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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 Lophiostomataceae s. 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.

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, fruitingbody 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 . Another important distinguishing character, ascus type, has been reported to be phylogenetically misleading in numerous natural groups (Schmitt & Lumbsch 2004, Wedin et al. 2005, Lumbsch et al. 2007. Indeed, several DNA sequence based phylogenetic reconstructions have shown that ascospore morphology has little phylogenetic significance at familial or generic level classification (Crous et al. 2003, Schmitt & Lumbsch 2004, Kodsueb et al. 2006, Wang et al. 2007, Zhang et al. 2009b. 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 et al. 2006), Melanommataceae (Liew et al. 2000, Wang et al. 2007) and genera Massariosphaeria (Wang et al. 2007), Melanomma (Wang et al. 2007), Massarina and Lophiostoma (Liew et al. 2002, Zhang et al. 2009b. 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 et al. 1989). 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 et al. 2008).
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 et al. 2008, Zhang et al. 2009a. 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(Simmons , 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) , 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 ).

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 et al. 1990). The fRPB2-5F and fRPB2-7cR primers were used for the amplification of the partial RNA polymerase second largest subunit (RPB2) (Liu et al. 1999). The EF1-Fα and EF1-Rα primers were used to amplify a region from the translation elongation factor 1-alpha gene (TEF1) (Schoch et al. 2006) and the RPB1-Ac and RPB1-Cr primers were used for RPB1 region ; 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 MgCl 2 , 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, Multi-locuS phylogeny of the PleosPorales: a taxonoMic, ecological and evolutionary re-evaluation 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 et al. 1999). 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 et al. 2007) as well as from previous publications (e.g. Lutzoni et al. 2004, Schoch et al. 2009b. Taxa was aligned by using default options for a simultaneous method of estimating alignments and tree phylogenies, SATé (Liu et al. 2009). 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 et al. 2009).
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 presentexcept for a set of nine taxa with closely related species needed to confirm their identity (Table 1 -see online Supplementary  Information).

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 et al. 2008), using the CIPRES 2.1 webportal (Miller et al. 2009) 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 .
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 et al. 2008). 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 et al. 2008).

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: . The highest scoring likely tree had a log likelihood of -107754.307532.
Phaeosphaeriaceae is an important family in the Pleosporales, comprising 19 genera and 394 species (Kirk et al. 2008), with many plant pathogens or forming associations with plants (Shoemaker & Babcock 1989, Carson 2005, Stukenbrock et al. 2006. 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 (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 et al. 2002).
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 et al. 2008).
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 Multi-locuS phylogeny of the PleosPorales: a taxonoMic, ecological and evolutionary re-evaluation 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.
The genera Ampelomyces, Coniothyrium, Phoma and Plenodomus are polyphyletic ). 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 ); however, its identity needs to be studied in more detail.
Pleosporaceae comprises 36 genera and 769 species (Kirk et al. 2008) 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, 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 et al. 2006). In this study however, the generic types of both Macroventuria (M. anomochaeta) and Leptosphaerulina (L. australis) cluster within the Didymellaceae, as previously recorded ).
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".
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 ). *Note: Genera lack generic types or other representative species in the clades are marked as "?" to indicate their uncertain status.

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).
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 et al. 2002), peridium structure (Khashnobish & Shearer 1996, Câmara et al. 2002 and host spectrum (Câmara et al. 2002) 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 et al. 2002, Voigt et al. 2005.
The genus Chaetodiplodia has been recorded as an anamorph of Leptosphaeria (Wehmeyer 1975), but not confirmed. A Chaetodiplodia sp. isolate clustered in the Leptosphaeriaceae ); however the identity of this strain is uncertain.
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 ). 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 , members of Didymellaceae are also mostly hemibiotrophic or saprobic, and have sometimes setose ascomata, persistent or deliquescing pseudoparaphyses and fusiform, symmetric or apiosporous ascospores.
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 ). The generic type, Phoma herbarum, grouped in the Didymellaceae, and therefore, Phoma species in the Didymellaceae are considered as Phoma s. str. ). The taxonomy of Phoma species in the Leptosphaeriaceae, Phaesphaeriaceae and Pleosporaceae needs further study.
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 Multi-locuS phylogeny of the PleosPorales: a taxonoMic, ecological and evolutionary re-evaluation 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 Aquaticus vel terrestris. Saprophyticus. Ascomata immersa, lenticulare, solitaria vel disseminata, nigra. Asci bitunicati, fissitunicati, clavati vel oblongati-cylindrici, 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. Notes: Lentithecium was introduced to accommodate some freshwater taxa with lenticular ascomata and hyaline, 1-septate ascospores (Zhang et al. 2009b). 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.
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.
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.
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 et al. 2001). 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 . 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 . 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 et al. 2006), 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 et al. 2006). The fact that this species produces ostensibly unitunicate asci within ascostromatic ascomata makes it especially interesting (Rogerson 1970, Parguey-Leduc & Janex-Favre 1981.

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 et al. 2008). 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 et al. 2000. The strains of M. pulvis-pyrius and P. siparia were verified by checking the voucher specimens connected to these cultures (Zhang et al. 2008a). 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 et al. 2000). 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.
The genus Pyrenochaeta is polyphyletic , 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 et al. 2008). 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 et al. 2006. 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.

Sporormiaceae
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.

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 et al. 2009a). 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 et al. 2008b). Subsequently, two additional new species of Amniculicola, i.e. A. immersa and A. parva were recovered from Denmark and France, respectively (Zhang et al. 2009a). 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 et al. 2009a). Metabolites have rarely been used in the phylogeny and taxonomy of Pleosporales, but it is widely used in the taxonomy of xylariaceous taxa (Stadler et al. 2004, Bitzer et al. 2008. In addition, all species in this clade are from freshwater environments, which may indicate this as a unique ecological habit for the Amniculicolaceae.  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 et al. 2009a). 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.
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).
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 slitelike 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.

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, Multi-locuS phylogeny of the PleosPorales: a taxonoMic, ecological and evolutionary re-evaluation pseudoparaphyses from abundant to rare, asci from cylindrical to clavate, ascospores from hyaline to reddish-brown, 1-or 3-septate.

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 et al. 2001. The peridium concept, however, is not supported by the lectotype specimen, which has a flattened, thin-walled base (Zhang et al. 2009b). Species with brown ascospores are found in Lophiotrema based on molecular phylogenetic results (Zhang et al. 2009b).
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.

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 et al. 2008. 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 et al. 2006, Schoch et al. 2006.

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.

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 et al. 2008. 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 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.

AcKNowledgeMeNts
The authors thank O.E. Eriksson for his comments on a draft version of this script, as well as two other anonymous reviewers. Dr Shaun Pennycook is thanked for his valuable suggestions on nomenclature. The University of Hong Kong is thanked for providing YZ with a postgraduate studentship. Work performed by CLS after 2008 was supported in part by the Intramural Research Program of the NIH, National Library of Medicine. This work was funded by a grant from NSF to JWS and to CLS until 2008 (DEB 0717476). 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.