cryptogamie mycologie volume 36 n°1 2015 contents Asanka R. BANDARA, Sylvie RAPIOR, Darbhe J. BHAT, Pattana KAKUMYAN, Sunita CHAMYUANG, Jianchu XU & Kevin D. HYDE — Polyporus umbellatus, an Edible-Medicinal Cultivated Mushroom with Multiple Developed Health-Care Products as Food, Medicine and Cosmetics: a review. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-42 Marisa de CAMPOS-SANTANA, Gerardo ROBLEDO, Cony DECOCK & Rosa Mara BORGES DA SILVEIRA — Diversity of the poroid Hymenochaetaceae (Basidiomycota) from the Atlantic Forest and Pampa in Southern Brazil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43-78 Rosa Emilia PÉREZ-PÉREZ, Gonzalo CASTILLO-CAMPOS & Marcela Eugenia da Silva CÁCERES — Diversity of corticolous lichens in cloud forest remnants in La Cortadura, Coatepec, Veracruz, México in relation to phorophytes and habitat fragmentation. . . . . . . . . . . . . . . . . . . . . . . . . . 79-92 Wen-Jing LI, Sajeewa S. N. MAHARACHCHIKUMBURA, Qi-Rui LI, D. Jayarama BHAT, Erio CAMPORESI, Qing TIAN, Indunil C. SENANAYAKE, Dong-Qing DAI, Putarak CHOMNUNTI & Kevin D. HYDE — Epitypification of Broomella vitalbae and introduction of a novel species of Hyalotiella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93-108 Michel Navarro BENATTI, Márcia Isabel KÄFFER, Suzana Maria de Azevedo MARTINS, Alessandra Bittencourt de LEMOS — Bulbothrix bulbillosa, a presumed Galapagos endemic, is common in Rio Grande do Sul State, Brazil (Parmeliaceae, lichenized Ascomycota) . . . . . . . . . . . . . . 109-114 Instructions aux auteurs / Instructions to authors . . . . . . . . . . . . . . . . . . . . 115-118 Cryptogamie, Mycologie, 2015, 36 (1): 93-108 © 2015 Adac. Tous droits réservés Epitypification of Broomella vitalbae and introduction of a novel species of Hyalotiella Wen-Jing LIa,b,c,d, Sajeewa S. N. MAHARACHCHIKUMBURAc,d, Qi-Rui LIh, D. Jayarama BHATc,e, Erio CAMPORESIf, Qing TIANc,d, Indunil C. SENANAYAKEc,d, Dong-Qing DAIc,d, Putarak CHOMNUNTIc,d & Kevin D. HYDEa,b,c,d, g,* aWorld Agroforestry Centre, East and Central Asia, 132 Lanhei Road, Kunming 650201, China bKey Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Lanhei Road No 132, Panlong District, Kunming, Yunnan Province, 650201, PR China cInstitute dSchool of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand eFormerly, fA.M.B. gBotany Department of Botany, Goa University, Goa 403206, India Gruppo Micologico Forlivese “Antonio Cicognani”, Via Roma 18, Forlì, Italy and Microbiology Department, College of Science, King Saud University, Riyadh, KSA 11442, Saudi Arabia hThe Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of China, Guizhou University, Guiyang 550025, Guizhou Province, People’s Republic of China Abstract – Broomella and Hyalotiella are poorly known genera in Amphisphaeriaceae. Both genera are known from morphological descriptions, but lack molecular data, thus their generic placement and relationships with other genera are unclear. Three collections of Amphisphaeriaceae were made from dead twigs of Clematis in Italy, and two from Spartium species and were identified as Broomella and Hyalotiella species. In order to obtain a phylogenetic understanding of Broomella and Hyalotiella within the family Amphisphaeriaceae, we carried out a phylogenetic analysis based on LSU gene data. Results show that these five isolates represent two distinct genera. Based on both morphological and phylogenetic data, the three isolates form Clematis are shown to be conspecific with Broomella vitalbae. In this paper we designated an epitype with a sexual and asexual morph for B. vitalbae to stabilize the understanding of the genus. The two strains from Spartium fit within the generic concepts of Hyalotiella, but sequence data for Hyalotiella species are presently lacking. Hyalotiella spartii sp. nov. is introduced based on its host association and morphological characters. Asexual morphs / Amphisphaeriaceae / Broomella / Hyalotiella / Phylogeny / epitypification * Corresponding author: Kevin D. Hyde, email address: kdhyde3@gmail.com doi/10.7872/crym.v36.iss1.2015.93 94 W.