Molecular Phylogenetics and Evolution 35 (2005) 60–75
www.elsevier.com/locate/ympev
Multi-gene phylogenies indicate ascomal wall morphology is a better
predictor of phylogenetic relationships than ascospore morphology
in the Sordariales (Ascomycota, Fungi)
Andrew N. Miller a,¤, Sabine M. Huhndorf b
a
b
Illinois Natural History Survey, Center for Biodiversity, 607 E. Peabody Dr., Champaign, IL 61820, USA
The Field Museum of Natural History, Botany Department, 1400 S. Lake Shore Dr., Chicago, IL 60605-2496, USA
Received 3 December 2003; revised 20 October 2004
Abstract
Ascospore characters have commonly been used for distinguishing ascomycete taxa, while ascomal wall characters have received
little attention. Although taxa in the Sordariales possess a wide range of variation in their ascomal walls and ascospores, genera have
traditionally been delimited based on diVerences in their ascospore morphology. Phylogenetic relationships of multiple representatives from each of several genera representing the range in ascomal wall and ascospore morphologies in the Sordariales were estimated using partial nuclear DNA sequences from the 28S ribosomal large subunit (LSU), -tubulin, and ribosomal polymerase II
subunit 2 (RPB2) genes. These genes also were compared for their utility in predicting phylogenetic relationships in this group of
fungi. Maximum parsimony and Bayesian analyses conducted on separate and combined data sets indicate that ascospore morphology is extremely homoplastic and not useful for delimiting genera. Genera represented by more than one species were paraphyletic or
polyphyletic in nearly all analyses; 17 species of Cercophora segregated into at least nine diVerent clades, while six species of Podospora occurred in Wve clades in the LSU tree. However, taxa with similar ascomal wall morphologies clustered in Wve well-supported
clades suggesting that ascomal wall morphology is a better indicator of generic relationships in certain clades in the Sordariales. The
RPB2 gene possessed over twice the number of parsimony-informative characters than either the LSU or -tubulin gene and consequently, provided the most support for the greatest number of clades.
 2005 Elsevier Inc. All rights reserved.
Keywords: Ascomycota; Bayesian inference; -Tubulin; LSU; Morphological characters; RPB2; Phylogenetics; Sordariales; Systematics
1. Introduction
The Sordariales is one of the most economically and
ecologically important groups within the ascomycetes in
that it contains species of Chaetomium, which are
responsible for the destruction of paper and fabrics, and
the “fruit Xies” of the fungal world (i.e., Neurospora
crassa, Podospora anserina, and Sordaria Wmicola). Taxa
within the order occur worldwide as saprobes on dung,
¤
Corresponding author. Fax: +1 217 333 4949.
E-mail address: [email protected] (A.N. Miller).
1055-7903/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2005.01.007
decaying wood, leaf litter, and soil (Lundqvist, 1972).
The Sordariales also was one of the most taxonomically
diverse orders being comprised of 114 genera divided
among 10 families (Eriksson and Hawksworth, 1998;
Eriksson et al., 2004), but recently has been reduced to
ca. 35 genera within three families, the Chaetomiaceae,
Lasiosphaeriaceae, and Sordariaceae (Huhndorf et al.,
2004). Since only one of these families (Sordariaceae)
was shown to be monophyletic by Huhndorf et al.
(2004), families within the Sordariales will not be further
discussed.
The Sordariales is one of several orders in the Class
Sordariomycetes (Eriksson et al., 2004). Taxa in the
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
Sordariomycetes (historically known as pyrenomycetes) usually form minute fruiting bodies ( D ascomata)
containing hymenial layers commonly composed of
sterile hyphae intermixed among asci (with single wall
layers) possessing ascospores (Alexopolous et al., 1996).
Few morphological characters exist with which to
delimit taxa in the Sordariomycetes most likely due to
their small stature and simple structure. Taxa within
the Sordariomycetes have traditionally been distinguished based on characters of the ascomata and
ascospores, although centrum and ascus morphologies
also have been used at higher taxonomic levels (Barr,
1990; Luttrell, 1951; Parguey-Leduc and Janex-Favre,
1981). Ascomata can have single- or multi-layered walls
and may possess various types of outside covering such
as tomentum, hairs, or setae. Although considerable
variation in ascomal wall morphology exists in the
Sordariomycetes, its potential use in systematics has
seldom been recognized (Jensen, 1985). Several workers, however, have noted similarities in ascomal wall
characters among taxa (von Arx et al., 1984; Barr, 1978;
Carroll and Munk, 1964; Jensen, 1985; Lundqvist,
1972).
Genera within the Sordariales have been delimited
primarily on diVerences in their ascospore morphology
(Lundqvist, 1972) (Fig. 1). While ascospore morphology
varies little within a genus, ascospores among genera in
the Sordariales range from a cylindrical, hyaline ascospore in Lasiosphaeria (Fig. 1A) to an ellipsoidal, brown
ascospore in Sordaria (Fig. 1I). Intermixed between these
two extremes are many genera which possess two-celled
ascospores with cylindrical to ellipsoidal, brown cells
and diVerent degrees of cylindrical to triangular (often
basal), hyaline cells (Figs. 1B–G) (Lundqvist, 1972). Several earlier workers (Boedijn, 1962; Chenantais, 1919;
Lundqvist, 1972; Munk, 1953) hypothesized that ascospore evolution within this group may have occurred
along this continuum either through the gain or loss of a
hyaline cell, resulting in either Lasiosphaeria or Sordaria
being the derived genus.
61
Ascomal wall morphology also has been suggested as
an alternative means of delimiting certain genera within
this group (Lundqvist, 1972) (Fig. 2). All members of
Bombardia and Bombardioidea possess a similar ascomal
wall referred to as a bombardioid wall, which contains a
putatively stromatic ( D arising from vegetative hyphae)
gelatinized layer composed of interwoven hyphae
(Lundqvist, 1972) (Fig. 2C). Three other genera (Arnium,
Cercophora, and Podospora) also contain species that
possess a similar gelatinized layer in their ascomal wall,
but since the wall is non-stromatic, it is termed pseudobombardioid (Miller, 2003) (Figs. 2A and B). However,
all of these species have been placed into diVerent genera
based primarily on diVerences in their ascospore morphologies (Fig. 1). Certain species of Cercophora and
Lasiosphaeria also have been placed into separate genera
based on diVerences in their ascospore morphologies
even though they possess similar three-layered ascomal
walls in which the outer layer is composed of hyphae
that form a tomentum (Fig. 2D). Finally, certain species
of Podospora possess ascomata with outer wall layers
that form swollen protruding cells or agglutinated hairs
(Fig. 2E), and some of these species have been transferred into a separate genus, Schizothecium (Lundqvist,
1972). These genera, which contain species that possess
ascomata with obvious morphological diVerences in
their ascomal walls, are the focus of this paper. Additional genera in the Sordariales (e.g., Apiosordaria, Jugulospora, and Triangularia), which contain species that
possess ascomata with morphologically simple ascomal
walls, require further study and will be treated in future
studies. Our study is the Wrst to evaluate ascomal wall
characters for their phylogenetic potential in delimiting
certain genera in the Sordariales.
Several nuclear and mitochondrial ribosomal and
protein-coding genes have been employed for assessing
phylogenetic relationships of Wlamentous ascomycetes.
Nuclear ribosomal genes such as 18S small subunit
(SSU) and 28S LSU are commonly used due to their
ease in ampliWcation resulting from their high copy num-
Fig. 1. Ascospores of representative genera in the Sordariales. (A) Lasiosphaeria. (B) Cercophora. (C) Podospora. (D) Apiosordaria. (E) Triangularia.
