Fungal Diversity (2014) 68:69–104 DOI 10.1007/s13225-014-0305-6 A molecular phylogenetic reappraisal of the Didymosphaeriaceae (= Montagnulaceae) Hiran A. Ariyawansa & Kazuaki Tanaka & Kasun M. Thambugala & Rungtiwa Phookamsak & Qing Tian & Erio Camporesi & Sinang Hongsanan & Jutamart Monkai & Dhanushka N. Wanasinghe & Ausanan Mapook & Ekachai Chukeatirote & Ji-Chuan Kang & Jian-Chu Xu & Eric H. C. McKenzie & E. B. Gareth Jones & Kevin D. Hyde Received: 18 July 2014 / Accepted: 25 August 2014 / Published online: 26 September 2014 # School of Science 2014 Abstract The ascomycetous families, Didymosphaeriaceae and Montagnulaceae, have been treated in Pleosporales, Dothideomycetes, and both include saprobes, endophytes and pathogens associated with a wide variety of substrates worldwide. Didymosphaeriaceae was characterized by 1septate ascospores and trabeculate pseudoparaphyses, mainly anastomosing above the asci, while species in Montagnulaceae had 1 to multi-septate ascospores and generally cellular pseudoparaphyses. In recent treatments, Bimuria, Didymocrea, Kalmusia, Karstenula, Montagnula, Paraphaeosphaeria, Paraconiothyrium and Letendraea were placed in Montagnulaceae, while only Didymosphaeria has been placed in Didymosphaeriaceae. New morphological and molecular data from recent collections have become available and thus the understanding of the families can be improved. Based on analyses of concatenated internal transcribed spacer (ITS) with LSU, SSU and β-tubulin gene sequences, the taxonomy of the genera classified in these families are reassessed. Our phylogenetic analyses conclude that, the recently introduced Didymosphaeria rubi-ulmifolii with Pa ra c o ni ot hy r iu m b r as il ie ns e s p e c i e s co m p l e x , Alloconiothyrium, Bimuria, Deniquelata, Didymocrea, Kalmusia, Karstenula, Letendraea, Montagnula, Neokalmusia, Paraconiothyrium, Paraphaeosphaeria, Phaeodothis and Tremateia, forms a robust clade named here D i dy m o s ph a e r i ac e ae . We t h e r e f or e s yn o n ym i z e Electronic supplementary material The online version of this article (doi:10.1007/s13225-014-0305-6) contains supplementary material, which is available to authorized users. H. A. Ariyawansa : J.<C. Xu : K. D. Hyde (*) Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China e-mail: kdhyde3@gmail.com H. A. Ariyawansa : K. M. Thambugala : R. Phookamsak : Q. Tian : S. Hongsanan : J. Monkai : D. N. Wanasinghe : A. Mapook : E. Chukeatirote : K. D. Hyde Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand H. A. Ariyawansa : J.<C. Xu : K. D. Hyde World Agroforestry Centre, East Asia and Central, Heilongtan, Kunming 650201, People’s Republic of China K. Tanaka Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan H. A. Ariyawansa : J.<C. Kang The Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of China, Guizhou University, Guiyang 550025, Guizhou Province, China E. Camporesi A.M.B. Gruppo Micologico Forlivese “Antonio Cicognani”, Via Roma 18, Forlì, Italy H. A. Ariyawansa : K. M. Thambugala : R. Phookamsak : Q. Tian : S. Hongsanan : J. Monkai : D. N. Wanasinghe : A. Mapook : E. Chukeatirote : K. D. Hyde School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand E. H. C. McKenzie Manaaki Whenua Landcare Research, Private Bag, 92170 Auckland, New Zealand E. B. G. Jones Department of Botany and Microbiology, King Saudi University, Riyadh, Saudi Arabia 70 Montagnulaceae under Didymosphaeriaceae which is the oldest name and has priority and provide a update account of the family. Didymosphaeria is represented by Didymosphaeria rubi-ulmifolii. A new genus, Neokalmusia, is introduced in the family to accommodate the b a m b u s i c o l o u s t a x a K a l m u s i a b re v i s p o r a a n d K. scabrispora. This introduction is based on molecular data c o u p l e d w i t h m o r p h o l o g y. O n e n e w s p e c i e s , Paraconiothyrium magnoliae and the sexual morph of Paraconiothyrium fuckelii, is also introduced. Julella and Barria are assigned to the family based on morphological similarity with Didymosphaeriaceae. Wilmia (previously referred to the Phaeosphaeriaceae) is synonymized under Letendraea (Didymosphaeriaceae). Furthermore, a new species, Letendraea cordylinicola, is introduced and placed in Didymosphaeriaceae based on phylogeny and morphology. The paraphyletic nature of Paraconiothyrium is partially resolved, but further sampling with fresh collections and molecular data of species in this genus are needed to obtain a natural classification. Keywords Didymosphaeriaceae . Montagnulaceae . New species . Phylogeny . rDNA . β-tubulin Introduction Munk A (1953) introduced the family Didymosphaeriaceae, typified by the genus Didymosphaeria Fuckel. The family was characterized by 1-septate ascospores and trabeculate pseudoparaphyses which anastomosed mostly above the asci (Aptroot 1995; Ariyawansa et al. 2014b). Based on its trabeculate pseudoparaphyses, Barr (1990) referred Didymosphaeriaceae to the order Melanommatales. However, the importance of trabeculate pseudoparaphyses was later disputed (Liew et al. 2000). The confusion surrounding Didymosphaeriaceae and Didymosphaeria was discussed in detail by Ariyawansa et al. (2014b). They explained that considering the morphological data only (peridium comprising flattened or irregular cells, 1-septate ascospores and trabeculate pseudoparaphyses mostly anastomosing above the asci) the family Didymosphaeriaceae appears to be distinct from Pleosporales. However, on the basis of molecular data the status of Didymosphaeriaceae as a distinct family was found to be doubtful as it clustered in Montagnulaceae (Ariyawansa et al. 2014b). Ariyawansa et al. (2014b) introduced a new species of Didymosphaeria, D. rubi-ulmifolii Ariyawansa et al., based on morphological comparison with the generic type, D. futilis (Berk. & Broome) Rehm. Sequences of Didymosphaeria rubi-ulmifolii clustered within the Montagnulaceae as a separate genus, thus suggesting that Montagnulaceae and Didymosphaeriaceae are synonymous. Fungal Diversity (2014) 68:69–104 Barr (2001) introduced the family Montagnulaceae with three genera viz. Kalmusia Niessl, Montagnula Berl., and Didymosphaerella Cooke, in the order Pleosporales, based on ascomata immersed under a clypeus, a pseudoparenchymatous peridium of small cells, cylindric or oblong, fissitunicate, pedicellate asci and multi-septate ascospores. Ascospores in Kalmusia have two to many transverse septa, in Montagnula they are muriform and in Didymosphaerella ascospore have a single transverse septum (Barr 2001). The family was thought to be closely related to Cucurbitariaceae, but differed in its obtuse ascomatal apex with a minute papillate ostiole and thin-walled, pale ascospores (Barr 2001). Species in the family Montagnulaceae are parasitic, saprotrophic or hemibiotropic and also include saprobic species in aquatic environments (Barr 2001; Zhang et al. 2012). Several recent studies have provided the groundwork for classification of the family Montagnulaceae (Ariyawansa et al. 2014b; Verkley et al. 2014). Even though there is a large body of work comprising taxonomic and phylogenetic studies, most of the genera regarded as members of Montagnulaceae remain under-studied and therefore poorly understood for a systematic treatment. Combined analyses of LSU, SSU, RBP2 and TEF1 gene data (Schoch et al. 2009; Zhang et al. 2012) showed the family Montagnulaceae to be well-resolved comprising the genera Bimuria D. Hawksw et al., Didymocrea Kowalski, Kalmusia Niessl, Karstenula Speg., Montagnula Berl., and Paraphaeosphaeria O.E. Erikss. Some species of Phaeosphaeria, Paraconiothyrium Verkley, and Letendraea Sacc., also clustered in Montagnulaceae (Schoch et al. 2009; Zhang et al. 2012). Paraconiothyrium was introduced by Verkley et al. (2004) to accommodate four species viz. Parac. estuarinum Verkley & Manuela Silva., Parac. brasiliense Verkley, Parac. cyclothyrioides Verkley, and Parac. fungicola Verkley & Wicklow. The sexual morphs of these species are unknown, but maximum parsimony analysis of ITS and partial SSU nrDNA sequences showed that these asexual genera clustered within Pleosporales and grouped in a clade including Paraphaeosphaeria in Montagnulaceae (Verkley et al. 2004). In the same study, Coniothyrium minitans W.A. Campb., C. sporulosum (W. Gams & Domsch) Aa, and C. minitans W.A. Campb., clustered in the same clade and were therefore referred to Paraconiothyrium. Following SSU and ITS analyses, Damm et al. (2008) suggested that Paraconiothyrium can be accommodated within Paraphaeosphaeria. Zhang et al. (2012) showed that the asexual morphs of Paraphaeosphaeria michotii (Westend.) O.E. Erikss. and Paraph. pilleata Kohlm et al. are representatives of Paraconiothyrium in their multi-gene phylogenetic analysis. Didymosphaerella has been excluded from the family Montagnulaceae and placed in Didymellaceae (Zhang et al. 2012). Schoch et al. (2009) and Suetrong et al. (2009) showed Fungal Diversity (2014) 68:69–104 that the marine ascomycete, Tremateia halophila nested in Montagnulaceae. Zhang et al. (2012) provided a description of Montagnula, the type of the family Montagnulaceae, which also included Bimuria, Didymocrea, Karstenula, Letendraea, Montagnula, Paraphaeosphaeria and Tremateia, while Ariyawansa et al. (2013c) added the new genus Deniquelata. Zhang et al. (2014) neotypified Kalmusia, with K. ebuli Niessl., to resolve phylogenetic placement of the species within Kalmusia. Zhang et al. (2014) concluded that Kalmusia species are polyphyletic within the Montagnulaceae, with K. ebuli being distant from K. brevispora (Nagas. & Y. Otani) Y. Zhang et al. and K. scabrispora (Teng) Kaz. Tanaka et al. The latter two species appear to represent a different genus. Verkley et al. (2014) re-assessed Coniothyrium-like taxa belonging to the Montagnulaceae, based on combine gene analyses of ITS, LSU, γ-actin and β-tubulin sequences and proposed Alloconiothyrium Verkley & Stielow, and Dendrothyrium Verkley & Stielow, as new genera. In the same study, one new species was described in Paraconiothyrium while two species so far classified in Paraconiothyrium were transferred to Paraphaeosphaeria (Verkley et al. 2014). We have been studying the families of Pleosporales basing on both morphology and molecular phylogeny in order to provide a natural classification of this large order (Ariyawansa et al. 2013a, b, c, 2014a, b, c, d; Hyde et al. 2013; Zhang et al. 2012). This paper aims to clarify the genetic and taxonomic relationships within the genera of the Didymosphaeriaceae and Montagnulaceae using a set of isolates that encompasses its genetic, biological and geographic diversity. Type specimens, or cultures derived from type specimens, have been examined wherever possible. Material and methods Specimen examination Fresh materials of Didymosphaeriaceae and Montagnulaceae species were collected in Thailand and Italy during 2011-2013. Specimens were taken to the laboratory in Ziplock plastic bags. The samples were processed and examined following the method described in Ariyawansa et al. (2013a, b, c). Fresh and herbarium materials were examined under a Motic SMZ 168 dissecting microscope to locate and isolate ascomata fruiting bodies. Hand sections of the fruiting structures were mounted in water for microscopic studies and photomicrography. The taxa were examined using a Nikon ECLIPSE 80i compound microscope and photographed with a Canon 450D digital camera fitted to the microscope. Measurements were made with the Tarosoft (R) Image Frame Work program and images used for figures processed with Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, USA). 71 Isolations were made from single ascospores, following a modified method of Ariyawansa et al. (2013b) and Chomnunti et al. (2014). Contents of the sectioned fruiting body were transferred to a drop of sterile water on a flame-sterilized slide. Drops of the spore suspension were pipetted and spread on a Petri-dish containing 2 % water agar (WA) and incubated at 25 °C. Germinated ascospores were transferred singly to MEA media (Alves et al. 2006). Herbarium specimens were obtained on loan from the Swedish Museum of Natural History (S) and the New York Botanical Garden (NY). Voucher specimens are deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand and New Zealand Fungarium, Landcare Research (PDD), New Zealand. The living cultures are deposited at the Mae Fah Luang University Culture Collection (MFLUCC), Biodiversity and Climate Research Centre (BiK-F) and International Collection of Microorganisms from Plants (ICMP) under Material Transfer Agreement No. 4/2010 (MTA). Each genus is listed along with a description of the type species, except in cases where there is only a single species in the genus. DNA extraction, PCR amplification and sequencing Single ascospore fungal isolates were grown on MEA or PDA for 28 days at 25 °C in the dark. Genomic DNA was extracted from the growing mycelium using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux®) following the manufacturer’s protocol (Hangzhou, P.R. China). Otherwise DNA extracted directly from ascomata using a DNA extraction kit (E.Z.N.A.® Forensic DNA kit, D3591- 01,Omega Bio-Tek) following Telle and Thines (2008). The amplification procedure was performed in a 50 μl reaction volume containing 5–10 ng DNA, 0.8 units Taq polymerase, 1X PCR buffer, 0.2 mM d’NTP, 0.3 μM of each primer with 1.5 mM MgCl2 (Cai et al. 2009). The PCR reactions for amplification of the recently ratified universal fungal barcode ITS1-5.8S-ITS2 of the nuclear ribosomal DNA operon (Verkley et al. 2014), were performed under standard conditions (White et al. 1990; Stielow et al. 2010). PCR conditions for amplifying the partial SSU and LSU rDNA followed the protocol of Phillips et al. (2008). Amplification of partial β-tubulin (TUB), covering the variable 5’-end containing four small introns, followed the protocol of Aveskamp et al. (2009) and Carbone and Kohn (1999). The PCR products were observed on 1 % agarose electrophoresis gels stained with ethidium bromide. Purification and sequencing of PCR products were carried at Shanghai Sangon Biological Engineering Technology and Services Co., (China). DNA sequence data was obtained from the internal transcribe spacer (ITS), small and large subunits of the nuclear 72 ribosomal RNA genes (SSU, LSU) and the protein coding gene, β-tubulin (TUB). Primer sets used for these genes were as follows: ITS: ITS5/ITS4 SSU: NS1/NS4; LSU: LR0R/ LR5; TUB4Rd/TUB4Fd (Aveskamp et al. 2009; Carbone and Kohn 1999). Primer sequences are available at the WASABI database at the AFTOL website (aftol.org). Sequences are deposited at NCBI GenBank under the accession numbers provided in Supplementary Table 1. Alignments are deposited in TreeBASE. Sequence alignment and phylogenetic analysis Combined ITS, LSU, SSU and TUB genes were used in the analysis. All sequences obtained from GenBank and used by Ariyawansa et al. (2014d), Boonmee et al. (2014), Hyde et al. (2013), Phookamsak et al. (2014) Schoch et al. (2009), Suetrong et al. (2009), Verkley et al. (2014), Wijayawardene et al. (2014) and Zhang et al. (2012) are listed in supplementary Table 1. Multiple sequence alignments were generated with MAFFT v. 6.864b (http://mafft.cbrc.jp/alignment/server/ index.html). All introns and exons were aligned separately. Regions containing many leading or trailing gaps were removed from the ITS, SSU and LSU alignments prior to tree building. The alignments were checked visually and improved manually where necessary. Concordance of the ITS, SSU, LSU and TUB genes data sets was estimated with the partition-homogeneity test implemented with PAUP v. 4. 