A molecular phylogenetic reappraisal of the Didymosphaeriaceae (= Montagnulaceae)

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, Didymosphaeriaceaeand Montagnulaceae, have been treated in Pleosporales,Dothideomycetes, and both include saprobes, endophytesand pathogens associated with a wide variety of substratesworldwide. Didymosphaeriaceae was characterized by 1-septate ascospores and trabeculate pseudoparaphyses, mainlyanas tomosing above the asc i , whi le species inMontagnulaceae had 1 to multi-septate ascospores and gen-erally cellular pseudoparaphyses. In recent treatments,Bimuria, Didymocrea, Kalmusia, Karstenula, Montagnula,Paraphaeosphaeria, Paraconiothyrium and Letendraea wereplaced in Montagnulaceae, while only Didymosphaeria hasbeen placed inDidymosphaeriaceae. New morphological and

molecular data from recent collections have become availableand 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, thetaxonomy of the genera classified in these families are re-assessed. Our phylogenetic analyses conclude that, the recent-ly introduced Didymosphaeria rubi-ulmifolii withParaconiothyr ium brasi l iense species complex,Alloconiothyrium, Bimuria, Deniquelata, Didymocrea,Kalmusia , Karstenula , Letendraea , Montagnula ,Neokalmusia, Paraconiothyrium, Paraphaeosphaeria,Phaeodothis and Tremateia, forms a robust clade named hereDidymosphaeriaceae. We therefore synonymize

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 Chinae-mail: [email protected]

H. A. Ariyawansa : J.<C. Xu :K. D. HydeWorld Agroforestry Centre, East Asia and Central, Heilongtan,Kunming 650201, People’s Republic of China

H. A. Ariyawansa : J.<C. KangThe Engineering and Research Center for SouthwestBio-Pharmaceutical Resources of National Education Ministry ofChina, Guizhou University, Guiyang 550025, Guizhou Province,China

H. A. Ariyawansa :K. M. Thambugala : R. Phookamsak :Q. Tian :S. Hongsanan : J. Monkai :D. N. Wanasinghe :A. Mapook :E. Chukeatirote :K. D. HydeSchool of Science, Mae Fah Luang University, Chiang Rai 57100,Thailand

H. A. Ariyawansa :K. M. Thambugala : R. Phookamsak :Q. Tian :S. Hongsanan : J. Monkai :D. N. Wanasinghe :A. Mapook :E. Chukeatirote :K. D. HydeInstitute of Excellence in Fungal Research, Mae Fah LuangUniversity, Chiang Rai 57100, Thailand

K. TanakaFaculty of Agriculture and Life Science, Hirosaki University, 3Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan

E. CamporesiA.M.B. Gruppo Micologico Forlivese “Antonio Cicognani”, ViaRoma 18, Forlì, Italy

E. H. C. McKenzieManaakiWhenua Landcare Research, Private Bag, 92170 Auckland,New Zealand

E. B. G. JonesDepartment of Botany and Microbiology, King Saudi University,Riyadh, Saudi Arabia

Fungal Diversity (2014) 68:69–104DOI 10.1007/s13225-014-0305-6

http://dx.doi.org/10.1007/s13225-014-0305-6

Montagnulaceae under Didymosphaeriaceae which is theoldest name and has priority and provide a update accountof the family. Didymosphaeria is represented byDidymosphaeria rubi-ulmifolii. A new genus, Neokalmusia,is introduced in the family to accommodate thebambus i co lous t axa Kalmus ia brev i spora andK. scabrispora. This introduction is based on molecular datacoup l ed w i t h mo rpho l ogy. One new spec i e s ,Paraconiothyrium magnoliae and the sexual morph ofParaconiothyrium fuckelii, is also introduced. Julella andBarria are assigned to the family based on morphologicalsimilarity with Didymosphaeriaceae. Wilmia (previously re-ferred to the Phaeosphaeriaceae) is synonymized underLetendraea (Didymosphaeriaceae). Furthermore, a new spe-cies, Letendraea cordylinicola, is introduced and placed inDidymosphaeriaceae based on phylogeny and morphology.The paraphyletic nature of Paraconiothyrium is partially re-solved, but further sampling with fresh collections and molec-ular data of species in this genus are needed to obtain a naturalclassification.

Keywords Didymosphaeriaceae .Montagnulaceae .

New species . Phylogeny . rDNA .β-tubulin

Introduction

Munk A (1953) introduced the family Didymosphaeriaceae,typified by the genusDidymosphaeria Fuckel. The family wascharacterized by 1-septate ascospores and trabeculatepseudoparaphyses which anastomosed mostly above the asci(Aptroot 1995; Ariyawansa et al. 2014b). Based on its trabec-u l a t e pseudopa raphyse s , Ba r r ( 1990 ) r e f e r r edDidymosphaeriaceae to the order Melanommatales.However, the importance of trabeculate pseudoparaphyseswas later disputed (Liew et al. 2000). The confusion surround-ing Didymosphaeriaceae and Didymosphaeria was discussedin detail by Ariyawansa et al. (2014b). They explained thatconsidering the morphological data only (peridium compris-ing flattened or irregular cells, 1-septate ascospores and tra-beculate pseudoparaphyses mostly anastomosing above theasci) the family Didymosphaeriaceae appears to be distinctfrom Pleosporales. However, on the basis of molecular datathe status of Didymosphaeriaceae as a distinct family wasfound to be doubtful as it clustered in Montagnulaceae(Ariyawansa et al. 2014b). Ariyawansa et al. (2014b) intro-duced a new species of Didymosphaeria, D. rubi-ulmifoliiAriyawansa et al., based on morphological comparison withthe generic type, D. futilis (Berk. & Broome) Rehm.Sequences of Didymosphaeria rubi-ulmifolii clustered withintheMontagnulaceae as a separate genus, thus suggesting thatMontagnulaceae and Didymosphaeriaceae are synonymous.

Barr (2001) introduced the family Montagnulaceae withthree genera viz. Kalmusia Niessl, Montagnula Berl., andDidymosphaerella Cooke, in the order Pleosporales, basedon ascomata immersed under a clypeus, a pseudoparenchy-matous 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 Didymosphaerellaascospore have a single transverse septum (Barr 2001). Thefamily was thought to be closely related to Cucurbitariaceae,but differed in its obtuse ascomatal apex with a minute papil-late ostiole and thin-walled, pale ascospores (Barr 2001).Species in the family Montagnulaceae are parasitic,saprotrophic or hemibiotropic and also include saprobic spe-cies in aquatic environments (Barr 2001; Zhang et al. 2012).Several recent studies have provided the groundwork forclassification of the family Montagnulaceae (Ariyawansaet al. 2014b; Verkley et al. 2014). Even though there is a largebody of work comprising taxonomic and phylogenetic stud-ies, most of the genera regarded as members ofMontagnulaceae remain under-studied and therefore poorlyunderstood for a systematic treatment.

Combined analyses of LSU, SSU, RBP2 and TEF1 genedata (Schoch et al. 2009; Zhang et al. 2012) showed the familyMontagnulaceae to be well-resolved comprising the generaBimuria D. Hawksw et al., Didymocrea Kowalski, KalmusiaNiessl, Karstenula Speg., Montagnula Berl., andParaphaeosphaeria O.E. Erikss. Some species ofPhaeosphaeria, Paraconiothyrium Verkley, and LetendraeaSacc., also clustered in Montagnulaceae (Schoch et al. 2009;Zhang et al. 2012). Paraconiothyrium was introduced byVerkley et al. (2004) to accommodate four species viz.Parac. estuarinum Verkley & Manuela Silva., Parac.brasiliense Verkley, Parac. cyclothyrioides Verkley, andParac. fungicola Verkley & Wicklow. The sexual morphs ofthese species are unknown, but maximum parsimony analysisof ITS and partial SSU nrDNA sequences showed that theseasexual genera clustered within Pleosporales and grouped in aclade including Paraphaeosphaeria in Montagnulaceae(Verkley et al. 2004). In the same study, Coniothyriumminitans W.A. Campb., C. sporulosum (W. Gams &Domsch) Aa, and C. minitans W.A. Campb., clustered in thesame clade and were therefore referred to Paraconiothyrium.Following SSU and ITS analyses, Damm et al. (2008) sug-gested that Paraconiothyrium can be accommodated withinParaphaeosphaeria. Zhang et al. (2012) showed that theasexual morphs of Paraphaeosphaeria michotii (Westend.)O.E. Erikss. and Paraph. pilleata Kohlm et al. are represen-tatives of Paraconiothyrium in their multi-gene phylogeneticanalysis.

Didymosphaerella has been excluded from the familyMontagnulaceae and placed in Didymellaceae (Zhang et al.2012). Schoch et al. (2009) and Suetrong et al. (2009) showed

70 Fungal Diversity (2014) 68:69–104

that the marine ascomycete, Tremateia halophila nested inMontagnulaceae. Zhang et al. (2012) provided a descriptionofMontagnula, the type of the familyMontagnulaceae, whichalso included Bimuria, Didymocrea, Karstenula, Letendraea,Montagnula, Paraphaeosphaeria and Tremateia, whileAriyawansa et al. (2013c) added the new genus Deniquelata.

Zhang et al. (2014) neotypified Kalmusia, with K. ebuliNiessl., to resolve phylogenetic placement of the specieswithin Kalmusia. Zhang et al. (2014) concluded thatKalmus ia spec i e s a re po lyphy le t i c wi th in theMontagnulaceae, with K. ebuli being distant fromK. brevispora (Nagas. & Y. Otani) Y. Zhang et al. andK. scabrispora (Teng) Kaz. Tanaka et al. The latter twospecies appear to represent a different genus.

Verkley et al. (2014) re-assessed Coniothyrium-like taxabelonging to the Montagnulaceae, based on combine geneanalyses of ITS, LSU, γ-actin and β-tubulin sequences andproposed Alloconiothyrium Verkley & Stielow, andDendrothyriumVerkley& Stielow, as new genera. In the samestudy, one new species was described in Paraconiothyriumwhile two species so far classified in Paraconiothyrium weretransferred to Paraphaeosphaeria (Verkley et al. 2014).

We have been studying the families of Pleosporales basingon both morphology and molecular phylogeny in order toprovide 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 geneticand taxonomic relationships within the genera of theDidymosphaeriaceae andMontagnulaceae using a set of iso-lates that encompasses its genetic, biological and geographicdiversity. Type specimens, or cultures derived from type spec-imens, have been examined wherever possible.

Material and methods

Specimen examination

Fresh materials of Didymosphaeriaceae and Montagnulaceaespecies 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 meth-od described in Ariyawansa et al. (2013a, b, c). Freshand herbarium materials were examined under a Motic SMZ168 dissecting microscope to locate and isolate ascomatafruiting bodies. Hand sections of the fruiting structures weremounted in water for microscopic studies and photomicrogra-phy. The taxa were examined using a Nikon ECLIPSE 80icompound microscope and photographed with a Canon 450Ddigital camera fitted to the microscope. Measurements weremade with the Tarosoft (R) Image Frame Work program andimages used for figures processed with Adobe Photoshop CS3Extended version 10.0 software (Adobe Systems, USA).

Isolations were made from single ascospores, following a mod-ified method of Ariyawansa et al. (2013b) and Chomnunti et al.(2014). Contents of the sectioned fruiting body were transferredto a drop of sterile water on a flame-sterilized slide. Drops of thespore suspension were pipetted and spread on a Petri-dishcontaining 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 theSwedish Museum of Natural History (S) and the New YorkBotanical Garden (NY).

Voucher specimens are deposited in the herbarium of MaeFah Luang University (MFLU), Chiang Rai, Thailand andNew Zealand Fungarium, Landcare Research (PDD), NewZealand. The living cultures are deposited at the Mae FahLuang University Culture Collection (MFLUCC),Biodiversity and Climate Research Centre (BiK-F) andInternational Collection of Microorganisms from Plants(ICMP) under Material Transfer Agreement No. 4/2010(MTA). Each genus is listed along with a description of thetype species, except in cases where there is only a singlespecies in the genus.

