Multi-locus phylogeny of Pleosporales: a taxonomic, ecological and evolutionary re-evaluation.

Five loci, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, are used for analysing 129 pleosporalean taxa representing 59 genera and 15 families in the current classification of Pleosporales. The suborder Pleosporineae is emended to include four families, viz.Didymellaceae, Leptosphaeriaceae, Phaeosphaeriaceae and Pleosporaceae. In addition, two new families are introduced, i.e. Amniculicolaceae and Lentitheciaceae. Pleomassariaceae is treated as a synonym of Melanommataceae, and new circumscriptions of Lophiostomataceaes. str., Massarinaceae and Lophiotrema are proposed. Familial positions of Entodesmium and Setomelanomma in Phaeosphaeriaceae, Neophaeosphaeria in Leptosphaeriaceae, Leptosphaerulina, Macroventuria and Platychora in Didymellaceae, Pleomassaria in Melanommataceae and Bimuria, Didymocrea, Karstenula and Paraphaeosphaeria in Montagnulaceae are clarified. Both ecological and morphological characters show varying degrees of phylogenetic significance. Pleosporales is most likely derived from a saprobic ancestor with fissitunicate asci containing conspicuous ocular chambers and apical rings. Nutritional shifts in Pleosporales likely occured from saprotrophic to hemibiotrophic or biotrophic.

INTRODUCTION

Pleosporales is the largest order in the class Dothideomycetes, with a reported 23 families, 332 genera and more than 4 700 species (Kirk ), or 19 families and 174 genera in Lumbsch & Huhndorf (2007)*. Members of Pleosporales can be endophytes or epiphytes (Huang , Sánchez Márquez , Tao ), parasitic on green leaves or stems (Wetzel , Solomon ), lichenicolous (Calatayud ), saprobic on dead leaves or stems in terrestrial or aquatic environments (Câmara , Ramesh 2003, Kodsueb , Zhang et al. 2008b, 2009a), or occur on animal dung (Kruys , Kruys & Wedin 2009).

The circumscription of Pleosporales has undergone great changes in the last half century. The name Pleosporales was first proposed in 1955 by Luttrell to accommodate members of Dothideomycetes having perithecioid ascomata with pseudoparaphyses amongst the asci, and seven families, i.e. Botryosphaeriaceae, Didymosphaeriaceae, Herpotrichiellaceae, Lophiostomataceae, Mesnieraceae, Pleosporaceae and Venturiaceae were included. Luttrell (1973) redefined the concept of Pleosporales based on ascomatal morphology, ascal arrangement in locules, presence or absence of hamathecial tissue, shape of papilla or ostioles, ascospore features and type of habitats, and added three more families, i.e. Dimeriaceae, Mycoporaceae and Sporormiaceae. The morphology of the pseudoparaphyses was given much importance at the ordinal level classification when Barr (1983) introduced Melanommatales to accommodate pleosporalean taxa with trabeculate pseudoparaphyses (Sporormia-type centrum development) as compared to cellular pseudoparaphyses (Pleospora-type centrum development) possessed by other members of Pleosporales. Due to the lack of a Latin description in the original publication, Pleosporales was formally established in 1987 (Barr 1987b), and was characterised by perithecioid ascomata, usually with a papillate apex, an ostiole with or without periphyses, cellular pseudoparaphyses, fissitunicate asci, and ascospores with various shapes, pigmentation and septation. Barr's concept included previous families, i.e. Botryosphaeriaceae, Dimeriaceae, Lophiostomataceae, Mesnieraceae, Pleosporaceae, Venturiaceae, plus 15 additional families, i.e. Arthopyreniaceae, Coccoideaceae, Cucurbitariaceae, Dacampiaceae, Hysteriaceae, Leptosphaeriaceae, Micropeltidaceae, Parodiellaceae, Phaeosphaeriaceae, Phaeotrichaceae, Pleomassariaceae, Polystomellaceae, Pyrenophoraceae, Tubeufiaceae and Vizellaceae. Recent phylogenetic analysis based on DNA sequence data however, have indicated that the Pleospora-type and Sporormia-type of centrum development (cellular versus trabeculate pseudoparaphyses) are not natural groupings, as taxa with these centrum types are dispersed in phylogenetic trees (Liew , Lumbsch & Lindemuth 2001). Thus members of Melanommatales were assigned to Pleosporales, and consequently, Melanommatales was treated as a synonym of Pleosporales (Eriksson 2006). Nineteen families have been assigned to Pleosporales in Kirk et al. (2001), 13 in Eriksson (2006), and 19 in Lumbsch & Huhndorf (2007).

One important reason for the unstable circumscriptions in the traditional classification of the Pleosporales is that the value given to the various morpho-characters, even those used at high-level classification, has proven to be overstated. For instance, fruiting-body shapes, i.e. cleistothecioid, perithecioid and apothecioid, previously considered sanctum at class level classification, were found to have undergone convergent evolution (Hawksworth & Lagreca 2007), as can be seen across Ascomycota (Schoch ). Another important distinguishing character, ascus type, has been reported to be phylogenetically misleading in numerous natural groups (Schmitt & Lumbsch 2004, Wedin , Lumbsch ). Indeed, several DNA sequence based phylogenetic reconstructions have shown that ascospore morphology has little phylogenetic significance at familial or generic level classification (Crous , Schmitt & Lumbsch 2004, Kodsueb , Wang , Zhang ). Consequently, an increasing number of taxa designated only by morphological characterisations in Pleosporales have been reported to be polyphyletic, such as the families Pleosporaceae (Kodsueb ), Melanommataceae (Liew , Wang ) and genera Massariosphaeria (Wang ), Melanomma (Wang ), Massarina and Lophiostoma (Liew , Zhang ).

Various anamorph genera have been recorded in Pleosporales and include both hyphomycetes and coelomycetes. Anamorph genera are often associated with multiple teleomorph genera, and in many cases anamorph relationships described in older literature have not yet been tested with DNA sequence data (Farr , de Gruyter ). In the few cases where this was done, anamorph genera such as Ampelomyces, Ascochyta, Coniothyrium and Phoma proved to be polyphyletic and associated with multiple teleomorphic genera (Aveskamp , de Gruyter ).

Besides the morphological characters used in traditional taxonomy, several other biological characters have been used to define families. For instance, metabolite production and substrate staining reactions have been shown to be phylogenetically informative in xylariaceous and pleosporalean taxa (Stadler et al. 2001, 2004, 2007, Stadler & Fournier 2006, Bitzer , Zhang ). Host spectrum has been used to distinguish between Phaeosphaeria and Leptosphaeria (Holm 1957, Shoemaker & Babcock 1989), and anamorphic stages have been used to distinguish Pleospora and Lewia (Simmons 1986, 2007).

Since the first attempts at a classification of the order Pleosporales it has been a challenge to address the enormous diversity in biology, morphology and ecology within a stable classification. Thus, in molecular studies comprehensive taxon sampling is essential in order to avoid biased conclusions. To counteract this, a large number of taxa from various families and habitats, in particular generic types were included in the present phylogenetic analysis. The aims of the present investigation are: 1) to build up an overall molecular phylogenetic framework based on a multi-gene analysis showing the interfamilial relationships in the Pleosporales; 2) to re-evaluate the significance of morphological or ecological characters used in phylogeny and taxonomy of the order; and 3) to redefine hypotheses for evolutionary trends in the Pleosporales.

MATERIALS AND METHODS

Collection and examination of specimens

Twenty-eight fresh specimens were collected in Europe (the majority from France) during 2004 to 2008 by J. Fournier, and returned to the laboratory for examination. In most cases ascomata were collected directly on natural wood without incubation. The samples were processed and examined following the method described in Tsui et al. (2000). Colonies were sub-cultured onto 2 % potato-dextrose agar (PDA), synthetic nutrient-poor agar (SNA), 2 % malt extract agar (MEA), and oatmeal agar (OA) (Crous ), and incubated under continuous near-UV light at 25 °C to promote sporulation. Observations and photographs were prepared from material mounted in water, congo red, cotton blue, chlorazol black, aqueous nigrosin, lactic acid or Indian ink. Additional cultures used in this study were obtained from the Centraalbureau voor Schimmelcultures (CBS) in Utrecht, the Netherlands. Nomenclatural novelties and descriptions were deposited in MycoBank (Crous ).

Fungal isolates and DNA extraction

Total genomic DNA was extracted from mycelia following the protocols as outlined by Cai et al. (2006) and Shenoy et al. (2007). A second set of DNA samples were obtained following DNA extraction protocols outlined in Schoch et al. (2007). In cases where no cultures could be obtained, a Forensic Kit (UltraClean™ Forensic Kit, Cambio) was used to extract DNA from specimens directly.

DNA amplification and sequencing

DNA amplification was performed by PCR. For partial large subunit (28S, LSU) nuclear rDNA amplification (nu-rDNA), LROR and LR5 primers (Vilgalys & Hester 1990) were used. Primer pairs NS1 and NS4 were used to amplify a region from the small subunit (18S, SSU) of the nu-rDNA (White ). The fRPB2-5F and fRPB2-7cR primers were used for the amplification of the partial RNA polymerase second largest subunit (RPB2) (Liu ). The EF1-Fα and EF1-Rα primers were used to amplify a region from the translation elongation factor 1-alpha gene (TEF1) (Schoch ) and the RPB1-Ac and RPB1-Cr primers were used for RPB1 region (Schoch et al. 2009; this volume). The amplification reaction for partial LSU, SSU and TEF1 nu-rDNA genes was performed in a 50 μL reaction volume as outlined by Jeewon et al. (2004) and Shenoy et al. (2007): 1 × PCR buffer, 0.2 mM dNTPs, 0.3 μM of each primer; 1.5 mM MgCl2, 0.8 units Taq polymerase and 5–10 ng gDNA. The PCR thermal cycle programme for partial LSU nu-rDNA amplication was as follows: 95 °C for 3 min, followed by 34 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C for 30 s and elongation at 72 °C for 1 min, with a final extension step of 72 °C for 10 min (Vilgalys & Hester 1990). The PCR thermal cycle programme for the partial RPB2 gene amplification consisted of 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 2 min and elongation at 72 °C for 90 s, with a final extension step of 72 °C for 10 min (Liu ). The PCR products, spanning approximately 700 bp (TEF1), 900 bp (partial LSU) and 1200 bp (partial SSU and RPB2), were checked on 1 % agarose electrophoresis gels stained with ethidium bromide. The PCR products were then purified using minicolumns, purification resin and buffer according to the manufacturer's protocols (GFX PCR DNA and Gel Band Purification Kit, Amersham Biosciences, Buckinghamshire, U.K.). DNA sequencing was performed using the above-mentioned primers in an Applied Biosystem 3730 DNA analyser at the Genome Research Centre, the University of Hong Kong.

Sequence alignment and phylogenetic analyses

Sequences were obtained from WASABI (Kauff ) as well as from previous publications (e.g. Lutzoni , Schoch ). Taxa was aligned by using default options for a simultaneous method of estimating alignments and tree phylogenies, SATé (Liu ). Protein coding fragments were translated in BioEdit v. 7.0.1 (Hall 2004) and aligned within SATé as amino acids. These were aligned with their respective DNA sequences using the RevTrans 1.4 Server (Wernersson & Pedersen 2003). Subsequently, newly generated sequences were added to this initial alignment with MAFFT v. 6.713 (Katoh ).

A supermatrix of five genes (LSU, SSU, TEF1, RPB1, RPB2) consisting of 47 % gaps and undetermined characters across 171 taxa was obtained. Most taxa had at least two genes present – except for a set of nine taxa with closely related species needed to confirm their identity (Table 1 - see online Supplementary Information).

Table 1.

Isolates used in this study and their GenBank accession numbers. Name changes from their originals are indicated in brackets and newly generated sequences are indicated in bold.