-J. Li et al. INTRODUCTION The family Amphisphaeriaceae was introduced by Winter (1887) with Amphisphaeria Ces. & De Not. as the generic type (Kang et al., 1999; Maharachchikumbura et al., 2014). Amphisphaeriaceae is a large heterogeneous family, with approximately 57 genera (Maharachchikumbura et al., 2015) and apparently comprises several families. Asexual morphs of Amphisphaeriaceae are largely coelomycetous with appendaged conidia (Nag Raj, 1993; Jeewon et al., 2002; Liu et al., 2015). Several members of this group are plant pathogens on a broad range of hosts, and responsible for numerous diseases, including leaf-spots, canker lesions, fruit rot and twig dieback (Espinoza et al., 2008, Eken et al., 2009; Maharachchikumbura et al., 2014). They are also endophytes which colonizing plant tissues without causing visible symptoms, or can be saprobic on terrestrial plants (Jeewon et al., 2002; Tanaka et al., 2011; Qadri et al,. 2014) and some genera are chemically highly creative (e.g. Pestalotiopsis, Xu et al., 2014). The inter- and intrageneric relationships of most amphisphaeriaceous asexual morphs are problematic, due to their simplicity, plasticity and variability of morphological characters (i.e. conidial size, septation, pigmentation, and presence or absence of appendages) (Jeewon et al., 2002). The classification and circumscription of these genera have been controversial in the past decades (Steyaert, 1949; Guba, 1961; Sutton, 1980; Nag Raj, 1993). Steyaert (1949) introduced the genus Truncatella with five species to accommodate species having 3-septate conidia, which previously, belonged to Pestalotia (Nag Raj, 1993; Lee et al., 2006). Guba (1961) adopted a broader concept by synonymising Truncatella with Pestalotia. Sutton (1980) disagreed with the synonymy and reinstated the genus considering that the species defined by Guba (1961) in Pestalotia (sect Quadriloculatae) and Monochaetia (sect. Quadriloculatae) should be relocated to Truncatella (Sutton, 1980; Lee et al., 2006). Nag Raj (1993) agreed with Sutton (1980) and accepted ten species in Truncatella, while another two species, namely T. pampeana and T. suffocata, still remained in Pestalotiopsis (Nag Raj, 1993; Jeewon et al., 2002; Lee et al., 2006). Through a study of LSU and ITS sequence data, and together with existing morphological data, Jeewon et al. (2002) showed that species of Truncatella appear to be a natural group, and are distinct from other pestalotioid taxa. This was confirmed by Maharachchikumbura et al. (2014). A search of Index Fungorum (2015) reveals 21 names in Truncatella, although five have been transfered to other genera, many to Pestalotiopsis. Maharachchikumbura et al. (2014) showed that the broad generic concept of Pestalotiopsis of Nag Raj (1993) is problematic and 11 species which have 3-septate conidia should be relocated in Truncatella. The sexual morph of Truncatella is associated with Broomella (Kang et al., 1999). According to Shoemaker & Müller (1963) and Yuan et al. (1992) there are six species of Broomella linked to the asexual morph Truncatella. The type species B. vitalbae (Berk. & Broome) Sacc. and B. excelsa Shoemaker & E. Müll. have a conidial morph with single apical and basal appendage, and this does not fit well with the concepts of Truncatella. Whether the link between of Broomella species and Truncatella species is correct requires further assessment using molecular data. Papendorf (1967) introduced the genus Hyalotiella based on H. transvalensis Papendorf, which somewhat morphologically resembles Truncatella. Both share similar characters in having 4-celled conidia, more than one branched apical appendage, and lack of basal appendages. However, Epitypification of Broomella vitalbae and introduction of a novel species of Hyalotiella 95 Hyalotiella is further characterized by vase-shaped pycnidia, with a long neck, and 3-septate, cylindrical conidia, with median cells that are