DNA extraction, PCR amplification and sequencing

Single ascospore fungal isolates were grown onMEA or PDAfor 28 days at 25 °C in the dark. Genomic DNAwas extractedfrom the growing mycelium using the Biospin FungusGenomic DNA Extraction Kit (BioFlux®) following the man-ufacturer’s protocol (Hangzhou, P.R. China). Otherwise DNAextracted 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 μlreaction volume containing 5–10 ng DNA, 0.8 units Taqpolymerase, 1X PCR buffer, 0.2 mM d’NTP, 0.3 μM of eachprimer with 1.5 mM MgCl2 (Cai et al. 2009). The PCRreactions for amplification of the recently ratified universalfungal barcode ITS1-5.8S-ITS2 of the nuclear ribosomalDNA operon (Verkley et al. 2014), were performed understandard conditions (White et al. 1990; Stielow et al. 2010).PCR conditions for amplifying the partial SSU and LSU r-DNA followed the protocol of Phillips et al. (2008).Amplification of partial β-tubulin (TUB), covering the vari-able 5’-end containing four small introns, followed the proto-col of Aveskamp et al. (2009) and Carbone and Kohn (1999).The PCR products were observed on 1 % agarose electropho-resis gels stained with ethidium bromide. Purification andsequencing of PCR products were carried at ShanghaiSangon Biological Engineering Technology and ServicesCo., (China).

DNA sequence data was obtained from the internal tran-scribe spacer (ITS), small and large subunits of the nuclear

Fungal Diversity (2014) 68:69–104 71

ribosomal RNA genes (SSU, LSU) and the protein codinggene, β-tubulin (TUB). Primer sets used for these genes wereas follows: ITS: ITS5/ITS4 SSU: NS1/NS4; LSU: LR0R/LR5; TUB4Rd/TUB4Fd (Aveskamp et al. 2009; Carboneand Kohn 1999). Primer sequences are available at theWASABI database at the AFTOL website (aftol.org).Sequences are deposited at NCBI GenBank under the acces-sion numbers provided in Supplementary Table 1. Alignmentsare deposited in TreeBASE.

Sequence alignment and phylogenetic analysis

Combined ITS, LSU, SSU and TUB genes were used in theanalysis. All sequences obtained from GenBank and used byAriyawansa 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), Wijayawardeneet al. (2014) and Zhang et al. (2012) are listed in supplemen-tary Table 1. Multiple sequence alignments were generatedwith 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 wereremoved from the ITS, SSU and LSU alignments prior totree building. The alignments were checked visually andimproved manually where necessary. Concordance of theITS, SSU, LSU and TUB genes data sets was estimated withthe partition-homogeneity test implemented with PAUP v. 4.0b10 (Swofford 2002).

Maximum parsimonious analysis was performed to obtainthe most parsimonious tree. Trees were inferred using theheuristic search option with 1,000 random sequence additionsand tree-bisection-reconnection (TBR) branch-swapping al-gorithm. All gaps were treated as missing data. Maxtrees weresetup to 50,000 and branches of zero length were collapsedand all multiple parsimonious trees were saved. Descriptivetree statistics for parsimony (Tree Length [TL], ConsistencyIndex [CI], Retention Index [RI], Relative Consistency Index[RC] and Homoplasy Index [HI] were calculated for treesgenerated under different optimality criteria. Kishino-Hasegawa tests (KHT) (Kishino and Hasegawa 1989) wereperformed in order to determine whether trees were signifi-cantly different. Maximum parsimony bootstrap values (MP)equal or greater than 50 % are given below or above eachnode.

Maximum likelihood analyses including 1,000 bootstrapreplicates were run using RAxML v. 7.2.6 (Stamatakis2006; Stamatakis et al. 2008) The online tool Findmodel(http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html) was used to determine the best nucleotidesubstitution model for each partition. The best scoring treewas selected with a final likelihood value of -19492.551787.The resulting replicates were plotted on to the best scoring treeobtained previously. Maximum Likelihood bootstrap values

(ML) equal or greater than 50 % are given below or aboveeach node in red (Fig. 1).

The model of evolution was performed by usingMrModeltest 2.2 (Nylander 2004). Posterior probabilities(PP) (Rannala and Yang 1996; Zhaxybayeva and Gogarten2002) were determined by Markov Chain Monte Carlo sam-pling (MCMC) in MrBayes v. 3.0b4 (Huelsenbeck andRonquist 2001). Six simultaneous Markov chains were runfor 3,000,000 generations and trees were sampled every 100thgeneration and 30,000 trees were obtained. The first 6,000trees, representing the burn-in phase of the analyses, werediscarded while the remaining trees were used for calculatingposterior probabilities in the majority rule consensus tree(critical value for the topological convergence diagnostic setto 0.01) (Crous et al. 2006). Bayesian Posterior Probabilities(BYPP) equal or greater than 0.90 is given below or aboveeach node (Fig. 1). Phylogenetic trees and data files wereviewed in MEGA v. 5 (Tamura et al. 2011), TreeView v.1.6.6 (Page 1996) and FigTree v. 1.4 (Rambaut andDrummond 2008).

Results

Phylogeny of combined ITS, LSU, SSU and TUB genedatasets

The combined ITS, LSU, SSU and TUB dataset comprises 76taxa. Halojulella avicenniae is selected as an outgroup taxon.Results of the partition-homogeneity test (P=0.107) indicatethat the ITS, LSU, SSU and TUB gene trees reflect the sameunderlying phylogeny. Therefore, these datasets were com-bined and analyzed by using several tree-building programs,the resulting trees compared and the best tree is presented inFig. 1. New sequences are deposited in GenBank (Table 1).

Phylogenetic analysis

The combined ITS, LSU, SSU and TUB gene dataset fromfive families in the Pleosporales is shown in Fig 1. All trees(ML, MP and BYPP) were similar in topology and not sig-nificantly different (data not shown). The maximum parsimo-ny dataset consists of 2,283 characters and of which 2,437were constant, 300 variable characters parsimony-uninformative and 776 characters parsimony-informative.

�Fig. 1 RAxML tree based on a combined dataset of SSU, LSU, RPB2and TEF1, bootstrap support values for maximum likelihood (blue),maximum parsimony (green) greater than 50 % and bayesian posteriorprobabilities greater than 0.90 (black) below and above the nodes.Halojulella avicenniae is the outgroup taxon. The original isolate num-bers are noted after the species names and ex-type strains are in bold. Thetype species of each genus is indicated in blue


http://mafft.cbrc.jp/alignment/server/index.html

http://mafft.cbrc.jp/alignment/server/index.html

http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html

http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html


Kishino-Hasegawa (KH) test showed length = 3,000 steps, CI= 0.532, RI = 0.673, RC = 0.358 and HI = 0.468. A bestscoring RAxML tree is shown in Fig. 1, with the value of -19492.551787. Phylogenetic trees obtained from MaximumLikelihood, Maximum Parsimony and Bayesian analysisyielded trees with similar overall topology at subclass andfamily relationships in agreement with previous work basedon 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 combineddataset shown in Fig. 1, several well-supported sub clades canbe identified in the family Didymosphaeriaceae, which areinterpreted as appropriate for the delimitation of genera, i.e.Alloconiothyrium, Bimuria, Deniquelata, Didymosphaeria,Didymocrea , Kalmusia , Karstenula , Letendraea ,Montagnu la , Neoka lmus ia , Paracon io thyr ium ,Paraphaeosphaeria, Phaeodothis and Tremateia. Thus theDidymosphaer iaceae i nco rpora t es member s o fMontagnulaceae. The phylogenetic data for each genus isfurther discussed after the genus notes.

The Didymosphaeria clade comprises the type strainD. rubi-ulmifolii (MFLUCC 14-0023) plus another strain ofD. rubi-ulmifolii (MFLUCC 14-0024), which clustered withthe type strain of Paraconiothyrium brasilienseVerkley (CBS100299) and synonymised underD. rubi-ulmifolii and definedhere as D. rubi-ulmifolii sensu stricto. Several strain collec-tions of Paraconiothyrium brasiliense (CBS 122319, CBS122320, CBS 587.84, CBS 115.92 and CBS 115.92) otherthan the type strain, form a distinct clade to D. rubi-ulmifoliiclade, thus treated here as D. rubi-ulmifolii sensu lato. Theclosely related Paraconiothyrium variabile (CBS 120014)forms a separate clade in Didymosphaeria clade which issynonymised under Didymosphaeria as Didymosphaeriavariabile. Another well-supported (100/1.00/98) cladeforming the major part of the ingroup of the tree comprisessix strains assigned to Paraphaeosphaeria, with three isolatesof Paraph. michotii, the type species of the genus, and therelatively highly supported clades of the following species:Paraph. angularis Verkley & van der Aa (CBS 167.70),Paraph. minitans (W.A. Campb.) Verkley & Stielow (CBS111750), Paraph. arecacearum Verkley & Stielow (CBS158.75) and Paraph. viridescens Verkley et al. (CBS854.73). A putative strain of Karstenula rhodostoma (CBS690.94) forms a distinct clade sister to Paraphaeosphaeriaclade.

The Paraconiothyrium clade comprises 13 strains withParac. estuarinum (CBS 109850T), the type species ofParaconiothyrium,: 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

(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, includingKalmusia ebuli (CBS 123120T), the type species of the genus,Kalmusia sp. (MFLUCC 14-0560), K. variisporum (CBS121517T) and K. longisporum (CBS 824.84, 582.83T). Themonotypic asexual genus Alloconiothyrium (CBS 980.95T,981.95) forms a robust clade sister to Kalmusia.

Letendraea forms a relatively well supported clade withinthe family Didymosphaeriaceae. This clade contains fivestrains with Letendraea eurotioides (CBS 212.31), the typespecies of Letendraea, and the relatively highly supportedclades of the following species: L. padouk (CBS 485.70),L. helminthicola (CBS 884.85) and the two isolates of thenovel species L. cordylinicola (MFLUCC11-0148T,MFLUCC 11-0150) which clusters with L. padouk. The newgenusNeokalmusia forms a robust clade sister toDeniquelata,Didymocrea and Phaeodothis. Neokalmusia comprises fivestrains N. brevispora (CBS 120248, NBRC 106240),N. scabrispora (CBS 120246, NBRC 106237) andNeokalmusia sp.. Another well-supported (70/0.91/74) cladeis formed by members of Montagnula, the basal clade in thefamily Didymosphaeriaceae, which comprises four stains in-cluding Montagnula opulenta (CBS 168.34), M. aloes (CPC19671), Montagnula sp. (Letendraea helminthicola) (CHTAR43) and M. anthostomoides (CBS 615.86).

Taxonomy

Didymosphaeriaceae Munk, Dansk bot. Ark. 15(no. 2): 128(1953), Facesoffungi number: FoF 00200.

= MontagnulaceaeM.E. Barr, Mycotaxon 77: 194 (2001)Saprobic or parasitic in terrestrial or aquatic environments.

Sexual state: Ascomata scattered or gregarious, immersed insubstrate, surrounded with hyphae forming an apical clypeus,dark brown to black, globose to rarely subglobose, medium tolarge sized, and coriaceous. Ostiole immersed to sub-im-mersed, periphysate when well-developed with a short apex,narrow or wide, usually circular in section. Peridium twolayered or often three layered in the upper region, composedof cells of textura angularis or textura intricata, dark brown toblack, coriaceous, with outermost layer consisting of black,highly pigmented thick-walled conglutinate cells or pseudo-parenchymatous cells, with middle layer less pigmented, cellsvisible via apex, with innermost layer comprising narrow,compressed rows of cells. Hamathecium of dense, septate,narrow, cellular or trabeculate pseudoparaphyses often in agelatinous 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


states: coelomycetous (Barr 2001), Kalmusia andParaphaeosphaeria have Coniothyrium-like, Cytoplea,Microsphaeropsis asexual stages. The generic type ofParaphaeosphaeria (P. michotii) was linked withConiothyrium scirpi Trail (Webster 1955). TheConiothyrium-like species were subdivided into four groupsby Sutton (1980), viz. Coniothyrium, Microsphaeropsis,Cyclothyrium and Cytoplea. Paraconiothyrium, which isclosely related to Paraphaeosphaeria based on SSUsequences phylogeny (Verkley et al. 2004), was introducedt o a ccommoda t e Con io th y r i um min i t an s andC. sporulosum.

Notes: The Didymosphaeriaceae is amended here to in-clude Didymosphaeria and genera from Montagnulaceae in-cluding asexual states. We synonymise Montagnulaceae un-der Didymosphaeriaceae which is the oldest name and haspriority and provide an updated account of the family.

Type: Didymosphaeria Fuckel, Jb. Nassau. Ver. Naturk.23-24: 140 (1870) [1869-70].

Genera accepted in DidymosphaeriaceaeAlloconiothyrium 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).BimuriaD. 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).

Type species: Karstenula rhodostoma (Alb. & Schwein.)Speg., Decades Mycologicae Italicae 7–12: no. 94 (1879).

Basionym: Sphaeria rhodostomaAlb. & 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 michotiiWestend., 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 halophilaKohlm. et al., Bot. Mar.