Classification	Species name	Culture/voucher1	SSU	LSU	RPB1	RPB2	TEF1
Aigialaceae	Aigialus grandis	JK 5244A	GU296131	GU301793		GU371762
	Astrosphaeriella aggregata	MAFF 239486	AB524450	AB524591	AF242264	AB539092	AB539105
	Rimora mangrovei (as Lophiostoma mangrovei)	JK 5246A	GU296193	GU301868		GU371759
Amniculicolaceae	Amniculicola immersa	CBS 123083	GU456295	FJ795498		GU456358	GU456273
	Amniculicola lignicola	CBS 123094	EF493861	EF493863		EF493862	GU456278
	Amniculicola parva	CBS 123092	GU296134	FJ795497			GU349065
	Neomassariosphaeria grandispora	CBS 613.86	GU296172	GU301842	GU357747	GU371725	GU349036
	Neomassariosphaeria typhicola	CBS 123126	GU296174	FJ795504		GU371795
	Murispora rubicunda	IFRD 2017	GU456308	FJ795507			GU456289
Delitschiaceae	Delitschia didyma 1	UME 31411		DQ384090
	Delitschia didyma 2 (duplicate)	UME 31411	AF242264	DQ384090		DQ677975	DQ677922
	Delitschia winteri	CBS 225.62	DQ678026	DQ678077		DQ677975	DQ677922
Didymellaceae	Ascochyta pisi	CBS 126.54	DQ678018	DQ678070		DQ677967	DQ677913
	Didymella exigua	CBS 183.55	GU296147		GU357800	GU371764
	Didymella bryoniae	CBS 133.96		GU456335		GU371767
	Leptosphaerulina argentinensis	CBS 569.94		AY849947	GU357759		GU349008
	Leptosphaerulina australis 1	CBS 311.51-T		FJ795500		GU456357	GU456272
	Leptosphaerulina australis 2	CBS 317.83	GU296160	GU301830		GU371790	GU349070
	Macroventuria anomochaeta	CBS 525.71	AY787936	GU456315		GU456346	GU456262
	Monascostroma innumerosum	CBS 345.50	GU296179	GU301850			GU349033
	Phoma complanata	CBS 268.92	EU754081	EU754180		GU371778	GU349078
	Phoma exigua	CBS 431.74	EU754084	EU754183		GU371780	GU349080
	Phoma glomerata	CBS 528.66	EU754085	EU754184		GU371781	GU349081
	Phoma herbarum	CBS 276.37	DQ678014	DQ678066	GU357792	DQ677962	DQ677909
	Phoma zeae-maydis	CBS 588.69	EU754093	EU754192		GU371782	GU349082
	Platychora ulmi	CBS 361.52	EF114726	EF114702
Lentitheciaceae	Katumotoa bambusicola	JCM 13131, MAFF 239641	AB524454	AB524595		AB539095	AB539108
	Keissleriella cladophila	CBS 104.55	GU296155	GU301822		GU371735	GU349043
	Lentithecium aquaticum	CBS 123099	FJ795477	FJ795434		FJ795455	GU349068
	Lentithecium arundinaceum 1	CBS 123131	GU456298	GU456320			GU456281
	Lentithecium arundinaceum 2	CBS 619.86	DQ813513	DQ813509		FJ795473
	Lentithecium fluviatile(as Massarina fluviatile)	CBS 122367	FJ795493	FJ795451			GU456290
	Ophiosphaerella sasicola	JCM 13134, MAFF 239644	AB524458	AB524599		AB539098	AB539111
	Stagonospora macropycnidia	OSC 100965	GU296198	GU301873			GU349026
	Wettsteinina lacustris	CBS 618.86	DQ678023			DQ677972	DQ677919
Leptosphaeriaceae	Coniothyrium palmarum	CBS 400.71	DQ678008	DQ767653		DQ677956	DQ677903
	Leptosphaeria biglobosa	CBS 303.51		GU301826			GU349010
	Leptosphaeria doliolum	CBS 505.75	GU296159	FJ795499			GU349069
	Leptosphaeria dryadis	CBS 643.86		GU301828		GU371733	GU349009
	Leptosphaeria maculans	DAOM 229267	DQ470993	DQ470946	DQ471136	DQ470894	DQ471062
	Neophaeosphaeria filamentosa	CBS 102202	GQ387516	GQ387577		GU371773	GU349084
	Phoma heteromorphospora	CBS 115.96	EU754089	EU754188		GU371775	GU349077
	Pyrenochaeta nobilis	CBS 407.76		DQ678096		DQ677991	DQ677936
Lophiostomataceae s. str.	Lophiostoma arundinis	CBS 621.86	DQ782383	DQ782384		DQ782386	DQ782387
	Lophiostoma caulium 1	CBS 623.86	FJ795479	FJ795436		FJ795456
	Lophiostoma caulium 2	CBS 624.86		GU301832			GU349007
	Lophiostoma compressum 1	IFRD 2014	FJ795480	FJ795437		FJ795457
	Lophiostoma compressum 2	IFRDCC2081	FJ795486	GU456321		GU456349	GU456264
	Lophiostoma crenatum	CBS 629.86	DQ678017	DQ678069		DQ677965	DQ677912
	Lophiostoma fuckelii	CBS 101952	FJ795496	DQ399531		FJ795472
	Lophiostoma macrostomoides	CBS 123097	FJ795482	FJ795439		FJ795458	GU456277
	Lophiostoma semiliberum	CBS 626.86	FJ795484	FJ795441		FJ795460
	Lophiostoma viridarium	IFRDCC2090	FJ795486	FJ795443		FJ795468
Massariaceae	Arthopyrenia salicis 1	CBS 368.94	AY538333	AY538339	GU371814
	Arthopyrenia salicis 2	1994Coppins		AY607730	AY607742
	Massaria inquinans	CBS 122369	GU456300	GU456322			GU456282
	Pleosporales sp. 1 (as Thelenella luridella)	CBS 101277	GU456309			GU456361
	Roussoella hysterioides 1	JCM 13126, MAFF 239636	AB524480	AB524621		AB539101	AB539114
	Roussoella hysterioides 2	CBS 125434	AB524481	AB524622		AB539102	AB539115
	Roussoella pustulans	JCM 13127, MAFF 239637	AB524482	AB524623		AB539103	AB539116
	Roussoellopsis tosaensis	NBRC 106245		AB524625		AB539104	AB539117
	Torula herbarum	CBS 379 58				GU456362
Massarinaceae	Byssothecium circinans	CBS 675.92	AY016339	AY016357		DQ767646
	Massarina cisti	CBS 266.62	FJ795490	FJ795447		FJ795464
	Massarina eburnea	CBS 473.64	AF164367	FJ795449	GU357755	FJ795466	GU349040
	Massarina igniaria	CBS 845.96	FJ795494	FJ795452		FJ795469
	Neottiosporina paspali	CBS 331.37	EU754073	EU754172		GU371779	GU349079
Melanommataceae	Beverwykella pulmonaria	CBS 283.53		GU301804		GU371768
	Herpotrichia diffusa	CBS 250.62	DQ678019	DQ678071		DQ677968	DQ677915
	Herpotrichia juniperi	CBS 200.31	DQ678029	DQ678080		DQ677978	DQ677925
	Melanomma pulvis-pyrius 1	CBS 109.77	FJ201987	FJ201986		GU456359	GU456274
	Melanomma pulvis-pyrius 2	CBS 124080	GU456302	GU456323		GU456350	GU456265
	Monotosporella tuberculata	CBS 256.84		GU301851			GU349006
	Pleomassaria siparia	CBS 279.74	DQ678027	DQ678078		DQ677976	AY544726
Sporormiaceae	Preussia funiculata	CBS 659.74	GU296187	GU301864		GU371799	GU349032
	Preussia lignicola (as Sporormia lignicola)	CBS 264.69	GU296197	GU301872		GU371765	GU349027
	Preussia terricola	DAOM 230091	AY544726	AY544686	DQ471137	DQ470895	DQ471063
	Sporormiella minima	CBS 524.50	DQ678003	DQ678056		DQ677950	DQ677897
	Westerdykella cylindrica	CBS 454.72	AY016355	AY004343	DQ471168	DQ470925	DQ497610
	Westerdykella ornata	CBS 379.55	GU296208	GU301880		GU371803	GU349021
Montagnulaceae	Bimuria novae-zelandiae	CBS 107.79	AY016338	AY016356	DQ471159	DQ470917	DQ471087
	Didymocrea sadasivanii	CBS 438.65	DQ384066	DQ384103
	Kalmusia scabrispora 1	NBRC 106237	AB524453	AB524594		AB539094	AB539107
	Kalmusia scabrispora 2	JCM 12851, MAFF 239517	AB524452	AB524593		AB539093	AB539106
	Karstenula rhodostoma	CBS 690.94	GU296154	GU301821		GU371788	GU349067
	Letendraea helminthicola	CBS 884.85	AY016345	AY016362
	Letendraea padouk	CBS 485.70	GU296162	AY849951
	Montagnula opulenta	CBS 168.34	AF164370	DQ678086		DQ677984
	Paraconiothyrium minitans	CBS 122788	EU754074	EU754173		GU371776	GU349083
	Paraphaeosphaeria michotii 1	CBS 652.86	GU456304	GU456325		GU456351	GU456266
	Paraphaeosphaeria michotii 2	CBS 591.73	GU456305	GU456326		GU456352	GU456267
	Phaeosphaeria brevispora 1	NBRC 106240	AB524459	AB524600		AB539100	AB539113
	Phaeosphaeria brevispora 2	MAFF 239276	AB524460	AB524601		AB539099	AB539112
Phaeosphaeriaceae	Chaetosphaeronema hispidulum	CBS 216.75	EU754045	EU754144		GU371777
	Entodesmium rude	CBS 650.86		GU301812			GU349012
	Leptosphaeria derasa	CBS 184.57	GU456299			GU456360	GU456275
	Ophiosphaerella herpotricha 1	CBS 620.86	DQ678010	DQ678062		DQ677958	DQ677905
	Ophiosphaerella herpotricha 2	CBS 240.31	DQ767650	DQ767656		DQ767645	DQ767639
	Phaeosphaeria ammophilae	CBS 114595	GU296185	GU301859	GU357746	GU371724	GU349035
	Phaeosphaeria avenaria	CBS 602.86	AY544725	AY544684		DQ677941	DQ677885
	Phaeosphaeria caricis	CBS 120249		GU301860			GU349005
	Phaeosphaeria elongata	CBS 120250	GU456306	GU456327	GU456340	GU456345	GU456261
	Phaeosphaeria eustoma	CBS 573.86	DQ678011	DQ678063		DQ677959	DQ677906
	Phaeosphaeria juncicola	CBS 595.86					GU456291
	Phaeosphaeria juncophila	CBS 575.86	GU456307	GU456328			GU456283
	Phaeosphaeria luctuosa	CBS 308.79		GU301861			GU349004
	Phaeosphaeria nigrans	CBS 576.86		GU456331		GU456356	GU456271
	Phaeosphaeria nodorum 1	CBS 259.49		GU456332			GU456285
	Phaeosphaeria nodorum 2	Genome (Broad)	Genome	Genome	Genome	Genome	Genome
	Phaeosphaeria spartinae (as Leptosphaeria albopunctata)	CBS 254.64	AF439506	GU456314	GU456337		GU456279
	Phaeosphaeria spartinicola	CBS 176.91		GU456333			GU456286
	Phaeosphaeria typharum	CBS 296.54		GU456334			GU456287
	Phoma radicina	CBS 111.79	EU754092	EU754191			GU349076
	Setomelanomma holmii	CBS 110217	GU296196	GU301871		GU371800	GU349028
Pleosporaceae	Allewia eureka	DAOM 195275	DQ677994	DQ678044		DQ677938	DQ677883
	Alternaria alternata	CBS 916.96	DQ678031	DQ678082		DQ677980	DQ677927
	Alternaria maritima	CBS 126.60	GU456294	GU456317		GU456347
	Cochliobolus heterostrophus	CBS 134.39	AY544727	AY544645		DQ247790	DQ497603
	Cochliobolus sativus	DAOM 226212	DQ677995	DQ678045		DQ677939
	Phoma betae	CBS 109410	EU754079	EU754178		GU371774	GU349075
	Pleospora herbarum	CBS 714.68	DQ767648	DQ678049	DQ471163	DQ677943	DQ677888
	Pyrenophora phaeocomes	DAOM 222769	DQ499595	DQ499596		DQ497614	DQ497607
	Pyrenophora tritici-repentis 1 (as Pyrenophora trichostoma)	OSC 100066		AY544672			DQ677882
	Pyrenophora tritici-repentis 2 (as Pyrenophora trichostoma)	CBS 392.54					GU349017
	Pyrenophora tritici-repentis 3	CBS 328.53					GU456292
	Scolecobasidium arenarium (as Dendryphiella arenaria)	CBS 181.58	DQ471022	DQ470971	GU349071	DQ470924	DQ677890
	Setosphaeria monoceras	CBS 154.26	AY016352	AY016368
Trematosphaeriaceae	Asteromassaria pulchra	CBS 124082	GU296137	GU301800		GU371772	GU349066
	Halomassarina thalassiae (as Massarina thalassiae)	JK 5262D		GU301816			GU349011
	Trematosphaeria pertusa 1	CBS 122368	FJ201991	FJ201990		FJ795476	GU456276
	Trematosphaeria pertusa 2	CBS 122371	FJ201992	FJ201993		GU371801	GU349085
Pleosporales Incertae sedis	Aglaospora profusa 1	CBS 123109	GU296130	GU301792			GU349062
	Aglaospora profusa 2	CBS 123129	GU456293	GU456316			GU456280
	Byssolophis sphaerioides	IFRDCC2053	GU296140	GU301805		GU456348	GU456263
	Lepidosphaeria nicotiae	CBS 101341		DQ678067		DQ677963	DQ677910
	Lophiotrema brunneosporum	CBS 123095	FJ795487	FJ795444			GU349071
	Lophiotrema lignicola	CBS 123094	FJ795488	FJ795445		FJ795462	GU349072
	Lophiotrema nucula	CBS 627.86	FJ795489	FJ795446		FJ795463	GU349073
	Massaria anomia	CBS 591.78	GU296169	GU301839		GU371769
	Massarina arundinariae 1	NBRC 106238	AB524455	AB524596		AB539097	AB539110
	Massarina arundinariae 2	MAFF 239461	AB524456	AB524597		AB539096	AB539109
	Massarina rubi	CBS 691.95	GU456301	FJ795453		FJ795470
	Massariosphaeria phaeospora	CBS 611.86	GU296173	GU301843		GU371794
	Munkovalsaria rubra	CBS 109505	GU456303	GU456324	GU456339	GU456344	GU456260
	Thyridaria rubronotata	CBS 419.85		GU301875		GU371728	GU349002
	Ulospora bilgramii	CBS 110021	DQ678025	DQ678076		DQ677974	DQ677921
	Valsaria insitiva	CBS 123098	GU456310	GU460204			GU456284
	Valsaria insitiva	CBS 123125	GU456311	GU460205		GU456353	GU456268
	Verruculina enalia	JK 5235A	DQ678028	DQ678079		DQ677977	DQ677924
	Xenolophium applanatum	CBS 123123	GU456312	GU456329		GU456354	GU456269
	Xenolophium applanatum	CBS 123127	GU456313	GU456330		GU456355	GU456270
Botryosphaeriales (outgroup)	Botryosphaeria dothidea	CBS 115476	DQ677998	DQ678051	GU357802	DQ677944	DQ767637
	Botryosphaeria tsugae	CBS 171.55	DQ678009	DQ678061		DQ677957	DQ677904
	Guignardia gaultheriae	CBS 447.70		DQ678089	GU357796	DQ677987
	Guignardia bidwellii	CBS 237.48	DQ678034	DQ678085	GU357794	DQ677983
Dothideales (outgroup)	Dothidea hippophaës	CBS 188.58	U42475	DQ678048	GU357801	DQ677942	DQ677887
	Phaeosclera dematioides	CBS 157.81	GU296184	GU301858	GU357764		GU349047
	Dothidea sambuci	DAOM 231303	AY544722	AY544681		DQ522854	DQ497606
Hysteriales (outgroup)	Psiloglonium clavisporum	CBS 123339	FJ161157	FJ167526		FJ161124	FJ161105
	Hysteriales sp. 1	CBS 243.34	GU456297	GU456319	GU456338	GU456343	GU456259
	Hysterium angustatum	CBS 236.34	GU397359	FJ161180	GU456341	FJ161117	FJ161096
Jahnulales (outgroup)	Jahnula seychellensis	SS2113.1	EF175644	EF175665
	Jahnula aquatica	R68-1	EF175633	EF175655
	Aliquandostipite khaoyaiensis	CBS 118232	AF201453	GU301796		FJ238360	GU349048
Mytilinidiales (outgroup)	Mytilinidion andinense	CBS 123562	FJ161159	FJ161199		FJ161125	FJ161107
	Lophium mytilinum	CBS 269.34	DQ678030	DQ678081	GU456342	DQ677979	DQ677926
Venturiaceae (outgroup)	Venturia pyrina	ATCC 38995		EF114714
	Venturia inaequalis	CBS 476.61		GU456336			GU456288
	Metacoleroa dickei	medipc		EF114695
Arthoniomycetes (outgroup)	Opegrapha dolomitica	DUKE 0047528	DQ883706		DQ883717	DQ883714	DQ883732
	Opegrapha varia	DUKE 0047526			FJ772242	FJ772243	FJ772244

