Mycol Progress DOI 10.1007/s11557-017-1292-2

ORIGINAL ARTICLE

Molecular taxonomy and morphological characterization reveal new species and new host records of Torula species (Torulaceae, Pleosporales) Jun-Fu Li 1,2,3,4 & Rungtiwa Phookamsak 1,2,3 & Rajesh Jeewon 5 & Darbhe J. Bhat 6 & Ausana Mapook 3,4 & