Berbee1993-Dating the Evolutionary Radiations of the True Fungi

8/20/2019 Berbee1993-Dating the Evolutionary Radiations of the True Fungi

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ating the evolutionary radiations of the true fungi

M A R Y

L

BE RBE E

Department of Botany The University of British Columbia 627 0 Urziversify Bolllevard

Vancouver BC V6T 124 Canada

A N D

J O H NW. TAYLOR

Department of Plutzt Biology Koshland Hull Room 11 1 University of Cali fornia Berkeley CA 9472 0 U.S.A.

Received Apri l 5, 1992

BERBEE,M . L . , a n d TAYLOR,.W. 1993. Dating the evolutionary radiations of the true fungi. C an . J . Bot.

7 :

11 14- 1127.

In this paper we construct a relative time scale for the origin and radiation of major lineages of the true fungi, using the

18 s ribosomal RN A gene sequence da ta of 37 fungal species , and then calibrate the time scale using fossil evidence. Of the

sequences, 28 were from the l i terature or data banks and the remaining 9 are new. T o est imate the order of origin of fungal

lineages we reconstructed the phylogeny of the fungi using aligned sequence data . T o compensate for the differences in

nucleotide substitution rates among various fungal lineages, we normalized the pairwise substitution data before estimating

the relative timing of fungal divergences. We divided the fungi into nine groups. We then calculated the average percent

substitution for each group, and also the average for all the groups, for the time period beginning when the fungi diverged

from a comm on ancestor and ending at the present . W e used the ratios of group-specif ic percent subst itut ions to the average

percent substitution to normalize our pairwise substitution data matrix. To infer the relative timing of the origin of lineages

we superimposed the normalized percentages of nucleotide substitutions onto the parsimony-based phylogeny. Calibrating

the rate of sequence chang e involved relating th e normalized percent su bstitution associated with phylogenetic events to fungal

fossils, the ages of fungus hosts, and ages of symbionts. These calibration points were consistent with a substitution rate of

1 per lineage per 100 Ma. Based on phylogeny and calibrated percent substitution, the terrestrial fungi diverged from the

chytrids approximately 550 Ma a go. A fter plants invaded the land approximately 40 0 Ma ag o, ascomycetes split from basidio-

mycetes. Mushrooms, many ascomycetous yeasts , and common molds in the genera Perlicilliurrl and Aspergillus may have

evolved after the origin of angiosperm plants and in the last 200 Ma.

Key words: fungus evolut ion, molecular clock, ascomycete phylogeny, basidiomycete phylogeny, 18 s rRNA.

BERBEE,M . L . , e t TAYLOR,.W. 1993. Dating the evolutionary radiations of the true fungi. Ca n. J. Bot. 7 11 14- 1127.

En utilisant les donnCes de la sCquence du gkne AR N ribosomique 18 s chez 37 espkces fong iques, puis en calibrant 1'Cchelle

temporelle l 'aide de fossiles, les auteu rs ont construit une Cchelle de temps relative pour I 'origine ci la radiation de s phylums

principaux d'eumyco ta. Parm i les sequences utiliskes, 2 8 proviennent d e la IittCrature ou de banques d e donnCes et les 9 autres

sont nouvelles. Afin d'estimer l 'ordre d'origine des phylums fongiques, les auteurs ont reconstruit la phylo gtnie des champ ig-

nons en utilisant I 'alignement d es donnCes de skquences. P our co mpe nser les differences des taux de substitution d es nuclCo-

tides entre les differentes IignCes de champignons, ils ont normalis6 les donnCes de substitution par paires avant d'estimer

le taux relatif des divergences chez les champignons. Les champignons ont Ctt divisks cn neuf groupes et les pourcentages

moyens de substitution pour chaque g roup e ont CtC calculCs ainsi qu e la nloyenne pou r tous les g roupe s, et ceci pour la periode

h partir du moment oh les champignons o nt commencC a diverger d 'un ancstre commun jusqu'h aujourd'hui . Pour normaliser

la

matrice d e donnCes des substitutions par paires, les auteur s ont utilisC les rapports de s pourcentages de substitution speci-

fiques aux groupes sur le pourcentage moyen de substitution. Pour dCduire le moment relatif de l 'origine des IignCes, les

auteurs ont sup erp ost les pourcentages normalisCs des substitutions de nuclCotides su r la phylogenic bas sur la parcim onie.

La cal ibrat ion du taux de changement des sequences nkcessi te une comparaison des pourcentages de subst itut ion normalises

associCs aux Cvknements phylogCnttiques, av ec les champignons fossiles, l 'bge de s hates de ces champign ons et de leurs sym -

biontes. Ce s points de calibration con cordent avec un taux de substitution de 1 par IignCe par 100 Ma. Su r la base de la phylo-

genie et de la calibration du pourcentage de substitution, les champignons terrestres auraient divergC des chytridiales il y a

environ 550 M a. Les basidiomycktes se sont stpares des ascomycktes aprks [ 'appari t ion des plantes sur le mil ieu terrestre,

il y a environ 400 Ma. I1 apparait que les macromycktes, plusieurs levures ascomycktes ainsi que les moisissures communes

des genres

Penicilliurrl

e t

Aspergillus

seraient apparus aprks les angiospermes, au cours des derniers 200 Ma.

Mots cl is

6volut ion des champignons, horloge m oltculaire, phylogknie de s ascomycktes, phylogenie de s basidiomycktes,

rARN 18s .

[Traduit par la redaction]

Introduction

The true fungi, the ascomycetes, basidiomycetes, zygo-

mycetes, and chytridiomycetes, form a monophyletic group

(Bowman et al. 1992), distinguished from slime molds and

algae by the presence of chitin in their cell walls and by an

unusual, multienzyme synthesis pathway for the amino acid

lysine (Bartnicki-Garcia 1970; Vogel 1964). Evidence of radi-

ation of major fungal groups is probably preserv ed in the fossil

record in the form of diversification of filament morphology

in samples of fossilized decaying vegetation, and in the diver-

Prinred

in anada

/

Ilnprim

au

anada

sification of spore morphology from pollen samples (Tiff

and Barghoorn 1974; Pirozynski 1976). How ever, interp

ing fungal fossils can be difficu lt; spores in fossil pollen s

ples or branching filaments from fossil plant parts often l

the diagnostic characteristics of modern taxa. We think

interpretation of some fossil fungi will benefit from at lea

rough estimate, gleaned from molecular data, of the timing

major events in the evolution of fungi.

D N A

clock evide

and fossil evidence have often proved congruent and u

mately mutually reinforc ing (Clegg 19 90; Knoll 19 92). Us


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BERBEE A N D TAYLOR 1115

sequence data, S imon et al. (1993) estimated the time of origin

of endomycorrhizal fungi in the order G lomales. In this paper,

we establish a relative time scale for the evolution of the fun gi

using a phylogeny from D NA seque nce data and then calibrate

the time scale using correlations between phylogenetic events

and events in the fossil record of fungi or fungal symbionts

(Ochman and Wilson 1987). This time scale, by predicting

when radiations of m ajor group s of fungi should be detectable

in the fossil record, will facilitate interpretation of fossil fungi

in palaeobotanical samples. More accurate interpretation of

fungal fossils will, in turn, allow for more accurate recalibra-

tion of the molecular clock (Taylor 1990).

