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Phylogenetic lineages in the Capnodiales

¹ CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD, Utrecht, The Netherlands
² Wageningen University and Research Centre (WUR), Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
³ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, 45 Center Drive, MSC 6510, Bethesda, Maryland 20892-6510, U.S.A.
⁴ School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand
⁵ ARC – Plant Protection Research Institute, P. Bag X5017, Stellenbosch, 7599, South Africa

^* Correspondence: Pedro W. Crous, p.crous{at}cbs.knaw.nl

	Abstract

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
The Capnodiales incorporates plant and human pathogens, endophytes, saprobes and epiphytes, with a wide range of nutritional modes. Several species are lichenised, or occur as parasites on fungi, or animals. The aim of the present study was to use DNA sequence data of the nuclear ribosomal small and large subunit RNA genes to test the monophyly of the Capnodiales, and resolve families within the order. We designed primers to allow the amplification and sequencing of almost the complete nuclear ribosomal small and large subunit RNA genes. Other than the Capnodiaceae (sooty moulds), and the Davidiellaceae, which contains saprobes and plant pathogens, the order presently incorporates families of major plant pathological importance such as the Mycosphaerellaceae, Teratosphaeriaceae and Schizothyriaceae. The Piedraiaceae was not supported, but resolves in the Teratosphaeriaceae. The Dissoconiaceae is introduced as a new family to accommodate Dissoconium and Ramichloridium. Lichenisation, as well as the ability to be saprobic or plant pathogenic evolved more than once in several families, though the taxa in the upper clades of the tree lead us to conclude that the strictly plant pathogenic, nectrotrophic families evolved from saprobic ancestors (Capnodiaceae), which is the more primitive state.

Taxonomic novelties: Brunneosphaerella Crous, gen. nov., B. jonkershoekensis (Marinc., M.J. Wingf. & Crous) Crous, comb. nov., B. protearum (Syd. & P. Syd.) Crous, comb. nov., Devriesia hilliana Crous & U. Braun, sp. nov., D. lagerstroemiae Crous & M.J. Wingf., sp. nov., D. strelitziicola Arzanlou & Crous, sp. nov., Dissoconiaceae Crous & de Hoog, fam. nov., Hortaea thailandica Crous & K.D. Hyde, sp. nov., Passalora ageratinae Crous & A.R. Wood, sp. nov., P. armatae Crous & A.R. Wood, sp. nov., Rachicladosporium cboliae Crous, sp. nov.

Keywords Ascomycetes / Brunneosphaerella / Capnodiales / DNA sequence comparisons / Mycosphaerella / novel primers / systematics

	INTRODUCTION

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
The Dothideomycetes encompasses plant and human pathogens, endophytes, saprobes and epiphytes. The class presently contains two subclasses, namely Pleosporomycetidae and Dothideomycetidae (Schoch et al. 2006, 2009a). Although the main orders, Pleosporales and Dothideales correlate with the presence or absence of pseudoparaphyses and other centrum characteristics, many orders remain unresolved. The Dothideomycetidae include the orders Dothideales, Capnodiales and Myriangiales, which lack paraphyses, pseudoparaphyses and periphysoids. Based on a multi-gene phylogeny, and the presence of ostiolar periphyses as possible synapomorphy, the Capnodiales were recognised as the order incorporating the Capnodiaceae, Davidiellaceae, Mycosphaerellaceae and Piedraiaceae (Schoch et al. 2006). However, several studies (Hunter et al. 2006, Crous et al. 2007a, b) showed the Mycosphaerellaceae to be polyphyletic, and to contain additional variation at the familial level, leading to the circumscriptions of the Teratosphaeriaceae and Schizothyriaceae. Crous et al. (2009b, c) again revealed Teratosphaeriaceae to be too widely defined, including some further unresolved families.

The present study focuses on the Capnodiales, which is based on the Capnodiaceae, representing a group of leaf epiphytes associated with honeydew of insects, usually visible as a black growth on leaf surfaces, fruit and twigs. Members of the Capnodiaceae form superficial ascomata with fasciculate asci, and hyaline to dark, septate ascospores. Anamorphs are dematiaceous, and include mycelial (phragmo- to dictyoconidia), spermatial and pycnidial synanamorphs (Hughes 1976, Cheewangkoon et al. 2009).

The Mycosphaerellaceae was treated as a family in the Dothideales by Hawksworth et al. (1995), while Kirk et al. (2001) introduced a separate order, the Mycosphaerellales for this family, and Kirk et al. (2008) again placed it in the Capnodiales. The Mycosphaerellaceae is recognised by having characteristic pseudothecial ascomata that can be immersed or superficial, embedded in host tissue or erumpent, having ostiolar periphyses, but lacking interascal tissue at maturity. Ascospores are hyaline, but in some cases slightly pigmented (Barr 1987), and predominantly 1-septate, although some taxa with 3-septate ascospores have been recorded (Crous et al. 2003). Although up to 30 anamorph genera have been linked to Mycosphaerella (Crous et al. 2000, 2001, 2007a, b, c, 2009a, b, c, Crous 2009), recent studies have shown this to be incorrect, and that the family in fact consists of numerous genera with morphologically conserved Mycosphaerella-like teleomorphs, and distinct anamorphs (Crous et al. 2007a, b, 2009b, c).

Families tentatively placed in the Capnodiales (Lumbsch & Huhndorf 2007, Kirk et al. 2008) include epiphytes (Antennulariellaceae, Capnodiaceae, Metacapnodiaceae) (Hughes 1976), saprobes and plant pathogens (Davidiellaceae, Dissoconiaceae, Mycosphaerellaceae, Schizothyriaceae, Teratosphaeriaceae) (Aptroot 2006, Crous 2009), and colonisers or hair shafts of mammals (Piedraiaceae) (de Hoog et al. 2000). To address the status of the Capnodiales as an order, and the intrafamilial relationships within this order, DNA sequences of the 18S, 5.8S and 28S nrRNA genes were generated for a set of specifically selected taxa. A further aim was to clarify genera within these families, and resolve anamorph-teleomorph relationships for the taxa investigated.

	MATERIALS AND METHODS

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
Isolates
Isolates were selected (Table 1 - see online Supplementary Information) that are representative of the Mycosphaerellaceae (Crous 1998, Crous et al. 2004a, c, 2006a, b, 2007a), Schizothyriaceae (Batzer et al. 2005, 2007), Teratosphaeriaceae (Crous et al. 2007a, 2008b, c, 2009a, b, c), Piedraiaceae (Kruys et al. 2006), Davidiellaceae (Braun et al. 2003, Schubert et al. 2007a, b), Capnodiaceae (Schoch et al. 2006), as well as numerous other genera for which the familial relationships have remained unclear, such as the Phaeophleospora complex (Crous et al. 1997, 2007a, 2009b, c, Andjic et al. 2007), Polythrincium (Simon et al. 2009), the Dissoconium complex (Crous et al. 2004c, 2007c, 2008b, Arzanlou et al. 2008b), and several less well-known genera represented by one or two species only. For fresh material excised leaf spots bearing ascomata were soaked in water for approximately 2 h, after which they were placed in the bottom of Petri dish lids, with the top half of the dish containing 2 % malt extract agar (MEA; Crous et al. 2009d). Ascospore germination patterns were examined after 24 h, and single-ascospore and conidial cultures established as described by Crous et al. (1991). Colonies were sub-cultured onto synthetic nutrient-poor agar (SNA), potato-dextrose agar (PDA), oatmeal agar (OA), MEA (Crous et al. 2009d), and incubated at 25 °C under continuous near-ultraviolet light to promote sporulation. Other cultures were obtained from the culture collection of the Centraalbureau voor Schimmelcultures (CBS-KNAW) in Utrecht, the Netherlands or the working collection of Pedro Crous (CPC).

DNA isolation, amplification and molecular phylogeny
Genomic DNA was extracted from mycelium taken from fungal colonies on MEA using the UltraClean^TM Microbial DNA Isolation Kit (Mo Bio Laboratories, Inc., Solana Beach, CA, U.S.A.). A part of the nuclear rDNA operon spanning the 3' end of the 18S rRNA gene (SSU), the first internal transcribed spacer (ITS1), the 5.8S rRNA gene, the second ITS region (ITS2) and the first 900 bp at the 5' end of the 28S rRNA gene (LSU) was amplified and sequenced as described by Cheewangkoon et al. (2008) standard for all strains included (Table 1). For selected strains (see Table 1), the almost complete SSU and LSU (missing the first and last 20–30 nucleotides) were amplified and sequenced using novel and previously published primers (Table 2; see below).