(F) ZopWella. (G) Jugulospora. (H) Bombardioidea. (I) Sordaria. Ascospore evolution has been hypothesized to have occurred through the loss
(A ! I) or gain (I ! A) of a hyaline tail resulting in either Sordaria or Lasiosphaeria being the derived genus. Ascospores not to scale.
62
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
Fig. 2. Ascomal walls occurring in members of the Wve wall clades (A–E); outer layer to the right. (A) Pseudo-bombardioid wall (Podospora Wmiseda).
(B) Pseudo-bombardioid wall (Cercophora scortea). (C) Bombardioid wall (Bombardioidea anartia). (D) Three-layered wall with outer layer of
hyphae forming tomentum (Lasiosphaeria ovina). (E) Wall with agglutinated hairs (Schizothecium vesticola). Ascomal walls not to scale.
ber and the availability of numerous universal primers
(Vilgalys and Hester, 1990; White et al., 1990). Most
studies utilize the Wrst 1100 bp of the 5⬘ end of LSU,
which contains three variable domains. Nuclear proteincoding genes such as -tubulin and RPB2 are increasingly being used in ascomycete phylogenetic studies
incorporating multiple, unlinked genes. The -tubulin
gene contains a highly variable intron-rich 5⬘ end and a
more conserved intron-poor 3⬘ end, the latter of which
has been used to recover higher-level relationships in
ascomycetes (Landvik et al., 2001). A paralogous copy
has been discovered in ascomycetes (Gold et al., 1991;
May et al., 1987; Panaccione and Hanau, 1990), but the
duplication event is believed to have occurred after the
divergence of the Sordariomycetes (Landvik et al., 2001).
The RPB2 gene is the second largest subunit of the ribosomal polymerase II gene and contains 12 conserved
sequence motifs interspersed among highly variable
regions (Liu et al., 1999). Although a second copy has
recently been found in plants (Oxelman and Bremer,
2000), RPB2 is believed to occur as a single orthologous
gene in fungi and is becoming increasingly popular in
studies of ascomycete phylogeny (Liu et al., 1999; Miller
and Huhndorf, 2004b; Reeb et al., 2004; Zhang and
Blackwell, 2002).
The primary purpose of this study was to test whether
ascospore morphology is phylogenetically informative for
predicting generic relationships within the Sordariales
using a multi-gene approach. Multiple species, which possess the range of ascospore morphologies known to occur
in the order, were sampled from each of several genera. To
determine the phylogenetic potential of ascomal wall morphology for delimiting certain genera in the Sordariales,
species with similar ascomal walls in several genera also
were included. Finally, a comparison of the LSU, -tubulin, and RPB2 genes was made for their utility in resolving
phylogenetic relationships within this group of fungi.
2. Methods
2.1. Taxon sampling
Taxa used in this study are listed in Table 1 along
with source information and origin for those specimens
sequenced in this study. Multiple representatives were
included from each of eight genera representing the
range in ascospore morphology in the Sordariales. Several of these taxa also possess similar ascomal wall morphologies. The full data sets contained 95, 83, and 68
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
63
Table 1
Taxa used in this study
Taxon
Annulatascus triseptatus
Annulatascus triseptatus
Annulatascus velatispora
Anthostomella sp.
Apiosordaria backusii
Apiosordaria verruculosa
Barrina polyspora
Bertia moriformis
Bombardia bombarda
Bombardia bombarda
Bombardia bombarda
Bombardioidea anartia
Botryosphaeria rhodina
Botryosphaeria ribis
Camarops amorpha
Camarops petersii
Camarops tubulina
Camarops ustulinoides
Capronia mansonii
Catabotrys deciduum
Cercophora sp.
Cercophora areolata
Cercophora atropurpurea
Cercophora atropurpurea
Cercophora caudata
Cercophora coprophila
Cercophora costaricensis
Cercophora lanuginosa
Cercophora macrocarpa
Cercophora aV. mirabilis
Cercophora aV. mirabilis
Cercophora newWeldiana
Cercophora newWeldiana
Cercophora rugulosa
Cercophora scortea
Cercophora sordarioides
Cercophora sparsa
Cercophora striata
Cercophora striata
Cercophora sulphurella
Cercophora terricola
Chaetomium elatum
Chaetomium globosum
Chaetomium microascoides
Chaetosphaerella phaeostroma
Chaetosphaeria innumera
Chaetosphaeria ovoidea
Coniochaeta ligniaria
Coniochaetidium savoryi
Copromyces sp.
Daldinia concentrica
Diaporthe phaseolorum
Diatrype disciformis
Dothidea insculpta
Duradens sp.
Eutypa sp.
Gelasinospora tetrasperma
Hypocrea pallida
Hypocrea schweinitzii
Sourcea,b
SMH2359
SMH4832
GenBank
SMH3101
ATCC34568
F-152365 (A-12907)
AWR9560A
SMH4320 (a)
AR1903
SMH3391
SMH4821
HHB99-1 (a)
GenBank
GenBank
SMH1450
JM1655 (a)
SMH4614 (a)
SMH1988 (a)
GenBank
SMH3436
SMH3200
UAMH7495
SMH2961
SMH3073
SMH3298
SMH3794 (a)
SMH4021 (a)
SMH3819
SMH2000
SMH4238
SMH4002 (a)
SMH2622
SMH3303
SMH1518
GJS L556
UAMH9301
JF00229 (a)
SMH3431 (a)
SMH4036 (a)
SMH2531
ATCC200395
GenBank
SMH4214b
F-153395 (A-12898)
SMH4585 (a)
SMH2748
SMH2605
SMH2569
TRTC51980
TRTC51747 (CBS386.78)
GenBank
FAU458
GenBank
GenBank
SMH1708
SMH3580
ATCC96230
GenBank
GenBank
Originc
Costa Rica
France
Puerto Rico
Japan
Spain
Texas
Michigan
New Zealand
Michigan
France
Alaska
Puerto Rico
Indiana
Denmark
Puerto Rico
Panama
Costa Rica
Canada
Puerto Rico
Puerto Rico
North Carolina
Puerto Rico
Costa Rica
North Carolina
Puerto Rico
Costa Rica
Costa Rica
Michigan
North Carolina
Puerto Rico
Louisiana
France
France
Panama
Costa Rica
Illinois
Japan
Jamaica
Spain
England
North Carolina
Michigan
Michigan
Malawi
Argentina
Mississippi
Puerto Rico
Panama
Canada
GenBank Accession No.d
LSU
-Tubulin
RPB2
AY346257
AY780049
AF132320
AY780050e
AY780051
AY346258
AY346261
AY695260
AY780052e
AY346263
AY780053
AY346264
—
AY004336
AY780054e
AY346265
AY346266
AY346267
AY004338
AY346268
AY780055e
AY587936
AY780056e
AY780057
AY436407
AY780058
AY780059
AY436412
AY780060e
AY780061
AY346271
AF064642
AY780062e
AY436414
AY780063
AY780064
AY587937
AY780065
AY780066
AY587938
AY780067
—
AY346272
AY346273
AY346274
AY017375
AF064641
AY346275
AY346276
AY346277
U47828
AY346279
U47829
—
AY780068e
AY346280
AY346281
—
—
AY780082
AY780083
—
AY780084
AY780085
AY780086
AY780087
AY780088
AY780089
AY780090
AY780091
AY780092
—
—
AY780093
AY780094
AY780095
AY780096
—
AY780097
AY780098
AY600252
AY780099
AY780100
AY780101
AY780102
AY780103
AY600262
—
AY780104
AY780105
AF466019
AY780106
AY600272
AY780107
—
AY600253
AY780108
—
AY600254
AY780109
—
AY780110
AY780111
AY780112
AF466018
AF466057
AY780113
AY780114
—
—
AY780115
—
—
AY780116
AY780117
AY780118
—
—
AY780148
—
—
—
AY780149
AY780150
—
AY780151
AY780152f
AY780153f
AY780154
AY780155
AF107802
—
AY780156f
—
AY780157
—
—
AY780158
AY780159f
AY600275
—
AY780160f
AY780161
AY780162
AY780163
AY600283
AY780164f
AY780165
—
AY780166f
AY780167
AY600294
AY780168f
—
—
AY780169
—
AY600276
AY780170
AF107791
—
AY780171
AY780172
—
AY780173f
—
AY780174
—
—
AY780175
—
AF107800
—
AY780176
AY780177f
AY015639
AY015636
(continued on next page)
64
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
Table 1 (continued)
Taxon
Sourcea,b
Hypocrea virens
Hypomyces luteovirens
Hypomyces odoratus
Induratia sp.
