Multi-gene phylogenies indicate ascomal wall morphology is ......were included. Finally, a comparison of the LSU, -tubu-lin, and RPB2 genes was made for their utility in resolving

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 Illinois Natural History Survey, Center for Biodiversity, 607 E. Peabody Dr., Champaign, IL 61820, USAb 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 receivedlittle attention. Although taxa in the Sordariales possess a wide range of variation in their ascomal walls and ascospores, genera havetraditionally been delimited based on diVerences in their ascospore morphology. Phylogenetic relationships of multiple representa-tives from each of several genera representing the range in ascomal wall and ascospore morphologies in the Sordariales were esti-mated using partial nuclear DNA sequences from the 28S ribosomal large subunit (LSU), �-tubulin, and ribosomal polymerase IIsubunit 2 (RPB2) genes. These genes also were compared for their utility in predicting phylogenetic relationships in this group offungi. Maximum parsimony and Bayesian analyses conducted on separate and combined data sets indicate that ascospore morphol-ogy is extremely homoplastic and not useful for delimiting genera. Genera represented by more than one species were paraphyletic orpolyphyletic in nearly all analyses; 17 species of Cercophora segregated into at least nine diVerent clades, while six species of Podos-pora occurred in Wve clades in the LSU tree. However, taxa with similar ascomal wall morphologies clustered in Wve well-supportedclades suggesting that ascomal wall morphology is a better indicator of generic relationships in certain clades in the Sordariales. TheRPB2 gene possessed over twice the number of parsimony-informative characters than either the LSU or �-tubulin gene and conse-quently, 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 andecologically important groups within the ascomycetes inthat it contains species of Chaetomium, which areresponsible for the destruction of paper and fabrics, andthe “fruit Xies” of the fungal world (i.e., Neurosporacrassa, Podospora anserina, and Sordaria Wmicola). Taxawithin 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 taxonomicallydiverse orders being comprised of 114 genera dividedamong 10 families (Eriksson and Hawksworth, 1998;Eriksson et al., 2004), but recently has been reduced toca. 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 furtherdiscussed.

The Sordariales is one of several orders in the ClassSordariomycetes (Eriksson et al., 2004). Taxa in the

mailto: [email protected]

mailto: [email protected]

A.N. Miller, S.M. Huhndorf / Molecular Phylogenetics and Evolution 35 (2005) 60–75 61

Sordariomycetes (historically known as pyrenomyce-tes) usually form minute fruiting bodies ( D ascomata)containing hymenial layers commonly composed ofsterile hyphae intermixed among asci (with single walllayers) possessing ascospores (Alexopolous et al., 1996).Few morphological characters exist with which todelimit taxa in the Sordariomycetes most likely due totheir small stature and simple structure. Taxa withinthe Sordariomycetes have traditionally been distin-guished based on characters of the ascomata andascospores, although centrum and ascus morphologiesalso have been used at higher taxonomic levels (Barr,1990; Luttrell, 1951; Parguey-Leduc and Janex-Favre,1981). Ascomata can have single- or multi-layered wallsand may possess various types of outside covering suchas tomentum, hairs, or setae. Although considerablevariation in ascomal wall morphology exists in theSordariomycetes, its potential use in systematics hasseldom been recognized (Jensen, 1985). Several work-ers, however, have noted similarities in ascomal wallcharacters among taxa (von Arx et al., 1984; Barr, 1978;Carroll and Munk, 1964; Jensen, 1985; Lundqvist,1972).

Genera within the Sordariales have been delimitedprimarily on diVerences in their ascospore morphology(Lundqvist, 1972) (Fig. 1). While ascospore morphologyvaries little within a genus, ascospores among genera inthe Sordariales range from a cylindrical, hyaline asco-spore in Lasiosphaeria (Fig. 1A) to an ellipsoidal, brownascospore in Sordaria (Fig. 1I). Intermixed between thesetwo extremes are many genera which possess two-celledascospores with cylindrical to ellipsoidal, brown cellsand diVerent degrees of cylindrical to triangular (oftenbasal), hyaline cells (Figs. 1B–G) (Lundqvist, 1972). Sev-eral earlier workers (Boedijn, 1962; Chenantais, 1919;Lundqvist, 1972; Munk, 1953) hypothesized that asco-spore evolution within this group may have occurredalong this continuum either through the gain or loss of ahyaline cell, resulting in either Lasiosphaeria or Sordariabeing the derived genus.

Ascomal wall morphology also has been suggested asan alternative means of delimiting certain genera withinthis group (Lundqvist, 1972) (Fig. 2). All members ofBombardia and Bombardioidea possess a similar ascomalwall referred to as a bombardioid wall, which contains aputatively 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 thatpossess a similar gelatinized layer in their ascomal wall,but since the wall is non-stromatic, it is termed pseudo-bombardioid (Miller, 2003) (Figs. 2A and B). However,all of these species have been placed into diVerent generabased primarily on diVerences in their ascospore mor-phologies (Fig. 1). Certain species of Cercophora andLasiosphaeria also have been placed into separate generabased on diVerences in their ascospore morphologieseven though they possess similar three-layered ascomalwalls in which the outer layer is composed of hyphaethat form a tomentum (Fig. 2D). Finally, certain speciesof Podospora possess ascomata with outer wall layersthat form swollen protruding cells or agglutinated hairs(Fig. 2E), and some of these species have been trans-ferred into a separate genus, Schizothecium (Lundqvist,1972). These genera, which contain species that possessascomata with obvious morphological diVerences intheir ascomal walls, are the focus of this paper. Addi-tional genera in the Sordariales (e.g., Apiosordaria, Jugu-lospora, and Triangularia), which contain species thatpossess ascomata with morphologically simple ascomalwalls, require further study and will be treated in futurestudies. Our study is the Wrst to evaluate ascomal wallcharacters for their phylogenetic potential in delimitingcertain genera in the Sordariales.