0b10 (Swofford 2002). Maximum parsimonious analysis was performed to obtain the most parsimonious tree. Trees were inferred using the heuristic search option with 1,000 random sequence additions and tree-bisection-reconnection (TBR) branch-swapping algorithm. All gaps were treated as missing data. Maxtrees were setup to 50,000 and branches of zero length were collapsed and all multiple parsimonious trees were saved. Descriptive tree statistics for parsimony (Tree Length [TL], Consistency Index [CI], Retention Index [RI], Relative Consistency Index [RC] and Homoplasy Index [HI] were calculated for trees generated under different optimality criteria. KishinoHasegawa tests (KHT) (Kishino and Hasegawa 1989) were performed in order to determine whether trees were significantly different. Maximum parsimony bootstrap values (MP) equal or greater than 50 % are given below or above each node. Maximum likelihood analyses including 1,000 bootstrap replicates were run using RAxML v. 7.2.6 (Stamatakis 2006; Stamatakis et al. 2008) The online tool Findmodel (http://www.hiv.lanl.gov/content/sequence/findmodel/ findmodel.html) was used to determine the best nucleotide substitution model for each partition. The best scoring tree was selected with a final likelihood value of -19492.551787. The resulting replicates were plotted on to the best scoring tree obtained previously. Maximum Likelihood bootstrap values Fungal Diversity (2014) 68:69–104 Fig. 1 RAxML tree based on a combined dataset of SSU, LSU, RPB2„ and TEF1, bootstrap support values for maximum likelihood (blue), maximum parsimony (green) greater than 50 % and bayesian posterior probabilities greater than 0.90 (black) below and above the nodes. Halojulella avicenniae is the outgroup taxon. The original isolate numbers are noted after the species names and ex-type strains are in bold. The type species of each genus is indicated in blue (ML) equal or greater than 50 % are given below or above each node in red (Fig. 1). The model of evolution was performed by using MrModeltest 2.2 (Nylander 2004). Posterior probabilities (PP) (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002) were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.0b4 (Huelsenbeck and Ronquist 2001). Six simultaneous Markov chains were run for 3,000,000 generations and trees were sampled every 100th generation and 30,000 trees were obtained. The first 6,000 trees, representing the burn-in phase of the analyses, were discarded while the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree (critical value for the topological convergence diagnostic set to 0.01) (Crous et al. 2006). Bayesian Posterior Probabilities (BYPP) equal or greater than 0.90 is given below or above each node (Fig. 1). Phylogenetic trees and data files were viewed in MEGA v. 5 (Tamura et al. 2011), TreeView v. 1.6.6 (Page 1996) and FigTree v. 1.4 (Rambaut and Drummond 2008). Results Phylogeny of combined ITS, LSU, SSU and TUB gene datasets The combined ITS, LSU, SSU and TUB dataset comprises 76 taxa. Halojulella avicenniae is selected as an outgroup taxon. Results of the partition-homogeneity test (P=0.107) indicate that the ITS, LSU, SSU and TUB gene trees reflect the same underlying phylogeny. Therefore, these datasets were combined and analyzed by using several tree-building programs, the resulting trees compared and the best tree is presented in Fig. 1. New sequences are deposited in GenBank (Table 1). Phylogenetic analysis The combined ITS, LSU, SSU and TUB gene dataset from five families in the Pleosporales is shown in Fig 1. All trees (ML, MP and BYPP) were similar in topology and not significantly different (data not shown). The maximum parsimony dataset consists of 2,283 characters and of which 2,437 were constant, 300 variable characters parsimonyuninformative and 776 characters parsimony-informative. Fungal Diversity (2014) 68:69–104 73 74 Kishino-Hasegawa (KH) test showed length = 3,000 steps, CI = 0.532, RI = 0.673, RC = 0.358 and HI = 0.468. A best scoring RAxML tree is shown in Fig. 1, with the value of 19492.551787. Phylogenetic trees obtained from Maximum Likelihood, Maximum Parsimony and Bayesian analysis yielded trees with similar overall topology at subclass and family relationships in agreement with previous work based on Maximum Likelihood analysis (Ariyawansa et al. 2014d; Boonmee et al. 2014; Hyde et al. 2013; Phookamsak et al. 2014; Schoch et al. 2009; Suetrong et al. 2009; Verkley et al. 2014; Wijayawardene et al. 2014 and Zhang et al. 2012). In the multi-locus phylogeny inferred from the combined dataset shown in Fig. 1, several well-supported sub clades can be identified in the family Didymosphaeriaceae, which are interpreted as appropriate for the delimitation of genera, i.e. Alloconiothyrium, Bimuria, Deniquelata, Didymosphaeria, Didymocrea, Kalmusia, Karstenula, Letendraea, Montagnula, Neokalmusia, Paraconiothyrium, Paraphaeosphaeria, Phaeodothis and Tremateia. Thus the Didymosphaeriaceae incorporates members of Montagnulaceae. The phylogenetic data for each genus is further discussed after the genus notes. The Didymosphaeria clade comprises the type strain D. rubi-ulmifolii (MFLUCC 14-0023) plus another strain of D. rubi-ulmifolii (MFLUCC 14-0024), which clustered with the type strain of Paraconiothyrium brasiliense Verkley (CBS 100299) and synonymised under D. rubi-ulmifolii and defined here as D. rubi-ulmifolii sensu stricto. Several strain collections of Paraconiothyrium brasiliense (CBS 122319, CBS 122320, CBS 587.84, CBS 115.92 and CBS 115.92) other than the type strain, form a distinct clade to D. rubi-ulmifolii clade, thus treated here as D. rubi-ulmifolii sensu lato. The closely related Paraconiothyrium variabile (CBS 120014) forms a separate clade in Didymosphaeria clade which is synonymised under Didymosphaeria as Didymosphaeria variabile. Another well-supported (100/1.00/98) clade forming the major part of the ingroup of the tree comprises six strains assigned to Paraphaeosphaeria, with three isolates of Paraph. michotii, the type species of the genus, and the relatively highly supported clades of the following species: Paraph. angularis Verkley & van der Aa (CBS 167.70), Paraph. minitans (W.A. Campb.) Verkley & Stielow (CBS 111750), Paraph. arecacearum Verkley & Stielow (CBS 158.75) and Paraph. viridescens Verkley et al. (CBS 854.73). A putative strain of Karstenula rhodostoma (CBS 690.94) forms a distinct clade sister to Paraphaeosphaeria clade. The Paraconiothyrium clade comprises 13 strains with Parac. estuarinum (CBS 109850T), the type species of Paraconiothyrium,: Parac. fuckelii (6), Parac. africanum (CBS 121166), Parac. hawaiiense (CBS 120025), Parac. fungicola (CBS 113269), Parac. estuarinum (CBS 109850), Parac. cyclothyrioides (CBS 972.95), Parac. archidendri Fungal Diversity (2014) 68:69–104 (CBS 168.77) and the new species Parac. magnoliae (MFLUCC 10-0278) which clusters with Parac. fungicola. Kalmusia (74/1.00/54) contains five strains, including Kalmusia ebuli (CBS 123120T), the type species of the genus, Kalmusia sp. (MFLUCC 14-0560), K. variisporum (CBS 121517T) and K. longisporum (CBS 824.84, 582.83T). The monotypic asexual genus Alloconiothyrium (CBS 980.95T, 981.95) forms a robust clade sister to Kalmusia. Letendraea forms a relatively well supported clade within the family Didymosphaeriaceae. This clade contains five strains with Letendraea eurotioides (CBS 212.31), the type species of Letendraea, and the relatively highly supported clades of the following species: L. padouk (CBS 485.70), L. helminthicola (CBS 884.85) and the two isolates of the novel species L. cordylinicola (MFLUCC11-0148 T, MFLUCC 11-0150) which clusters with L. padouk. The new genus Neokalmusia forms a robust clade sister to Deniquelata, Didymocrea and Phaeodothis. Neokalmusia comprises five strains N. brevispora (CBS 120248, NBRC 106240), N. scabrispora (CBS 120246, NBRC 106237) and Neokalmusia sp.. Another well-supported (70/0.91/74) clade is formed by members of Montagnula, the basal clade in the family Didymosphaeriaceae, which comprises four stains including Montagnula opulenta (CBS 168.34), M. aloes (CPC 19671), Montagnula sp. (Letendraea helminthicola) (CHTA R43) and M. anthostomoides (CBS 615.86). Taxonomy Didymosphaeriaceae Munk, Dansk bot. Ark. 15(no. 2): 128 (1953), Facesoffungi number: FoF 00200. = Montagnulaceae M.E. Barr, Mycotaxon 77: 194 (2001) Saprobic or parasitic in terrestrial or aquatic environments. Sexual state: Ascomata scattered or gregarious, immersed in substrate, surrounded with hyphae forming an apical clypeus, dark brown to black, globose to rarely subglobose, medium to large sized, and coriaceous. Ostiole immersed to sub-immersed, periphysate when well-developed with a short apex, narrow or wide, usually circular in section. Peridium two layered or often three layered in the upper region, composed of cells of textura angularis or textura intricata, dark brown to black, coriaceous, with outermost layer consisting of black, highly pigmented thick-walled conglutinate cells or pseudoparenchymatous cells, with middle layer less pigmented, cells visible via apex, with innermost layer comprising narrow, compressed rows of cells. Hamathecium of dense, septate, narrow, cellular or trabeculate pseudoparaphyses often in a gelatinous matrix. Asci 2–8-spored, bitunicate, fissitunicate, cylindric or oblong, pedicellate, with an ocular chamber. Ascospores uniseriate or biseriate, oblong to narrowly oblong, hyaline, brown to reddish-brown or dark yellowish-brown, one to several septate (phragmosporous or muriform), verruculose, with or without a gelatinous sheath. Asexual Fungal Diversity (2014) 68:69–104 states: coelomycetous (Barr 2001), Kalmusia and Paraphaeosphaeria have Coniothyrium-like, Cytoplea, Microsphaeropsis asexual stages. The generic type of Paraphaeosphaeria (P. michotii) was linked with Coniothyrium scirpi Trail (Webster 1955). The Coniothyrium-like species were subdivided into four groups by Sutton (1980), viz. Coniothyrium, Microsphaeropsis, Cyclothyrium and Cytoplea. Paraconiothyrium, which is closely related to Paraphaeosphaeria based on SSU sequences phylogeny (Verkley et al. 2004), was introduced to accommodate Coniothyrium minitans and C. sporulosum. Notes: The Didymosphaeriaceae is amended here to include Didymosphaeria and genera from Montagnulaceae including asexual states. We synonymise Montagnulaceae under Didymosphaeriaceae which is the oldest name and has priority and provide an updated account of the family. Type: Didymosphaeria Fuckel, Jb. Nassau. Ver. Naturk. 23-24: 140 (1870) [1869-70]. Genera accepted in Didymosphaeriaceae Alloconiothyrium Verkley et al., Persoonia, 32: 33 (2014). Type species: Alloconiothyrium aptrootii Verkley et al., Persoonia, 32: 33 (2014). Barria Z.Q. Yuan, Mycotaxon 51: 313 (1994). Type species: Barria piceae Z.Q. Yuan, Mycotaxon 51: 314 (1994). Bimuria D. Hawksw. et al., N.Z. Jl. Bot. 17(3): 267 (1979). Type species: Bimuria novae-zelandiae D. Hawksw. et al., N.Z. Jl. Bot. 17(3): 268 (1979). Deniquelata Ariyawansa & K.D. Hyde, Phytotaxa 105(1): 13 (2013). Type species: Deniquelata barringtoniae Ariyawansa & K.D. Hyde, Phytotaxa 105(1): 15 (2013). Didymocrea Kowalski, Mycologia 57(3): 405 (1965). Type species: Didymocrea sadasivanii (T.K.R. Reddy) Kowalski, Mycologia 57(3): 405 (1965). Basionym: Didymosphaeria sadasivanii K.R.C. Reddy, Mycologia 53(5): 471 (1962) [1961]. Didymosphaeria Fuckel, Jb. Nassau. Ver. Naturk. 23–24: 140 (1870) [1869-70]. Type species: Didymosphaeria futilis (Berk & Broome) Rehm, Hedwigia 18: 167 (1879). Basionym: Sphaeria futilis Berk. & Broome, Ann. Mag. nat. Hist., Ser. 2 9: 326 (1852). Julella Fabre, Annls Sci. Nat., Bot., sér. 6, 9: 113. 1879 [1878]. MycoBank MB 2539. Type species: Julella buxi Fabre, Annls Sci. Nat., Bot., sér. 6, 9: 113. 1878. Kalmusia Niessl, Verh. nat. Ver. Brünn 10: 204 (1872). Type species: Kalmusia ebuli Niessl, Verh. nat. Ver. Brünn 10: 204 (1872). Karstenula Speg., Decades Mycologicae Italicae 7–12: no. 94 (in sched.) (1879). 75 Type species: Karstenula rhodostoma (Alb. & Schwein.) Speg., Decades Mycologicae Italicae 7–12: no. 94 (1879). Basionym: Sphaeria rhodostoma Alb. & Schwein., Consp. fung. (Leipzig): 43 (1805). Letendraea Sacc., Michelia 2(6): 73 (1880). Type species: Letendraea eurotioides Sacc., Michelia 2(no. 6): 73 (1880). Montagnula Berl., Icon. fung. (Abellini) 2: 68 (1896). Type species: Montagnula infernalis (Niessl) Berl., Icon. fung. (Abellini) 2(2–3): 68 (1896). Basionym: Leptosphaeria infernalis Niessl, Inst. Coimbra 31: 13 (1883). Neokalmusia Kaz. Tanaka et al. (In this study) Type species: Neokalmusia brevispora (Nagas. & Y. Otani) Tanaka et al., Paraconiothyrium Verkley, Stud. Mycol. 50(2): 327 (2004) Type species: Paraconiothyrium estuarinum Verkley et al., Stud. Mycol. 50(2): 327 (2004). Paraphaeosphaeria O.E. Erikss., Ark. Bot., Ser. 2 6: 405 (1967). Type species: Paraphaeosphaeria michotii (Westend.) O.E. Erikss., Cryptogams of the Himalayas 6: 405 (1967). Basionym: Sphaeria michotii Westend., Bull. Acad. R. Sci. Belg., Cl. Sci., sér. 2 7(5): 87 (1859). Phaeodothis Syd. & P. Syd., Annls mycol. 2(2): 166 (1904). Type species: Phaeodothis tricuspidis Syd. & P. Syd., Annls mycol. 2(2): 166 (1904). Tremateia Kohlm. et al., Bot. Mar. 38(2): 165 (1995). Type species: Tremateia halophila Kohlm. et al., Bot. Mar. 38(2): 166 (1995). Key to the genera accepted in Didymosphaeriaceae 1. Parasitic on living leaves.................................. Deniquelata 1. Saprobic on dead wood or leaves.......................................... 2 2. Asci with 2 spores.................................................................... 3 2. Asci with more than 2 spores................................................. 