Novel primers were designed using a variety of complete SSU and LSU sequences obtained from the GenBank sequence database (www.ncbi.nlm.nih.gov/). The selection was not limited only to fungi belonging to the Dothideomycetes but encompassed as many as possible full sequences in order to make the primers as robust as possible. We aimed to keep the melting temperature (Tm) of the novel primers at 40–45 °C and the GC content to approximately 50 % to keep them as compatible as possible to existing published primers. Primer parameters were calculated using the OligoAnalyzer tool on the web site of Integrated DNA Technologies (http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/) with the "Oligo Conc" parameter set at 0.2 mM and the "Na+ Conc" parameter set at 16 mM. A framework of existing and novel primers was then aligned onto the sequence of Magnaporthe grisea (GenBank accession AB026819 [GenBank] ) to derive primer positions (Table 2) and evaluate coverage over the gene regions. These primers were amplified and sequenced in the following overlapping sections to cover the almost complete SSU and LSU for the selected strains (Table 2): SSU1Fd or SSU6Fm with SSU2Rd, SSU2Fd with SSU3Rd, SSU7Fm with SSU4Rd or SSU6Rm, SSU4Fd with 5.8S1Rd, V9G or LSU1Fd with LSU3Rd, LSU8Fd with LSU8Rd, LSU4Fd with LSU5Rd, and LSU5Fd with LSU7Rd. For some strains (Table 3) it was necessary to add an additional overlap for SSU4Fd with 5.8S1Rd (using SSU4Fd with SSU7Rm and SSU8Fm with 5.8S1Rd), for LSU8Fd with LSU8Rd (using LSU8Fd with LSU3Rd and LSU3Fd with LSU8Rd), and for LSU5Fd with LSU7Rd (using LSU5Fd with LSU6Rd and LSU6Fd with LSU7Rd) to complete the gaps due to large insertions.

The internal transcribed spacer regions, as well as all insertions (Table 3) were excluded from all analyses. Sequence data were deposited in GenBank (Table 1) and alignments in TreeBASE (www.treebase.org). Two separate analyses were performed: The first using only partial LSU data due to the limited number of complete LSU sequences available and the second using the almost complete SSU, 5.8S nrDNA and LSU alignment.

Maximum likelihood analyses (ML) were conducted in RAxML v. 7.0.4 (Stamatakis 2006) for the partial LSU alignment. A general time reversible model (GTR) with a discrete gamma distribution and four rate classes was applied. A tree was obtained by simultaneously running a fast bootstrap search of 1000 pseudoreplicates (Stamatakis et al. 2008) followed by a search for the most likely tree. Maximum Likelihood bootstrap value (MLBP) equal or greater than 70 % are given at the nodes (Fig. 1).

View larger version (195K): [in this window] [in a new window]   Fig. 1. RAxML tree using only the partial LSU alignment with bootstrap values after 1 000 pseudorepetitions on the nodes. Type strains and novel species described in this study are indicated in bold.

View larger version (143K): [in this window] [in a new window]   Fig. 2. RAxML tree using the SSU, 5.8S nrDNA and LSU alignment with bootstrap values after 500 pseudorepetitions on the nodes.

	RESULTS

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
DNA amplification and phylogeny
Amplification products of approximately 1 700 bases were obtained for the standard amplification of the isolates listed in Table 1. The LSU region of these sequences was used to obtain additional sequences from GenBank that were added to the partial LSU alignment. We expected a total size of approximately 5 500 bp for the concatenated SSU, ITS1, 5.8S nrDNA, ITS2 and LSU at the start of the study; however, our alignment totalled about 12 000 bp due to numerous insertions (most likely group 1 introns) encountered for several strains (Table 3). These insertions frequently resulted in products too large to amplify or sequence effectively and sometimes required us to design additional novel primers in extra overlapping steps to complete these gaps (see Materials and Methods for details). Searching the GenBank database using these insertions had varied success (Table 3). Sequences of the 18S nrDNA are more abundant in the database whereas sequences of the second half to two-thirds of the 28S nrDNA are mostly absent. This also evident in Table 3, where insertions in the SSU more frequently found with similarity sequences in the database and insertions in the LSU (e.g. those between positions 5018–5019 and 5424–5425) frequently did not retrieve any significant similarity. Although there were some exceptions (e.g. the insertion between 1820 and 1821 in the SSU of Batcheloromyces leucadendri), most of the insertions in the SSU obtained hits with SSU sequences of species of Capnodiales in the database. In one case, between 954 and 955 for the SSU sequence of Teratosphaeria mexicana (both strains), a partial hit was obtained with an LSU sequence of Lipomyces spencermartinsiae (GenBank DQ518980 [GenBank] ). Many of the insertions in the LSU sequences did not retrieve significant hits in the database and those that did were with unrelated taxa. It is quite possible that this is an artifact of the poor representation of full-length LSU sequences in the database, especially for members of the Capnodiales. In some cases, an LSU insertion retrieved a hit with SSU sequences in the database, e.g. the insertion between 5538 and 5539 in Batcheloromyces proteae and between 3560 and 3561 and 4875 and 4876 in Teratosphaeria mexicana strain CBS 120744. In two cases (Mycosphaerella latebrosa and Phaeophleospora eugeniicola), an insertion was either lost or gained between two strains of the same species. The primers designed in this study allowed us to effectively amplify and sequence the SSU and LSU for the selected isolates. Althought these primers were not tested on taxa outside of the Capnodiales (except for one of the outgroups, Neofusicoccum australe), we attempted to design them as robust as possible using degeneracy if needed. We therefore expect that these primers will have wider applicability than just the Capnodiales in cases where other published primers fail to amplify or amplify poorly.

The RAxML search of the partial LSU alignment yielded a most likely tree (Fig. 1) with a log likelihood -13397.994021. The matrix had 395 distinct alignment patterns, with 6 % completely undetermined characters in the alignment. The manually adjusted alignment contained 295 sequences (including the outgroup sequence, Dothidea insculpta GenBank DQ247802 [GenBank] ) and 763 characters including alignment gaps. The RAxML search of the almost complete SSU, 5.8S nrDNA and LSU alignment yielded a most likely tree (Fig. 2) with a log likelihood -39022.881140. The matrix had 1211 alignment patterns with 0.01 % of the characters consisting of gaps or undetermined characters. The manually adjusted alignment contained 205 sequences (including the outgroup sequences, Neofusicoccum australe CPC 10899 and Magnaporthe grisea GenBank AB026819 [GenBank] ) and 5110 characters including alignment gaps. The obtained phylogenies (Figs 1, 2) are discussed in the Taxonomy section below.

Taxonomy
Several well-supported clades could be distinguished in the present study (Figs 1, 2), correlating to families in the Capnodiales. These families, and several new genera and species, are treated below.

Treatment of phylogenetic clades
Capnodiales Woron. Ann. Mycol. 23: 177. 1925.

Data obtained from multi-gene phylogenies prompted Schoch et al. (2006) to merge Mycosphaerellales with Capnodiales. Although the present study included numerous additional isolates, the orders remain problematic. Although there is support for the Mycosphaerellales as an order, additional families such as the Schizothyriaceae and Dissoconiaceae (see below) would have to also be elevated to order level, which would result in orders containing a single family, while Teratosphaeriaceae appears to comprise unresolved lineages. For this reason it was decided to retain these families within Capnodiales, but noting that as more families are added and better circumscribed, it is quite possible that the Mycosphaerellales would again be resurrected.

Mycosphaerellaceae Lindau, In: Engler & Prantl, Nat. Pflanzenfamilien 1(1): 421. 1897.

Type species: Mycosphaerella punctiformis (Pers.: Fr.) Starbäck, Bih. Kongl. Svenska Vetensk.-Akad. Handl. 15(3, 2): 9. 1889.

Notes: The Mycosphaerellaceae contains numerous genera, 20 of which are listed by Crous (2009), with many names under consideration (Crous et al. 2009b, c). From these data it is clear that genera such as Passalora, Pseudocercospora, Pseudocercosporella, Septoria, Zasmidium and Ramichloridium are paraphyletic (Hunter et al. in prep.). Well-resolved genera include Cercospora, Cercosporella, Ramularia, Ramulispora, Sonderhenia and Polythrincium. One particularly problematic clade contains Periconiella, Ramichloridium, Verrucisporota and Zasmidium, along with "Mycosphaerella" and Rasutoria teleomorphs. Barr (1987) erected Rasutoria for species with brown ascospores occurring on Gymnospermae. Rasutoria clusters in a clade adjacent to "Mycosphaerella" species with hyaline ascospores, such as M. aleuritidis and Mycosphaerella daviesiicola (Verrucisporota daviesiae) (Beilharz & Pascoe 2002).

The genus Phaeophleospora (1916) clusters with Lecanosticta acicola. The genus Lecanosticta (1922) has typical Phaeophleospora-like conidia, except that its conidiomata are acervular, and not pycnidial. If the type of Lecanosticta, L. pini also clusters in this clade, the generic concept Phaeophleospora may have to be widened to include Lecanosticta, as was done with Kirramyces to include Colletogloeopsis (Cortinas et al. 2006a, b).