38(2): 166 (1995).

Key to the genera accepted in Didymosphaeriaceae

1. Parasitic on living leaves..................................Deniquelata1. Saprobic on dead wood or leaves.......................................... 22. Asci with 2 spores.................................................................... 32. Asci with more than 2 spores................................................. 43. Asci with short knob-like pedicel and brown, muriform

ascospores................................................................... Bimuria3. Asci without short knob-like pedicel and hyaline, muriform

ascospores....................................................................... Julella4. Peridium composed of thin pseudoparenchymatous cells of

textura intricata..............................................Didymosphaeria4. Peridium composed of pseudoparenchymatous cells of

textura angularis or textura prismatica................................. 55. Ascus with a long, furcate pedicel........................................ 65. Ascus with a short furcate pedicel.......................................... 76. Clavate ascus with narrowly ovoid to clavate, pale brown, 3-septate ascospores..................................................... Kalmusia

6. Cylindro-clavate to clavate asci with oblong to narrowlyoblong, reddish-brown to dark yellowish-brown, muriformor phragmosporous ascospores............................Montagnula

7. Ascomatal wall composed only cells of textura intricata.........8


7. Ascomatal wall composed both cells of textura angularis andtextura prismatica............................................................ Barria

8. Strictly in marine environments................................ Tremateia8. In terrestrial environments......................................................... 99. Hamathecium consisting of filiform pseudoparaphyses

.................................................................................. Didymocrea9. Hamathecium consisting of cellular pseudoparaphyses........... 1010. Ascomata with a pore-like ostiole......................................... 1110. Ascomata with short papillate ostiole.................................. 1211. Cylindrical asci with short pedicel and fusiform with rounded

ends, olivaceous-brown, phragmosporous asco-spores...................................................................... Phaeodothis

11. Cylindrical to cylindro-clavate asci with short furcate pediceland ellipsoid to fusoid, reddish-brown to dark brownmuriform ascospores.............................................. Karstenula

12. Ascomata with hyaline periphyses.................... Neokalmusia12. Ascomata without hyaline periphyses.........Paraphaeosphaeria

Key to asexual genera of Didymosphaeriaceae

1. Conidiomata pycnidial and with a single cavity, or eustromaticwith several cavities............ Alloconiothyrium

1. Conidiomata eustromatic, simple or complex, rarely pyc-nidial........................................................... 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: un-

known (Verkley et al. 2014). Asexual state: Conidiomata300–450μm diam, pycnidial and with a single cavity, oreustromatic and consisting of complexes reaching 1mm diam.,with several cavities, with outer surface black, glabrous orcovered by grey mycelium. Conidiomatal wall composed ofan outer layer of brown, thick-walled cells of texturaangularis and an inner layer of hyaline, thick-walled cells oftextura angularis, the outer surface sometimes covered by adiffuse web of brown hyphae. Conidiogenous cells 4–9×3–4μm, discrete, often positioned on clumps of cells that pro-trude into the cavity, broadly ampulliform, holoblastic,annellidic, often with an elongated neck showing severaldistinct percurrent proliferations. Conidia 3–4(–5)×2–3(–3.5) μm, globose to irregularly ellipsoid, aseptate, initiallyhyaline, after secession olivaceous-brown, mature conidialwall orange-brown, the outer surface verruculose giving theconidium an irregular outline, with 1 large oil-droplet 1–1.5μm diam (description from Verkley et al. 2014).

Notes: Alloconiothyrium was introduced to accommodateConiothyrium-like species in the Montagnulaceae (Verkleyet al. 2014). Alloconiothyrium aptrootii is known from a soilsample collected in Papua New Guinea, and all other

Coniothyrium-like taxa studied in Verkley et al. (2014) wererelatively distantly related. The annellidic conidiogenous cellsand the verruculose conidia resemble Coniothyriumpalmarum, the type species of Coniothyrium, but that speciesis characterized by 2-celled conidia and is also geneticallydistinct, belonging in the family Leptosphaeriaceae (deGruyter et al. 2009). In our phylogeny, Alloconiothyriumforms a robust clade, sister to the Kalmusia clade. Thus, basedon morphology coupled with DNA data, we confirm that thisgenus is well resolved and should be classified underDidymosphaeriaceae.

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, im-mersed, visible as black spots on host surface, uniloculate,globose, brown to dark brown, with centrally opening ostiole.Ostiole papillate to depressed, ostiolar canal filled withperiphyses. Peridium 10–23μm wide, composed of two celltypes, outer layers comprising 3–5 layers, thin-walled, darkbrown to black, pseudoparenchymatous cells of texturaangularis 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 andembedded in a gelatinous matrix. Asci (126–) 130–170(–185)×(15–)17–20(–23) μm (x=151.2×19μm, n=25), 8-spored, bitunicate, fissitunicate, clavate to cylindric-clavate,shortly acute pedicel or sub-sessile, apically rounded with awell-developed ocular chamber, arising from the base of theascoma. Ascospores (18.5–)20–24.5(–27)×9–12.5μm(x=22×10.9μm, n=30), overlapping, 1–2-seriate, ellipsoidalto broadly fusiform, initially hyaline, becoming brown to darkbrown at maturity, 1-septate, constricted at the septum,smooth to rough-walled, with small guttules, comprising twolayers, endospore thin-walled, epispore thick-walled, mostlywith upper cell larger than lower cell, surrounded by a distinctmucilaginous sheath. Asexual state: unknown.

Material examined: CHINA, Xinjiang, Urumqi, TianshanMountain, on leaves of Picea schrenkiana Fisch. & C.A. Mey(Pinaceae), 1 July 1992, Z.Q. Yuan (NY 00164027, isotype).

Notes: Themonotypic genusBarria shares similarities withDidymopleella in ascomatal structure, asci and ascosporescharacters, but differs in having a peridium of texturaprismatica and strongly unequal ascospore cells (Munk1957; Yuan 1994). Recently, Zhang et al. (2012) tentativelyassigned Barria in Phaeosphaeriaceae based on theascomata, colour and shape of ascospores. Barria differs fromPhaeosphaeria in having ascomata with thick peridium, cla-vate, short-pedicellate asci, and didymosporous ascospores,while Phaeosphaeria has broadly cylindrical to cylindric-


clavate asci with sub-sessile pedicellate and phragmosporesascospores.

Didymosphaeria shows similarities with Barria in havingimmersed to slightly erumpent ascomata under a clypeus,hyaline pseudoparaphyses, anastomosing frequently above

the asci and 1-septate, ellipsoid ascospores. However, Barriadiffers from Didymosphaeria in having brown to dark brownascomata comprising several layers of textura angularis totextura prismatica cells in the peridium, cellularpseudoparaphyses and asci having well-developed ocular

Fig. 2 Barria piceae (NY 00164027, isotype). a Herbarium label andspecimens of Barria piceae. bAppearance of ascomata on host surface. cVertical section through ascoma d Section through peridium. ePseudoparaphyses in Congo red reagent. f Asci with pseudoparaphyses.

g, h-i Asci. j Ocular chamber in congo red reagent. k-n Ellipsoidal tobroadly fusiform ascospores with a sheath. Scale bars: c=100μm, d-i=20μm, j-m=10μm


chamber bearing ellipsoidal to broadly fusiform, initially as-cospores hyaline, becoming brown to dark brown at maturity,1 -sep ta te wi th a dis t inc t muci lag inous shea th .Didymosphaeria has hyaline to pale brown or (rarely) blackperidium, consisting of an internal and external layers, flat-tened or elongated hyphae, textura intricata with trabeculatepseudoparaphyses and distoseptate ascospores without as h e a t h (Ap t r o o t 1 9 9 5 ) . We p l a c e Ba r r i a i nDidymosphaeriaceae because of its similarities with othergenera in Didymosphaeriaceae. However, fresh collectionsof the type species of the genus are needed so that moleculardata can be obtained to verify the natural taxonomic affinitiesof 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 superfi-cial, solitary, scattered, globose, hyaline when young, turningdark brown to black when mature, ostiolate, the ostiole moreor less sessile or raised into a very short neck. Peridium 5–8μm diam., comprising 2–3 layers of compressed pseudopar-enchymatous 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

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=75×29μm, n = 10), (1-)2(-3)-spored, bitunicate,fissitunicate, broadly clavate, with a short and small knob-like pedicel, ocular chamber best seen in immature asci.Ascospores 55–68×25–28μm (x =59×26μm, n=10), accu-mulating in a subglobose black shiny mass adhering togetheroutside the ostiole, broadly ellipsoid but becoming narrowedtowards the poles, muriform with (5-) 7 transverse septa, cellswith (0-)l(-2) longitudinal septa in each cell, not constricted atthe septa, dark brown, apical cells paler and lacking longitu-dinal septa, verruculose. Asexual state: unknown.

Material examined: NEW ZEALAND, North Island,Wairarapa District, Nutty Farm, isolated from soil, 3 March1978, C.C. Yen and J.E. Sheridan (CBS 107.79, isotype).

Notes: Bimuria is a monotypic genus characterized by avery thin peridium, mostly 2-spored, fissitunicate asci andmuriform, dark brown, verrucose ascospores (Hawksworthet al. 1979). Because of its unique morphological characters,the familial placement of this genus has been debatable, thusinitially Hawksworth et al. (1979) placed the genus inPleosporaceae. Later Barr (1987) transferred Bimuria toPhaeosphaeriaceae while Lumbsch and Huhndorf (2007)placed it in Melanommataceae. Morphologically, Bimuria ismost 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 likepedicel. h-j Dark brown, muriform ascospores. Scale bars: a-b=100μm, c=5μm, d=5μm, e-g=30μm, i-j=10μm


However, the thick carbonaceous peridium distinguishesMontagnula from Bimuria (Hawksworth et al. 1979). Inaddition, the ascospores of Montagnula are dischargedforcibly through the ostiole instead of forming a massoutside 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 Hyde1999). Hawksworth et al. (1979) suggested that the mor-phological characters of Bimuria, such as ascospore releaseand large, thick-walled ascospores may be an adaptation toits soil-borne habitat.

Phylogenetic analysis based on combined genes of SSU,LSU, RPB2 and TEF1 concluded that the type strain ofBimuria novae-zelandiae (CBS 107.79) nested in theMontagnulaceae (Schoch et al. 2009). Our phylogenetic studyalso showed similar results, with Bimuria forming a mono-phyletic clade sister to Tremateia. Therefore we includeBimuria as a well-established genus in Didymosphaeriaceaebased 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 de-pressed, elongate ostiole. Peridium 9–17μm diam. (x=12μm,n=10), composed of 3–5 layers of brown to black, darklypigmented, small, thick-walled, 2–5μm wide cells of texturaangularis, with outer wall fused with the host cells, inner wallconsists of 2-layers of polygonal to rectangular, light brown-hyaline cells 1–4μm diam. Hamathecium composed of dense,1–3μm diam. (x=2μm, n=20), broad, hyaline, septatepseudoparaphyses, surrounding the numerous asci and em-bedded in a gelatinous matrix. Asci (-60) 68–80×10–15μm(x=72×13μm, n=20), 8-spored, bitunicate, fissitunicate, cla-vate 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 ordistichously arranged, partially overlapping, oblong to nar-rowly oblong, straight or somewhat curved, reddish-brown,with three transverse septa and 1–2 vertical septa in the centralcells, constricted at the primary and secondary septa at matu-rity, verruculose, straight or slightly inequilateral, without agelatinous sheath. Asexual state: unknown.

Material examined: THAILAND, Chiang Rai Province.,Muang District, Bandu, Baan Khuakhae, on leaf ofBarringtonia asiatica (L.) Kurz (Lecythidaceae), 18September 2011, K.D. Hyde (MFLU 12-0303, holotype).