4 3. Asci with short knob-like pedicel and brown, muriform ascospores................................................................... Bimuria 3. Asci without short knob-like pedicel and hyaline, muriform ascospores....................................................................... Julella 4. Peridium composed of thin pseudoparenchymatous cells of textura intricata.............................................. Didymosphaeria 4. Peridium composed of pseudoparenchymatous cells of textura angularis or textura prismatica................................. 5 5. Ascus with a long, furcate pedicel........................................ 6 5. Ascus with a short furcate pedicel.......................................... 7 6. Clavate ascus with narrowly ovoid to clavate, pale brown, 3septate ascospores..................................................... Kalmusia 6. Cylindro-clavate to clavate asci with oblong to narrowly oblong, reddish-brown to dark yellowish-brown, muriform or phragmosporous ascospores............................ Montagnula 7. Ascomatal wall composed only cells of textura intricata.........8 76 7. Ascomatal wall composed both cells of textura angularis and textura prismatica............................................................ Barria 8. Strictly in marine environments................................ Tremateia 8. In terrestrial environments......................................................... 9 9. Hamathecium consisting of filiform pseudoparaphyses .................................................................................. Didymocrea 9. Hamathecium consisting of cellular pseudoparaphyses........... 10 10. Ascomata with a pore-like ostiole......................................... 11 10. Ascomata with short papillate ostiole.................................. 12 11. Cylindrical asci with short pedicel and fusiform with rounded ends, olivaceous-brown, phragmosporous ascospores...................................................................... Phaeodothis 11. Cylindrical to cylindro-clavate asci with short furcate pedicel and ellipsoid to fusoid, reddish-brown to dark brown muriform ascospores.............................................. Karstenula 12. Ascomata with hyaline periphyses.................... Neokalmusia 12. Ascomata without hyaline periphyses.........Paraphaeosphaeria Key to asexual genera of Didymosphaeriaceae 1. Conidiomata pycnidial and with a single cavity, or eustromatic with several cavities............ Alloconiothyrium 1. Conidiomata eustromatic, simple or complex, rarely pycnidial........................................................... Paraconiothyrium Alloconiothyrium Verkley et al., Persoonia, 32: 33 (2014), Facesoffungi number: FoF 00028. Type species: Alloconiothyrium aptrootii Verkley et al., Persoonia, 32: 33 (2014), Facesoffungi number: FoF 00029. Saprobic in terrestrial environments. Sexual state: unknown (Verkley et al. 2014). Asexual state: Conidiomata 300–450μm diam, pycnidial and with a single cavity, or eustromatic and consisting of complexes reaching 1mm diam., with several cavities, with outer surface black, glabrous or covered by grey mycelium. Conidiomatal wall composed of an outer layer of brown, thick-walled cells of textura angularis and an inner layer of hyaline, thick-walled cells of textura angularis, the outer surface sometimes covered by a diffuse web of brown hyphae. Conidiogenous cells 4–9×3– 4μm, discrete, often positioned on clumps of cells that protrude into the cavity, broadly ampulliform, holoblastic, annellidic, often with an elongated neck showing several distinct percurrent proliferations. Conidia 3–4(–5)×2–3(– 3.5) μm, globose to irregularly ellipsoid, aseptate, initially hyaline, after secession olivaceous-brown, mature conidial wall orange-brown, the outer surface verruculose giving the conidium an irregular outline, with 1 large oil-droplet 1– 1.5μm diam (description from Verkley et al. 2014). Notes: Alloconiothyrium was introduced to accommodate Coniothyrium-like species in the Montagnulaceae (Verkley et al. 2014). Alloconiothyrium aptrootii is known from a soil sample collected in Papua New Guinea, and all other Fungal Diversity (2014) 68:69–104 Coniothyrium-like taxa studied in Verkley et al. (2014) were relatively distantly related. The annellidic conidiogenous cells and the verruculose conidia resemble Coniothyrium palmarum, the type species of Coniothyrium, but that species is characterized by 2-celled conidia and is also genetically distinct, belonging in the family Leptosphaeriaceae (de Gruyter et al. 2009). In our phylogeny, Alloconiothyrium forms a robust clade, sister to the Kalmusia clade. Thus, based on morphology coupled with DNA data, we confirm that this genus is well resolved and should be classified under Didymosphaeriaceae. Barria Z.Q. Yuan, Mycotaxon 51: 313 (1994). Type species: Barria piceae Z.Q. Yuan, Mycotaxon 51: 314 (1994) (Fig 2), Facesoffungi number: FoF 00031. Parasitic on Picea schrenkiana. Sexual state: Ascomata 240–300×270–330μm, solitary, sometimes gregarious, immersed, visible as black spots on host surface, uniloculate, globose, brown to dark brown, with centrally opening ostiole. Ostiole papillate to depressed, ostiolar canal filled with periphyses. Peridium 10–23μm wide, composed of two cell types, outer layers comprising 3–5 layers, thin-walled, dark brown to black, pseudoparenchymatous cells of textura angularis or textura prismatica, inner layers composed of 2– 3 layers of hyaline, thin-walled cells of textura angularis. Hamathecium composed of 1.5–3μm wide, broad, hyaline, septate pseudoparaphyses, surrounding the numerous asci and embedded in a gelatinous matrix. Asci (126–) 130–170(– 185)× (15–)17–20(–23) μm (x= 151.2× 19μm, n =25), 8spored, bitunicate, fissitunicate, clavate to cylindric-clavate, shortly acute pedicel or sub-sessile, apically rounded with a well-developed ocular chamber, arising from the base of the ascoma. Ascospores (18.5–)20–24.5(–27) × 9–12.5 μm (x =22×10.9μm, n=30), overlapping, 1–2-seriate, ellipsoidal to broadly fusiform, initially hyaline, becoming brown to dark brown at maturity, 1-septate, constricted at the septum, smooth to rough-walled, with small guttules, comprising two layers, endospore thin-walled, epispore thick-walled, mostly with upper cell larger than lower cell, surrounded by a distinct mucilaginous sheath. Asexual state: unknown. Material examined: CHINA, Xinjiang, Urumqi, Tianshan Mountain, on leaves of Picea schrenkiana Fisch. & C.A. Mey (Pinaceae), 1 July 1992, Z.Q. Yuan (NY 00164027, isotype). Notes: The monotypic genus Barria shares similarities with Didymopleella in ascomatal structure, asci and ascospores characters, but differs in having a peridium of textura prismatica and strongly unequal ascospore cells (Munk 1957; Yuan 1994). Recently, Zhang et al. (2012) tentatively assigned Barria in Phaeosphaeriaceae based on the ascomata, colour and shape of ascospores. Barria differs from Phaeosphaeria in having ascomata with thick peridium, clavate, short-pedicellate asci, and didymosporous ascospores, while Phaeosphaeria has broadly cylindrical to cylindric- Fungal Diversity (2014) 68:69–104 77 Fig. 2 Barria piceae (NY 00164027, isotype). a Herbarium label and specimens of Barria piceae. b Appearance of ascomata on host surface. c Vertical section through ascoma d Section through peridium. e Pseudoparaphyses in Congo red reagent. f Asci with pseudoparaphyses. g, h-i Asci. j Ocular chamber in congo red reagent. k-n Ellipsoidal to broadly fusiform ascospores with a sheath. Scale bars: c=100μm, d-i= 20μm, j-m=10μm clavate asci with sub-sessile pedicellate and phragmospores ascospores. Didymosphaeria shows similarities with Barria in having immersed to slightly erumpent ascomata under a clypeus, hyaline pseudoparaphyses, anastomosing frequently above the asci and 1-septate, ellipsoid ascospores. However, Barria differs from Didymosphaeria in having brown to dark brown ascomata comprising several layers of textura angularis to textura prismatica cells in the peridium, cellular pseudoparaphyses and asci having well-developed ocular 78 chamber bearing ellipsoidal to broadly fusiform, initially ascospores hyaline, becoming brown to dark brown at maturity, 1-septate with a distinct mucilaginous sheath. Didymosphaeria has hyaline to pale brown or (rarely) black peridium, consisting of an internal and external layers, flattened or elongated hyphae, textura intricata with trabeculate pseudoparaphyses and distoseptate ascospores without a s h e a t h ( A p t r o o t 1 9 9 5 ) . We p l a c e B a r r i a i n Didymosphaeriaceae because of its similarities with other genera in Didymosphaeriaceae. However, fresh collections of the type species of the genus are needed so that molecular data can be obtained to verify the natural taxonomic affinities of this genus. Bimuria D. Hawksw et al., N. Z. Jl. Bot. 17: 268 (1979). Type species: Bimuria novae-zelandiae D. Hawksw. et al., N.Z. Jl. Bot. 17(3): 268 (1979), (Fig 3), Facesoffungi number: FoF 00033. Saprobic in terrestrial habitats. Sexual state: Ascomata (185-)200×310(-330) μm diam., semi-immersed or superficial, solitary, scattered, globose, hyaline when young, turning dark brown to black when mature, ostiolate, the ostiole more or less sessile or raised into a very short neck. Peridium 5– 8μm diam., comprising 2–3 layers of compressed pseudoparenchymatous cells, outer layer comprising sub-hyaline at first, but becoming pale brown with age cells of textura angularis, inner wall composed of hyaline radially compressed cells of Fungal Diversity (2014) 68:69–104 textura angularis. Hamathecium of dense, 2.5–4μm broad, septate, cellular pseudoparaphyses, embedded in mucilage, rarely anastomosing and branching. Asci 82–95×20–33μm (x = 75 × 29 μm, n = 10), (1-)2(-3)-spored, bitunicate, fissitunicate, broadly clavate, with a short and small knoblike pedicel, ocular chamber best seen in immature asci. Ascospores 55–68×25–28μm (x =59×26μm, n=10), accumulating in a subglobose black shiny mass adhering together outside the ostiole, broadly ellipsoid but becoming narrowed towards the poles, muriform with (5-) 7 transverse septa, cells with (0-)l(-2) longitudinal septa in each cell, not constricted at the septa, dark brown, apical cells paler and lacking longitudinal septa, verruculose. Asexual state: unknown. Material examined: NEW ZEALAND, North Island, Wairarapa District, Nutty Farm, isolated from soil, 3 March 1978, C.C. Yen and J.E. Sheridan (CBS 107.79, isotype). Notes: Bimuria is a monotypic genus characterized by a very thin peridium, mostly 2-spored, fissitunicate asci and muriform, dark brown, verrucose ascospores (Hawksworth et al. 1979). Because of its unique morphological characters, the familial placement of this genus has been debatable, thus initially Hawksworth et al. (1979) placed the genus in Pleosporaceae. Later Barr (1987) transferred Bimuria to Phaeosphaeriaceae while Lumbsch and Huhndorf (2007) placed it in Melanommataceae. Morphologically, Bimuria is most similar to Montagnula (Hawksworth et al. 1979). Fig. 3 Bimuria novae-zelandiae (CBS 107.79, isotype). a-c Close up of ascoma. d Hyaline, pseudoparaphyses. e-g Two-spored asci with knob like pedicel. h-j Dark brown, muriform ascospores. Scale bars: a-b=100μm, c=5μm, d=5μm, e-g=30μm, i-j=10μm Fungal Diversity (2014) 68:69–104 However, the thick carbonaceous peridium distinguishes Montagnula from Bimuria (Hawksworth et al. 1979). In addition, the ascospores of Montagnula are discharged forcibly through the ostiole instead of forming a mass outside of the ostiole as in Bimuria (Hawksworth et al. 1979). Ascomauritiana lignicola V.M. Ranghoo & K.D. Hyde has somewhat similar ascospores in 4-spored asci, but this taxon has unitunicate asci (Ranghoo and Hyde 1999). Hawksworth et al. (1979) suggested that the morphological characters of Bimuria, such as ascospore release and large, thick-walled ascospores may be an adaptation to its soil-borne habitat. Phylogenetic analysis based on combined genes of SSU, LSU, RPB2 and TEF1 concluded that the type strain of Bimuria novae-zelandiae (CBS 107.79) nested in the Montagnulaceae (Schoch et al. 2009). Our phylogenetic study also showed similar results, with Bimuria forming a monophyletic clade sister to Tremateia. Therefore we include Bimuria as a well-established genus in Didymosphaeriaceae based on both morphology and phylogeny. Deniquelata Ariyawansa & K.D. Hyde, Phytotaxa 105(1): 13 (2013), Facesoffungi number: FoF 00034. Type species: Deniquelata barringtoniae Ariyawansa & K.D. Hyde, Phytotaxa 105(1): 15 (2013) (Fig 4), Facesoffungi number: FoF 00035. Pathogenic on living leaves of Barringtonia asiatica. Sexual state: Ascomata 150–180×164–190μm (x=175× 167μm, n=10), immersed, scattered, globose to subglobose, black to dark brown, smooth-walled, with a papillate to depressed, elongate ostiole. Peridium 9–17μm diam. (x=12μm, n=10), composed of 3–5 layers of brown to black, darkly pigmented, small, thick-walled, 2–5μm wide cells of textura angularis, with outer wall fused with the host cells, inner wall consists of 2-layers of polygonal to rectangular, light brownhyaline cells 1–4μm diam. Hamathecium composed of dense, 1–3 μm diam. (x= 2 μm, n = 20), broad, hyaline, septate pseudoparaphyses, surrounding the numerous asci and embedded in a gelatinous matrix. Asci (-60) 68–80×10–15μm (x=72×13μm, n=20), 8-spored, bitunicate, fissitunicate, clavate to broadly-clavate, with a 9–17μm, short, broad, furcate, long pedicel, rounded at apex, with an ocular chamber. Ascospores 14–16×5–7μm (x=15×6μm, n=40), biseriate or distichously arranged, partially overlapping, oblong to narrowly oblong, straight or somewhat curved, reddish-brown, with three transverse septa and 1–2 vertical septa in the central cells, constricted at the primary and secondary septa at maturity, verruculose, straight or slightly inequilateral, without a gelatinous sheath. Asexual state: unknown. Material examined: THAILAND, Chiang Rai Province., Muang District, Bandu, Baan Khuakhae, on leaf of Barringtonia asiatica (L.) Kurz (Lecythidaceae), 18 September 2011, K.D. Hyde (MFLU 12-0303, holotype). 79 Notes: Deniquelata is a monotypic genus characterized by immersed, dark brown to black ascomata, with bitunicate asci and brown, muriform ascospores. Deniquelata differs considerably from the type of Montagnula (M. infernalis) both in phylogeny and morphology (Ariyawansa et al. 2013c). Deniquelata is a pathogenic genus with fruiting bodies scattered in the necrotic host tissues. Ariyawansa et al. (2013c) concluded that phylogenetic analysis of the nucleotide sequences of combined 18S and 28S nrDNA provided good evidence that Deniquelata belongs in Montagnulaceae. In the present study Deniquelata forms a distinct clade sister to Bimuria and Tremateia. Thus we accept this genus under Didymosphaeriaceae. Didymosphaeria Fuckel, Jb. nassau. Ver. Naturk. 