Public culture collections and herbaria ATCC: American Type Culture Collection, Virginia, U.S.A.; 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.; IFRD: International Fungal Research & Development Centre, Chinese Academy of Forestry, Kunmin, People's Republic of China; JCM: Japan Collection of Microorganism, RIKEN BioResource Center, Japan; MAFF: Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan; OSC: Oregon State University Herbarium, Corvallis, Oregon, U.S.A.; NBRC: National Institute of Technology and Evaluation, Chiba, Japan; UME: Umeå University Herbarium, Umeå, Sweden.

Conflict tests

Conflict tests were conducted by selecting single gene data sets and doing comparisons on a gene-by-gene basis applying the bootstrapping criterion in RAxML v. 7.0.4 (Stamatakis ), using the CIPRES 2.1 webportal (Miller ) to produce trees of comparative gene sets where all taxa have the gene present. Comparisons between two sets of gene trees were done using a script (compat.py; Kauff & Lutzoni 2002) obtained through the Lutzoni lab website (www.lutzonilab.net/downloads/index.shtml) to detect taxa within clades with a cut-off value of 70 %. This is also performed as in Schoch et al. (2009).

A phylogenetic analysis was performed using RAxML v. 7.2.2 (Stamatakis 2006) applying unique model parameters for each gene and codon. The data set was thus partitioned in 11 partitions as previously done in Schoch et al. (2009b). In addition a general time reversible model (GTR) was applied with a discrete gamma distribution and four rate classes. One hundred successive most likely tree searches were done in RAxML under the same model, each one starting from a randomised tree with joint branch length optimisation and a rapid hill climbing option. Bootstrap pseudoreplicates were performed 145 times using the fast bootstrapping option and a frequency-based bootstrapping criterion (Stamatakis ). These were plotted above the nodes in the most likely tree obtained earlier. The values below the nodes are percentages of 500 jacknife resamplings performed in TNT for MS windows with a new technology search set to 20 (Goloboff ).

RESULTS AND DISCUSSION

DNA phylogeny

The tree presented in Fig. 1 represents the most complete phylogeny of Pleosporales produced to date. In addition it contains the members of other potential orders in Pleosporomycetidae and Dothideomycetes for outgroup comparisons. The tree was rooted with two Arthoniomycetes as outgroups, Opegrapha varia and O. dolomitica (not shown). The supermatrix analysed in this study produced 4 290 distinct alignment patterns distributed as follows across the various partitions: SSU – 563, LSU – 807, RPB1 codon1 – 232, RPB1 codon2 – 198, RPB1 codon3 – 333, RPB2 codon1 – 467, RPB2 codon2 – 404, RPB2 codon3 – 614, TEF1 codon1 – 185, TEF1 codon2 – 176 and TEF1 codon3 – 311. The highest scoring likely tree had a log likelihood of -107754.307532.

Fig 1.

RAxML tree with bootstrap values after 1000 pseudo repetitions on the nodes. The values below the nodes are percentages of 500 jacknife resamplings. Pleosporalean leaves highlighted in red and bold are marine or maritime taxa, in blue and bold are freshwater taxa, and others are terrestrial ones. Relevant biological or morphological characters plotted on the leaves are abbreviated as follows: Biology: Mono – monocotyledons; Dico – dicotyledons; Gy – Gymnosperm; SF – Stream foam;? – unknown; X – no information. Morphology: SZ – size, OS – ostiole, SP – shape, ST – septum, CL – colour; Ascoma size: S – small (diam < 300 μm), M–medial (300 μm < diam < 600 μm), L – large (diam > 600 μm); ostiole: P – pore-like ostiole, Sl – slite-like ostiole, Nil – no opening. Ascospore shape: Fi – filiform, Fu – fusiform, NF – narrowly fusiform, BF – broadly fusiform, Cy – cylindrical; ascospore septum: 1 – one transverse septum, 2 – two transverse septa, 3 – three transverse septa, >3 – more than three transverse septa, M– muriform, Ap – apiosporous; ascospore colour: H – hyaline, B – brown, PB – pale brown, RB – reddish brown, DB – dark brown, Y – yellow, PY – pale yellow.? – characters unknown. - – anamorph strain.

Families of Pleosporales

In total, 151 taxa (171 strains) of Ascomycota (including the outgroups Opegrapha dolomitica and O. varia) were included in the analysis. It comprises 149 taxa (169 strains) of Dothideomycetes, of which 129 taxa (148 strains) were Pleosporales. The Pleosporales formed a well-supported clade (Fig. 1). The pleosporalean taxa comprised of representatives from 59 pleosporalean genera out of about 200 known genera (ca. 30 %), with 39 generic types of Pleosporales included in the analysis. As shown in Fig. 1, Pleosporales can be subdivided into 17 clades with more than 70 % ML bootstrap (MLB) or 65 % Jacknife (JK); 15 representing familial ranks, i.e. Aigialaceae, Delitschiaceae, Didymellaceae, Leptosphaeriaceae, Lophiostomataceae s. str., Massarinaceae, Melanommataceae, Montagnulaceae, Phaeosphaeriaceae, Pleosporaceae, Sporormiaceae, Trematosphaeriaceae and Massariaceae (Lumbsch & Huhndorf 2007, Kirk ), as well as Amniculicolaceae and Lentitheciaceae, which are newly introduced in this paper. Based on the multi-gene phylogenetic data generated here, a new circumscription of Pleosporales is given as follows:

Luttr. ex M.E. Barr, Prodromus to class Loculoascomycetes: 67. 1987. emend.

Hemibiotrophic, saprobic, hypersaprobic, or lichenised. Habitats in freshwater, marine or terrestrial environment. Ascomata perithecioid, rarely cleistothecioid, immersed, erumpent to superficial, globose to subglobose, or lenticular to irregular, with or without conspicuous papilla or ostioles. Ostioles with or without periphyses. Peridium usually composed of a few layers of cells with various shapes and structures. Hamathecium persistent, filamentous, very rarely decomposing. Asci bitunicate, fissitunicate, cylindrical, clavate to obclavate, with or without pedicel. Ascospores hyaline or pigmented, ellipsoidal, broadly to narrowly fusoid or filiform, mostly septate.

