A class-wide phylogenetic assessment of Dothideomycetes.
Journal: 2011/July - Studies in Mycology
ISSN: 1872-9797
Abstract:
We present a comprehensive phylogeny derived from 5 genes, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, for 356 isolates and 41 families (six newly described in this volume) in Dothideomycetes. All currently accepted orders in the class are represented for the first time in addition to numerous previously unplaced lineages. Subclass Pleosporomycetidae is expanded to include the aquatic order Jahnulales. An ancestral reconstruction of basic nutritional modes supports numerous transitions from saprobic life histories to plant associated and lichenised modes and a transition from terrestrial to aquatic habitats are confirmed. Finally, a genomic comparison of 6 dothideomycete genomes with other fungi finds a high level of unique protein associated with the class, supporting its delineation as a separate taxon.
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Studies in Mycology. Dec/31/2008; 64: 1-15-S10

A class-wide phylogenetic assessment of Dothideomycetes

+45 authors

Abstract

We present a comprehensive phylogeny derived from 5 genes, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, for 356 isolates and 41 families (six newly described in this volume) in Dothideomycetes. All currently accepted orders in the class are represented for the first time in addition to numerous previously unplaced lineages. Subclass Pleosporomycetidae is expanded to include the aquatic order Jahnulales. An ancestral reconstruction of basic nutritional modes supports numerous transitions from saprobic life histories to plant associated and lichenised modes and a transition from terrestrial to aquatic habitats are confirmed. Finally, a genomic comparison of 6 dothideomycete genomes with other fungi finds a high level of unique protein associated with the class, supporting its delineation as a separate taxon.

INTRODUCTION

Multi laboratory collaborative research in various biological disciplines is providing a high level of interaction amongst researchers with diverse interests and backgrounds. For the mycological community, the “Assembling the Fungal Tree of Life” project (AFTOL) provided the first DNA-based comprehensive multigene phylogenetic view of the fungal Kingdom (Lutzoni et al. 2004, James et al. 2006). This has also made it possible to revise the classification of the fungi above the ordinal level (Hibbett et al. 2007). Subsequent work is focused on elucidating poorly resolved nodes that were highlighted in the initial DNA-based phylogeny (McLaughlin et al. 2009).

At the other end of the scale from the tree of life projects, taxon sampling with relatively small numbers of sequence characters are also progressing in various barcoding projects (Seifert et al. 2007, Chase et al. 2009, Seifert 2009). It remains important to link these two ends of the spectrum by also sampling intensively at foci of interest between barcoding and the tree of life. With this in mind it is the aim of this paper and subsequent ones in this volume to provide a broadly sampled phylogeny at class level and below for Dothideomycetes. This result is combined efforts and data from a diverse group of researchers to focus on systematic sampling, therefore developing a more robust fungal class wide phylogeny of Dothideomycetes. This is especially important as a framework for comprehending how fungi have evolved as they shift ecological habitats and adapt to new environments and nutritional modes.

It is apparent that the assemblage of fungi, now defined as Dothideomycetes, exemplifies a dynamic evolutionary history. This is by far the largest and arguably most phylogenetically diverse class within the largest fungal phylum, Ascomycota (Kirk et al. 2008). It contains a heterogeneous group of fungi that subsist in the majority of the niches where fungi can be found. The best-known members of the group are plant pathogens that cause serious crop losses. Species in the genera Cochliobolus, Didymella, Phaeosphaeria, Pyrenophora, Venturia, Mycosphaerella and Leptosphaeria, or their anamorphs, are major pathogens of corn, melons, wheat, barley, apples, bananas and brassicas respectively, in most areas of the world where they are cultivated. Other species are important pathogens in forestry e.g. species in the genera Botryosphaeria and Mycosphaerella and their anamorphs that attack economically important tree species.

Despite a large body of work containing taxonomic, phytopathological, genetic and genomic research, the majority of fungi hypothesised to be members of Dothideomycetes remain under-sampled within a systematic framework. Several studies performed during the course of the last four years have advanced our understanding of these fungi, but phylogenetic relationships of the saprobes, aquatic, asexual and lichenised species remain particularly poorly studied. Indeed, their conspicuous absence in phylogenetic analyses frustrates a broader understanding of dothideomycete evolution.

Dothideomycetes share a number of morphological characters with other fungal classes. It was recently formally described (Eriksson & Winka 1997) replacing in part the long-recognised loculoascomycetes (Luttrell 1955). This redefinition of the loculoascomycetes was mainly prompted by DNA sequencing comparisons of ribosomal RNA genes (Berbee & Taylor 1992, Spatafora et al. 1995) that was subsequently expanded and confirmed (Berbee 1996, Silva-Hanlin & Hanlin 1999, Lindemuth et al. 2001, Lumbsch & Lindemuth 2001). These early phylogenetic studies demonstrated that loculoascomycetes, as it was defined, is not monophyletic, although contrary views exist (Liu & Hall 2004). Nevertheless the majority of analyses have shown that some loculoascomycete taxa, such as the “black yeasts” in Chaetothyriales as well as the lichenised Verrucariales, reside within Eurotiomycetes as subclass Chaetothyriomycetidae (Spatafora et al. 1995, Winka et al. 1998, Geiser et al. 2006, Gueidan et al. 2008). The majority of the remaining loculoascomycete species are now placed in Dothideomycetes. Although finer morphological distinctions between the distantly related members of loculoascomycetes can be made, their synapomorphies remain elusive (Lumbsch & Huhndorf 2007). These findings all point to the fact that a number of loculoascomycete morphological characters are either retained ancestral traits or that they exhibit convergence due to similar selection pressures.

Traditionally the most important morphological characters used to define major groups in Ascomycota were the type of ascus, septation of ascospores, the morphology and development of the ascoma, as well as the structure and organisation of the centrum. Dothideomycetes (and previously, loculoascomycetes) have fissitunicate (or functionally bitunicate) asci, that emerge from ascolocular development in preformed locules within vegetative tissue, that represents the ascoma. The reproductive structures in ascolocular development are derived from cells before fusion of opposing mating types occurs and can contain one or several locules. This form of ascolocular development is in contrast to the ascohymenial development found in most other fungal classes. During ascohymenial development asci are generated in a hymenium and the reproductive structure is derived from cells after fusion of opposing mating types. The fissitunicate ascus has been described for more than a century, but the importance of ascolocular development was first emphasised in 1932 (Nannfeldt 1932). Importantly Nannfeldt's concepts were also the basis for the Santesson's integration of lichens into the fungal classification (Santesson 1952). In fissitunicate asci, generally, the ascospores are dispersed by the rupture of the thick outer layers (ectotunica) at its apex, allowing the thinner inner layer (endotunica) to elongate similar to a “jack in a box”. The elongated endotunica ruptures apically and releases the ascospores forcefully through the ascoma opening. The spores are then released in the air, or in aquatic species, under water. Building on this work and that of others (Miller 1949), Luttrell proposed Loculoascomycetes, synonymous to Nannfeldt's “Ascoloculares” (Luttrell 1955). Importantly, he proposed a correlation between fissitunicate asci and ascolocular development, also emphasising the importance of ascus morphology and dehiscence as well as the development of surrounding elements within the ascoma.

Although the concept of a group of fungi (including the Dothideomycetes) with fissitunicate asci and ascolocular development has been accepted by several authors, much less agreement could be found on ordinal definitions in the era before molecular characters. This ranged from proposing a single order (von Arx & Müller 1975) to three (Müller & von Arx 1962), five (Luttrell 1951, 1955) six (Barr 1979), or seven (Barr 1987). Luttrell initially described a number of important development types centered on descriptions of all tissues inside the ascoma (the centrum concept) and combined this with ascoma structure to define his five orders (Luttrell 1951, 1955). Of Luttrell's initial centrum concepts three are applicable to the Dothideomycetes as they are presently defined. Thus, the Pleospora type, the Dothidea type and the Elsinoë type centra correspond to the dothideomycete orders Pleosporales, Dothideales and Myriangiales, respectively. An important refinement to Luttrell's ideas was introduced with the concept of the hamathecium by Eriksson (Eriksson 1981). This is defined as a neutral term for sterile hyphae or other tissues between the asci in the ascoma (Kirk et al. 2008). For example, hamathecial types can include the presence or absence of pseudoparaphyses, which are sterile cells that extend down from the upper portion of the ascomatal cavity. They become attached at both ends, although the upper part may become free at maturity. Other important concepts introduced by Müller and von Arx (Müller & von Arx 1962) focused on the morphology of the ascoma opening and ascus shape. The Dothidea type centrum in the type species of Dothidea, D. sambuci illustrates several typical dothideomycete morphologies (Fig. 1). These include the thick-walled fissitunicate asci produced within a multilocular stroma.

The most recent dothideomycete class-wide morphological assessments were carried out by Barr (Barr 1979, 1987). Her subclasses were determined based on characters in the centrum, including the absence, presence and types of hamathecial tissues. Consistent with several earlier authors, Barr's ordinal classifications were based on ascomatal shape (perithecioid or apothecioid) and manner in which nutrients are obtained by the fungus (Barr 1987). In addition to these characters she emphasised the importance of finer distinctions in the hamathecium such as the shape and structure of the pseudoparaphyses (Barr 1979, 1987).