Notes: Deniquelata is a monotypic genus characterized byimmersed, dark brown to black ascomata, with bitunicate asciand brown, muriform ascospores. Deniquelata differs consider-ably from the type of Montagnula (M. infernalis) both in phy-logeny andmorphology (Ariyawansa et al. 2013c).Deniquelatais a pathogenic genus with fruiting bodies scattered in thenecrotic host tissues. Ariyawansa et al. (2013c) concluded thatphylogenetic analysis of the nucleotide sequences of combined18S and 28S nrDNA provided good evidence that Deniquelatabelongs in Montagnulaceae. In the present study Deniquelataforms a distinct clade sister to Bimuria and Tremateia. Thus weaccept 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 andleaves. Sexual state: Ascomata solitary, scattered, or in smallgroups, immersed to erumpent, globose to ovoid, ostiolate.Ostiole papillate with a pore-like opening, ostiolar canal filledwith hyaline cells (periphyses). Peridium 1-layered, thin,composed of brown pseudoparenchymatous cells of texturain t r i ca ta . Hamathec ium of dense , t r abecu la te ,pseudoparaphyses, anastomosing mostly above the asci. Asci8-spored, bitunicate, fissitunicate, cylindrical, with a furcatedpedicel, apically rounded with an indistinct ocular chamber.Ascospores uniseriate, ellipsoid, brown, 1-septate, wall withdifferent 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 deadwood. Sexual state: Ascomata 110–140×120–160μm (x=130×140μm, n=10), scattered, or in smallgroups, immersed to slightly erumpent, rarely nearly superfi-cial, under a clypeus, globose to subglobose, membraneous,papillate. Papilla black, with a pore-like ostiole, ostiolar canalfilled with periphyses. Peridium 10–20 μm (x=15μm, n=10)wide, 1-layered, composed of hyaline pseudoparenchymatouscompressed cells of textura intricata, fusing at the outsidewith the host. Hamathecium of dense, 0.5–1μm (x =0.8μm,n=20) broad, long, trabeculate pseudoparaphyses, anastomos-ing frequently above the asci, embedded inmucilage. Asci 75–85×4–6μm (x=78×6μm, n=20), 8-spored, bitunicate,fissitunicate, cylindrical, pedicellate, rounded at the apex withan indistinct ocular chamber. Ascospores 7–10×3–5μm (x =9×5μm, n=40), uniseriate, slightly overlapping, ellipsoidwith obtuse ends, brown, 1-septate, slightly to not constrictedat the septum, with distinctly spinulose ornamentation.Asexual state: unknown.

Material examined: UK. England: Norfolk, on dead stemsof Rosa sp. (Rosaceae), March 1850, M.J. Berkeley (K147683, holotype of Sphaeria futilis).


Notes: Didymosphaeria sensu lato, introduced for threespecies of ascomycetes with 2-celled ascospores, comprisespecies having a wide distribution and a broad host range.Saccardo (1882) restricted the genus to only those species withbrown ascospores. Aptroot (1995) included over 400 epithets of

Didymosphaeria in his monograph of the genus, after examin-ing over 3,000 species, but only seven species were accepted.

The placement of Didymosphaeria is confused as de-scribed in Ariyawansa et al. (2014b). Sivanesan (1984) re-ported that Didymosphaeria has Ascochyta and Periconia

Fig. 4 Deniquelata barringtoniae (MFLU 12-0303, holotype) a-b Fun-gus on host. c-d Section of ascoma (TS). e Section of peridium compris-ing a few layers of cells. f Hyaline pseudoparaphyses g-i Eight-sporedasci with short, broad pedicel at the base. j Reddish-brown, muriform,

ascospores. k Ascospores stained in Indian ink. l Germ tubes developingfrom ascospore cells. Scale bars: c-d=100μm, e=5μm, f=5μm, g-i=20μm, j-k=10μm, l=20μm


asexual states, while Kirk et al. (2008) reported Fusicladiella-like and Phoma-like species. Linking Didymosphaeria toasexual states should be treated with caution until the type ofD. futilis has been sequenced. Ariyawansa et al. (2014b) foundthat two strains of the D. rubi-ulmifolii clustered in the familyMontagnulaceae, but were separated from other genera of thefamily with high bootstrap support. Comparison of the generictype, D. futilis, and D. rubi-ulmifolii shows that they havesimilar morphology and thus representDidymosphaeria. ThusAriyawansa et al. (2014b) suggested that, based on the avail-able molecular data and morphology, Didymosphaeria can bereferred toMontagnulaceae with Didymosphaeriaceae as theprobable synonym of Didymosphaeriaceae. In this paper, weformally treatDidymosphaeriaceae as a separate family in theorder Pleosporales and with Montagnulaceae as its synony-my. The generic type of Didymosphaeria, D. futilis needs tobe recollected, epitypified and sequenced so that phylogeneticanalysis can be used to confirm family relationships withinPleosporales.

In the present study, we observed that the type strain ofParaconiothyrium brasiliense (CBS 100299) strain forms arobust clade with D. rubi-ulmifolii. Paraconiothyriumbrasiliense was isolated from coffee fruits in Brazil, but thisspecies has since been reported from various habitats in othercontinents, on woody and herbaceous host plants, such asPrunus spp. in South Africa (Damm et al. 2008). Near-identical ITS sequences have been deposited in GenBank forendophytes isolated from Ginkgo biloba (DQ094168),Juniperus virginiana (Hoffman and Arnold 2008), andUlmus davidiana var. japonica (AB665311), and also fromthe herb Alliaria petiolata (EF432267). Strain CBS 395.87from soil sampled in Italy was identified as Parac. brasilienseby Verkley et al. (2014). Verkley et al. (2014) showed that theACTand TUB sequences of several Parac. brasiliense strainsused in their study were more variable than in other relatedspecies, suggesting that Parac. brasiliense could be a speciescomplex. In this study we consider that Parac. brasiliense andDidymosphaeria rubi-ulmifolii are the same species as they

Fig. 5 Didymosphaeria futilis (K 147683, holotype of Sphaeria futilis)a-b Fungus on the host. c Section through ascoma. d Close up ofperidium. e Broad, long trabeculate pseudoparaphyses, anastomosing

mostly above the asci. f-h Cylindrical asci with an indistinct ocularchamber. i-l Ascospores with distinct spinulose ornamentation. Scalebars: c=100μm, d=10μm, e, f-h=20μm, i-l=5μm


cluster with a high bootstrap support. We thereforesynonymise these species. The only available name for thisspecies is Didymosphaeria rubi-ulmifolii becauseDidymosphaeria brasiliensis is already in use.

Didymosphaeria rubi-ulmifolii Ariyawansa et al.,Phytotaxa.176 (1)111

Basionym: Paraconiothyrium brasiliense Verkley, inVerkley 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-ulmifoliisensu stricto, thus here we decide to treat it as a separatespecies in Didymosphaeria (Didymosphaeria sp.) but withoutdesignating a new species because the morphology is notavailable. Paraconiothyrium variabile (CBS 120014) formsa well-supported clade in Didymosphaeria, thus here wesynonymise Paracon io thyr ium var iabi l e underDidymosphaeria

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: Ascomataimmersed, becoming erumpent to nearly superficial,sphaeroid, black, coriaceous, ostiolate, formed under a clype-us. Ostiole usually widely porate, with a short neck, ostiolarcanal filled with periphyses. Peridium two layered, outer wallcomposed of small, dark brown to black, heavily pigmented,thick-walled cells of textura angularis and fusing with thehost tissue to the outside, inner wall comprising broadyellowish-brown cells of textura angularis, thick at the apexand thinner at the base. Hamathecium of dense broad, septate,cellular, pseudoparaphyses anastomosing and branching andembedded in mucilage. Asci 2-spored, bitunicate,fissitunicate, clavate to cylindrical, with a short, broad,furcated or knob-like pedicel, rounded at apex and without adistinct ocular chamber. Ascospores biseriate or partiallyuniseriate, 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), immersedbecoming erumpent to nearly superficial, sphaeroid, black,coriaceous, ostiolate, formed under a clypeus. Ostiole usuallywidely porate, with a short neck, ostiolar canal filled withperiphyses. Peridium two layered, outer wall composed ofsmall, dark brown to black, heavily pigmented, thick-walledcells of textura angularis and fusing with the host to the

outside, inner wall comprising broad yellowish-brown cellsof textura angularis, thick at the apex and thinner at the base.Hamathecium of dense , 2–4 μm broad , sep ta te ,pseudoparaphyses anastomosing and branching above the asciand embedded inmucilage. Asci 90–130×25–35μm (x=110×30μm, n=10), two-spored, bitunicate, fissitunicate, clavate tocylindrical, 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), biseriateor partially uniseriate, asymmetric or nearly symmetric, hya-line, obovoid, fusoid, or elongate, multi-septate, muriform,verruculose, thin-walled, surrounded by a mucilaginoussheath, guttulate, constricted at the septa. Asexual state:unknown.

Material examined: FRANCE, Serignan, on Buxussempervirens L. (Buxaceae), H. Fabre (S-F5992, holotype).

Notes: Julella has been confused, with some species beingsaprotrophic on bark, while others are lichenized, and thedelineation of the genus is poorly defined. Currently, 46epithets are listed in Index Fungorum (2014). Many of thespecies assigned to the genus are facultatively lichenized withthe alga Trentepohlia as the photobiont (Purvis et al. 1992).Julella shares many common features with Peltosphaeria butdiffers in having bisporous asci (Barr 1985). Barr (1985)proposed Peltosphaeria as a synonym of Julella. Treatmentsof Julella are by Barr (1985) who lists three species and onevariety and Aptroot and van den Boom (1995) who consid-ered Julella a predominantly tropical group of barksaprotrophs, and recognized the species: J. lactea (A.Massal.) M.E. Barr, J. sericea (A. Massal.) Coppins(=J. fallcaiosa (Stizenb. ex Arnold) R.C. Harris) andJ. vitrispora (Cooke & Harkn.) M.E. Barr (=J. sublactea),and regarded other species as synonyms or assigned them toother genera. After examine the generic type of Julella,Ariyawansa et al. (2013a) introduced Halojulellaceae andHalojullela to accommodate J. avicenniae, a marine speciesin the suborder Pleosporineae, order Pleosporales,Dothideomycetes. Justification for the new family was basedon combined gene analysis of the large and small subunits ofthe nuclear ribosomal RNA genes (LSU, SSU) and two pro-tein coding genes RPB2 and TEF1, as well as morphologicalcharacters (Ariyawansa et al. 2013a). Combined gene analysisof nuSSU, nuLSU, mtSSU and TEF1 has shown that theputative strains of Julella fallaciosa (MPN141 andMPN547) forms a separate clade in Trypetheliales (Nelsenet al. 2011), and are therefore excluded from our analysis.

Zhang et al. (2012) suggested that with the exception ofhyaline ascospores, most of the characters of Julella are com-patible with Montagnulaceae. Julella is compatible withDidymosphaeriaceae in having immersed ascomata formedunder a clypeus, short neck, 2-layered peridium composed ofcells of textura angularis and cylindric or oblong, pedicellate,often with an ocular chamber and oblong to narrowly oblong


muriform ascospores but differing in having hyaline asco-spores. We add Julella to Didymosphaeriaceae but this hasto be confirmed by molecular data.

Kalmusia Niessl, Verh. nat. Ver. Brünn 10: 204 (1872),Facesoffungi number: FoF 00040

Saprobic on terrestrial habitats. Sexual state: Ascomatasmall- to medium-sized, solitary, scattered or in small groups,immersed to erumpent, globose or subglobose, often laterallyflattened, coriaceous, black-walled, with or without papilla.Hamathecium of dense, filliform, delicate, septatepseudoparaphyses, branching and anastomosing betweenand above asci, embedded in mucilage. Asci bitunicate, cla-vate with a long, furcate pedicel. Ascospores narrowly ovoidto 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 orsubglobose, 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 prismaticato textura angularis. Hamathecium of dense, 1.5 μm wide,delicate pseudoparaphyses, 1–septate, branching and anasto-mosing between and above asci, embedded in mucilage. Asci75–125×10–15μm (x=90×12μm, n=10), 8-spored,bitunicate, fissitunicate, clavate, with a long, narrow, furcatepedicel 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, withoutconstriction, smooth-walled. Asexual state: unknown.

Material examined: MORAVIA, Bruno, on dead stems.(LUX 045054, isotype)


Fig. 6 Julella buxi (S-F5992, holotype). a-b Surface view of ascomataon host substrate (bark). c Longitudinal section of ascoma with clypeus. dClose up of the peridium. e Cellular pseudoparaphyses. f-g Two spored

asci lacking an ocular chamber. h-j Muriform ascospores. m Ascosporemounted in Indian ink to show mucilaginous sheath. Scale bars: c=80μm, d=10μm, e=5μm, f-g=25μm, h-j=15μm

Notes: Kalmusia was characterized as “immersed,sphaeroid ascoma with central, stout papilla, surrounded byhyphae in the substrate, stipitate asci with septatepseudoparaphyses, and brown, 3-septate, inequilateral asco-spores” by Barr (1992). The most morphologically compara-ble genus to Kalmusia is Thyridaria, which had been treatedas a subgenus under Kalmusia (Lindau 1897), and was sub-sequently transferred to Platystomaceae in Melanommatales(Barr 1987, 1990). After comparing their morphological char-acters, Diapleella and Dendropleella were also consideredsynonyms of Kalmusia.