Considerable controversy has surrounded the coelomycetes that Crous et al. (1997) placed in Phaeophleospora. Based on DNA phylogenetic data, it has now been shown that Kirramyces anamorphs (Walker et al. 1992), including those accommodated in Colletogloeopsis (Crous & Wingfield 1996, Crous et al. 2004c, 2006c, Cortinas et al. 2006a, b), are linked to Teratosphaeria (Andjic et al. 2007, Crous et al. 2009b, c). Crous et al. (2007a) showed Phaeophleospora to reside in the Mycosphaerellaceae and Kirramyces in the Teratosphaeriaceae, respectively. However, most taxa investigated to date were collected from Eucalyptus. As shown in the present study, Phaeophleospora atkinsonii, a pathogen of Hebes spp. (Wu et al. 1996, Pennycook & McKenzie 2002), clusters distant from Phaeophleospora s. str., while the same is true for Phaeophleospora concentrica, which is a pathogen of Protea spp. (Taylor et al. 2001a), and Phaeophleospora stonei, a pathogen of Eucalyptus (Crous et al. 2007c, 2009c). These taxa thus clearly represent yet another two genera in the Phaeophleospora complex. An older name that would potentially be available is Scoleciasis. However, when B. Sutton examined exsiccati of the type species, S. aquatica, only ascomata of a Leptosphaeria species were found (Crous et al. 1997). The association of S. aquatica with the Leptosphaeria was also noted in the original description, and this may indicate that Scoleciasis is allied to taxa in the Phaeosphaeriopsis/Phaeoseptoria complex (Arzanlou & Crous 2006). Both P. atkinsonii and P. concentrica have a typical Kirramyces morphology, namely brown, percurrently proliferating conidiogenous cells, and brown, obclavate, verruculose, transversely euseptate conidia. Further species thus need to be included in analyses before these generic concepts can be clarified.

During the course of this study several fresh collections of Leptosphaeria protearum were obtained. Leptosphaeria protearum is a major leaf spot and blight pathogen of Protea spp. (Knox-Davies et al. 1987), and causes severe losses in plantations of South African Protea spp. in Hawaii, and has been recorded in many countries where South African proteas are cultivated (Taylor & Crous 1998, Taylor et al. 2001b, Crous et al. 2004a). Cultures of this pathogen were found to cluster in the Mycosphaerellaceae, where they represent an undescribed genus, characterised by having bitunicate asci without pseudoparaphyses, brown, 3-septate ascospores, and a Coniothyrium-like anamorph. Its close phylogenetic relationship to Phaeophloeospora concentrica (Fig. 1) suggests that they could be congeneric, and that in future more Phaeophloeospora-like anamorphs may be found to cluster in this clade. We propose a new genus to accommodate Leptosphaeria protearum below.

Brunneosphaerella Crous, gen. nov. MycoBank MB514694.

Etymology: Brunneus + Sphaerella = is after its brown ascospores and Sphaerella-like morphology.

Mycosphaerellae similis, sed ascosporis brunneis, 3-septatis.

Ascomata amphigenous, immersed to semi-immersed, black, single, gregarious, substomatal, pyriform or globose with a papillate, periphysate ostiole. Peridium consisting of three strata of slightly compressed textura angularis, an outer stratum of dark brown, thick-walled cells, becoming paler in the central stratum, and hyaline, thin-walled in the inner stratum. Asci clavate to cylindro-clavate, often curved, tapering to a pedicel, narrowing slightly to a rounded apex with an indistinct ocular chamber, 8-spored, bitunicate with fissitunicate dehiscense. Pseudoparaphyses absent. Ascospores biseriate, fusiform, broader at the apical end, initially hyaline and 1-septate, becoming yellow-brown and 3-septate at maturity, slightly constricted at median to supra-median septum.

Type species: Brunneosphaerella protearum (Syd. & P. Syd.) Crous, comb. nov.

Brunneosphaerella jonkershoekensis (Marinc., M.J. Wingf. & Crous) Crous, comb. nov. MycoBank MB514695. Fig. 3.

View larger version (117K): [in this window] [in a new window]   Fig. 3. Brunneosphaerella jonkershoekensis. A–B. Vertical sections through ascomata showing wall structure. C–D, G. Bitunicate asci. E–F. Ascospores. Scale bars: A, C = 50 µm, B = 20 µm, D, G = 10 µm, E–F = 5 µm (from Marincowitz et al. 2008).

Ascomata pseudothecial, subepidermal, immersed, obpyriform, papillate, 180–205 x 160–235 µm. Peridium 20–30 µm thick, composed of relatively large cells, 11–15 x 2.5–5.5 µm; cells arranged in three strata; outer stratum consisting of 3–5 layers of dark brown, very thick-walled cells; middle stratum transient, consisting of a few layers of pale brown, thick-walled, compressed cells; inner stratum consisting of 1–2 layers of thin-walled, very compressed cells. Pseudoparaphyses absent. Asci bitunicate, inflated cylindrical to clavate, 81–95 x 13–15 µm, ocular chamber dome-shaped, indistinct. Ascospores pale brown, fusoid to ellipsoidal, tapering towards the base, (25–)29–34(–36) x (5–)6–7(–9) µm (av. 31.4 x 6.7 µm), apical cell the shortest, upper hemispore slightly larger than lower, at times slightly curved, 3-septate, smooth, guttulate (adapted from Marincowitz et al. 2008).

Host range and geographic distribution: Protea repens (South Africa, Western Cape) (Marincowitz et al. 2008).

Specimen examined: south Africa, Western Cape Province, Jonkershoek Nature Reserve, leaf litter of Protea repens, 6 Jun. 2000, S. Marincowitz, PREM 59447 holotype.

Notes: Although no culture is presently available for this species, it clearly represents a species of Brunneosphaerella, characterised by its bitunicate asci, and brown, 3-septate ascospores, as well as the absence of pseudoparaphyses. Brunneosphaerella jonkershoekensis can easily be distinguished from B. protearum based on its much larger ascospores (Crous et al. 2004a).

Brunneosphaerella protearum (Syd. & P. Syd.) Crous, comb. nov. MycoBank MB514696. Fig. 4.

View larger version (129K): [in this window] [in a new window]   Fig. 4. Brunneosphaerella protearum. A–D. Leaf spots on different Protea spp. E. Close up of leaf spot showing ascomata. F. Substomatal ascomata. G–H. Vertical sections though ascomata, showing wall structure. I–K. Germinating ascospores on MEA. L–M, R. Bitunicate asci. N–Q, S. Juvenile to mature ascospores. Scale bars: G = 75 µm, H = 10 µm.

Anamorph: "Coniothyrium" protearum Joanne E. Taylor & Crous, IMI Descriptions of Fungi and Bacteria No. 1343. 1998.

Leaf spots circular to irregular, discrete to confluent, variable in size, under conditions favourable to disease symptoms more similar to a blight than a leaf spot, necrotic, sunken with a raised dark brown margin and with conspicuous black ascomata in the dead tissue, 4–30 mm diam. Ascomata pseudothecial, substomatal, amphigenous, immersed to semi-immersed, not erumpent, black, single, gregarious, 180–320 µm diam; in section, substomatal, subepidermal, pyriform or globose with a papillate, periphysate ostiole, immersed in a stroma consisting of deteriorated host mesophyll cells filled with fungal hyphae, (210–)230–264(–288) µm high, (180–)200–255(–300) µm diam. Peridium consisting of three strata of slightly compressed textura angularis, an outer stratum of dark brown, thick-walled cells, becoming paler in the central stratum, and hyaline, thin-walled in the inner stratum, altogether (20–)24.5–37.5(–50) µm thick. Asci clavate to cylindro-clavate, often curved, tapering to a pedicel, narrowing slightly to a rounded apex with an indistinct ocular chamber, 8-spored, bitunicate with fissitunicate dehiscense, (70–)80–87.5(–105) x (13.5–)14.5–16(–21.5) µm. Pseudoparaphyses absent. Ascospores biseriate, fusiform, broader at the apical end, initially hyaline and 1-septate, becoming yellow-brown and 3-septate at maturity, slightly constricted at median to supra-median septum, (21.5–)27.5–29.5(–37.5) x (6.3–)7.5–8(–10) µm in water mounts, (21–)25.5–27.5(–31) x (5.5–)6–7(–8) µm in lactophenol. Conidiomata barely visible and interspersed between ascomata, pycnidial, subepidermal, substomatal, separate, globose to pyriform, occasionally with well-developed papilla, dark brown, < 200 µm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells discrete, smooth, hyaline, doliiform to ampulliform, holoblastic, proliferating 1–2 times percurrently, 4–6 x 3–4 µm. Conidia pale brown to medium brown, thick-walled on maturity, smooth to finely verruculose, eguttulate, ellipsoidal to globose, often truncate at one end, 5–10 x 3–7 µm (adapted from Crous et al. 2004a).