Several nuclear and mitochondrial ribosomal andprotein-coding genes have been employed for assessingphylogenetic relationships of Wlamentous ascomycetes.Nuclear ribosomal genes such as 18S small subunit(SSU) and 28S LSU are commonly used due to theirease 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

ber and the availability of numerous universal primers(Vilgalys and Hester, 1990; White et al., 1990). Moststudies utilize the Wrst 1100 bp of the 5� end of LSU,which contains three variable domains. Nuclear protein-coding genes such as �-tubulin and RPB2 are increas-ingly being used in ascomycete phylogenetic studiesincorporating multiple, unlinked genes. The �-tubulingene contains a highly variable intron-rich 5� end and amore conserved intron-poor 3� end, the latter of whichhas been used to recover higher-level relationships inascomycetes (Landvik et al., 2001). A paralogous copyhas been discovered in ascomycetes (Gold et al., 1991;May et al., 1987; Panaccione and Hanau, 1990), but theduplication event is believed to have occurred after thedivergence of the Sordariomycetes (Landvik et al., 2001).The RPB2 gene is the second largest subunit of the ribo-somal polymerase II gene and contains 12 conservedsequence motifs interspersed among highly variableregions (Liu et al., 1999). Although a second copy hasrecently been found in plants (Oxelman and Bremer,2000), RPB2 is believed to occur as a single orthologousgene in fungi and is becoming increasingly popular instudies of ascomycete phylogeny (Liu et al., 1999; Millerand Huhndorf, 2004b; Reeb et al., 2004; Zhang andBlackwell, 2002).

The primary purpose of this study was to test whetherascospore morphology is phylogenetically informative forpredicting generic relationships within the Sordarialesusing a multi-gene approach. Multiple species, which pos-sess the range of ascospore morphologies known to occurin the order, were sampled from each of several genera. Todetermine the phylogenetic potential of ascomal wall mor-phology for delimiting certain genera in the Sordariales,species with similar ascomal walls in several genera alsowere included. Finally, a comparison of the LSU, �-tubu-lin, and RPB2 genes was made for their utility in resolvingphylogenetic relationships within this group of fungi.

2. Methods

2.1. Taxon sampling

Taxa used in this study are listed in Table 1 alongwith source information and origin for those specimenssequenced in this study. Multiple representatives wereincluded from each of eight genera representing therange in ascospore morphology in the Sordariales. Sev-eral of these taxa also possess similar ascomal wall mor-phologies. The full data sets contained 95, 83, and 68

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 ofhyphae forming tomentum (Lasiosphaeria ovina). (E) Wall with agglutinated hairs (Schizothecium vesticola). Ascomal walls not to scale.


Table 1Taxa used in this study

Taxon Sourcea,b Originc GenBank Accession No.d

LSU �-Tubulin RPB2

Annulatascus triseptatus SMH2359 Costa Rica AY346257 AY780082 AY780148Annulatascus triseptatus SMH4832 France AY780049 AY780083 —Annulatascus velatispora GenBank AF132320 — —Anthostomella sp. SMH3101 Puerto Rico AY780050e AY780084 —Apiosordaria backusii ATCC34568 Japan AY780051 AY780085 AY780149Apiosordaria verruculosa F-152365 (A-12907) Spain AY346258 AY780086 AY780150Barrina polyspora AWR9560A Texas AY346261 AY780087 —Bertia moriformis SMH4320 (a) Michigan AY695260 AY780088 AY780151Bombardia bombarda AR1903 New Zealand AY780052e AY780089 AY780152f

Bombardia bombarda SMH3391 Michigan AY346263 AY780090 AY780153f

Bombardia bombarda SMH4821 France AY780053 AY780091 AY780154Bombardioidea anartia HHB99-1 (a) Alaska AY346264 AY780092 AY780155Botryosphaeria rhodina GenBank — — AF107802Botryosphaeria ribis GenBank AY004336 — —Camarops amorpha SMH1450 Puerto Rico AY780054e AY780093 AY780156f

Camarops petersii JM1655 (a) Indiana AY346265 AY780094 —Camarops tubulina SMH4614 (a) Denmark AY346266 AY780095 AY780157Camarops ustulinoides SMH1988 (a) Puerto Rico AY346267 AY780096 —Capronia mansonii GenBank AY004338 — —Catabotrys deciduum SMH3436 Panama AY346268 AY780097 AY780158Cercophora sp. SMH3200 Costa Rica AY780055e AY780098 AY780159f

Cercophora areolata UAMH7495 Canada AY587936 AY600252 AY600275Cercophora atropurpurea SMH2961 Puerto Rico AY780056e AY780099 —Cercophora atropurpurea SMH3073 Puerto Rico AY780057 AY780100 AY780160f

Cercophora caudata SMH3298 North Carolina AY436407 AY780101 AY780161Cercophora coprophila SMH3794 (a) Puerto Rico AY780058 AY780102 AY780162Cercophora costaricensis SMH4021 (a) Costa Rica AY780059 AY780103 AY780163Cercophora lanuginosa SMH3819 North Carolina AY436412 AY600262 AY600283Cercophora macrocarpa SMH2000 Puerto Rico AY780060e — AY780164f

Cercophora aV. mirabilis SMH4238 Costa Rica AY780061 AY780104 AY780165Cercophora aV. mirabilis SMH4002 (a) Costa Rica AY346271 AY780105 —Cercophora newWeldiana SMH2622 Michigan AF064642 AF466019 AY780166f

Cercophora newWeldiana SMH3303 North Carolina AY780062e AY780106 AY780167Cercophora rugulosa SMH1518 Puerto Rico AY436414 AY600272 AY600294Cercophora scortea GJS L556 Louisiana AY780063 AY780107 AY780168f