longer than the ends cells. With the exception of the apical cell, the other cells in the conidia are almost colourless to pale brown (Nag Raj, 1993). In addition, species of Hyalotiella have conidia bearing 3-4-branched appendages, which arise from the apical cell, and lack a basal appendage (Nag Raj, 1993). According to the Index Fungorum (2015) there are four names in Hyalotiella. However, H. subramanianii Agnihothr. & Luke was transferred to Hyalotiopsis by Nag Raj (1993) based on conidia characters and H. orientalis was synonymised under H. americana (Speg.) Nag Raj (Nag Raj 1993). Therefore, to date, only two species H. americana and H. transvalensis are accepted in Hyalotiella. However, none of these are presently known from culture or to have DNA sequence data. The aim of the present paper is to designate an epitype for the type species of Broomella, B. vitalbae, in order to stabilize the understanding of Broomella (Amphisphaeriaceae) with morphological and molecular characterization. In addition, a new species of Hyalotiella is introduced. MATERIAL AND METHODS Collection and examination of specimens Fresh specimens were collected in Italy from dead stems of Clematis vitalba L. and Spartium junceum L., dried and sent to Thailand for examination. Type specimen was borrowed from K (M). Samples were examined and pure cultures obtained by single spore isolation, following the method described in Chomnunti et al. (2014). The colonies were transferred to 2% potato-dextrose agar (PDA) and incubated at 25°C. The colony characters and growth rates were determined after one to four weeks. The pure cultures from our study are deposited at Mae Fah Luang University Culture Collection (MFLUCC). Duplicate cultures are deposited in International Collection of Microorganisms from Plants, Landcare Research, New Zealand (ICMP). The holotype is deposited at the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand, and the isotype specimens are deposited at the herbarium of Kunming institute of Botany Chinese Academy of Sciences (KUN). DNA extraction, PCR amplification and sequencing Isolates were grown on PDA plates in darkness at 25°C until completely covering the agar surface. The mycelium (about 50 mg) was scraped off and collected in a 1.5 ml micro centrifuge tube. Genomic DNA was extracted from fresh mycelium, following the specification of Biospin Fungus Genomic DNA Extraction Kit (BioFlux®). The primer pairs LROR and LR5 as defined by Vilgalys and Hester (1990) were used to amplify a segment of the large subunit rDNA. DNA amplification was performed by polymerase chain reaction (PCR). The sequencing of PCR products were carried on to Beijing Bai Mai Hui Kang Biological Engineering Technology Co. Ltd (Beijing, P. R. China). 96 W.-J. Li et al. DNA sequence data analysis The newly generated sequences were analyzed with sequences obtained from GenBank (Table 1). Sequences were aligned using Bioedit v. 7.0.9 (Hall, 1999) and Clustal X v. 1.83 (Thompson et al., 1997). The alignments were checked visually and improved manually where necessary. A maximum likelihood (ML) analysis was performed with raxmlGUI version 1.3 (Silvestro & Michalak, 2011). The optimal ML tree search was conducted with 1000 separate runs, using the default algorithm of the program from a random starting tree for each run. The final tree was selected among suboptimal trees from each run by comparing likelihood scores under the GTRGAMMA substitution model. The resulting trees were printed with TreeView v. 1.6.6 (Page, 1996). RESULTS Phylogenetic analyses Partial sequences of the large subunit rRNA (LSU) were used to resolve the generic placement of species of Amphisphaeriaceae. The alignment dataset is comprised of 50 taxa, with Rostrohypoxylon terebratum (CBS 119137) as the outgroup taxon. The maximum likelihood dataset consists of 795 characters. The best scoring RAxML tree generated from maximum likelihood analysis is shown in Figure 1. Sequence analysis of LSU data was used to link sexual and asexual