23-24: 140 (1870) [1869–70], Facesoffungi number: FoF 00036. Saprobic on woody branches and herbaceous stems and leaves. Sexual state: Ascomata solitary, scattered, or in small groups, immersed to erumpent, globose to ovoid, ostiolate. Ostiole papillate with a pore-like opening, ostiolar canal filled with hyaline cells (periphyses). Peridium 1-layered, thin, composed of brown pseudoparenchymatous cells of textura intricata. Hamathecium of dense, trabeculate, pseudoparaphyses, anastomosing mostly above the asci. Asci 8-spored, bitunicate, fissitunicate, cylindrical, with a furcated pedicel, apically rounded with an indistinct ocular chamber. Ascospores uniseriate, ellipsoid, brown, 1-septate, wall with different ornamentations. Asexual state: see notes below. Type species: Didymosphaeria futilis (Berk. & Broome) Rehm, Hedwigia 18: 167 (1879) (Fig 5), Facesoffungi number: FoF 00037. Basionym: Sphaeria futilis Berk. & Broome, Ann. Mag. nat. Hist., Ser. 2 9: 326 (1852). Saprobic on dead wood. Sexual state: Ascomata 110–140× 120–160μm (x=130×140μm, n=10), scattered, or in small groups, immersed to slightly erumpent, rarely nearly superficial, under a clypeus, globose to subglobose, membraneous, papillate. Papilla black, with a pore-like ostiole, ostiolar canal filled with periphyses. Peridium 10–20 μm (x=15μm, n=10) wide, 1-layered, composed of hyaline pseudoparenchymatous compressed cells of textura intricata, fusing at the outside with the host. Hamathecium of dense, 0.5–1μm (x =0.8μm, n=20) broad, long, trabeculate pseudoparaphyses, anastomosing frequently above the asci, embedded in mucilage. Asci 75– 85 × 4–6 μm (x= 78 × 6 μm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical, pedicellate, rounded at the apex with an indistinct ocular chamber. Ascospores 7–10×3–5μm (x = 9×5μm, n=40), uniseriate, slightly overlapping, ellipsoid with obtuse ends, brown, 1-septate, slightly to not constricted at the septum, with distinctly spinulose ornamentation. Asexual state: unknown. Material examined: UK. England: Norfolk, on dead stems of Rosa sp. (Rosaceae), March 1850, M.J. Berkeley (K 147683, holotype of Sphaeria futilis). 80 Fungal Diversity (2014) 68:69–104 Fig. 4 Deniquelata barringtoniae (MFLU 12-0303, holotype) a-b Fungus on host. c-d Section of ascoma (TS). e Section of peridium comprising a few layers of cells. f Hyaline pseudoparaphyses g-i Eight-spored asci with short, broad pedicel at the base. j Reddish-brown, muriform, ascospores. k Ascospores stained in Indian ink. l Germ tubes developing from ascospore cells. Scale bars: c-d=100μm, e=5μm, f=5μm, g-i= 20μm, j-k=10μm, l=20μm Notes: Didymosphaeria sensu lato, introduced for three species of ascomycetes with 2-celled ascospores, comprise species having a wide distribution and a broad host range. Saccardo (1882) restricted the genus to only those species with brown ascospores. Aptroot (1995) included over 400 epithets of Didymosphaeria in his monograph of the genus, after examining over 3,000 species, but only seven species were accepted. The placement of Didymosphaeria is confused as described in Ariyawansa et al. (2014b). Sivanesan (1984) reported that Didymosphaeria has Ascochyta and Periconia Fungal Diversity (2014) 68:69–104 81 Fig. 5 Didymosphaeria futilis (K 147683, holotype of Sphaeria futilis) a-b Fungus on the host. c Section through ascoma. d Close up of peridium. e Broad, long trabeculate pseudoparaphyses, anastomosing mostly above the asci. f-h Cylindrical asci with an indistinct ocular chamber. i-l Ascospores with distinct spinulose ornamentation. Scale bars: c=100μm, d=10μm, e, f-h=20μm, i-l=5μm asexual states, while Kirk et al. (2008) reported Fusicladiellalike and Phoma-like species. Linking Didymosphaeria to asexual states should be treated with caution until the type of D. futilis has been sequenced. Ariyawansa et al. (2014b) found that two strains of the D. rubi-ulmifolii clustered in the family Montagnulaceae, but were separated from other genera of the family with high bootstrap support. Comparison of the generic type, D. futilis, and D. rubi-ulmifolii shows that they have similar morphology and thus represent Didymosphaeria. Thus Ariyawansa et al. (2014b) suggested that, based on the available molecular data and morphology, Didymosphaeria can be referred to Montagnulaceae with Didymosphaeriaceae as the probable synonym of Didymosphaeriaceae. In this paper, we formally treat Didymosphaeriaceae as a separate family in the order Pleosporales and with Montagnulaceae as its synonymy. The generic type of Didymosphaeria, D. futilis needs to be recollected, epitypified and sequenced so that phylogenetic analysis can be used to confirm family relationships within Pleosporales. In the present study, we observed that the type strain of Paraconiothyrium brasiliense (CBS 100299) strain forms a robust clade with D. rubi-ulmifolii. Paraconiothyrium brasiliense was isolated from coffee fruits in Brazil, but this species has since been reported from various habitats in other continents, on woody and herbaceous host plants, such as Prunus spp. in South Africa (Damm et al. 2008). Nearidentical ITS sequences have been deposited in GenBank for endophytes isolated from Ginkgo biloba (DQ094168), Juniperus virginiana (Hoffman and Arnold 2008), and Ulmus davidiana var. japonica (AB665311), and also from the herb Alliaria petiolata (EF432267). Strain CBS 395.87 from soil sampled in Italy was identified as Parac. brasiliense by Verkley et al. (2014). Verkley et al. (2014) showed that the ACT and TUB sequences of several Parac. brasiliense strains used in their study were more variable than in other related species, suggesting that Parac. brasiliense could be a species complex. In this study we consider that Parac. brasiliense and Didymosphaeria rubi-ulmifolii are the same species as they 82 cluster with a high bootstrap support. We therefore synonymise these species. The only available name for this species is Didymosphaeria rubi-ulmifolii because Didymosphaeria brasiliensis is already in use. Didymosphaeria rubi-ulmifolii Ariyawansa et al., Phytotaxa.176 (1)111 Basionym: Paraconiothyrium brasiliense Verkley, in Verkley et al., Stud. Mycol. 50(2): 329 (2004), Furthermore, in our phylogeny several Parac. brasiliense, strains form a separate clade sister to the D. rubi-ulmifolii sensu stricto, thus here we decide to treat it as a separate species in Didymosphaeria (Didymosphaeria sp.) but without designating a new species because the morphology is not available. Paraconiothyrium variabile (CBS 120014) forms a well-supported clade in Didymosphaeria, thus here we synonymise Paraconiothyrium variabile under Didymosphaeria Didymosphaeria variabile (Riccioni et al.) Ariyawansa & K.D. Hyde, comb. nov., Index Fungorum number: IF550707. Basionym: Paraconiothyrium variabile Riccioni et al. Persoonia 20: 13 (2008). Julella Fabre, Annls Sci. Nat., Bot., sér. 6 9: 113 (1879) [1878], Facesoffungi number: FoF 00038. Saprobic or possibly lichenized. Sexual state: Ascomata immersed, becoming erumpent to nearly superficial, sphaeroid, black, coriaceous, ostiolate, formed under a clypeus. Ostiole usually widely porate, with a short neck, ostiolar canal filled with periphyses. Peridium two layered, outer wall composed of small, dark brown to black, heavily pigmented, thick-walled cells of textura angularis and fusing with the host tissue to the outside, inner wall comprising broad yellowish-brown cells of textura angularis, thick at the apex and thinner at the base. Hamathecium of dense broad, septate, cellular, pseudoparaphyses anastomosing and branching and embedded in mucilage. Asci 2-spored, bitunicate, fissitunicate, clavate to cylindrical, with a short, broad, furcated or knob-like pedicel, rounded at apex and without a distinct ocular chamber. Ascospores biseriate or partially uniseriate, asymmetric or nearly symmetric, hyaline, obovoid, fusoid, or elongate, multi-septate, muriform, verruculose, thin-walled, surrounded by a mucilaginous sheath, guttulate, constricted at the septa. Asexual state: unknown. Type species: Julella buxi Fabre, Annls Sci. Nat., Bot., sér. 6 9: 113 (1878) (Fig 6), Facesoffungi number: FoF 00039. Saprobic on woody substrates. Sexual state: Ascomata 200–350×190–250μm (x=300×230μm, n=10), immersed becoming erumpent to nearly superficial, sphaeroid, black, coriaceous, ostiolate, formed under a clypeus. Ostiole usually widely porate, with a short neck, ostiolar canal filled with periphyses. Peridium two layered, outer wall composed of small, dark brown to black, heavily pigmented, thick-walled cells of textura angularis and fusing with the host to the Fungal Diversity (2014) 68:69–104 outside, inner wall comprising broad yellowish-brown cells of textura angularis, thick at the apex and thinner at the base. Hamathecium of dense, 2–4 μm broad, septate, pseudoparaphyses anastomosing and branching above the asci and embedded in mucilage. Asci 90–130×25–35μm (x=110× 30μm, n=10), two-spored, bitunicate, fissitunicate, clavate to cylindrical, with a short, broad, furcated or knob-like pedicel, rounded at apex and without a distinct ocular chamber. Ascospores 30–35×10–15μm (x=32×30μm, n=13), biseriate or partially uniseriate, asymmetric or nearly symmetric, hyaline, obovoid, fusoid, or elongate, multi-septate, muriform, verruculose, thin-walled, surrounded by a mucilaginous sheath, guttulate, constricted at the septa. Asexual state: unknown. Material examined: FRANCE, Serignan, on Buxus sempervirens L. (Buxaceae), H. Fabre (S-F5992, holotype). Notes: Julella has been confused, with some species being saprotrophic on bark, while others are lichenized, and the delineation of the genus is poorly defined. Currently, 46 epithets are listed in Index Fungorum (2014). Many of the species assigned to the genus are facultatively lichenized with the alga Trentepohlia as the photobiont (Purvis et al. 1992). Julella shares many common features with Peltosphaeria but differs in having bisporous asci (Barr 1985). Barr (1985) proposed Peltosphaeria as a synonym of Julella. Treatments of Julella are by Barr (1985) who lists three species and one variety and Aptroot and van den Boom (1995) who considered Julella a predominantly tropical group of bark saprotrophs, and recognized the species: J. lactea (A. Massal.) M.E. Barr, J. sericea (A. Massal.) Coppins (=J. fallcaiosa (Stizenb. ex Arnold) R.C. Harris) and J. vitrispora (Cooke & Harkn.) M.E. Barr (=J. sublactea), and regarded other species as synonyms or assigned them to other genera. After examine the generic type of Julella, Ariyawansa et al. (2013a) introduced Halojulellaceae and Halojullela to accommodate J. avicenniae, a marine species in the suborder Pleosporineae, order Pleosporales, Dothideomycetes. Justification for the new family was based on combined gene analysis of the large and small subunits of the nuclear ribosomal RNA genes (LSU, SSU) and two protein coding genes RPB2 and TEF1, as well as morphological characters (Ariyawansa et al. 2013a). Combined gene analysis of nuSSU, nuLSU, mtSSU and TEF1 has shown that the putative strains of Julella fallaciosa (MPN141 and MPN547) forms a separate clade in Trypetheliales (Nelsen et al. 2011), and are therefore excluded from our analysis. Zhang et al. (2012) suggested that with the exception of hyaline ascospores, most of the characters of Julella are compatible with Montagnulaceae. Julella is compatible with Didymosphaeriaceae in having immersed ascomata formed under a clypeus, short neck, 2-layered peridium composed of cells of textura angularis and cylindric or oblong, pedicellate, often with an ocular chamber and oblong to narrowly oblong Fungal Diversity (2014) 68:69–104 83 Fig. 6 Julella buxi (S-F5992, holotype). a-b Surface view of ascomata on host substrate (bark). c Longitudinal section of ascoma with clypeus. d Close up of the peridium. e Cellular pseudoparaphyses. f-g Two spored asci lacking an ocular chamber. h-j Muriform ascospores. m Ascospore mounted in Indian ink to show mucilaginous sheath. Scale bars: c= 80μm, d=10μm, e=5μm, f-g=25μm, h-j=15μm muriform ascospores but differing in having hyaline ascospores. We add Julella to Didymosphaeriaceae but this has to be confirmed by molecular data. Kalmusia Niessl, Verh. nat. Ver. Brünn 10: 204 (1872), Facesoffungi number: FoF 00040 Saprobic on terrestrial habitats. Sexual state: Ascomata small- to medium-sized, solitary, scattered or in small groups, immersed to erumpent, globose or subglobose, often laterally flattened, coriaceous, black-walled, with or without papilla. Hamathecium of dense, filliform, delicate, septate pseudoparaphyses, branching and anastomosing between and above asci, embedded in mucilage. Asci bitunicate, clavate with a long, furcate pedicel. Ascospores narrowly ovoid to clavate, pale brown, 3-distoseptate. Asexual state: unknown. Type species: Kalmusia ebuli Niessl, Verh. nat. Ver. Brünn 10: 204 (1872) (Fig 7), Facesoffungi number: FoF 00041. Saprobic on dead stems. Sexual state: Ascomata 290– 360×300–520μm (x=300×430μm, n=10), solitary, scattered, or in small groups, immersed to erumpent, globose or subglobose, coriaceous, wall black, with or without papilla, ostiolate. Papilla small, with small ostioles. Peridium 15– 40μm wide, comprising one cell type of small, pigmented, dark brown to black, thick-walled cells of textura prismatica to textura angularis. Hamathecium of dense, 1.5 μm wide, delicate pseudoparaphyses, 1–septate, branching and anastomosing between and above asci, embedded in mucilage. Asci 75–125 × 10–15 μm (x = 90 × 12 μm, n = 10), 8-spored, bitunicate, fissitunicate, clavate, with a long, narrow, furcate pedicel up to 45 μm long, and a shallow ocular chamber. Ascospores 15–18×5–7μm (x=16×6μm, n=10), biseriate, narrowly ovoid to clavate, pale brown, 3-distoseptate, without constriction, smooth-walled. Asexual state: unknown. Material examined: MORAVIA, Bruno, on dead stems. (LUX 045054, isotype) 84 Fungal Diversity (2014) 68:69–104 Fig. 7 Kalmusia ebuli (LUX 045054, isotype) a Hebarium packet. b-c Surface view of ascomata on host substrate (bark). d Longitudinal section ascoma with clypeus. e Close up of the peridium. f Broad, septate pseudoparaphyses. g Long, narrow asci with furcate pedicel. h-i Pale brown ascospores with 3-distosepta. Scale bars: d=80μm, e=10μm, f= 5μm, g =25μm, h-i=15μm Notes: Kalmusia was characterized as “immersed, sphaeroid ascoma with central, stout papilla, surrounded by hyphae in the substrate, stipitate asci with septate pseudoparaphyses, and brown, 3-septate, inequilateral ascospores” by Barr (1992). The most morphologically comparable genus to Kalmusia is Thyridaria, which had been treated as a subgenus under Kalmusia (Lindau 1897), and was subsequently transferred to Platystomaceae in Melanommatales (Barr 1987, 1990). After comparing their morphological characters, Diapleella