Cercophora sordarioides UAMH9301 France AY780064 — —Cercophora sparsa JF00229 (a) France AY587937 AY600253 —Cercophora striata SMH3431 (a) Panama AY780065 AY780108 AY780169Cercophora striata SMH4036 (a) Costa Rica AY780066 — —Cercophora sulphurella SMH2531 Illinois AY587938 AY600254 AY600276Cercophora terricola ATCC200395 Japan AY780067 AY780109 AY780170Chaetomium elatum GenBank — — AF107791Chaetomium globosum SMH4214b Jamaica AY346272 AY780110 —Chaetomium microascoides F-153395 (A-12898) Spain AY346273 AY780111 AY780171Chaetosphaerella phaeostroma SMH4585 (a) England AY346274 AY780112 AY780172Chaetosphaeria innumera SMH2748 North Carolina AY017375 AF466018 —Chaetosphaeria ovoidea SMH2605 Michigan AF064641 AF466057 AY780173f

Coniochaeta ligniaria SMH2569 Michigan AY346275 AY780113 —Coniochaetidium savoryi TRTC51980 Malawi AY346276 AY780114 AY780174Copromyces sp. TRTC51747 (CBS386.78) Argentina AY346277 — —Daldinia concentrica GenBank U47828 — —Diaporthe phaseolorum FAU458 Mississippi AY346279 AY780115 AY780175Diatrype disciformis GenBank U47829 — —Dothidea insculpta GenBank — — AF107800Duradens sp. SMH1708 Puerto Rico AY780068e AY780116 —Eutypa sp. SMH3580 Panama AY346280 AY780117 AY780176Gelasinospora tetrasperma ATCC96230 Canada AY346281 AY780118 AY780177f

Hypocrea pallida GenBank — — AY015639Hypocrea schweinitzii GenBank — — AY015636

(continued on next page)


taxa for the LSU, �-tubulin, and RPB2 genes, respec-tively, while reduced data sets sampled the same 58 taxafor each of the three genes and were subsequently used

in the combined analyses. Based on results from previ-ous phylogenetic analyses (Huhndorf et al., 2004; Liu et al.,1999; Miller and Huhndorf, 2004a), two representatives of

Table 1 (continued)

a (a) D DNA extracted from ascomata; all others were extracted from cultures.b 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, Wil-liam 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.

Taxon Sourcea,b Originc GenBank Accession No.d


Hypocrea virens GenBank — AY158203 —Hypomyces luteovirens GenBank AF160237 — —Hypomyces odoratus GenBank — Y12256 —Induratia sp. SMH1255 Puerto Rico AY780069 AY780119 —Jugulospora rotula ATCC38359 N/A AY346287 AY780120 AY780178Lasiosphaeria glabrata TL4529 (a) Denmark AY436410 AY600255 AY600277Lasiosphaeria hirsuta SMH1543 Wisconsin AY436417 AY780121 AY780179Lasiosphaeria hispida SHM3336 North Carolina AY436419 AY780122 AY780180f

Lasiosphaeria immersa SMH4104 Wisconsin AY436409 AY780123 AY780181Lasiosphaeria ovina SMH1538 Illinois AF064643 AF466046 AY600287Lasiosphaeria sorbina GJS L555 Louisiana AY436415 AY600273 AY600295Lasiosphaeriella nitida SMH1664 Puerto Rico AY346289 AY780124 AY780182Leptosphaeria maculans GenBank — AF257329 —Linocarpon appendiculatum ATCC90499 Brunei AY346291 AY780125 AY780183f

Melanochaeta hemipsila SMH2125 Puerto Rico AY346292 AF466049 AY780184Microascus trigonosporus GenBank — — AF107792Nectriopsis violacea GenBank AF193242 — —Neurospora crassa GenBank AF286411 M13630 AF107789Neurospora pannonica TRTC51327 Hungary AY780070 AY780126 AY780185f

Nitschkia grevillei SMH4663 (a) Illinois AY346294 AY780127 —Ophioceras tenuisporum SMH1643 Puerto Rico AY346295 AY780128 —Pleospora herbarum GenBank — Y17077 —Podospora anserina GenBank — — AF107790Podospora appendiculata CBS212.97 New Zealand AY780071 AY780129 AY780186f

Podospora comata ATCC36713 Venezuela AY780072 — —Podospora decipiens CBS258.64 Wyoming AY780073 AY780130 AY780187Podospora Wbrinocaudata TRTC48343 California AY780074e AY780131 AY780188Podospora Wmbriata CBS144.54 N/A AY780075 AY780132 AY780189f

Podospora Wmiseda CBS990.96 New Zealand AY346296 AY780133 AY780190f

Poroconiochaeta discoidea SANK12878 Japan AY346297 AY780134 AY780191Pseudohalonectria lignicola SMH2440 Costa Rica AY346299 AY780135 —Schizothecium curvisporum ATCC36709 Kenya AY346300 AY780136 AY780192Schizothecium vesticola SMH3187 Indiana AY780076e — —Sinosphaeria bambusicola SMH1999 Puerto Rico AY780077e AY780137 AY780193Sordaria humana ATCC22796 Oklahoma AY780078 — —Sordaria Wmicola SMH4106 (a) Wisconsin AY780079 AY780138 AY780194Sordaria lappae SMH4107 (a) Wisconsin AY780080 AY780139 —Sordaria macrospora Buck s.n. Canada AY346301 AY780140 AY780195f

Strattonia carbonaria ATCC34567 Japan AY346302 AY780141 AY780196f

Striatosphaeria codinaeaphora SMH1524 Puerto Rico AF466088 — —Triangularia mangenotii ATCC38847 Japan AY346303 AY780142 —Triangularia tanzaniensis TRTC51981 Tanzania AY780081e AY780143 AY780197Valsa ceratosperma AR3426 Austria AF408387 AY780144 AY780198Valsonectria pulchella SMH1193 Puerto Rico AY346304 AY780145 AY780199f

Xylaria hypoxylon GenBank U47841 — —ZopWella ebriosa CBS111.75 N/A AY346305 AY780146 AY780200Zygopleurage zygospora SMH4219 Texas AY346306 AY780147 —


loculoascomycetes were used as outgroups for the fulldata sets, while a member of the Xylariales was used toroot trees in the reduced data sets. All voucher speci-mens are deposited in the Field Museum Mycology Her-barium (F).