Materials and methods

Sources of fungal sequences used are listed in Table 1. Most of the

sequences are either published or are available in the GenBank or

EMBL sequence data bases. For this study, we determined the

sequences of the 18s rRNA gene of nine additional fungi. We ampli-

fied the 1 8s ribosomal RNA gene from extracted, partially purified,

total genomic DNA, using the polymerase chain reaction, and then

performed a series of overlapping, asymmetrical amplifications to

generate the single-stranded DNA that we sequenced (Lee and Taylor

1990; White et al. 1990).

We sequenced over 1720 nucleotides from each of the seven fungi,

the entire gene except for about 50 nucleotides at the 5' end, and

about 20 nucleotides at the 3' end that we could not reach using o ur

primers. The sequence is based on both strands of DNA except for

about 30 nucleotides 5' to NS 8, (the primer nearest the 3 ' end of the

noncoding strand of the gene) and a region in the center of the gene

approximately 100 nucleotides long, near NS 5 (White et al. 1990).

For Taphrina deformans we sequenced the 18s rRNA gene from

three isolates. The results in this paper are from the completed

sequence of the first strain, ATCC 34556. To confirm the identity of

the fungus, we obtained partial sequence from two other strains,

ATCC 34557, and a cu lture kindly donated by X.X. Phaff, University

of California. The three strains had identical sequences.

The 18 s sequences from the 37 fungi were aligned by first using

the Genalign option in the computer package

INTELLIGENETICS

5.4

for UNIX (700 East El Camino Real, Mountain View, Calif.) and

then visually changing the positions of the gaps to maximize aligned

sites. To align all of the 37 fungi, including the most distantly related

chytrids, zygomycetes, and basidiomycetes, we had to exclude over

100 bp of ambiguously aligned sequenc e data, s o that 158 9 aligned

sites were analyzed. Gaps were consistently associated with ambigui-

ties and were excluded from the phylogenetic analysis.

To estimate the phylogeny of the fungi, we used the sequences of

33 fungi, excluding 4, very closely related chytrids from the analysis

to speed computations. For phylogenetic reconstruction, we used par-

simony algorithms

in

the computer package P A U P 3.0 (Swofford

1991) and neighbo r joining from the pac kage PHYLIP 3.4 (Felsenstein

1992). The two methods yielded topologically similar phylogenetic

trees from the aligned data set.

Distance matrices were generated from aligned sequen ce data using

the maximum likelihood option of

D N A D IS T

in the computer package

PHYLIP

3.4 (Felsenstein 1992).

ssumptions

Correction for group-spec@ rates of nucleotide substitution

All lineages of true fungi alive today a rose from a comm on

ancestor and therefore, h ave been evolving for the sa me num-

ber of years. Some lineages have accumulated more nucleotide

substitutions than other lineages in the time since the fungi

diverged from the common ancestor. These differences in per-

cent substitution from lineage to lineage must be due to differ-

ent rates of substitution. If all fungi were evolving a t the sam e

rate, the percent sequence difference between the outgroup

Chytridium and each of the ascomycetes and basidiomycetes

would be about the same. In fact, the percent substitution

ranges from 13.2 between

Chytridium confervae

and the

basidiomycete

Leucosporidium scottii, to 7 .0 , f rom Chytrid-

ium confervae to Schizosaccharomyces pombe. In general,

basidiomycetes including the rusts had the highest substitution

rates, while the basal lineage of ascomycete yeasts evolved

most slowly (Fig. 1).

This rate variation between different fungal groups limits

the potential accuracy of molecular clock estimates of diver-

gence times. To compensate for obvious substitution rate dif-

ferences, we divided the fungi into groups, determined how

quickly each group had evolved relative to the average of all

group s, and imposed correction factors based on the observed

rate differences.

Definition of groups

W e divided taxa into nine groups to estimate the lineage-

specific nucleotide substitution rates (Fig. 2). Where possible,

the groups represent monophyletic lineages of phylogeneti-

cally related taxa. Three such lineages received bootstrap sup-

port over 90 (Fig. l ), and three other groups appeared in the

sequence-based parsimony trees even when different out-

groups were added to or removed from the data set. Glomus

intraradices is the sole representative of its group. In the last

two groups, however, we lumped taxa with similar branch

lengths (Fig. 1). The morel group consists of filamentous

ascomycetes for which fine-scale phylogenetic relationships

are unclear, including

Aureobasidium pullulans, Pleospora

rudis, and Morchella esculenta. The smuts Ustilago hordei

and Tilletia caries are of uncertain position within the basidio-

myc etes, and w e arbitrarily placed them toge ther with the rusts

(Figs. 1 and 2) to estimate their substitution rates.

Calculation of group-spec ic relative rates

T o calculate substitution rates for each of our nine groups

of species we approximated fungal evolutionary history with

the polychotomy in Fig. 2, as if all nine groups had been inde-

pendent since the original divergence of chytrids from terres-

trial fungi. Using the relative rate test (Fig.

3 (Li and Graur

1991) and a distance matrix with a maximum likelihood cor-

rection for multiple mutations at the same site, we calculated

the average percent substitution that each of the nine groups

has accumulated since the time when the chytrids diverged

from the terrestrial fungi. W e also calculated a total average

percent substitution for all groups of fungi. The relative rate

of substitution for each of the nine groups was then the ratio

of the average percent substitution for the gro up to the average

percent for all l ineages (Fig. 2 ). T o keep the time period cons-

tant, we had to compare the percent substitution that each

group had accumulated since it diverged from the common

ancestor to all other lineages. F rom o ur phylogeny (Fig.

l ) ,

we know that our nine groups were not independent lineages

for the first part of their evolutionary history. We had to

ignore the fact that groups with a common ancestor w ere origi-

nally evolving at a shared, common rate. For example, the

group including the rust and the fungus Leucosporidium

(L. scottii, Fig. 1) has an unusually high number of substitu-

tions. Our calculated percent substitution for a L. scottii

includes all the substitutions along lineages leading to

L. scottii

beginning with the time when the chytrids diverged from the

terrestrial fungi. The percent substitution for the rust group

fungus L. scottii includes the substitutions occurring on the

branches leading to pre-ascomycetes and pre-basidiomycetes,


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C A N .

J.

BOT.

VOL. 71,

1993

TABLE

. Sources of sequence data

Fungus and strain

GenBank

Sourcea accession No.

Reference

Ascomycotina

Ascosphaera apis

Aureobasidium pullulans

Candida albicans

Colletotrichum gloeosporoides

Dipodascopsis uninucleata

Endomyces geotrichum

Eremascus albus

Eurotium rubrum

Hypomyces chrysospermus

Kluyveromyces lactis

Leucostoma persoonii

Morchella esculenta

Neurospora crassa

Ophiostoma

ulmi

Pleospora rudis

Pneumocystis carinii

Saccharomyces cerevisiae

Schizosaccharornyces pombe

Taphrina deforman s

Talaromyces flavus

Basidiomycotina

Athelia bombacina

Pers.

Cronartium ribicola

Leucosporidium scottii

Spongipellis unicolor

Tilletia caries

Tremella globospora

Tremella moriformis

Ustilago hordii

Zygomy cetes

Glomus intraradices

Chytridiomycetes

Blastocladiella emersonii

Caecomyces Shaerornonas) communis

Chytridium confervae

Neocallimastix

sp.