morph of Broomella vitalbae and to resolve its placement within the family Amphisphaeriaceae. The three strains (strain MFLUCC 13-0798 for the sexual morph, strains MFLUCC 14-1000 and MFLUCC 15-0023 for the asexual morph) of Broomella formed a well-supported clade (BS=100%). Based on morphology and sequence data, our fresh collection (MFLU 15-0065) from Clematis vitalba is epitypified. Based on morphology and LSU sequence data, a new species Hyalotiella spartii, isolated from Spartium junceum, is also introduced. ITS (KP757756), SSU (KP757760) and EF1-α (KP757764) sequences data for this taxon have also been deposited in GenBank for the benefit of future study. TAXONOMY Broomella vitalbae (Berk. & Broome) Sacc., Syll. fung. (Abellini) 2: 558 (1883) Fig. 2-4 (1859) ≡ Hypocrea vitalbae Berk. & Broome, Ann. Mag. nat. Hist., Ser. 33: 363 Epitypification identifier: IF551046; Facesoffungi number: FoF 00589 Epitype: MFLU 15-0065, designated here. Epitypification of Broomella vitalbae and introduction of a novel species of Hyalotiella 97 Table 1. Collection details and GenBank accession number of isolates includes in this study. The newly generated sequences are indicated with an asterisk. T signifies ex-type/ex-epitype isolates Species Culture accession No. Host/Substrate Location GenBank Accession LSU Adisciso tricellulare NBRC 32705T T Rhododendron indicum Japan AB593728 Symplocos prunifolia Japan AB593721 Adisciso yakushimense MAFF 242774 Broomella vitalbae MFLUCC 13–0798T Clematis vitalba Italy KP757749* Broomella vitalbae MFLUCC 14–1000 Clematis vitalba Italy KP757750* Broomella vitalbae MFLUCC 15–0023 Clematis vitalba Italy KP757751* Amphisphaeria umbrina HKUCC 994 Tilia sp. Switzerland AF452029 Bartalinia bischofiae HKUCC 6534 Unidentified dead leaf Hong Kong AF382367 Bartalinia laurina HKUCC 6537 Unidentified dead leaf Hong Kong AF382369 Ciliochorella castaneae HHUF 28799 – Japan AB433277 Discosia artocreas NBRC 8975 Poa pratensis – AB593705 Discosia pini MAFF 410149 Pinus densiflora Japan AB593708 Discosia sp. MAFF 238070 Fallopia japonica Japan AB593720 Discostroma fuscellum NBRC 32680 Ribes sp. – AB593739 Rosa canina – AB593726 T Discostroma fuscellum NBRC 32625 Discostroma tostum NBRC 32626 – – AB593727 Dyrithiopsis lakefuxianensis HKUCC 7303 – – AF452047 Ellurema indica ATCC 22062 – – MIU43478 Spartium junceum Italy KP757752* Spartium junceum Italy KP757753* Immersidiscosia eucalypti MAFF 242781 Decayed leaves Japan AB593725 Immersidiscosia eucalypti NBRC 104195 Quercus myrsinifolia Japan AB593722 Hyalotiella spartii MFLUCC 13–0397 Hyalotiella spartii MFLUCC 15–0024 T Lepteutypa cupressi IMI 052255 Cupressus forbesii Kenya AF382379 Monochaetia kansensis PSHI2004Endo1031 – China DQ534036 Monochaetia kansensis PSHI2004Endo1030 – China DQ534035 Monochaetia kansensis PSHI2004Endo1032 – China DQ534037 Neopestalotiopsis formicarum CBS 115.83 Plant debris Cuba KM116255 Neopestalotiopsis formicarum CBS 362.72T Dead Formicidae Ghana KM116248 98 W.-J. Li et al. Table 1. Collection details and GenBank accession number of isolates includes in this study. The newly generated sequences are indicated with an asterisk. T signifies ex-type/ex-epitype isolates (continued) Species Culture accession No. Host/Substrate Location GenBank Accession LSU T Neopestalotiopsis protearum CBS 114178 Neopestalotiopsis rosae CBS 101057T Rosa sp. New Zealand KM116245 Pestalotiopsis knightiae CBS 114138 Knightia sp. New Zealand KM116227 Macaranga triloba Malaysia KM116238 Protea neriifolia × susannae Australia KM116219 Cocos nucifera Indonesia KM116276 Camellia sinensis Thailand KM116282 Pestalotiopsis malayana CBS 102220 Pestalotiopsis telopeae CBS 114137 Leucospermum cuneiforme Zimbabwe T Pseudopestalotiopsis cocos CBS 272.29T Pseudopestalotiopsis theae MFLUCC 12–0055 T JN712564 Robillarda sessilis BCC13393 Eucalyptus camaldulensis Thailand FJ825378 Rostrohypoxylon terebratum CBS 119137 Lithocarpus sp. Thailand DQ840069 Seimatosporium elegans NBRC 32674 Melaleuca ericifolia Seimatosporium