Received 18April 2002 Accepted 26 June 2002 Publishedonline 12September 2002

Evolutionof complexfruiting-bo dymorpholog ies inhomobasidi omycetes David S.Hibbett * and Manfred Binder BiologyDepartment, Clark University, 950Main Street,Worcester, MA 01610,USA The fruiting bodiesof homobasidiomycetes include some of the most complex formsthat have evolved in thefungi, such as gilled mushrooms,bracket fungi andpuffballs (‘pileate-erect’) forms.Homobasidio- mycetesalso includerelatively simple crust-like‘ resupinate’forms, however, which accountfor ca. 13– 15% ofthedescribed species in thegroup. Resupinatehomobasidiomycetes have beeninterpreted either asa paraphyletic grade ofplesiomorphic formsor apolyphyletic assemblage ofreducedforms. The former view suggeststhat morphological evolutionin homobasidiomyceteshas been marked byindependentelab- oration in many clades,whereas the latter view suggeststhat parallel simpliŽcation has beena common modeof evolution.To infer patternsof morphological evolutionin homobasidiomycetes,we constructed phylogenetic treesfrom adatasetof 481 speciesand performed ancestral statereconstruction (ASR) using parsimony andmaximum likelihood (ML)methods. ASR with both parsimony andML implies that the ancestorof the homobasidiomycetes was resupinate, and that therehave beenmultiple gains andlosses ofcomplex formsin thehomobasidiomycetes. We also usedML toaddresswhether there is an asymmetry in therate oftransformations betweensimple andcomplex forms.Models of morphological evolution inferredwith MLindicatethat therate oftransformations from simple tocomplex formsis about three tosix times greater than therate oftransformations in thereverse direction. A null model ofmorphological evolution,in which thereis noasymmetry in transformation rates,was rejected. These results suggest that thereis a‘driven’trend towards the evolution of complex formsin homobasidiomycetes. Keywords: comparative methods;corticioid fungi;molecular phylogeny

1. INTRODUCTION natefruiting bodiesare oftenproduced on the underside ofwoody substrates, where they are easily overlooked. Complex multicellular formshave arisen independentlyin There is general agreement among mycologists that several cladesof eukaryotes, including fungi,plants, ani- resupinatehomobasidiomycetes are notmonophyletic mals andstramenopiles. The repeatedevolution of com- (Donk1964, 1971; Ju¨lich 1981; Parmasto 1986), buttheir plex formshas beentaken as evidence that natural preciserelationships are notwell resolved.Some authors selectiontends to favour morphological elaboration have suggestedthat resupinateforms represent a polyphy- (Bonner1988). Alternatively, it has beensuggested that letic assemblage ofspecies that have beenderived by theoverall increasein thecomplexity ofbiological forms reductionfrom pileate-erect forms( Ju¨lich 1981; Corner has occurredsimply becausethere is alower limit ofallow- 1991), butothers have suggestedthat resupinateforms able complexity, representedby unicellular forms,but no constitutea paraphyletic grade, from which pileate-erect upper limit oncomplexity. If so,an overall increasein formshave repeatedly arisen (Oberwinkler 1985; Parmasto complexity couldoccur by a‘passive’process, which can 1995). Recentphylogenetic studieshave conŽrmed that beconceptualized as diffusion through morphospace resupinatetaxa are intermingled with pileate-erect taxa in (McShea1994, 1996). Muchof the debate concerning anumberof cladesof homobasidiomycetes (Hibbett et al. trendsin theevolution of organismal complexity resides 1997; Hibbett &Thorn 2001; Langer 2002), butso far in thepalaeontological literature andconcerns morpho- therehas not,to our knowledge, been an analysis with logical evolutionin animals (e.g.Gould 1988; Wagner sufŽciently broad sampling toresolve theoverall pattern 1996; Sidor 2001). ofevolution of fruiting-body forms. Within thefungi, some of the most conspicuous and Our studyhad threemain objectives:(i) toinfer broad elaborate formsthat have evolved are thefruiting bodies phylogenetic relationships among resupinateand pileate- ofhomobasidiomycetes. Familiar examples includegilled erecthomobasidiomycetes; (ii) toestimate theancestral mushrooms,polypores, coral fungi,puffballs andstink- fruiting-body morphology ofthe homobasidiomycetes; horns(hereafter, ‘ pileate-erect’forms). Nevertheless, and(iii) todetermine whether the rate oftransformations homobasidiomycetesalso producerelatively simple from resupinateto pileate-erect formsis different from the ‘resupinate’forms, which lie at ontheir substrates. rate oftransformations in thereverse direction. Resupi- Resupinatefruiting bodiesrange from ‘athelioid’forms, nateforms are morphologically simple relative topileate- which consistonly ofsparsenetworks of fertile hyphae, to erectforms because they are notdivided into a cap and more robust,crust-like or eshyforms that have smooth, stalk or other discreteparts, andthey have simple ridged,toothed or poroid spore-bearing surfaces.Resupi- ontogeniesthat donot include the production of veils or other protective tissuesthat are commonamong pileate- erectforms. We used maximum likelihood (ML)toesti- *Authorfor correspondence ([email protected]). mate asimple modelof evolution of homobasidiomycete