Jugulospora rotula
Lasiosphaeria glabrata
Lasiosphaeria hirsuta
Lasiosphaeria hispida
Lasiosphaeria immersa
Lasiosphaeria ovina
Lasiosphaeria sorbina
Lasiosphaeriella nitida
Leptosphaeria maculans
Linocarpon appendiculatum
Melanochaeta hemipsila
Microascus trigonosporus
Nectriopsis violacea
Neurospora crassa
Neurospora pannonica
Nitschkia grevillei
Ophioceras tenuisporum
Pleospora herbarum
Podospora anserina
Podospora appendiculata
Podospora comata
Podospora decipiens
Podospora Wbrinocaudata
Podospora Wmbriata
Podospora Wmiseda
Poroconiochaeta discoidea
Pseudohalonectria lignicola
Schizothecium curvisporum
Schizothecium vesticola
Sinosphaeria bambusicola
Sordaria humana
Sordaria Wmicola
Sordaria lappae
Sordaria macrospora
Strattonia carbonaria
Striatosphaeria codinaeaphora
Triangularia mangenotii
Triangularia tanzaniensis
Valsa ceratosperma
Valsonectria pulchella
Xylaria hypoxylon
ZopWella ebriosa
Zygopleurage zygospora
GenBank
GenBank
GenBank
SMH1255
ATCC38359
TL4529 (a)
SMH1543
SHM3336
SMH4104
SMH1538
GJS L555
SMH1664
GenBank
ATCC90499
SMH2125
GenBank
GenBank
GenBank
TRTC51327
SMH4663 (a)
SMH1643
GenBank
GenBank
CBS212.97
ATCC36713
CBS258.64
TRTC48343
CBS144.54
CBS990.96
SANK12878
SMH2440
ATCC36709
SMH3187
SMH1999
ATCC22796
SMH4106 (a)
SMH4107 (a)
Buck s.n.
ATCC34567
SMH1524
ATCC38847
TRTC51981
AR3426
SMH1193
GenBank
CBS111.75
SMH4219
Originc
GenBank Accession No.d
Puerto Rico
N/A
Denmark
Wisconsin
North Carolina
Wisconsin
Illinois
Louisiana
Puerto Rico
Brunei
Puerto Rico
Hungary
Illinois
Puerto Rico
New Zealand
Venezuela
Wyoming
California
N/A
New Zealand
Japan
Costa Rica
Kenya
Indiana
Puerto Rico
Oklahoma
Wisconsin
Wisconsin
Canada
Japan
Puerto Rico
Japan
Tanzania
Austria
Puerto Rico
N/A
Texas
LSU
—
AF160237
—
AY780069
AY346287
AY436410
AY436417
AY436419
AY436409
AF064643
AY436415
AY346289
—
AY346291
AY346292
—
AF193242
AF286411
AY780070
AY346294
AY346295
—
—
AY780071
AY780072
AY780073
AY780074e
AY780075
AY346296
AY346297
AY346299
AY346300
AY780076e
AY780077e
AY780078
AY780079
AY780080
AY346301
AY346302
AF466088
AY346303
AY780081e
AF408387
AY346304
U47841
AY346305
AY346306
-Tubulin
AY158203
—
Y12256
AY780119
AY780120
AY600255
AY780121
AY780122
AY780123
AF466046
AY600273
AY780124
AF257329
AY780125
AF466049
—
—
M13630
AY780126
AY780127
AY780128
Y17077
—
AY780129
—
AY780130
AY780131
AY780132
AY780133
AY780134
AY780135
AY780136
—
AY780137
—
AY780138
AY780139
AY780140
AY780141
—
AY780142
AY780143
AY780144
AY780145
—
AY780146
AY780147
RPB2
—
—
—
—
AY780178
AY600277
AY780179
AY780180f
AY780181
AY600287
AY600295
AY780182
—
AY780183f
AY780184
AF107792
—
AF107789
AY780185f
—
—
—
AF107790
AY780186f
—
AY780187
AY780188
AY780189f
AY780190f
AY780191
—
AY780192
—
AY780193
—
AY780194
—
AY780195f
AY780196f
—
—
AY780197
AY780198
AY780199f
—
AY780200
—
a
(a) D DNA extracted from ascomata; all others were extracted from cultures.
ATCC, American Type Culture Collection; CBS, Centraalbureau voor Schimmelcultures, Netherlands; TRTC, Royal Ontario Museum,
Toronto, Canada; UAMH, University of Alberta Microfungus Collection and Herbarium; AR, Amy Rossman; AWR, A. W. Ramaley; Buck, William Buck; FAU, Francis A. Uecker; GJS, Gary J. Samuels; HHB, Harold H. Burdsall; JF, Jacques Fournier; JM, Jack Murphy; SMH, Sabine M.
Huhndorf; TL, Thomas Læssøe.
c
Origin not given for taxa obtained from GenBank.
d
Dashes indicate gene was not sequenced for taxon.
e
For these taxa, although 1100 bp were used in the analyses, 1300 bp were sequenced and deposited in GenBank.
f
For these taxa, although a 1200 bp region between conserved motifs 5 and 7 (Liu et al., 1999) was used in the analyses, an 1800 bp region between
conserved motifs 3 and 7 was sequenced and deposited in GenBank.
b
taxa for the LSU, -tubulin, and RPB2 genes, respectively, while reduced data sets sampled the same 58 taxa
for each of the three genes and were subsequently used
in the combined analyses. Based on results from previous phylogenetic analyses (Huhndorf et al., 2004; Liu et al.,
1999; Miller and Huhndorf, 2004a), two representatives of
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
loculoascomycetes were used as outgroups for the full
data sets, while a member of the Xylariales was used to
root trees in the reduced data sets. All voucher specimens are deposited in the Field Museum Mycology Herbarium (F).
2.2. Morphological analyses
All taxa in which voucher specimens were available
(i.e., AR, AWR, GJS, HHB, JF, JM, SMH, and TL specimens) were used in morphological analyses (Table 1).
Ascospore morphology was observed from squash
mounts of ascomata made in water, while ascomal wall
morphology was determined from sections made at ca.
30 m following the techniques of Miller (2003). Images
were captured using diVerential interference (DIC)
microscopy from a Dage DC-330 video system mounted
on a Zeiss Axioskop and processed using Adobe Photoshop 3.0 and 5.5 (Adobe Systems).