Compared to Thyridaria, Kalmusia has sphaeroidascomata, a peridium of small pseudoparenchymatous cells,basal asci and very thin pseudoparaphyses, thus it wasassigned to Phaeosphaeriaceae by Barr (1990), and the genusis 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 andK. coniothyrium (Fuckel) Huhndorf (Barr 1992). But thisproposal is questionable because the clavate asymmetric as-cospores as well as the clavate asci with long pedicels ofK. ebuli do not fit the concept of the Phaeosphaeriaceae asdefined by Zhang et al. (2012) and is excluded from the familyby Phookamsak et al. (2014)

Most recent phylogenetic studies indicated that some spe-c i es of Kalmus ia res ide outs ide of the fami lyPhaeosphaeriaceae (Zhang et al. 2009). Kalmusia ebuli, thetype species of Kalmusia, lacks type material and as such itsphylogenetic position has remained unresolved. As a conse-quence, the familial position of Kalmusia is based on mor-phology and molecular phylogeny of species other than thetype (Zhang et al. 2014). Recently, a fresh collection of


Fig. 7 Kalmusia ebuli (LUX 045054, isotype) a Hebarium packet. b-cSurface view of ascomata on host substrate (bark). d Longitudinal sectionascoma with clypeus. e Close up of the peridium. f Broad, septate

pseudoparaphyses. g Long, narrow asci with furcate pedicel. h-i Palebrown ascospores with 3-distosepta. Scale bars: d=80μm, e=10μm, f=5μm, g =25μm, h-i=15μm

K. ebuli was obtained from decorticated wood of Populustremula in the foothills of the French Pyrenees (Zhang et al.2014). The new collection was designated as neotype tostabilize 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 with-in the family Montagnulaceae, with K. ebuli distantly placedfrom K. brevispora and K. scabrispora, which appear torepresent a different genus. During our phylogenetic analysis,we also obtained similar results, with K. ebuli being distantlyplaced from K. brevispora and K. scabrispora and formed adistinct clade sister to a newly introduced asexual genusAlloconiothyrium in Didymosphaeriaceae. Furthermore, a re-cently introduced asexual genus, Dendrothyrium (typifiedwith D. variisporum (CBS 121517T) and another species ofDendrothyrium, D. longisporum (CBS 582.83T), clusteredwithin the Kalmusia ebuli clade. Therefore, we propose tosynonymize Dendrothyrium under Kalmusia by giving prior-ity to the older name and combine two species underKalmusia.

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-brownto dark brown small cells, with inner layer of pale compressedcells.Hamathecium of dense, cellular pseudoparaphyses. Ascibitunicate, fissitunicate, cylindrical to cylindro-clavate withshort furcate pedicel. Ascospores ellipsoid to fusoid, reddish-brown to dark brownmuriform.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 rhodostomaAlb. & 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 whichsometimes sloths off, globose or subglobose, black, flattenedtop often white or reddish and sometimes slightly protruding

out of the substrate surface, usually with a wide openingostiole after removing the cover, coriaceous. Peridium 30–40μm wide, comprising two cell types, outer region singlelayered, composed of relatively small heavily pigmentedthick-walled compressed cells, inner layer cells larger andwall thinner, comprising cells of textura angularis, mergingwith pseudoparaphyses. Hamathecium of dense, 2–3.5μmwide, 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 is12–35μm long, and with an ocular chamber. Ascospores 20–26×7.5–10μm (x=22×8μm, n=20), obliquely uniseriate andpartially overlapping, ellipsoid, reddish-brown, with 3-transverse septa and a vertical septum in one or two centralcells, 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 beensynonymised under Pleomassaria by several authors (Lindau1897; Winter 1885). Karstenula shares similarities withDidymosphaeria in having ascomata seated in a subiculumor beneath a clypeal thickening and sometimes apical cellsbecome reddish or orange-brown (Barr 1990). Barr (1990)modified the concept of Karstenula (sensu lato), which en-compasses some species of Thyridium. However, Barr (1990)treated Karstenula as having trabeculate pseudoparaphysesand this is clearly not the case. In most cases, the ascosporesare brown with transverse septa and sparse longitudinal septa.The ascomata of Karstenula rhodostoma are similar to thosefound in Byssosphaeria and Herpotrichia, especially in thepaler area around the ostiole and even in peridium and devel-opment under a subiculum. The numerous wide cellularpseudoparaphyses and cylindrical asci (in Herpotrichia) arealso similar. The main difference between Karstenula and theother two genera is the 3-septate ascospores with rare longi-tudinal septa (1-septate in Byssosphaeria and Herpotrichia).

Recent phylogenetic studies have shown that the putativestrain ofKarstenula rhodostoma (CBS 690.94) forms a robustclade with Phaeodothis winteri (Niessl) Aptroot, Didymocreasadasivanii, 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 Zhanget al . (2012) tentatively included Karstenula inMontagnulaceae.

Our phylogenetic analysis reveals that the putative strain ofKarstenula rhodostoma (CBS 690.94) resides inDidymosphaeriaceae, sister to the Paraphaeosphaeria clade.The r e f o r e we t en t a t i v e l y r e f e r Kars t enu la t oDidymosphaeriaceae. However, the placement of Karstenulain Didymosphaeriaceae can only be confirmed by phylogenetic


work including sequencing the generic type of Karstenula(K. rhodostoma).

Letendraea Sacc., Michelia 2(6): 73 (1880), Facesoffunginumber: FoF 00044.

Saprobic in terrestrial habitats. Sexual state: Ascomatararely small, usually medium-sized, immersed usually undera thin clypeus, scattered to gregarious, with flattened top androunded pore-like ostiole, coriaceous. Peridium 2-layered,outer layer composed of reddish-brown to dark brown smallcells, inner layer of pale compressed cells. Hamathecium ofdense, cellular pseudoparaphyses. Asci cylindrical to cylindro-

clavate with short furcate pedicel. Ascospores one septate,ellipsoid to fusoid, reddish-brown to dark brown. Asexualstate: 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 tosubglobose, solitary, bright coloured, with papillate, centralostiole (from illustration). Hamathecium of dense, filliform,hyaline pseudoparaphyses. Asci (63-)73–75×13–18μm (x =

Fig. 8 Karstenula rhodostoma (PH 01048835, holotype of Sphaeriarhodostoma). a-b Herbarium packet and specimen. c. Ascomata onsubstrate. d Section thought ascoma. e Asci arrangement. f Close up ofthe peridium. g Hamathecium comprising cellular pseudoparaphyses. h-i

Asci when immature. j Ascus at maturity. k-m Ellipsoid, reddish-brownascospores. Scale bars: d=100μm, e, h–j=50μm. f, k–m=20μm, g=10μm


71×15μm, n=8), 8-spored, bitunicate, fissitunicate, cylindrical-clavate, slightly curved, sessile. Ascospores 13–16×5–6μm(x = 14×5μm, n=20), 1–2-seriate, ellipsoidal to fusoid, upper-cell wider toward base narrow and rounded ends, 1-septate,olivaceous brown, guttulate, smooth. Asexual state: unknown.

Material examined: USA, on dead branches of Rubifruticosi (NY0091436, lectotype).

Notes: Letendraea is characterized by superficial, globoseto conical ascomata, filliform pseudoparaphyses, obclavate tocylindrical, 8-spored asci, and fusoid to oblong, 1-septateascospores (Saccardo 1880). Because of its similar morphol-ogy with Karstenula rhodostoma, Kodsueb et al. (2006b)assigned Letendraea to Melanommataceae.

Subsequent multigene phylogenetic analysis indicated thatboth L. helminthicola (Berk. & Broome) Weese. andL. padouk Nicot & Parg. -Leduc. nes ted withinMontagnulaceae (Schoch et al. 2009; Zhang et al. 2009), thusZhang et al. (2012) referred Letendraea to Montagnulaceae.In our phylogeny we obtained the same results and apart fromthe putative strains of L. helminthicola (CBS 884.85) andL. padouk (CBS 485.70), we included the putative strain ofL. eurotioides (CBS 212.31), the generic type of Letendraeain our analysis. Therefore, we treat Letendraea as a separategenus in Didymosphaeriaceae.

During our study we collected a Dothideomycetes species(Letendraea cordylinicola) from living and dead leaves ofCordyline sp. from Thailand, associated with leaf spotdisease, and showing similar morphology to Wilmiabrasiliensis. Dianese et al. (2001) referred Wilmia inPhaeosphaeriaceae based on morphological characters.Wilmia is similar to other genera in Phaeosphaeriaceae viz.Barria, Lautitia and Metameris, with its didymosporous as-cospores and coelomycete asexual morph (Dianese et al.2001; Zhang 2012). Zhang et al. (2012) tentatively placedWilmia in Phaeosphaeriaceae as the genus is poorly knownand has no supporting molecular phylogenetic evidence,although Zhang et al. (2012) mentioned that the genus mightbe accommodated in Leptosphaeriaceae based on its dicoty-ledonous host habit.

Our phylogenetic analysis showed that a novel Letendraeaspecies (Letendraea cordylinicola) nested within Letendraeain the family Didymosphaeriaceae. Letendraea and Wilmiashare similar characters in having immersed to superficial,g l o b o s e - s u bg l o bo s e a s c oma t a w i t h f i l l i f o rmpseudoparaphyses and ellipsoidal to fusoid 1-septate, oliva-ceous brown ascospores with smooth wall. Therefore, basedon morphology of the type species of Wilmia, W. brasiliensisand novel Wilmia-like (Letendraea cordylinicola) species,

Fig. 9 Letendraea eurotioides(NY0091436, lectotype) a Semiimmersed to superficial, globose-subglobose ascomata. bHamathecium. c Young andimmature aci. d-f Asci with eightspores. Scale bars: a-b=20μm,c=50μm, d-f=10μm, g=20μm


coupled with molecular data we exclude Wilmia fromPhaeosphaeriaceae and transfer it to Didymosphaeriaceae.Furthermore, we propose to synonymize Wilmia underLetendraea by giving priority to the older name. The typespecies, Letendraea brasiliensis and the novel speciesL. cordylinicola are described below.

Letendraea brasiliensis (Dianese et al.), Phookamsak,Ariyawansa & K.D. Hyde comb. nov., Index Fungorumnumber: IF550698 (Fig 10), Facesoffungi number: FoF00046.

Basionym: Wilmia brasiliensis Dianeseet al., Mycologia93(5): 1014 (2001).

Fig. 10 Letendraea brasiliensis (UB 8438, holotype of Wilmiabrasiliensis) a Herbarium label and specimens. b Ascomata on hostsurface. c Section through ascomata stained by cotton blue reagent. dSection through peridium. e Pseudoparaphyses stained by Congo red

reagent. f-h Ascospores. i-k Asci. l Section through conidiomata stainedby 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


Parasitic on Memora pedunculata. Sexual state:Ascomata 140–200×90–125μm, scattered, rarely clustered,immersed to erumpent through host surface, globose tosubglobose, brown to dark brown, central ostiole. Ostiolepapillate with elongated periphyses. Peridium 6–21μm wide,composed of 2–5 layers, thin-walled with equal thickness,small, flattened pseudoparenchymatous, brown to dark browncells of textura angularis to textura prismatica. Hamatheciumdense of 1.5–3μm wide, cellular pseudoparaphyses,unbranched, not -constricted at the septa, hyaline, embeddedin a mucilaginous matrix. Asci 70–90(-98)×(11-)13–15(-17)μm (x=82.9×13.8μm, n=30), 8-spored, bitunicate,fissitunicate, cylindric-clavate, short-pedicellate, apicallyrounded with indistinctly ocular chamber. Ascospores(18-)20–24×6–7(-7.5) μm (x=21.9×6.9μm, n=30), over-lapping, 1–2-seriate, didymosporous, fusiform with ob-tuse 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. Conidiomata80–220μm high, 100–165μm diam., pycnidial, scatteredto clustered, solitary, immersed to erumpent throughhost surface as black dots, visible as slightly raised orbumped, small, pale brown to brown spots on hostsurface, uniloculate, globose to sub globose, brown todark brown, central ostiole, papillate with largeperiphyses. Conidiomata walls 8–14μm wide, composedof 2–5 layers, thin-walled cells with equal thickness,small, flattened pseudoparenchymatous cells of texturaangularis 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 tolageniform, hyaline, smooth-walled, aseptate. Conidia(12-)14–20(-26.5)×5–7μm (x =15.9×6.4μm, n=30),oblong to obovoid or ellipsoidal with rounded or obtuseends, hyaline, indistinctly 1-distoseptate, rough-walled.