eucalypti CBS 115131 AB593733 Eucalyptus smithii South Africa JN871209 Seimatosporium glandigenum NBRC 32677 Fagus sylvatica – AB593735 Seimatosporium hypericinum NBRC 32647 Hypericum sp. – AB593737 Seiridium cardinale CBS 172.56 – – AF382376 Seiridium cardinale ICMP 7323 Cupressocyparis leylandii New Zealand AF382377 Seiridium papillatum CBS 340.97 – – DQ414531 Seiridium phylicae CPC 19970 Phylica arborea United Kingdom KC005810 Seiridium phylicae CPC 19965 Phylica arborea United Kingdom KC005809 Truncatella angustata ICMP 7062 Malus X domestica New Zealand AF382383 Truncatella hartigii CBS118148 Restio egregius South Africa DQ278928 Truncatella laurocerasi ICMP 11214 Prunus persica New Zealand AF382385 Truncatella restionacearumCMW 18755T Ischyrolepis cf. gaudichaudiana South Africa DQ278929 Truncatella sp. HKUCC 7987 Leucospermum sp. South Africa AF382382 Zetiasplozna acaciae CPC 23421 Acacia melanoxylon – KJ869206 Epitypification of Broomella vitalbae and introduction of a novel species of Hyalotiella 99 Fig. 1. Best scoring RAxML tree of Amphisphaeriaceae strains obtained from dataset of LSU sequence alignment. RAxML bootstrap support values (equal to or greater than 50% based on 1.000 replicates) are shown at the nodes. The ex-types (ex-epitype strain) are in bold; the new isolates are in blue. The tree is rooted to Rostrohypoxylon terebratum CBS 119137. 100 W.-J. Li et al. Fig. 2. Broomella vitalbae (MFLU 15-0065, epitype) a-b. Black ascomata on the host. c-d. Vertical section of ascomata. e, g. Section of peridium. f. Ostiole. h. Germinating spore. i. Paraphyses. j-l. Asci with ascospores. m-r. Ascospores. Scale bars: a = 500 µm, b-c = 200 cm, d-e = 100 µm, f-g = 20 µm, h-r =10 µm. Saprobic on the dead stem of Clematis viltalba forming conspicuous rounded, black dots, ascomata initially immersed between the vascular stands of the host stem and later becoming partly exposed on twig-surface when the bark shreds out. Sexual morph: Ascomata 300-550 µm high, 250-300 µm diam., solitary to gregarious, uniloculate, glabrous, globose to subglobose, papillate. Ostiole Epitypification of Broomella vitalbae and introduction of a novel species of Hyalotiella 101 Fig. 3. Broomella vitalbae (MFUL 15-0054) a-b. Black coniodiomata on the host. c. Section of peridium. d. Vertical section of conidiomata. e. Developing conidia are growing in conidiaomata. f, g. Conidiophores, conidiogenous cells and developing conidia. h-j. Conidia. k. Germinating spore. l-m. Culture on PDA. Scale bars: a= 200 µm, b = 500 µm, c = 50 µm, d = 100 µm, e = 50 µm, f-k = 10 µm, l-m = 25 mm. 60-90 µm long, 40-80 µm wide, central located, composed of longitudinally aligned cells, and internally lined with hyaline periphyses. Peridium 20-80 µm wide, composed of light yellow, thick-walled cells of textura prismatica in the upper part and surrounding the ostiole, thin-walled, hyaline to pale brown in the most part. Hamathecium comprising numerous, 3-6 µm wide, cylindrical, hypha-like, septate, paraphyses, tapering towards the ends. Asci 100-160 × 10-20 µm (› = 120 × 15 µm; n = 30), 8-spored, unitunicate, cylindrical to cylindrical-clavate, pedicellate, apically rounded, with a J-, apical area (ring). Ascospores 30-40 × 6.5-10 µm 102 W.-J. Li et al. Fig. 4. Broomella vitalbae (holotype) a. Herbarium package. b. Herbarium material. c. Ascomata on the surface of host. d. Section of ascoma. e. Peridium. f, g. Ascus apex with a J- subapical ring. h-k. Asci with ascospores (j, k stained in Melzer’s). l-n. Ascospores. Scale bars: b = 5 mm, c = 200 µm, d = 20 µm, e = 10 µm, f, g = 5 µm, h-n = 10 µm. Epitypification of Broomella vitalbae and introduction of a novel species of Hyalotiella 103 (› = 35 × 8 µm; n = 50), biseriate or overlapping tri-seriate, fusiform, glabrous, straight or inequilaterally curved, 3-septate, constricted at the septa, thick walled, pale greyish brown, with doliform median cells, with conic, yellowish to pale grayish brown ends cells, each bearing 10-20 µm long, unbranched, terminal