The introduction of molecular phylogenies for Dothideomycetes (Berbee 1996) provided an opportunity to verify the significance of various morphological characters used in the aforementioned classifications. The clearest correlation with a DNA sequence-based phylogeny was for the presence or absence of pseudoparaphyses, largely agreeing with the first orders proposed by Luttrell (Liew et al. 2000, Lumbsch & Lindemuth 2001). Barr's concept of applying the shape of the pseudoparaphyses to define orders was rejected by molecular phylogenies (Liew et al. 2000). This set the stage for more comprehensive analyses incorporating protein data, and resulted in the definition of two subclasses, Pleosporomycetidae (pseudoparaphyses present) and the Dothideomycetidae (pseudoparaphyses absent; Schoch et al. 2006). Numerous orders and other taxa remained unresolved outside of these two subclasses.

Fig. 1.

Dothidea sambuci. A–B. Appearance of ascomata on the host surface. C, F. Asci in cotton blue reagent. D. Vertical section through ascomata illustrating the mutilocule at the upper layer. E. Vertical section through ascomata in cotton blue reagent illustrating the locule. G–H. Ascospores in cotton blue reagent. Scale bars: B = 1000 μm; C = 500 μm; E = 100 μm; F–H = 10 μm.

The most recent class level phylogenetic analyses combining sequences from protein coding genes with ribosomal RNA sequences fortified the view that Dothideomycetes is a monophyletic group (Schoch et al.2009a, b). Furthermore, strong support was found for a sister relationship between Dothideomycetes and the lichenised class Arthoniomycetes (Lumbsch et al. 2005, Spatafora et al. 2006, Schoch et al. 2009a). This clade was recently defined as a rankless taxon “Dothideomyceta” (Schoch et al.2009a, b). The Arthoniomycetes consists of a single order (Arthoniales) of lichens and lichenicolous fungi (Ertz et al. 2009) that produce bitunicate asci in ascohymenial apothecia and was proposed as an intermediate group or “Zwischengruppe” (Henssen & Thor 1994). This placement raises intriguing questions regarding the origins of ascolocular development and further illustrates the importance of including lichen-forming fungi in dothideomycete phylogenies.

While considerable progress has been made in defining these fungi the placement of Dothideomycetes in relation to the majority of other Ascomycota classes remains unresolved. Here, greater clarity would likely require a huge increase of characters from genome projects. In this regard, the first phylogenomic studies have shown low resolution for this relationship (Fitzpatrick et al. 2006, Kuramae et al. 2006, Robbertse et al. 2006). This could indicate a rapid radiation event, but more likely suggests taxon sampling bias. This latter view is supported by the fact that none of these studies has included lichenised species that represent about 25 % of the number of species in Ascomycota.

The authors of this volume have focused on two primary goals. These are to considerably expand the taxon sampling of existing orders by including saprobes, asexual species and other poorly sampled groups. Secondly we aim to sample widely within specific environmental niches and present a multigene phylogeny that exposes the highly diverse nature of Dothideomycetes.

MATERIAL AND METHODS

DNA extraction, amplification and sequencing

The majority of fungal cultures were obtained from the CBS culture collection and additional sources mentioned in other papers of this volume. DNA was also provided by authors of several papers presented in this volume and the reader is referred to Boehm et al. (2009a), Crous et al. (2009a), Suetrong et al. (2009) and Zhang et al. (2009). For additional details see Table 1 - see online Supplementary Information. Fungal genomic DNA was obtained by scraping mycelium from PDA plates. Samples were subsequently pulverised and the DNA was extracted using the FastDNA® kit and the FastPrep® instrument from MPI Biochemicals (Irvine, CA, U.S.A.). DNA amplifications were completed using Taq polymerase (GenScript, Piscataway, NJ, U.S.A.), with FailSafe™ PCR 2× PreMix E (Epicentre, San Diego, CA, U.S.A.). Primers were used as noted in the Assembling the Fungal Tree of Life project (AFTOL; Schoch et al. 2009a). This resulted in DNA sequence data obtained from the small and large subunits of the nuclear ribosomal RNA genes (SSU, LSU) and three protein coding genes, namely the translation elongation factor-1 alpha (TEF1) and the largest and second largest subunits of RNA polymerase II (RPB1, RPB2). Primer sets used for these genes were as follows: SSU: NS1/NS4; LSU: LR0R/LR5; TEF1 983/2218R (initially obtained from S. Rehner: ocid.nacse.org/research/deephyphae/EF1primer.pdf); RPB2: fRPB2-SF/fRPB2-7cR; RPB1: RPB1-Ac/RPB1-Cr (obtained from V. Hofstetter). Primer sequences are available at the WASABI database at the AFTOL website (aftol.org). PCRs for these genes were performed in various laboratories of the coauthors mentioned but the majority of reactions were run under conditions described previously (Lutzoni et al. 2004, Schoch et al. 2009a). Two duplicate sets of sequences were inadvertently included in the analysis (indicated in Table 1).

Table 1.

Isolates of Dothideomycetes included in this study. Newly deposited sequences are shown in bold.