Neocallinlastix fronta lis

Neocallimastix joynii

Piromyces Pirom onas) comrnunis

Spizellomyces acurninatus

UCB 61.016

UCD yeasts

UCB 88.016

UCB 75-001

ATCC 34556

OSU, D. Mills

UCD, K. Wells

UCD,

K.

Wells

OSU, D. Mills

Berbee and Taylor 1992a

Illingworth et al. 1991

Hendriks et al. 1989


New sequence

New sequence


New sequence

Berbee and Taylor 19926

Maleszka and Clarkwalker 1990

Berbee and Taylor 19926

A Gargas and J.W. Taylor, unpublished d

Sogin et al. 1986


New sequence

Edman et al. 1988

Rubtsov et al. 1980, Mankin et al. 1986

Bruns et al. 1992

New sequence



Bruns et al. 1993

Hendriks et al. 1991

Bowman et al. 1992

New sequence

New sequence

New sequence

New sequence

Simon et al. 1992

Forster et a]. 1990

Dore and Stahl 1991

Bowman et al. 1992

Bowman et al. 1992

Dore and Stahl 1991

Dore and S tahl' 199

Dore and Stahl 1991

Bowman et al. 1992

Source information for new sequences only: ATCC, American type

culture

collection: OSU, Oregon

State

University; UCB. University of California. Berkeley

microgar

UCBH. University

of

California, Berkeley herbar ium; UCD,

University of

California, Davis.

to the basidiomycetes, and to the first divergence in the basid-

iomycetes, along with the substitutions specific to the rust

lineage (Figs.

1

and 3 . By including the shared branches in

our estimates of the relative rate, we underestimated the actual

rate differences between taxa on the terminal branches and

brought the taxa evolving mo re quickly or more slowly closer

to the average rates of the ancestral taxa.

Normalizing pairwise percent substitution

We could then use the relative amount of substitution (or

relative rate of substitution, since each group is assumed to

have evolved over the same period of time) shown in Fig.

2

to normalize the rate of substitution for each fungus to the

average for all the nine groups.

For taxa within on e of the nine group s, only the one rate cor-

rection factor specific to the group is needed. For e xamp le, in

the time the fungi have been evolving, members of the r

group accumulated 1.47 times as many substitutions as

average fungus. The corrected distance from the comm

node to either the rust group fungus L. scottii or the r

Cronartium ribicola would be found by taking half of the pa

wise percent substitution between the two tax a and dividin

by the rust group relative rate, or 1.47 (Fig.

2).

In contrast, we had to take the substitution rates of b

groups into account when we compared pairs of taxa fr

different groups. The computation is much simpler if the c

rection can be applied to the

pairwise distances between ta

which are available in the distance matrix, rather than to

amount of substitution on each branch.

Th e algebraic logic leading to a relationship between s l ,

average number of substitutions for the group containing

individual fungi and

S

the pairwise distances, follows.


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BERBEE AND TAYLOR

Morchella

Aureobasidium

Pleospora

Leucostoma

Ophiostoma

7

1

Hypomyces

Colletotrichum

Neurospora

I I Q

r

remascus

Ascosphaera

urotium

Dipodascus

Kluyveromyces

Saccharomyces

Candida

Schizosaccharomyces

Taphrina

Pneumocystis

Tremella glob.

Tremella mori.

Tilletia

Ustilago

100 Cronartium

Leucosporidium

Neocallimas tix sp.

Spizellomyces

Chytridium

FIG 1. This consensus tree shows the relationships among 3 3 fungi as inferred from 18 s rRNA gene sequence data. To facilitate analysis,

three of the four very sim ilar rumen chytrid sequences in our data set were not included in this tree. The numbers on the branches ar e bootstrap

percentages from 500 bootstrap replicates using the heuristics option in PAUP version 3.0 Swofford 1991). Percentages over 90 indicate support

for the branch from the data set. Names of taxa are truncated; for complete species names, see Table 1. If a molecular clock were working

perfectly in this gene, a line could be drawn through the end points of branches to terminal taxa from Glomus through Morchella. The end

points of terminal branches are not aligned, evidence that substitution rates vary.

the variables s and s2 represent the number of nucleotide

and

substitutions along the branches from node

0

where fungus

species and 2 diverged, to species

1

and 2 respectively. Let

[ l b ] s 2 = r 2 t 2

the variables t and t2 represent the time of evolution of

S1 S2

taxons and 2 respectively. S the percentage of nucleotide

[ l c l t

=

t2

=

substitutions between taxons and 2 comes from the distance

r r2

matrix. Values of r and r2 represent the rates of substitution

and because the two extant taxa diverged from a common

for the two lineages. ancestor,

[ l a ]

s l

=

r l t l

[2a]

t l = t2


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VOL. 71

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I 32

Colletotrichum

I ipodascus

lastocladiella

Saccharomyces X0 87

Schizosaccharomyces

I

0 71

Cronartium

Leucosporidium I 47

o us us I

0 96

F I G 2

TO calculate correction s for variation in substitution rates we assumed as represented by the polychotomy in this figure that

groups of higher fungi were independent since the original divergence of the chytrids from the terrestrial fu ngi. We then calculated the ave

percent substitution per lineage for each of the nine fungal groups since their divergence from the common ancestor of hytridiurn and

terrestrial fungi. The relative rates given to the right of the groups are the ratios of the averages of the percent substitutions per lineage

taxa in the group divided by the average percent substitution of all nine lineages.

Substituting slr from [ l c ] or tl and t2 ,

Substituting the equation for s2 from

[3b]

into

[ 2 c ] ,

X0 60

Chytridium

pizellomyces

and

~

[2c]

s l r 2

=

s2r l

t 3f1 s l r2 r l ) = S ~ I

The total number of substitutions between the pair of taxa is

the sum of the substitutions on each lineage.

S ~ I

[3gl S I

=

Neocallima stix fron.

aecomyces

Neocallimastix sp.

[3a] S = S , s2

and

L

I

and

Neocallimastix joy.

Piromyces


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BERBEE

A N D

TAYLOR

The rates r l and r2 are equal to an average rate R, identical

for all taxa, mu ltiplied by the relative rates, r i and r;, which

are specific to taxons 1 and 2, respectively.

Then

and from [3h] and [4a]

Both sides of [3h] can be multiplied by R

Equation 3h perm its estimation of the substitutions per b ranch ,

given the relative rate of substitution of the two groups.

