution of changes/site was similar

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above and are unlikely to be resolved confidently without additional data or novel insights into the evolution of the small subunit gene.

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ACKNOWLEDGMENTS SchlzoMwllum y

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Blastomyces cotxldlomes Aspe@llus Penicllllum AutwbasMium Colkwtrkhum PodoSpota Neumspom Candl& Sacchanwnyces KluVveromVce Schkoeaccharomvces

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We thank Barbara Bowman, Mary Berbee, and John Taylor for prepublication access to their sequence data and manuscripts; and John Taylor and Brent Mishler for useful comments on earlier drafts of the manuscript. Funding was provided in part by NSF Grants BSR-8918454 and BSR-8700391 to T. D. Bruns, NIH Grant GM32964 to M. L. Sogin, and the NSF Grant BSR-88-06655 and USDA Grant 88-37234-4038 to R. Vilgalys.

REFERENCES 1 .? 3 45 6 7 6 changes/site

9,0,,,2,3

Mucor Blastocladlella

FIG. 7. Distance and homoplasy contributed by sites that are invariant in the Ascomycota. Sites invariant in the Ascomycota were superimposed on tree topology shown in Fig. 2B. Bar graph shows the distribution of the changes/site for the same tree. Only the black sites contribute to the distance in this tree.

to that of all sites. This pattern suggests that the rate of change for many sites is not independent of lineage. This result is consistent with the ideas of Shoemaker and Fitch (19891, who suggested that sites are not independent of each other but instead are covariant, and that even if the fraction of variable sites remains relatively constant across lineages, the rate of change at any particular site may be highly variable. The examples they chose were protein genes, but one could easily envision covariation based on secondary structural constraints of rRNA or the interaction of the small subunit with other rRNA subunits or with ribosomal associated proteins. CONCLUSIONS Despite the problems of alignment and level of homoplasy, the small subunit rRNA gene provides a view of fungal evolution that is largely compatible with traditional ideas. The strongly supported branches are robust to method of analysis, minor changes in alignment, and various weighting schemes. These results demonstrate that the level of divergence within the gene is well matched to phylogenetic analyses of many fungal groups. The weakly supported branches, however, are more sensitive to the parameters mentioned

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