appendage. Asexual morph: Coelomycetous. Conidiomata 300-400 µm high, 350-410 µm diam., stromatic, pycnidioid, scattered to gregarious, immersed to semi-immersed, rounded, oval or elongated in outline, black, unilocular, papilate, glabrous. Peridium 40-60 µm wide, composed of 8-10-cell layers, with thick-walled cells of textura globulosa to textura angularis, pale brown to brown in the outer layers, merging with relatively thin-walled and colourless cells in the inner layers. Conidiophores 10-15 wide, arising all around the cavity of conidioma, short, branched, septate, hyaline, cylindrical. Conidiogenous cells 10-15 µm wide, integrated, cylindrical, long, phialidic, percurrently proliferating 1-2-times, hyaline, smooth. Conidia 35-45 × 4-9 µm (› = 45 × 6.5 µm; n = 50), fusiform to aciculate, with acute ends, 3-septate, with single apical and basal appendages; conidial cells unequal in size; basal cell obconic, pale brown, with 2 doliform median cells, verruculose, thick walled, brown, constricted at septa, with conic apical cell, thin-walled, pale brown; with an 6-15 µm long tubular, unbranched, filiform, flexuous, hyaline appendage. Culture characteristics: Colonies fast growing on PDA, reaching 40 mm diam. after one week at 20-25with crenate edge, whitened to pale pink, flattened, felt-like, with filamentous, dense, aerial mycelium on the surface, reverse similar in colour. Material examined: UK, England, Batheaston, on dead stems of Clematis vitalba, 15 January 1859, C.E. Broome & M.J. Berkeley, (K(M) 190800, holotype); ITALY. Province of Forlì-Cesena [FC], Modigliana, Montebello, on dead stem of Clematis vitalba, 23 February 2013, Erio Camporesi, IT-1079 (MFLU 15-0065, epitype designated here); ex-type living culture, MFLUCC 13-0798, ICMP; ITALY. Province of Arezzo [AR], Montemezzano, on dead stem of Clematis vitalba, 25 August 2013, Erio Camporesi, IT-1430 (MFLU 15-0054); living culture, MFLUCC 14-1000, ICMP; ibid. IT-1430B (MFLU 15-0064); living culture, MFLUCC 15-0023, ICMP. Notes: Broomella was introduced by Saccardo (1883) and typified by B. vitalbae (Berk. & Broome) Sacc. It was later placed in Amphisphaeriaceae (Kang et al., 1999). The genus is characterized by unitunicate, cylindrical-elongate asci, with a J-, discoid ring at the apex, and ellipsoid-fusiform, straight or inequilaterally curved, 3-septate ascospores with two brown median cells, lighter terminal cells, and single, centric appendages arising from the ends (Shoemaker & Müller, 1963). Broomella has been shown to be linked to pestalotiod-like asexual morph (Shoemaker et al., 1989; Yuan et al., 1992; Kang et al., 1999). Shoemaker & Müller (1963) introduced two new species in Broomella, namely B. acuta Shoemaker & E. Müll. and B. excelsa Shoemaker & E. Müll. Subsequently, the genus was expanded to include three more species, viz. B. montaniensis (Ellis & Everh.) E. Müll. & S. Ahmad, B. tianshanica Z.Q. Yuan & Z.Y. Zhao and B. verrucosa Shoemaker et al.. Presently, there are 20 species epithets listed under Broomella in Index Fungorum (2015), however, none of these are studied using DNA sequence data. The Broomella species and their truncatella-like asexual morphs differ in various ways from the type species of Broomella and its asexual morph and are probably not congeneric. Broomella vitalbae was originally collected on a dead stem of Clematis vitalba L. in Batheaston (England). We re-examined the type specimen, and 104 W.