2.2. Morphological analyses

All taxa in which voucher specimens were available(i.e., AR, AWR, GJS, HHB, JF, JM, SMH, and TL spec-imens) were used in morphological analyses (Table 1).Ascospore morphology was observed from squashmounts of ascomata made in water, while ascomal wallmorphology was determined from sections made at ca.30 �m following the techniques of Miller (2003). Imageswere captured using diVerential interference (DIC)microscopy from a Dage DC-330 video system mountedon a Zeiss Axioskop and processed using Adobe Photo-shop 3.0 and 5.5 (Adobe Systems).

2.3. DNA extraction, ampliWcation, and sequencing

A DNeasy Mini Plant extraction kit (Qiagen) wasused for extracting DNA from either dried ascomata orcultures following the manufacturer’s protocols excepttissues were ground in 100�L AP1 buVer instead of liq-uid nitrogen. The relative quantity of total genomic DNAwas observed on a 1% TBE agarose gel stained with ethi-dium bromide. Gene fragments were PCR-ampliWed oneither a MJ Research PTC 200 or PTC 220 Dyad thermocycler using the following oligonucleotide primers:LSU D LROR–LR7 (Rehner and Samuels, 1995; Vilgalysand Hester, 1990), �-tubulin D BT1819R–BT2916 (Table2), and RPB2D fRPB2-5f–RPB2AM-7R (Liu et al., 1999;Table 2). The LSU was ampliWed using the followingthermocycling parameters: initial denaturation at 94 °Cfor 2 min followed by 35–40 cycles of 94 °C for 30 s, 47 °Cfor 15 s, and 72 °C for 1 min with a Wnal extension step of

72 °C for 10 min. Parameters for amplifying the protein-coding genes were identical except annealing was con-ducted at 50 °C for �-tubulin and at 50 °C for 10 cyclesfollowed by 20–30 cycles at 54–58 °C for RPB2. Ready-To-Go PCR beads (Amersham–Pharmacia Biotech)were occasionally used to amplify diYcult taxa accordingto the manufacturer’s instructions. In rare cases of weakampliWcation, a punch of the PCR product was takenfrom the gel, suspended in 50–150�L double distilledsterile water, melted at 70 °C, and 1�L of this dilutionwas reampliWed using the thermocycling parametersabove except the annealing temperature was increased 3–5 °C. After veriWcation on an ethidium bromide-stained1% TBE agarose gel, PCR products were gel-puriWed ona 1% TALE agarose gel using GELase Agarose Gel-Digesting Preparation (Epicentre Technologies). A Big-Dye Terminator Cycle Sequencing Kit (ABI PRISM,Perkin–Elmer Biosystems) was used to sequence bothstrands using a combination of the following primers:LSUD LROR, LRFF1, LRAM1, LR3, LR3R, LR5, andLR6 (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); andRPB2D fRPB2-5f, RPB2AM-6R, RPB2AM-1f,RPB2AM-1R, RPB2AM-1bf, RPB2AM-1bR, andRPB2AM-7R (Table 2). Sequences were generated on anApplied Biosystems 3100 automated DNA sequencer.Each sequence fragment was subjected to a blast searchto verify its identity. Sequences were assembled andaligned with Sequencher 4.1 (Gene Codes), optimized byeye, and manually corrected when necessary.

2.4. Phylogenetic analyses

2.4.1. SaturationA considerable number of changes occur in the third

codon positions compared to the Wrst and second

Table 2Primers developed in this study for PCR ampliWcation and sequencing

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.

Name Primer sequence Positiona

BT1819Rb 5�-TTC CGT CCC GAC AAC TTC GT-3� 1131–1150BT1283c 5�-CGC GGG AAG GGC ACC ATG TTG-3� 1643–1663BT1283Rb 5�-CAA CAT GGT GCC CTT CCC GCG-3� 1643–1663BT2916b 5�-CTC AGC CTC AGT GAA CTC CAT-3� 2151–2171BTAM1f 5�-GTT CGA CCC CAA GAA CAT GAT GGC YGC-3� 1757–1783BTAM1R 5�-GCA GCC ATC ATG TTC TTG G-3� 1765–1783RPB2AM-6R 5�-TTG ACC AGA CCR CAA GCC TG-3� 985–1004RPB2AM-1f 5�-GAG TTC AAG ATY TTC TCK GAT GC-3� 1261–1283RPB2AM-1R 5�-GCA TCM GAG AAR ATC TTG AAC TC-3� 1261–1283RPB2AM-1bf 5�-CCA AGG TBT TYG TSA ACG G-3� 1127–1145RPB2AM-1bR 5�-GGY CTC ATR ACR CGR CCR GC-3� 1282–1301RPB2AM-7R 5�-GAA TRT TGG CCA TGG TRT CCA T-3� 1783–1804

http://www.lutzonilab.net/pages/primer.shtml

http://www.lutzonilab.net/pages/primer.shtml


positions in the two protein-coding genes, especially �-tubulin (Table 3), suggesting these sites may be saturatedand, thus, represent noise rather than phylogenetic sig-nal. Therefore, analyses were conducted on the full �-tubulin and RPB2 data sets to determine if the Wrst,second, and third codon positions were saturated byconstructing scatter plots which compare time ofsequence divergence to pairwise transition and pairwisetransversion divergences (Hackett, 1996). Uncorrectedpairwise sequence divergence (uncorrected “p”) wasused as an approximation of divergence time. Transi-tions and transversions at each of the codon positionswere determined to be saturated if the scatter of pointsappeared to level oV as sequence divergence increased.