2.3. DNA extraction, ampliWcation, and sequencing
A DNeasy Mini Plant extraction kit (Qiagen) was
used for extracting DNA from either dried ascomata or
cultures following the manufacturer’s protocols except
tissues were ground in 100 L AP1 buVer instead of liquid nitrogen. The relative quantity of total genomic DNA
was observed on a 1% TBE agarose gel stained with ethidium bromide. Gene fragments were PCR-ampliWed on
either a MJ Research PTC 200 or PTC 220 Dyad thermo
cycler using the following oligonucleotide primers:
LSU D LROR–LR7 (Rehner and Samuels, 1995; Vilgalys
and Hester, 1990), -tubulin D BT1819R–BT2916 (Table
2), and RPB2 D fRPB2-5f–RPB2AM-7R (Liu et al., 1999;
Table 2). The LSU was ampliWed using the following
thermocycling parameters: initial denaturation at 94 °C
for 2 min followed by 35–40 cycles of 94 °C for 30 s, 47 °C
for 15 s, and 72 °C for 1 min with a Wnal extension step of
65
72 °C for 10 min. Parameters for amplifying the proteincoding genes were identical except annealing was conducted at 50 °C for -tubulin and at 50 °C for 10 cycles
followed by 20–30 cycles at 54–58 °C for RPB2. ReadyTo-Go PCR beads (Amersham–Pharmacia Biotech)
were occasionally used to amplify diYcult taxa according
to the manufacturer’s instructions. In rare cases of weak
ampliWcation, a punch of the PCR product was taken
from the gel, suspended in 50–150 L double distilled
sterile water, melted at 70 °C, and 1 L of this dilution
was reampliWed using the thermocycling parameters
above except the annealing temperature was increased 3–
5 °C. After veriWcation on an ethidium bromide-stained
1% TBE agarose gel, PCR products were gel-puriWed on
a 1% TALE agarose gel using GELase Agarose GelDigesting Preparation (Epicentre Technologies). A BigDye Terminator Cycle Sequencing Kit (ABI PRISM,
Perkin–Elmer Biosystems) was used to sequence both
strands using a combination of the following primers:
LSU D LROR, LRFF1, LRAM1, LR3, LR3R, LR5, and
LR6 (Huhndorf et al., 2004; Rehner and Samuels, 1995;
Vilgalys and Hester, 1990); -tubulin D BT1819R, Bt1a,
BT1283, BT1283R, BTAM1f, BTAM1R, and BT2916
(Glass and Donaldson, 1995; Table 2); and
RPB2 D fRPB2-5f,
RPB2AM-6R,
RPB2AM-1f,
RPB2AM-1R, RPB2AM-1bf, RPB2AM-1bR, and
RPB2AM-7R (Table 2). Sequences were generated on an
Applied Biosystems 3100 automated DNA sequencer.
Each sequence fragment was subjected to a blast search
to verify its identity. Sequences were assembled and
aligned with Sequencher 4.1 (Gene Codes), optimized by
eye, and manually corrected when necessary.
2.4. Phylogenetic analyses
2.4.1. Saturation
A considerable number of changes occur in the third
codon positions compared to the Wrst and second
Table 2
Primers developed in this study for PCR ampliWcation and sequencing
Name
Primer sequence
Positiona
BT1819Rb
BT1283c
BT1283Rb
BT2916b
BTAM1f
BTAM1R
RPB2AM-6R
RPB2AM-1f
RPB2AM-1R
RPB2AM-1bf
RPB2AM-1bR
RPB2AM-7R
5⬘-TTC CGT CCC GAC AAC TTC GT-3⬘
5⬘-CGC GGG AAG GGC ACC ATG TTG-3⬘
5⬘-CAA CAT GGT GCC CTT CCC GCG-3⬘
5⬘-CTC AGC CTC AGT GAA CTC CAT-3⬘
5⬘-GTT CGA CCC CAA GAA CAT GAT GGC YGC-3⬘
5⬘-GCA GCC ATC ATG TTC TTG G-3⬘
5⬘-TTG ACC AGA CCR CAA GCC TG-3⬘
5⬘-GAG TTC AAG ATY TTC TCK GAT GC-3⬘
5⬘-GCA TCM GAG AAR ATC TTG AAC TC-3⬘
5⬘-CCA AGG TBT TYG TSA ACG G-3⬘
5⬘-GGY CTC ATR ACR CGR CCR GC-3⬘
5⬘-GAA TRT TGG CCA TGG TRT CCA T-3⬘
1131–1150
1643–1663
1643–1663
2151–2171
1757–1783
1765–1783
985–1004
1261–1283
1261–1283
1127–1145
1282–1301
1783–1804
Letters follow standard IUPAC–IUBMB ambiguity codes.
a
Relative to Neurospora crassa as sequenced in Orbach et al. (1986) for -tubulin (M13630) and in Liu et al. (1999) for RPB2 (AF107789).
b
Developed by Valérie Reeb in Lutzoni lab, Biology Dept., Duke University (http://www.lutzonilab.net/pages/primer.shtml).
c
Developed by Fernando Fernández in Huhndorf lab, Botany Dept., The Field Museum of Natural History.
66
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
Table 3
Comparison of data sets and trees in phylogenetic analyses
Data sets
Genes
Combined
-Tubulin
LSU
No. of total sitesa
No. of ambiguous sites
No. of constant sites
No. of polymorphic sites
No. of parsimony-informative sitesb
Percent of total sites that are
parsimony-informative
No. of MP trees
Length of MP trees
Consistency index
Retention index
Rescaled consistency index
No. of clades with 770% bootstrap support
No. of clades with 795% Bayesian post. prob.
a
b
RPB2
Full
Reduced Full
Reduced
Full
Reduced
1084
222
524
338
251
23
1039
126
599
314
228
22
939
0
568
371
41, 16, 254 (311)
33
924
0
591
333
27, 8, 231 (266)
29
1200
222
372
606
163, 84, 312 (559)
47
1197
222
407
568
153, 75, 310 (538)
45
3153
348
1596
1214
1032
33
2
3070.46
0.370
0.632
0.234
39
35
1
2070.52
0.402
0.583
0.235
22
26
2
4532.81
0.207
0.509
0.105
30
27
1
3164.12
0.248
0.471
0.117
19
20
1
12751.79
0.174
0.449
0.078
32
39
1
10242.09
0.195
0.467
0.091
29
40
1
15660.21
0.231
0.475
0.110
35
40
Excluding sites in 5⬘ and 3⬘ ends and introns.
Divided into Wrst, second, and third codon positions for -tubulin and RPB2; total shown in parentheses.
positions in the two protein-coding genes, especially tubulin (Table 3), suggesting these sites may be saturated
and, thus, represent noise rather than phylogenetic signal. Therefore, analyses were conducted on the full tubulin and RPB2 data sets to determine if the Wrst,
second, and third codon positions were saturated by
constructing scatter plots which compare time of
sequence divergence to pairwise transition and pairwise
transversion divergences (Hackett, 1996). Uncorrected
pairwise sequence divergence (uncorrected “p”) was
used as an approximation of divergence time. Transitions and transversions at each of the codon positions
were determined to be saturated if the scatter of points
appeared to level oV as sequence divergence increased.
2.4.2. Maximum parsimony and Bayesian analyses
Maximum parsimony (MP) analyses were performed
on each of the three full data sets and on the equal-sized
(58 taxa) reduced data sets to assess the amount of
incongruence among data partitions (see below) and to
compare the relative utility of the three genes in resolving relationships. Portions of the 5⬘ and 3⬘ ends of each
data set were excluded from all analyses due to missing
data in most taxa. Fifteen and seven ambiguously
aligned regions were delimited in the full and reduced
LSU data sets, respectively, and these regions along with
three introns were excluded from all analyses. Single
introns in the -tubulin and RPB2 data sets also were
excluded from all analyses. Several taxa in the RPB2
data sets contained amino acid indels in the highly variable region between conserved motifs 6 and 7 (Liu et al.,
1999). These regions were so variable that even amino
acids could not be unambiguously aligned, so these
regions were excluded from all analyses. Unequally
weighted MP analyses were performed with 1000 random addition heuristic searches and TBR branch-swap-
ping using PAUP* 4.0b10 (SwoVord, 2002).