Anamorphs: Acroconidiellina, Alternaria, Aposphaeria, Ascochyta, Ascochytella, Bipolaris, Ceratophoma, Coniothyrium, Corynespora, Curvularia, Cytoplea, Drechslera, Exserohilum, Hendersonia, Leptophoma, Metabotryon, Microsphaeropsis, Myxocyclus, Nigrolentilocus, Nimbya, Phoma, Pithomyces, Pleurophomopsis, Prosthemium, Pseudospiropes, Pyrenochaeta, Scolecosporiella, Scolicosporium, Shearia, Sphaerellopsis, Stagonospora, Steganosporium, Stemphylium and Tiarospora (www.cbs.knaw.nl/databases/anateleo.htm 04-2009, www.indexfungorum.org/ 12-2009, www.mycobank.org/DefaultPage.aspx 12-2009). It should be noted that these anamorphs are based on literature data, and the anamorph-teleomorph relations based on in vitro studies or molecular data are provided in the following families.

Pleosporineae

Pleosporales contains many notorious plant pathogens, most belonging to one of four families, viz. Didymellaceae, Leptosphaeriaceae, Phaeosphaeriaceae and Pleosporaceae. These four families cluster together with high support (MLB = 99 %, JK = 92 %) (Fig. 1). Most taxa in these families are associated with living plants and many are serious plant pathogens (Shoemaker & Babcock 1989, Ueng , Rouxel & Balesdent 2005). Examples of important plant pathogens representing the different families are Cochliobolus heterostrophus (Pleosporaceae), the cause of southern corn leaf blight on maize (White 1999), Phaeosphaeria nodorum (anamorph Stagonospora nodorum) the cause of wheat glume blotch (Vergnes ), Didymella pisi (Didymellaceae), the cause of Ascochyta blight of pea (Chilvers ) and Leptosphaeria maculans (Leptosphaeriaceae) the cause of stem canker on Brassica crops (Rouxel & Balesdent 2005). Because of their economic importance, members of Pleosporineae have already been subject to extensive molecular phylogenetic and pathogenic investigations over several decades (Wehmeyer 1961, Shoemaker 1976, 1984a, Shoemaker & Babcock 1985, Simmons 1986, Barr 1992). This includes studies on taxonomy, fungus-host interactions, biochemistry and genomics. Recently, the production of full genome data sets have spurred renewed interest in species such as Stagonospora nodorum (Solomon , Hane ), Leptosphaeria maculans (Rouxel & Balesdent 2005), and Alternaria brassicicola (Pedras ). The designation of Pleosporineae was first proposed by Barr (1979) to accommodate fungi having “globose, depressed, conic or vertically elongated ascomata, with a peridium equal in thickness or thickened at the lower sides”. Six families were included, viz. Mesnieraceae, Phaeosphaeriaceae, Pleosporaceae, Pyrenophoraceae, Tubeufiaceae and Venturiaceae (Barr 1979). The findings here support previous phylogenetic studies in concluding that the ordinal type, Pleosporaceae, and the families Phaeosphaeriaceae, Leptosphaeriaceae and Didymellaceae form a robust clade, and consistently occupy the terminal branches of pleosporalean dendrograms (Liew , Kodsueb , Kruys , Schoch , de Gruyter ). Thus Pleosporineae is emended here to accommodate these four families. Many anamorphic stages of the Pleosporineae are coelomyceteous genera, which includes Ascochyta, Chaetosphaeronema, Coniothyrium, Microsphaeropsis, Pleurophoma, Phoma, and Stagonospora (de Gruyter ). However, hyphomyceteous anamorphs such as Bipolaris, Alternaria or Stemphylium are also included (Simmons 1986).

Barr, Mycologia 71: 947. 1979. emend.

Mostly hemibiotrophic or saprobic, rarely symbiotic. Ascomata perithecioid, immersed, erumpent to superficial; globose to subglobose, ovoid or obpyriform. Hamathecium broadly to narrowly trabeculate or cellular pseudoparaphyses, rarely deliquescing at maturity. Asci bitunicate, fissitunicate, usually basal, rarely extending laterally, cylindrical, clavate to oblong. Ascospores mostly pigmented, rarely hyaline, one- to multi-septate or muriform, symmetrical or rarely assymmetrical.

Anamorphs: Acroconidiellina, Alternaria, Ascochyta, Ascochytella, Bipolaris, Coniothyrium, Curvularia, Drechslera, Exserohilum, Leptophoma, Metabotryon, Nimbya, Phoma, Pithomyces, Scolecosporiella, Stagonospora, Stemphylium and Tiarospora (www.cbs.knaw.nl/databases/anateleo.htm 04-2009, www.indexfungorum.org/ 12-2009, www.mycobank.org/DefaultPage.aspx 12-2009).

Clade I Phaeosphaeriaceae

The clade of Phaeosphaeriaceae (MLB = 92 %, JK = 83 %) comprises 19 taxa including the generic types of Amarenomyces (A. ammophilae), Entodesmium (E. rude) and Setomelanomma (S. holmii), as well as the species Leptosphaeria derasa, Ophiosphaerella herpotricha and some other Phaeosphaeria species, such as P. avenaria, P. eustoma and P. nodorum (Fig. 1). This clade could be further subdivided into four subclades, i.e. I-A–D. Of these, I-A comprises species of Ophiosphaerella and Phaeosphaeria; and I-B–D Phaeosphaeria species.

Phaeosphaeriaceae is an important family in the Pleosporales, comprising 19 genera and 394 species (Kirk ), with many plant pathogens or forming associations with plants (Shoemaker & Babcock 1989, Carson 2005, Stukenbrock ). Phaeosphaeriaceae was introduced by Barr (1979) based on a pseudoparenchymatous peridium almost equal in thickness, and narrowly fusiform or filiform, hyaline, pale brown or rarely dark brown ascospores, and was assigned under Pleosporales sensu Barr. The anamorphs are coelomycetes. Fourteen genera were included, viz. Comoclathris, Didymella, Eudarluca, Heptameria, Leptosphaeria, Loculohypoxylon, Metameris, Microthelia, Nodulosphaeria, Ophiobolus, Paraphaeosphaeria, Rhopographus, Scirrhodothis and Teichospora (Barr 1979). Subsequent phylogenetic studies indicated that the Phaeosphaeriaceae is heterogeneous, and Leptosphaeriaceae was introduced to accommodate species related to Leptosphaeria (Barr 1987a), which is supported by subsequent phylogenetic results (Fig. 1; Khashnobish & Shearer 1996, Câmara , de Gruyter ).

Phaeosphaeria, as the familial type of Phaeosphaeriaceae, was first introduced by Miyake (1909), but was regarded as a synonym of Leptosphaeria for a long time. Holm (1957) noticed the presence of pseudoparaphyses in the generic type of Phaeosphaeria (P. oryzae), reinstated Phaeosphaeria, assigned some Leptosphaeria (s. l.) species with relatively small ascomata which occurred on monocotyledons to Phaeosphaeria, and treated 17 species. Subsequently, more species and information were added (Hedjaroude 1968, Leuchtmann 1984, Shoemaker & Babcock 1989). In a world monograph, 114 species of Phaeosphaeria were treated, and they were further divided into 6 subgenera, viz. Ovispora, Fusispora, Phaeosphaeria, Spathispora, Vagispora and Sicispora, based on differences in ascospore shape and the number of septa (Shoemaker & Babcock 1989). Many species of Phaeosphaeria have characteristic gelatinous sheaths on spores, and some are dictyosporous (Eriksson 1967). Currently, ca. 80 species are accepted under Phaeosphaeria, and many of them have Stagonospora anamorphs (Kirk ).

Two of the three strains in subclade I-B are isolated from maritime environments; e.g. P. ammophilae from beach grass Ammophila arenaria and Phaeosphaeria spartinae from stems of Spartina alterniflora in estuarine salt marshes. A strain of Phaeosphaeria caricis (CBS 120249) used here was isolated from Typha latifolia occurring in or near freshwater. All species in the other three subclades (I-A, C–D, Fig. 1) are associated with terrestrial or near freshwater grasses such as P. elongata with Miscanthus sinensis, P. juncophila with Juncus articulatus and Ophiosphaerella herpotricha with Bromus erectus. The only exception is Phaeosphaeria spartinicola, which was isolated from salt marsh grass (Spartina alterniflora).

Amarenomyces was separated from Phaeosphaeria (as Amarenomyces ammophilae) based on its multilayered endotunica and large and thick-walled, sheathed ascospores (Eriksson 1981). However, its relationship with other Phaeosphaeria species is supported in this study. Thus Amarenomyces is treated as a synonym of Phaeosphaeria. Entodesmium is exclusively associated with legumes, and is traditionally assigned to Lophiostomataceae based on its periphysate papilla (Eriksson & Hawksworth 1990, Barr 1992). But its immersed ascomata, non-compressed papilla and thin peridium, plus the multiseptate, lightly pigmented ascospores, which break up into part-spores support its inclusion in Phaeosphaeriaceae. In particular, Entodesmium multiseptatum and E. niessleanum were originally described as a Leptosphaeria species (Shoemaker 1984b), indicating their similarity with Phaeosphaeria which is commonly confused with Leptosphaeria (Shoemaker 1984a, Shoemaker & Babcock 1989).

Notes: Although members of the Phaeosphaeriaceae are usually known as saprobes or parasites of plants or other fungi, the strain of Phaeosphaeria luctuosa (CBS 308.79) in this clade is recorded as an endophyte in Zea mays. In addition, the inclusion of Entodesmium rude in this clade indicates the ascospores of this family can be filiform.

Currently accepted genera:?* Ophiosphaerella,? Phaeosphaeria, Entodesmium and Setomelanomma.

Anamorphs: Ampelomyces, Chaetosphaeronema, Coniothyrium, Phoma, Plenodomus, Stagonospora and Wojnowicia (Leuchtmann 1984, de Gruyter ).

The genera Ampelomyces, Coniothyrium, Phoma and Plenodomus are polyphyletic (de Gruyter ). The generic type species Ampelomyces quisqualis clustered in the Phaeosphaeriaceae, whereas A. quercinus grouped in the Didymellaceae. The type species of the genera Phoma, Coniothyrium and Pleonodomus clustered in the Didymellaceae and Leptosphaeriaceae respectively. Although Chaetosphaeronema was associated with Ophiobolus (Petrak 1944), this teleomorph-anamorph relation has not been confirmed. An isolate preserved as Trematophoma sp. was found in the Phaeosphaeriaceae (de Gruyter ); however, its identity needs to be studied in more detail.

Clade II Pleosporaceae

Pleosporaceae (Clade II), including the generic type of Pleospora — P. herbarum, forms a robust clade (MLB = 100 %, JK = 100 %), and comprises four subclades as well, i.e. II-A–D. Clade II-A, including the generic type – Cochliobolus heterostrophus represents Cochliobolus, II-B comprises two taxa, i.e. Pleospora herbarum and the anamorphic Dendryphiella arenaria (Scolecobasidium arenarium), which represents Pleospora, II-C represents anamorphic fungi – Alternaria, and II-D contains the generic type – Pyrenophora phaeocomes, represents Pyrenophora.