and Dendropleella were also considered synonyms of Kalmusia. Compared to Thyridaria, Kalmusia has sphaeroid ascomata, a peridium of small pseudoparenchymatous cells, basal asci and very thin pseudoparaphyses, thus it was assigned to Phaeosphaeriaceae by Barr (1990), and the genus is utilized to accommodate K. ebuli and K. clivensis (Berk. & Broome) M.E. Barr, as well as closely related species, i.e. K. utahensis (Ellis and Everh.) Huhndorf & M.E. Barr and K. coniothyrium (Fuckel) Huhndorf (Barr 1992). But this proposal is questionable because the clavate asymmetric ascospores as well as the clavate asci with long pedicels of K. ebuli do not fit the concept of the Phaeosphaeriaceae as defined by Zhang et al. (2012) and is excluded from the family by Phookamsak et al. (2014) Most recent phylogenetic studies indicated that some species of Kalmusia reside outside of the family Phaeosphaeriaceae (Zhang et al. 2009). Kalmusia ebuli, the type species of Kalmusia, lacks type material and as such its phylogenetic position has remained unresolved. As a consequence, the familial position of Kalmusia is based on morphology and molecular phylogeny of species other than the type (Zhang et al. 2014). Recently, a fresh collection of Fungal Diversity (2014) 68:69–104 K. ebuli was obtained from decorticated wood of Populus tremula in the foothills of the French Pyrenees (Zhang et al. 2014). The new collection was designated as neotype to stabilize the application of the species and genus name. Zhang et al. (2014) also observed the holotype of K. ebuli f. sarothamni, and it was found to be a synonym of K. ebuli. They concluded that the genus Kalmusia is polyphyletic within the family Montagnulaceae, with K. ebuli distantly placed from K. brevispora and K. scabrispora, which appear to represent a different genus. During our phylogenetic analysis, we also obtained similar results, with K. ebuli being distantly placed from K. brevispora and K. scabrispora and formed a distinct clade sister to a newly introduced asexual genus Alloconiothyrium in Didymosphaeriaceae. Furthermore, a recently introduced asexual genus, Dendrothyrium (typified with D. variisporum (CBS 121517T) and another species of Dendrothyrium, D. longisporum (CBS 582.83T), clustered within the Kalmusia ebuli clade. Therefore, we propose to synonymize Dendrothyrium under Kalmusia by giving priority to the older name and combine two species under Kalmusia. Kalmusia longispora (Verkley & Stielow) Ariyawansa & K.D. Hyde, comb. nov., Index Fungorum number: IF550696. Basionym: Dendrothyrium longisporum Verkley & Stielow, Persoonia 32: 35 (2014). Kalmusia variispora (Verkley & Stielow) Ariyawansa & K.D. Hyde, comb. nov., Index Fungorum number: IF550697. Basionym: Dendrothyrium variisporum Verkley & Stielow, Persoonia, 32: 36 (2014). Karstenula Speg., Decades Mycologicae Italicae ad no. 94 (in sched.) (1879), Facesoffungi number: FoF 00042. Saprobic on dead wood and stems in terrestrial habitats. Sexual state: Ascomata rarely small, usually medium-sized, immersed usually under thin clypeus, scattered to gregarious, with flattened top and rounded pore-like ostiole, coriaceous. Peridium 2-layered, outer layer composed of reddish-brown to dark brown small cells, with inner layer of pale compressed cells. Hamathecium of dense, cellular pseudoparaphyses. Asci bitunicate, fissitunicate, cylindrical to cylindro-clavate with short furcate pedicel. Ascospores ellipsoid to fusoid, reddishbrown to dark brown muriform. Asexual state: Microdiplodia (Constantinescu 1993). Type species: Karstenula rhodostoma (Alb. & Schwein.) Speg., Decades Mycologicae Italicae no. 94. (1879) (Fig 8), Facesoffungi number: FoF 00043. Basionym: Sphaeria rhodostoma Alb. & Schwein., Consp. fung. (Leipzig): 43 (1805). Saprobic on dead stems in terrestrial habitats. Sexual state: Ascomata 250–430×450–650μm (x=310×500μm, n=10), scattered or gregarious, immersed in the subiculum which sometimes sloths off, globose or subglobose, black, flattened top often white or reddish and sometimes slightly protruding 85 out of the substrate surface, usually with a wide opening ostiole after removing the cover, coriaceous. Peridium 30– 40μm wide, comprising two cell types, outer region single layered, composed of relatively small heavily pigmented thick-walled compressed cells, inner layer cells larger and wall thinner, comprising cells of textura angularis, merging with pseudoparaphyses. Hamathecium of dense, 2–3.5μm wide, long cellular, septate pseudoparaphyses. Asci 150– 210×12–15μm (x=182×13μm, n=20), 8-spored, bitunicate, fissitunicate, cylindrical, with a broad, furcate pedicel which is 12–35μm long, and with an ocular chamber. Ascospores 20– 26×7.5–10μm (x=22×8μm, n=20), obliquely uniseriate and partially overlapping, ellipsoid, reddish-brown, with 3transverse septa and a vertical septum in one or two central cells, constricted at the septa, verruculose. Asexual state: unknown. Material examined: SWEDEN, on dead stems, 1834, E. Fries (PH 01048835, holotype of Sphaeria rhodostoma Alb. & Schwein.). Notes: Karstenula is an ambiguous genus, which has been synonymised under Pleomassaria by several authors (Lindau 1897; Winter 1885). Karstenula shares similarities with Didymosphaeria in having ascomata seated in a subiculum or beneath a clypeal thickening and sometimes apical cells become reddish or orange-brown (Barr 1990). Barr (1990) modified the concept of Karstenula (sensu lato), which encompasses some species of Thyridium. However, Barr (1990) treated Karstenula as having trabeculate pseudoparaphyses and this is clearly not the case. In most cases, the ascospores are brown with transverse septa and sparse longitudinal septa. The ascomata of Karstenula rhodostoma are similar to those found in Byssosphaeria and Herpotrichia, especially in the paler area around the ostiole and even in peridium and development under a subiculum. The numerous wide cellular pseudoparaphyses and cylindrical asci (in Herpotrichia) are also similar. The main difference between Karstenula and the other two genera is the 3-septate ascospores with rare longitudinal septa (1-septate in Byssosphaeria and Herpotrichia). Recent phylogenetic studies have shown that the putative strain of Karstenula rhodostoma (CBS 690.94) forms a robust clade with Phaeodothis winteri (Niessl) Aptroot, Didymocrea sadasivanii, Bimuria novae-zelandiae, Montagnula opulenta, Curreya pityophila (J.C. Schmidt & Kunze) Arx & E. Müll., and some species of Letendraea and Paraphaeosphaeria (Kodsueb et al. 2006a; Zhang et al. 2009a). Thus Zhang et al. (2012) tentatively included Karstenula in Montagnulaceae. Our phylogenetic analysis reveals that the putative strain of Karstenula rhodostoma (CBS 690.94) resides in Didymosphaeriaceae, sister to the Paraphaeosphaeria clade. Therefore we tentatively refer Karstenula to Didymosphaeriaceae. However, the placement of Karstenula in Didymosphaeriaceae can only be confirmed by phylogenetic 86 Fungal Diversity (2014) 68:69–104 Fig. 8 Karstenula rhodostoma (PH 01048835, holotype of Sphaeria rhodostoma). a-b Herbarium packet and specimen. c. Ascomata on substrate. d Section thought ascoma. e Asci arrangement. f Close up of the peridium. g Hamathecium comprising cellular pseudoparaphyses. h-i Asci when immature. j Ascus at maturity. k-m Ellipsoid, reddish-brown ascospores. Scale bars: d=100μm, e, h–j=50μm. f, k–m=20μm, g= 10μm work including sequencing the generic type of Karstenula (K. rhodostoma). Letendraea Sacc., Michelia 2(6): 73 (1880), Facesoffungi number: FoF 00044. Saprobic in terrestrial habitats. Sexual state: Ascomata rarely small, usually medium-sized, immersed usually under a thin clypeus, scattered to gregarious, with flattened top and rounded pore-like ostiole, coriaceous. Peridium 2-layered, outer layer composed of reddish-brown to dark brown small cells, inner layer of pale compressed cells. Hamathecium of dense, cellular pseudoparaphyses. Asci cylindrical to cylindro- clavate with short furcate pedicel. Ascospores one septate, ellipsoid to fusoid, reddish-brown to dark brown. Asexual state: unknown. Type species: Letendraea eurotioides Sacc., Michelia 2(6): 73 (1880) (Fig 9), Facesof fungi number: FoF 00045. Saprobic in terrestrial habitats. Sexual state: Ascomata 100–130μm diam., semi-immersed to superficial, globose to subglobose, solitary, bright coloured, with papillate, central ostiole (from illustration). Hamathecium of dense, filliform, hyaline pseudoparaphyses. Asci (63-)73–75×13–18μm (x = Fungal Diversity (2014) 68:69–104 87 Fig. 9 Letendraea eurotioides (NY0091436, lectotype) a Semi immersed to superficial, globosesubglobose ascomata. b Hamathecium. c Young and immature aci. d-f Asci with eight spores. Scale bars: a-b=20μm, c=50μm, d-f=10μm, g=20μm 71×15μm, n=8), 8-spored, bitunicate, fissitunicate, cylindricalclavate, slightly curved, sessile. Ascospores 13–16×5–6μm (x = 14×5μm, n=20), 1–2-seriate, ellipsoidal to fusoid, uppercell wider toward base narrow and rounded ends, 1-septate, olivaceous brown, guttulate, smooth. Asexual state: unknown. Material examined: USA, on dead branches of Rubi fruticosi (NY0091436, lectotype). Notes: Letendraea is characterized by superficial, globose to conical ascomata, filliform pseudoparaphyses, obclavate to cylindrical, 8-spored asci, and fusoid to oblong, 1-septate ascospores (Saccardo 1880). Because of its similar morphology with Karstenula rhodostoma, Kodsueb et al. (2006b) assigned Letendraea to Melanommataceae. Subsequent multigene phylogenetic analysis indicated that both L. helminthicola (Berk. & Broome) Weese. and L . p a d o u k N i c o t & P a rg . - L e d u c . n e s t e d w i t h i n Montagnulaceae (Schoch et al. 2009; Zhang et al. 2009), thus Zhang et al. (2012) referred Letendraea to Montagnulaceae. In our phylogeny we obtained the same results and apart from the putative strains of L. helminthicola (CBS 884.85) and L. padouk (CBS 485.70), we included the putative strain of L. eurotioides (CBS 212.31), the generic type of Letendraea in our analysis. Therefore, we treat Letendraea as a separate genus in Didymosphaeriaceae. During our study we collected a Dothideomycetes species (Letendraea cordylinicola) from living and dead leaves of Cordyline sp. from Thailand, associated with leaf spot disease, and showing similar morphology to Wilmia brasiliensis. Dianese et al. (2001) referred Wilmia in Phaeosphaeriaceae based on morphological characters. Wilmia is similar to other genera in Phaeosphaeriaceae viz. Barria, Lautitia and Metameris, with its didymosporous ascospores and coelomycete asexual morph (Dianese et al. 2001; Zhang 2012). Zhang et al. (2012) tentatively placed Wilmia in Phaeosphaeriaceae as the genus is poorly known and has no supporting molecular phylogenetic evidence, although Zhang et al. (2012) mentioned that the genus might be accommodated in Leptosphaeriaceae based on its dicotyledonous host habit. Our phylogenetic analysis showed that a novel Letendraea species (Letendraea cordylinicola) nested within Letendraea in the family Didymosphaeriaceae. Letendraea and Wilmia share similar characters in having immersed to superficial, globose-subglobose ascomata with filliform pseudoparaphyses and ellipsoidal to fusoid 1-septate, olivaceous brown ascospores with smooth wall. Therefore, based on morphology of the type species of Wilmia, W. brasiliensis and novel Wilmia-like (Letendraea cordylinicola) species, 88 Fungal Diversity (2014) 68:69–104 coupled with molecular data we exclude Wilmia from Phaeosphaeriaceae and transfer it to Didymosphaeriaceae. Furthermore, we propose to synonymize Wilmia under Letendraea by giving priority to the older name. The type species, Letendraea brasiliensis and the novel species L. cordylinicola are described below. Letendraea brasiliensis (Dianese et al.), Phookamsak, Ariyawansa & K.D. Hyde comb. nov., Index Fungorum number: IF550698 (Fig 10), Facesoffungi number: FoF 00046. Basionym: Wilmia brasiliensis Dianeseet al., Mycologia 93(5): 1014 (2001). Fig. 10 Letendraea brasiliensis (UB 8438, holotype of Wilmia brasiliensis) a Herbarium label and specimens. b Ascomata on host surface. c Section through ascomata stained by cotton blue reagent. d Section through peridium. e Pseudoparaphyses stained by Congo red reagent. f-h Ascospores. i-k Asci. l Section through conidiomata stained by cotton blue reagent. m Conidiogenous cells. n Conidia. o-q Conidia. Scale bars: c=100μm, l=50μm, d-k=20μm, m=10μm, f-n=5μm, o-q= 2μm Fungal Diversity (2014) 68:69–104 Parasitic on Memora pedunculata. Sexual state: Ascomata 140–200×90–125μm, scattered, rarely clustered, immersed to erumpent through host surface, globose to subglobose, brown to dark brown, central ostiole. Ostiole papillate with elongated periphyses. Peridium 6–21μm wide, composed of 2–5 layers, thin-walled with equal thickness, small, flattened pseudoparenchymatous, brown to dark brown cells of textura angularis to textura prismatica. Hamathecium dense of 1.5–3 μm wide, cellular pseudoparaphyses, unbranched, not -constricted at the septa, hyaline, embedded in a mucilaginous matrix. Asci 70–90(-98)×(11-)13–15(-17) μm (x = 82.9 × 13.8 μm, n = 30), 8-spored, bitunicate, fissitunicate, cylindric-clavate, short-pedicellate, apically rounded with indistinctly ocular chamber. Ascospores (18-)20–24×6–7(-7.5) μm (x=21.9×6.9μm, n=30), overlapping, 1–2-seriate, didymosporous, fusiform with obtuse ends, brown, 1-septate, constricted at the septum, smooth-walled, with indistinct appendage at the ends, surrounded by mucilaginous sheath. Asexual state: cohabit with sexual state on leaf lesions. Conidiomata 80–220μm high, 100–165μm diam., pycnidial, scattered to clustered, solitary, immersed to erumpent through host surface as black dots, visible as slightly raised or bumped, small, pale brown to brown spots on host surface, uniloculate, globose to sub globose, brown to dark brown, central ostiole, papillate with large periphyses. Conidiomata walls 8–14μm wide, composed of 2–5 layers, thin-walled cells with equal thickness, small, flattened pseudoparenchymatous cells of textura angularis to textura prismatica, brown to dark brown. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 5–10×(4.5-) 5–7(-10) μm (x =7× 6.4μm, n=20), enteroblastic, phialidic, ampulliform to lageniform, hyaline, smooth-walled, aseptate. Conidia (12-)14–20(-26.5) × 5–7 μm (x = 15.9 × 6.4 μm, n = 30), oblong to obovoid or ellipsoidal with rounded or obtuse ends, hyaline, indistinctly 1-distoseptate, rough-walled. Material examined: BRAZIL, Distrito Federal Vargem Bonita, Fazenda Áqua Limpa, on leaves of Memora pedunculata Vell. (Bignoniaceae), 18 May 1995, C.A. Inácio (UB 8438, holotype of Wilmia brasiliensis). Letendraea cordylinicola Phookamsak, Ariyawansa & K.D. Hyde, sp. nov., Index Fungorum number: IF550695 (Fig 11), Facesoffungi number: FoF 00047. Etymology: The specific epithet cordylinicola is based on the host genus from which the fungus was isolated. Saprobic or parasitic on Cordyline sp. Sexual state: Ascomata 85–115 × 100–160 μm, scattered, immersed to semi-immersed, pale yellowish to reddish-brown on leaf lesions, globose to subglobose, pale yellowish to brown, central ostiole. Ostiole papillate and ostiolar canal filled with long periphyses. Peridium 9.5–20μm wide, composed of 2–5 l a y e r s , t h i n - w a l l e d w i t h e q u a l t h i c k n e s s , l a rg e 89 pseudoparenchymatous cells of textura angularis to textura prismatica, hyaline to pale yellowish. Hamathecium dense of 2–3(-4) μm wide, numerous, broadly cellular pseudoparaphyses, unbranched, anastomosing, indistinctly constricted at the septum, embedded in a mucilaginous matrix. Asci (51-) 55–60(-68)×(8-) 10–12μm (x=59.1×10.4μm, n= 20), 8-spored, bitunicate, fissitunicate, cylindric-clavate, short pedicellate, apically rounded with an indistinct ocular chamber, arising from the base of the ascoma. Ascospores 13–15× 3–4μm (x =13.9×3.4μm, n=30), overlapping, 1–3-seriate, didymosporous, fusiform, pale brown to brown, 1-septate, constricted at the septum, often enlarged near septum in the upper cell, smooth-walled, with distinct appendage at both ends, surrounded by a mucilaginous sheath. Asexual state: unknown. Culture characters: Colonies on potato dextrose agar (PDA) 73–82mm diam. after 30 days at 25-30 °C, white to pale orange at the margin, white to orange or grey at the centre; reverse white orangish to pale orange at the margin, with zonate brown to black in the centre, medium dense, irregular shape, flatten, separate from agar, dull with undulate edges, fluffy to velvety, margin with well-defined to slightly radiating, forming ascomata on colony after 8 weeks, producing light brown pigment tinted agar. Material examined: THAILAND, Mae Jai District, Pa Faek village, on dead leaves of Cordyline sp., 5 September 2010, R. Phookamsak, RP0064 (MFLU 11-0184, holotype), ex-type living culture = MFLUCC 11-0148; Chiang Rai, Muang District, Pakha village, on living Cordyline sp., 15 August 2010, S. Wikee, RP0066 (MFLU 11-0186), living culture = MFLUCC 11-0150; Phan District, Muang Phan, on living leaves of Cordyline sp. 7 November 2010, K. Niraphai, RP0097 (MFLU 11-0217), living culture = MFLUCC 11-0181. Notes: Letendraea brasiliensis and the generic type L. eurotioides share similar characters such as having globose-subglobose ascomata, cylindric-clavate asci with 1septate brown ascospores, but differ in the nature of the pseudoparaphyses (cellular versus filliform), size of the asci (small versus large) and nature of the ascospores (with distinct appendage versus without distinct appendage). Letendraea cordylinicola shares similarities with L. brasiliensis in having immersed to semi-immersed ascomata with central ostiole filled with long periphyses, cylindric-clavate, short pedicellate asci bearing fusiform, 1septate, brown ascospores and both cause leaf spots on their host. However, L. brasiliensis differs from L. cordylinicola in having large ascomata and indictinct appendages and sheath while L. cordylinicola has small ascomata, nice appendages and a sheath. Based on morphology coupled with DNA sequence data, we accept L. brasiliensis and L. cordylinicola in the genus Letendraea because of their similarities with other species in this genus. 90 Fungal Diversity (2014) 68:69–104 Fig. 11 Letendraea cordylinicola (MFLU11-0184, holotype) a Ascomata on host surface. b Section through ascomata. c Section through peridium. d Pseudoparaphyses stained by cotton blue reagent. e Asci embedded in pseudoparaphyses. f-h Asci. i-k Ascospores with polar appendages that eventually expand into a sheath. l Ascospore stained in Indian ink with a prominent sheath. m Germination of ascospore. Scale bars: b=50μm, c=20μm, d-h, m=10μm, i-l=5μm Montagnula Berl., Icon. fung. (Abellini) 2: 68 (1896), Facesoffungi number: FoF 00048. Saprobic on dead wood and leaves in terrestrial habitats. Sexual state: Ascomata small to medium size, immersed to erumpent, gregarious or grouped, globose to subglobose, black. Hamathecium of dense, narrowly cellular, septate pseudoparaphyses. Asci bitunicate, fissitunicate, usually cylindric-clavate to clavate with a long pedicel. Ascospores oblong to narrowly oblong, straight or somewhat curved, reddish-brown to dark yellowish-brown, muriform or phragmosporous. Asexual state: unknown. Type species: Montagnula infernalis (Niessl) Berl., Icon. fung. (Abellini).2: 68 (1896) (Fig 12), Facesoffungi number: FoF 00049. Basionym: Leptosphaeria infernalis Niessl, Inst. Coimbra 31: 13 (1883). Saprobic on dead leaves in terrestrial habitats. Sexual state: Ascomata 220–280×250–310μm (x = 250×280μm, n=5), immersed to erumpent, gregarious or clustered, globose to subglobose, sometimes triangular in dried material, short ostiole always filled with hyaline closely adhering cells. Peridium 40–55μm thick at sides, up to 80μm thick near the apex, 3-layered, outer layer composed of heavily pigmented thick-walled small cells of textura angularis, apex thicker with smaller cells and thicker cell wall, thinner near the base; mid layer less pigmented, innermost layer of narrow compressed rows of cells, merging with pseudoparaphyses. Hamathecium of dense, 2–4.5μm broad, narrow, septate cellular pseudoparaphyses. Asci 153–170×18–22μm (x = 157× Fungal Diversity (2014) 68:69–104 91 Fig. 12 Montagnula infernalis (M 1183, holotype of Leptosphaeria infernalis). a Fungus on the herbarium material. b-c Dry black ascomata on host surface. d Ascoma in horizontal section. e Layers of peridium. f Cellular pseudoparaphyses g immature ascus. h mature ascus with long pedicel. g-h Eight spored asci not visible. i-j Asci bearing muriform ascospores. Scale bars: a-c=0.5mm, d=100μm, e-f=10μm, g-j=20μm 20μm, n=10), 8-spored, bitunicate, fissitunicate, cylindroclavate to clavate, pedicel 28–60μm long, with an ocular chamber best seen in immature ascus. Ascospores 24–29×9– 11μm (x = 26×10μm, n=20), biseriate, oblong to narrowly oblong, straight or somewhat curved, reddish-brown to dark yellowish-brown, verruculose, with five transverse septa and one vertical septum in each middle cells, constricted at the primary and secondary primary septa. Asexual state: unknown. Material examined: PORTUGAL, Coimbra lusitania, on leaves of Fourcroya longava (Agavoideae), February 1881, Moller (M 1183, holotype of Leptosphaeria infernalis). Notes: Montagnula was introduced by Berlese (1896) in order to separate two dictyosporous species, M. infernalis (Niessl) Berl., and M. gigantean (Mont.) Berl. from Pleospora, based on the presence of hyphal stromatic tissue over the ascomata and asci with a long pedicel (Barr 2001). Wehmeyer (1957) had placed Montagnula as a subgenus of Pleospora. Crivelli (1983) again treated Montagnula as a separate genus and divided the genus into two sub genera, i.e. Montagnula and Rubiginospora, which are distinguished based on dark brown ascospores located on Agavaceae and reddish-brown ascospores on Poaceae, respectively (Barr 2001), but this proposal was not accepted by many mycologists. Subsequently, Leuchtmann (1984) and Aptroot (1995) included some phragmosporous and didymosporous species in the genus and eventually it became heterogenic (Zhang et al. 2012). Apart from the type species, some Montagnula species produce Aschersonia Mont. asexual morphs (Hyde et al. 2011). The genus presently has 28 epithets (Index Fungorum 2014). GenBank has 19 hits for the genus including putative strains of M. opulenta (De Not.) Aptroot (CBS 168.34), M. aloes Crous et al. (CPC 19671), M. rhodophaea (Bizz.) Leuchtm. (CBS 616.86), M. dura (Niessl) Crivelli (CBS 380.54), M. spartii (Fabre) Aptroot (CBS 183.58) and M. anthostomoides (Rehm) Leuchtm. (CBS 615.86). One striking character of Montagnula infernalis is the very long ascal pedicel which develops once it is released from the ascomata. However, this character appears to have evolved more than once and can be found in Kirschsteiniothelia elaterascus Shearer which clusters with Helicascus (Shearer et al. 2009). The same character is also found in Xenolophium and Ostropella in the Platystomaceae (Mugambi and Huhndorf 2009). Montagnula opulenta is a didymosporous species, but phylogenetically closely related to those dictyosporous (Karstenula rhodostoma) and phragmosporous (Paraphaeosphaeria michotii) members of Montagnulaceae (Zhang et al. 2009). This might indicate that compared to other morphological characters, ascospore type is not a good character at the family level classification. Recent phylogeny based on multi-gene analysis has shown that the putative strain of M. opulenta forms a robust phylogenetic clade with species of Bimuria, Curreya, Didymocrea, Letendraea, Paraphaeosphaeria, Phaeodothis and Karstenula, which 92 might represent a familial group (Schoch et al. 2006; Zhang et al. 2009, 2012). Our phylogenetic data also shows that the putative strains of Montagnula rhodophaea (CBS 616.86) and M. spartii ( C B S 1 8 3. 58 ) cl us t e r e d i n L e n t i t h e c i a c e a e an d Massarinaceae, respectively. The morphology and identification of these putative strains in GenBank as far as we can ascertain, cannot be checked, as they are not linked to any herbarium material, therefore we placed them in Lentitheciaceae incertae sedis and Massarinaceae incertae sedis, respectively. Montagnula dura (CBS 380.54) was excluded from our final analysis because in the preliminary analysis we observed that this strain clustered within Pleosporaceae (details not shown). Our phylogenetic results indicated that the putative strains of M. opulenta (CBS 168.34), M. aloes (CPC 19671) and M. anthostomoides (CBS 615.86) nested within Montagnulaceae and form a separate clade sister to the Kalmusia and Alloconiothyrium clades. The strain named Letendraea helminthicola (CHTA R43) resides in Montagnula. This is probably a misidentification of Montagnula sp. as Letendraea helminthicola. The morphology and identification of the putative strain (CHTAR43) of Letendraea helminthicola in GenBank as far as we can determine, cannot be checked, as they are not linked to any herbarium material thus preliminarily reidentified as Montagnula sp. Therefore based on morphology, coupled with available molecular data, we keep Montagnula as a distinct genus in Didymosphaeriaceae. Fresh collections of the M. infernalis and further molecular and morphological studies are desirable to confirm our results. Neokalmusia Kaz. Tanaka et al., gen. nov., Index Fungorum number: IF550700, Facesoffungi number: FoF 00050. Etymology: The generic epithet, neo (Lat., new), refers to the similarity to Kalmusia. Saprobic on culms of bamboo in terrestrial habitats. Sexual state: Ascomata immersed under black clypeus-like structure composed of host epidermis and fungal mycelia, subglobose to oblong on host surface, hemispherical, with several perithecia arranged in single or double rows along the long axis of the culms. Ostiole absent or short papillate, with numerous periphyses. Peridium at side composed of several layers of polygonal, thin-walled, brown cells; at the rim composed of vertically orientated, hyaline, cells of textura angularis. Hamathecium composed of numerous, broadly cellular pseudoparaphyses, branching, anastomosing, with slime coating. Asci (4–) 8-spored, bitunicate, fissitunicate, cylindricclavate, with a long stipe, apically rounded with welldeveloped ocular chamber, arising from basal ascomata. Ascospores overlapping, uniseriate or biseriate, phragmospores, fusiform, slightly curved, initially hyaline, becoming yellowish-brown to reddish-brown at maturity, 3– Fungal Diversity (2014) 68:69–104 5-septate, thick-walled, verrucose, with a sheath. Asexual state: unknown. Type species: Neokalmusia brevispora (Nagas. & Y. Otani) Kaz. Tanaka, Ariyawansa & K. D. Hyde, comb. nov., Index Fungorum number: IF550701. (Fig. 13), Facesoffungi number: FoF 00051. Basionym: Phaeosphaeria arundinacea var. brevispora Nagas. & Y. Otani, Rep. Tottori Mycol. Inst. 15: 38 (1977). Saprobic on culms of Sasa spp. Sexual state: Ascomata 200–370(–400)×190–330(–440) μm immersed under black clypeus-like structure composed of host epidermis and fungal mycelium, scattered to grouped, subglobose to oblong on host surface, 1–3.5mm long, 0.4–0.7mm wide. Ostiole absent or short papillate, 50–75(-110) μm long, with hyaline periphyses. Peridium 15–20μm diam., composed of 2- layers, outer layer composed of 2–4 layers, light brown to dark brown pseudoparenchymatous cells of textura angularis, inner wall consist of 1–2 layers, hyaline cells of textura angularis. Hamathecium of 1.5–2.5μm dense thick, cellular, hyaline, septate pseudoparaphyses. Asci 80–118×10.5–15μm (x= 100.2×13.4μm, n=32), 4–8-spored, cylindrical to clavate, bitunicate, fissitunicate, with a 10–25(–30) μm (x =19.8μm, n=30) a short pedicel. Ascospores 18–24(-26.5)×4–7μm (x= 20.1×5.2μm, n=76), uniseriate or partially overlapping, fusiform, 3-septate, with a mostly submedian (0.48–0.56; x=0.52, n=46) primary septum, reddish-brown, thick-walled, verrucose, with 2–5μm thick sheath. Asexual state: unknown. Material examined: JAPAN, Fukushima, Minamiaizu, Ose pond, on dead twigs of Sasa sp. (Poaceae), 30 Aug. 2003, N. Asama, (KT 1466 = HHUF 28229), living culture CBS 120248 = JCM 13543 = MAFF 239276; Hokkaido, Isl. Rishiri, Afutoromanai river, on dead twigs of Sasa kurilensis, 29 July 2007, K. Tanaka & G. Sato, (KT 2313 = HHUF 30016), living culture = NBRC 106240. Notes: A new genus Neokalmusia is established to accommodate two bambusicolous taxa, N. brevispora and N. scabrispora. These species have been previously placed in Kalmusia (Tanaka et al. 2005; Zhang et al. 2009), but subsequent molecular studies (Hyde et al. 2013) as well as in the present study (Fig. 1), do not support this placement. Morphological studies of Kalmusia based on the holotype of K. ebuli (type of this genus) indicated that the genus is characterized by solitary sphaeroid ascomata, a peridium of small pseudoparenchymatous cells, clavate basal asci with very long pedicels, very thin pseudoparaphyses and distoseptate, smooth-walled ascospores (Zhang et al. 2012, this study). The two Neokalmusia species on bamboo have subglobose to oblong ascomata including several perithecia in a row, clypeus-like structure composed of thinwalled cells and verrucose ascospores. Neokalmusia scabrispora (Teng) Kaz. Tanaka et al., comb. nov., Index Fungorum number: IF550702. (Fig. 14), Facesoffungi number: FoF 00052 Fungal Diversity (2014) 68:69–104 93 Fig. 13 Neokalmusia brevispora (KT 1466). a-b Ascomata on host surface. c-d Vertical section through ascomata. e Section through peridium. f Pseudoparaphyses. g-h Asci. i Ocular chamber. j Germinating ascospore. k-o Ascospores. Scale bars: a-b=1mm, c-d=100μm, e-o=10μm Basionym: Leptosphaeria scabrispora Teng, Sinensia, Shanghai 4: 378 (1934). Saprobic on culms of bamboo. Sexual state: Ascomata 200–300×130–500μm, immersed under black clypeus-like structure composed of host epidermis and fungal mycelium, subglobose to oblong on host surface scattered to grouped. Ostiole absent or slightly papillate, about 85–100 μm long, with numerous periphyses. Peridium 7.5–20μm thick at sides, composed of 3–6 layers of brown polygonal thin-walled cells of 5–10×2.5–6.5μm, surrounded by wedge-shaped stromatic region (250–400μm wide at sides) composed of vertically orientated hyaline cells of textura angularis. Hamathecium 1–2μm wide narrowly cellular, numerous, septate, branched and anastomosing, embedded in a mucilaginous matrix. Asci 123.5–160×(15.5-)17–22μm (x =142.1×1 8.8μm, n=50), 8spored, bitunicate, clavate, rounded at the apex and with an apical chamber, with a short stipe. Ascospores 29–40.5×7– 10μm (x =34.8×8.5μm, n=68), biseriate, fusiform to ellipsoid, slightly curved, 5 (rarely 7) -septate, with a median primary septum 0.48–0.53μm (x =0.50μm, n=57) wide, slightly constricted at the septa, with third cell from the apex enlarged, penultimate cells shortest, brown to yellowishbrown, verrucose, with 10–20μm wide sheath. Asexual state: unknown. Material examined: JAPAN, Tochigi, Kanuma, near Ooashi river, on dead twigs of Phyllostachys bambusoides (Poaceae), 6 March 2003, N. Asama (KT 1023 = HHUF 28608), living culture CBS 120246 = JCM 12851 = MAFF 239517; Kagoshima, Kumagegun, Isl. Yakushima, Miyanoura river (riverbank), on dead twigs of Phyllostachys bambusoides (Poaceae), 17 Mar. 2007, K. Tanaka & H. Yonezawa (KT 2202 = HHUF 30013), living cultures = NBRC 106237. 