Proc.R. Soc.Lond. B (2002) 269, 1963–1969 1963 Ó 2002 TheRoyal Society DOI10.1098/ rspb.2002.2123 1964D. S.Hibbettand M. Binder Evolution of complex morphologies in fungi *

Christiansenia pallida Tremellales * Boletus retipes (a) Guepinia spathularia Xerocomus chrysenteron Femsjonia sp. Boletus satanas Dacryomitra pusilla Phylloporus rhodoxanthus

* Ditiola radicata Paragyrodon sphaerosporus Dacryopinax spathularia Calostoma cinnabarina Calocera cornea Dacrymycetales Scleroderma citrinum Cerinomyces grandinioides Suillus luteus Dacrymyces chrysospermus Suillus sinuspaulianus Bolete Dacrymyces sp. Suillus cavipes Dacrymyces stillatus Chroogomphus vinicolor clade Basidiodendron caesiocinereum # Gomphidius glutinosus Basidiodendron sp. Rhizopogon subcaerulescens Bourdotia sp. # Coniophora arida # Heterochaete sp. Auriculariales Coniophora puteana Serpula himantioides # * Auricularia auricula judae Exidia thuretiana # Tapinella atrotomentosa Gastrosporium simplex Tapinella panuoides Anthurus archeri Pseudocolus fusiformis Jaapia argillacea Sphaerobolus stellatus Dendrocorticium polygonioides # Jaapia Geastrum saccatum Dendrocorticium roseocarenum Geastrum sessile Gomphoid Punctularia strigoso zonata # Dendrocorticium Ramaria stricta Vuilleminia comedens Clavariadelphus pistillaris Galzinia incrustans clade Ramaricium alboflavescens –Phalloid clade Tomentella ferruginea #

* Gautieria otthii Tomentella stuposa Gomphus floccosus Tomentella coerulea * Ramaria formosa Thelephora sp. Ramaria obtussisima Thelephora palmata Uthatobasidium fusisporum # Thelephora vialis Thelephoroid Uthatobasidium sp. Hydnellum sp. Thanatephorus practicola # Sarcodon imbricatus clade Tulasnella sp. Bankera fuligineoalba Tulasnella pruinosa Phellodon tomentosus Tulasnella sp. Pseudotomentella mucidula Botryobasidium candicans Pseudotomentella nigra # Botryobasidium vagum # Pseudotomentella ochracea Botryobasidium subcoronatum Gloeophyllum sepiarium Botryobasidium isabellinum # Cantharelloid Heliocybe sulcata Gloeophyllum clade Botryobasidium sp. clade Neolentinus dactyloides Botryobasidium sp. # Hyphodontia gossypina Clavulina cinerea Subulicystidium longisporum # Paullicorticium Sistotrema eximum # Tubulicium vermiculare Sistotrema sernanderi Paullicorticium niveocremeum # clade Multiclavula mucida Sistotremastrum sp. Sistotrema brinkmannii # Bjerkandera adusta Sistotremastrum niveocremeum Phanerochaete chrysosporium # Hydnum repandum Phanerochaete sordida Hydnum rufescens Sistotrema musicola # Cantharellus cibarius Pulcherricium caeruleum Cantharellus tubaeformis Phlebiopsis gigantea # Craterellus cornucopioides Ceraceomyces serpens * Phlebia albomellea # Gerronema marchantiae Ceriporiopsis subvermispora

Sphaerobasidium minutum # * Cystidiodontia isabellina # *

Resinicium bicolor * Ceriporia purpurea Hyphodontia alutaria # Byssomerulius sp.

Oxyporus populinus * Gloeoporus taxicola Subulicium sp. # * Ceriporia viridans Tubulicrinis sp. Ceraceomyces eludens # Hyphodontia pallidula # Ceraceomyces microsporus Schizopora flavipora Lindtneria trachyspora #

* Hyphodontia cineracea Grifola frondosa

* Hyphodontia alutacea # Gelatoporia pannocincta Hyphodontia palmae Candelabrochaete septocystidia * Basidioradulum radula # Climacodon septentrionalis * Tubulicrinis gracillimus Phlebia radiata # * Tubulicrinis subulatus # Mycoacia aurea Coltricia perennis Phlebiella griseofulva Phellinus gilvus Hymenochaetoid Mycoacia aff fuscoatra # * Hymenochaete corrugata clade Peniophora sp. # Hymenochaete rhabarbarina Scopuloides hydnoides Inonotus hispidus Phanerochaete chrysorhiza # Phellinus igniarius Candelabrochaete africana # Phylloporia ribis Phanerochaete sanguinea # Fibricium rude # Steccherinum fimbriatum Trichaptum abietinum Junghuhnia nitida Hyphodontia aff. breviseta Antrodiella romellii # Hyphodontia nespori # Albatrellus syringae