Pleosporaceae comprises 36 genera and 769 species (Kirk ) and is the largest family in Pleosporales. Members have been reported as plant parasites or saprobes occurring on herbaceous or woody plant leaves or stems (Sivanesan 1984). Pleosporaceae was introduced by Nitschke (1869), which had been assigned to Sphaeriales based on the immersed ascomata and presence of pseudoparaphyses, then to Pseudosphaeriales (Theissen & Sydow 1917, Wehmeyer 1975), and the name of Pseudosphaeriales subsequently was replaced by Pleosporales (Luttrell 1955). Morphology of ascospores, i.e. shape, colour, septation and presence or absence of sheaths has been emphasised in defining the circumscriptions of genera under Pleosporaceae (Luttrell 1955, 1973, Wehmeyer 1961, 1975, von Arx & Müller 1975, Sivanesan 1984, Barr 1987b, Abler 2003). The polyphyletic nature of Pleosporaceae has been indicated in previous investigations, and some genera have been assigned to other families, such as Leptosphaerulina to Leptosphaeriaceae, and Macroventuria to Phaeosphaeriaceae (Kodsueb ). In this study however, the generic types of both Macroventuria (M. anomochaeta) and Leptosphaerulina (L. australis) cluster within the Didymellaceae, as previously recorded (de Gruyter ).

The current clade of Pleosporaceae, comprising the generic types of Cochliobolus (C. heterostrophus), Pleospora (P. herbarum) and Pyrenophora (P. phaeocomes), represents the core members of Pleosporaceae, and are mostly plant pathogens (Fig. 1). Species in subclades II-A and II-D are exclusively associated with monocotyledons, such as Pyrenophora tritici-repentis with wheat and P. phaeocomes with Festuca rubra. Pleospora herbarum (Clade II-B) has been recorded as associates of numerous monocotyledons and dicotyledons, while the strain of Dendryphiella arenaria is from the root zone soil of beachgrass (Ammophila arenaria). Subclade II-C comprises two Alternaria species and one Allewia species, of which Alternaria maritima was isolated from submerged wood in seawater, A. alternata is generally occurring on all kinds of substrates, and Allewia eureka is associated with terrestrial dicotyledons.

Notes: Members of this clade mostly have middle-sized ascomata, and the hyaline and filiform ascospores possessed by Setosphaeria monoceras expanded the familial concept from “brown” by Cannon & Kirk (2007) to “hyaline or brown”.

Currently accepted genera:? Allewia,? Lewia, Cochliobolus, Pleospora, Pyrenophora and? Setosphaeria.

Anamorphs: Alternaria, Ascochyta, Bipolaris, Curvularia, Drechslera, Embellisia, Exserohilum, Phoma and Stemphylium (Simmons 1986, 1989, 1990, Cannon & Kirk 2007, Aveskamp , de Gruyter ).

Most of the anamorphs in the Pleosporaceae are hyphomycetes. Both Ascochyta and Phoma species have been described in the Pleosporaceae. However, the generic type species, Ascochyta pisi and Phoma herbarum, belong to the Didymellaceae (de Gruyter ).

Clade III Leptosphaeriaceae

The clade containing members of Leptosphaeriaceae is sister to the Pleosporaceae, but receives poor statistical support (Fig. 1), indicating the need for more thorough analysis. It comprises the generic types of Leptosphaeria (L. doliolum) and Neophaeosphaeria (N. filamentosa), as well as other taxa from numerous groups, such as Coniothyrium palmarum, L. maculans (Leptosphaeriaceae) and Pyrenochaeta nobilis (Herpotrichia, Melanommataceae).

The Leptosphaeriaceae is likely paraphyletic (Schoch ; this volume). This taxon was separated from the Pleosporaceae and formally introduced by Barr (1987a) based on its “coelomycetous anamorphs” and “narrower and thinner-walled asci” (Barr 1987b), and supported by phylogenetic data (Dong ). Initially, five genera, i.e. Curreya, Didymolepta, Heptameria, Leptosphaeria and Ophiobolus, were accepted under Leptosphaeriaceae (Barr 1987b), while Eriksson & Hawksworth (1990) only accepted Leptosphaeria and Ophiobolus under this family. The Leptosphaeriaceae only comprises some species of Leptosphaeria and Neophaeosphaeria filamentosa, as well as the anamorph Coniothyrium palmarium. Pyrenochaeta nobilis also clustered in the Leptosphaeriaceae. However, this species probably represents a closely related subclade (de Gruyter ).

Morphologically, Leptosphaeriaceae is mostly comparable with Phaeosphaeriaceae, and numerous characters have been used to distinguish them at generic or family level. For instance, anamorphic states (Câmara ), peridium structure (Khashnobish & Shearer 1996, Câmara ) and host spectrum (Câmara ) have all been proposed in distinguishing Leptosphaeria s. str. and Phaeosphaeria. Of these characters, the host preference of Leptosphaeria on dicotyledons in contrast to Phaeosphaeria on monocotyledons has been widely reported (Eriksson 1967, Hedjaroude 1968, Eriksson 1981, Shoemaker & Babcock 1989). Currently, six of the eight species included in Leptosphaeriaceae (Fig. 1) have dicotyledonous hosts, while Coniothyrium palmarum is associated with palms. Thus present results further support the fact that the host spectrum has phylogenetic significance to some degree (Câmara , Voigt ).

Currently accepted genera: Leptosphaeria and Neophaeosphaeria.

Anamorphs: Chaetodiplodia, Coniothyrium, Phoma, Plectophomella and Pyrenochaeta (Wehmeyer 1975, de Gruyter ).

The genus Chaetodiplodia has been recorded as an anamorph of Leptosphaeria (Wehmeyer 1975), but not confirmed. A Chaetodiplodia sp. isolate clustered in the Leptosphaeriaceae (de Gruyter ); however the identity of this strain is uncertain.

Clade IV Didymellaceae

The Didymellaceae (Clade IV) receives high bootstrap support, and includes the generic types of Didymella (D. exigua), Macroventuria (M. anomochaeta), Monascostroma (M. innumerosum), Leptosphaerulina (L. australis) and Platychora (P. ulmi), as well as some species of Phoma and Ascochyta (Fig. 1).

This family was introduced to accommodate some species of Phoma and their phylogenetically closely related anamorphic taxa, as well as teleomorphs such as Didymella and Leptosphaerulina (de Gruyter , Woudenberg ). The generic types of Platychora, Monascostroma and Macroventuria are also located in Clade IV. In particular, both Platychora ulmi and Monascostroma innumerosum have immersed ascomata and clavate asci with lightly pigmented, 1-septate ascospores, and they form a robust subclade (Fig. 1), which most likely represents a single genus. When compared with M. innumerosum, the apiosporous ascospores are the most striking character of Platychora ulmi. Thus the symmetry of ascospores might have no phylogenetic significance at the generic level.

What is most interesting is that Leptosphaerulina argentinensis forms a robust clade with two strains of L. australis. Although L. argentinensis can be distinguished from L. australis by its larger ascospores, their morphological similarity can not be ignored (Graham & Luttrell 1961). Thus this subclade most likely represents a species complex for L. australis.

Most species in this clade are associated with dicotyledons, such as Macroventuria anomochaeta with Medicago sativa, Phoma cucurbitacearum with Cucurbita spp., Didymella exigua with Rumex arifolius, Leptosphaerulina argentinensis with Lonicera periclymenum and Ascochyta pisi with Pisum sativum, while Leptosphaerulina australis and Phoma herbarum are associated with a wide range of hosts including dicotyledons and monocotyledons.

Notes: Besides the characters described by de Gruyter et al. (2009), members of Didymellaceae are also mostly hemibiotrophic or saprobic, and have sometimes setose ascomata, persistent or deliquescing pseudoparaphyses and fusiform, symmetric or apiosporous ascospores.

Currently accepted genera: Didymella, Leptosphaerulina, Macroventuria, Monascostroma and Platychora.

Anamorphs: Chaetasbolisia, Diplodina, Microsphaeropsis and Phoma (Aveskamp , de Gruyter ).

The genus Phoma is subdivided in nine sections with teleomorphs in the genera Didymella, Leptosphaeria, Mycosphaerella and Pleospora (Boerema 1997). Molecular studies confirmed the polyphylectic character of Phoma in the Pleosporineae (de Gruyter ). The generic type, Phoma herbarum, grouped in the Didymellaceae, and therefore, Phoma species in the Didymellaceae are considered as Phoma s. str. (de Gruyter ). The taxonomy of Phoma species in the Leptosphaeriaceae, Phaesphaeriaceae and Pleosporaceae needs further study.

Clade V Lentitheciaceae

The clade of Lentitheciaceae comprises the generic type Lentithecium fluviatile, as well as L. arundinaceum, Stagonospora macropycnidia, Wettsteinina lacustris, Keissleriella cladophila, and the bambusicolous species Katumotoa bambusicola and Ophiosphaerella sasicola, which receives high bootstrap support (MLB = 100 %, JK = 100 %). The teleomorphs have lenticular ascomata, trabeculate to broadly cellular pseudoparaphyses, cylindrical to clavate asci with short pedicels, uni-, 3- to multiseptate, fusiform or filiform ascospores. Based on morphological characters and current molecular phylogenetic results, a new family — Lentitheciaceae is introduced to accommodate them.

This clade is further subdivided into two groups. One subclade comprises Lentithecium arundinaceum, Katumotoa bambusicola, W. lacustris, Ophiosphaerella sasicola and Stagonospora macropycnidia (Clade V-A), while the other subclade (Clade V-B) comprises L. fluviatile and L. aquaticum with Keissleriella cladophila basal to both. Species of Clade V-A exclusively occur on monocotyledons, such as Lentithecium arundinaceum and Stagonospora macropycnidia which are isolated from Phragmites sp., and Wettsteinina lacustris which is recorded on Schoenoplectus sp. The strain of W. lacustris (CBS 618.86) used here was isolated from Schoenoplectus lacustris, and both Ophiosphaerella sasicola (from Sasa senanensis) and Katumotoa bambusicola (from Sasa kurilensis) are bambusicolous. In contrast, species of Clade V-B seem to be exclusively associated with dicotyledonous woody substrates in freshwater environments, i.e. L. aquaticum and L. fluviatile are from submerged wood of Fraxinus sp. and Populus sp. from France, respectively. The habit details of the Keissleriella cladophila strain (CBS 104.55) used here are unknown, but it was isolated from dicotyledonous woody plants (Smilax parvifolia) in Pakistan.

The relatively larger ascomata (500–600 vs. 300–400 μm) and the sheathed ascospore of Ophiosphaerella sasicola make it readily distinguishable from O. herpotricha, and the latter is morphologically similar to the generic type of Ophiosphaerella (O. graminicola). The identification of the strain of Wettsteinina lacustris (CBS 618.86) used here could not be verified. According to Shoemaker & Babcock (1989, p. 1596) however, the collections studied by Leuchtmann (collector of CBS 618.86) under this name, represent “a good Massarina”, which is “not conspecific with Wettsteinina”. Thus the strain of CBS 618.86 most likely is of Massarina s. l., which is closely related to Lentithecium. Both Ophiosphaerella sasicola and Katumotoa bambusicola are bambusicolous, and they have lenticular ascomata with a simple peridium structure, as well as numerous persistent pseudoparaphyses. All of these characters fit in the traditional concept of Lentithecium. However, their ascospores are asymmetrical (K. bambusicola) or filiform (Ophiosphaerella sasicola), which differs from the symmetrical and cylindrical to fusiform ascospores possessed by other species of Lentithecium (Nagasawa & Otani 1997, Tanaka & Harada 2005a).

Yin. Zhang, C.L. Schoch, J. Fourn., Crous & K.D. Hyde, fam. nov. MycoBank MB515470.

Aquaticus vel terrestris. Saprophyticus. Ascomata immersa, lenticulare, solitaria vel disseminata, nigra. Asci bitunicati, fissitunicati, clavati vel oblongaticylindrici, pedicellati. Ascosporae cylindrica vel fusiforme vel filiforme, uniseptatae vel aliquando 3-septatae cum supra-maturae, parce multiseptatum, hyalinae vel fulvum.