94 Fungal Diversity (2014) 68:69–104 Fig. 14 Neokalmusia scabrispora (KT 1023) a-b Ascomata on host surface. c-d Vertical section through ascomata. e Section through peridium. f Pseudoparaphyses. g-h Asci. i Ocular chamber. j Germinating ascospore. k-o Ascospores. a-b, f-o from KT 2202, c-e from KT 1023. Scale bars: a-b=1mm, c-d=100μm, e-o=10μm Notes: This fungus was originally described as a species of Leptosphaeria (Teng 1934) and later transferred to Massariosphaeria (Shoemaker and Babcock 1989) or Kalmusia (Tanaka et al. 2005). Neokalmusia scabrispora, however, does not belong to the Leptosphaeriaceae typified by Leptosphaeria doliolum or to the Thyridariaceae encompassing the type species of Massariosphaeria (M. phaeospora) (Hyde et al. 2013). Neokalmusia scabrispora shares similar characters to Neokalmusia brevispora in having immersed ascomata under black clypeus-like, cellular pseudoparaphyses, 8-spored, bitunicate, clavate, asci rounded at the apex and fusiform to ellipsoid, brown to yellowish-brown, verrucose ascospores with a thick sheath. Neokalmusia scabrispora differs in the thickness of the peridium (7.5–20 μm versus 15–20 μm) and the number of septa in ascospores (5–7 versus 3). Paraconiothyrium Verkley, Stud. Mycol. 50(2): 327 (2004), Facesoffungi number: FoF 00053. Saprobic or parasitic in terrestrial habitats. Sexual state: unknown (Verkley et al. 2004). Asexual state: Conidiomata Fungal Diversity (2014) 68:69–104 eustromatic, simple or complex, rarely pycnidial, Conidiogenous cells discrete or integrated, phialidic, sometimes percurrent. Conidia aseptate, sometimes 1-septate, thinwalled, smooth-walled or minutely warted, hyaline when liberated, later brown (Verkley et al. 2004). Type species: Paraconiothyrium estuarinum Verkley & Manuela Silva, in Verkley et al., Stud. Mycol. 50(2): 327 (2004), Facesoffungi number: FoF 00054. Saprobic in terrestrial environments. Asexual state: Conidiomata 0.2–0.5(-1) mm diam., mostly submerged in the agar, but also superficial and on the aerial mycelium, eustromatic, globose or flattened, dark brown to black, with several merging cavities, ostioles absent, opening by dissolution of upper cells. Conidiomatal wall 30–45μm thick, composed of outer layer of isodiametric or more flattened cells with hyaline to reddish-brown walls thickened up to 1.5μm, lined by a 35–60(-75) μm thick inner layer of textura angularis, surface of the conidiomatal wall often covered under brown entangling hyphae. Conidiogenous cells 4– 6.5×2.5–3.5(-4) μm, discrete, assembled into protruding masses of cells, or integrated in very compact conidiophores, ampulliform to subcylindrical, hyaline, indeterminate, phialidic with an inconspicuous periclinal thickening and collarette, later often with a single percurrent proliferation, mostly. Conidia narrowly ellipsoidal or short-cylindrical, straight or slightly curved, rounded at both ends, 1-celled, with one or two small, polar guttules, and with thin and smooth walls that are hyaline at secession, but soon becoming olivaceous- or yellowish-brown. Sexual state: unknown (Verkley et al. 2004). Notes: Paraconiothyrium was introduced by Verkley et al. (2004) to accommodate four new species, Parac. estuarinum, Parac. brasiliense, Parac. cyclothyrioides, and Parac. fungicola. Based on the combination of ITS and partial SSU nrDNA sequences Verkley et al. (2004), included Coniothyrium minitans and the ubiquitous soil fungus Coniothyrium sporulosum in Paraconiothyrium. In the same study, the asexual morphs of Paraphaeosphaeria michotii and P. p i l l e a t a w e r e r e g a r d e d a s r e p r e s e n t a t i v e o f Paraconiothyrium, but remained formally unnamed. Paraconiothyrium species are phylogenetically distant from typical members of the other coelomycete genera such as Coniothyrium, Microsphaeropsis, Cyclothyrium, and Cytoplea. In Paraconiothyrium, the conidiomata generally are complex, eustromatic and relatively thick-walled. They may appear as simple pycnidia, but then they usually lack a well differentiated ostiole. In Coniothyrium, the conidiomata are true pycnidia, which may merge in vitro but then always produce well-developed, sometimes even papillate ostioles. The most distinctive Coniothyrium feature is the annellidic conidiogenous cells, which percurrently proliferate after secession of each conidium. Coniothyrium conidia are thick- 95 walled and verruculose, with a truncate base and sometimes a basal frill (Sutton 1980). Microsphaeropsis species are also pycnidial, but their conidiogenous cells are discrete, Phomalike phialides, which only rarely proliferate percurrently. Microsphaeropsis olivacea, the type species, has pale brown, 1-celled, thin- and smooth-walled conidia (Verkley et al. 2004). Câmara et al. (2001) assessed morphological data in relation to ITS sequences for nine species of Paraphaeosphaeria, and identified three lineages, which were later confirmed by SSU data (Câmara et al. 2003). They found that only one species, Paraph. pilleata, was congeneric with the type species Paraph. michotii (Verkley et al. 2004). Thus, only two species were retained in Paraphaeosphaeria sensu stricto. For the other species the genera Phaeosphaeriopsis and Neophaeosphaeria were erected. Following multi-locus DNA analysis and detailed morphological study, Verkley et al. (2014) were able to delimit and formally propose nine new species and two new genera among the fungi in the Montagnulaceae formerly recognizable as Coniothyrium-like asexual morphs. Verkley et al. (2014) suggested that the genus Paraconiothyrium appears to be paraphyletic within the family Montagnulaceae but proposed that it should not be split up to further genera. In the present study, we also observed similar phylogeny, where Paraconiothyrium appears to be paraphyletic within the family Didymosphaeriaceae. Several sexual stages of some Paraconiothyrium species were reported during the study, i.e. the sexual state of Paraconiothyrium fuckelii (MFLUCC 13-0043) and novel species of Paraconiothyrium, Parac. magnoliaee introduced from Thailand and are described below. Paraconiothyrium fuckelii (Sacc.) Verkley & de Gruyter, in Gruyter et al., Stud. Mycol. 75: 25 (2012) (Fig. 15), Facesoffungi number: FoF 00055. Basionym: Coniothyrium fuckelii Sacc., Fungi venet. nov. vel. Crit., Sér. 5: 200 (1878). Saprobic on dead wood in terrestrial habitats. Sexual state: Ascomata 290–360×300–520μm (x=300×430μm, n=10), solitary, scattered, or in small groups, immersed to erumpent, globose or subglobose, coriaceous, wall black, with or without papilla, ostiolate. Papilla small, with small ostioles. Peridium 15– 40 μm wide, comprising one cell type of small, pigmented, thick-walled cells of textura prismatica to textura angularis. Hamathecium of dense, 1.5μm broad, delicate pseudoparaphyses, 1-septate, branching and anastomosing between and above asci, embedded in mucilage. Asci 75– 125×10– 15μm (x =90×12μm, n=10), 8-spored, bitunicate, fissitunicate, clavate, with a long, narrowed, furcate pedicel which is up to 45 μm long, and a low ocular chamber. Ascospores 15–18×5–7μm (x =16×6μm, n=10), biseriate, narrowly ovoid to clavate, pale brown, 3-septate, constricted at the middle septum, smooth-walled. Asexual state: 96 Fungal Diversity (2014) 68:69–104 Fig. 15 Paraconiothyrium fuckelii. (MFLU 14-0305) a-b Appearance of ascomata on the host surface. c Section of an ascoma. d Section of peridium. e Clavate ascus with a short, narrow pedicel. f-h Subglobose to ellipsoid or obovoid ascospores. i-j Conidiogenous cells. k Orangebrown conidia. Scale bars: c=100μm, d=25μm, e=10μm, f-k=5μm Conidiomata pycnidial 300–400μm diam. and with a single cavity, more often eustromatic. Conidiomatal wall 3-layered, outer layer composed cells of textura angularis with somewhat thickened, brown walls, and an inner layer composed cells of textura angularis-globulosa with somewhat thickened, hyaline walls. Conidiogenous cells 4–10(–13)×3– 5μm, discrete or integrated in short, simple, 1–2-septate conidiophores, broadly ampulliform to globose, holoblastic, often annellidic with 1 or 2 percurrent proliferations noticeable by the distinct scars on a somewhat elongated neck, hyaline. Conidia 3–4×2–3(–3.5) μm variable in shape, subglobose to ellipsoid or obovoid, rarely more cylindrical, initially hyaline, olivaceous-brown soon after secession, smooth, orangebrown, aseptate. Material examined: THAILAND, Chiang Rai, Bandu, on dead wood, 28 September 2012, K.M. Thambugala (MFLU 14-0305), living culture = MFLUCC 13-0043. Paraconiothyrium magnoliae Monkai, Ariyawansa & K.D. Hyde, sp. nov., Index Fungorum number: IF550703 (Fig. 16), Facesoffungi number: FoF 00056. Etymology: The specific epithet magnoliae is based on the host genus from which the fungus was isolated. Saprobic on dead leaves in terrestrial environments. Sexual state: Ascomata 130–200×150–250 μm (x = 150×320μm, n=10) small to medium-sized, immersed to semi-immersed, depressedglobose, ostiolate. Ostiole papillate, black, smooth, with beak, ostiolar canal lined without hyaline periphyses. Peridium 10–17μm (x =14μm, n=20) wide, usually with 3- Fungal Diversity (2014) 68:69–104 97 Fig. 16 Paraconiothyrium magnolia (MFLU 12-1414, holotype) a-b Fungus on host substrate. c Section of an ascoma. d Cellular, hyaline, septate, broad, pseudoparaphyses. e-g Cylindrical asci with a short, broad pedicel. h-j Yellowishbrown ascospores with bipolar appendage. k Germinating ascospores. Scale bars: c=50μm, d=5μm, e-g=30μm, h-j=5μm, k=10μm 5 layers, composed cells of textura prismatica. Hamathecium of dense 2–4μm (x =2μm, n=20) wide, cellular, septate, hyaline, pseudoparaphyses. Asci 16–27×4–6μm (x =23× 5μm, n=30), 8-spored, bitunicate, fissitunicate, cylindrical with a short, broad pedicel with a minute ocular chamber. Ascospores 15–30×4–7μm (x = 24×5, n=40), uniseritate or partially overlapping, broadly elliptical, yellowish-brown, 3septate (rarely 4-septate), with small guttules, with bipolar appendage at the tip and one polar appendage on the base, surrounded by a gelatinous sheath. Asexual state: unknown. Material examined: THAILAND, Chiang Mai, Doi Suthep-pui, on dead leaves of Magnoliae liliifera (Magnoliaeceae) 18 September 2011, J. Monkai (MFLU 140306, holotype), ex-type living culture = MFLUCC 10-0278 Notes: Paraconiothyrium magnoliae resembles many species of Phaeosphaeria in having 3-septate ascospores with a gelatinous sheath. Phaeosphaeria eustoma and Phaeosphaeria parvula have ascospores which are very similar in size to the morphology of P. magnoliaee. In addition, Parac. magnoliae is similar to Phaeosphaeria typharum and Phaeosphaeria barriae in having gelatinous sheathed ascospores (with appendaged ascospores only in Phaeosphaeria barriae), but differ in the shape of the ascospores. The phylogenetic analysis of combined ITS, LSU, SSU nrDNA and TUB sequences provides strong evidence that Parac. magnoliae belongs in Didymosphaeriaceae, where it forms a separate clade along with Parac. fungicola within the clade of Paraconiothyrium with relatively high bootstrap support (Fig 1), thus a new species is proposed. Paraphaeosphaeria O.E. Erikss., Ark. Bot., Ser. 2 6: 405 (1967), Facesoffungi number: FoF 00057. Saprobic in terrestrial habitats. Sexual state: Ascomata small to medium-sized, immersed to semi-immersed, depressed, globose, ostiolate. Ostiole with a short beak, without periphyses. Peridium usually with 3-5 layers, composed cells of textura prismatica. Hamathecium composed of filamentous, hyaline, septate, broad, dense pseudoparaphyses. Asci 8spored, bitunicate, fissitunicate, cylindrical with a short pedicel. Ascospores uniseritate or partially overlapping, multi septate, broadly elliptical, yellowish-brown, with small guttules, smooth, with a thin uniform sheath. Asexual state: Coniothyrium-like, Conidiomata eustromatic or pycnidial. Conidiogenous cells discrete or integrated, phialidic, or annellidic with one or two percurrent proliferations. Conidia aseptate or 1-septate, smooth to verrucose (Verkley et al. 2014). Type species: Paraphaeosphaeria michotii (Westend.) O.E. Erikss., Cryptogams of the Himalayas 6: 405 (1967) (Fig. 17), Facesoffungi number: FoF 00058. Basionym: Sphaeria michotii Westend., Bull. Acad. R. Sci. Belg., Cl. Sci., sér. 2 7(5): 87 (1859). Saprobic on dead leaves of Poaceae. Sexual state: Ascomata 130–200×150–250μm (x = 150×320μm, n=10) 98 Fungal Diversity (2014) 68:69–104 Fig. 17 Paraphaeosphaeria michotii. (MFLU 12-2210, epitype) a Ascomata on host substrate. b Section of ascoma. c Close up of the peridium. d Ascus surrounded by cellular pseudoparaphyses. e-g Asci with short, broad pedicel bearing 8 spores. h-j Mature ascospores with thin uniform sheath. k Ascospores mounted in Indian ink. Scale bars: b=100μm, c=50μm, d=20μm, f-h=60 μm, i-k=10μm small to medium-sized, immersed to semi immersed, depressed globose, ostiolate. Ostiole papillate, black, smooth, with beak and without periphyses. Peridium 