Conceptually, n ormalizing the substitutions for the lineages

involves dividing the amount of substitution on branches lead-

ing to individual fungi by the relative rate for that group,

resulting in a corrected amount of nucleotide substitution, s',

Replacing s l r i using [4c] ,

As an example of how correction works, Chytridium con-

fervae shou ld-be equally distant from each ascomycete or

basidiomycete, if lineages evolved at equal rates. However,

the uncorrected percen t substitution in the chytrid

L

scottii

pair (13.20 for the pair, o r 6. 6 for each lineage) is almost

twice the percentage in the chytrid

S

pombe pair (7.01 ,

or 3.51 per lineage). Corrected for group-specific rate dif-

ferences, the percent substitution per lineage of chytrid

L

scottii becomes 6.3 8 (calculated by dividing 13.2 0 , the

uncorrected percent substitution for the pair, by 0.6 0 1.4 7,

the sum of the relative rates of the chytrid group and the rust

lineage). T his is quite close to the corrected estimate of 5.3 5

substitutions per lineage (calculated as 7.01/(0 .60 0.7 1))

from the chytrid

S

pombe pair . To establish an average

percent substitution per lineage accumulated since chytrids

diverged from terrestrial fungi, we averaged the corrected per-

cent substitutions per lineage calculated from the Chytridium

confervae

L

scottii, Chytridium confervae Schizosac-

charomyces pombe pairs as well as all the other chytrid-

ascomycete or chytrid-basidiomycete pairs that mark the

same divergence.

W e present estimates of the percent substitution per lineage

corresponding to the divergence of fungal lineages from both

corrected and uncorrected substitution data (Table 2). 'The

average percent substitutions marking the origins of major

lineages are similar with and w ithout lineage-specific c orrec-

tion, but estimates without correction are more frequently in

conflict with phylogeny (assuming that percent substitution

increases with time). F or example-, from -the phylogeny, the

basal ascomycete yeasts diverged before the ascomycetous

true yeasts, and so the basal ascomycete lineage should have

more substitutions than the true y east lineage. 1n fact, because

of their slow evolution, the basal ascomycetes accumulated

FIG 3

An example of estimating the average percent substitution

per lineage from a pairwise distance matrix using the relative rate

test. To calculate the number of substitutions from the node along

the branch to a yeast, we used the following formula based on pair-

wise percent substitutions for the yeast,

Blastocladiella

and a chytrid:

yeas . chytrid Dyeast Blasloclad. chytrid. Blasloclad.

D O y ea st

2

We repeated the sam e calculation for each of the five true yeasts that

together represent a monophyletic group of ascomycetes . The results,

when averaged, yielded the percent substitution typical of the true

yeast lineage.

only 3.0 substitution since their origin , whereas the true

yeasts averag e 3.1 0 substitution. Following correction for

differences in rates, the basal ascomycetes exhibit the higher

percent substitution predicted by phylogeny. In general, the

rate correction reduces the stand ard deviation for the estimates

of percent divergence (Table 2). Consequently, we used the

rate-corrected estimates for further analysis.

Calib ration of the molecu lar clock

W e make the assumption that our phylogeny of the fungi

(Fig. 1) is true. Consistent with evidence from Bruns et al.

(1992) the tree is rooted so that the water m olds Chytridium

confervae and Blastocladiella emersonii are basal to the pre-

dominantly terrestrial ascomycetes and basidiomycetes. Fol-

lowing logic outlined by Hennig (1966) and O chman and W ilson

(1987), we used fossil evidence and ages of fungal hosts and

symbionts to calibrate the rate of substitutions in fungal line-

ages (Fig. 5).

Among the oldest fungal fossils are spheroidal, thick-walled

resistant spores from 390 Ma old Lower Devonian fossilized

rhizomes of vascular plants in the genus Rhynia (Kidston and

Lang 1921; Pirozynski and Dalpt 1989). The fungal spores

probably represent early members of the Glomaceae, the

family that now includes G. intraradices (Pirozynski and

DalpC 1989), and the time of the appearance of these spores

in the fossil record defines the latest possible date for the ori-

gin of the Glomaceae lineage.

An average rate-corrected 4. 9 substitution per lineage has

accumulated since G. intraradices diverged from the lineage

of terrestrial fungi that gave rise to the ascom ycetes and basid-

iomycetes.

Regularly septate filaments are also present in the Rhynie

plants (Kidston and Lang 1921). The Glomaceae and the line-

age ancestral to ascom ycetes and basidiomycetes originated at

the same time, having diverged from a common ancestor.

While mem bers of the Glomaceae, an d almost all basal terres-

trial fungi, produce aseptate filaments, regularly septate fila-

ments characterize the ascomycetes and basidiomycetes. The

regularly septate filaments in the fossil record are evidence

that the lineage immediately an cestral to the ascomycetes and


7/14

TAB LE Nucleotide substitutions since divergences

No rate correction

With rate correction

Event

Taxa diverging

Avg.

substitution

Origin terrestrial fungi

Origin rumen chytrids

Radiation of rumen chytrids

Origin Glomaceae

Origin ascomycetes and basidiomycetes

First basidios divergence

Origin smut and rust lineages

Earliest possible homobasidiomycetes

Radiation of homobasidiomycetes

First ascomycete radiation

Origin true yeast

Radiation of true yeast

Radiation 1-cell true yeast

Origin discomycetes and loculoascomycetes

Origin of plectomycetes -pyrenomycetes

Pyrenomycete radiation

Plectomycete radiation

Aspergillus and Penicillium

Chytrid errestrial fungi

Spizello chytrid rumen chytrids

Caeco myce s eocallirnastix Piromyces

Neocallirnastix sp. N. joynii N. frontalis Piromyces

Glomus pre-ascomycetes and basidiomycetes

Ascomycetes asidiomycetes

Simp le septate dolipore basidiomycetes

Tilletia Ustilago Cronartium Leucosporidiurn

Cronartium -Leucosporidium

Tilletia Ustilago

Tremellas omobasidiomycetes

Athelia -Spongipellis

Tremella globospora T. morifonn is

Basal ascos filamentous ascos yeasts

Pneumocystis aphrina Schizosaccharomyces

Taphrina -Schizosaccharomyces

Yeasts filamentous ascos

Dipodascopsis higher yeasts

Endomyces

higher yeasts

Candida luyveromyces Saccharomyces

Kluyveromyces -Saccharom yces

Morchella group other ascomycetes

Pleospora M orchella Aureobasidium

Morchella -Aureobasidium

Plectomycetes yrenomycetes

Ophiostorna Leucostoma-Neurospora Hypomyces Colletotrichum

Neurospora Hypomyces Colletotrichum

Hypomyces Colletotrichum

Ophiostoma eucostom

Ascosphaera Eremascus urotium Talaromyces

Eurotium alaromyces

Ascosphaera remscus

Avg.

substitution SD

5 5 0 77

4 0 1 40

0 8 0 11

0 2 0 03

4 9 0 41

3 9 0 60

3 4 0 84

3 8 0 61

3 1

2 3

2 2 0 24

1 2

0 8

3 3 0 47

3 0

2 4

3 1 0 41

1 9 0 19

2 4 0 16

1 3

0 7

2 4 0 28

1 8

1 o

2 8 0 20

1 7 0 16

1 7

1 2

1 2

1O 0 04

0 5

0 3


8/14

L N TAYLOR

1121

basidiomycetes had diverged from the Glomaceae before the

Lower D evonian.

The percentage of nucleotide substitution accumulated since

the origin of septation should be less than the 4 .9 per lineage

accumulated since aseptate

G.

intraradices split from the pre-

ascomycete basidiomycete lineage, but more than the 3.9

per lineage accumulated since the divergence of septate

ascomycetes and septate basidiomycetes.