Taxonvoucher/culture1SSULSURPB1RPB2TEF1
Acanthostigma perpusillumUAMHAY856937AY856892
Aglaospora profusaGU296130GU301792GU349062
Aigialus grandis 12QGU296132GU301794GU349063
Aigialus grandis 2JK 5244AGU296131GU301793GU371762
Aigialus parvusA6GU296133GU301795GU371771GU349064
Aliquandostipite khaoyaiensisAF201453GU301796FJ238360GU349048
Alternaria alternataDQ678031DQ678082DQ677980DQ677927
Amniculicola parvaGU296134FJ795497GU349065
Anteaglonium abbreviatum 1ANM 925.1GQ221877GQ221924
Anteaglonium abbreviatum 2GKM 1029GQ221878GQ221915
Anteaglonium globosum 1SMH 5283GQ221911GQ221919
Anteaglonium globosum 2ANM 925.2GQ221879GQ221925
Anteaglonium latirostrumL100N 2GQ221876GQ221938
Anteaglonium parvulumSMH 5210GQ221907GQ221917
Apiosporina collinsiiGU296135GU301798GU357778GU349057
Apiosporina morbosadimospEF114694
Arthopyrenia salicis 11994 CoppinsAY607730AY607742
Arthopyrenia salicis 2AY538333AY538339GU371814
Ascochyta pisiDQ678018DQ678070DQ677967DQ677913
Ascocratera manglicolaJK 5262CGU296136GU301799GU371763
Asteromassaria pulchraGU296137GU301800GU371772GU349066
Astrosphaeriella aggregataMAFF 239486AB524450AB524591AB539105AB539092
Astrosphaeriella bakerianaGU301801GU357752GU349015
Astrothelium cinnamomeumDUKE 0000007AY584652DQ782896
Aulographina pinorum 1GU371766
Aulographina pinorum 2GU296138GU301802GU357763GU371737GU349046
Aureobasidium pullulansDQ471004DQ470956DQ471148DQ470906DQ471075
Bagnisiella examinansGU296139GU301803GU357776GU371746GU349056
Batcheloromyces proteaeAY251102EU019247
Beverwykella pulmonariaGU301804GU371768
Bimuria novae-zelandiaeAY016338AY016356DQ471159DQ470917DQ471087
Botryosphaeria dothideaDQ677998DQ678051GU357802DQ677944DQ767637
Botryosphaeria tsugaeAF271127DQ767655DQ767644DQ677914
Byssolophis sphaerioidesIFRDCC2053GU296140GU301805GU456348GU456263
Byssothecium circinansAY016339AY016357DQ767646GU349061
Camarosporium quaternatumGU296141GU301806GU357761GU349044
Capnobotryella renisporaAY220613GQ852582
Capnodium coffeaeDQ247808DQ247800DQ471162DQ247788DQ471089
Capnodium salicinumDQ677997DQ678050DQ677889
Catenulostroma abietis (as Trimmatostroma abietis)DQ678040DQ678092GU357797DQ677933
Catenulostroma elginenseGU214517EU019252
Catinella olivaceaUAMH 10679DQ915484EF622212
Cenococcum geophilum 1HUNT A1L76616
Cenococcum geophilum 2CGMONTL76617
Cenococcum geophilum 310L76618
Cercospora beticolaDQ678039DQ678091DQ677932
Chaetosphaeronema hispidulumEU754045EU754144GU357808GU371777
Cladosporium cladosporioidesDQ678004DQ678057GU357790DQ677952DQ677898
Cladosporium iridis (teleomorph Davidiella macrospora)DQ008148
Clathrospora elynaeGU296142GU323214
Cochliobolus heterostrophusAY544727AY544645DQ247790DQ497603
Cochliobolus sativusDAOM 226212DQ677995DQ678045DQ677939
Columnosphaeria fagiAY016342AY016359DQ677966
Comminutispora agavaciensisY18699EU981286
Conidioxyphium gardeniorumCPC 14327GU296143GU301807GU357774GU371743GU349054
Coniothyrium palmarumDQ678008DQ767653DQ677956DQ677903
Corynespora cassiicola 1GU296144GU301808GU357772GU371742GU349052
Corynespora cassiicola 2CCPGU296145
Corynespora olivaceaGU301809GU349014
Corynespora smithiiCABI 5649bGU323201GU371804GU371783GU349018
Cryptothelium amazonum47GU327713GU327731
Cryptothelium pulchrum63CGU327714
Cystocoleus ebeneus 1L348EU048573EU048580
Cystocoleus ebeneus 2L315EU048572
Davidiella tassianaDQ678022DQ678074GU357793DQ677971DQ677918
Delitschia cf. chaetomioides 1GKM 3253.2GU390656
Delitschia cf. chaetomioides 2GKM 1283GU385172
Delitschia didyma 1 (duplicate)UME 31411DQ384090
Delitschia didyma 2UME 31411AF242264DQ384090
Delitschia winteriDQ678026DQ678077DQ677975DQ677922
Delphinella strobiligenaDQ470977DQ471175DQ677951DQ471100
Devriesia staurophoraEF137359DQ008151
Devriesia strelitziaeGU296146GU301810GU371738GU349049
Didymella bryoniae (as Phoma cucurbitacearum)GU301863GU371767
Didymella exiguaGU296147GU357800GU371764
Didymocrea sadasivaniiDQ384066DQ384103
Diplodia mutila (teleomorph Botryosphaeria stevensii)DQ678012DQ678064DQ677960DQ677907
Dissoconium aciculareGU214523GQ852587
Dissoconium commune (teleomorph Mycosphaerella communis)GU214525GQ852589
Dissoconium dekkeri (teleomorph Mycosphaerella lateralis)GU214531GU214425
Dothidea hippophaësU42475DQ678048GU357801DQ677942DQ677887
Dothidea insculptaDQ247810DQ247802DQ471154AF107800DQ471081
Dothidea sambuciDAOM 231303AY544722AY544681DQ522854DQ497606
Dothiora cannabinaeDQ479933DQ470984DQ471182DQ470936DQ471107
Dothiora ellipticaGU301811GU349013
Dothistroma septosporum 1 (teleomorph Mycosphaerella pini)GU301853GU371730
Dothistroma septosporum 2GU214533GQ852597
Elsinoë centrolobiDQ678041DQ678094GU357798DQ677934
Elsinoë phaseoliDQ678042DQ678095GU357799DQ677935
Elsinoë venetaDQ767651DQ767658DQ767641
Endosporium aviariumUAMH 10530EU304349EU304351
Endosporium populi-tremuloidisUAMH 10529EU304346_EU304348
Entodesmium rudeGU301812GU349012
Falciformispora lignatilis 1BCC 21118GU371835GU371827GU371820
Falciformispora lignatilis 2BCC 21117GU371834GU371826GU371819
Farlowiella carmichaeliana 2GU296148
Farlowiella carmichealiana 1 (as anamorph Acrogenospora sphaerocephala)GU296129GU301791GU357780GU371748GU349059
Floricola striataJK 56781GU296149GU301813GU371758
Friedmanniomyces endolithicusCCFEE 522DQ066715
Friedmanniomyces simplexDQ066716
Gibbera confertaGU296150GU301814GU357758GU349041
Gloniopsis arciformisGKM L166AGU323180GU323211
Gloniopsis praelonga 1FJ161134FJ161173FJ161113FJ161090
Gloniopsis praelonga 2FJ161154FJ161195FJ161103FJ161103
Gloniopsis subrugosaFJ161170FJ161210GU371808FJ161131
Glonium circumserpens 1FJ161168FJ161208
Glonium circumserpens 2FJ161160FJ161200GU371806FJ161126FJ161108
Glonium stellatumFJ161140FJ161179FJ161095
Guignardia bidwelliiDQ678034DQ678085GU357794DQ677983
Guignardia citricarpaGU296151GU301815GU357773GU349053
Guignardia gaultheriaeDQ678089GU357796DQ677987
Halomassarina ramunculicola 1 (as Massarina ramunculicola)BCC 18404GQ92538GQ925853
Halomassarina ramunculicola 2 (as Massarina ramunculicola)BCC 18405GQ925839GQ925854
Halomassarina thalassiae (as Massarina thalassia)JK 5262DGU301816GU349011
Helicomyces roseusDQ678032DQ678083DQ677981DQ677928
Hortaea acidophilaGU323202GU357768
Hortaea werneckiiGU296153GU301818GU357779GU371747GU349058
Hortaea werneckiiGU296152GU301817GU371739GU349050
Hysterium angustatumFJ161167FJ161207FJ161129FJ161111
Hysterium barrianum 1ANM 1495GU323182GQ221885
Hysterium barrianum 2ANM 1442GU323181GQ221884
Hysterobrevium mori 1FJ161164FJ161204
Hysterobrevium mori 2SMH 5273GU301820GQ221936
Hysterobrevium mori 3GKM 1013GU301819GU397338
Hysterobrevium smilacis 1FJ161135FJ161174GU357806FJ161114FJ161091
Hysterobrevium smilacis 2SMH 5280GU323183GQ221912GU371810GU371784
Hysteropatella clavisporaDQ678006AY541493DQ677955DQ677901
Hysteropatella ellipticaEF495114DQ767657DQ767647DQ767640
Jahnula aquaticaR68-1EF175633EF175655
Jahnula bipileataF49-1EF175635EF175657
Jahnula seychellensisSS2113.1EF175644EF175665
Julella avicenniae 1BCC 18422GU371831GU371823GU371787GU371816
Julella avicenniae 2BCC 20173GU371830GU371822GU371786GU371815
Kabatiella caulivoraEU167576EU167576GU357765
Kalmusia scabrispora 1MAFF 239517AB524452AB524593AB539093AB539106
Kalmusia scabrispora 2NBRC 106237AB524453AB524594AB539094AB539107
Karstenula rhodostomaGU296154GU301821GU371788GU349067
Katumotoa bambusicolaMAFF 239641AB524454AB524595AB539095AB539108
Keissleriella cladophilaGU296155GU301822GU371735GU349043
Kirschsteiniothelia elaterascusA22-5A / HKUCC7769AF053727AY787934
Kirschsteiniothelia maritimaGU323203GU349001
Laurera megaspermaAFTOL 2094FJ267702
Lentithecium aquaticumGU296156GU301823GU371789GU349068
Lentithecium arundinaceumGU296157GU301824FJ795473
Lentithecium fluviatileGU296158GU301825GU349074
Lepidosphaeria nicotiaeDQ678067DQ677963DQ677910
Leptosphaeria biglobosaGU301826GU349010
Leptosphaeria doliolumGU296159GU301827GU349069
Leptosphaeria dryadisGU301828GU371733GU349009
Leptosphaerulina argentinensisGU301829GU357759GU349008
Leptosphaerulina australisGU296160GU301830GU371790GU349070
Leptosphearia maculansDAOM 229267DQ470993DQ470946DQ471136DQ470894DQ471062
Leptoxyphium fumagoGU296161GU301831GU357771GU371741GU349051
Letendraea helminthicolaAY016345AY016362
Letendraea padoukGU296162AY849951
Lindgomyces breviappendiculataHHUF 28193AB521733AB521748
Lindgomyces ingoldianusATCC_200398AB521719AB521736
Lindgomyces rotundatusHHUF_27999AB521723AB521740
Lophiostoma alpigenumGKM 1091bGU385193
Lophiostoma arundinisDQ782383DQ782384DQ782386DQ782387
Lophiostoma caulium 1GU296163GU301833GU371791
Lophiostoma caulium 2GU301832GU349007
Lophiostoma compressumIFRD 2014GU296164GU301834FJ795457
Lophiostoma crenatumDQ678017DQ678069DQ677965DQ677912
Lophiostoma fuckeliiGKM 1063GU385192
Lophiotrema brunneosporumGU296165GU301835GU349071
Lophiotrema lignicolaGU296166GU301836GU349072
Lophiotrema nuculaGU296167GU301837GU371792GU349073
Lophium elegansEB 0366GU323184GU323210
Lophium mytilinum 1EF596819EF596819
Lophium mytilinum 2DQ678030DQ678081DQ677979DQ677926
Loratospora aestuariiJK 5535BGU296168GU301838GU371760
Macrophomina phaseolinaDQ678037DQ678088DQ677986DQ677929
Macrovalsaria megalospora 1178150FJ215707FJ215701
Macrovalsaria megalospora 2178149FJ215706FJ215700
Massaria anomiaGU296169GU301839GU371769
Massaria plataniDQ678013DQ678065DQ677961DQ677908
Massarina arundinariae 1MAFF 239461AB524455AB524596AB539096AB524817
Massarina arundinariae 2NBRC 106238AB524456AB524597AB539097AB524818