10–17μm (x= 14μm, n=20) wide, usually with 3–5 layers, composed of cells textura prismatica. Hamathecium of dense 2–3μm (x= 2 μm, n = 20) filamentous, hyaline, septate, broad, pseudoparaphyses. Asci 60–85×12–28μm (x=77×20μm, n=20), 8-spored, bitunicate, fissitunicate, cylindrical with a short, broad pedicel with minute ocular chamber. Ascospores 15–30×4–7μm (x = 24×5, n=40), uniseritate or partially overlapping, 2-septate, broadly elliptical, yellowish-brown, with small guttules, smooth, with a thick uniform sheath. Asexual state: not observed in the culture. Material examined: ITALY, Forlì-Cesena, Montevescovo, on dead leaves of Poaceae, 3 February 2012, E. Camporesi (MFLU 12-2210, epitype), ex-type living culture = MFLUCC 13-0349. Notes: Paraphaeosphaeria has been separated from Leptosphaeria (Eriksson 1967), and it is quite comparable with Phaeosphaeria. Paraphaeosphaeria can be distinguished from Phaeosphaeria by its ascospores. Ascospores of Paraphaeosphaeria michotii have two septa, and they are biseriate, straight, subcylindrical with broadly rounded ends, rather dark brown and punctate. The primary septum is laid down closer to the distal end than to the proximal, and the larger, proximal hemispore is divided by one transverse septum. There are more septa in the proximal hemispore of other species such as Paraph. castagnei (Durieu & Mont.) O.E. Erikss., Paraph. obtusispora (Speg.) O.E. Erikss. and Paraph. vectis (Berk. & Broome) Hedjar. Asexual characters can also distinguish Paraphaeosphaeria and Phaeosphaeria. Paraphaeosphaeria produces Coniothyrium-like asexual states, but Phaeosphaeria has Hendersonia-Phaeoseptoria asexual states (Eriksson 1967). Câmara et al. (2001) provided descriptions of sexual and asexual morphs of Paraph. michotii and Paraph. pilleata, while other species treated under Paraphaeosphaeria were transferred later to Neophaeosphaeria and Phaeosphaeriopsis (Câmara et al. 2003). None of the amerosporic coniothyrium-like species associated with these sexual morphs has been assigned with a formal name. Shoemaker and Babcock (1985) redescribed some Canadian and extralimital species, and excluded Paraph. longispora (Wegelin) Crivelli and Paraph. oblongata (Niessl) Crivelli from Paraphaeosphaeria based on their longitudinal septa as well as beak-like papilla and wall structures. Molecular phylogenetic results based on a multi-gene study indicate that Paraphaeosphaeria should belong to Montagnulaceae (Zhang et al. 2009). Recently, Ariyawansa et al. (2014d) epitypified Paraph. michotii from a fresh collection and thus the placement of the Paraphaeosphaeria in Montagnulaceae is confirmed. In our phylogenetic analysis, Paraphaeosphaeria forms a well-supported clade sister to Fungal Diversity (2014) 68:69–104 Didymosphaeria, thus Paraphaeosphaeria is treated as a separate genus in the Didymosphaeriaceae. Phaeodothis Syd. & P. Syd., Annls mycol. 2(2): 166 (1904), Facesoffungi number: FoF 00059. Saprobic or parasitic on dead wood or grasses in terrestrial environments. Sexual state: Ascostromata subglobose to hemispherical, immersed, scattered to gregarious, dark brown to black. Peridium comprising several layers of hyaline compressed cells. Hamathecium of dense, cellular pseudoparaphyses often in a gelatinous matrix. Asci 8-spored, bitunicate, fissitunicate, cylindrical, short pedicellate. Ascospores fusiform with rounded ends, olivaceousbrown. Asexual state: unknown. Type species: Phaeodothis tricuspidis Syd. & P. Syd., Annls mycol. 2(2): 166 (1904) (Fig. 18), Facesoffungi number: FoF 00060. Saprobic on dead wood or grasses. Sexual state: Ascomata 235–290×120−180μm (x=254×137μm, n=10), immersed to nearly superficial under clypeus, scattered to gregarious, subglobose to hemispherical, dark brown to black, papillate, Fig. 18 Phaeodothis tricuspidis (S F125876, holotype) a Herbarium packet and specimen. b Close up of ascomata. c-d Sections through ascomata. e Peridium comprising hyaline compressed cells. f-h Mature and immature asci surrounded by pseudoparaphyses. i-l Fusiform, olivaceous brown ascospores. Scale bars: c-d=100μm, e= 50μm, f-h=25μm, i-l=5μm 99 smooth. Papilla black, with pore-like ostioles, ostiolar canal filled with periphyses. Peridium 15−28μm (x=22μm, n=20) wide, comprising several layers of hyaline compressed cells. Hamathecium of dense, 2−4μm (x=2.5μm, n=20), aseptate, cellular pseudoparaphyses often in a gelatinous matrix. Asci 55 − 80 × 7 − 12 μm (x =68 × 10 μm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical, short pedicellate, apically rounded with a minute ocular chamber. Ascospores 14.5−17.5×4−5μm (x=15.8×4.4μm, n=30), biseriate and partially overlapping, fusiform with rounded ends, olivaceous brown, 1-septate, slightly constricted at the septa. Asexual state: unknown. Material examined: ARGENTINA, San José, Salta, 12 February 1873, P.G. Lorentz (SF125876, holotype). Notes: Phaeodothis is characterized by its immersed to nearly superficial ascomata, a sparse hamathecium consisting of cellular pseudoparaphyses and 1-septate ascospores (Aptroot 1995). Phaeodothis was placed in Didymosphaeria by von Niessl (1875), but Aptroot (1995) transferred it to Phaeosphaeriaceae. Phaeodothis is similar to genera in the 100 Didymosphaeriaceae in having ascomata immersed under a clypeus, a pseudoparenchymatous peridium with small cells, cylindrical asci and brown ascospores. However, Phaeodothis differs from other genera of Didymosphaeriaceae in having a hamathecium consisting of sparse pseudoparaphyses and 1septate ascospores. Phylogenetic analysis based on LSU, SSU, RBP1, RBP2 and EF-1 sequences concluded that a strain named Phaeodothis winteri (a synonym of P. tricuspidis Syd. & P. Syd.) clustered within the clade of Montagnulaceae (Schoch et al. 2009). Currently 27 epithets are listed for the genus in Index Fungorum (2014), while GenBank has seven hits for the genus including the putative strain of P. winteri (CBS 182.58). Based on the above morphological characters and available molecular data, we suggest that Phaeodothis can be referred to Didymosphaeriaceae. Tremateia Kohlm., Volkm.-Kohlm. & O.E. Erikss., Bot. Mar. 38(2): 165 (1995). Facesoffungi number: FoF 00223 Type species: Tremateia halophila Kohlm et al., Bot. Mar. 38(2): 166 (1995) (Fig. 19), Facesoffungi number: FoF 00062. Saprobic on dead wood in marine habitats. Sexual state: Ascomata 225–320×330−410μm (x=260×355μm, n=10), solitary, scattered, brown to black, immersed in the host tissue, globose to subglobose. Peridium 15–25μm diam., composed of two layers, outer layer composed of irregular, thick-walled, brown cells of textura angularis. The inner layer composed of hyaline, larger cells of textura angularis. Hamathecium dense of 2–2.5 μm cellular, septate long, hyaline pseudoparaphyses. Asci 120–170 × 24 − 30 μm (x= 150 × 26 μm, n = 20), 4–8spored, bitunicate, clavate to broadly clavate, short pedicellate, thin-walled, apically rounded with a broad ocular chamber. Ascospores 25–42×12−20μm (x=30×16μm, n=20), uniseriate or biseirate, ellipsoid to fusiform, light brown to brown, muriform, constricted at central septum, with 2layered cells, sometimes rounded at both sides with a 15– 25μm wide, large uniform sheath. Asexual state: Phoma-like (Kohlmeyer et al. 1995). Material examined: USA, North Carolina: Broad Creek, on senescent culms of Juncus roemerianus Scheele (Juncaceae), 28 November, 1993, J. J. Kohlmeyer (NY 01353617, isotype). Notes: Tremateia, introduced as a facultative marine genus (Kohlmeyer et al. 1995), is characterized by depressed globose, immersed ascomata, numerous cellular pseudoparaphyses, fissitunicate and clavate asci, ellipsoid muriform ascospores, and a Phoma-like asexual stage (Kohlmeyer et al. 1995). Based on these morphological characters, Kohlmeyer et al. (1995) referred Tremateia to Pleosporaceae. Furthermore, Kohlmeyer et al. (1995) suggested that Lewia M.E. Barr & E.G. Simmons and Diademosa Shoemaker & C.E. Babc., are superficially similar Fungal Diversity (2014) 68:69–104 to Tremateia. DNA sequences based phylogenies of recent literatures, and also in the present study, have shown that T. halophila nested in Montagnulaceae sister to Bimuria novae-zelandiae. (Schoch et al. 2009; Suetrong et al. 2009), thus familial status of Tremateia is verified. Discussion Molecular data play a pivotal role in modern mycological taxonomy, but have some constraints in application (Ariyawansa et al. 2014a; Boonmee et al. 2014; Hyde et al. 2014; Nilsson et al. 2014; Phookamsak et al. 2014; Schoch et al. 2014; Thambugala et al. 2014; Wijayawardene et al. 2014). The most significant and unsettled problem is that the phylogeny inferred from any gene may not reveal the evolution history of the organism (Uilenberg et al. 2004). It is therefore better to incorporate a polyphasic taxonomy including genotypical and phenotypical characteristics in all future studies (Ariyawansa et al. 2014a; Udayanga et al. 2014; Uilenberg et al. 2004). The genome also needs to be evaluated (Ariyawansa et al. 2014a; Uilenberg et al. 2004). There have been only a few molecular investigations of Didymosphaeriaceae compared to morphological studies (Ariyawansa et al. 2014b). Genera with trabeculate pseudoparaphyses and 1-septate, brown ascospores viz Bicrouania, Caryosporella, Lineolata, Phaeodothis, Roussoella and Verruculina, were generally classified under Didymosphaeriaceae. Molecular studies have shown that these particular morphological characters have evolved in different families (Ariyawansa et al. 2014b). The position to the taxa treated in the present study, it is required to first ascertain the phylogenetic placement of the fungus in the Montagnulaceae, as similar fungi occur in other Pleosporales as well; LSU can be used to verify the order and mostly also the family and genus to which the fungus belongs. ITS alone might be adequate for a precise identification of most species, as it is adequately variable among most closely associated taxa in Montagnulaceae, but it fails to distinguish all species. Furthermore we used TUB gene for our combine gene phylogeny, which was proposed by Verkley et al. (2014) to resolve the molecular taxonomy of taxa in Montagnulaceae. By combining multi-locus DNA sequencing with detailed morphological analyses, we were able to delimit and formally propose one new species, one sexual morph and one new genus among the taxa in the family Didymosphaeriaceae, previously classified in different families and genera in the order Pleosporales. Importantly, we synonymise Montagnulaceae under Didymosphaeriaceae by giving the Fungal Diversity (2014) 68:69–104 Fig. 19 Tremateia halophila (NY 01353617, isotype) a-b, d Vertical hand sections of ascomata. c, e Vertical hand section of ostiole and peridium. f-j Asci with muriform ascospores. k Cellular 101 pseudoparaphyses. l-q Fusiform, light brown to brown, muriform ascospores. Scale bars: a-d=50μm, c, e=20μm, f-i, j-k=10μm, l-k=5μm 102 priority to the oldest name. The paraphyletic nature of Paraconiothyrium is partially resolved by classifying some species with their sexual states and combining both morpholo g y a n d p h y l o g e n y. B e c a u s e t h e b r a n c h e s o f Paraconiothyrium clade that conflict with its monophyly are insufficiently supported, it was decided not to split it up into further genera. Morphological characters traditionally used to define genera in coelomycetes include conidiomatal structure, structure of the conidiophores, conidiogenesis and conidial characters such as pigmentation, septal structure and number, and conidial appendages (Sutton 1980; Nag Raj 1993). Recent molecular studies have shown that these features are not always suitable in delimiting genera as natural entities, and they may vary even between sibling species (Crous et al. 2012). Generic boundaries drawn in the present study are based primarily on statistically well-supported branches in a multi-locus phylogeny. Some of the characters mentioned above are thus overlapping between the accepted genera. For example, phialidic and annellidic conidiogenesis occur both in Paraconiothyrium and Paraphaeosphaeria (Verkley et al. 2004, 2014; Damm et al. 2008). Recent studies based on multi gene analysis have proven that the morphology of the Coniothyrium-like scatter throughout the order Pleosporales i.e. species with setose pycnidia and conidiogenesis with elongated conidiophores occur in other genera, such as, Cucurbitariaceae, Didymellaceae, Phaeosphaeriaceae, Leptosphaeriaceae, Montagnulaceae and Sporormiaceae, indicating convergent evolution (Gruyter et al. 2013). Neokalmusia was introduced in this study to accommodate Kalmusia brevispora and K. scabrispora based on molecular analysis in combination with morphology and a review of the literature. By introducing the new genus Neokalmusia, we were able to sort out the paraphyletic nature of Kalmusia in the family Didymosphaeriaceae. Furthermore, Barria included in the family Didymosphaeriaceae, have been previously referred to Phaeosphaeriaceae. Wilmia was synonymized under Letendraea based on the fresh collection of new species Letendraea cordylinicola (MFLU11-0184). A confusing fact is that the putatively named strains of Montagnula, M. spartii (CBS 183.58) and M. rhodophaea (CBS 616.86) obtained from GenBank, clustered in Massarinaceae and Lentitheciaceae, respectively. This is an example of the problems of sequence data in GenBank and other public databases. The morphology and identification of the putative strains of M. spartii (CBS 183.58) and M. rhodophaea (CBS 616.86) in GenBank cannot be checked, as they are not linked to any herbarium material. Acknowledgments MFLU grant number 56101020032 is thanked for supporting studies on Dothideomycetes. We are also grateful to the Mushroom Research Foundation, Chiang Rai, Thailand for funding. Kevin D. Hyde thanks the Chinese Academy of Sciences, project number 2013T2S0030, for the award of Visiting Professorship for Senior International Scientists at Kunming Institute of Botany. Jian-Chu Xu would Fungal Diversity (2014) 68:69–104 like to thank Humidtropics, a CGIAR Research Program that aims to develop new opportunities for improved livelihoods in a sustainable environment, for partially funding this work. H.A Ariyawansa and J.C. Kang are grateful to the Agricultural Science and Technology Foundation Of Guizhou Province (Nos. NY[2013]3042), the International Collaboration Plan of Guizhou Province (No. G [2012]7006) and the innovation team construction For Science and Technology Of Guizhou Province (No. [2012]4007) from the Science and Technology Department of Guizhou Province, China. K. Tanaka would like to thank the Japan Society for the Promotion of Science (JSPS, 25440199) for financial support. 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