Host range and geographic distribution: Protea cynaroides, P. `Susara' (Portugal, Madeira) (Moura & Rodrigues 2001); P. caffra, P. compacta, P. cynaroides, P. gaguedi, P. grandiceps, P. lacticolor, P. laurifolia, P. lepidocarpodendron, P. lorifolia, P. magnifica, P. nitida, P. punctata, P. repens, P. `Sheila', Protea spp. (South Africa); P. cynaroides, P. laurifolia, P. neriifolia, P. `Ivory Musk', P. `Mink', P. `Pink Ice', P. `Rose Mink', P. susannae, Protea sp. (U.S.A., Hawaii) (Taylor et al. 2001b); P. cynaroides, P. gaguedi, P. neriifolia, Protea sp. (Zimbabwe, Inyanga) (Masuka et al. 1998).

Specimens examined: south Africa, Western Cape Province, Bettys' Bay, leaf litter of Protea magnifica, 11 Jul. 2000, S. Marincowitz, PREM 59448; Helderberg Nature Reserve, leaf litter of Protea laurifolia, 14 Aug. 2000, S. Marincowitz, PREM 59482; Helderberg Nature Reserve, leaf litter of Protea obtusifolia, 14 Aug. 2000, S. Marincowitz, PREM 59495; Jonkershoek Nature Reserve, leaf litter of Protea nitida, 6 Jun. 2000, S. Marincowitz, PREM 59442; Jonkershoek Nature Reserve, leaf litter of Protea repens, 6 Jun. 2000, S. Marincowitz, PREM 59450; Jonkershoek Nature Reserve, S33°59'11.2" E18°57'14.7" leaves of Protea sp., 1 Apr. 2007, P.W. Crous, CBS H-20330, cultures CPC 13914–13916; Jonkershoek Nature Reserve, S33°59'26.1" E18°57'59.5" leaves of Protea repens, 1 Apr. 2007, P.W. Crous, CBS H-20331, cultures CPC 13911–13913; Jonkershoek Nature Reserve, leaves of Protea sp., 1 Apr. 2007, P.W. Crous, CBS H-20332, cultures CPC 13908–13910; Jonkershoek Nature Reserve, "Tweede Waterval", leaves of Protea sp., 1 Apr. 2007, P.W. Crous, CBS H-20333, cultures CPC 13902–13907; Jonkershoek Nature Reserve, leaves of Protea nitida, 12 Apr. 2008, L. Mostert, CBS H-20334, cultures CPC 15231–15233; Kirstenbosch Botanical Garden, leaves of Protea sp., 13 Jan. 2009, P.W. Crous, CBS H-20335, culture CPC 16338.

Notes: Although Taylor & Crous (1998) reported a Coniothyrium-like anamorph to develop in culture, none of the cultures examined in the present study on MEA, PDA or OA could be induced to sporulate, though spermatogonia and ascomatal initials were commonly observed.

The fact that cultures of Leptosphaeria protearum, which represents a well-known and serious pathogen of Proteaceae, clustered in the Mycosphaerellaceae, was totally unexpected. A further surprise was the fact that this species appears to represent a complex of several cryptic taxa. Whether these taxa can be correlated with differences in host range and geographic distribution can only be resolved once more collections have been obtained for study. Although the genus Sphaerulina, which represents Mycosphaerella-like taxa with 3-septate, hyaline ascospores, is part of the Mycosphaerellaceae (Crous et al., unpubl data), the type species, S. myriadea, clusters in the Septoria clade, and is thus unavailable for the species occurring on Proteaceae. Morphologically Brunneosphaerella is also distinct in that ascospores are always brown at maturity, and anamorphs have brown, percurrently proliferating conidiogenous cells, appearing Phaeophleospora-like. The recognition of Brunneosphaerella as a distinct genus in the Mycosphaerellaceae also raises the intriguing possibility that many phytopathogenic species of the Leptosphaeria-complex with brown, 3-septate ascospores, but lacking paraphyses, actually belong to Brunneosphaerella.

Passalora ageratinae Crous & A.R. Wood, sp. nov. MycoBank MB514697. Fig. 5.

View larger version (96K): [in this window] [in a new window]   Fig. 5. Passalora ageratinae. A. Leaf spots. B. Close up of leaf spot with fruiting structures. C–D. Conidiophores. E–J. Conidia. Scale bars = 10 µm.

Passalorae assamensis similis, sed coloniis amphigenis, sine mycelio externo, conidiophoris brevioribus, 15–40 x 3–4.5 µm.

Leaf spots amphigenous, angular to irregular, 2–8 mm diam, medium brown, frequently with pale to grey-brown central part, and raised, dark brown border; pale to medium brown in reverse, with raised, dark brown border. Mycelium internal, consisting of smooth, branched, pale brown, 2–3 µm wide hyphae. Caespituli fasciculate, amphigenous, medium brown, arising from a brown, erumpent stroma, up to 80 µm wide, 40 µm high. Conidiophores subcylindrical, straight to geniculous-sinuous, unbranched, medium brown, finely verruculose, 1–3-septate, 15–40 x 3–4.5 µm. Conidiogenous cells terminal, pale to medium brown, finely verruculose with terminal, sympodial conidiogenous loci that are 1–2 µm diam, slightly thickened, darkened and refractive, 10–20 x 3–4 µm. Conidia in unbranched chains, pale brown, smooth, finely to prominently guttulate, subcylindrical to narrowly obclavate, apex obtuse, base long obconically subtruncate, (0–)1–3(–5)-septate, (20–)30–60(–80) x (3–)4(–4.5) µm; hila 1–1.5 µm wide, somewhat thickened, darkened and refractive.

Culture characteristics: On MEA erumpent, with uneven, folded surface, lobate margin, and moderate aerial mycelium; centre pale mouse-grey with patches of cinnamon, outer margin olivaceous-grey; reverse olivaceous-grey with patches of cinnamon; reaching 15 mm diam; on PDA spreading, with cinnamon to cream patches in centre, becoming umber towards smooth margins, with diffuse red pigment in agar; reverse olivaceous-grey, with patches of red, reaching 15 mm diam; on OA flat, spreading, up to 30 mm diam, iron-grey, with white, solitary mycelia strands, though aerial mycelium generally absent, reaching 30 mm diam.

Host range and geographic distribution: Ageratina adenophora, Australia, South Africa.

Specimen examined: south Africa, KwaZulu-Natal Province, Hilton, on leaves of Ageratina adenophora, 28 May 2008, A.R. Wood, CBS H-20336 holotype, cultures ex-type CPC 15365 = CBS 125419, CPC 15366, 15367.

Notes: Ageratina adenophora (crofton weed; Asteraceae), which is indigenous to Mexico, has invaded many countries as a rapidly growing weed, forming dense thickets (Morris 1989, Parsons & Cuthbertson 1992, Wagner et al. 1999, Zhu et al. 2007, Muniappan et al. 2009). It is considered a serious weed in agriculture and forestry (Bess & Haramoto 1958, Sharma & Chhetri 1977, Kluge 1991), often replacing more-desired vegetation or native species.

A leaf spot pathogen, originally misidentified as Cercospora eupatorii (this species is currently known as Pseudocercospora eupatorii), was found to infect plants in Australia where a stem galling fly (Procecidochares utilis; Tephritidae) was introduced from Hawaii as a biological control agent (Dodd 1961). Presumably the fungus was introduced together with the flies originally from Mexico to Hawaii and then to Australia. Subsequently this same fungus was obtained from Australia and released in South Africa after host specificity testing indicated it was restricted to A. adenophora (Morris 1989). The fungus causes partial defoliation of mature plants (Dodd 1961, Auld 1969), though the impact depends on environmental conditions (Dodd 1961). Seedlings are however killed rapidly (Wang et al. 1997).

This fungus, which has hitherto been known simply as "Phaeoramularia" sp., still lacks a name and proper description. The genus Phaeoramularia is treated as a synonym of Passalora (Crous & Braun 2003), and hence the species is named in the latter genus as P. ageratinae. Interestingly, this species appears to be closely related to Passalora fulva, which is a serious pathogen of tomato (Solanaceae) (Thomma et al. 2005).

Passalora armatae Crous & A.R. Wood, sp. nov. MycoBank MB514698. Fig. 6.

View larger version (105K): [in this window] [in a new window]   Fig. 6. Passalora armatae. A. Fruiting in vivo. B–C. Caespituli with prominent basal stroma. D. Sporulation on MEA. E. Conidiogenous cells giving rise to conidia. F–G. Conidia. Scale bars: B = 125 µm, C–E = 10 µm.

Passaloraea dalbergiicolae similis, sed conidiophoris in synnematibus densis, conidiis ad basim obconice truncatis, apice rostrato.