Material examined: BRAZIL, Distrito Federal VargemBonita, Fazenda Áqua Limpa, on leaves of Memorapedunculata 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 onthe host genus from which the fungus was isolated.

Saprobic or parasitic on Cordyline sp. Sexual state:Ascomata 85–115×100–160μm, scattered, immersed tosemi-immersed, pale yellowish to reddish-brown on leaf le-sions, globose to subglobose, pale yellowish to brown, centralostiole. Ostiole papillate and ostiolar canal filled with longperiphyses. Peridium 9.5–20μm wide, composed of 2–5layers , th in-wal led wi th equal th ickness , la rge

pseudoparenchymatous cells of textura angularis to texturaprismatica, hyaline to pale yellowish. Hamathecium dense of2–3( -4 ) μm wide , numerous , b road ly ce l l u l a rpseudoparaphyses, unbranched, anastomosing, indistinctlyconstricted at the septum, embedded in a mucilaginousmatrix.Asci (51-) 55–60(-68)×(8-) 10–12μm (x=59.1×10.4μm, n=20), 8-spored, bitunicate, fissitunicate, cylindric-clavate, shortpedicellate, apically rounded with an indistinct ocular cham-ber, 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 theupper cell, smooth-walled, with distinct appendage at bothends, 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 topale orange at the margin, white to orange or grey at thecentre; 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 undulateedges, fluffy to velvety, margin with well-defined to slightlyradiating, forming ascomata on colony after 8 weeks, produc-ing light brown pigment tinted agar.

Material examined: THAILAND, Mae Jai District, PaFaek village, on dead leaves of Cordyline sp., 5 September2010, R. Phookamsak, RP0064 (MFLU 11-0184, holotype),ex-type living culture = MFLUCC 11-0148; Chiang Rai,Muang District, Pakha village, on living Cordyline sp., 15August 2010, S. Wikee, RP0066 (MFLU 11-0186), livingculture = 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 typeL. eurotioides share similar characters such as havingglobose-subglobose ascomata, cylindric-clavate asci with 1-septate brown ascospores, but differ in the nature of thepseudoparaphyses (cellular versus filliform), size of the asci(small versus large) and nature of the ascospores (with dis-tinct appendage versus without distinct appendage).Letendraea cordylinicola shares similarities withL. brasiliensis in having immersed to semi-immersedascomata with central ostiole filled with long periphyses,cylindric-clavate, short pedicellate asci bearing fusiform, 1-septate, brown ascospores and both cause leaf spots on theirhost. However, L. brasiliensis differs from L. cordylinicola inhaving large ascomata and indictinct appendages and sheathwhile L. cordylinicola has small ascomata, nice appendagesand a sheath. Based on morphology coupled with DNAsequence da t a , we accep t L . bras i l i ens i s andL. cordylinicola in the genus Letendraea because of theirsimilarities with other species in this genus.


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 toerumpent, gregarious or grouped, globose to subglobose,black. Hamathecium of dense, narrowly cellular, septatepseudoparaphyses. Asci bitunicate, fissitunicate, usuallycylindric-clavate to clavate with a long pedicel. Ascosporesoblong to narrowly oblong, straight or somewhat curved,reddish-brown to dark yellowish-brown, muriform orphragmosporous. Asexual state: unknown.

Type species:Montagnula infernalis (Niessl) Berl., Icon. fung.

(Abellini).2: 68 (1896) (Fig 12), Facesoffungi number: FoF00049.

Basionym: Leptosphaeria infernalis Niessl, Inst. Coimbra31: 13 (1883).

Saprobic on dead leaves in terrestrial habitats. Sexualstate: Ascomata 220–280×250–310μm (x = 250×280μm,n=5), immersed to erumpent, gregarious or clustered, globoseto subglobose, sometimes triangular in dried material, shortostiole always filled with hyaline closely adhering cells.Peridium 40–55μm thick at sides, up to 80μm thick near theapex, 3-layered, outer layer composed of heavily pigmentedthick-walled small cells of textura angularis, apex thickerwith smaller cells and thicker cell wall, thinner near the base;mid layer less pigmented, innermost layer of narrow com-pressed rows of cells, merging with pseudoparaphyses.Hamathecium of dense, 2–4.5μm broad, narrow, septate cel-lular pseudoparaphyses. Asci 153–170×18–22μm (x= 157×

Fig. 11 Letendraea cordylinicola (MFLU11-0184, holotype) aAscomata on host surface. b Section through ascomata. cSection through peridium. d Pseudoparaphyses stained by cotton bluereagent. e Asci embedded in pseudoparaphyses. f-h Asci. i-k Ascospores

with polar appendages that eventually expand into a sheath. l Ascosporestained in Indian ink with a prominent sheath. m Germination of asco-spore. Scale bars: b=50μm, c=20μm, d-h, m=10μm, i-l=5μm


20μm, n=10), 8-spored, bitunicate, fissitunicate, cylindro-clavate to clavate, pedicel 28–60μm long, with an ocularchamber best seen in immature ascus. Ascospores 24–29×9–11μm (x = 26×10μm, n=20), biseriate, oblong to narrowlyoblong, straight or somewhat curved, reddish-brown to darkyellowish-brown, verruculose, with five transverse septa andone vertical septum in each middle cells, constricted at theprimary and secondary primary septa. Asexual state:unknown.

Material examined: PORTUGAL, Coimbra lusitania, onleaves of Fourcroya longava (Agavoideae), February 1881,Moller (M 1183, holotype of Leptosphaeria infernalis).

Notes: Montagnula was introduced by Berlese (1896) inorder to separate two dictyosporous species, M. infernalis(Niessl) Berl., and M. gigantean (Mont.) Berl. fromPleospora, based on the presence of hyphal stromatic tissueover the ascomata and asci with a long pedicel (Barr 2001).Wehmeyer (1957) had placed Montagnula as a subgenus ofPleospora. Crivelli (1983) again treated Montagnula as aseparate genus and divided the genus into two sub genera,i.e. Montagnula and Rubiginospora, which are distinguishedbased on dark brown ascospores located on Agavaceae andreddish-brown ascospores on Poaceae, respectively (Barr2001), but this proposal was not accepted by many mycolo-gists. Subsequently, Leuchtmann (1984) and Aptroot (1995)included some phragmosporous and didymosporous speciesin the genus and eventually it became heterogenic (Zhang

et al. 2012). Apart from the type species, some Montagnulaspecies produce Aschersonia Mont. asexual morphs (Hydeet al. 2011). The genus presently has 28 epithets (IndexFungorum 2014). GenBank has 19 hits for the genus includ-ing putative strains of M. opulenta (De Not.) Aptroot (CBS168.34), 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) andM. anthostomoides (Rehm) Leuchtm. (CBS 615.86).

One striking character ofMontagnula infernalis is the verylong ascal pedicel which develops once it is released from theascomata. However, this character appears to have evolvedmore than once and can be found in Kirschsteiniotheliaelaterascus Shearer which clusters with Helicascus (Sheareret al. 2009). The same character is also found in Xenolophiumand Ostropella in the Platystomaceae (Mugambi andHuhndorf 2009). Montagnula opulenta is a didymosporousspecies, but phylogenetically closely related to thosedictyosporous (Karstenula rhodostoma) and phragmosporous(Paraphaeosphaeria michotii) members of Montagnulaceae(Zhang et al. 2009). This might indicate that compared toother morphological characters, ascospore type is not a goodcharacter at the family level classification. Recent phylogenybased on multi-gene analysis has shown that the putativestrain of M. opulenta forms a robust phylogenetic clade withspecies of Bimuria, Curreya, Didymocrea, Letendraea,Paraphaeosphaeria, Phaeodothis and Karstenula, which


Fig. 12 Montagnula infernalis (M 1183, holotype of Leptosphaeriainfernalis). a Fungus on the herbarium material. b-c Dry black ascomataon host surface. d Ascoma in horizontal section. e Layers of peridium. f

Cellular pseudoparaphyses g immature ascus. h mature ascus with longpedicel. g-h Eight spored asci not visible. i-j Asci bearing muriformascospores. Scale bars: a-c=0.5mm, d=100μm, e-f=10μm, g-j=20μm

might represent a familial group (Schoch et al. 2006; Zhanget al. 2009, 2012).

Our phylogenetic data also shows that the putative strainsof Montagnula rhodophaea (CBS 616.86) and M. spartii(CBS 183.58) clus tered in Lent i theciaceae andMassarinaceae, respectively. The morphology and identifica-tion of these putative strains in GenBank as far as we canascertain, cannot be checked, as they are not linked to anyherbarium material, therefore we placed them inLentitheciaceae incertae sedis and Massarinaceae incertaesedis, respectively. Montagnula dura (CBS 380.54) was ex-cluded from our final analysis because in the preliminaryanalysis we observed that this strain clustered withinPleosporaceae (details not shown). Our phylogenetic resultsindicated that the putative strains of M. opulenta (CBS168.34), M. aloes (CPC 19671) and M. anthostomoides(CBS 615.86) nested within Montagnulaceae and form aseparate clade sister to the Kalmusia and Alloconiothyriumclades. The strain named Letendraea helminthicola (CHTAR43) resides in Montagnula. This is probably a misidenti-fication of Montagnula sp. as Letendraea helminthicola.The morphology and identification of the putative strain(CHTAR43) of Letendraea helminthicola in GenBank asfar as we can determine, cannot be checked, as they are notlinked to any herbarium material thus preliminarily re-identified as Montagnula sp. Therefore based on morphol-ogy, coupled with available molecular data, we keepMontagnula as a distinct genus in Didymosphaeriaceae.Fresh collections of theM. infernalis and further molecularand morphological studies are desirable to confirm ourresults.

Neokalmusia Kaz. Tanaka et al., gen. nov., IndexFungorum number: IF550700, Facesoffungi number: FoF00050.

Etymology: The generic epithet, neo (Lat., new), refers tothe similarity to Kalmusia.

Saprobic on culms of bamboo in terrestrial habitats. Sexualstate: Ascomata immersed under black clypeus-like structurecomposed of host epidermis and fungal mycelia, subgloboseto oblong on host surface, hemispherical, with several peri-thecia arranged in single or double rows along the long axis ofthe culms. Ostiole absent or short papillate, with numerousperiphyses. Peridium at side composed of several layers ofpolygonal, thin-walled, brown cells; at the rim composed ofvertically orientated, hyaline, cells of textura angularis.Hamathecium composed of numerous, broadly cellularpseudoparaphyses, branching, anastomosing, with slime coat-ing. Asci (4–) 8-spored, bitunicate, fissitunicate, cylindric-clavate, with a long stipe, apically rounded with well-developed ocular chamber, arising from basal ascomata.Ascospores overlapping, uniser iate or biser iate ,phragmospores, fusiform, slightly curved, initially hyaline,becoming yellowish-brown to reddish-brown at maturity, 3–

5-septate, thick-walled, verrucose, with a sheath. Asexualstate: unknown.

Type species:Neokalmusia brevispora (Nagas. &Y. Otani) Kaz. Tanaka,

Ariyawansa & K. D. Hyde, comb. nov., Index Fungorumnumber: IF550701. (Fig. 13), Facesoffungi number: FoF00051.

Basionym: Phaeosphaeria arundinacea var. brevisporaNagas. & Y. Otani, Rep. Tottori Mycol. Inst. 15: 38 (1977).

Saprobic on culms of Sasa spp. Sexual state: Ascomata200–370(–400)×190–330(–440) μm immersed under blackclypeus-like structure composed of host epidermis and fungalmycelium, scattered to grouped, subglobose to oblong on hostsurface, 1–3.5mm long, 0.4–0.7mm wide. Ostiole absent orshort papillate, 50–75(-110) μm long, with hyaline periphyses.Peridium 15–20μm diam., composed of 2- layers, outer layercomposed of 2–4 layers, light brown to dark brown pseudo-parenchymatous cells of textura angularis, inner wall consistof 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, fusi-form, 3-septate, with a mostly submedian (0.48–0.56; x=0.52,n=46) primary septum, reddish-brown, thick-walled, verru-cose, with 2–5μm thick sheath. Asexual state: unknown.

Material examined: JAPAN, Fukushima, Minamiaizu, Osepond, on dead twigs of Sasa sp. (Poaceae), 30 Aug. 2003, N.Asama, (KT 1466 = HHUF 28229), living culture CBS120248 = JCM 13543 = MAFF 239276; Hokkaido, Isl.Rishiri, Afutoromanai river, on dead twigs of Sasa kurilensis,29 July 2007, K. Tanaka & G. Sato, (KT 2313 = HHUF30016), living culture = NBRC 106240.