Massarina eburneaGU296170GU301840GU357755GU371732GU349040
Massarina igniariaGU296171GU301841GU371793
Massariosphaeria grandisporaGU296172GU301842GU357747GU371725GU349036
Massariosphaeria phaeosporaGU296173GU301843GU371794
Massariosphaeria typhicola 1GU296174GU301844GU371795
Massariosphaeria typhicola 2KT 797AB521730AB521747
Mauritiana rhizophorae 1BCC 28866GU371832GU371824GU371796GU371817
Mauritiana rhizophorae 2BCC 28867GU371833GU371825GU371797GU371818
Melanomma pulvis-pyrius 1SMH 3291GU385197
Melanomma pulvis-pyrius 2GU301845GU371798GU349019
Melanomma rhododendriANM 73GU385198
Microthyrium microscopicumGU296175GU301846GU371734GU349042
Microxyphium aciculiformeGU296176GU301847GU357762GU371736GU349045
Microxyphium citriGU296177GU301848GU357750GU371727GU349039
Microxyphium theaeGU296178GU301849GU357781GU349060
Monascostroma innumerosumGU296179GU301850GU349033
Monotosporella tuberculataGU301851GU349006
Montagnula opulentaAF164370DQ678086DQ677984
Mycosphaerella endophyticaGU214538DQ246255
Mycosphaerella eurypotamiJK 5586JGU301852GU371722
Mycosphaerella graminicola 1DQ678033DQ678084DQ677982
Mycosphaerella graminicola 2GU214540GU214436
Mycosphaerella heimiiGU214541GQ852604
Mycosphaerella latebrosaDQ848331GU214444
Mycosphaerella marksiiGU214549GQ852612
Mycosphaerella punctiformis (anamorph Ramularia endophylla)DQ471017DQ470968DQ471165DQ470920DQ471092
Myriangium duriaeiAY016347DQ678059DQ677954DQ677900
Myriangium hispanicumGU296180GU301854GU357775GU371744GU349055
Mytilinidion acicolaEB 0349GU323185GU323209GU371757
Mytilinidion andinenseFJ161159FJ161199FJ161125FJ161107
Mytilinidion californicumEB 0385GU323186GU323208
Mytilinidion mytilinellumFJ161144FJ161184GU357810FJ161119FJ161100
Mytilinidion resinicolaFJ161145FJ161185FJ161101FJ161101FJ161120
Mytilinidion rhenanumEB 0341GU323187GU323207
Mytilinidion scolecosporumFJ161146FJ161186GU357811FJ161121FJ161102
Mytilinidion thujarumEB 0268GU323188GU323206
Mytilinidion tortileEB 0377GU323189GU323205
Neofusicoccum ribis (teleomorph Botryosphaeria ribis)DQ678000DQ678053GU357789DQ677947DQ677893
Neophaeosphaeria filamentosaGQ387516GQ387577GU357803GU371773GU349084
Neottiosporina paspaliEU754073EU754172GU357812GU371779GU349079
Oedohysterium insidens 1FJ161142FJ161182FJ161118FJ161097
Oedohysterium insidens 2ANM 1443GU323190GQ221882GU371811GU371785
Oedohysterium sinenseFJ161169FJ161209GU371807FJ161130
Opegrapha dolomiticaDUKE 0047528DQ883706DQ883717DQ883714DQ883732
Ophiosphaerella herpotrichaDQ678010DQ678062DQ677958DQ677905
Ophiosphaerella sasicolaMAFF 239644AB524458AB524599AB539098AB539111
Otthia spiraeae 1EF204515EF204498
Otthia spiraeae 2EF204516EF204499GU357777
Paraconiothyrium minitansEU754074EU754173GU357807GU371776GU349083
Patellaria atrataGU296181GU301855GU357749GU371726GU349038
Patellaria cf. atrata 1BCC 28876GU371836GU371828
Patellaria cf. atrata 2BCC 28877GU371837GU371829
Phacellium paspaliGU214669GQ852627
Phaeocryptopus gaeumannii 1GU357766GU371740
Phaeocryptopus gaeumannii 2EF114722EF114698GU357770
Phaeocryptopus nudusGU296182GU301856GU357745GU349034
Phaeodothis winteriGU296183GU301857DQ677917
Phaeosclera dematioidesGU296184GU301858GU357764GU349047
Phaeosphaeria ammophilaeGU296185GU301859GU357746GU371724GU349035
Phaeosphaeria avenariaDAOM 226215AY544725AY544684DQ677941DQ677885
Phaeosphaeria brevispora 1NBRC 106240AB524460AB524601AB539100AB539113
Phaeosphaeria brevispora 2MAFF 239276AB524459AB524600AB539099AB539112
Phaeosphaeria caricisGU301860GU349005
Phaeosphaeria eustomaDQ678011DQ678063DQ677959DQ677906
Phaeosphaeria juncicolaGU349016
Phaeosphaeria luctuosaGU301861GU349004
Phaeosphaeria nodorumBroadGenomeGenomeGenomeGenomeGenome
Phaeosphaeriopsis musaeGU296186GU301862GU357748GU349037
Phaeotrichum benjaminiiAY016348AY004340GU357788DQ677946DQ677892
Phoma betaeEU754079EU754178GU357804GU371774GU349075
Phoma complanataEU754081EU754180GU357809GU371778GU349078
Phoma exiguaEU754084EU754183GU357813GU371780GU349080
Phoma glomerataEU754085EU754184GU371781GU349081
Phoma herbarumDQ678014DQ678066GU357792DQ677962DQ677909
Phoma heteromorphosporaEU754089EU754188GU371775GU349077
Phoma radicinaEU754092EU754191GU357805GU349076
Phoma zeae-maydisEU754093EU754192GU357814GU371782GU349082
Piedraia hortaeAY016349AY016366DQ677990
Pleomassaria sipariaDQ678027DQ678078DQ677976DQ677923
Pleospora ambiguaAY787937GU357760
Pleospora herbarumDQ247812DQ247804DQ471163DQ247794DQ471090
Polyplosphaeria fuscaMAFF 239685AB524463AB524604
Polythrincium trifolii (as Cymadothea trifolii)133EU167612EU167612
Preussia funiculataGU296187GU301864GU371799GU349032
Preussia lignicola (as Sporormia lignincola)GU296197GU301872GU371765GU349027
Preussia terricolaDAOM 230091AY544726AY544686DQ471137DQ470895DQ471063
Pseudocercospora fijiensis (teleomorph Mycosphaerella fijiensis)OSC 100622DQ767652DQ678098DQ677993
Pseudocercospora griseola f. griseolaCPC 10461GU323191GU348997
Pseudocercospora vitisCPC 11595DQ289864GU214483
Pseudotetraploa curviappendiculataMAFF 239495AB524467AB524608
Psiloglonium araucanumFJ161133FJ161172GU357743FJ161112FJ161089
Psiloglonium clavisporum 1FJ161156FJ161197FJ161123
Psiloglonium clavisporum 2GKM L172AGU323192GU323204
Psiloglonium simulansFJ161139FJ161178FJ161116FJ161094
Pyrenochaeta nobilisDQ678096DQ677991DQ677936
Pyrenophora phaeocomesDAOM 222769DQ499595DQ499596DQ497614DQ497607
Pyrenophora tritici-repentis 1OSC 100066AY544672DQ677882
Pyrenophora tritici-repentis 2GU349017
Quadricrura septentrionalisAB524474AB524615
Quintaria lignatilisGU296188GU301865GU371761
Quintaria submersaGU301866GU357751GU349003
Racodium rupestre 1L423EU048576EU048581
Racodium rupestre 2L424EU048577EU048582
Ramichloridium apiculatumGU296189GU371770
Ramichloridium cerophilumGU296190EU041855
Rasutoria tsugaeratstkEF114730EF114705GU371809
Rhytidhysterium rufulum 2GU296191FJ469672FJ238444GU349031
Rhytidhysteron rufulum 1GKM 361AGU296192GU301867
Rimora mangroveiJK 5246AGU296193GU301868GU371759
rock isolate TRN 111GU323193GU323220GU357783GU371751GU349088
rock isolate TRN 123GU323194GU323219GU357784GU371753
rock isolate TRN 137GU323195GU323218GU357782GU371749
rock isolate TRN 138GU323196GU323217GU371750
rock isolate TRN 152GU323197GU323223GU371752
rock isolate TRN 211GU323198GU323222GU357785GU371754
rock isolate TRN 235GU323199GU357787GU371756GU349087
rock isolate TRN 43GU323200GU323221GU357786GU371755GU349086
Roussoella hysterioides 1MAFF 239636AB524480AB524621AB539101AB539114
Roussoella hysterioides 2AB524481AB524622AB539102AB539115
Roussoella pustulansMAFF 239637AB524482AB524623AB539103AB539116
Roussoellopsis tosaensisMAFF 239638AB524625AB539104AB539117
Saccharata proteaeGU296194GU301869GU357753GU371729GU349030
Saccothecium sepincolaGU296195GU301870GU371745GU349029
Schismatomma decoloransDUKE 0047570AY548809AY548815DQ883715DQ883725
Schizothyrium pomi 1EF134949EF134949
Schizothyrium pomi 2EF134948EF134948
Schizothyrium pomi 3EF134947EF134947
Scorias spongiosaDQ678024DQ678075DQ677973DQ677920
Setomelanomma holmiiGU296196GU301871GU371800GU349028
Setosphaeria monocerasAY016368AY016368
Spencermartinsia viticola (teleomorph Botryosphaeria viticola)DQ678036DQ678087GU357795DQ677985
Sporormiella minimaDQ678003DQ678056DQ677950DQ677897
Stagonospora macropycnidiaGU296198GU301873GU349026
Stylodothis puccinioidesAY004342FJ238427DQ677886
Sydowia polysporaDQ678005DQ678058GU357791DQ677953DQ677899
Teratosphaeria associata (as Teratosphaeria jonkershoekensis)GU296200GU301874GU357744GU371723GU349025
Teratosphaeria cryptica (as Mycosphaerella cryptica)GU214602GQ852682
Teratosphaeria fibrillosa 1GU296199GU323213GU357767
Teratosphaeria fibrillosa 2CPC 1876GU214506
Teratosphaeria stellenboschiana (as Colletogloeopsis stellenboschiana)GU214583EU019295
Teratosphaeria suberosa (as Mycosphaerella suberosa)CPC 11032GU214614GQ852718
Tetraplosphaeria sasicolaMAFF 239677AB524490AB524631
Thyridaria rubronotataGU301875GU371728GU349002
Tremateia halophilaJK 5517JGU296201GU371721
Trematosphaeria pertusaGU348999GU301876GU371801GU349085
Trichodelitschia bisporula 1GU349000GU348996GU371812GU371802GU349020
Trichodelitschia bisporula 2 (duplicate)GU296202
Trichodelitschia munkiiKruys201DQ384070DQ384096
Triplosphaeria maximaMAFF 239682AB524496AB524637
Trypethelium nitidiusculum 1139GU327728GU327732
Trypethelium nitidiusculum 2AFTOL 2099FJ267701
Trypethelium tropicum25GU327730
Tubeufia cereaDQ471034DQ470982DQ471180DQ470934DQ471105
Tubeufia paludosaGU296203GU301877GU357754GU371731GU349024
Tubeufia paludosa (as anamorph Helicosporium phragmitis)DQ767649DQ767654DQ767643DQ767638
Tyrannosorus pinicolaDQ471025DQ470974DQ471171DQ470928DQ471098
Ulospora bilgramiiDQ678025DQ678076DQ677974DQ677921
Venturia inaequalis 1GU296205GU301879GU357757GU349022
Venturia inaequalis 2GU296204GU301878GU357756GU349023
Venturia inaequalis 3 (as Spilocaea pomi)GU348998GU349089
Venturia populinaGU296206GU323212GU357769
Verrucisporota daviesiaeGU296207GQ852730
Verruculina enaliaJK 5253ADQ678028DQ678079DQ677977DQ677924
Westerdykella angulata (as Eremodothis angulata)DQ384067DQ384105GU371805GU371821
Westerdykella cylindricaAY016355AY004343DQ471168DQ470925DQ497610
Westerdykella ornataGU296208GU301880GU371803GU349021
Wettsteinina lacustrisDQ678023DQ677972DQ677919
Wicklowia aquaticaAF289-1GU045446
Wicklowia aquaticaGU266232GU045445GU371813
Zasmidium cellareEF137362EU041878
Zopfia rhizophilaDQ384086DQ384104