Leaf spots amphigenous, on upper surface visible as red-brown, irregular to subcircular spots with indistinct margins, 0.5–2 mm diam; in reverse indistinct, chlorotic to medium or red-brown. Mycelium internal, consisting of smooth, branched, pale brown, 2–3 µm wide hyphae. Caespituli hypophyllous, fasciculate to synnematous, up to 200 µm high and 250 µm wide, situated on a prominently erumpent, pale brown stroma, up to 100 µm high and wide. Conidiophores subcylindrical, unbranched, flexuous, guttulate, pale to medium brown, smooth, 120–180 x 4–6 µm, 2–6-septate. Conidiogenous cells terminal, subcylindrical, guttulate, pale to medium brown, finely verruculose, becoming somewhat swollen, appearing slightly clavate, 25–70 x 6–8 µm; conidiogenous loci 4–20 per conidiogenous cell, sympodial, round, darkened, thickened, refractive, prominent, 2–3 µm wide, up to 1 µm high. Conidia (27–)30–40(–45) x 9–10(–12) µm, pale to medium brown, smooth to finely verruculose, granular to guttulate, thin-walled, ellipsoidal to obovoid, transversely 2–4-euseptate, widest in middle of basal cell, or middle of conidium, tapering to an obconically truncate base; hilum thickened, darkened and refractive; apical cell conical, elongating to an apical beak up to 20 µm long. When cultivated conidia remain attached to conidiogenous cells, giving conidiophores the appearance of small tufts which is very characteristic, and not commonly observed in Passalora.

Culture characteristics: On MEA slow growing, erumpent, with dense white aerial mycelium, which becomes mouse-grey, reaching 5 mm diam after 1 wk; on PDA mouse-grey (surface), iron-grey (reverse), with diffuse red pigment in agar; on OA similar to PDA, also with diffuse red pigment in agar.

Host range and geographic distribution: Dalbergia armata, South Africa.

Specimen examined: south Africa, KwaZulu-Natal Province, South Coast, Mpenjati Nature Reserve, between Ramsgate and Port Edward, on leaves of Dalbergia armata, 28 May 2008, A.R. Wood, CBS H-20337 holotype, cultures ex-type CPC 15419 = CBS 125420, CPC 15420, 15421.

Notes: Passalora dalbergiae, which occurs on Dalbergia sissoo (Fabaceae) in India, is distinct from P. armatae in having superficial mycelium and solitary conidiophores (Hernández-Gutiérrez & Dianese 2009). The previously described Passalora dalbergiicola is similar to P. armatae in conidial dimensions (3-septate, 25–45 x 7–10 µm; Ellis 1976), but distinct in that conidiophores are not in dense synnemata, conidiogenous cells can have single apical loci, and conidia have a less prominent basal taper, and lack the apical beaks typical of P. armatae (in vivo and in vitro).

Schizothyriaceae Höhn. ex Trotter, Sacc., D. Sacc. & Traverso, In: Saccardo, Syll. Fung. 24(2): 1254. 1928.

Type species: Schizothyrium acerinum Desm., Ann. Sci. Nat. Bot. 11: 360. 1849.

Notes: Members of the Schizothyriaceae are associated with flyspeck symptoms on apples and pear fruit. The fungi grow superficially on the epicuticular wax, thereby reducing the marketability of the fruit, but do not penetrate the cuticle (Belding et al. 2000). Batzer et al. (2005, 2007) reported a range of diverse fungi to be associated with flyspeck symptoms on apples, the most prominent being species of Schizothyrium.

Dissoconiaceae Crous & de Hoog, fam. nov. MycoBank MB514699.

Ascomata pseudotheciales, immerse, globosa, uniloculares. Sine pseudoparaphysibus. Asci fasciculati, octospori, bitunicati. Ascosporae ellipsoideae-fusiformes, 1-septatae, hyalinae. Conidiophora separata, ex hyphis oriunda, subcylindrica, subulata, lageniformia vel cylindrica, apicem versus attenuata, apice obtuse rotundato vel truncate, recta vel semel geniculata, laevia, modice brunnea, 0–pluriseptata, locis terminalibus vel lateralibus, rhachidi cum cicatricibus leniter incrassates, fuscatis. Conidia solitaria, pallide olivaceo-brunnea, laevia, ellipsoidea, obclavata vel globosa, 0–1-septata, hilis aliquantum fuscatis. Conidia secundaria nulla vel formata ad conidia primaria, pallide olivacea vel subhyalina, aseptata, pyriformia; conidiis impigre vel passive emittentibus.

Ascomata pseudothecial, immersed, globose, unilocular, papillate, ostiolate, canal periphysate; wall consisting of 3–4 layers of brown textura angularis; inner layer of flattened, hyaline cells. Pseudoparaphyses absent. Asci fasciculate, 8-spored, bitunicate. Ascospores ellipsoid-fusoid, 1-septate, hyaline, with or without mucoid sheath. Mycelium internal and external, consisting of branched, septate, smooth, hyaline to pale brown hyphae. Conidiophores separate, arising from hyphae, subcylindrical, subulate or lageniform to cylindrical, tapering to a bluntly rounded or truncate apex, straight to once geniculate, smooth, medium brown, 0–multi-septate; loci terminal and lateral, visible as slightly thickened, darkened scars on a rachis. Conidia solitary, pale olivaceous-brown, smooth, ellipsoid to obclavate or globose, 0–1-septate; hila somewhat darkened. Secondary conidia present or absent; developing adjacent to primary conidia, pale olivaceous to subhyaline, aseptate, pyriform; conidium discharge active or passive.

Type species: Dissoconium aciculare de Hoog, Oorschot & Hijwegen, Proc. K. Ned. Akad. Wet., Ser. C, Biol. Med. Sci. 86(2): 198. 1983.

Notes: Species of Dissoconium have Mycosphaerella-like teleomorphs (Crous et al. 2004c). The genus is characterised by forming conidia in pairs that are forcefully discharged, which is quite unique in the Capnodiales (de Hoog et al. 1983). Although D. aciculare, the type species of Dissoconium, was originally assumed to be hyperparasitic on powdery mildew (de Hoog et al. 1983), Jackson et al. (2004) revealed that another species, D. dekkeri, could act as a foliar pathogen of Eucalyptus. Dissoconium dekkeri is, however, most commonly found in leaf spots in association with other species of Teratosphaeria and Mycosphaerella. Species of Dissoconium remain commensalists, and frequently occur asexually on lesions associated with pathogenic species of Capnodiales (Crous unpubl. data). They are ecologically and morphologically quite distinct from other members of the Capnodiales, and hence a separate family, the Dissoconiaceae, is herewith introduced to accommodate them. Ramichloridium forms brown, solitary conidiophores with a rachis and apical loci similar to that observed on Dissoconium, and primary conidia that are pale brown, 0–1-septate, with slightly thickened hila, but lacks secondary conidia (Arzanlou et al. 2008b). Both Dissoconium and Ramichloridium have in the past been reported as hyperparasitic on powdery mildews on various hosts (Hijwegen & Buchenauer 1984), which suggests that they share a similar ecology.

Teratosphaeriaceae Crous & U. Braun, Stud. Mycol. 58: 8. 2007.

Type species: Teratosphaeria fibrillosa Syd. & P. Syd., Ann. Mycol. 10: 40. 1912.

Notes: Since the family was established by Crous et al. (2007a) it has been shown to be too widely defined, incorporating many diverse genera (Crous et al. 2009b, c), and even families such as the Piedraiaceae (Fig. 1). The node as such is not well supported, suggesting that as more taxa are added, further families remain to be separated from the Teratosphaeriaceae. Presently it incorporates diverse elements, and even lichens such as Cystocoleus ebeneus and Anisomeridium consobrinum. The identity of the latter strain (CBS 101364) needs to be confirmed, as its position in the tree appears doubtful.

The genus Catenulostroma, which is associated with numerous diverse substrates and habitats (Crous et al. 2007a), is typified by C. protearum, for which an epitype is designated in the present study. Several strains isolated from rock surfaces (Guiedan et al. 2008, Ruibal et al. 2008, 2009, this volume) cluster with Catenulostroma (Fig. 1), and appear to represent undescribed species of the latter. Of interest is the fact that the type species of Aulographina, A. pinorum (CBS 302.71, CBS 174.90), which has hysterothecia, clusters in a clade with Catenulostroma microsporum, which has a Teratosphaeria-like teleomorph with pseudothecia (Taylor & Crous 2000, Crous et al. 2004a, 2007a). Isolates of A. pinorum were found to produce a Catenulostroma anamorph in culture. This raises two possibilities, namely that either the incorrect fungus was originally isolated from pine needles (namely Catenulostroma abietis), or that this is a species complex, in which A. pinorum resides. If these strains are indeed confirmed to represent A. pinorum, then it reveals the genus Aulographina to be heterogeneous, as A. eucalypti, which is a major leaf spot pathogen of Eucalyptus (Crous et al. 1989, Park et al. 2000, Carnegie & Keane 2003), clusters distant from A. pinorum. The taxonomy of these taxa is currently being addressed, and will be reported on elsewhere (Cheewangkoon et al., in prep.). During the course of this study some new members of the Teratosphaeriaceae were collected, which are described below: Catenulostroma protearum (Crous & M.E. Palm) Crous & U. Braun, Stud. Mycol. 58: 17. 2007. Fig. 7.

View larger version (84K): [in this window] [in a new window]   Fig. 7. Catenulostroma protearum. A. Colony on OA. B–G. Sporulating colony, with variable muriform to transversely septate conidia. Scale bars = 10 µm.