Notes: A new genus Neokalmusia is established to accom-modate two bambusicolous taxa, N. brevispora andN. scabrispora. These species have been previously placed inKalmusia (Tanaka et al. 2005; Zhang et al. 2009), but subsequentmolecular studies (Hyde et al. 2013) as well as in the presentstudy (Fig. 1), do not support this placement. Morphologicalstudies of Kalmusia based on the holotype of K. ebuli (type ofthis genus) indicated that the genus is characterized by solitarysphaeroid ascomata, a peridium of small pseudoparenchymatouscells, clavate basal asci with very long pedicels, very thinpseudoparaphyses and distoseptate, smooth-walled ascospores(Zhang et al. 2012, this study). The two Neokalmusia specieson bamboo have subglobose to oblong ascomata including sev-eral perithecia in a row, clypeus-like structure composed of thin-walled cells and verrucose ascospores.

Neokalmusia scabrispora (Teng) Kaz. Tanaka et al., comb.nov., Index Fungorum number: IF550702. (Fig. 14),Facesoffungi number: FoF 00052


Basionym: Leptosphaeria scabrispora Teng, Sinensia,Shanghai 4: 378 (1934).

Saprobic on culms of bamboo. Sexual state: Ascomata200–300×130–500μm, immersed under black clypeus-likestructure 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 cellsof 5–10×2.5–6.5μm, surrounded by wedge-shaped stromaticregion (250–400μm wide at sides) composed of verticallyorientated hyaline cells of textura angularis. Hamathecium1–2μm wide narrowly cellular, numerous, septate, branchedand anastomosing, embedded in a mucilaginous matrix. Asci123.5–160×(15.5-)17–22μm (x=142.1×1 8.8μm, n=50), 8-spored, 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 ellip-soid, slightly curved, 5 (rarely 7) -septate, with a medianprimary septum 0.48–0.53μm (x =0.50μm, n=57) wide,slightly constricted at the septa, with third cell from the apexenlarged, penultimate cells shortest, brown to yellowish-brown, verrucose, with 10–20μmwide sheath.Asexual state:unknown.

Material examined: JAPAN, Tochigi, Kanuma, near Ooashiriver, on dead twigs of Phyllostachys bambusoides (Poaceae),6 March 2003, N. Asama (KT 1023 = HHUF 28608), livingculture 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 (KT2202 = HHUF 30013), living cultures = NBRC 106237.


Fig. 13 Neokalmusia brevispora (KT 1466). a-b Ascomata on host surface. c-d Vertical section through ascomata. e Section through peridium. fPseudoparaphyses. g-h Asci. i Ocular chamber. j Germinating ascospore. k-o Ascospores. Scale bars: a-b=1mm, c-d=100μm, e-o=10μm

Notes: This fungus was originally described as a species ofLeptosphaeria (Teng 1934) and later transferred toMassariosphaeria (Shoemaker and Babcock 1989) orKalmusia (Tanaka et al. 2005). Neokalmusia scabrispora,however, does not belong to the Leptosphaeriaceae typifiedby Leptosphaeria doliolum or to the Thyridariaceaeencompassing the type species of Massariosphaeria(M. phaeospora) (Hyde et al. 2013). Neokalmusiascabrispora shares similar characters to Neokalmusiabrevispora 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 thicksheath. Neokalmusia scabrispora differs in the thickness ofthe peridium (7.5–20 μm versus 15–20 μm) and the numberof 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

Fig. 14 Neokalmusia scabrispora (KT 1023) a-b Ascomata on hostsurface. c-d Vertical section through ascomata. e Section through perid-ium. 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


eustromatic, simple or complex, rarely pycnidial,Conidiogenous cells discrete or integrated, phialidic, some-times percurrent. Conidia aseptate, sometimes 1-septate, thin-walled, smooth-walled or minutely warted, hyaline whenliberated, 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 inthe agar, but also superficial and on the aerial mycelium,eustromatic, globose or flattened, dark brown to black, withseveral merging cavities, ostioles absent, opening by dissolu-tion of upper cells. Conidiomatal wall 30–45μm thick, com-posed of outer layer of isodiametric or more flattened cellswith hyaline to reddish-brown walls thickened up to 1.5μm,lined by a 35–60(-75) μm thick inner layer of texturaangularis, surface of the conidiomatal wall often coveredunder brown entangling hyphae. Conidiogenous cells 4–6.5×2.5–3.5(-4) μm, discrete, assembled into protrudingmasses of cells, or integrated in very compact conidiophores,ampulliform to subcylindrical, hyaline, indeterminate,phialidic with an inconspicuous periclinal thickening andcollarette, 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 andsmooth walls that are hyaline at secession, but soon becomingolivaceous- 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 SSUnrDNA sequences Verkley et al. (2004), includedConiothyrium minitans and the ubiquitous soil fungusConiothyrium sporulosum in Paraconiothyrium. In the samestudy, the asexual morphs of Paraphaeosphaeria michotii andP. p i l l ea ta were regarded as represen ta t ive ofParaconiothyrium, but remained formally unnamed.Paraconiothyrium species are phylogenetically distant fromtypical members of the other coelomycete genera such asConiothyrium, Microsphaeropsis, Cyclothyrium, andCytoplea. In Paraconiothyrium, the conidiomata generallyare complex, eustromatic and relatively thick-walled. Theymay appear as simple pycnidia, but then they usually lack awell differentiated ostiole. In Coniothyrium, the conidiomataare true pycnidia, which may merge in vitro but then alwaysproduce well-developed, sometimes even papillate ostioles.The most distinctive Coniothyrium feature is the annellidicconidiogenous cells, which percurrently proliferate after se-cession of each conidium. Coniothyrium conidia are thick-

walled and verruculose, with a truncate base and sometimes abasal frill (Sutton 1980). Microsphaeropsis species are alsopycnidial, but their conidiogenous cells are discrete, Phoma-like 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 rela-tion to ITS sequences for nine species of Paraphaeosphaeria,and identified three lineages, which were later confirmed bySSU data (Câmara et al. 2003). They found that only onespecies, Paraph. pilleata, was congeneric with the type spe-cies Paraph. michotii (Verkley et al. 2004). Thus, only twospecies were retained in Paraphaeosphaeria sensu stricto. Forthe other species the genera Phaeosphaeriopsis andNeophaeosphaeria were erected. Following multi-locusDNA analysis and detailed morphological study, Verkleyet al. (2014) were able to delimit and formally propose ninenew species and two new genera among the fungi in theMontagnulaceae formerly recognizable as Coniothyrium-likeasexual morphs. Verkley et al. (2014) suggested that the genusParaconiothyrium appears to be paraphyletic within the fam-ilyMontagnulaceae but proposed that it should not be split upto further genera.

In the present study, we also observed similar phylogeny,where Paraconiothyrium appears to be paraphyletic withinthe family Didymosphaeriaceae. Several sexual stages ofsome Paraconiothyrium species were reported during thestudy, i.e. the sexual state of Paraconiothyrium fuckelii(M F LUCC 1 3 - 0 0 4 3 ) a n d n o v e l s p e c i e s o fParaconiothyrium, Parac. magnoliaee introduced fromThailand and are described below.

Paraconiothyrium fuckelii (Sacc.) Verkley & de Gruyter, inGruyter 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 withoutpapilla, ostiolate. Papilla small, with small ostioles. Peridium15– 40μm wide, comprising one cell type of small,pigmented, thick-walled cells of textura prismatica to texturaangularis. Hamathecium of dense, 1.5μm broad, delicatepseudoparaphyses, 1-septate, branching and anastomosingbetween 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 pedicelwhich is up to 45μm 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, constrictedat the middle septum, smooth-walled. Asexual state:


Conidiomata pycnidial 300–400μm diam. and with a singlecavity, more often eustromatic. Conidiomatal wall 3-layered,outer layer composed cells of textura angularis with some-what thickened, brown walls, and an inner layer composedcells of textura angularis-globulosa with somewhat thick-ened, hyaline walls. Conidiogenous cells 4–10(–13)×3–5μm, discrete or integrated in short, simple, 1–2-septate co-nidiophores, broadly ampulliform to globose, holoblastic, of-ten annellidic with 1 or 2 percurrent proliferations noticeableby the distinct scars on a somewhat elongated neck, hyaline.Conidia 3–4×2–3(–3.5) μm variable in shape, subglobose toellipsoid or obovoid, rarely more cylindrical, initially hyaline,olivaceous-brown soon after secession, smooth, orange-brown, aseptate.

Material examined: THAILAND, Chiang Rai, Bandu, ondead wood, 28 September 2012, K.M. Thambugala (MFLU14-0305), 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 epithetmagnoliae is based on thehost genus from which the fungus was isolated.

Saprobic on dead leaves in terrestrial environments. Sexualstate: 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-

Fig. 15 Paraconiothyrium fuckelii. (MFLU 14-0305) a-b Appearanceof ascomata on the host surface. c Section of an ascoma. d Section ofperidium. e Clavate ascus with a short, narrow pedicel. f-h Subglobose to

ellipsoid or obovoid ascospores. i-j Conidiogenous cells. k Orange-brown conidia. Scale bars: c=100μm, d=25μm, e=10μm, f-k=5μm


5 layers, composed cells of textura prismatica. Hamatheciumof 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, cylindricalwith a short, broad pedicel with a minute ocular chamber.Ascospores 15–30×4–7μm (x = 24×5, n=40), uniseritate orpartially overlapping, broadly elliptical, yellowish-brown, 3-septate (rarely 4-septate), with small guttules, with bipolarappendage at the tip and one polar appendage on the base,surrounded by a gelatinous sheath. Asexual state: unknown.

Material examined: THAILAND, Chiang Mai, DoiSuthep-pui, on dead leaves of Magnoliae liliifera(Magnoliaeceae) 18 September 2011, J. Monkai (MFLU 14-0306, holotype), ex-type living culture = MFLUCC 10-0278

Notes: Paraconiothyrium magnoliae resembles many spe-cies of Phaeosphaeria in having 3-septate ascospores with agela t inous sheath . Phaeosphaer ia eus toma andPhaeosphaeria parvula have ascospores which are very sim-ilar in size to the morphology of P. magnoliaee. In addition,Parac. magnoliae is similar to Phaeosphaeria typharum andPhaeosphaeria barriae in having gelatinous sheathed asco-spores (with appendaged ascospores only in Phaeosphaeriabarriae), but differ in the shape of the ascospores. The phy-logenetic analysis of combined ITS, LSU, SSU nrDNA andTUB sequences provides strong evidence that Parac.magnoliae belongs in Didymosphaeriaceae, where it forms aseparate 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: Ascomatasmall to medium-sized, immersed to semi-immersed, de-pressed, globose, ostiolate. Ostiole with a short beak, withoutperiphyses. Peridium usually with 3-5 layers, composed cellsof textura prismatica. Hamathecium composed of filamen-tous, hyaline, septate, broad, dense pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, cylindrical with a short pedi-cel. Ascospores uniseritate or partially overlapping, multiseptate, broadly elliptical, yellowish-brown, with smallguttules, smooth, with a thin uniform sheath. Asexual state:Coniothyrium-like, Conidiomata eustromatic or pycnidial.Conidiogenous cells discrete or integrated, phialidic, orannellidic with one or two percurrent proliferations. Conidiaaseptate 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 michotiiWestend., 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)

Fig. 16 Paraconiothyriummagnolia (MFLU 12-1414,holotype) a-b Fungus on hostsubstrate. c Section of an ascoma.dCellular, hyaline, septate, broad,pseudoparaphyses. e-gCylindrical asci with a short,broad pedicel. h-j Yellowish-brown ascospores with bipolarappendage. k Germinatingascospores. Scale bars: c=50μm,d=5μm, e-g=30μm, h-j=5μm,k=10μm


small to medium-sized, immersed to semi immersed, de-pressed 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 ofcells textura prismatica. Hamathecium of dense 2–3μm (x=2μm, n= 20) filamentous, hyaline, septate, broad,pseudoparaphyses. Asci 60–85×12–28μm (x=77×20μm,n=20), 8-spored, bitunicate, fissitunicate, cylindrical with ashort, broad pedicel with minute ocular chamber. Ascospores15–30×4–7μm (x = 24×5, n=40), uniseritate or partiallyoverlapping, 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 =MFLUCC13-0349.