1BCC: Belgian Coordinated Collections of Microorganisms; CABI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, U.K.; CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; DAOM: Plant Research Institute, Department of Agriculture (Mycology), Ottawa, Canada; DUKE: Duke University Herbarium, Durham, North Carolina, U.S.A.; HHUF: Herbarium of Hirosaki University, Japan; IFRDCC: Culture Collection, International Fungal Research & Development Centre, Chinese Academy of Forestry, Kunming, China; MAFF: Ministry of Agriculture, Forestry and Fisheries, Japan; NBRC: NITE Biological Resource Centre, Japan; OSC: Oregon State University Herbarium, U.S.A.; UAMH: University of Alberta Microfungus Collection and Herbarium, Edmonton, Alberta, Canada; UME: Herbarium of the University of Umeå, Umeå, Sweden; Culture and specimen abbreviations: ANM: A.N. Miller; CPC; P.W. Crous; EB: E.W.A. Boehm; EG: E.B.G. Jones; GKM: G.K. Mugambi; JK: J. Kohlmeyer; KT: K. Tanaka; SMH: S.M. Huhndorf.

Sequence alignment and phylogenetic analyses

Sequences were obtained from WASABI (Kauff et al. 2007) as well as from previous publications (e.g.Lutzoni et al. 2004, Schoch et al. 2009a). Introns were removed and an initial core set of 171 taxa were aligned by using default options for a simultaneous method of estimating alignments and tree phylogenies, SATé (Liu et al. 2009). In order to consider codons without the insertion of unwanted gaps, protein coding fragments were translated in BioEdit v. 7.0.1 (Hall 2004) and aligned within SATé as amino acids. These were then realigned with their respective DNA sequences using the RevTrans 1.4 Server (Wernersson & Pedersen 2003). After the removal of intron sequences the alignment was examined manually in BioEdit with a shade threshold of 40 % and regions with high amounts of gap characters were excluded. This resulted in a reduction of 99 columns in the LSU data set, 118 in RPB1 and 162 in RPB2, for a total of 379. Nothing was removed for TEF1. In order to allow for the extension of our alignment as newly generated sequences became available from other studies in this volume, these were subsequently added to this core alignment with MAFFT v. 6.713 (Katoh et al. 2009). The E-INS-i setting, focused on high accuracy with a high percentage of unalignable regions such as introns, was applied and the SATé alignment was used as a seed. This resulted in a supermatrix of five genes (LSU, SSU TEF1, RPB1, RPB2) consisting of 52 % gaps and undetermined characters out of a total of 6 582 characters. GenBank accession numbers are shown in Table 1.

Conflict tests

Conflict tests on the initial core set of 204 taxa were conducted by selecting single gene data sets and doing comparisons on a gene by gene basis. This was done using the “bootstopping” criterion in RAxML v. 7.0.4 (Stamatakis et al. 2008) under the CIPRES v. 2.1 webportal to produce trees of comparative gene sets where all taxa have the gene present. Comparisons between all potential sets of gene trees with no missing taxa were done using a script (Kauff & Lutzoni 2002) obtained through the Lutzoni lab website and to detect present or absent taxa within clades with a cut-off bootstrap value of 70 %. This is described in more detail elsewhere (Miadlikowska et al. 2006, Schoch et al. 2009a).

Phylogeny

A phylogenetic analysis was performed using RAxML v. 7.0.4 (Stamatakis 2006) applying unique model parameters for each gene and codon. The dataset was divided in 11 partitions as previously described in Schoch et al. (2009a). A general time reversible model (GTR) was applied with a discrete gamma distribution and four rate classes following procedures laid out in Schoch et al. (2009). Ten thorough maximum likelihood (ML) tree searches were done in RAxML v. 7.0.4 under the same model, each one starting from a randomised tree. Bootstrap pseudo replicates were performed 2000 times using the fast bootstrapping option and the best scoring tree form 10 separate runs were selected. The resulting trees were printed with TreeDyn v. 198.3 (Chevenet et al. 2006). All alignments are deposited in TreeBASE. Additionally, the data sets were analyzed in GARLI v. 0.96 (Zwickl 2006) using the GTR-gamma-invariant model. In this case 200 bootstraps were run under default conditions.

Fig. 2A–C.
Best scoring ML tree with RAxML and GARLI bootstrap values respectively above (green) and below (red) the nodes. Values below 50 % were removed and branches with more than 90 % bootstrap for both methods are thickened without values. Environmental sources relevant to the papers in this volume are indicated in the key (R-Rock; M-Marine; F-Freshwater; D-Dung; B-Bamboo). Nutritional characters are indicated by colour as per the key.

Ancestral reconstruction

Ancestral reconstructions were performed in Mesquite v. 2.6 with character states traced over 2000 bootstrapped trees obtained with RAxML-MPI v. 7.0.4 (Stamatakis 2006). Following the phylogeny presented (Fig. 2) this reconstruction was performed with a maximum-likelihood criterion using the single parameter Mk1 model. Ancestral states were assigned to a node if the raw likelihood was higher by at least 2 log units than the likelihood value of the other ancestral state(s) according to default settings. Character states were also mapped using TreeDyn v. 198.3 (Chevenet et al. 2006), shown in Fig. 3. This is presented as a clockwise circular tree, starting with outgroup taxa. Only clades with more than two taxa of the same state are shown and bootstrap recovery was not considered in assigning character states. In applying the character states of saprobes (including rock heterotrophs), plant associated fungi (including pathogens, endophytes and mycorrhizae) and lichenised fungi the broad concepts presented were followed as laid out in Schoch et al. (2009a). Some character assessments were taken from Zhang et al. (2009; this volume). Ecological characters of sampling sources, terrestrial, fresh water and marine were assessed based on papers elsewhere in this volume (Suetrong et al. 2009, Shearer et al. 2009).

Genome analyses

A MCL (Markov Cluster Algorithm) protein analysis of 52 fungi and one metazoan (Drosophila melanogaster) (Table 2 - see online Supplementary Information) and the phylogenetic placement of these species was used to characterise the phylogenetic profile of each cluster. Chytridiomycota and Mucoromycotina each were represented by one and two species, respectively. In Dikarya, Basidiomycota and Ascomycota were represented by 8 and 40 species respectively. The Pezizomycotina (filamentous ascomycetes) was presented by 26 species in four classes [Sordariomycetes (12), Leotiomycetes (2), Dothideomycetes (6) and Eurotiomycetes (6)].

Table 2.

Genomes used for phylogenetic profile. All are opisthokonts; remaining classifications used in Fig. 4 are indicated in columns: Do – Dothideomycetes, ED - Eurotiomycetes & Dothideomycetes, S – Saccharomyceta, A – Ascomycota, Di — Dikarya, MD - Mucoromycotina & Dikarya, CMD - Chytridiomycota, F - Fungi.