Hyphodontia crustosa * Meripilus giganteus Hyphodontia sambuci # Hyphoderma setigerum # Hyphodontia nudiseta # Hyphoderma nudicephalum Hyphodontia aspera Hyphoderma definitum # Hyphodontia serpentiformis # Panus rudis Schizopora paradoxa Spongipellis pachyodon Hyphodontia niemelaei Hypochnicium sp. # Hyphodontia radula # Hypochnicium geogenium Polyporoid Gloeocystidiellum leucoxantha # Hypochnicium eichleri # Stereum annosum Hypochnicium polonense clade Aleurodiscus botryosus Abortiporus biennis

* Acanthophysium cerrusatus Podoscypha petalloides Acanthophysium sp. # Datronia mollis * Stereum armeniacum Polyporus squamosus

Stereum hirsutum * Polyporus tenuiculus Heterobasidion annosum Polyporus tuberaster Bondarzewia berkeleyi Polyporus varius Bondarzewia montana * Polyporus melanopus

Aleurodiscus scutellatus * Cryptoporus volvatus Auriscalpium vulgare Perenniporia medulla-panis Fomes fomentarius * Gloiodon strigosus *

* Creolophus cirrhatus Daedaleopsis confragosa Hericium erinaceum Grammothele fuligo # Hericium coralloides Ganoderma australe Laxitextum bicolor Ganoderma lucidum Cymatoderma caperatum Ganoderma applanatum Dentipellis separans # Russuloid Pycnoporus cinnabarinus Polyporoletus sublividus Lentinus tigrinus

Albatrellus fletti clade * Polyporus arcularius Albatrellus skamanius Physalacria inflata Amylostereum laevigatum # Wolfiporia cocos # Amylostereum chailettii Trametes suaveolens Echinodontium tinctorium Gloeophyllum odoratum Laurilia sulcata Trametes versicolor Lactarius volemus Junghunia subundata Lactarius corrugis Lenzites betulina Russula earlei Dendrodontia sp. # Russula virescens Dentocorticium sulphurellum Russula romagnesii Laetiporus sulphureus Russula compacta Phaeolus schweinitzii Russula mairei Tyromyces chioneus Amphinema byssoides Diplomitoporus lindbladii # Dichostereum pallescens Climacocystis sp. Vararia insolita # Postia balsamea Peniophora nuda # Postia leucomallela Coronicium alboglaucum Oligoporus rennyi # Asterostroma andinum Amylocystis lapponica Trechispora farinacea # * Cyphella digitalis * Scytinostroma alutum Ischnoderma benzoinum Scytinostroma portentosum # Antrodia carbonica # Antrodia xantha Parmastomyces transmutans # Auriporia aurea Pycnoporellus fulgens * Fomitopsis pinicola Piptoporus betulinus Daedalea quercina Antrodia serialis # Neolentiporus maculatissimus

Figure 1. Phylogenetic relationships of homobasidiomycetes inferred withEP analysis. Tree 1/10 000. Branch shading indicates ASRwithparsimony: red, resupinate; black, pileate-erect; green, uncertain. Nodes thatcollapse in thestrict consensus tree are marked withasterisks. Resupinate taxathat were deleted from the ‘pruned’ trees are indicated bya hash sign. Bracketed groups are discussed in thetext.

fruiting-body forms,in whichthere are twocharacter 2. METHODS states(resupinate and pileate-erect) andtwo parameters that specifythe rates offorward andbackward transform- (a) Taxonsampling andsequence data ationsbetween the states (Pagel 1997). If thevalues of Weassembled a dataset that contains464 species of homo- theseparameters couldbe shown to be signi Ž cantly differ- basidiomycetesand 17species of ‘jelly fungi’ (hetero- ent,then this wouldindicate the existence of a ‘driven’ basidiomycetes pro parte),includingsix speciesof Auriculariales, trendin theevolution of complex formsin fungi. 10species of Dacrymycetalesand onespecies of Tremellales,