2.4.2. Maximum parsimony and Bayesian analysesMaximum 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 ofincongruence among data partitions (see below) and tocompare the relative utility of the three genes in resolv-ing relationships. Portions of the 5� and 3� ends of eachdata set were excluded from all analyses due to missingdata in most taxa. Fifteen and seven ambiguouslyaligned regions were delimited in the full and reducedLSU data sets, respectively, and these regions along withthree introns were excluded from all analyses. Singleintrons in the �-tubulin and RPB2 data sets also wereexcluded from all analyses. Several taxa in the RPB2data sets contained amino acid indels in the highly vari-able region between conserved motifs 6 and 7 (Liu et al.,1999). These regions were so variable that even aminoacids could not be unambiguously aligned, so theseregions were excluded from all analyses. Unequallyweighted MP analyses were performed with 1000 ran-dom addition heuristic searches and TBR branch-swap-

ping using PAUP* 4.0b10 (SwoVord, 2002).Unambiguously aligned characters in the LSU data setsand each of the three codon positions in the �-tubulinand RPB2 data sets were subjected to a symmetricstepmatrix generated using STMatrix ver. 2.2 (FrançoisLutzoni and Stefan Zoller, Biology Department, DukeUniversity), which calculates the costs for changesamong character states based on the negative naturallogarithm of the percentages of reciprocal changesbetween any two character states. The phylogenetic sig-nal from 11 of the 15 ambiguous regions in the full LSUdata set and Wve of the seven regions in the reduced LSUdata set was recovered using INAASE (Lutzoni et al.,2000) and analyzed in the MP analyses. The remainingLSU ambiguous regions and the two RPB2 ambiguousregions were excluded because their recoded characterscontained more than 32 character states, which is notallowed in PAUP*. Branch support for all MP analyseswas estimated by performing 1000 bootstrap replicates(Felsenstein, 1985), each consisting of 100 random addi-tion heuristic searches and TBR branch-swapping.

MODELTEST 3.06 (Posada and Crandall, 1998) wasused to determine the best-Wt model of evolution foreach data set. Bayesian analyses employing a Markovchain Monte Carlo (MCMC) method were performedusing MrBayes 3.0b4 (Huelsenbeck and Ronquist, 2001)as an additional means of assessing branch support. Thebest-Wt model of evolution was implemented for eachdata set in the separate analyses and for each partition(i.e., separate models for LSU and for each of the threecodon positions in �-tubulin and RPB2) in the combinedanalyses. Constant characters were included and fourMCMC chains were ran simultaneously for 5,000,000generations with trees sampled every 100th generationresulting in 50,000 total trees. The MCMC chains alwaysachieved stationarity after the Wrst 20,000–150,000 gen-

Table 3Comparison of data sets and trees in phylogenetic analyses

a Excluding sites in 5� and 3� ends and introns.b Divided into Wrst, second, and third codon positions for �-tubulin and RPB2; total shown in parentheses.

Data sets Genes Combined


Full Reduced Full Reduced Full Reduced

No. of total sitesa 1084 1039 939 924 1200 1197 3153No. of ambiguous sites 222 126 0 0 222 222 348No. of constant sites 524 599 568 591 372 407 1596No. of polymorphic sites 338 314 371 333 606 568 1214No. of parsimony-informative sitesb 251 228 41, 16, 254 (311) 27, 8, 231 (266) 163, 84, 312 (559) 153, 75, 310 (538) 1032Percent of total sites that are

parsimony-informative23 22 33 29 47 45 33

No. of MP trees 2 1 2 1 1 1 1Length of MP trees 3070.46 2070.52 4532.81 3164.12 12751.79 10242.09 15660.21Consistency index 0.370 0.402 0.207 0.248 0.174 0.195 0.231Retention index 0.632 0.583 0.509 0.471 0.449 0.467 0.475Rescaled consistency index 0.234 0.235 0.105 0.117 0.078 0.091 0.110No. of clades with 770% bootstrap support 39 22 30 19 32 29 35No. of clades with 795% Bayesian post. prob. 35 26 27 20 39 40 40


erations ( D 200–1500 trees), so the Wrst 10,000 trees,which extended well beyond the burn-in phase in eachanalysis, were discarded. Posterior probabilities weredetermined from a consensus tree generated from theremaining 40,000 trees. This analysis was repeated threetimes starting from diVerent random trees to ensure treesfrom the same tree space were being sampled duringeach analysis.

2.4.3. CombinabilityThe validity of the incongruence length diVerence

(ILD) test for determining whether multiple data setsshould be combined has recently been questioned(Barker and Lutzoni, 2002; Yoder et al., 2001) and, thus,other methods should be explored. One method ofassessing combinability of data sets, and the oneadopted in this study, is by simply comparing highlysupported clades among trees generated from diVerentdata sets to detect conXict (de Queiroz, 1993; Mason-Gamer and Kellogg, 1996). High support typically refersto bootstrap support values 770% and Bayesian poster-ior probabilities 795% (Alfaro et al., 2003). If no con-Xict exists between the highly supported clades in treesgenerated from these diVerent data sets, this suggests thegenes share similar phylogenetic histories and phyloge-netic resolution and support could ultimately beincreased by combining the data sets.

3. Results

3.1. Phylogenetic analyses

3.1.1. SaturationExcept for third position transitions in the RPB2

gene, no evidence of saturation was detected in any ofthe codon positions since scatter plots clearly show anincrease when pairwise sequence divergence is plottedagainst pairwise transition/transversion divergence (Fig.3). Only third positions were plotted for the �-tubulingene since very few changes occur in the Wrst and secondcodon positions (Table 3). The scatter plot of third posi-tion transitions in the RPB2 gene appears to be levelingoV slightly suggesting a low level of saturation may beoccurring at these sites (Fig. 3). As expected, the scatterof points in this area (points at the far right of the graph)primarily represent pairwise comparisons between theSordariales ingroup taxa and the more distant loculoas-comycetes outgroups. Therefore, additional MP analyseswere conducted on the RPB2 full data set in which thirdposition transitions were arbitrarily down-weighted by afactor of 2, 10, and 100 relative to transversions using astepmatrix. Third position transitions and transversionswere weighted approximately equally in the original ratesubstitution 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

1.86). A single most-parsimonious tree with an identicaltopology to that in Fig. 6 was estimated when transitionswere down-weighted by 2. However, phylogenetic reso-lution was substantially decreased in analyses in whichtransitions were down-weighted by 10 and 100 in thatoutgroup taxa (Coniochaetales and Chaetosphaeriales)occurred within the ingroup (Sordariales) (data notshown). This suggests that while some third position

Fig. 3. Saturation plots relating uncorrected pairwise sequence diver-gence to pairwise transition/transversion divergence. Only third posi-tion changes are shown for �-tubulin (A), whereas Wrst, second, andthird position transitions (B), and transversions (C) are shown forRPB2.


transitions are becoming saturated, a majority of thesesites still possess phylogenetic signal which is necessaryto accurately estimate phylogenies.