GenomesClassifications
Alternaria brassicicolaDoEDSADiMDCMDF
Cochliobolus heterostrophusDoEDSADiMDCMDF
Mycosphaerella fijiensisDoEDSADiMDCMDF
Mycosphaerella graminicolaDoEDSADiMDCMDF
Pyrenophora tritici-repentisDoEDSADiMDCMDF
Stagonospora nodorumDoEDSADiMDCMDF
Aspergillus fumigatusEDSADiMDCMDF
Aspergillus nidulansEDSADiMDCMDF
Aspergillus terreusEDSADiMDCMDF
Coccidioides immitisEDSADiMDCMDF
Histoplasma capsulatumEDSADiMDCMDF
Uncinocarpus reesiiEDSADiMDCMDF
Ashbya gossypiiSADiMDCMDF
Botrytis cinereaSADiMDCMDF
Candida albicansSADiMDCMDF
Candida glabrataSADiMDCMDF
Candida guilliermondiiSADiMDCMDF
Candida lusitaniaeSADiMDCMDF
Chaetomium globosumSADiMDCMDF
Debaryomyces hanseniiSADiMDCMDF
Fusarium graminearumSADiMDCMDF
Fusarium oxysporumSADiMDCMDF
Fusarium verticillioidesSADiMDCMDF
Kluyveromyces lactisSADiMDCMDF
Laccaria bicolorSADiMDCMDF
Lodderomyces elongisporusSADiMDCMDF
Magnaporthe griseaSADiMDCMDF
Nectria haematococcaSADiMDCMDF
Neurospora crassaSADiMDCMDF
Pichia stipitisSADiMDCMDF
Podospora anserinaSADiMDCMDF
Saccharomyces cerevisiaeSADiMDCMDF
Sclerotinia sclerotiorumSADiMDCMDF
Sporobolomyces roseusSADiMDCMDF
Trichoderma atrovirideSADiMDCMDF
Trichoderma reseeiSADiMDCMDF
Trichoderma virensSADiMDCMDF
Verticillium dahliaeSADiMDCMDF
Yarrowia lipolyticaSADiMDCMDF
Schizosaccharomyces japonicusADiMDCMDF
Schizosaccharomyces octosporusADiMDCMDF
Schizosaccharomyces pombeADiMDCMDF
Coprinus cinereusDiMDCMDF
Cryptococcus neoformansDiMDCMDF
Phanerochaete chrysosporiumDiMDCMDF
Postia placentaDiMDCMDF
Puccinia graminis f. sp. triticiDiMDCMDF
Ustilago maydisDiMDCMDF
Phycomyces blakesleeanusMDCMDF
Rhizopus oryzaeMDCMDF
Batrachochytrium dendrobatidisCMDF
Encephalitozoon cuniculiF
Drosophila melanogaster

RESULTS AND DISCUSSION

Taxon sampling

The phylogram presented in Fig. 2 represents the largest ever phylogenetic assessment of Dothideomycetes to date. Here the focus has been on expanding taxon diversity in the class while specifically avoiding a small number of taxa that other analyses suggest reside on long unstable branches. This still allowed for an extensive sweep of dothideomycete taxon diversity; in doing so we followed the premise of allowing for missing data in our supermatrix (Wiens 2006). An effort was made to intersperse taxa with poor character sampling amongst those having better sampling throughout the tree, but the inclusion of missing characters could still have unanticipated effects on phylogenetic assessments (Lemmon et al. 2009). While recognising this caveat, a recent expansive data set covering all of Ascomycota noted very little changes in major nodes even after the removal of taxa with high proportions of missing characters (Schoch et al. 2009a). The phylogeny presented here agrees well with broad phylogenies in this volume and elsewhere (Schoch et al. 2006, Crous et al. 2007a, Zhang et al. 2008, Crous et al. 2009b). After all introns and 379 ambiguous character positions were removed, the matrix consisted of 52 % missing and indeterminate characters. This maximum-likelihood analysis had 5 069 distinct alignment patterns and produced a best known likely tree with a log likelihood of -207247.761117.

Evolution of nutritional modes

The ancestral reconstructions in Fig. 3 indicate that phytopathogenicity can be confined to a number of terminal clades throughout the tree and that these always reside within saprobic lineages. A maximum of seven transitions likely occurred in several lineages of the orders Pleosporales, Capnodiales and singular lineages in Myriangiales, Botryosphaeriales and Venturiaceae (also see in this volume; Crous et al. 2009a, Zhang et al. 2009). Several transitions to lichenisation have also occurred, although phylogenetic uncertainty may limit this to a minimum of two. Due to the use of lichenised Arthoniomycetes as outgroup a broader assessment is required to determine whether the Dothideomycetes evolved from a lichenised ancestor. Previous studies suggested that the saprobic habit is an ancestral trait but only with marginal support (Schoch et al. 2009a). Similar conclusions can be reached for the aquatic ecological characters – the majority of fresh water and marine clades reside within terrestrial clades as has been shown previously e.g. (Spatafora et al. 1998, Vijaykrishna et al. 2006). Transitions from a terrestrial life style to fresh water likely occurred at least three times and transitions to marine environments up to six times. Phylogenetic uncertainty for the placement of some marine clades can limit this to a minimum of four times (Fig. 2). Reversions from aquatic to terrestrial environments are rare, with one possible exception in the Lentitheciaceae where bambusicolous saprobes reside, nested within several fungi occurring in freshwater habitats (for additional details see Zhang et al. 2009; this volume). Phylogenetic resolution will have to improve to test this further.

An analysis of recently released genomes was compared to consider whether genome composition reinforces phylogenetic support for Dothideomycetes (Fig. 4). Relative to a clustering analysis of proteins from 52 sequenced fungi and Drosophila melanogaster, about 5 515 protein coding genes from Dothideomycetes shared protein clusters with proteins from other dothideomycete fungi only. This comprises roughly 8–11 % of the protein coding genes in each of six sequenced Dothideomycetes. The species profile of each protein cluster was used to assign a phylogenetically informed designation. The profiles most frequently seen were those of the most conserved proteins, namely clusters designated as having a shared Ophistokont phylogenetic profile. Among the more derived nodes of the Dothideomycetes, protein clusters were observed that had a species composition that could reflect the result of selection pressure on more distantly related fungi that share the same niche.

A phylogenomic profile (Fig. 4) of the proteins from six Dothideomycetes from the two largest orders seen in Fig. 1 is presented (Mycosphaerella graminicola, Mycosphaerella fijiensis, Phaeosphaeria nodorum, Alternaria brassicicola, Pyrenophora tritici-repentis, Cochliobolus heterostrophus). The highest percentage of proteins (excluding species specific proteins) were conserved outside kingdom Fungi (Ophistokont node, 23 %), followed by proteins specific for the Dikarya (14 %) and the Pezizomycotina (13 %). This breakdown was also prevalent within other Pezizomycotina classes. Approximately 8 % of the proteins from the six Dothideomycetes were conserved across and within derived nodes in this class. Relative to this analysis 28 % of the proteins were specific to the Dothideomycetes (including species specific proteins). The other class containing loculoascomyetes, Eurotiomycetes, had 19.5 % proteins characterised as class specific. This means the percentage dothideomycete specific proteins were about 8.5 % more. Eurotiomycetes in the analysis were mostly human pathogens, with most having no known sexual state whereas the Dothideomycetes in the analysis were all plant pathogens and mostly with known sexual states. This breakdown of nutritional modes, although not comprehensive for these two classes, is somewhat representative. In Eurotiomycetes human pathogens are more diverse and plant pathogens uncommon, with the converse being true for Dothideomycetes. Both classes contain melanised species with similar morphologies and more comprehensive comparative studies need to expand sampling to incorporate species from the different nutritional modes for both classes.

Fig. 3.

Simplified ancestral state reconstructions, showing potential transitions between character states. The same phylogeny as in Fig. 2A–C is shown, with the outgroups positioned at twelve o' clock and subsequent clades arranged in a clockwise manner. Characters were traced over 2 000 bootstrap trees and those that were recovered in the majority are coloured on the nodes. In the case of equivocal construction no colour was used (white). To simplify the figure, only clades with two or more neighbouring character states are shown.

Fig. 4.
Pie chart showing relative numbers of unique proteins per genome according to taxonomic classification.