Unambiguously aligned characters in the LSU data sets
and each of the three codon positions in the -tubulin
and RPB2 data sets were subjected to a symmetric
stepmatrix generated using STMatrix ver. 2.2 (François
Lutzoni and Stefan Zoller, Biology Department, Duke
University), which calculates the costs for changes
among character states based on the negative natural
logarithm of the percentages of reciprocal changes
between any two character states. The phylogenetic signal from 11 of the 15 ambiguous regions in the full LSU
data set and Wve of the seven regions in the reduced LSU
data set was recovered using INAASE (Lutzoni et al.,
2000) and analyzed in the MP analyses. The remaining
LSU ambiguous regions and the two RPB2 ambiguous
regions were excluded because their recoded characters
contained more than 32 character states, which is not
allowed in PAUP*. Branch support for all MP analyses
was estimated by performing 1000 bootstrap replicates
(Felsenstein, 1985), each consisting of 100 random addition heuristic searches and TBR branch-swapping.
MODELTEST 3.06 (Posada and Crandall, 1998) was
used to determine the best-Wt model of evolution for
each data set. Bayesian analyses employing a Markov
chain Monte Carlo (MCMC) method were performed
using MrBayes 3.0b4 (Huelsenbeck and Ronquist, 2001)
as an additional means of assessing branch support. The
best-Wt model of evolution was implemented for each
data set in the separate analyses and for each partition
(i.e., separate models for LSU and for each of the three
codon positions in -tubulin and RPB2) in the combined
analyses. Constant characters were included and four
MCMC chains were ran simultaneously for 5,000,000
generations with trees sampled every 100th generation
resulting in 50,000 total trees. The MCMC chains always
achieved stationarity after the Wrst 20,000–150,000 gen-
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
67
erations ( D 200–1500 trees), so the Wrst 10,000 trees,
which extended well beyond the burn-in phase in each
analysis, were discarded. Posterior probabilities were
determined from a consensus tree generated from the
remaining 40,000 trees. This analysis was repeated three
times starting from diVerent random trees to ensure trees
from the same tree space were being sampled during
each analysis.
2.4.3. Combinability
The validity of the incongruence length diVerence
(ILD) test for determining whether multiple data sets
should be combined has recently been questioned
(Barker and Lutzoni, 2002; Yoder et al., 2001) and, thus,
other methods should be explored. One method of
assessing combinability of data sets, and the one
adopted in this study, is by simply comparing highly
supported clades among trees generated from diVerent
data sets to detect conXict (de Queiroz, 1993; MasonGamer and Kellogg, 1996). High support typically refers
to bootstrap support values 770% and Bayesian posterior probabilities 795% (Alfaro et al., 2003). If no conXict exists between the highly supported clades in trees
generated from these diVerent data sets, this suggests the
genes share similar phylogenetic histories and phylogenetic resolution and support could ultimately be
increased by combining the data sets.
3. Results
3.1. Phylogenetic analyses
3.1.1. Saturation
Except for third position transitions in the RPB2
gene, no evidence of saturation was detected in any of
the codon positions since scatter plots clearly show an
increase when pairwise sequence divergence is plotted
against pairwise transition/transversion divergence (Fig.
3). Only third positions were plotted for the -tubulin
gene since very few changes occur in the Wrst and second
codon positions (Table 3). The scatter plot of third position transitions in the RPB2 gene appears to be leveling
oV slightly suggesting a low level of saturation may be
occurring at these sites (Fig. 3). As expected, the scatter
of points in this area (points at the far right of the graph)
primarily represent pairwise comparisons between the
Sordariales ingroup taxa and the more distant loculoascomycetes outgroups. Therefore, additional MP analyses
were conducted on the RPB2 full data set in which third
position transitions were arbitrarily down-weighted by a
factor of 2, 10, and 100 relative to transversions using a
stepmatrix. Third position transitions and transversions
were weighted approximately equally in the original rate
substitution stepmatrix (A M C D 1.84, A M G D 1.77,
A M T D 1.86, C M G D 1.82, C M T D 1.63, and G M T D
Fig. 3. Saturation plots relating uncorrected pairwise sequence divergence to pairwise transition/transversion divergence. Only third position changes are shown for -tubulin (A), whereas Wrst, second, and
third position transitions (B), and transversions (C) are shown for
RPB2.
1.86). A single most-parsimonious tree with an identical
topology to that in Fig. 6 was estimated when transitions
were down-weighted by 2. However, phylogenetic resolution was substantially decreased in analyses in which
transitions were down-weighted by 10 and 100 in that
outgroup taxa (Coniochaetales and Chaetosphaeriales)
occurred within the ingroup (Sordariales) (data not
shown). This suggests that while some third position
68
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
transitions are becoming saturated, a majority of these
sites still possess phylogenetic signal which is necessary
to accurately estimate phylogenies.
3.1.2. Maximum parsimony and Bayesian analyses
The best-Wt maximum-likelihood model of evolution
for the LSU data sets was the TIMef model (Rodríguez
et al., 1990). The best-Wt model for the -tubulin data
sets and the full RPB2 data set was the GTR model
(Rodríguez et al., 1990), while the TrN model (Tamura
and Nei, 1993) was selected as the best-Wt model for the
reduced RPB2 data set. A proportion of sites were
invariable while the remaining sites were subjected to a
gamma distribution shape parameter in all models.
Applying separate models to each of the three codon
positions for the -tubulin and RPB2 data sets had little
eVect on the Bayesian posterior probabilities in the combined analyses (data not shown).
Maximum parsimony analyses of the LSU and tubulin full data sets each generated two trees. The LSU
trees diVered only slightly in relationships among species
of Sordaria, whereas the -tubulin trees diVered in relationships among species of Neurospora. One of the trees
from each data set is shown in Figs. 4 and 5. A single
most-parsimonious tree was generated in analyses of the
RPB2 full data set (Fig. 6). Data set and tree statistics
are listed in Table 3. Overall relationships among the
major ordinal and familial lineages diVered between the
most-parsimonious trees generated from the full data
sets, although most of these relationships were not supported (Figs. 4–6).
3.1.3. Combinability
Separate analyses of the three reduced data sets produced single most-parsimonious trees (data not shown).
Data set and tree statistics are listed in Table 3 for the
reduced data sets. Although minor diVerences occurred
among most-parsimonious trees in the reduced data sets
(data not shown), only one instance of highly supported
conXict existed between phylogenies. In the LSU tree, the
Coronophorales were a sister clade to the Hypocreales
with 77% bootstrap support, whereas this order was
placed within the Boliniales with 79% bootstrap support
in the -tubulin tree (data not shown). This conXict was
not supported by Bayesian posterior probabilities and is
most likely due to poor taxon sampling in these orders.
Since little evidence exists against combining these data
sets, they were analyzed simultaneously in a combined
analysis, which produced a single most-parsimonious tree
(Fig. 7). Combined data statistics are given in Table 3.
dariales in all analyses (Figs. 4–7). For example, 17 species of Cercophora segregate into at least nine diVerent
clades, while six species of Podospora occur in Wve clades
in the LSU tree (Fig. 4). Multiple species were sampled
from six to nine genera (Apiosordaria, Cercophora, Chaetomium, Lasiosphaeria, Neurospora, Podospora, Schizothecium, Sordaria, and Triangularia) in the full data sets
and all genera occur as paraphyletic or polyphyletic in at
least one of the most-parsimonious trees (Figs. 4–6).
Combined analyses of the reduced data sets corroborate
these results for four (Cercophora, Lasiosphaeria, Neurospora, and Podospora) of the six genera represented by
multiple species (Fig. 7). While morphology suggests
that ascospore evolution in the Sordariales may have
occurred directionally along a continuum (Fig. 1), our
molecular data do not support this in that species with
vastly diVerent ascospore morphologies occur in several
well-supported clades throughout the Sordariales (Figs.