3.1.2. Maximum parsimony and Bayesian analysesThe best-Wt maximum-likelihood model of evolution

for the LSU data sets was the TIMef model (Rodríguezet al., 1990). The best-Wt model for the �-tubulin datasets and the full RPB2 data set was the GTR model(Rodríguez et al., 1990), while the TrN model (Tamuraand Nei, 1993) was selected as the best-Wt model for thereduced RPB2 data set. A proportion of sites wereinvariable while the remaining sites were subjected to agamma distribution shape parameter in all models.Applying separate models to each of the three codonpositions for the �-tubulin and RPB2 data sets had littleeVect on the Bayesian posterior probabilities in the com-bined analyses (data not shown).

Maximum parsimony analyses of the LSU and �-tubulin full data sets each generated two trees. The LSUtrees diVered only slightly in relationships among speciesof Sordaria, whereas the �-tubulin trees diVered in rela-tionships among species of Neurospora. One of the treesfrom each data set is shown in Figs. 4 and 5. A singlemost-parsimonious tree was generated in analyses of theRPB2 full data set (Fig. 6). Data set and tree statisticsare listed in Table 3. Overall relationships among themajor ordinal and familial lineages diVered between themost-parsimonious trees generated from the full datasets, although most of these relationships were not sup-ported (Figs. 4–6).

3.1.3. CombinabilitySeparate analyses of the three reduced data sets pro-

duced single most-parsimonious trees (data not shown).Data set and tree statistics are listed in Table 3 for thereduced data sets. Although minor diVerences occurredamong most-parsimonious trees in the reduced data sets(data not shown), only one instance of highly supportedconXict existed between phylogenies. In the LSU tree, theCoronophorales were a sister clade to the Hypocrealeswith 77% bootstrap support, whereas this order wasplaced within the Boliniales with 79% bootstrap supportin the �-tubulin tree (data not shown). This conXict wasnot supported by Bayesian posterior probabilities and ismost likely due to poor taxon sampling in these orders.Since little evidence exists against combining these datasets, they were analyzed simultaneously in a combinedanalysis, which produced a single most-parsimonious tree(Fig. 7). Combined data statistics are given in Table 3.

3.2. Ascospore morphology

Ascospore morphology is shown to be extremelyhomoplastic in that multiple species in the same genusoccur in diVerent clades scattered throughout the Sor-

dariales in all analyses (Figs. 4–7). For example, 17 spe-cies of Cercophora segregate into at least nine diVerentclades, while six species of Podospora occur in Wve cladesin the LSU tree (Fig. 4). Multiple species were sampledfrom six to nine genera (Apiosordaria, Cercophora, Chae-tomium, Lasiosphaeria, Neurospora, Podospora, Schizo-thecium, Sordaria, and Triangularia) in the full data setsand all genera occur as paraphyletic or polyphyletic in atleast one of the most-parsimonious trees (Figs. 4–6).Combined analyses of the reduced data sets corroboratethese results for four (Cercophora, Lasiosphaeria, Neu-rospora, and Podospora) of the six genera represented bymultiple species (Fig. 7). While morphology suggeststhat ascospore evolution in the Sordariales may haveoccurred directionally along a continuum (Fig. 1), ourmolecular data do not support this in that species withvastly diVerent ascospore morphologies occur in severalwell-supported clades throughout the Sordariales (Figs.4–7). Three well-supported clades exist which containspecies of Cercophora that cluster with species of Apios-ordaria or Triangularia (Figs. 4 and 5), taxa which pos-sess ascospore morphologies that do not occur along theputative evolutionary transition. The most extremeexample is found in the well-supported clade that con-tains Bombardia and Bombardioidea (Figs. 4–7). WhileBombardia possesses Cercophora-like ascospores at oneend of the continuum (Fig. 1B), Bombardioidea possessesellipsoidal, brown ascospores at the opposite end of thecontinuum (Fig. 1H).

3.3. Ascomal wall morphology

Although many of the clades within the Sordariales areunsupported, Wve well-supported clades, which containspecies with similar ascomal wall morphologies, occur inall 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 andB), while species of Bombardia and Bombardioidea withsimilar bombardioid walls (Fig. 2C) occur in a third clade(wall clade C). A fourth clade (wall clade D) is repre-sented by species of Cercophora and Lasiosphaeria whichpossess a similar three-layered ascomal wall in which theouter wall layer is composed of hyphae that form atomentum (Fig. 2D), while the Wfth clade (wall clade E) isrepresented by species of Podospora/Schizothecium whichpossess an outer ascomal wall layer that forms swollenprotruding cells or agglutinated hairs (Fig. 2E).

4. Discussion

4.1. Ascospore morphology

The Sordariales has recently been redeWned to includegenera which possess ascospores that share a similar


developmental pattern (i.e., cylindrical, hyaline ascosp-ores that develop apical, brown heads and basal, hyalinetails) (Huhndorf et al., 2004). Seventeen genera wereincluded in the LSU data set which possess ascosporesthat vary along these developmental lines and all Wndtheir placement in the monophyletic Sordariales (Fig. 4).The �-tubulin and RPB2 data sets, which included 16and 15 genera, respectively, corroborate these results

(Figs. 5 and 6). These genera possess ascospores whichform a continuum from a cylindrical, hyaline ascosporein Lasiosphaeria to an ellipsoidal, brown ascospore inSordaria with numerous genera which possess ascosp-ores with brown heads and hyaline tails intermixedbetween (Fig. 1). This hypothesized trend in ascosporeevolution is, however, not supported by our moleculardata (Figs. 4–7). Since diVerent taxa occur as the basal

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 supportbased on 1000 replicates. Thickened branches represent signiWcant posterior probabilities (795%) generated from Bayesian analyses. Shaded boxesindicate the Wve well-supported ascomal wall clades discussed in the text. ClassiWcation following Huhndorf et al. (2004) is shown along the right.


group of the order in diVerent trees, any trend, if oneeven exists, in ascospore evolution is presently unclear.