Proc.R. Soc.Lond. B (2002) Evolution of complex morphologies in fungi D.S.Hibbettand M. Binder1965

with the estimatedproportions ofdescribedspecies in eachclade (b) Plicaturopsis crispa Radulomyces molaris Deflexula subsimplex (table1). Parmasto (1997)recognized 1733 described species Stephanospora caroticolor Phlebiella sp. # Athelia fibulata ofcorticioidhomobasidiomycetes, which includethe majorityof Athelia arachnoidea Hyphoderma praetermissum # Hygrocybe citrinopallida resupinateforms. Based on Parmasto ’s Ž gures,we estimatethat Humidicutis marginata Chrysomphalina chrysophylla Chrysomphalina grossula ca. 13–15%of describedspecies of homobasidiomycetesare Hygrophorus bakerensis Hygrophorus sordidus Clitocybe lateritia resupinate.Our dataset includes144 resupinate species (27%), Caulorhiza hygrophoroides Conchomyces bursaeformis Lentaria albovinacea which meansthat these formsmay beover-represented. Arrhenia auriscalpium Arrhenia lobata Resupinatus trichotis Taxa inourdataset arerepresented by oneto fourmolecular Resupinatus sp. Resupinatus alboniger Resupinatus dealbatus regions,including nuclear and mitochondrialsmall- and large- Clitocybe clavipes Hohenbuehelia sp. Euagarics Hohenbuehelia tristis subunit ribosomalDNA (rDNA)regions. The nuclearsmall Pleurotus purpureoolivaceus clade Pleurotus tuberregium Pleurotus dryinus subunit rDNA isanearlyfull-length sequence (1.8 kb), whereas Pleurotus smithii Pleurotus cystidiosus Pleurotus cornucopiae the other regionsare represented by partial sequencesthat have Pleurotus calyptratus Pleurotus djamor Pleurotus abieticola beendescribed elsewhere (White et al. 1990;Bruns & Szaro Pleurotus populinus Pleurotus pulmonarius Pleurotus ostreatus 1992;Moncalvo et al. 2000).One hundered and seventeenspec- Pleurotus eryngii Pleurotus fossulatus Limnoperdon incarnatum iesare represented by allfour regions, 78 speciesare represented Pluteus primus Pluteus sp. Entoloma odorifer by threeregions and 12specieshave two regions.All species in Entoloma strictius Tricholoma sp. Tricholoma giganteum the dataset have the nuclearlarge-subunit (nuc-lsu)rDNA Clitopilus prunulus Callistosporium luteoolivaceum Crucibulum laeve region (ca.1.0kb). Sequences were obtained inour laboratory Cyathus striatus Dermocybe marylandensis Cylindrobasidium laeve # using establishedprotocols, or were downloaded from GenBank Cortinarius sp. Cylindrobasidium sp. * Rozites caperatus Cylindrobasidium evolvens * Laccaria bicolor Rhodotus palmatus (http://www.ncbi.nih.gov/genbank/).The studiesof Moncalvo et Laccaria amethystina Flammulina velutipes Laccaria pumila Gloiocephala menieri Cortinarius iodes Marasmius pyrocephalus al. (2000)and Langer (2002)provided 174 (36%) of the nuc- * Limacella glioderma Limacella glischra Cyptotrama asprata * Amanita jacksonii Strobilurus trullisatus lsurDNA sequences.One hundred and Ž fty sevennew Amanita farinosa Xerula furfuracea Amanita muscaria Xerula megalospora Amanita solitariiformis Oudemansiella mucida sequenceswere generated in this study and have beendeposited Amanita virosa Armillaria tabescens Armillariella ostoyae Amanita citrina var grisea Physalacria bambusae * Amanita flavoconia inGenBank (accession numbers AF518568 –AF518724).A Bolbitius vitellinus Physalacria maipoensis Conocybe rickenii Collybia dryophila * Collybia polyphylla Inocybe geophylla Lentinula edodes completelist of speciesand GenBanknumbers of allsequences Inocybe sp. Lentinula lateritia * Panaeolina foenisecii Marasmiellus ramealis Anellaria semiovata Rhodocollybia maculata analysedare available on requestfrom D.S.H. Panaeolus acuminatus Marasmius alliaceus * Hebeloma crustuliniforme

* Micromphale perforans Cortinarius stuntzii Henningsomyces candidus Psilocybe stuntzii Rectipilus fasciculatus Kuehneromyces mutabilis Chondrostereum purpureum * Psilocybe silvatica Campanella junghuhnii (b) Phylogenetic analyses * Pholiota squarrosoides Campanella subdendrophora Hypholoma sublateritium * Hypholoma subviride Lampteromyces japonicus Sequenceswere aligned by eyein M acClade v. 4.0 Stropharia rugosoannulata Nothopanus eugrammus Ripartitella brasiliensis Omphalotus nidiformis Coprinus nudiceps Cyphellopsis anomala (Maddison& Maddison2000) or P aup¤ v. 4.0(Swofford 2001) Lacrymaria velutina Cyphellopsis anomala Psathyrella delineata Merismodes fasciculatus Psathyrella gracilis Dendrothele acerina and regionsthat weredeemed too divergentto alignwere * Coprinus bisporus Lachnella villosa Psathyrella candolleana Flagelloscypha minutissima Coprinus quadrifidus Halocyphina villosa excludedfrom analysis. The data matrix isavailable on request Coprinus atramentarius Nia vibrissa Coprinus sp. Calathella mangrovei * Coprinus cinereus Favolaschia intermedia fromD.S.H. Phylogeneticanalyses inP aup¤ usedequally- Coprinus kimurae Cyphelopsis sp Tulostoma macrocephala Gerronema strombodes Tulostoma sp. Gerronema subclavatum weighted parsimony (EP)ordifferentially weighted parsimony Coprinus sterquilinus * Crinipellis campanella Montagnea arenaria Crinipellis maxima Podaxis pistillaris Marasmius delectans (WP).The latterused a step-matrix of transformation costs that Lepiota cristata Marasmius capillaris