Phylogenetic relationships

In the phylogram presented (Fig. 2) the two dothideomycete subclasses previously described based on presence or absence of pseudoparaphyses (Schoch et al. 2006) could be recovered with varying levels of bootstrap representation. Subclass Pleosporomycetidae previously included Pleosporales plus a single species, representing Mytilinidiaceae, namely Lophium mytilinum (Schoch et al. 2006). Taxon sampling for the Mytilinidiaceae was considerably expanded by Boehm et al. (2009b), with the addition of a number of new taxa, leading to the establishment of the Mytilinidiales. Likewise, extensive taxon sampling for the family Hysteriaceae led to a newly redefined Hysteriales also included in this subclass (Boehm et al. 2009a; this volume). It appears that persistent, hysteriaceous carbonaceous ascomata that dehisce via a longitudinal slit (e.g., hysterothecia) have evolved multiple times within Pleosporomycetidae (Mugambi & Huhndorf 2009,Mugambi & Huhndorf 2009). Pleosporomycetidae can be expanded to tentatively include Jahnulales (Fig. 2B) based on strong bootstrap support from RAxML analyses and morphology. Perithecioid ascomata and a hamathecium of wide cellular pseudoparaphyses are characteristic of Jahnulales (Inderbitzin et al. 2001, Pang et al. 2002; Shearer et al. 2009; this volume) and agree with diagnostic features for Pleosporomycetidae. We also recommend that the definition of the subclass be reassessed with more inclusive character sets. Also, Leptosphaerulina species characterised by the absence of pseudoparaphyses reside within the pseudoparaphysate Pleosporales (Fig. 2C; Silva-Hanlin & Hanlin 1999, Kodsueb et al. 2006), indicating that pseudoparaphyses could have been lost multiple times. It should be noted that the maturity of ascomata may play an important role in these assessments. Immature specimens may contain pseudoparaphyses that dehisce when mature and these characteristics need to be evaluated with more complete sampling of the numerous aparaphysate taxa still listed as incertae sedis. The second subclass, Dothideomycetidae, previously circumscribed based on the absence of pseudoparaphyses remains well supported (Fig. 2C).

The results of this study provided continued support for ten orders within class Dothideomycetes, namely Pleosporales, Hysteriales, Mytilinidiales, Patellariales, Botryosphaeriales, Jahnulales, Dothideales, Capnodiales, Myriangiales and Trypetheliales. The latter order was recently proposed (Aptroot et al. 2008) and represents the largest lichen forming clade in Dothideomycetes. Another recently proposed order, Botryosphaeriales includes only the single family, Botryosphaeriaceae. The analysis (Fig. 2B), however, shows strong support for a narrower interpretation of the Botryosphaeriaceae, typified by Botryosphaeria dothidea and related genera, excluding a separate clade of species residing in Guignardia (with Phyllosticta anamorphs). Bagnisiella examinens and Saccharata protea did not reside in either of the above clades, placed on early diverging branches. A more extensive taxon sampling is required to address the diversity in this order, which most likely will validate the separation of additional families. Another currently accepted order, Microthyriales, consisting of species occurring as saprobes or epiphytes on stems and leaves is represented in this study by only a single sample, Microthyrium microscopicum (Fig. 2C). Members of this order are poorly represented in culture and have unusual thyrothecial ascomata that have a scutate covering comprising a thin layer of radiating cells. This structure is generally lacking a basal layer and is quite unlike any morphologies in other orders. This positioning adjacent to the plant parasitic Venturiaceae and coprophilic Phaeotrichaceae, is unexpected but since the single representative of the Microthyriales is on a long branch this is a relationship that will require more intensive taxon sampling.

Additional families that could not be placed in an order are Tubeufiaceae and Gloniaceae (Fig. 2B). Species in Tubeufiaceae have superficial clustered ascomata and characteristic bitunicate asci with relatively long ascospores, often with helicosporous anamorphs (Kodsueb et al. 2008). Members of Tubeufiaceae, which frequently occur in freshwater habitats include anamorph genera, such as Helicoon and Helicodendron, and are ecologically classified as aeroaquatic species. A few teleomorph taxa such as Tubeufia asiana occur on submerged wood (Tsui et al. 2007), and Tubeufia paludosa occur on herbaceous substrates in wet habitats (Webster 1951). The Gloniaceae are saprobic, have dichotomously branched, laterally anastomosed pseudothecia that form radiating pseudo-stellate composites and dehisce by an inconspicuous, longitudinal, but evaginated slit. They reside sister to the saprobic Mytilinidiales but due to conspicuous morphological differences and moderate statistical support they are placed in Pleosporomycetidae incertae sedis (Boehm et al. 2009a, this volume).

Several other well supported clades representing families were evident in this study (Fig. 2). These include several families in Pleosporales, treated elsewhere (Zhang et al. 2009; this volume). Other clades have lower levels of support. For example Leptosphaeriaceae (Fig. 2A) have moderate bootstrap support and it is treated in the very broad sense here. There was also support for several newly described families treated in different papers within this volume. In Pleosporales these include Amniculicolaceae and Lentitheciaceae (Zhang et al. 2009; this volume). The Lindgomycetaceae (Shearer et al. 2009; this volume, Hirayama et al. 2010) encompassing a majority of species isolated from fresh water habitats. Two other novel families, Aigialaceae and Morosphaeriaceae include mainly marine species (Suetrong et al. 2009; this volume). In addition to these, the sampling of a wide diversity of fungi on bamboo yielded the description of Tetraplosphaeriaceae (Tanaka et al. 2009; this volume). Another novel family, Dissoconiaceae, is proposed by Crous et al. 2009 (this volume) for foliicolous commensalists on Eucalyptus leaves, some of which are putative hyper parasites and reside in Capnodiales.

Results of this study suggest that sampling within existing families also requires continued expansion as familial definitions in Dothideomycetes remains problematic. A paper focused on two families, with poor representation in molecular data sets, Melanommataceae and Lophiostomataceae addresses this in more detail (Mugambi & Huhndorf 2009,Mugambi & Huhndorf 2009; this volume). Numerous other clades in our tree remain without familial placement. This includes a diverse group in Capnodiales (Fig. 2C, clade C) a newly described group of hysteriaceous fungi in Pleosporales (Fig. 2A, clade G) and additional marine lineages (clades H, L, Fig. 2A). An interesting clade tentatively circumdescribed by Zhang et al. (2009; this volume) as Massariaceae contains bambusicolous fungi and appears related to the lichenised Arthopyreniaceae (Fig. 2A).

Finally, a clade including Corynespora anamorphs (clade K, Fig. 2A) is placed for the first time, but without clear relationship to any other currently defined families. The genus Corynespora includes anamorphic fungi with tretic, percurrent, and acropetal conidiogenesis. The melanised, pseudoseptate conidia have a pronounced hilum from which the conidial germ tube emerges and are borne apically from solitary, melanised conidiophores. Though nearly 100 species are described based on differences in morphology, considerable phenotypic plasticity within individual isolates complicates species recognition, and molecular analyses that may result in taxonomic clarification have not been done. Corynespora species fill a diversity of roles as saprobes, pathogens, and endophytes on and in woody and herbaceous plants, other fungi, nematodes, and human skin (Dixon et al. 2009). One of the species represented here, C. cassiicola is an important pathogen of rubber. The teleomorphic fungi Pleomassaria swidae (Pleomassariaceae; Tanaka et al. 2005) and Corynesporasca caryotae (Corynesporascaceae; Sivanesan 1996) have unnamed Corynespora species as anamorphs. In this study, species currently placed in Corynespora are not monophyletic and are positioned in at least two families: Massarinaceae and Clade K (Fig. 2A).

Anamorph taxa

The previously mentioned Dissoconiaceae relies on taxonomic descriptions based on anamorph characters. This is a theme that is expected to continue for mitosporic taxa in Dothideomycetes as molecular data accelerates their integration. The artificial nature of the “higher” taxa of anamorphs e.g., deuteromycetes (Kirk et al. 2001) is now well recognised, but the integration of anamorphs into the phylogenetic classification of teleomorphs remains a significant challenge in fungal systematics (Shenoy et al. 2007). The correlation of teleomorphs and anamorphs (Seifert et al. 2000) is not always predictive but it has been applied in some genera within Dothideomycetes, e.g. Botryosphaeria and Mycosphaerella (Crous et al.2006, 2009b). However, numerous examples underscoring anamorph convergence can be found throughout the class e.g. Dictyosporium (Tsui et al. 2006, Kodsueb et al. 2008), Sporidesmium (Shenoy et al. 2006), Cladosporium (Crous et al. 2007b) and Phoma (Fig. 2A; Aveskamp et al. 2009, de Gruyter et al. 2009, Woudenberg et al. 2009) as well as Fusicoccum and Diplodia (Crous et al. 2006, Phillips et al. 2008). The use of large multigene phylogenies will be essential to bring taxonomic order to cryptic anamorph lineages.

Ecological diversity

Besides the unclassified diversity found in anamorphic genera, numerous ecological niches contain diverse lineages of fungi lacking systematically sampled molecular characters. Several examples of this knowledge gap can be found in papers in this volume. In this regard, the rock inhabiting fungi are amongst the least understood. These fungi exist ubiquitously as melanised, slow growing colonies and that usually do not produce generative structures. They subsist on bare rock surfaces and are consequently highly tolerant of the environmental stresses induced by lack of nutrients, water and extremes in radiation and temperature (Palmer et al. 1990, Sterflinger 1998, Ruibal 2004, Gorbushina et al. 2008). Members of this ecological guild are diverse and occur in two classes – Eurotiomycetes and Dothideomycetes. Ruibal et al. 2009 (this volume) present the results of an expanded sampling of rock-inhabiting fungi that include lineages residing within Dothideomycetes and sister class Arthoniomycetes. These rock inhabiting fungi can be placed in Capnodiales, Pleosporales, Dothideales and Myriangiales, as well as some unclassified lineages of Dothideomycetes. Interestingly, some associated lineages were without clear placement within either Arthoniomycetes or Dothideomycetes. The rock isolates included in Fig. 2C illustrate a subsection of genetic diversity seen in these extremophiles, in particular for the Capnodiales, with two rock isolates-rich lineages Teratosphaeriaceae and Clade C (Fig. 2C). A more detailed analysis (Ruibal et al. 2009; this volume) allows for the presentation of hypotheses related to evolution of pathogenicity and lichenisation because these modes of nutrition are often found in close proximity of rock inhabiting fungal lineages.