Genera within the Sordariales have been delimitedprimarily on diVerences in their ascospore morphology(Lundqvist, 1972). While our data conWrm the Wndingsof Huhndorf et al. (2004) that ascospore morphology is agood indicator of whether a taxon belongs in the Sordar-iales, these data indicate that ascospore morphology isextremely homoplastic and a poor predictor of generic

relationships within the group. When more than one spe-cies from the same genus was included, most did notresolve along current generic lines, but were scatteredthroughout several clades (Figs. 4–7). Dettman et al.(2001) also found ascospore morphology to be a poorpredictor of phylogenetic relationships in Neurosporaand Gelasinospora. Analyses of four nuclear genesrevealed that the two genera conventionally distin-guished by diVerences in ascospore ornamentation did

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.


not represent two distinct monophyletic groups butinstead were a polyphyletic group of closely related,morphologically similar taxa. The two species of Neuros-pora included in our study never occurred as a mono-phyletic clade in any analysis (Figs. 4–7).

The Sordariales is not the only group where asco-spore morphology has been used for delimiting genera.For example, generic delimitation within the Amphisph-aeriaceae (Xylariales) has been based on ascospore pig-mentation, septation, and shape and, in some cases,ornamentation (Barr, 1994; Kang et al., 1999). Genera ofascosporogenous yeasts also have been based primarily

on ascospore shape and ornamentation and, as in ourstudy, LSU sequences suggest that ascospore morphol-ogy is a poor predictor of generic relationships (Kurtz-man and Robnett, 1994). Additional studies are neededto determine whether ascospore morphology is phyloge-netically informative for delimiting genera in othergroups of ascomycetes.

4.2. Ascomal wall morphology

Although ascospore morphology is a poor predictorof generic relationships in the Sordariales, ascomal wall

Fig. 6. Single most-parsimonious tree based on the RPB2 data set of 68 taxa. Support values, shading, and classiWcation as in Fig. 4.


morphology may serve as an alternative means of delim-iting certain genera. Five highly supported clades (wallclades A–E) are found in all trees (Figs. 4–7) and four ofthese clades (wall clades A–D) contain taxa with diVer-ent ascospore morphologies but similar ascomal wallmorphologies. Because taxon sampling within each ofthese Wve wall clades is nearly complete, it is unlikelythat the addition of more Sordarialean taxa will altercurrent hypotheses of relationships in these clades.Therefore, discussions of their homologous charactersare appropriate. However, taxon sampling for otherwell-supported clades throughout the Sordariales ispresently incomplete and relationships may change withthe addition of more taxa. In addition, taxa in theseclades possess relatively simple 2- to 3-layered ascomalwalls, which possess no obvious morphological charac-ters with which to distinguish them at the present time.

Additional taxa must be included and further examina-tion of their ascomal walls must be completed beforetheir homologous characters can be adequately discussed.

Three well-supported clades (wall clades A–C) con-tain taxa possessing an ascomal wall with a gelatinizedlayer (Figs. 2A–C). Wall clades A and B contain speciesof Cercophora and Podospora which possess a pseudo-bombardioid wall (Figs. 2A and B), while wall clade Ccontains species of Bombardia and Bombardioidea whichhave a bombardioid wall (Miller, 2003) (Fig. 2C).Ascospores in Cercophora are initially cylindrical buteventually develop a swollen head and long tail (Fig.1B), whereas those in Podospora are initially clavatebefore developing a swollen head and short tail (Fig.1C). The pseudo-bombardioid wall morphology isslightly homoplasious in the Sordariales in that itappears to have arisen independently in two distantly

Fig. 7. Single most-parsimonious tree based on the combined data set of 58 taxa. Support values, shading, and classiWcation as in Fig. 4.


related groups, although relationships among thesegroups are unsupported at this time (Figs. 4–7). How-ever, taxa in wall clade B can be diVerentiated fromthose in wall clade A in possessing short, brown, hyaline-tipped setae. Species of Bombardia and Bombardioideaare morphologically similar in all characters except theirascospores, which occur near the extremes of the mor-phological ascospore continuum. Ascospores in Bombar-dia are identical to those in Cercophora (Fig. 1B),whereas those in Bombardioidea are ellipsoidal andbrown (Fig. 1H). However, these species form a highlysupported clade (wall clade C) united by a bombardioidascomal wall (Fig. 2C), which occurs as a homologouscharacter in the Sordariales (Figs. 4–7).

A fourth wall clade (wall clade D) contains species ofCercophora and Lasiosphaeria. While both genera pos-sess similar ascospores that are cylindrical and hyaline,those in Cercophora eventually swell at one end and turnbrown (Fig. 1B). However, species in this clade possess asimilar three-layered ascomal wall in which the outerlayer is composed of hyphae that form a tomentum (Fig.2D). This ascomal wall morphology is slightly homo-plastic in that three additional taxa (C. coprophila, C.sparsa, and C. sulphurella), which also possess morpho-logically similar walls, are not found within this well-supported tomentum clade (Figs. 4–7). Cercophoracoprophila occurs well outside this clade, while C. sparsaand C. sulphurella occur as unsupported sister taxa tothis clade. However, the wall in C. coprophila has beeninterpreted by some to be slightly areolate (Lundqvist,1972) and this distinction may separate it from species inwall clade D.