* Lepiota clypeolaria Physalacria sp. * Cystolepiota cystidiosa Baeospora myosura wereestimated with ML(HKY85model of evolution,with Baeospora myriadophylla Leucoagaricus rubrotinctus Hydropus scabripes

* Leucocoprinus fragilissimus Calvatia gigantea Pleurotopsis longinqua empiricalbase frequencies,transition –transversionbias 2,four Lycoperdon perlatum Fistulina pallida Lycoperdon sp. Porodisculus pendulus Lepiota procera Fistulina hepatica rate classesmodelled on discretegamma distribution,shape Macrolepiota procera Fistulina antarctica Lepiota acutesquamosa Auriculariopsis ampla Leucoagaricus naucinus * Schizophyllum commune parameter a = 0.5)on a treederived from EP analysis.Trans- Leucocoprinus cepaestipes Termitomyces cylindricus Chlorophyllum molybdites Termitomyces heimii Macrolepiota rachodes Podabrella microcarpus formationprobabilities were scaled to approximate integer Agaricus campestris Lyophyllum decastes Agaricus arvensis Xeromphalina cauticinalis valuesand adjusted inP to avoid violationof the triangle Agaricus bisporus Favolaschia sp. aup¤ * Favolaschia sp. * Clitocybe connata Mycena rutilanthiformis Mycena haematopoda inequality.Transformation costs inWP wereas follows:A – Dendrocollybia racemosa Panellus stipticus * * Clitocybe ramigena Hypsizygus ulmarius Resinomycena acadiensis Tricholoma caligatum Mycena clavicularis G = 4, A–C = 10, A–T = 8, C–G = 12, C–T = 2, T–G = 10. Tricholoma intermedium Mycena flavoalba Tricholoma portentosum Panellus serotinus EPanalysis useda two-step searchprotocol. The Ž rst step Tricholoma subaureum Macrotyphula cf. juncea Tricholoma focale Typhula phacorhiza Tricholoma imbricatum Bulbillomyces farinosus # used1000 heuristic searches with randomtaxon addition Tricholoma myomyces Phyllotopsis nidulans Tricholoma atroviolaceum Henningsomyces candidus Tricholoma vernaticum Pleurocybella porrigens sequencesand treebisection and reconnection(TBR) branch Tricholoma pardinum Tricholoma venenatum swapping, keepingtwo treesper replicate. The secondstep used Figure 1. (Continued.) the shortest treesfound inthe Ž rst step as starting treesfor TBR branch swapping, with M axTrees set to 10000.The WPanaly- sisused the sameprotocol, except that only100 searches were Christianseniapallida ,which was usedfor rooting purposes. The done in the Ž rst step and M axTrees was set to 1000in the jellyfungi plushomobasidiomycetes make up amonophyletic secondstep. group that has beentermed the Hymenomycetes(Swann & Inaddition to the unconstrainedanalyses describedabove, we Taylor 1995).Homobasidiomycetes include 96% of the species performedthree constrained EP analyses to explorealternative inour dataset, which iscomparable with the proportion of topologies suggested by apreviousphylogenetic study (Binder& describedspecies of homobasidiomycetesin the Hymenomy- Hibbett 2002).The study fromwhich the constraint topologies cetes(98%) (Hawksworth et al. 1995). weredrawn included93 species that area subset of the 481 The homobasidiomycetesinclude about 13500described speciesin the presentanalysis and that wererepresented by all species(Hawksworth et al. 1995),which aredistributed across fourof the rDNA regionsused in the presentstudy (i.e.there at leasteight majorclades (Hibbett &Thorn 2001).Our dataset wereno missing data). Treesderived from EP and MLanalyses includesless than 4% of the describedspecies of homobasidio- of the 93-speciesdataset wereloaded as backbone constraint mycetes.Nevertheless, all of the majorclades of homobasidio- trees(trees used as constraints 1 –3areshown inBinder & mycetesare represented, in proportions that arecomparable Hibbett (2002), Ž gs 1,3and 5,respectively)and analyses were

Proc.R. Soc.Lond. B (2002) 1966D. S.Hibbettand M. Binder Evolution of complex morphologies in fungi

Table1. Taxasampled.