Freshwater or terrestrial habitat. Saprobic. Ascomata immersed, lenticular, solitary or scattered. Peridium comprising a few layers of thin-walled cells. Asci bitunicate, fissitunicate, cylindro-clavate to cylindro-oblong, short pedicellate. Ascospores fusiform or filiform, hyaline to pale yellow, 1-septate, constricted at the septum, sometimes becoming 3-septate when mature, rarely multiseptate.

Type genus: Lentithecium K.D. Hyde, J. Fourn. & Yin. Zhang.

Notes: Lentithecium was introduced to accommodate some freshwater taxa with lenticular ascomata and hyaline, 1-septate ascospores (Zhang ). Wettsteinina lacustris, Ophiosphaerella sasicola, and the anamorphic Stagonospora macropycnidia, as well as Keissleriella cladophila and Katumotoa bambusicola are also included in this clade. The strain of Wettsteinina lacustris used here may be misidentified (see comments above). However, they all have immersed and lenticular ascomata, with thin peridium usually almost equal in thickness, short pedicellate asci and fusiform or filiform, hyaline or rarely lightly pigmented, 1- to multi-septate ascospores. Phylogenetically, they form a robust clade separating them from all other pleosporalean families. Thus a new family, Lentitheciaceae, is introduced to accommodate these species of Massarina s. l., a “genus” which should contain species from numerous genera.

Currently accepted genera: Lentithecium, Katumotoa and? Keissleriella.

Anamorph:? Stagonospora macropycnidia.

The genus Stagonospora is polyphyletic and considered as the anamorph of Phaeosphaeria (Leuchtmann 1984), while a strain of Stagonospora macropycnidia used here clusters in Lentitheciaceae in this study.

Clade VI Massarinaceae

The Massarinaceae clade comprises the generic types of Massarina (M. eburnea) and Byssothecium (B. circinans), as well as M. cisti and M. igniaria, and receives high bootstrap support (MLB = 100 %, JK = 97 %). Massarinaceae was introduced to accommodate species having immersed, flattened or sphaerical ascomata with or without clypeus, trabeculate or cellular pseudoparaphyses, clavate to cylindro-clavate asci, hyaline, fusiform to narrowly fusiform, 1- to 3-septate ascospores with or without sheath. Five genera were accepted, i.e. Keissleriella, Massarina, Metasphaeria, Pseudotrichia and Trichometasphaeria (Munk 1956). This family name has not been commonly used and the familial type — Massarina has usually been placed under the Lophiostomataceae (Bose 1961, Eriksson & Yue 1986, Barr 1987b, 1990). The polyphyletic nature of Massarina has been noted (Liew , Zhang ), and a narrow concept of Massarina was accepted, which comprises the generic type (M. eburnea) and morphologically similar species (e.g. M. cisti) (Zhang ). The strain of Byssothecium circinans (CBS 675.92) in this clade is unverified, thus its status remains unresolved (see comments by Zhang ). Massarina s. str. comprising M. cisti, M. eburnea and M. igniaria is confirmed based on these five nuclear loci, which represents a separate branch in Pleosporales.

Munk, Friesia 5: 305. 1956. emend.

Terrestrial habitat. Saprobic. Ascomata immersed, erumpent to superficial with small to wide papilla, solitary or scattered. Pseudoparaphyses cellular to narrowly cellular. Asci clavate to cylindrical, with short pedicels. Ascospores fusiform to broadly fusiform, hyaline or brown, 1- to 3-septate, with or without sheaths.

Currently accepted genera:? Byssothecium and Massarina.

Anamorph: Periconia.

The hyphomycete genus Periconia is polyphyletic, and in the Massarinaceae associated with Didymosphaeria (Booth 1968). The coelomycete genus Neottiosporina has not been associated with a teleomorph. In this study however, a strain of N. paspali grouped in the Massarinaceae.

Clade VII Montagnulaceae

The well-supported clade of Montagnulaceae (MLB = 100 %, JK = 100 %) comprises the generic types of Bimuria (B. novae-zelandiae), Didymocrea (D. sadasivanii), Karstenula (K. rhodostoma) and Paraphaeosphaeria (P. michotii), as well as some species of Kalmusia, Paraconiothyrium, Letendraea and Montagnula. Based on the morphological and ecological similarities, Phaeosphaeria brevispora was assigned to Kalmusia (see comments below). Species in this clade can be saprobic (Kalmusia scabrispora, Phaeosphaeria brevispora and Bimuria novae-zelandiae), plant pathogenic (Paraphaeosphaeria michotii) or mycoparasitic (Paraconiothyrium minitans) (Fukuhara 2002, Verkley ). Montagnulaceae was introduced by promoting the heterogeneric Montagnula to familial level, which contains species with three types of ascospores, i.e. muriform (Montagnula), phragmosporous (Kalmusia) and didymosporous (Didymosphaerella) (Barr 2001).

Paraphaeosphaeria has been treated as a segregate of Leptosphaeria based on its swollen cell above the A1 septum and a longer more highly septate upper part and Coniothyrium s. l. anamorphs (Eriksson 1967). By analysing the ITS and 18S rDNA sequences, Paraphaeosphaeria was shown to be polyphyletic, and a narrow generic concept accepted (Câmara ). The familial placement of Paraphaeosphaeria under Montagnulaceae is verified in this study.

Remarkably, our phylogenetic results indicated that the generic type of Bimuria, B. novae-zelandiae is included in this group. Bimuria novae-zelandiae was initially isolated from soil in a barley field in New Zealand, and is characterised by a very thin peridium, mostly 2-spored, fissitunicate asci and muriform, dark brown, verrucose ascospores, which is considered somewhat comparable with Montagnula (Hawksworth ). The thick carbonaceous peridium, however, distinguishes Montagnula from Bimuria. In addition, the ascospores of Montagnula are discharged forcibly through the ostiole instead of simply deliquescing and gathering at the apex of the ascomata as happens in Bimuria (Hawksworth ). Because of its unique morphological characters, the familial placement of this genus has been debatable and it has been placed in Pleosporaceae by Hawksworth et al. (1979), in Phaeosphaeriaceae by Barr (1987b) and in Melanommataceae by Lumbsch & Huhndorf (2007). In agreement with previous phylogenetic studies (Schoch ), its affinity to other members of Montagnulaceae is noted here.

The generic type of Karstenula (K. rhodostoma) clusters in this group, which is characterised by immersed ascomata, usually with a wide ostiolar opening, narrowly cellular pseudoparaphyses, cylindrical asci with short pedicels, and reddish-brown, muriform ascospores (information obtained from type material). Traditionally, Karstenula has been assigned to Melanommataceae, but the immersed ascomata, narrowly cellular pseudoparaphyses and reddish-brown, muriform ascospores fit the definition of Montagnulaceae (Barr 2001), and this placement is confirmed by the present phylogenetic data (Fig. 1). The clade also contains sequences of Didymocrea sadasivanii (Zopfiaceae) obtained from GenBank, confirming the polyphyly of Zopfiaceae, and its placement in relation to Bimuria, as noted before (Kruys ). The fact that this species produces ostensibly unitunicate asci within ascostromatic ascomata makes it especially interesting (Rogerson 1970, Parguey-Leduc & Janex-Favre 1981).

Notes: The 2- or 3-spored asci possessed by Bimuria novae-zelandiae is another unique character in Montagnulaceae.

Currently accepted genera: Bimuria, Didymocrea,? Kalmusia, Karstenula,? Letendraea,? Montagnula and Paraphaeosphaeria.

Anamorph: Paraconiothyrium (Verkley ).

(Nagas. & Y. Otani) Yin. Zhang, Kaz. Tanaka, C.L. Schoch, comb. nov. MycoBank MB515474. Basionym: Phaeosphaeria arundinacea var. brevispora Nagas. & Y. Otani, Rep. Tottori Mycol. Inst. 15: 38. 1977.

≡ Phaeosphaeria brevispora (Nagas. & Y. Otani) Shoemaker & C.E. Babc., Canad. J. Bot. 67: 1523. 1989.

Notes: Morphological characters of Phaeosphaeria brevispora, such as the immersed ascomata with clypei, thin peridium, clavate asci with relatively long pedicels, and the reddish-brown, verrucose ascospores constricted at the primary septum, fit Kalmusia well. Phylogenetically, P. brevispora and K. scabrispora form a robust clade. In particular, both of these two species occur on Sasa sp. (Tanaka & Harada 2004, Tanaka et al. 2005b).

Clade VIII Trematosphaeriaceae

The generic type of Trematosphaeria (T. pertusa) and the marine fungus, Halomassarina thalassiae, form a well supported clade (MLB = 100 %, JK = 100 %), and represent a pleosporalean family, Trematosphaeriaceae. Details of this family are addressed by Suetrong ; this volume).

Clade IX Melanommataceae (syn. Pleomassariaceae)

The generic types of Melanomma (M. pulvis-pyrius) and Pleomassaria (P. siparia), and some other species, e.g. Monotosporella tuberculata, Herpotrichia diffusa and H. juniperi, representing Melanommataceae, form a well-supported clade (MLB = 100 %, JK = 100 %). The Melanommataceae is one of the largest families in Pleosporales, which comprises 21 genera and 265 species (Kirk ). Traditionally, Melanommataceae comprises immersed, erumpent to superficial, gregarious and black, mostly thick-walled ascomata, trabeculate pseudoparaphyses, and cylindrical asci, brown, septate or muriform ascospores. Presence of trabeculate pseudoparaphyses have been emphasised in Melanommataceae and several related families, but this proposal was not supported by molecular phylogenetic results (Barr 1990, Liew ). The strains of M. pulvis-pyrius and P. siparia were verified by checking the voucher specimens connected to these cultures (Zhang ). As the familial type, Pleomassaria is characterised by its cellular pseudoparaphyses (Sivanesan 1984). This study further indicates that morphology of pseudoparaphyses has little significance at familial level classification (Liew ). Herein Pleomassariaceae is treated as a synonym of Melanommataceae.

Differing from other terrestrial members of this clade, both Beverwykella pulmonaria and Monotosporella tuberculata are from freshwater. A Phoma-like anamorph (Aposphaeria?) has been reported for Melanomma pulvis-pyrius (Chesters 1938, Sivanesan 1984). Both Beverwykella pulmonaria and Monotosporella tuberculata are aquatic hyphomycetous fungi isolated from Europe (Netherlands and U.K., respectively), which indicates that the anamorphs of Melanommataceae should include hyphomycetes as well.

Genera currently accepted:? Herpotrichia, Melanomma and Pleomassaria.

Anamorphs: Aposphaeria (or Phoma-like according to Chesters 1938), Beverwykella pulmonaria, Monotosporella tuberculata, Prosthemium and? Pyrenochaeta (Sivanesan 1984, Paavolainen ).

The genus Pyrenochaeta is polyphyletic (de Gruyter ), and the generic type species P. nobilis grouped in the Leptosphaeriaceae in this study.

Clade X Sporormiaceae

The Sporormiaceae including the generic types of Preussia (P. funiculata) and Westerdykella (W. ornata), and some other species such as Sporormiella minima, Preussia lignicola, P. terricola and Westerdykella cylindrica form a well-supported clade (MLB = 98 %, JK = 82 %). The Sporormiaceae is the largest coprophilous family of Pleosporales, which contains 10 genera and 143 species (Kirk ). The absence of periphyses and well-developed apical rings together with ascomata with or without ostioles, ascospores with or without germ slits have been used to distinguish the Sporormiaceae from other coprophilous families, such as the Delitschiaceae and the Phaeotrichaceae (Barr 2000, Kruys ). Phylogenetic analysis based on ITS-nLSU rDNA, mtSSU rDNA and ß-tubulin sequences indicated that compared to the shape of the asci or ascospores, the substrate choice, presence or absence of ostiole, and presence or absence of germ slits have less phylogenetic significance within Sporormiaceae (Kruys & Wedin 2009). In particular, the presence of periphyses was verified in the generic type of Sporormiella (S. nigropurpurea, type, NY), which belongs in Sporormiaceae (as Preussia) (Kruys & Wedin 2009). Currently, after modifying their concept, three genera, i.e. Sporormia, Preussia and Westerdykella are accepted under Sporormiaceae (Kruys & Wedin 2009).