Basionym: Trimmatostroma protearum Crous & M.E. Palm, Mycol. Res. 103: 1303. 1999.

Culture characteristics: On MEA spreading, erumpent, with folded surface, and unevenly lobed, smooth margins; aerial mycelium sparse; surface iron-grey to greenish black, reverse greenish black; reaching 15 mm diam after 2 wk; similar on PDA and OA.

Host range and geographic distribution: Protea, Leucadendron and Hakea spp., South Africa.

Specimens examined: south Africa, on leaves of Protea grandiceps, L. Schroeder, 15 Sept. 1986, holotype BPI 1107849; south Africa, Western Cape Province, Stellenbosch, Assegaibos, on leaves of Leucadendron tinctum, F. Roets, 16 Apr. 2008, epitype designated here CBS H-20338, culture ex-epitype, CPC 15369, 15370 = CBS 125421; ditto, on leaves of Hakea sericea, CBS H-20339, single ascospore culture CPC 15368.

Notes: Catenulostroma protearum was originally described from dead leaves of Protea grandiceps collected in South Africa (Crous & Palm 1999). Unfortunately the cultures died before they could be deposited, and hence the phylogenetic position of Catenulostroma remained uncertain. This proved to be problematic, as the genus was later shown to be heterogeneous (Crous et al. 2007a). The designation of the epitype in the present study clarifies the phylogenetic position of the genus, and reveals Catenulostroma s. str. to represent species that occur in extreme environments, on rocks, or on hard, leathery leaves such as Proteaceae and Gymnospermae.

Devriesia hilliana Crous & U. Braun, sp. nov. MycoBank MB514700. Fig. 8.

View larger version (116K): [in this window] [in a new window]   Fig. 8. Devriesia hilliana. A. Sporulating colony on OA. B–D. Conidiophores giving rise to catenulate conidia. E–G. Fragmenting conidial segments from aerial hyphae. Scale bars = 10 µm.

Devriesiae strelitziae similis, sed conidiis minoribus, (5–)7–10(–12) x (2–)2.5(–3) µm.

Colonies sporulating on MEA. Mycelium consisting of branched, septate, pale brown, smooth, 2–3 µm wide hyphae. Conidiophores solitary, erect on creaping hyphae, unbranched, medium brown, smooth, flexuous, thick-walled, 15–50 x 2–3 µm, 3–11-septate. Conidiogenous cells terminal, medium brown, subcylindrical, smooth, 5–20 x 2–3 µm; proliferating sympodially; hila flattened, unthickened, somewhat darkened, 1–1.5 µm wide. Conidia medium brown, smooth, subcylindrical to narrowly fusoid-ellipsoidal or obclavate, apical conidium with obtuse apex, additional conidia with truncate ends, somewhat darkened, 1–1.5 µm wide; conidia straight to irregularly bent, mostly in unbranched chains, (5–)7–10(–12) x (2–)2.5(–3) µm.

Culture characteristics: On MEA erumpent, spreading, with folded surface, and smooth margins with sparse aerial mycelium; surface mouse-grey, with thin, olivaceous-grey margin; reverse iron-grey, reaching 8 mm diam; on PDA similar, up to 8 mm diam, centre mouse-grey, margin and reverse iron-grey; on OA erumpent with moderate mouse-grey aerial mycelium, and iron-grey margin.

Host range and geographic distribution: Macrozamia communis, Auckland, New Zealand.

Specimen examined: New Zealand, Auckland, Auckland University Campus, Princes Street, on Macrozamia communis, C.F. Hill, 20 Apr. 2008, CBS H-20340 holotype, culture ex-type CPC 15382 = CBS 123187.

Devriesia lagerstroemiae Crous & M.J. Wingf., sp. nov. MycoBank MB514701. Fig. 9.

View larger version (111K): [in this window] [in a new window]   Fig. 9. Devriesia lagerstroemiae. A. Leaves and flowers of Lagerstroemia indica. B. Leaf spots. C. Colony on OA. D–H. Conidiophores giving rise to branched conidial chains. Scale bars = 10 µm.

Devriesiae strelitziae similis, sed conidiis latioribus, (5–)7–10(–12) x (2–)2.5(–3) µm.

Colonies sporulating on OA. Mycelium consisting of smooth, branched, septate, 2–3 µm wide hyphae. Conidiophores rarely micronematous, predominantly macronematous, erect on creeping hyphae, brown, cylindrical with swollen basal cell, thick-walled, smooth, flexuous, 20–90 x 3–4 µm, 5–20-septate. Conidiogenous cells terminal, cylindrical to clavate, polyblastic, pale to medium brown, 5–10 x 2–3(–4) µm; scars somewhat thickened and darkened, not refractive. Ramoconidia medium brown, smooth, subcylindrical, 9–15 x 3–5 µm, (0–)1(–2)-septate, but with clavate apex and several flattened loci that are somewhat darkened and thickened, 1 µm diam. Conidia in branched chains of up to 10, pale brown, smooth, narrowly ellipsoid, 0–1-septate, (5–)8–12(–15) x 2–3(–4) µm; apical conidium with rounded apex, the rest with flattened loci that are somewhat darkened and thickened, not refractive, 0.5–1 µm diam.

Culture characteristics: On MEA erumpent, spreading, with sparse aerial mycelium and irregular margin; surface olivaceous-grey, with patches of iron-grey; reverse iron-grey, reaching 10 mm diam; on PDA similar, but on OA iron-grey, reaching 15 mm diam.

Host range and geographic distribution: Lagerstroemia indica, U.S.A., Louisiana.

Specimen examined: U.S.A., Louisiana, Baton Rouge, Cod & Cook Centre, N30°24'50.3" W91°10'6.6", on Lagerstroemia indica, P.W. Crous & M.J. Wingfield, holotype CBS H-20341, culture ex-type CPC 14403 = CBS 125422.

Notes: Devriesia lagerstroemiae clusters close to D. hilliana. As far as we know, neither species is heat-resistant, nor forms chlamydospores, and hence the placement in Devriesia is more due to phylogenetic similarity than their ecology.

Devriesia strelitziicola Arzanlou & Crous, sp. nov. MycoBank MB514702. Fig. 10.

View larger version (148K): [in this window] [in a new window]   Fig. 10. Devriesia strelitziicola. A. Strelitzia sp. with dead leaves. B. Colony on OA. C–G. Conidiophores giving rise to conidia. H–M. Conidia. Scale bars = 10 µm.

Devriesiae strelitziae similis, sed conidiis majoribus, (7–)25–45(–100) x (2–)2.5(–3) µm.

Colonies sporulating on OA. Mycelium consisting of medium brown, smooth, septate, branched, 2–3 µm wide hyphae; chlamydospores not observed. Conidiophores dimorphic. Microconidiophores reduced to conidiogenous cells on hyphae, erect, cylindrical, medium brown, smooth with truncate ends, proliferating sympodially, 4–7 x 2–3 µm. Macroconidiophores erect, cylindrical, straight to geniculate-sinuous, medium brown, smooth, unbranched or branched above, 30–100 x 2.5–3 µm, 3–10-septate. Conidiogenous cells terminal or lateral on branched conidiophores, medium brown, smooth, cylindrical, proliferating sympodially, 7–15 x 2.5–3 µm; loci truncate, inconspicuous, 1–1.5 µm wide. Conidia medium brown, smooth, guttulate, subcylindrical to narrowly obclavate, apex obtuse to truncate, base truncate, occurring in branched chains, widest at the basal septum, (7–)25–45(–100) x (2–)2.5(–3) µm, (0–)3–6(–13)-septate; hila inconspicuous to somewhat darkened and thickened, not refractive, 1–1.5 µm wide.

Culture characteristics: On MEA erumpent, slow growing, with moderate aerial mycelium and smooth margins; surface mouse-grey, reverse iron-grey, reaching 8 mm diam after 2 wk; similar on PDA and OA.

Host range and geographic distribution: Strelitzia sp., South Africa.

Specimen examined: south Africa, KwaZulu-Natal, Durban, Botanical Garden near Reunion, on leaves of Strelitzia sp., 5 Feb. 2005, W. Gams & H. Glen, CBS H-20342, holotype, culture ex-type X1045 = CBS 122480.

Notes: Devriesia strelitziicola is the second Devriesia species to be described from this host (Arzanlou et al. 2008a). The genus Devriesia was originally established to accommodate a group of heat-resistant, Cladosporium-like fungi (Seifert et al. 2004), and it appears that a different generic name will have to be introduced to accommodate those taxa occurring on plants. Further collections are required, however, to clarify the generic boundaries of Devriesia (Crous et al. 2007b).

Hortaea thailandica Crous & K.D. Hyde, sp. nov. MycoBank MB514703. Fig. 11.

View larger version (130K): [in this window] [in a new window]   Fig. 11. Hortaea thailandica. A. Cercosporoid leaf spots on Syzygium siamense, in which H. thailandica occurred. B. Colonies on OA. C–E. Hyphae with conidiogenous loci (arrows). F–H. Conidia. Scale bars = 10 µm.