clade number of speciessampled estimated number of described speciesin clade a

homobasidiomycetes 464 13 497 Bolete clade 18 (4%) 840 (6%) Cantharelloid clade 23 (5%) 170 (1%) Euagarics clade 214 (46%) 8425 (62%) Gomphoid–Phalloid clade 13 (3%) 350 (3%) Hymenochaetoid clade 34 (7%) 630 (5%) Polyporoid clade 98 (21%) 1350 (10%) Russuloid clade 42 (9%) 1000 (7%) Thelephoroid clade 13 (3%) 240 (2%) other minor clades 9 (2% ) — a Estimatednumbers of speciesin eachgroup basedon Ž gures from Hawksworth et al. (1995) and Hibbett &Thorn (2001).

performedusing the samesettings as inthe unconstrainedEP 3. RESULTS analyses. The datasethas 3977 bp ofaligned sequence,of which (c) Analyses ofcharacter evolution 177 bp weretoo divergent tobeincluded in ouranalyses. Wescored fruiting-body morphology as resupinate(0) or There are 2262 variable positionsand 1605 parsimony- pileate-erect(1) (effused-re  exedtaxa, which have both resupi- informative positions.The EPandWP analyseseach reco- nate and pileateparts of the fruitingbody, werescored as vered10 000 trees(EP: 23 536 steps,consistency index pileate-erect)and performedancestral state reconstruction (CI) = 0.174, retentionindex (RI) = 0.584; WP:125 982 (ASR)using EPoptimizationin M acClade,onall of the EP, steps, CI = 0.175, RI = 0.592). The constrainedEP analy- WPand constrainedEP trees.We also inferred the ancestral sesrecovered trees that are 69 –75 stepslonger than the fruiting-body morphology of the homobasidiomyceteswith ML, unconstrainedtrees (23 605 –23 611 steps,CI = 0.173, using the ‘local’ method of Pagel(1999), which was RI = 0.583). Despitethe large numberof equally parsi- implementedin D iscrete.To runthe MLtests of ancestral monioustrees, the strict consensustrees in eachanalysis states, we Ž xed the ancestralnode of the homobasidiomycetes are highly resolved(only theEP treeis shownin detail; as resupinateand obtained the likelihoodof the data; next, we Ž gure 1). Ž xed the ancestralnode as pileate-erectand obtained the likeli- The Ž ve phylogenetic analysesthat weperformed indi- hood again. FollowingPagel (1999) and others (Mooers& catedifferent patterns of higher-order relationships among Schluter1999), we useda differenceof two unitsof log likeli- themajor cladesof homobasidiomycetes ( Ž gure 2). hood as the criterionfor ‘strong’ support of oneancestral state Nevertheless,the eight major cladesof homobasidio- overanother. Weperformed ML tests of ancestralstates using mycetesrecognized by Hibbett &Thorn (2001) were ninedifferent trees that variedin topology, branch-length esti- resolvedin all trees,as well asseveral independentminor mates and sampling regimes,including (i) one tree each from clades,including the Gloeophyllum clade(three species), the EP,WPand constrainedEP analyses,with branch lengths Dendrocorticium clade (Ž ve species)and Jaapiaargillacea estimatedwith MLfromthe nuc-lsurDNA only(which is (Ž gures1 and2). Acladeof Ž ve speciesincluding shared by allspecies); (ii) ‘punctuational ’ versionsof the uncon- Paullicorticiumniveocremeum (the Paullicorticium clade) strainedEP and WPtrees,in which allbranch lengths wereset jumpedbetween the Polyporoid clade(EP, constrained to have the samevalue; and (iii) ‘pruned’ versionsof the uncon- EPanalysis 2), Russuloidclade (WP) andpositions close strainedEP and WPtrees,in which the numberof resupinate tothe Auriculariales (constrainedEP analyses1 and3), taxa was reducedby half, with the deletionsspread across the butthe composition of major cladesin thehomobasidio- tree(with MLbranch lengths).The prunedtrees include 72 myceteswas otherwise stable acrossthe different analyses (17%)resupinate species, which may bea morerepresentative (Ž gure 2). Resupinateforms occur in eachof the eight samplethan that inthe unprunedtrees (Hawksworth et al. 1995; major cladesof homobasidiomycetes, as well asthe Den- Parmasto 1997). drocorticium clade, Jaapiaargillacea , and the Paullicorticium To test whether the rate of transformations fromresupinate clade (Ž gure 1). to pileate-erectforms is signi Ž cantlydifferent from the rate of Parsimony-based ASRson theEP, WPandconstrained transformations inthe reversedirection (these transformations EPtreesindicate that therehave been50 –54 transform- arehereafter called gains and losses,and theirrate parameters ationsbetween resupinate and pileate-erect forms( Ž gure are called a and b,respectively),we usedML analysis inD is- 1; table 2). Onaverage, theASRs on theEP treesindicate crete,with the samenine trees as wereused in ML tests of aslight preponderanceof gains relative tolosses (29.3 ancestralstates. To runthe test, we Ž rst estimated a and b with- gains versus24.7 losses),but WP treesindicate a roughly out restrictionon their values and obtained the likelihood;next, equal numberof gains andlosses, and constrained EP we restrictedthe valuesof a and b to beequal and repeatedthe treesindicate a preponderanceof losses (table 2). analysis.The test statistic isequalto twicethe differencein log Weexamined parsimony-based ASRsin detail onone likelihoodsand is x2-distributedwith onedegree of freedom treeeach from theEP, WPandconstrained EP analyses. (Pagel1997, 1999). The various topologies imply differentpatterns of changes,