4–7). Three well-supported clades exist which contain
species of Cercophora that cluster with species of Apiosordaria or Triangularia (Figs. 4 and 5), taxa which possess ascospore morphologies that do not occur along the
putative evolutionary transition. The most extreme
example is found in the well-supported clade that contains Bombardia and Bombardioidea (Figs. 4–7). While
Bombardia possesses Cercophora-like ascospores at one
end of the continuum (Fig. 1B), Bombardioidea possesses
ellipsoidal, brown ascospores at the opposite end of the
continuum (Fig. 1H).
3.3. Ascomal wall morphology
Although many of the clades within the Sordariales are
unsupported, Wve well-supported clades, which contain
species with similar ascomal wall morphologies, occur in
all trees (Figs. 4–7). Species of Cercophora and Podospora,
which possess similar pseudo-bombardioid walls (Figs.
2A and B), occur in two of these clades (wall clades A and
B), while species of Bombardia and Bombardioidea with
similar bombardioid walls (Fig. 2C) occur in a third clade
(wall clade C). A fourth clade (wall clade D) is represented by species of Cercophora and Lasiosphaeria which
possess a similar three-layered ascomal wall in which the
outer wall layer is composed of hyphae that form a
tomentum (Fig. 2D), while the Wfth clade (wall clade E) is
represented by species of Podospora/Schizothecium which
possess an outer ascomal wall layer that forms swollen
protruding cells or agglutinated hairs (Fig. 2E).
4. Discussion
3.2. Ascospore morphology
4.1. Ascospore morphology
Ascospore morphology is shown to be extremely
homoplastic in that multiple species in the same genus
occur in diVerent clades scattered throughout the Sor-
The Sordariales has recently been redeWned to include
genera which possess ascospores that share a similar
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
69
Fig. 4. One of two most-parsimonious trees based on the LSU data set of 95 taxa. Numbers above or below the branches indicate bootstrap support
based on 1000 replicates. Thickened branches represent signiWcant posterior probabilities (795%) generated from Bayesian analyses. Shaded boxes
indicate the Wve well-supported ascomal wall clades discussed in the text. ClassiWcation following Huhndorf et al. (2004) is shown along the right.
developmental pattern (i.e., cylindrical, hyaline ascospores that develop apical, brown heads and basal, hyaline
tails) (Huhndorf et al., 2004). Seventeen genera were
included in the LSU data set which possess ascospores
that vary along these developmental lines and all Wnd
their placement in the monophyletic Sordariales (Fig. 4).
The -tubulin and RPB2 data sets, which included 16
and 15 genera, respectively, corroborate these results
(Figs. 5 and 6). These genera possess ascospores which
form a continuum from a cylindrical, hyaline ascospore
in Lasiosphaeria to an ellipsoidal, brown ascospore in
Sordaria with numerous genera which possess ascospores with brown heads and hyaline tails intermixed
between (Fig. 1). This hypothesized trend in ascospore
evolution is, however, not supported by our molecular
data (Figs. 4–7). Since diVerent taxa occur as the basal
70
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
Fig. 5. One of two most-parsimonious trees based on the -tubulin data set of 83 taxa. Support values, shading, and classiWcation as in Fig. 4.
group of the order in diVerent trees, any trend, if one
even exists, in ascospore evolution is presently unclear.
Genera within the Sordariales have been delimited
primarily on diVerences in their ascospore morphology
(Lundqvist, 1972). While our data conWrm the Wndings
of Huhndorf et al. (2004) that ascospore morphology is a
good indicator of whether a taxon belongs in the Sordariales, these data indicate that ascospore morphology is
extremely homoplastic and a poor predictor of generic
relationships within the group. When more than one species from the same genus was included, most did not
resolve along current generic lines, but were scattered
throughout several clades (Figs. 4–7). Dettman et al.
(2001) also found ascospore morphology to be a poor
predictor of phylogenetic relationships in Neurospora
and Gelasinospora. Analyses of four nuclear genes
revealed that the two genera conventionally distinguished by diVerences in ascospore ornamentation did
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
71
Fig. 6. Single most-parsimonious tree based on the RPB2 data set of 68 taxa. Support values, shading, and classiWcation as in Fig. 4.
not represent two distinct monophyletic groups but
instead were a polyphyletic group of closely related,
morphologically similar taxa. The two species of Neurospora included in our study never occurred as a monophyletic clade in any analysis (Figs. 4–7).
The Sordariales is not the only group where ascospore morphology has been used for delimiting genera.
For example, generic delimitation within the Amphisphaeriaceae (Xylariales) has been based on ascospore pigmentation, septation, and shape and, in some cases,
ornamentation (Barr, 1994; Kang et al., 1999). Genera of
ascosporogenous yeasts also have been based primarily
on ascospore shape and ornamentation and, as in our
study, LSU sequences suggest that ascospore morphology is a poor predictor of generic relationships (Kurtzman and Robnett, 1994). Additional studies are needed
to determine whether ascospore morphology is phylogenetically informative for delimiting genera in other
groups of ascomycetes.
4.2. Ascomal wall morphology
Although ascospore morphology is a poor predictor
of generic relationships in the Sordariales, ascomal wall
72
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
Fig. 7. Single most-parsimonious tree based on the combined data set of 58 taxa. Support values, shading, and classiWcation as in Fig. 4.
morphology may serve as an alternative means of delimiting certain genera. Five highly supported clades (wall
clades A–E) are found in all trees (Figs. 4–7) and four of
these clades (wall clades A–D) contain taxa with diVerent ascospore morphologies but similar ascomal wall
morphologies. Because taxon sampling within each of
these Wve wall clades is nearly complete, it is unlikely
that the addition of more Sordarialean taxa will alter
current hypotheses of relationships in these clades.
Therefore, discussions of their homologous characters
are appropriate. However, taxon sampling for other
well-supported clades throughout the Sordariales is
presently incomplete and relationships may change with
the addition of more taxa. In addition, taxa in these
clades possess relatively simple 2- to 3-layered ascomal
walls, which possess no obvious morphological characters with which to distinguish them at the present time.
Additional taxa must be included and further examination of their ascomal walls must be completed before
their homologous characters can be adequately discussed.
Three well-supported clades (wall clades A–C) contain taxa possessing an ascomal wall with a gelatinized
layer (Figs. 2A–C). Wall clades A and B contain species
of Cercophora and Podospora which possess a pseudobombardioid wall (Figs. 2A and B), while wall clade C
contains species of Bombardia and Bombardioidea which
have a bombardioid wall (Miller, 2003) (Fig. 2C).
Ascospores in Cercophora are initially cylindrical but
eventually develop a swollen head and long tail (Fig.
1B), whereas those in Podospora are initially clavate
before developing a swollen head and short tail (Fig.
1C). The pseudo-bombardioid wall morphology is
slightly homoplasious in the Sordariales in that it
appears to have arisen independently in two distantly
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
related groups, although relationships among these
groups are unsupported at this time (Figs. 4–7). However, taxa in wall clade B can be diVerentiated from
those in wall clade A in possessing short, brown, hyalinetipped setae. Species of Bombardia and Bombardioidea
are morphologically similar in all characters except their
ascospores, which occur near the extremes of the morphological ascospore continuum. Ascospores in Bombardia are identical to those in Cercophora (Fig. 1B),
whereas those in Bombardioidea are ellipsoidal and
brown (Fig. 1H). However, these species form a highly
supported clade (wall clade C) united by a bombardioid
ascomal wall (Fig. 2C), which occurs as a homologous
character in the Sordariales (Figs. 4–7).
A fourth wall clade (wall clade D) contains species of
Cercophora and Lasiosphaeria. While both genera possess similar ascospores that are cylindrical and hyaline,
those in Cercophora eventually swell at one end and turn
brown (Fig. 1B). However, species in this clade possess a
similar three-layered ascomal wall in which the outer
layer is composed of hyphae that form a tomentum (Fig.