Munk, Dansk Bot. Ark. 17: 450. 1957.

Note: Although strains of Eremodothis and Pycnidiophora are not included in current analysis, their familial status in Sporormiaceae has been well demonstrated (Kruys & Wedin 2009), and the cleistothecioid ascomata of Eremodothis and Pycnidiophora is another striking character of this family.

Currently accepted genera:? Sporormia (including Sporormiopsis), Preussia (including Sporormiella and Spororminula) and Westerdykella (including Eremodothis and Pycnidiophora) (Kruys & Wedin 2009).

Anamorphs: Phoma-like (von Arx 1974).

Clade XI Amniculicolaceae

Amniculicolaceae (clade XI) comprises all three species of Amniculicola together with Murispora rubicunda, Neomassariosphaeria grandispora and N. typhicola, and receives high bootstrap support (MLB = 100 %, JK = 100 %). This clade is closely related to Anguillospora longissima, Spirosphaera cupreorufescens and Repetophragma ontariense (Zhang ). Compared with Massariosphaeria grandispora (as N. grandispora) and M. typhicola (as N. typhicola), the generic type of Massariosphaeria (M. phaeospora) cluster with Thyridaria rubronotata, and its familial status is undetermined (Fig. 1). Amniculicola was first introduced to accommodate the freshwater fungus A. lignicola isolated from France, which is characterised by its ascomata with slit-like ostioles, thin, branching and anastomosing hamathecium, cylindrical asci, and hyaline, 1–3-septate ascospores (Zhang ). Subsequently, two additional new species of Amniculicola, i.e. A. immersa and A. parva were recovered from Denmark and France, respectively (Zhang ). In particular, the paraphyletic nature of Amniculicola was revealed in Fig. 1, which indicated that more genes or phylogenetic analyses are needed to separate those genera. All three species were collected in Europe, and stain the woody substrate purple, which could be indicative of metabolite activity (Zhang ). Metabolites have rarely been used in the phylogeny and taxonomy of Pleosporales, but it is widely used in the taxonomy of xylariaceous taxa (Stadler , Bitzer ). In addition, all species in this clade are from freshwater environments, which may indicate this as a unique ecological habit for the Amniculicolaceae.

Yin. Zhang, C.L. Schoch, J. Fourn., Crous & K.D. Hyde, fam. nov. MycoBank MB515469.

Aquaticus. Saprobicus. Ascomata globosa vel subglobosa vel lenticular, nigra, solitaria, immersa vel partim immersa vel superficialia. Apex productum. Peridium exilis. Trabeculae, hyalinae, gelatina circumdatae. Asci, 8-spori, cylindrico vel clavati, fissitunicati, breve pedicellati. Ascosporae, fusiforme vel peranguste fusiforme, uniseptatae vel multiseptatae vel muriforme, hyalinae vel pallide brunneus vel rufobrunneus, tunica gelatinosa praeditae. Substratum malvaceo purpureus.

Freshwater habitat. Saprobic. Ascomata solitary, scattered, or in small groups, immersed, erumpent, or nearly superficial, globose, subglobose to lenticular; surface black, roughened; apex elongated. Peridium thin. Pseudoparaphyses trabeculate, embedded in mucilage. Asci 8-spored, bitunicate, fissitunicate, cylindrical to clavate, short pedicellate, with an ocular chamber. Ascospores fusiform or narrowly fusiform, hyaline, pale or reddish-brown, one to multi-septate or muriform, constricted at the median septum, usually surrounded by an irregular, hyaline gelatinous sheath. Ascomata usually stain the woody substrate in shades of purple.

Type genus: Amniculicola Yin. Zhang & K.D. Hyde.

Currently accepted genera: Amniculicola, Murispora and Neomassariosphaeria.

Anamorphs:? Anguillospora longissima, Spirosphaera cupreorufescens and Repetophragma ontariense (Zhang ).

Yin. Zhang, J. Fourn. & K.D. Hyde, gen. nov. MycoBank MB515472.

Etymology: Named after its muriform ascospores.

Aquaticus. Saprobicus. Ascomata immersa vel partim immersa vel superficialia. Peridium exilis. Trabeculae, hyalinae, gelatina circumdatae. Asci, 8-spori, clavati vel late clavati, fissitunicati, breve pedicellati. Ascosporae, fusiforme, muriforme, brunneus, tunica gelatinosa praeditae. Substratum malvaceo purpureus.

Freshwater habitat. Saprobic. Ascomata scattered, or in small groups, immersed, erumpent, or nearly superficial, globose to subglobose, wall black, roughened; apex weakly papillate, conical to laterally flattened. Peridium thin. Pseudoparaphyses trabeculate, embedded in mucilage. Asci 8-spored, bitunicate, fissitunicate, oblong to clavate, short pedicellate, with an ocular chamber. Ascospores fusiform, pale or reddish brown, muriform, constricted at the median septum, usually surrounded by an irregular, hyaline, gelatinous sheath. Ascomata stain the woody substrate purple.

Type species: Murispora rubicunda (Niessl) Yin. Zhang, J. Fourn. & K.D. Hyde.

Note: The studied specimens from which the cultures were obtained are identified in the sense used by Webster (1957), who studied the type specimens, while they might be referred to Pleospora rubelloides sensu Crivelli (1983).

(Niessl) Yin. Zhang, J. Fourn. & K.D. Hyde, comb. nov. MycoBank MB515477. Basionym: Pleospora rubicunda Niessl, Notiz. Pyr.: 31. 1876.

≡ Massariosphaeria rubicunda (Niessl) Crivelli, Über die Heterogene Ascomycetengattung Pleospora Rabh.: 144. 1983.

≡ Karstenula rubicunda (Niessl) M.E. Barr, N. Amer. Fl., Ser. 2 (New York): 52. 1990.

Yin. Zhang, J. Fourn. & K.D. Hyde, gen. nov. MycoBank MB515473.

Etymology: “Neo-” meaning “new”, named after its similarity with Massariosphaeria.

Aquaticus. Saprophyticus. Ascomata dispergere vel gregariculus, immersa vel partim immersa. Apex productum. Peridium exilis. Trabeculae, hyalinae, gelatina circumdatae. Asci, 8-spori, clavati vel late clavati, fissitunicati, breve pedicellati. Ascosporae, peranguste fusiforme, multiseptatae, hyalinae vel rufobrunneus, tunica gelatinosa praeditae. Substratum plerumque purpureus.

Aquatic. Saprobic. Ascomata scattered or in small groups, immersed to erumpent, subglobose to lenticular; wall black, apex elongated. Peridium thin. Pseudoparaphyses trabeculate, embedded in mucilage. Asci 8-spored, bitunicate, fissitunicate, clavate to broadly clavate, short pedicellate. Ascospores narrowly fusiform, hyaline to reddish brown, multi-septate, constricted at the median septum, usually surrounded by an irregular, hyaline, gelatinous sheath. Ascomata or hyphae usually stain the woody substrate or cultural medium purple.

Type species: Neomassariosphaeria typhicola (P. Karst.) Yin. Zhang, J. Fourn. & K.D. Hyde.

(P. Karst.) Yin. Zhang, J. Fourn. & K.D. Hyde, comb. nov. MycoBank MB515479. Basionym: Leptosphaeria typhicola P. Karst., Bidrag Kännedom Finlands Natur Folk. 23: 100. 1873.

≡ Phaeosphaeria typhicola (P. Karst.) Hedjar., Sydowia 22: 86. 1969.

≡ Massariosphaeria typhicola (P. Karst.) Leuchtm., Sydowia 37: 168. 1984.

≡ Chaetomastia typhicola (P. Karst.) M.E. Barr, Mycotaxon 34: 514. 1989.

(Sacc.) Yin. Zhang, J. Fourn. & K.D. Hyde, comb. nov. MycoBank MB515478. Basionym: Leptosphaeria grandispora Sacc., Michelia 1: 341. 1878.

≡ Metasphaeria grandispora (Sacc.) Sacc., Syll. Fung. 2: 181. 1883.

≡ Massariosphaeria grandispora (Sacc.) Leuchtmann, Sydowia 37: 172. 1984.

≡ Lophiotrema grandispora (Sacc.) Shoemaker & C.E. Babc., Sydowia 37: 172. 1989.

Notes: Although the living habit of Neomassariosphaeria grandispora (CBS 613.86) can not be clarified, the freshwater habit of species under this clade seems characteristic (see comments by Zhang ). In addition, the ascomata of telemorphs usually stain the woody substrate purple. Their morphological characters, however, vary greatly. For instance, Amniculicola species have cylindrical asci, while N. grandispora, N. typhicola and Murispora rubicunda have clavate asci. Amniculicola species have hyaline, fusiform 1- or rarely 3-septate ascospores, while the ascospores of N. typhicola and N. grandispora are narrowly fusiform and multiseptate, but ascospores of N. typhicola are brown and N. grandispora are hyaline. The ascospores of M. rubicunda are brown and muriform. Based on their phylogenetic affinity and morphological distinctions, two new genera, i.e. Murispora (based on Pleospora rubicunda) and Neomassariosphaeria (based on Massariosphaeria typhicola) and a new family, Amniculicolaceae, are introduced.

Clade XII Lophiostomataceae (uncertain)

The Lophiostomataceae comprises some Lophiostoma species, such as L. caulium, L. semiliberum, L. arundinis, L. crenatum, L. compressum, L. viridarium and L. macrostomoides (MLB = 100 %, JK = 89 %) while L. fuckelii is basal (MLB = 94 %, JK = 77 %), as previously reported (Tanaka & Hosoya 2008, Zhang ). Traditionally, Lophiostomataceae comprised some other genera with various morphological characters, such as Entodesmium and Lophionema with filiform ascospores, and Herpotrichia and Lophiotrema with fusiform, brown or hyaline, 1-septate ascospores are usually multiseptate when senescent (Sivanesan 1984, Holm & Holm 1988). The present phylogeny does not support their placement in Lophiostomataceae. The paraphyletic nature of Lophiostomataceae has been previously noted (Schoch ), and Clade XII is likely to represent the narrow concept of Lophiostomataceae, although it is still too early to draw this conclusion until verified sequences of the generic type of Lophiostoma (L. macrostomum) are obtained (see comments by Zhang ).

Geographically, most species used in this study are from European locations such as Switzerland (Lophiostoma caulium, L. arundinis and L. crenatum), Sweden (L. semiliberum) and France (L. viridarium, L. compressum and L. macrostomoides). Lophiostoma fuckelii, the only strain from South Africa, diverged earlier than all other members (Fig. 1).

Sacc., Syll. Fung. 2: 672. 1883. emend.

Terrestrial or aquatic habitat. Saprobic. Ascomata perithecioid, medium to large-sized, solitary or scattered, immersed to erumpent or rarely superficial with protruding, compressed papilla and slite-like ostioles. Pseudoparaphyses numerous, narrowly cellular. Asci cylindrical to cylindro-clavate, with short pedicels. Ascospores fusiform to narrowly fusiform, and mostly multiseptate and heavily pigmented, sometimes with longitudinal septa in one or two cells, rarely 1-septate and hyaline, with or without sheath.

Currently accepted genus: Lophiostoma s. str.

Anamorphs: Reported as Pleurophomopsis-like (Leuchtmann 1985).