-J. Li et al. Table 2. Comparison of demension of ascomata, asci and ascospores of type Broomella vitalbae. Location Ascomata size (µm) Asci size (µm) Ascoapore size (µm) Reference Batheaston 160-210 µm diam., 170-230 µm high 98.5-133.5 µm long × 10-14 µm wide 26-32 µm long × 5-8 µm wide From this study Italy 250-300 µm diam., 300-550 µm high 100-160 µm long × 10-20 wide 30-40 µm long × 6.5-10 µm wide From this study 85-120 µm long × 8-12 µm wide 20-30 µm long × 4-5 µm wide Shoemaker & Müller 1963 South France 350-400 µm diam., 200-250 µm high compared it morphologically with our collection (MFLU 15-0065). Our collection (MFLU 15-0065) largely resembles the B. vitalbae in form of ascomata, asci and ascospores, and the only distinguishing character is the dimension of the asci and ascospores. It should be noted that this character may not be very reliable in B. vitalbae. The three specimens of B. vitalbae collected from different areas (i.e. Batheaston, south France and Italy) show different size in asci and ascospores (Table 2). In addition, the asexual morph (i.e. conidiomata structure, the number of apical appendage of conidia) is also used as criteria to distinguish the species in Broomella (Shoemaker et al., 1989; Yuan et al., 1992). According to Shoemaker & Müller (1963), the asexual morph of B. vitalbae is characterized by fusiform, 3-septate conidia with two doliform, verruculose, thick walled, brown median cells, and hyaline end cells, with one simple appendage arising from the ends, and cylindrical to subcylindrical, annelidic conidiogenous cells (Shoemaker & Müller, 1963). A comparison of morphological characters of the asexual morph of collection (MFLU 15-0054) with previously known species of Broomella, shows that the description of collection (MFLU 15-0054) is in accordance with the concept of B. vitalbae (Shoemaker & Müller 1963; Yuan et al. 1992). The phylogeny of the Amphisphaeriaceae is reconstructed based on sequence data from the LSU gene, showing that strain (MFULCC 13-0798), strain (MFLUCC 14-1000) and strain (MFLUCC 15-0023) form a distinct clade with high support (100%), and is sister to the H. spartii, Truncatella angustata (Pers.) S. Hughes and T. laurocerasi (Westend.) Steyaert (Fig.1). Based on molecular data coupled with morphological information, we confirm that the strain MFULCC 13-0798, MFLUCC 14-1000 and MFLUCC 15-0023 are conspecific with B. vitalbae, but they are the asexual morph. Because of the lack of detailed cultures and DNA sequence data from the type specimen, we therefore use our collection to epitypify Broomella vitalbae. Furthermore, the sexual and asexual morphs Broomella vitalbae are described and illustrated. Hyalotiella spartii W.J. Li, Camporesi & K.D. Hyde, sp. nov. Index Fungorum number: IF551047 Facesoffunginumber: FoF00590, Fig. 5. Etymology: Named after the host genus, Spartium, from which the species was isolated. Holotype: MFLU 15-0055 Saprobic on dead stems of Spartium junceum, forming conspicuous rounded, black conidiomata. Sexual morph: Undetermined. Asexual morph: coelomycetous. Conidiomata 250-300 µm high, 200-250 µm diam., stromatic, pycnidial, vase-shaped, scattered to gregarious, epidermal to subepidermal in origin, globose to subglobose, semi-immersed, unilocular with the locule occasionally convoluted or irregularly divided, glabrous, brown to dark brown, Epitypification of Broomella vitalbae and introduction of a novel species of Hyalotiella 105 Fig. 5. Hyalotiella spartii (MFLU 15-0055, holotype). a. Material specimen. b-c. Black coniodiomata on the host. d. Vertical section of conidioma. e. Ostiole. f, h-j. Conidiogenous cells and developing conidia. g. Section of peridium. k-l. Conidia. m. Germinating spore. n. Culture on PDA. Scale bars: c = 200 µm. d = 100 µm, e = 50 µm, f, h-j = 10 µm, g = 10 µm, k-l = 10 µm, n = 25 mm. ostiolate. Ostiole lageniform, single, centrally located, with a well-developed neck, thick-walled. Peridium 30-50 µm wide, composed of 7-8-cell layers, with thickwalled cells of textura angularis, brown to dark brown. Conidiophores arising all around the cavity of the conidioma, short, often reduced to conidiogenous cells. Conidiogenous cells 10-15 µm wide, integrated, cylindrical, long, phialidic, hyaline, 106 W.