The lichenised fungi allied with the Dothideomycetes represent another poorly sampled group of fungi. Several lichenised species remain enigmatically placed after they were confirmed as members of Dothideomycetes based on DNA sequence data (Lumbsch et al. 2005, Del Prado et al. 2006). Although the number of species is comparatively small, their placement can play an important link in determining how transitions to and from lichenisation influenced dothideomycete evolution. Trypetheliaceae known for its anastomosing, branched pseudoparaphyses was until very recently still placed within Pyrenulales, an ascohymenial order in Eurotiomycetes, based on bitunicate asci and lense-shaped lumina in the ascospores (Del Prado et al. 2006). Attempts to resolve members of this family remain challenging as they tend to occur on long, rapidly evolving branches in our phylogenetic analyses, which often lead to artifacts. Nelsen et al. 2009 (this volume) demonstrate the occurrence of two additional lichen-forming lineages within Dothideomycetes representing the families Strigulaceae and Monoblastiaceae. The delineation of lichenised family Arthopyreniaceae should continue to be assessed given their placement with a clade containing bambusicolous fungi (Tanaka et al. 2009; this volume) and their non monophyly is also confirmed elsewhere (Nelsen et al. 2009; this volume). The relationship between the lichenised groups and bambusicolous genera Roussoella and Roussoellopsis (Didymosphaeriaceae;Ju et al. 1996, Lumbsch & Huhndorf 2007) is strongly supported, but their affinity is not fully understood due to their considerable morphological differences.

The fungi collected from marine and freshwater habitats contain yet more varied species that have not been assessed well within a molecular based framework. Their diversity is supported by the fact that whole orders (Jahnulales) and several families, already mentioned, almost exclusively consist of species collected from these environments. A recent assessment of marine fungi tallied a number of more than 500 species with more than a fifth of these suggested to reside in Dothideomycetes (Jones et al. 2009). The number for fungi from fresh water habitats is somewhat lower (about 170 taxa).

Despite similarities in their preferred medium for spore dispersal (water) an examination of phylogenetic diversity within Dothideomycetes indicates that these groups of fungi tend to reside in divergent parts of the tree (Figs 2, 3). However, some exceptions may occur: For example, members of Aigialaceae are weakly supported to share ancestry with members of freshwater clade Lindgomycetaceae (Raja et al. 2010). The Jahnulales represents another recently delineated aquatic lineage with an interesting mixture of fresh water and marine taxa. It was delineated based on molecular and morphological data (Inderbitzin et al. 2001, Pang et al. 2002) and now contains four genera and several species (Campbell et al. 2007). Previously, two anamorphic species in the Jahnulales, Xylomyces rhizophorae (described from mangrove wood of Rhizophora) and X. chlamydosporus have been reported from mangroves and thus saline habitats (Kohlmeyer & Volkmann-Kohlmeyer 1998). It has further been documented that X. chlamydosporus is the anamorph of Jahnula aquatica, a freshwater species (Sivichai, pers. comm.).

Marine Dothideomycetes generally exist in association with algae and plants in marine and brackish environments, usually with intertidal or secondary marine plants (e.g., mangroves). The majority of these fungi have been classified in families and genera that comprise mostly terrestrial species (e.g., Pleospora) and no definitive clades of marine Dothideomycetes have been identified. Here we find support for diverse aquatic lineages similar to the situation in Sordariomycetes. Papers by Suetrong et al. 2009 (this volume) and Shearer et al. 2009 (this volume) continue to address this disparity by using multigene phylogenies to describe several lineages within a class wide context. In contrast, many marine members of the Dothideomycetes await interrogation at the DNA sequence level, especially the genera Belizeana, Thalassoascus, Lautospora and Loratospora, all exclusively marine taxa.

The final environmentally defined group sampled in this volume is the bambusicolous fungi. More than 1 100 fungal species have been described or recorded worldwide from bamboo (Hyde et al. 2002). Furthermore, their ecological specialisation as pathogens, saprophytes, and endophytes has been relatively well documented (e.g.Hino 1961). However, relatively few studies based on DNA sequence comparisons have been undertaken for many bambusicolous fungi. Several unique lineages, e.g. the Katumotoa bambusicola-Ophiosphaerella sasicola clade in a freshwater lineage (Lentitheciaceae) and the Roussoella-Roussoellopsis clade close to lichen-forming families could be found (Fig. 2). Particularly, a new family Tetraplosphaeriaceae including five new genera characterised by a Tetraploa anamorph s. l. is introduced as a lineage of fungi with bamboo habitat (Tanaka et al. 2009; this volume). It is clear that much additional diversity within this group of fungi remains to be sampled using DNA sequence data

A number of other niches remain poorly discussed in this volume. Coprophilous fungi occur in three families Delitschiaceae, Phaeotrichaceae, and Sporormiaceae (Figs 2A, C). These families are not closely related and it is clear that the fimicolous life style has arisen more than once in the Dothideomycetes. Also, many species from these groups are not strictly dung-inhabiting, but can be found on other substrates like soil, wood, and plant-debris. Interestingly, some are human pathogens, plant endophytes and lichenicolous fungi. As is true throughout the Ascomycota, a change in substrate is apparently not a substantial evolutionary step in these taxa (Kruys & Wedin 2009).

Additional observations

Several orders e.g. Dothideales, Myriangiales and Microthyriales have not been treated using the extensive systematic sampling that is true for studies treated in this volume. However, individual smaller studies continue to provide interesting and surprising results. One such example is the first described meristematic and endoconidial species residing in Myriangiales (Fig. 2C) reported by Tsuneda et al. (2008). These Endosporium species were isolated from very different substrates such as: poplar twigs and a dead bird. They also have a close relationship to a single lineage of rock inhabiting fungi. The nutritional shifts represented by these closely related species correlate well with scenarios described by Ruibal et al. (2009; this volume) for rock inhabiting fungi. Another melanised meristematic fungus, Sarcinomyces crustaceus, isolated from pine trees appears in a similar position in a phylogeny presented in the aforementioned paper (Ruibal et al. 2009; this volume).

Another unusual species, Catinella olivacea is included in Fig. 2C, but without any clearly resolved position, diverging early to Dothideomycetidae. This species was initially placed in Leotiomycetes, due to their flattened apothecia, found on the underside of moist, well-decayed logs of hardwood. Asci are unitunicate but they appear to form after ascolocular development. As in the previous analysis, it was not possible to identify relationships between this species and any known order, although there are indications of a close relationship with the Dothideomycetidae (Greif et al. 2007).

The placement of the single asexual mycorrhizal lineage representing Cenococcum geophilum in the Dothideomycetes (LoBuglio et al. 1996), allied to members of the saprobic Gloniaceae is intriguing (Fig. 2B; Boehm et al. 2009a; this volume). No resolved placement for this species in Dothideomycetes has been possible in the past. The results of this study were also unexpected because no biological data suggest a connection to the family. Cenococcum is a fungus that is intensively used in environmental studies and this could suggest a very interesting biology for members of the ostensibly saprobic Gloniaceae. Results of this study advocate a more expansive sampling of Cenococcum in order to confirm this intriguing result.

CONCLUSIONS

One of the major obstacles in dothideomycete systematics remains the lack of a clear understanding of what species are members of the class based on morphology alone. Throughout most of the 20th Century, comparative morphological studies have been the only character on which to base phylogenetic relationships. The advent of large DNA-sequence data sets should allow for a substantially improved interpretation of morphological characters for this class of fungi. Studies in this volume and elsewhere have provided a clear understanding that many of the characters classically used in taxonomy and systematics of the group are homoplastic and not helpful for reconstructing phylogenetic relationships. Dothideomycete taxonomy also needs to keep pace with the rapid advances being made in phylogenetics, genomics and related fields. The important principle here is that our classification should communicate diversity accurately and allow dothideomycete biologists from disparate fields to have access to an agreed upon set of taxonomic names to aid communication. In addition, it should allow for a focus on under-sampled groups and clades (i.e. poorly sampled saprobes and others). A major task ahead will be to add asexual genera to present phylogenetic schemes, and integrate these into the existing familial and ordinal classification. As most of these asexual genera are in fact poly- and paraphyletic, their type species will need to be recollected to clarify their phylogenetic position. In addition to this, it appears that even some concepts of teleomorphic taxa will require extensive reconsideration. Finally, we should attempt to incorporate valuable biological information from past workers, such as the three mycologists to which this volume is dedicated, by reliably assessing culture and sequence identity. It is hoped that the papers in this volume will make a meaningful contribution towards these goals.

Acknowledgments

Authors from individual papers in this volume contributed to this work and specific acknowledgements to that regard can be found in individual papers. Work performed for this paper by the first author after 2008 was supported in part by the Intramural Research Program of the NIH, National Library of Medicine. Part of this work was also funded by grants from NSF (DEB-0717476) to J. W. Spatafora (and C.L. Schoch until 2008) and (DEB-0732993) to J.W. Spatafora and B. Robbertse.

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