Hortaeae werneckii similis, sed conidiis brunneis, verruculosis, majoribus, (9–)10–13(–15) x (4–)5–6(–7) µm.

Colonies sporulating on MEA. Mycelium consisting of pale brown, smooth, septate, branched, 3–4 µm wide hyphae that become darker and thick-walled in the conidiogenous region. Conidiogenous cells integrated, intercalary on hyphae, reduced to short cylindrical loci, 2–2.5 µm wide, 1–4 µm tall; collarettes inconspicuous to minute; proliferating 1–2 times percurrently at apex. Conidia ellipsoid, aseptate, pale to medium brown, (4–)5–7(–9) x (2.5–)3 µm, verruculose, apex obtuse, base subtruncate with minute collarette; becoming swollen and elongate at maturity, with 1–4 transverse and 1–2 oblique septa; (9–)10–13(–15) x (4–) 5–6(–9) µm; hila inconspicuous, up to 2 µm wide, frequently with visible marginal frill; microcyclic conidiation commonly observed on OA, MEA and PDA.

Culture characteristics: On MEA erumpent, spreading; surface irregular, folded, greenish black, with sparse olivaceous-grey aerial mycelium and smooth, lobed, margins; reverse greenish black; reaching 12 mm diam after 2 wk; similar on OA and PDA.

Host range and geographic distribution: Syzygium siamense, Thailand.

Specimen examined: Thailand, Khao Yai National Park, N14°14'42.6" E101°22'15.7", on leaves of Syzygium siamense, in lesions with a cercosporoid fungus, 27 Mar. 2009, P.W. Crous & K.D. Hyde, holotype in BBH, isotype CBS H-20343, culture ex-type CPC 16652, 16651 = CBS 125423, also in BCC.

Notes: Similar to Hortaea werneckii, which is also frequently isolated from lesions in association with plant pathogenic fungi, H. thailandica occurred in leaf spots in association with a cercosporoid fungus. It is distinct from H. werneckii by forming larger conidia that turn medium brown and verruculose with age. Several other taxa are newly placed in the Teratosphaeriaceae in the present study that require further evaluation. Xenomeris juniperi, a bitunicate ascomycete on Jupinerus with pseudothecia associated with a stroma, and pigmented, 1-septate ascospores, clusters close to Teratosphaeria species occurring on Protea and Eucalyptus, where the ascomata are also associated with stromatic tissue (Taylor & Crous 2000, Crous et al. 2006c). Fresh collections of this fungus would be required, however, to resolve its status. The occurrence of Sporidesmium species in the Teratosphaeriaceae should be interpreted with care, as the genus is polyphyletic, and further studies are required to resolve its status (Shenoy et al. 2006, Crous et al. 2008a, Yang et al., in prep.).

Davidiellaceae C.L. Schoch, Spatafora, Crous & Shoemaker, Mycologia 98: 1048. 2006.

Type species: Davidiella tassiana (De Not.) Crous & U. Braun, Mycol. Progr. 3: 8. 2003.

Notes: The Davidiellaceae was introduced for the genus Davidiella, which has Cladosporium anamorphs. As shown in the present analysis, however, allied genera such as Toxicocladosporium, Verrucocladosporium, Rachicladosporium and Graphiopsis also belong in this family. Of interest is the position of Melanodothis caricis in Cladosporium s. str. This fungus, which infects florets of Carex and Kobresia, forms a stroma that gives rise to several immersed ascomata with bitunicate, oblong asci that are aparaphysate, and 0–(2)-septate, hyaline, 9–14.5 x 2–4 µm ascospores. In culture, a hyaline, Ramularia-like anamorph developed, with sympodial proliferation, catenulate conidia, with thickened, darkened loci (Arnold 1971). Although these characteristics are atypical of the Davidiella/Cladosporium species in this clade, the position of Melanodothis caricis in this family cannot simply be disregarded. However, the ex-type culture of this fungus (CBS 860.72) proved to be sterile.

A further unconfirmed sequence (CBS 354.29, culture sterile, but fast growing, grey-brown, Cladosporium-like), is that submitted as Sphaerulina polyspora. The culture was accessioned in 1929, deposited by A.E. Jenkins, and there is reason to believe that it was derived from BPI 623724!, which is authentic for the species, and collected by F.A. Wolf in May 1924. Wolf (1925) described this fungus from twigs of Oxydendron arboretum with die-back disease symptoms, collected in Raleigh, North Carolina. Sphaerulina polyspora (623723 = Type!) has pseudothecia with aparaphysate, bitunicate asci, and ascospores that are hyaline, 3–5-septate, 20–24 x 6–7 µm. On the host it was linked to a Phoma-like anamorph, which also grew similar in culture (yeast-like budding), and has hyaline conidia which are ellipsoidal, 7–8 x 3.8–4 µm.

Colonies were reported as slow-growing, grey, appressed, with germinating ascospores forming yeast-like budding cells, and rarely having hyphae that extended from the margin of the colonies. The link between Sphaerulina-like species, with Selenophoma and Aureobasidium synanamorphs was recently illustrated by Cheewangkoon et al. (2009). Although members of the Dothideomycetes, these taxa do not cluster in the Davidiellaceae, and hence it seems a fair assumption that CBS 354.29 is not representative of Sphaerulina polyspora.

Rachicladosporium cboliae Crous, sp. nov. MycoBank MB514704. Fig. 12.

View larger version (142K): [in this window] [in a new window]   Fig. 12. Rachicladosporium cboliae. A. Front Royal collection site in Virginia. B–E, G. Conidiophores with branched conidial chains. F. Hyphal coil. H–I. Chlamydospores in chains. J. Conidia. Scale bars = 10 µm.

Rachicladosporio americano similis, sed conidiophoris dense fasciculatis et conidiis minoribus.

Colonies sporulating on OA. Mycelium consisting of branched, septate hyphae, pale brown, smooth, 1.5–3 µm wide, frequently constricted at septa, forming hyphal coils, but characteristically also forming intercalary and terminal clusters of chlamydospores that are brown, thick-walled, up to 6 µm diam. Conidiophores forming laterally on creeping hyphae, erect, visible as densely branched tufts on agar surface; conidiophores medium brown, smooth, thick-walled with bulbous base, lacking rhizoids, cylindrical, unbranched, flexuous, up to 250 µm long, 4–6 µm wide, 10–20-septate. Conidiogenous cells terminal, medium brown, smooth, polyblastic, subcylindrical, 10–20 x 3–4 µm; loci terminal, thickened, darkend, refractive, 1 µm diam. Ramoconidia 0(–1)-septate, subcylindrical, medium brown, smooth, 7–12 x 3–4 µm. Conidia 0(–1)-septate, in branched chains of up to 10, ellipsoid, pale brown, smooth, (6–)7–8(–10) x (2–)2.5(–3) µm; hila thickened, darkened and refractive, up to 1 µm diam.

Culture characteristics: On MEA spreading with sparse aerial mycelium and smooth margins; surface folded, centre pale mouse-grey to mouse-grey, margin iron-grey; reverse greenish black, reaching 15–20 mm diam after 2 wk; on PDA spreading with moderate aerial mycelium and smooth margins; surface olivaceous-grey, margin mouse-grey, reverse olivaceous-grey; reaching 30 mm diam; on OA spreading, folded with moderate aerial mycelium; surface pale mouse-grey (centre) to olivaceous-grey at margin, reaching 20 mm diam.

Host range and geographic distribution: Twig litter, Virginia, U.S.A.

Specimen examined: U.S.A., Virginia, Front Royal, N38°53'35" W78°10'50", on twig debris, 14 May 2007, P.W. Crous, holotype CBS H-20344, cultures ex-type CPC 14034 = CBS 125424, CPC 14035, 14036.

Notes: Rachicladosporium cboliae is a cryptic species close to R. americanum, which was collected at the same site. They can be distinguished on the litter in that R. cboliae has conidiophores with densely branched tufts of conidia, in contrast to the more sparsely branched conidiophores of R. americanum. Furthermore, R. cboliae also forms prominent chains of chlamydospores in culture, which lacks in R. americanum. Finally, R. cboliae has smaller ramoconidia and conidia than those found in R. americanum (ramoconidia 13–23 x 3–4 µm; conidia 10–18 x 3–4 µm; Cheewangkoon et al. 2009).

	DISCUSSION

TOP Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
The class Dothideomycetes incorporates fungal taxa exhibiting a wide range of nutritional modes, and results in these fungi being found in many diverse niches (Fig. 13). The two largest orders Pleosporales (Zhang et al. 2009; this volume) and Capnodiales encapsulate this diversity. Here we continue to expand sampling within the Capnodiales in order to provide a well founded phylogenetic scaffold for taxonomic classification, informative genomic sampling, ecological studies and evolutionary evaluations.