Clade XIII Massariaceae

The well-supported clade of the Massariaceae comprises the generic type of Massaria (M. inquinans) as well as species of Roussoella and Arthopyrenia that form a robust clade. The phylogeny in Fig. 1 includes the generic type of Massaria — M. inquinans. Morphologically, all of them have immersed ascomata, pseudoparaphyses from abundant to rare, asci from cylindrical to clavate, ascospores from hyaline to reddish-brown, 1- or 3-septate.

Traditionally, Massariaceae (Melanommatales) is defined as having large ascomata, a peridium comprising compact, small cells, trabeculate pseudoparaphyses, large, and symmetric distoseptate ascospores usually surrounded with a sheath (Barr 1979). Based on these characters, six genera were included, i.e. Aglaospora, Caryospora, Dothivalsaria, Massaria, Titanella and Zopfia (Barr 1979). Massaria inquinans and Aglaospora profusa are the generic types of Massaria and Aglaospora respectively, and they share numerous morphological characters, such as the large, immersed ascomata, trabeculate pseudoparaphyses, cylindrical asci with large and conspicuous apical rings and large, reddish-brown, 3-distoseptate ascospores (Shoemaker & Leclair 1975). The phylogenies here exclude the placement of Aglaospora under Massariaceae, and the placement of other four traditional genera under Massariaceae, i.e. Caryospora, Dothivalsaria, Titanella and Zopfia can not be verified here either.

Nitschke, Verh. Naturhist. Vereines Preuss. Rheinl. 26: 73. 1869.

Note: Members of this clade are mostly saprobic.

Currently accepted genera:? Arthopyrenia, Massaria,? Roussoella.

Anamorph:? Torula herbarum.

Clade XIV

The current phylogenetic data show that Lophiotrema as well as the generic types of Lophiotrema (L. nucula), Verruculina (V. enalia), Ulospora (U. bilgramii), Lepidosphaeria (L. nicotiae) and Xenolophium (X. applanatum) cluster apart from the clade of Lophiostomataceae s. str. Members of this clade are all saprobes, but have diverse morphological characters. Lophiotrema was introduced as a genus closely related to Lophiostoma, but having hyaline ascospores, and was assigned to Lophiostomataceae (Saccardo 1878, Holm & Holm 1988). The relatively smaller ascomata, peridium of almost equal in thickness, and the hyaline, 1-septate ascospores have been used to distinguish Lophiotrema from Lophiostoma (Holm & Holm 1988, Yuan & Zhao 1994, Kirk ). The peridium concept, however, is not supported by the lectotype specimen, which has a flattened, thin-walled base (Zhang ). Species with brown ascospores are found in Lophiotrema based on molecular phylogenetic results (Zhang ).

Lepidosphaeria, Ulospora and Verruculina are all genera of the Testudinaceae, which is characterised by the cleistothecioid ascomata, 1-septate, brown, glabrous or ornamented ascospores (von Arx 1971). The size, shape and ornamentation of the ascospores serve as the distinguishing character between different genera (von Arx 1971, von Arx & Müller 1975, Hawksworth 1979). Based on the present phylogenetic result, these three genera of Testudinaceae are closely related. In addition, the non-ostiolate ascomata of the Testudinaceae provides evidence that taxa with cleistothecioid fruiting bodies have evolved from taxa with perithecioid ones in the Pleosporales.

The diverse morphological characters possessed by members of clade XIV might indicate that they are from more than one family. A more firmly stated hypothesis can only be obtained by further phylogenetic study which should include more genera and related species.

Sacc., Michelia 1: 338, 1878. emend.

Saprobic. Ascomata perithecioid, mostly immersed, rarely erumpent; globose, subglobose or ovoid. Hamathecium of broadly to narrowly trabeculate or cellular pseudoparaphyses, persistent. Asci bitunicate, fissitunicate, cylindrical to clavate. Ascospores mostly hyaline, rarely brown, 1-septate, smooth.

Anamorphs: unknown.

(Ellis & Everh.) Yin. Zhang, Kaz. Tanaka & K.D. Hyde, comb. nov. MycoBank MB515475. Basionym: Didymosphaeria arundinariae Ellis & Everh., N. Amer. Pyren. (Newfield): 732. 1892.

≡ Microthelia arundinariae (Ellis & Everh.) Kuntze, Revis. gen. pl. (Leipzig) 3: 498. 1898.

≡ Massarina arundinariae (Ellis & Everh.) M.E. Barr, Mycotaxon 45: 211. 1992.

≡ Lophiostoma arundinariae (Ellis & Everh.) Aptroot & K.D. Hyde, in Hyde, Wong & Aptroot, Fungal Diversity Res. Ser. 7: 107. 2002.

Note: To avoid the duplication with Lophiotrema arundinariae Rehm, a new name – Lophiotrema neoarundinaria is proposed here.

(Fuckel) Yin. Zhang, C.L. Schoch & K.D. Hyde, comb. nov. MycoBank MB515476. Basionym: Massaria rubi Fuckel, Jahrb. Nassauischen Vereins Naturk. 25–26: 303. 1871.

≡ Massarina rubi (Fuckel) Sacc., Syll. Fung. (Abellini) 2: 155. 1883.

= Didymellina rhaphithamni Keissl., Nat. Hist. Juan. Fernandez Easter Lsl. 2: 480. 1927.

= Mycosphaerella rhaphithamni (Keissl.) Petr., Ann. Mycol. 38: 221. 1940.

= Massarina emergens (P. Karst.) L. Holm, Les Pleosporaceae: 149. 1957.

≡ Lophiostoma rubi (Fuckel) E.C.Y. Liew, Aptroot & K.D. Hyde, Mycologia 94: 812. 2002.

Clade XV Aigialaceae

The generic type of Aigialus (A. grandis) and Lophiostoma mangrovei form a well-supported cluster, which represents a marine pleosporalean family, Aigialaceae. This new family is addressed by Suetrong et al. (2009; this volume).

Clade XVI Delitschiaceae

The generic type of Delitschia (D. didyma) and D. winteri, represent Delitschiaceae and form a robust clade that diverges before all other members of Pleosporales. The Delitschiaceae is a small group of coprophilous fungi, which comprises three genera (i.e. Delitschia, Ohleriella and Semidelitschia) and 54 species (Barr 2000, Kirk ). This family was introduced to accommodate coprophilous pleosporalean species with periphysate ostiole, wide ascus endotunica, conspicious apical ring and heavily pigmented 1- to multiseptate ascospore with germ slits in each cell (Barr 2000).

The presence of a large ocular chamber with an apical ring in the ascus is the most striking character of most members of Delitschiaceae as well as species in clade XVII, Aglaospora profusa. These two clades are consistently the earliest diverging lineage in Pleosporales as in several other phylogenies (Kruys , Schoch ).

SUMMARY

Phylogeny

The results presented here indicate that nutritional modes and environmental habits may have phylogenetic significance in Pleosporales, although more extensive statistical analyses remain to be done. Host spectrum (monocotyledon/dicotyledon) appears closely related to the phylogeny of plant associated fungi or plant pathogens (e.g. in Pleosporineae). Of the morphological characters, the size, shape and immersion degree of ascomata, ostiole characters and ascal shape can be of phylogenetic significance to varying degrees. The purple staining nature of the substrate found in some Amniculicolaceae might indicate that secondary metabolites have phylogenetic significance for this group.

However, even closely related species can exhibit diverse morphologies. Ascospores can vary from 1- to multiseptate to even muriform, hyaline to pigmented in many families, such as Amniculicolaceae (given as an example in Fig. 2), Lophiostomaceae s. str., Melanommataceae and Didymellaceae. From an evolutionary perspective, the “bipolar symmetrical ascospore tends to be correlated to passive dispersal”, and “the colour, size, shape and texture of spores should be viewed as probable functional adaptations modified in evolution by requirements of liberation, of flotation in fluids, and ultimately of deposition and survival” (Ingold 1971, Gregory 1973, Hawksworth 1987). Thus ascospore shape should be viewed as a highly adaptive character that can obscure underlying relationships.

Fig. 2.

A. Xenolophium applanatum. Ascomata on the host surface. Note the slit-like ostiole. B. Trematosphaeria pertusa. Ascomata on the host surface. Note the pore-like ostiole. C, E, H. Murispora rubicunda. C. Ascomata on the host surface. Note the purple woody substrate. E. Clavate 8-spored asci with short pedicels in pseudoparaphyses. H. Muriform ascospore with wide mucilaginous sheath. D. I. Trematosphaeria sp. D. Fusiform mature or immature 8-spored asci with pseudoparaphyses. I. multisepate dark brown ascospore. F. Neomassariosphaeria grandispora. Ascospores with sheath. G. Aglospora profusa. Apical aparatus. Note the conspicuous apical ring. J. Amniculicola immersa. Hyaline fusiform ascospores in ascus. Scale bars; A–C = 100 μm, D–J = 20 μm.

Evolutionary trends

Most plant pathogens in Pleosporales belong to Pleosporineae, which tends to occupy the terminal branches on the Pleosporales tree (Fig. 1). On the other hand, a clade of coprophilous fungi — Delitschiaceae — consistently occurs as an early-diverged lineage compared to all other pleosporalean members, with numerous other saprotrophic members interspersed. Parasitic fungi are usually considered as “highly specialised”, and may require nutritional shifts from several other modes (Cain 1972, Heath 1987, Berbee 2001, Sung ). This may indicate that Pleosporales originated from saprotrophic fungi, and that the transition from saprotrophic to necrotrophic and hemibiotrophic (or biotrophic) is likely, in agreement with earlier ideas (Lewis 1974, Cooke 1977, Cooke & Whipps 1986), also mirroring what is seen in the Capnodiales phylogeny (Crous , Schoch ; this volume).

It is remarkable that as with the Delitschiaceae, Aglaospora profusa is also an early diverging lineage. Members of both Delitschiaceae and Aglaospora have a striking morphological character in having a large apical apparatus, which is rare in Pleosporales. According to the hypothesis of Hawksworth (1987), “......foremost of these trends is the loss of apical apparatus associated with a change from active to passive discharge of the ascospores......”. Thus this striking apical apparatus might further indicate the plesiomorphic status of both Delitschiaceae and Aglaospora, supporting the premise that the ancestor of Pleosporales was saprobic with a well-developed apical ring.

Shortcomings and further work

Attempts to write a familial dichotomous key based on the present phylogenetic data has proven to be unsuccesful. The traditional keys rely on single morpho-characters, which are polyphyletic. Thus it appears to be impossible to find any single criterion which can be used to key out a family in such a way as to include all genera or species belonging to it, without incorporating the genus or species in several places in the key, as have been mentioned by Cain (1972).

Compared with the ca. 3 000 reported species in Pleosporales, the 130 species (< 5 %) used in present investigation are far from sufficient to obtain a comprehensive phylogenetic survey for the genetic diversity in the order, but will hopefully provide a framework for directing further work. Members of some families, such as Cucurbitariaceae and Diademaceae, are absent from our analysis, thus their status remains unresolved. In particular, erroneous strains or names in databases and culture collections necessitate verification, and circumscriptions of families within the clades currently remain preliminary. Importantly, this data set is geographically biased as most strains originated from temperate areas in the Northern Hemisphere, mainly Europe. Obtaining correctly identified fungal strains from various locations is crucial for further molecular phylogenetic investigations, necessitating the consistent analysis and interperpretation of large taxon datasets. It seems clear that most morphological criteria used by traditional taxonomy for Pleosporales at various taxonomic levels (such as genus or family) do not strictly correlate with distinct evolutionary groups. We will therefore have to rely on expanding our base of knowledge in ecology, biochemistry and other biological fields, to supplement the genetic information. The expected expansion in pleosporalean genome sequences makes this especially important.