2D). This ascomal wall morphology is slightly homoplastic in that three additional taxa (C. coprophila, C.
sparsa, and C. sulphurella), which also possess morphologically similar walls, are not found within this wellsupported tomentum clade (Figs. 4–7). Cercophora
coprophila occurs well outside this clade, while C. sparsa
and C. sulphurella occur as unsupported sister taxa to
this clade. However, the wall in C. coprophila has been
interpreted by some to be slightly areolate (Lundqvist,
1972) and this distinction may separate it from species in
wall clade D.
The Wfth wall clade (wall clade E) includes species of
Podospora/Schizothecium which possess an outer ascomal wall layer that forms swollen protruding cells or
agglutinated hairs (Fig. 2E). Other species of Podospora
included in this study possess glabrous ascomata or are
covered with short to long, Xexuous hairs or stiV setae
(Lundqvist, 1972). Schizothecium can be further distinguished from Podospora by the absence of typical interascal paraphyses and ascospores which become septate
at a very early stage in their development (Lundqvist,
1972), but some believe these characters do not warrant
generic distinction (Bell and Mahoney, 1995). Although
wall clade E may presently be delimited by a combination of ascomal wall, centrum, and ascospore characters,
additional species putatively belonging in Schizothecium
should be included in future analyses to test the signiWcance of these characters in this clade.
While studying the surface morphology of outer ascomal walls, Jensen (1985) found similarities among representative species from several orders and families of
Sordariomycetes. He also noted that three genera in the
Sordariaceae (i.e., Gelasinospora, Neurospora, and Sordaria) possessed virtually identical outer ascomal wall
layers. These genera also possess similar membraneous,
73
3- to 4-layered ascomal walls and ellipsoidal, brown
ascospores (Lundqvist, 1972). Representatives of these
genera always formed a highly supported clade in our
study (Figs. 4–7). However, since these ascomal wall and
ascospore morphologies occur in several other taxa
throughout the Sordariales (Lundqvist, 1972), it is presently unclear which characters are phylogenetically
informative for delimiting this clade.
One may argue that only characters which are putatively homologous ( D synapomorphies) should be used
for delimiting taxa. However, this argument ignores the
fact that there are diVerent degrees of homoplasy. Characters can range from slightly homoplasious (arising in
only two distantly related groups) to extremely homoplasious (arising in several groups throughout a tree)
with certain levels of the former still contributing some
amount of phylogenetic structure to the data set. At
what level homoplasy stops becoming partially informative and becomes merely noise is presently unclear.
Although ascomal wall morphology is slightly homoplasious in some groups (i.e., tomentum wall, pseudobombardioid wall), ascospore morphology is extremely
homoplasious throughout this group. Thus, while ascospore morphology cannot be used for delimiting genera,
ascomal wall morphology alone or in combination with
other characters is still useful at some level for distinguishing taxa.
4.3. Comparison of genes
All three genes were compared using the same 58
taxa in the reduced data sets, which contain approximately the same number of total sites (i.e., 924–
1197 bp) (Table 3). However, RPB2 contains over
twice the number of parsimony-informative sites (538)
as LSU (228) or -tubulin (266) (Table 3) resulting in
longer branch lengths and increased support for
clades. RPB2 contains more clades with signiWcant
bootstrap support (29) and Bayesian posterior probabilities (40) than either LSU (22, 26) or -tubulin (19,
20). LSU possesses numerous indels in its Wrst three
domains thereby reducing the number of parsimonyinformative sites after ambiguous regions are
removed. Most of the phylogenetic signal from tubulin comes from third position changes (87%),
whereas third positions account for only a little over
half (57%) of the signal in the RPB2 gene. Despite the
extreme bias towards changes in third position sites in
-tubulin, these sites showed no evidence of saturation
for either transitions or transversions (Fig. 3).
Although changes are more evenly distributed
throughout Wrst, second, and third codon positions in
RPB2, a low level of saturation was detected in third
position transitions. Saturation of third position transitions and transversions in RPB2 also was found by
Reeb et al. (2004) in their study of euascomycetes.
74
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
Future studies employing RPB2 for estimating fungal
phylogenies should determine the level of saturation
at third positions and its eVect on the resolution of the
resulting phylogeny.
as two anonymous reviewers for their comments which
improved this paper. Sequences were generated in the
Pritzker Laboratory for Molecular Systematics and Evolution at FMNH. This work represents a portion of a thesis in partial fulWllment of the requirements for the
doctoral degree at the Graduate College of UIC.
5. Conclusion
This study contributes to the understanding of the
evolution of morphological characters in ascomycetes.
Based on results from MP and Bayesian analyses of
three genes, ascospores with similar morphologies
appear to have evolved independently numerous times
throughout the Sordariales, whereas ascomal walls
appear to be less homoplasious in this group. Relationships among taxa outside the wall clades are mostly
unresolved and most relationships among well-supported clades are currently unsupported making it diYcult to draw conclusions regarding the evolution of
ascomal wall and ascospore characters. Increased taxon
sampling will improve resolution in many clades, while
support for relationships will be increased through the
incorporation of additional genes. While genera cannot
be recognized based on ascospore morphology, they
may be delimited in certain clades based on ascomal wall
morphology. However, we are not advocating simply
replacing one form of a one-character taxonomy based
on ascospores with another based on ascomal walls;
redeWned genera would need to be delimited by a unique
combination of characters primarily involving, but not
limited to, those found in ascomal walls.
Acknowledgments
This work was supported in part by a NSF DDIG
Grant (Doctoral Dissertation Improvement Grant, DEB0105077) to ANM through the University of Illinois at
Chicago (UIC) and in part by NSF PEET Grants (Partnerships for Enhancing Expertise in Taxonomy, DEB9521926 and DEB-0118695) to SMH through the Field
Museum of Natural History (FMNH). ANM also was
supported during this study by a Lester Armour Graduate Fellowship from FMNH. Fieldwork for ANM was
supported in part by an ASPT (American Society of Plant
Taxonomists) Graduate Student Research Grant and an
UIC Provost Award. Fieldwork for SMH was supported
in part by the National Research Council Resident
Research Associate Post-doctoral Program in cooperation with the USDA Forest Service, Madison, WI. The
authors are most grateful to G. Bills, W. Buck, H. Burdsall, M. Calduch, F. Fernández, J. Fournier, J. Krug, T.
Læssøe, A. Rossman, G. Samuels, and A. Stchigel for providing specimens or cultures. V. Reeb and F. Fernández
graciously allowed us to use their unpublished primers.
We also wish to thank A. Mitchell and G. Mueller as well
References
Alexopolous, C.J., Mims, C.W., Blackwell, M., 1996. Introductory
Mycology, fourth ed. John Wiley, New York.
Alfaro, M.E., Zoller, S., Lutzoni, F., 2003. Bayes or bootstrap. A simulation study comparing the performance of Bayesian Markov chain
Monte Carlo sampling and bootstrapping in assessing phylogenetic
conWdence. Mol. Biol. Evol. 20, 255–266.
von Arx, J.A., Dreyfuss, M., Müller, E., 1984. A revaluation of Chaetomium and the Chaetomiaceae. Persoonia 12, 169–179.
Barker, F.K., Lutzoni, F.M., 2002. The utility of the incongruence
length diVerence test. Syst. Biol. 51, 625–637.
Barr, M.E., 1978. The Diaporthales in North America. Mycol. Memoirs 7, 1–232.
Barr, M.E., 1990. Prodromus to nonlichenized, pyrenomycetous members of Class Hymenoascomycetes. Mycotaxon 39, 43–184.
Barr, M.E., 1994. Notes on Amphisphaeriaceae and related families.
Mycotaxon 51, 191–224.
Bell, A., Mahoney, D.P., 1995. Coprophilous fungi in New Zealand. I.