Clam p connections, hook-shaped structures that straddle the

septa in fungal filaments, first appear in the fossil record in

woody tissue from a 290 Ma old Carboniferous fern (Dennis

1970). Basidiomycetes a re the only fungi with clam p connec-

tions, so the presence of clamps in 290 Ma old coal balls pro-

vides a latest possible date for basidiomycete origins. All of

the basal basidiomycetes are either parasitic or saprop hytic on

land plants, so we assum e that basidiomycetes and their clamp

connections evolved after the earliest land plants radiation, in

the Mid-Ordovician, about 460 Ma ago (Gray 1985). Phylo-

genetically, clamp connections probably occur in both of the

two most divergent

basidiomycete lineages, the yeast-like

members of the rust lineage, and in the mushroom lineage.

Th e percent substitutions per lineage corresponding to the ori-

gin of clamp connections should fall between the 3.9 aver-

age of branches dating from the ascomycete basidiomycete

split, and 3.4 of the deepest divergence within the basidio-

mvcetes.

The progenitors of the ascomycete genus Ophiostoma, the

genus containing the fungus causing Dutch elm disease, lost

the ability to discharge ascospores forcibly when they became

dependent on bark beetles for spore dispersal. Bark beetles

have a reciprocal dependence on fungi (although they a re not

limited to the genus Oplziostoma), and many genera of bark

beetles have specialized body parts, or mycangia, for trans-

porting, sometimes nourishing, and disseminating fungal spores

(Wood 1982). The distinctive galleries that bark beetles

produce in wood date back to the Cretaceous, indicating that

bark beetles had probably originated by the Lower Creta-

ceous, perhaps 140 Ma (Wood 1982; Bright 1993). The genus

~ ~ h i o s t o m aa y have diverged, from related genera with

forcible spore discharge, around the time of origin of bark

beetles. O n the other hand , although loss of forcible disch arge

may have been dependent on the-origin of bark beetles, the

Leucostoma and Ophiostotna lineages could have diverged

well before bark beetles, limiting the reliability of this calibra-

tion point. Th e rate-corrected percent substitution co rrespond-

ing to the loss of forcible discharge and evolution of insect

dispersal must be less than the 1.2 accumulated since Ophio-

stoma split from Leucostoma.

Th e oldest fossil shelf fungus, Ph ellinites, is about 165 Ma

old (Singer and Archangelsky 1958). Phylogenetically, the

split between the jelly fungi (in the genus Tremella) and the

mushroom-like basidiomycetes (holobasidiomycetes) probably

occurred before the first recorded fossil shelf fungus. Th e cor-

responding percent substitution should be below the 2 .2 sub-

stitution accumulated since the Tremella species diverged from

the homobasidiomycetes.

Chytrids ar e phylogenetically old, but at least one group of

chytrids, those associated with mammal

hindguts and with

the stomachs of ruminants, must have undergone a relatively

recent radiation. The chytrids Caecomyces (formerly Sph aero-

monas) communis and Piromyces (Piromonas) communis are

found in the hindguts of diverse mammals as well as the

stomachs of ruminants, whereas related chytrids in the genus

mammal hindguts

[

5 4 3 2 1

Millions of years

FIG.

4.

Substitution rates in fung i are calibrated by relating fossil

evidence and ages of fungal hosts and symbionts to percent substi-

tutions on branches and nodes of the sequence-based phylogeny

(Ochman and Wilson 198 7). The shaded calibration areas relate time

to percent substitution. For ex amp le, a 290 Ma old fossil basidiomy-

cete clamp connections provides a firm upper time limit fo r the evolu-

tion of basidiomycete clamps that we correlated to a possible range

of percent substitution from the phylogeny. The logic unde rlying the

other calibration points is similar and is described in the text. These

calibration points are consistent with an average nucleotide substitu-

tion rate of 1 per lineage per 1 Ma.

Neocallimastix are restricted to the stomachs of ruminants

(A. B rown lee, personal comm unication). 'The split between

Caecomyces communis and the genera Piromy ces and Neocal-

limastix corresponds to 0. 8 substitution per lineage. Th e

split probably occurred after the origin of the mammallineage

ancestral to the placental and marsupial mammals, about

20 0- 150 Ma (G raves 1987), but before the time of origin and

initial radiation of ruminants about 40 Ma (Janis 1976).

Neocallimastix species in ruminants probably radiated less

than 4 0 M a, or af ter the origin of ruminants. Th e correspond-

ing percent substitution should be between 0 .8 , accumulated

since the Caecomyces communis diverged from the rumen

chytrids, and 0. 2 , accumulated since the first split among

the obligate rumen chytrid species.

Results and discussion

The above calibration points are consistent with an average

corrected or uncorrected nucleotide substitution rate of

substitution per lineage per 1 00 M a (Fig. 4) . This nucleotide

substitution rate is similar to the rate for green plants (0.8

per 100 Ma) (from Fig. 3b , Wolfe et al. 1989) and for verte-

brates (1.3 per 100 Ma) (Hedges et al. 19 90), but it is lower

than the 2 per 10 0 Ma rate reported for bacteria (Ochman

and Wilson 1987). For the true fungi to be older than ou r esti-

mates, their nucleotide substitution rates would have to be

even lower than the rates we have estimated. We hope that the

calibration curve will be tested with additional calibration

points from new fossil evidence or reinterpretation of existing

data. Because we are unable to test the relationship between


9/14

Ascosphaera

Eremascus

Eurotium

Talaromyces

a

Ophiostoma

I

eucostonla

Hy omyces

m o~etotmhum

m

Neurospora

Aureobasidium

Pleospora

Morchella

Kluyveromyces

Saccharomyces

Candida

Dipodascus

Endomyces

Taphrina

Schizosaccharomyces

Pneumocystis

. .

Athelia

:r;:lr;:b

b

Tremella mori

Cronattium

Leucosporidium

Ustilago

Tilletia

I

Glomus

Neocallimastix joynii

Neocallimastix frontalis

Piromyces

Neocallimastix sp

I

a

Caecomyces

6QO 5QO 400 300 200 100

Millions

of years

FIG.

5.

This tree shows estimated fungal divergence times superimposed on the phylogeny from Fig. 1 Letters on branches indicate the origin of fungus morphological features

diagrammed in Fig. 6. Branch lengths on the tree are proportional to the average percent nucleotide substitutions corrected for lineage-specific differences in substitution rates given

in Table

2

When the phylogeny was in conflict with branch lengths connecting branches have broken lines.

common

molds,

filamentous

ascomycetes

yeasts

basal

ascom ycetes

mushroom

group

V

a

z

V

a

z

.-

V

a

m

Jurassic creta ceou s Tertiary

Triassic

Cambrian

I I

Silurian Permian

Carboniferous

Ordovician

Devonian


10/14

BERBEE

A N D

TAYLOR

23

substitution rates and time, placing a statistical confidence

living 290 Ma in the Carboniferous may resemble their extant

interval around our estimates of divergence times is not pos-

relatives as little as a 30 m tall, coal age, arborescent lycopod

sible in a rigorous sense. T he ranges of times presented in this

Lepidodendron resembles a modern 0.1 m tall quillwort in the

text are based on the variation in percent substitution among genus

Isoetes.

lineages and correspond to two standard deviations in both

directions from the average percent substitution accumulated

since the origin of lineages (Table 2).