The Wfth wall clade (wall clade E) includes species ofPodospora/Schizothecium which possess an outer asco-mal wall layer that forms swollen protruding cells oragglutinated hairs (Fig. 2E). Other species of Podosporaincluded in this study possess glabrous ascomata or arecovered with short to long, Xexuous hairs or stiV setae(Lundqvist, 1972). Schizothecium can be further distin-guished from Podospora by the absence of typical inte-rascal paraphyses and ascospores which become septateat a very early stage in their development (Lundqvist,1972), but some believe these characters do not warrantgeneric distinction (Bell and Mahoney, 1995). Althoughwall clade E may presently be delimited by a combina-tion of ascomal wall, centrum, and ascospore characters,additional species putatively belonging in Schizotheciumshould be included in future analyses to test the signiW-cance of these characters in this clade.

While studying the surface morphology of outer asco-mal walls, Jensen (1985) found similarities among repre-sentative species from several orders and families ofSordariomycetes. He also noted that three genera in theSordariaceae (i.e., Gelasinospora, Neurospora, and Sorda-ria) possessed virtually identical outer ascomal walllayers. These genera also possess similar membraneous,

3- to 4-layered ascomal walls and ellipsoidal, brownascospores (Lundqvist, 1972). Representatives of thesegenera always formed a highly supported clade in ourstudy (Figs. 4–7). However, since these ascomal wall andascospore morphologies occur in several other taxathroughout the Sordariales (Lundqvist, 1972), it is pres-ently unclear which characters are phylogeneticallyinformative for delimiting this clade.

One may argue that only characters which are puta-tively homologous ( D synapomorphies) should be usedfor delimiting taxa. However, this argument ignores thefact that there are diVerent degrees of homoplasy. Char-acters can range from slightly homoplasious (arising inonly two distantly related groups) to extremely homo-plasious (arising in several groups throughout a tree)with certain levels of the former still contributing someamount of phylogenetic structure to the data set. Atwhat level homoplasy stops becoming partially informa-tive and becomes merely noise is presently unclear.Although ascomal wall morphology is slightly homo-plasious in some groups (i.e., tomentum wall, pseudo-bombardioid wall), ascospore morphology is extremelyhomoplasious throughout this group. Thus, while asco-spore morphology cannot be used for delimiting genera,ascomal wall morphology alone or in combination withother characters is still useful at some level for distin-guishing taxa.

4.3. Comparison of genes

All three genes were compared using the same 58taxa in the reduced data sets, which contain approxi-mately the same number of total sites (i.e., 924–1197 bp) (Table 3). However, RPB2 contains overtwice the number of parsimony-informative sites (538)as LSU (228) or �-tubulin (266) (Table 3) resulting inlonger branch lengths and increased support forclades. RPB2 contains more clades with signiWcantbootstrap support (29) and Bayesian posterior proba-bilities (40) than either LSU (22, 26) or �-tubulin (19,20). LSU possesses numerous indels in its Wrst threedomains thereby reducing the number of parsimony-informative sites after ambiguous regions areremoved. Most of the phylogenetic signal from �-tubulin comes from third position changes (87%),whereas third positions account for only a little overhalf (57%) of the signal in the RPB2 gene. Despite theextreme bias towards changes in third position sites in�-tubulin, these sites showed no evidence of saturationfor either transitions or transversions (Fig. 3).Although changes are more evenly distributedthroughout Wrst, second, and third codon positions inRPB2, a low level of saturation was detected in thirdposition transitions. Saturation of third position tran-sitions and transversions in RPB2 also was found byReeb et al. (2004) in their study of euascomycetes.


Future studies employing RPB2 for estimating fungalphylogenies should determine the level of saturationat third positions and its eVect on the resolution of theresulting phylogeny.

5. Conclusion

This study contributes to the understanding of theevolution of morphological characters in ascomycetes.Based on results from MP and Bayesian analyses ofthree genes, ascospores with similar morphologiesappear to have evolved independently numerous timesthroughout the Sordariales, whereas ascomal wallsappear to be less homoplasious in this group. Relation-ships among taxa outside the wall clades are mostlyunresolved and most relationships among well-sup-ported clades are currently unsupported making it diY-cult to draw conclusions regarding the evolution ofascomal wall and ascospore characters. Increased taxonsampling will improve resolution in many clades, whilesupport for relationships will be increased through theincorporation of additional genes. While genera cannotbe recognized based on ascospore morphology, theymay be delimited in certain clades based on ascomal wallmorphology. However, we are not advocating simplyreplacing one form of a one-character taxonomy basedon ascospores with another based on ascomal walls;redeWned genera would need to be delimited by a uniquecombination of characters primarily involving, but notlimited to, those found in ascomal walls.

Acknowledgments

This work was supported in part by a NSF DDIGGrant (Doctoral Dissertation Improvement Grant, DEB-0105077) to ANM through the University of Illinois atChicago (UIC) and in part by NSF PEET Grants (Part-nerships for Enhancing Expertise in Taxonomy, DEB-9521926 and DEB-0118695) to SMH through the FieldMuseum of Natural History (FMNH). ANM also wassupported during this study by a Lester Armour Gradu-ate Fellowship from FMNH. Fieldwork for ANM wassupported in part by an ASPT (American Society of PlantTaxonomists) Graduate Student Research Grant and anUIC Provost Award. Fieldwork for SMH was supportedin part by the National Research Council ResidentResearch Associate Post-doctoral Program in coopera-tion with the USDA Forest Service, Madison, WI. Theauthors are most grateful to G. Bills, W. Buck, H. Burd-sall, M. Calduch, F. Fernández, J. Fournier, J. Krug, T.Læssøe, A. Rossman, G. Samuels, and A. Stchigel for pro-viding specimens or cultures. V. Reeb and F. Fernándezgraciously allowed us to use their unpublished primers.We also wish to thank A. Mitchell and G. Mueller as well

as two anonymous reviewers for their comments whichimproved this paper. Sequences were generated in thePritzker Laboratory for Molecular Systematics and Evo-lution at FMNH. This work represents a portion of a the-sis in partial fulWllment of the requirements for thedoctoral degree at the Graduate College of UIC.

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Multi-gene phylogenies indicate ascomal wall morphology is ......were included. Finally, a comparison of the LSU, -tubu-lin, and RPB2 genes was made for their utility in resolving