Podospora species with swollen agglutinated perithecial hairs. Mycologia 87, 375–396.
Boedijn, K.B., 1962. The Sordariaceae of Indonesia. Persoonia 2, 305–
320.
Carroll, G.C., Munk, A., 1964. Studies on lignicolous Sordariaceae.
Mycologia 56, 77–98.
Chenantais, J.-E., 1919. Études sur les Pyrenomycètes. Bull. Soc. Mycol.
Fr. 35, 46–98.
de Queiroz, A., 1993. For consensus (sometimes). Syst. Biol. 42, 368–372.
Dettman, J.R., Harbinski, F.M., Taylor, J.W., 2001. Ascospore morphology is a poor predictor of the phylogenetic relationships of
Neurospora and Gelasinospora. Fung. Genet. Biol. 34, 49–61.
Eriksson, O.E., Hawksworth, D.L., 1998. Outline of the ascomycetes—
1998. Systema Ascomycetum 16, 83–296.
Eriksson, O.E., Baral, H.-O., Currah, R.S., Hansen, K., Kurtzman, C.P.,
Rambold, G., Laessøe, T. (Eds.), 2004. Outline of Ascomycota—
2004. Myconet, vol. 10, pp. 1–99.
Felsenstein, J., 1985. ConWdence intervals on phylogenies: An
approach using the bootstrap. Evolution 39, 783–791.
Glass, N.L., Donaldson, G.C., 1995. Development of primer sets
designed for use with the PCR to amplify conserved genes from
Wlamentous ascomycetes. Appl. Environ. Microbiol. 61, 1323–1330.
Gold, S.E., Casale, W.L., Keen, N.T., 1991. Characterization of 2 betatubulin genes from Geotrichum candidum. Mol. Gen. Genet. 230 (12), 104–112.
Hackett, S.J., 1996. Molecular phylogenetics and biogeography of tanagers in the genus Ramphocelus (Aves). Mol. Phylogenet. Evol. 5,
368–382.
Huelsenbeck, J.P., Ronquist, F.R., 2001. MrBayes: Bayesian inference
of phylogenetic trees. Bioinformatics 17, 754–755.
Huhndorf, S.M., Miller, A.N., Fernández, F.A., 2004. Molecular systematics of the Sordariales: The order and the family Lasiosphaeriaceae redeWned. Mycologia 96 (2), 368–387.
Jensen, J.D., 1985. Peridial anatomy and pyrenomycete taxonomy.
Mycologia 77, 688–701.
Kang, J.C., Hyde, K.D., Kong, R.Y.C., 1999. Studies on Amphisphaeriales: The Amphisphaeriaceae (sensu stricto). Mycol. Res. 103, 53–
64.
A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75
Kurtzman, C.P., Robnett, C.J., 1994. Orders and families of ascosporogenous yeasts and yeast-like taxa compared from ribosomal RNA
sequence similarity. In: Hawksworth, D.L. (Ed.), Ascomycete Systematics: Problems and Perspectives in the Nineties. Plenum, New
York, pp. 249–258.
Landvik, S., Eriksson, O.E., Berbee, M.L., 2001. Neolecta—A fungal
dinosaur. Evidence from beta-tubulin amino acid sequences. Mycologia 93, 1151–1163.
Liu, Y.J., Whelen, S., Hall, B.D., 1999. Phylogenetic relationships
among ascomycetes: Evidence from an RNA Polymerase II Subunit. Mol. Biol. Evol. 16, 1799–1808.
Lundqvist, N., 1972. Nordic Sordariaceae s. lat. Symb. Bot. Ups. 20, 1–
374.
Luttrell, E.S., 1951. Taxonomy of the pyrenomycetes. University of
Missouri Studies 24, 1–120.
Lutzoni, F., Wagner, P., Reeb, V., Zoller, S., 2000. Integrating ambiguously aligned regions of DNA sequences in phylogenetic analyses without violating positional homology. Syst. Biol. 49, 628–
651.
Mason-Gamer, R.J., Kellogg, E.A., 1996. Testing for phylogenetic conXict among molecular data sets in the tribe Triticeae (Gramineae).
Syst. Biol. 45, 524–545.
May, G.S., Tsang, M.L., Smith, H., Fidel, S., Morris, N.R., 1987. Aspergillus nidulans beta-tubulin genes are unusually divergent. Gene 55
(2–3), 231–243.
Miller, A.N., 2003. A reinterpretation of the pseudo-bombardioid
ascomal wall in taxa in the Lasiosphaeriaceae. Sydowia 55 (2),
267–273.
Miller, A.N., Huhndorf, S.M., 2004a. A natural classiWcation of Lasiosphaeria based on nuclear LSU rDNA sequences. Mycol. Res. 108
(1), 26–34.
Miller, A.N., Huhndorf, S.M., 2004b. Using phylogenetic species recognition to delimit species boundaries within Lasiosphaeria. Mycologia 96, 1106–1127.
Munk, A., 1953. The system of the Pyrenomycetes. Dansk Bot. Ark. 15
(2), 1–163.
Orbach, M.J., Porro, E.B., Yanofsky, C., 1986. Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant
mutant of Neurospora crassa and its use as a dominant selectable
marker. Mol. Cell. Biol. 6 (7), 2452–2461.
75
Oxelman, B., Bremer, B., 2000. Discovery of paralogous nuclear gene
sequences coding for the second-largest subunit of RNA polymerase II (RPB2) and their phylogenetic utility in Gentianales of the
Asterids. Mol. Biol. Evol. 17, 1131–1145.
Panaccione, D.G., Hanau, R.M., 1990. Characterization of two divergent beta-tubulin genes from Colletotrichum graminicola. Gene 86
(2), 163–170.
Parguey-Leduc, A., Janex-Favre, M.C., 1981. The ascocarps of ascohymenial Pyrenomycetes. In: Reynolds, D. (Ed.), Ascomycete Systematics. Springer-Verlag, New York, pp. 102–123.
Posada, D., Crandall, K.A., 1998. Modeltest: Testing the model of
DNA substitution. Bioinformatics 49, 817–818.
Reeb, V., Lutzoni, F., Roux, C., 2004. Contribution of RPB2 to multilocus phylogenetic studies of the euascomycetes (Pezizomycotina,
Fungi) with special emphasis on the lichen-forming Acarosporaceae and evolution of polyspory. Mol. Phylogenet. Evol. 32, 1036–
1060.
Rehner, S.A., Samuels, G.L., 1995. Molecular systematics of the Hypocreales: A teleomorph gene phylogeny and the status of their anamorphs. Can. J. Bot. 73 (Suppl. 1), S816–S823.
Rodríguez, F., Oliver, J.F., Marín, A., Medina, J.R., 1990. The general
stochastic model of nucleotide substitution. J. Theor. Biol. 142,
485–501.
SwoVord, D.L., 2002. PAUP*. Phylogenetic analysis using parsimony
(¤ and other methods). Version 4.0b10. Sinauer, Sunderland, MA.
Tamura, K., Nei, M., 1993. Estimation of the number of nucleotide
substitutions in the control region of mitochondrial DNA in
humans and chimpanzees. Mol. Biol. Evol. 10, 512–526.
Vilgalys, R., Hester, M., 1990. Rapid identiWcation and mapping of
enzymatically ampliWed ribosomal DNA from several Crytococcus
species. J. Bacteriol. 172, 4238–4246.
White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. AmpliWcation and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Innis, M.A. (Ed.), PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, pp. 315–322.
Yoder, A.D., Irwin, J.A., Payseur, B.A., 2001. Failure of the ILD to
determine data combinability for slow loris phylogeny. Syst. Biol.
50, 408–424.
Zhang, N., Blackwell, M., 2002. Molecular phylogeny of Melanospora
and similar pyrenomycetous fungi. Mycol. Res. 106, 148–155.