View larger version (122K): [in this window] [in a new window]   Fig. 13. Members of Capnodiales exhibiting different ecological growth habits. A–C. Mycosphaerella marksii (plant pathogen). A. Leaf spot on Eucalyptus. B. Homothallic colony on MEA. C. Asci. D. Conidiophore of Cladosporium sphaerospermum (saprobe). E–G. Ascomata and asci of Davidiella macrocarpa (saprobe). H–J. Dissoconium dekkeri (plant pathogen, commensalist). H. Colony sporulating on MEA, with discharged conidia at the margin. I. Asci. J. Primary and secondary conidia attached to conidiophore. K–L. Dissoconium proteae (commensalist). K. Sporulation on MEA with microsclerotia. L. Two conidial types attached to conidiophore (arrow). M–Q. Conidioxyphium gardeniorum (sooty mold). M. Sporulation on MEA. N–P. Elongated, branched conidiomata with apical ostiolar hyphae. Q. Conidia. R–T. Leaf spot, ascus and verruculose ascospores of Teratosphaeria fibrillosa (plant pathogen). U–X. Schizothyrium pomi (epiphyte). U. Thyrothecia occurring on a Rhus stem. V. Ascomatal initials forming on OA. W. Asci. X. Conidiophore and conidia in vitro. Scale bars: E = 200 µm, M–O = 50 µm, all others = 10 µm.

One of the main aims of the present study was to resolve the status of the Capnodiales and Mycosphaerellales. Although we were able to distinguish a clear, well resolved node for the Mycosphaerellales (incl. Mycosphaerellaceae), this node was not well supported, and elevating it to ordinal level would mean that additional orders need to be introduced to accommodate several families outside the Capnodiales s. str. This finding led us to conclude that it is best to retain all families within a single, diverse order, namely the Capnodiales.

Evolution of nutritional modes and ecological growth habits
The ancestral state of the present assemblage of taxa is likely to be saprobic, as Phaeotheca (Sigler et al. 1981, de Hoog et al. 1997, Tsuneda et al. 2004), and Comminutispora (Ramaley 1996) represent the earliest diverging lineages. This was similarly found for a majority of lineages in the larger context of Ascomycota (Schoch et al. 2009a, b). These taxa were not only all isolated from dead materials or substrates, but they also share the same unique mode of conidiogenesis, namely endoconidia, and a "black-yeast" appearance in culture. Phaeotheca, which is strongly halophilic (Zalar et al. 1999) is closely related to the lichen Racodium rupestre, which forms an association with Trentepohlia algae, in which the filamentous algae is enclosed by melanised hyphae of the fungus. This feature is also shared by another lichen, namely Cystocoleus ebeneus (Teratosphaeriaceae) (Muggia et al. 2008). The Capnodiaceae (sooty molds) that also cluster in a basal position in the tree are epiphytes, growing on insect exudates (honey dew). The Capnodiaceae are related to the Davidiellaceae, which represent Cladosporium and allied genera. This family contains a wide range of ecological adaptations, from primary plant pathogens, such as Graphiopsis chlorocephala on Paeonia (Schubert et al. 2007a, Braun et al. 2008), "Mycosphaerella" iridis on Iris (David 1997), to taxa opportunistic on humans, Cladosporium bruhnei (Schubert et al. 2007b), to halotolerant taxa, Cladosporium sphaerospermum (Zalar et al. 2007, Dugan et al. 2008), to saprobes, C. herbarum, C. cladosporioides (Schubert et al. 2007b).

The Teratosphaeriaceae contains several disjunct elements, many of which may still eventually be removed from the family as more taxa and additional sequence data are added, providing a better resolution to some of these clades. In its widest sense, the family contains lichens (Anisomeridium, Cystocoleus), saprobes (Catenulostroma spp.), and halophilic, hyperhydrotic or lipophilic species that have been reported from humans (Piedraia, Hortaea, Penidiella, Stenella) (de Hoog et al. 2000, Bonifaz et al. 2008, Plemenita et al. 2008), with the most derived clades tending to contain plant pathogens (Readeriella, Teratosphaeria).

Dissoconiaceae is an early diverging lineage to the Mycosphaerellaceae and Schizothyriaceae. Whereas most members of Dissoconiaceae appear to be commensalists, there is evidence that some species could be plant pathogenic (Jackson et al. 2004), while the Schizothyriaceae contains epiphytes (Batzer et al. 2007). The Mycosphaerellaceae contains species that are biotrophic (Polythrincium; Simon et al. 2009), necrotrophic plant pathogens (Brunneosphaerella, Cercospora, Dothistroma, Pseudocercospora, Pseudocercosporella, Ramularia, and Septoria), as well as some species that are saprobic (Passalora, Pseudocercospora, Ramichloridium and Zasmidium; Arzanlou et al. 2007), or endophytic (Pseudocercosporella endophytica; Crous 1998).

Within the Capnodiales, the positioning of saprobes such as Phaeotheca and Comminutispora and the sooty moulds (Capnodiaceae) may represent the more primitive state, from where transitions occurred to more lichenised, saprobic, biotrophic and nectrotrophic, plant pathogenic members of the order (Fig. 13). This appears to mirror the other large and diverse order in the class, the Pleosporales (Zhang et al. 2009; this volume). Lichenisation, as well as the ability to be saprobic or plant pathogenic evolved more than once, though the taxa in the later diverging clades of the tree tend to be strictly nectrotrophic plant pathogens. This should be interpreted with care, however, as Polythrincium is presently the only biotrophic member included in this analysis, and other biotrophic members of the Capnodiales may end up clustering here, among the presently dominant nectrotropic plant pathogens. One important and recent addition to Capnodiales diversity is the rock-inhabiting fungi (Ruibal et al. 2008, 2009; this volume). Although so far mainly isolated from sources in Antarctica and the Mediterranean area, it is clear that they are a ubiquitous group of fungi likely found throughout the globe. Their genetic diversity is underscored by the fact that rock inhabiting fungi of convergent morphology are also placed in other ascomycotan classes and orders (Gueidan et al. 2008). The fact that many of these species have reduced morphologies and are slow growers make their taxonomy challenging, but their phylogenetic placement within Teratosphaeriaceae and several other lineages within Capnodiales makes their inclusion in subsequent phylogenetic assessments of this order essential.

For this study, we designed novel primers to supplement primers presently available in literature. Although primers are usually designed for the genus or family of interest, they frequently tend to have a wider application. Therefore, we attempted to design our primers using a wide range of sequences from the GenBank sequence database, in the hope that these primers will eventually find application outside of the Capnodiales as well. Although this remains to be tested, we expect it to be the case. Our sequencing of the complete SSU and LSU for the selected members of the Capnodiales had a surprisingly large number of insertions present for numerous strains. Although some of these insertions were anticipated based on data already present in GenBank's database, the insertions in the LSU were not expected based on the sequences used for primer design. However, this could be a result of the fewer complete LSU sequences available in the database rather than a deviation on the part of members of the Capnodiales. More complete LSU sequences are needed from diverse orders to test whether this is the case or not. Some of the taxa sequenced during this study had insertions present at almost all of the possible insertion positions, e.g. Mycosphaerella latebrosa, Septoria quercicola and Teratosphaeria mexicana. These taxa are distributed throughout the tree, and do not only cluster in a basal position, and therefore it is difficult to predict why so many insertions were present. If these insertions were all present in a basal position, it would have been possible to argue that the higher number of insertions represents the ancestral condition, and that these insertions are lost during evolution. However, this proved not to be the case, and it could be that these taxa accumulated these insertions.

Although the present study adds significantly to our knowledge of the Capnodiales, the Capnodiaceae are still underrepresented, and probably consist of numerous diverse lineages that can be elevated to family level once our phylogenies become more resolved. Regardless of this fact, the Mycosphaerellaceae clade appears to be quite robust. It seems likely that further sampling of the diverse Teratosphaeriaceae will necessitate further taxonomic changes. The fact that the saprobic and plant pathogenic and endophytic modes have evolved several times in different families, suggest that many taxa can still easily adapt to changing environments. A focus on adding more lichenicolous taxa, and taxa occurring on non-plant substrates is crucial to provide further insight into the ecological adaptations occurring in the Capnodiales.

	Acknowledgments

 
Several colleagues have helped to collect material studied here, without which this work would not have been possible. Drs E.H.C. Mckenzie and S.R. Pennycook (Landcare New Zealand) are specifically thanked for recollecting Phaeophleospora atkinsonii. We are grateful to BIOTEC and Dr Lily Eurwilaichitr (Director, Bioresources unit, BIOTEC) for assisting with a collection trip in Thailand under the collaborative BIOTEC-CBS memorandum of understanding. We thank Miss Marjan Vermaas for preparing the photographic plates, and M. Starink-Willemse, and A. van Iperen, for assistance with DNA sequencing and fungal cultures. Work performed for this paper by the second author after 2008 was supported in part by the Intramural Research Program of the NIH, National Library of Medicine. Before 2008 work was funded by a grant from NSF (DEB-0717476). We are grateful to Drs Roland Kirschner, Alan J. Phillips and Treena I. Burgess for their critical comments on this script. The views expressed, however, are those of the authors.

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