Proc.R. Soc.Lond. B (2002) Evolution of complex morphologies in fungi D.S.Hibbettand M. Binder1967

(a) Polyp. clade + (b) Paullicort. clade Polyp. clade Euag. clade Russ. clade + Bol. clade Paullicort. clade Jaapia Hymeno. clade Dendrocort. clade Euag. clade Theleph. clade Bol. clade Jaapia Gloeoph. clade * Theleph. clade Russ. clade Dendrocort. clade Hymeno. clade Gloeoph. clade Canth. clade Canth. clade Gomph.–Phall. clade Gomph.–Phall. clade Auriculariales Auriculariales Dacrymycetales Dacrymycetales Christiansenia Christiansenia

(c) (d) (e) Polyp. clade Polyp. clade Polyp. clade + Russ. clade Russ. clade Paullicort. clade Euag. clade Hymeno. clade Euag. clade Bol. clade Euag. clade Bol. clade Jaapia Jaapia Bol. clade Gloeoph. clade Gomph.–Phall. clade Jaapia Russ. clade Gloeoph. clade Theleph. clade Hymeno. clade Theleph. clade Hymeno. clade Gomph.–Phall. clade Canth. clade Gomph.–Phall. clade Canth. clade Canth. clade Dendrocort. clade * Gloeoph. clade Theleph. clade Dendrocort. clade Dendrocort. clade Paullicort. clade Paullicort. clade Auriculariales Auriculariales Auriculariales Dacrymycetales Dacrymycetales Christiansenia Christiansenia Dacrymycetales Christiansenia

Figure 2. Higher-level relationships of homobasidiomycetes inferred with( a) EP, (b) WP and (c–e)constrained EPanalyses ((c)constrained analysis1; ( d)constrained analysis2; ( e)constrained analysis3). Theancestor of thehomobasidiomycetes is indicated withan arrowhead. Ancestral statesat nodes inferred withparsimony on one tree from eachanalysis are indicated by shaded circles: white, resupinate; black, pileate-erect; grey, uncertain. Labelled terminal groups are thesame as in Ž gure 1; asterisksdenote nodes thatcollapse in thestrict consensus tree.

Table2. Numbers of transformations in fruiting-body form (0, resupinate; 1, pileate-erect) estimated withparsimony.

number of gains (0 1) Number of losses (1 0) analysis total steps minimum–maximum (average)! minimum –maximum (average)!

EP 54 16–37 (29.3) 17–38 (24.7) WP 50–51 20–30 (25.0) 20–31 (25.8) EPconstrained analysis1 50–51 15–24 (19.4) 27–36 (31.6) EPconstrained analysis2 53 19–24 (20.9) 29–34 (32.1) EPconstrained analysis3 53–54 11–37 (25.7) 17–43 (28.2)

asoptimized usingparsimony ( Ž gure 2). For example, the MLanalysesindicate that therate oftransformations ancestral stateof the polyporoid cladeis resolved as from resupinateforms to pileate-erect formsis greater resupinatein theEP andWP trees,but it isresolved as than therate oftransformations in thereverse direction. pileate-erect in constrainedEP analyses1 and2 andit is In thetrees with all 481 speciesincluded (seven trees equivocal in constrainedEP analysis 3( Ž gure 2). Never- tested),the unrestricted value of a is about threeto four theless,in all thetrees that weexamined,the optimal ASR times greater than that of b (table 3). In thepruned trees, indicatesthat theancestor of the homobasidiomycetes was theasymmetry iseven more pronounced,with a being resupinate( Ž gure 2). Maximum likelihood analysesof about Ž ve tosix times greater than b (table 3). In all trees, ancestral statesalso indicatethat theancestor of the therestricted model, in which a and b are forcedto take homobasidiomyceteswas resupinate ( DlogL . 2 in all thesame value, is signi Ž cantly lesslikely than theuncon- ninetrees). strainedmodel ( p , 0.001; table 3).