Our calculations of percent substitution per lineage, with

and without correction factors, a re presented in Table 2. When

lineage-specific rate co rrections are used in an attempt to refine

the estimates of divergence times, the standard deviations of

the numbers of substitutions between the most divergent line-

ages, for example, between ascomycetes and basidiomycetes,

are reduced so that divergence times are generally consistent

with phylogeny (Tab le 2). Without lineage-specific rate corre c-

tions, the standard deviations of the numbers of substitutions

between deeply divergent taxa are high, and the divergence

times sometimes conflict with phylogeny, although the amount

of conflict is not significant compared with the large standard

deviations from the average num ber of substitutions (Table 2).

With or without lineage-specific corre ctions, w e found a simi-

lar pattern in the timing of origin of fungal groups. For exam-

ple, without correction for rate variation, an average of 4.7

(SD 1. 0 ) of the nucleotides have been substituted on the

branches leading from the chytrid to the terrestrial fungi

(Table 2). From o ur calibration of 1 substitution per 100 Ma ,

the divergence of chytrids from terrestrial fungi occurred

470 M a ago. Adding o r subtracting one standard deviation from

this estimate gives a range of 370-5 70 Ma. Th e rate-corrected

estimate for the same divergence was 5. 5 substitution and

would suggest that the chytrihs branched from terrestrial fungi

550 Ma ago. The rate-corrected estimate, although not identi-

cal to the-uncorrected estima te, falls within the range of esti-

mates from the uncorrected data.

To draw the tree in Fig. 5 , we took the branching order from

the tree in Fig. 1, which is based on parsimony analy sis, and

used the dis tances in Table 2 to ~rovidehe rate-corrected

lengths of branches, corresponding to the time scale.

Our estimate of divergence times using molecular clock

assumptions, with or without lineage-specific corrections,

leads to the conclusion that the main lineage of terrestrial fungi

diverged from aquatic chytrids roughly 550 Ma ago (400-

700 Ma), perhaps 50- 150 Ma before vascular plants colo-

nized the land (Taylor 1988 ). Possibly the terre strial radiation

of true fungi had its origin from groups of

Chytridium-like

freshwater fungi associated with green algae.

The terrestrial fungi are not motile, having lost flagella and

centrioles. Terrestrial fungi include the endomycorrhizal

Glomaceae, the ascomycetes, and the basidiornycetes. Based

on nucleotide substitution numbers, the Glomaceae diverged

from the progenitor of ascomycete and basidiomycete lineages

in the Ordovician, about 490 Ma ago (410-580 Ma) (Figs. 5

and 6). This is fairly consistent with Simon et al.'s (1993)

estimate of 353 -462 Ma for the divergence between the endo-

mycorrhizal genera

Glomus

and

Endogone.

The first terres-

trial fungal radiation may have been correlated with an adaptive

radiation of nonvascular land plants in the Mid-Ordovician,

about 460 Ma (Gray 1985). Land plants were well established

by the end of the Silurian, about 395 Ma (Gray 1985), by the

time ascomycetes and basidiomycetes lineages diverged . Major

lineages within the ascomycetes and basidiomycetes had evolved

by the end of the Carboniferous (Figs. 5 and 6). Although the

lineages were established, the ancestors of the modern fungi

The molecular clock and the fin gu s fossil record: conflicts

correlations and predictions

Precambrian jimgi?

Som e of the oldest, 900-380 0 Ma old, putative eukaryotic

fossils resemble modern true fungi (Schopf and Barghoorn

1969; Hallbauer et al. 1977; Pflug 1978). Molecular data

render it most improbable that these organisms are true fungi.

1 8 s ribosomal D NA sequence data suggest that the true fungi

originated at about the same time as the animals and the green

plants diverge d, possibly coinciding with a radiation of eukary-

otic fossil forms from 1 O to 1.2 Ga ago (Sogin 1989; Knoll

1992). The ascus-like elongate structure reported from 0.9 to

1.05 Ga sediments by Schopf and Barghoorn (1969) is, as

Mendelson and Schopf (1992) later suggested, most likely a

blue-green alga. We estimate that fungi producing elongate

asci containing eight ascospores arose after filamentous fungi

diverged from true yeasts, less than 3 10 Ma ago (230 90 Ma).

A 2.5-Ga lichen-like fossil from Witwatersrand, South Africa

(Hallbauer et al. 1977) exceeds our estimates of the probable

age of ascomycetes and basidiomycetes by 2 Ga . (Lichens in

current ecosystems are usually symbioses between ascomy-

cetes and algae, occasionally between basidiomycetes and

algae.) Yeast-like fossils from 3.4- and 3.8-Ga chert (Pflug

1978) are about 3 Ga older than ascomycetous o r basidiomy-

cetous yeasts. A ges of both the lichen and the yeast-like fossils

exceed the age of unambiguous fossil or biogeochemical evi-

dence for the presence of eukaryotes (Knoll 1992).

Ascomycetes

Ascomycetes fall phylogenetically into three groups estab-

lished perhaps during the coal age, from 330 (240-430 Ma)

to 310 Ma (230-3 90 Ma) (Fig. 5). 'The basal group includes

the peach leaf curl fungus

Taphrina deforman s

the mammal

lung pathogen Pneumocystis carinii and the saprobic fission

yeast

Schizosaccharomyces pombe.

Th e phylogeny of th e basal

ascomycetes is not resolved with statistical support (Fig. I),

and the three fungi have few unifying, advanced morphologi-

cal or ecological characters. As fossils, basal ascomycetes

would be hard to detect. The poorly resolved phylogeny pro-

vides little grounds for guessing w hich basal ascom ycete most

resembles the common ancestor to the group.

The second group, the true yeasts, form a well-supported

monophyletic group. The true yeasts are filamentous or uni-

cellular ascomycetes without fruiting bodies that diverged

from the lineage of filamentous ascomycetes w ith fruiting bod-

ies about 3 10 Ma ag o (230-390 Ma). From the phylogeny

(Fig. l), the first true yeasts were filamentous, giving-rise

to Dipodascopsis uninucleata and Endomyces geotrichum. The

lineage of unicellular yeasts, represented in the phylogeny by

Kluyveromyces lactis

and baker's yeast,

Saccharomyces cere-

visiae

diverged from the filamentous types roughly 240 Ma

ago (210-280 Ma). In the fossil record, true ascomycetous

yeast filaments and unicells would be difficult to distinguish

from similar structures made by zygomycetes, basidiomy-

cetes, or other ascomycetes (Tiffney and Barghoorn 1974).

The third group, the sister group to the true yeasts, com-

prises the filamentous ascomvcetes that enclose their sexual

spore sacs in fruiting bodies. At least three and probably m ore

lineages of filamentous ascomycetes radiated beginning approx-


11/14

CAN. J BOT.

VOL. 71

993

FIG. 6. This diagram illustrates so me milestones in the elaboration of fungal forms co rresponding to the tree and time scale in Fig . 5. B ra

ing, aseptate fungal filaments b ) originated after terrestrial higher fungi diverged from water molds

a ) .

Septate filaments

c )

evolved

the pre-basidiomycetes -ascomycetes diverged from the Glomaceae about 500 Ma ago. Clamp connections d)mark early basidiomycetes.

ual structures, asci

g )

and basidia

e ) ,

probably evolved before ascomycetes and basidiomycetes radiated. Asexual spores f ) and com

fruiting bodies

h )

probably increased as filamentous ascomycetes radiated. Mushrooms j ) , haring basidial type

i )

and other morpholo

features with

Athelia

and

Spongipellis,

probably radiated about

130

Ma ago.