-J. Li et al. smooth. Conidia 25-28 × 3-4 µm (› = 26 × 3.5 µm; n = 20), fusiform, 3-septate, bearing apical appendages; basal cell obconic with an obtuse base, almost colourless, with 2 cylindrical to subcylindrical, thick-walled, yellowish to brown, median cells constricted at septa, with conic, thin-walled, hyaline apical cell; apical appendages 14-20 µm long, tubular, acellular, often irregularly or dischotomously branched at the base, filiform, flexuous, hyaline. Culture characteristics: Colonies slow growing on PDA, reaching 15 mm diam. after 2 weeks at 20-25 with circular, whitened to pale yellow, dense, aerial mycelium on the surface, reverse similar in colour. Material examined: ITALY. Province of Forlì-Cesena [FC], Santa Sofia, Collina di Pondo, on dead stem of Spartium junceum, 16 October 2012, Erio Camporesi, IT-812 (MFLU 15-0055 holotype); ex-type living culture, MFLUCC 13-0397, ICMP 20788; ibid. IT-812B (MFLU 15–0066); living culture, MFLUCC 15-0024, ICMP. Notes: Hyalotiella is a poorly known genus both in morphology and phylogeny. The genus comprises 2 species, viz. H. americana and H. transvalensis, and both taxa lack sequence data. In the present phylogenetic study, both strains of H. spartii formed a distinct branch (Fig. 1). Comparative morphological study of H. spartii with other genera of coelomycetes showed that H. spartii fits well within the generic concept of Hyalotiella (Nag Raj, 1993). Hyalotiella spartii is similar to H. transvalensis, type species of the genus, in its conidiogenous cells and conidia, but can be easily distinguished in dimension and shape of conidiomata (Nag Raj 1993). Hyalotiella spartii has vase-shaped, ostiolate conidiomata that are smaller than those in H. transvalensis which has globose to cupulate, and irregularly-lobed conidiomata. In addition, H. spartii is similar to H. americana in having vase-shaped conidiomata and ampulliform to cylindrical conidiogenous cells, as well as cylindrical to fusiform conidia. However, recognizable differences between those two species can to be observed in the septation and number of conidial appendages. Hyalotiella spartii has 3-septate conidia, bearing 5-6-branched appendages, whereas H. americana has 3-septate conidia (occasionally 4-septate) with 2-4, mostly 3, branched appendages. Based on distinct morphology, H. spartii is introduced as a new species in Hyalotiella. DISCUSSION Epitypification is necessary to fix taxonomic problems and to stabilize the understanding of species, genera, families or orders (Hyde et al., 2008; Ariyawansa et al., 2014; Boonmee et al., 2014). Maharachchikumbura et al. (2012) epitypified three Pestalotiopsis species (P. adusta (Ellis & Everh.) Steyaert, P. clavispora (G.F. Atk.) Steyaert and P. foedans (Sacc. & Ellis) Steyaert), and this helped to resolve the natural classification within the genus. In this study, we use morphology coupled with sequence data from fresh collections, as well as sequence data downloaded from GenBank to place Broomella in the family Amphisphaeriaceae and link the sexual and asexual morph of Broomella. By designating an epitype with molecular data, we are able to confirm the placement of Broomella, so that related species and asexual morphs can be placed in this genus in future studies. Nevertheless, the classification of many genera in Amphisphaeriaceae is problematic. For example, our phylogenetic analysis (Fig. 1) suggests that there may be two distinct clades for Truncatella. Unfortunately, the ex-type culture of this genus (T. angustata) is unavailable, and Epitypification of Broomella vitalbae and introduction of a novel species of Hyalotiella 107 therefore it is difficult to recognize the type lineage of Truncatella. Thus recollecting material from type localities and isolating the organism into a pure culture are essential in order to provide further taxonomy and phylogeny studies of Truncatella. Acknowledgments. We would like to thank Humidtropics, a CGIAR Research Program that aims to develop new opportunities for improved livelihoods in a sustainable environment, for partially funding this work. KD Hyde thanks The Chinese Academy of Sciences, project number 2013T2S0030, for the award of Visiting Professorship for Senior International Scientists at Kunming Institute of Botany. 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