Proc.R. Soc.Lond. B (2002) 1968D. S.Hibbettand M. Binder Evolution of complex morphologies in fungi

Table3. MLtestsof asymmetriesin transformation rates.

unrestricted ( a Þ b) restricted ( a = b)

analysis a (0 1) b (1 0) 2logL a (0 1) b (1 0) 2logL 2DlogL¤ ! ! ! ! EP 4.385 1.379 405.013 2.392 2.392 414.590 19.154 WP 4.063 1.242 397.893 2.142 2.142 407.738 19.690 EPconstrained analysis 1 4.426 1.428 404.353 2.456 2.456 413.347 17.988 EPconstrained analysis 2 4.296 1.559 410.438 2.450 2.450 418.114 15.352 EPconstrained analysis 3 4.860 1.326 408.931 2.549 2.549 419.371 20.880 EPpunctuational analysis 28.772 7.095 379.925 13.911 13.911 390.344 20.838 WPpunctuational analysis 24.458 6.945 369.603 12.947 12.947 378.254 17.302 EP pruned 6.898 1.172 326.292 2.406 2.406 339.504 26.424 WP pruned 6.583 1.019 311.456 2.176 2.176 327.503 32.094

¤ p , 0.001.

4. DISCUSSION homobasidiomycetes.The samesources of error that affectASR also affectestimation ofevolutionary models, Resupinatehomobasidiomycetes have presentedsig- butagain ourresults were consistent across all ofthe trees niŽ canttaxonomic challenges becauseof their morpho- that wetested.The optimal modelsof morphological evol- logical simplicity. The Ž rstobjective of our study was to utionindicate that therate oftransformations from resupi- determinethe phylogenetic distribution ofresupinate nateto pileate-erect formsexceeds the rate of homobasidiomycetes.Our analysesresolve the placements transformations in thereverse direction by afactor ofat ofmany resupinateforms, con Ž rming that they are scat- least three(table 3). In other words,resupinate forms teredthroughout thehomobasidiomycetes, as has been appear tobe evolutionarily more labile than pileate-erect suggested(Donk 1964, 1971; Ju¨lich 1981; Parmasto forms,which may explain why pileate-erect formshave 1986, 1995; Corner1991). The taxonomic implications cometo predominate in homobasidiomycetes. ofthese analyses will bepresented elsewhere (M. Binder At a Ž rstglance, it might appear that thereis acon  ict andD. S.Hibbett,unpublished data). The secondobjective of our study was to infer the betweenthe results of the ML analyses,which indicatea ancestral morphology ofthehomobasidiomycetes. Ances- signiŽ canttrend towards evolution of pileate-erect forms, tral statereconstruction has many potential sourcesof andthose of parsimony analyses,which reveal noconsist- error, including error in phylogenetic reconstructionand entpattern oflosses of resupinate forms outnumbering biasedor incompletetaxon sampling (Cunningham1999; gains (tables 2and3). Amajor differencebetween these Mooers& Schluter1999; Omland 1999; Ree& Donoghue methodsof analysis, however,is that underparsimony, a 1999; Salisbury &Kim 2001). The MLmethodof ASR modelof evolution in which therates of losses and gains isalso sensitiveto error in branch-length estimates(Ree & are equal is implicit, whereasunder ML theseparameters Donoghue1999). Conversely,it is astrength ofthe ML are estimateddirectly from thephylogeny andare allowed methodthat it is able toincorporate information about tovary. In this case,likelihood-ratio testsrejected models branch lengths intoestimates of ancestral states.We ofevolution in which lossesand gains have equal rates, explored thesensitivity ofourresults to each of these fac- indicating that ancestral statereconstructions based on torsby performing ASRusingdifferent tree topologies, equally weightedparsimony may notbe reliable. One sampling regimes andbranch lengths.On all thetrees that potential application ofthe ML analysesis to use the wetested,parsimony andML analysesboth indicatethat transformation rates inferredwith MLtodevelop trans- theancestor of thehomobasidiomycetes had aresupinate formation costs(step-matrix values) for usein weighted fruiting body.These results are partially consistentwith parsimony analysis offruiting-body morphology. theview that resupinatehomobasidiomycetes make upa The analysespresented here employed asimple model paraphyletic grade ofplesiomorphic forms,as suggested offruiting-body evolution,in whichthere are only two by Parmasto (1995) andOberwinkler (1985). Neverthe- character states,and a uniform processof evolution is less,parsimony analysis also implies that therehave been assumedto operate acrossthe entire phylogeny. In future multiple reversals from pileate-erect formsto resupinate analyses,we will explore multi-state character codings, forms(table 2). The precisenumber of transformations which may betterre  ectthe diversity offruiting-body andthe states of many internal nodesaccording toparsi- forms,and we will testthe assumption of process homo- mony are, however,ambiguous ( Ž gure 2; table 2). geneity,for example through analysesof character corre- The Ž nal objectiveof ourstudy was to addresswhether lations (e.g.Hibbett &Donoghue2001). Suchanalyses thereis an asymmetry in therate oftransformations will involve modelswith many more parameters than the betweenresupinate and pileate-erect fruiting bodiesin modelsused here, and may require larger, more densely

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