Notes: Paraphaeosphaeria has been separated fromLeptosphaeria (Eriksson 1967), and it is quite comparablewith Phaeosphaeria. Paraphaeosphaeria can be distin-guished from Phaeosphaeria by its ascospores. Ascosporesof Paraphaeosphaeria michotii have two septa, and they arebiseriate, straight, subcylindrical with broadly rounded ends,rather dark brown and punctate. The primary septum is laiddown closer to the distal end than to the proximal, and thelarger, proximal hemispore is divided by one transverse

septum. There are more septa in the proximal hemispore ofother species such as Paraph. castagnei (Durieu & Mont.)O.E. Erikss., Paraph. obtusispora (Speg.) O.E. Erikss. andParaph. vectis (Berk. & Broome) Hedjar. Asexual characterscan also distinguish Paraphaeosphaeria and Phaeosphaeria.Paraphaeosphaeria produces Coniothyrium-like asexualstates, but Phaeosphaeria has Hendersonia-Phaeoseptoriaasexual states (Eriksson 1967). Câmara et al. (2001) provideddescriptions of sexual and asexual morphs ofParaph. michotiiand Paraph. pilleata, while other species treated underParaphaeo sphae r i a wer e t r a n s f e r r e d l a t e r t oNeophaeosphaeria and Phaeosphaeriopsis (Câmara et al.2003). None of the amerosporic coniothyrium-like speciesassociated with these sexual morphs has been assigned witha formal name. Shoemaker and Babcock (1985) redescribedsome Canadian and extralimital species, and excludedParaph. longispora (Wegelin) Crivelli and Paraph. oblongata(Niessl) Crivelli from Paraphaeosphaeria based on their lon-gitudinal septa as well as beak-like papilla and wall structures.Molecular phylogenetic results based on a multi-gene studyindicate that Paraphaeosphaeria should belong toMontagnulaceae (Zhang et al. 2009). Recently, Ariyawansaet al. (2014d) epitypified Paraph. michotii from a fresh col-lection and thus the placement of the Paraphaeosphaeria inMontagnulaceae is confirmed. In our phylogenetic analysis,Paraphaeosphaeria forms a well-supported clade sister to

Fig. 17 Paraphaeosphaeriamichotii. (MFLU 12-2210,epitype) a Ascomata on hostsubstrate. b Section of ascoma. cClose up of the peridium. dAscussurrounded by cellularpseudoparaphyses. e-g Asci withshort, broad pedicel bearing 8spores. h-j Mature ascosporeswith thin uniform sheath. kAscospores mounted in Indianink. Scale bars: b=100μm,c=50μm, d=20μm, f-h=60 μm,i-k=10μm


Didymosphaeria, thus Paraphaeosphaeria is treated as a sep-arate 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 terrestrialenvironments. Sexual state: Ascostromata subglobose tohemispherical, immersed, scattered to gregarious, dark brownto black. Peridium comprising several layers of hyaline com-pressed ce l l s . Hamathec ium of dense , ce l lu la rpseudoparaphyses 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 tonearly superficial under clypeus, scattered to gregarious,subglobose to hemispherical, dark brown to black, papillate,

smooth. Papilla black, with pore-like ostioles, ostiolar canalfilled 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. Asci55− 80× 7− 12μm (x =68×10μm, n=20), 8-spored,bitunicate, fissitunicate, cylindrical, short pedicellate, api-cally rounded with a minute ocular chamber. Ascospores14.5−17.5×4−5μm (x=15.8×4.4μm, n=30), biseriate andpartially overlapping, fusiform with rounded ends, oliva-ceous brown, 1-septate, slightly constricted at the septa.Asexual state: unknown.

Material examined: ARGENTINA, San José, Salta, 12February 1873, P.G. Lorentz (SF125876, holotype).

Notes: Phaeodothis is characterized by its immersed tonearly superficial ascomata, a sparse hamathecium consistingof cellular pseudoparaphyses and 1-septate ascospores(Aptroot 1995). Phaeodothis was placed in Didymosphaeriaby von Niessl (1875), but Aptroot (1995) transferred it toPhaeosphaeriaceae. Phaeodothis is similar to genera in the

Fig. 18 Phaeodothis tricuspidis(S F125876, holotype) aHerbarium packet and specimen.b Close up of ascomata. c-dSections through ascomata. ePeridium comprising hyalinecompressed cells. f-h Mature andimmature asci surrounded bypseudoparaphyses. i-l Fusiform,olivaceous brown ascospores.Scale bars: c-d=100μm, e=50μm, f-h=25μm, i-l=5μm


Didymosphaeriaceae in having ascomata immersed under aclypeus, a pseudoparenchymatous peridium with small cells,cylindrical asci and brown ascospores. However, Phaeodothisdiffers from other genera of Didymosphaeriaceae in having ahamathecium consisting of sparse pseudoparaphyses and 1-septate ascospores. Phylogenetic analysis based on LSU,SSU, RBP1, RBP2 and EF-1 sequences concluded that astrain named Phaeodothis winteri (a synonym ofP. tricuspidis Syd. & P. Syd.) clustered within the clade ofMontagnulaceae (Schoch et al. 2009). Currently 27 epithetsare listed for the genus in Index Fungorum (2014), whileGenBank has seven hits for the genus including the putativestrain of P. winteri (CBS 182.58). Based on the above mor-phological characters and available molecular data, we sug-gest that Phaeodothis can be referred toDidymosphaeriaceae.

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., composedof two layers, outer layer composed of irregular, thick-walled,brown cells of textura angularis. The inner layer composed ofhyaline, larger cells of textura angularis. Hamathecium denseof 2–2.5 μm cellular, septate long, hyaline pseudoparaphyses.Asci 120–170×24−30μm (x=150×26μm, n=20), 4–8-spored, bitunicate, clavate to broadly clavate, short pedicel-late, thin-walled, apically rounded with a broad ocular cham-ber. Ascospores 25–42×12−20μm (x=30×16μm, n=20),uniseriate or biseirate, ellipsoid to fusiform, light brown tobrown, muriform, constricted at central septum, with 2-layered 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, onsenescent 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 glo-bo s e , imme r s ed a s coma t a , nume rou s c e l l u l a rpseudoparaphyses, fissitunicate and clavate asci, ellipsoidmuriform ascospores, and a Phoma-like asexual stage(Kohlmeyer et al. 1995). Based on these morphological char-acters, Kohlmeyer et al. (1995) referred Tremateia toPleosporaceae. Furthermore, Kohlmeyer et al. (1995) sug-gested that Lewia M.E. Barr & E.G. Simmons andDiademosa Shoemaker & C.E. Babc., are superficially similar

to Tremateia. DNA sequences based phylogenies of recentliteratures, and also in the present study, have shown thatT. halophila nested in Montagnulaceae sister to Bimurianovae-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 mycologicaltaxonomy, 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; Schochet al. 2014; Thambugala et al. 2014; Wijayawardene et al.2014). The most significant and unsettled problem is that thephylogeny inferred from any gene may not reveal the evolu-tion history of the organism (Uilenberg et al. 2004). It istherefore better to incorporate a polyphasic taxonomy includ-ing genotypical and phenotypical characteristics in all futurestudies (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 ofDidymosphaeriaceae compared to morphological studies(Ariyawansa et al. 2014b). Genera with trabeculatepseudoparaphyses and 1-septate, brown ascospores vizBicrouania, Caryosporella, Lineolata, Phaeodothis,Roussoella and Verruculina, were generally classified underDidymosphaeriaceae. Molecular studies have shown that the-se particular morphological characters have evolved in differ-ent families (Ariyawansa et al. 2014b). The position to thetaxa treated in the present study, it is required to first ascertainthe phylogenetic placement of the fungus in theMontagnulaceae , as similar fungi occur in otherPleosporales as well; LSU can be used to verify the orderand mostly also the family and genus to which the fungusbelongs. ITS alone might be adequate for a preciseidentification of most species, as it is adequately variableamong most closely associated taxa in Montagnulaceae, butit fails to distinguish all species. Furthermore we used TUBgene for our combine gene phylogeny, which was proposedby Verkley et al. (2014) to resolve the molecular taxonomy oftaxa in Montagnulaceae.

By combining multi-locus DNA sequencing with detailedmorphological analyses, we were able to delimit and formallypropose one new species, one sexual morph and one newgenus among the taxa in the family Didymosphaeriaceae,previously classified in different families and genera in theorder Pleosporales . Importantly, we synonymiseMontagnulaceae under Didymosphaeriaceae by giving the


Fig. 19 Tremateia halophila (NY 01353617, isotype) a-b, d Verticalhand sections of ascomata. c, e Vertical hand section of ostiole andperidium. f-j Asci with muriform ascospores. k Cellular

pseudoparaphyses. l-q Fusiform, light brown to brown, muriform asco-spores. Scale bars: a-d=50μm, c, e=20μm, f-i, j-k=10μm, l-k=5μm


priority to the oldest name. The paraphyletic nature ofParaconiothyrium is partially resolved by classifying somespecies with their sexual states and combining both morphol-ogy and phy logeny. Because the b r anche s o fParaconiothyrium clade that conflict with its monophyly areinsufficiently supported, it was decided not to split it up intofurther genera. Morphological characters traditionally used todefine genera in coelomycetes include conidiomatal structure,structure of the conidiophores, conidiogenesis and conidialcharacters such as pigmentation, septal structure and number,and conidial appendages (Sutton 1980; Nag Raj 1993). Recentmolecular studies have shown that these features are notalways suitable in delimiting genera as natural entities, andthey may vary even between sibling species (Crous et al.2012). Generic boundaries drawn in the present study arebased primarily on statistically well-supported branches in amulti-locus phylogeny. Some of the characters mentionedabove are thus overlapping between the accepted genera.For example, phialidic and annellidic conidiogenesis occurboth in Paraconiothyrium and Paraphaeosphaeria (Verkleyet al. 2004, 2014; Damm et al. 2008). Recent studies based onmulti gene analysis have proven that the morphology of theConiothyrium-like scatter throughout the order Pleosporalesi.e. species with setose pycnidia and conidiogenesis withelongated conidiophores occur in other genera, such as,Cucurbitariaceae, Didymellaceae, Phaeosphaeriaceae,Leptosphaeriaceae, Montagnulaceae and Sporormiaceae, in-dicating convergent evolution (Gruyter et al. 2013).Neokalmusia was introduced in this study to accommodateKalmusia brevispora and K. scabrispora based on molecularanalysis in combination with morphology and a review of theliterature. By introducing the new genus Neokalmusia, wewere able to sort out the paraphyletic nature of Kalmusia inthe family Didymosphaeriaceae. Furthermore, Barria includ-ed in the family Didymosphaeriaceae, have been previouslyreferred to Phaeosphaeriaceae. Wilmia was synonymizedunder Letendraea based on the fresh collection of new speciesLetendraea cordylinicola (MFLU11-0184).

A confusing fact is that the putatively named strains ofMontagnula, M. spartii (CBS 183.58) and M. rhodophaea(CBS 616.86) obtained from GenBank, clustered inMassarinaceae and Lentitheciaceae, respectively. This is anexample of the problems of sequence data in GenBank andother public databases. The morphology and identification ofthe putative strains of M. spartii (CBS 183.58) andM. 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 forsupporting studies on Dothideomycetes. We are also grateful to theMushroom Research Foundation, Chiang Rai, Thailand for funding.Kevin D. Hyde thanks the Chinese Academy of Sciences, project number2013T2S0030, for the award of Visiting Professorship for Senior Inter-national Scientists at Kunming Institute of Botany. Jian-Chu Xu would

like to thank Humidtropics, a CGIAR Research Program that aims todevelop new opportunities for improved livelihoods in a sustainableenvironment, for partially funding this work. H.A Ariyawansa and J.C.Kang are grateful to the Agricultural Science and Technology FoundationOf Guizhou Province (Nos. NY[2013]3042), the International Collabo-ration Plan of Guizhou Province (No. G [2012]7006) and the innovationteam construction For Science and Technology Of Guizhou Province(No. [2012]4007) from the Science and Technology Department ofGuizhou Province, China. K. Tanaka would like to thank the JapanSociety for the Promotion of Science (JSPS, 25440199) for financialsupport. Gareth Jones is supported by the Distinguished Scientist Fellow-ship Program (DSFP), King Saud University, Saudi Arabia. HiranAriyawansa is grateful to A.D Ariyawansa, D.M.K AriyawansaDhanuska Udayanaga and Dimuth Manamgoda for their valuablesuggestions.

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http://dx.doi.org/10.1007/s13225-014-0303-8

http://dx.doi.org/10.1007/s13225-014-0297-2

http://dx.doi.org/10.1007/s13225-014-0297-2

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A molecular phylogenetic reappraisal of the Didymosphaeriaceae (= Montagnulaceae)

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