5 4 3 2 1 Millions

ima te ly 280 M a ago 180-320 Ma) Be rbee and Tay lo r

1992a, 19926; A. Gargas and J.W. Taylor , unpubl ished da ta)

Fig. 5 ) . Frui t ing bodies, because they a re present in a l l the

l ineages, most l ike ly evolved before these la te coal age to

Early Permian f i lamentous fungus divergences.

Flask-like, perithecial fruit ing bodies chara cterize a l ineage

of f i lamentous ascomycetes inc luding Ophiostorna ul rni and

Neurospora cras sa . T he f lask-shaped frui t ing bodies presum-

ably evolved before the first radiations of perithecial fungi

a r ou n d 1 7 0 M a a g o T a b l e 2 ; F i g . 5 ) .

Fr om molecular c lock evid ence , c losed , c le is tothecia l f rui t-

ing bodies or igina ted more recent ly . The fami ly Tricho

maceae , which inc ludes Talarornyces f lavus and Eurot

rubrurn comm on molds wi th Penic il l iurn and Asperg

s t at e s) , i s l ess t han 100 M a o ld F ig . 5 ) . L ik e members o f

T r i chocomaceae , foss il fo rm gene ra T raqua i r i a , Mycoca rp

Dub iocarbo n, e tc . , produce c losed f rui t ing bodies Stub

f ie ld and Taylor 1983; Stubblef ie ld e t a l . 1983) . Howe

these foss il s da t e back t o t he coa l age , 200 M a be fo re t he e

mated t ime of or igin of the i r puta tive fami ly . T he resolu

of th is confl ic t lies in the morphologica l fea tures of these fo

genera , which are mo re consistent wi th sporocarps in the zy

I

I

of years

Silurian

Ordovlclan

Devonian

Paleozoic Mesozoic Cenozoic

Permian

arbonlferous

Jurassic

retaceous Tertiary

Triassic


12/14

BERBEE A N D

TAYLOR

1125

mycete families Glomaceae or Endogonaceae than with asco-

mycete fruiting bodies (Pirozynski 1976; White and Taylor

1991). Th e fossil fruiting body form s can be empty or contain

large spheres, irregular in size. The spheres, too large to be

ascospores, have occasionally been interpreted as asci (Stubble-

field et al. 1983). However, asci of the Trichocomaceae are

uniform in size. The delicate Trichocomaceae asci release

spores into the central cavity of the fruiting body soon after

ascus formation. Unlike the fossils, fruiting bodies of the

Trichocomaceae are more frequently full of thick-walled,

resistant ascospores than with thin-walled and short-lived asci.

Convincing Paleozoic ascomycete fossil fruiting bodies have

yet to be found (Pirozynski and Weresub 1979), perhaps

because of the technical difficulties involved in finding small,

fossilized reproductive structures.

Compared with sexual fruiting bodies, asexual spores of

filamentous ascomycetes are abundant and widely dispersed.

Comp ared with the propagules from the basal ascomycetes or

the yeast groups , fossil filamentous ascomycete asexual sp ores

are morphologically distinctive. Consequen tly, the fossil record

of asexual spores is richer than the record of fruiting bodies

and more interpretable than the record of yeast groups. Con-

sistent with the radiation of mold s pores predicted from m olec-

ular clock considerations, Pirozynski (1976) and Pirozynski

and Were sub (1979) found diverse ascomycete asexual propa-

gules from the Lower Cretaceous (Figs. 5 and 6). Pirozynski

and Weresub (1979) argue that the ascomycete lineages arose

in the Mesozoic.

Inconsistent with molecular clock estimates of origins of

filamentous ascomycetes are structures that resemble propa-

gules from filamentous ascomycetes, from 400-Ma, Silurian

sediments (Sherwood-Pike and Gray 1985). These spo res are

almost 120 Ma (range 80-220 Ma) older than the estimated

radiation of filamentous ascomycetes.

Several explanations could account for this discrepancy

between molecular and fossil data: (i) The fossils could be

modern fungal contaminants of Silurian samples, a possibility

Sherwood-Pike and Gray (1985) evaluate and reject. (ii) The

fossils may be Silurian but may represent a group of nonfungi

or extinct fungi such as pre-ascomycetes, distantly related to

extant filamentous ascomycetes. From molecular clock esti-

mates, w e predict that filamentous ascomycetes did not evolve

until the Carboniferous but that the common ancestor to the

ascomycetes and basidiomycetes was present in the Silurian,

and we find appealing the possibility that the Silurian spores

were produced by the ancestors to the higher fungi. (iii) The

molecular clock estimate of the age of the filamentous ascomy-

cetes may be too low because w e have overestimated the rates

of nucleotide substitution in the fungi. W e accept that a

molecular clock may not be accurate to within 25 Ma, but

hope that it is usually accurate to within 1 00 Ma. If the Silurian

fossils do indicate that filamentous ascomycetes had not only

originated but had radiated by the Silurian, ou r estimates of the

age of fungal lineages should be increased by one-third. The

origin of Glomaceae, the Ascomycotina, and the Basidiomy-

cotina would be in the Precambrian and likely in the seas, so

that the major lineages colonized land independently (not a

particularly parsimonious assumption).

The general lack of ascomycete propagules from strati-

graphic series between the Silurian and the Low er Cretaceous

(Pirozynski and Weresub 1979) argues against a filamentous

ascomycete affinity for the 400-Ma fossil spores. T he Silurian

spores vary in septation and shape. If they were ascom ycetous,

they came from several different genera. Having radiated to

such an extent in th e Silurian, it is difficult to understand why

the filamentous ascomycete spores would vanish from the fos-

sil record for the next 280 Ma.

Basidiomycetes

If, as we estimate, the first basidiomycetes diverged from

ascomycetes 390 Ma ago (270-510 Ma), then thread-like

fungal filaments associated with decayed Callixylon wood

from Upper Devonian, about 350 Ma, could represent ear ly

saprobic basidiomycetes (Stubblefield et al. 1985). At about

340 Ma (170-5 00 Ma ), shortly after their split from the

ascomycetes, the basidiomycetes sep arated into three lineages

(Fig. 1). Th e smuts and rusts, extant descendants of two of the

early basidiomycete lineages, have simple septal pores. Septa

in the third lineage (which in cludes mushroom s) have layered

membranes and complex septal swellings surrounding their

pores.

Among the simple-pored groups, the divergence of the rust

lineage from related basidiomycetes about 310 M a ago is con-

sistent with Savile s (1955) speculation that rusts a rose as

parasites of early vascular plants. The simple-pored smut

genera Ustilago and Tilletia may have diverged from a com-

mon ancestor about 230 M a ago. A high proportion of species

in both smut genera are obligate parasites of monocots. The

age of their divergence suggests that they may have evolved

as pathogens on the very earliest monocots.

The basidiomycetes with complex septal swellings form a

monophyletic group including jelly fun gi, mushrooms , an d the

shelf fungi. The jelly fungi diverged f rom the holobasidiomy-

cetes, fungi with undivided basidia including the mushrooms

and shelf fungi, about 220 Ma ago (170-270 Ma). Th e earli-

est fossil holobasidiomycete, a convincing silicified shelf

fungus from the upper Middle Jurassic , f rom 165 Ma (Singer

and Archangelsky 1958), served as one of our calibration points.

Some of the basidiomycetes with comp lex septa can degrade

lignin to produce a characteristic white pocket rot. White

pocket rotters are fairly common among the holobasidiomy-

cetes, but they are also found in the Dacrymycetales (Seifert

1983), a lineage of jelly fungi that originated before Tremella

diverged from the holobasidiomycetes (Swann and Taylor

1992). Since it occurs in two of the most divergent lineages,

the ability to cause white pocket rot may have evolved early

in the history of com plex-pored basidiornycetes. Stubblefield

and Taylor (1986) report paleobotanical evidence of white

pocket rots from the Permian, about 290 Ma ago.

Fungal decay is more evident in Cretaceous and Tertiary

coals than in older Carboniferous coals (Robinson 1990). This

may reflect differences in the env ironments in which Paleozoic

and Mesozoic coals were formed (Schopf 19 52), but it may

also reflect the Mesozoic radiation of the holobasidiomycetes,

the most aggressive wood rotters in extant environments

(Robinson 1990).

From the molecular clock evidence, the holobasidiomycete

group including mushrooms radiated in the Cretaceous approxi-

mately 130 Ma, after angiosperms had become an important

part of the flora. M ost mushroo ms are ephem eral and disinte-

grate rather than fossilize. Mushrooms probably originated

well before Coprinites dominicana, the earliest known fossil

mushroom, was trapped in amber approximately 40 Ma ago

(Poinar and Singer 1990).

Many of the same basidiomycetes that form mush rooms also

form symbioses with the fine roots of trees, modifying the


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1126

C A N .

I

BOT. VOL. 71

1993

r oo ts in to shor t , th ick , and d ichotomous ly br anched s t r uc -

tur es . Recognizab le ec tomycor r h iza l f os s i l s should pe r haps

da te back to the mushr oo m r ad ia tion of about 130 Ma . Foss i l

ec tomycor r h izae f r om the Pa leozoic or Mesozoic have ye t to

be f ound Taylor 1990) .

Fossils versus molecules: limitations

Fo r sever a l r easons , i t i s d i ff i cul t to ex t r ac t an or de r ly s e r ie s

of phylogene t ic events f r om the f ungus f os s i l r ecor d . F i r s t ,

s exua l s ta te s tha t have a l l the char ac te r s needed f or accur a te

iden ti f ica t ion F ig . 6 ) a r e

so

s m a l l a n d e p h e m e r a l a p a r t o f t h e

na tura l b iomass tha t f ind ing them as f os s i ls has been ex t r emely

di f fi cu l t. Th ey a r e un l ike ly to a ppear in the f os s i l r ecor d un t i l

many m i l lions of yea r s a fte r they became co mmo n in na tur e .

Second , the vege ta t ive s ta te s o f f ungi tha t a r e commo n both in

na tur e F ig . 6 ) and in the f oss i l r ecor d lack char ac te r s needed

f or r e l iab le t axonomic de te r mina t ion . Th i r d , a lgae a nd pr o to-

zoans make s t r uc tur es s imi la r to f ungi . Four th , f ungi g r ow

in to r ocks long a f te r r ock depos i t ion , and moder n f unga l

spor es a r e ub iqu i tous in the a i r and w a te r , compl ica t ing the

task of distinguishing mode rn contaminants fro m genuin e fossils.

O nly th r ough f os s i l ev idence , ho w ever , w i l l the mor phology

and eco logy of ances t r a l f ungi be r econs t r uc ted . Because the

molecula r phylogeny pr esen ted he r e i s cons i s ten t w i th mor -

phology , w e be l ieve tha t our e s t ima te of the or de r o f r ad ia t ion

of major g r oups of f ungi i s l a r ge ly cor r ec t . But even i f our

know ledge o f the phylogeny w er e pe r f ec t and even i f mor pho-

log ical evolu t ion w er e a lw ays pa r s imonious , r econs t r uc t ion of

the mor phologica l cha r ac te r s o f ances t r a l f ungi f r om seq uence

da ta a lone w ould f a i l . Thi s i s because the d ive r si ty of cha r ac -

te r s exhib i ted by ex tan t l ineages l eads to mul t ip le equa l ly pa r -

s imonious poss ib le char ac te r - s ta tes f or the ances t r a l nodes in

the phylogene t ic t r ee . Sequence da ta sugg es t tha t a f i l amentous

pre-ascomycete-basidiomycete

exis ted

4

M a a g o b u t n o t

how the f ungus spor u la ted or w he the r i t w as sapr ophyt ic o r

parasitic.

Molecula r c lock da tes a r e sub jec t to tw o impor tan t sour ces

of e r r or . W e cannot eva lua te the ex ten t o f the f ir s t sour ce of

e r r or , w hich s tems f r om ambigui t i e s in the ca l ib r a t ion cur ve

F ig . 4 ) . W e can es t ima te the ex ten t o f the second sou r ce of

e r r or , w hich s tems f r om var ia t ion in nuc leo t ide subs t i tu t ion

rates Tab le 2) . Assuming that a c lock- l ike substi tut ion rate

w ould have been w i th in tw o s tandar d devia t ions in e i the r

d i r ec t ion of the obse r ved pe r cen t subs t i tu t ions , w e es t ima te

t h at o u r t i m e s c a l e m a y b e a c c u r a t e t o p l u s o r m i n u s 1 0 0 -

200 M a f or the deepes t f ungal d ive r gences . W e an t ic ipa te tha t

addi tiona l ev idence f r o m o the r genes and f r om addi tiona l ca l i -

brat ion points wil l substant ia l ly ref ine these ini t ia l es t imates .

Acknowledgements

Thank s to D r . Phaf f f o r cu l tu r es of Taphrina deformans t o

D r . D . M i l l s a n d B . R u s s e ll f o r Tilletia caries and Ustilago

hordei

c u l t u r e s a n d D N A , t o D r . K . W e l l s f o r

Tremella

globospora a n d T. moriformis a n d t o

L

Sig le r f o r he lp in

se lec t ing s t r a ins of f ungi f or ana lys i s . W e thank D r . T Br uns ,

T . S z a r o , D r . A . G a r g a s , a n d D r . M . S o g i n f o r u n p u b l is h e d

D N A s e q u e n ce d a t a . D r . T . W h i t e p r o v id e d c r i ti c al c o m m e n t s

and sugg es ted us ing r umen chyt r id hos t s a s ca l ib r a t ion po in t s ,

A lan Br ow nlee pr ovided in f or mat ion about the d i s tr ibu tion of

chyt r id spec ies among mammal hos t s , and Jes s ica Theodor

suppl ied in f or mat ion about the ages of mamm al hos t s . Than ks

t o D r . R . B a n d o n i , D r . G . R o u s e , a n d D r . K . A . P i r o z y n s k i

f or c r i ti ca l comments . Th anks a l so to

H .

Phil l ippe, Univers i tC

Par i s X I , f o r comments concer n ing molecula r c lock cor r

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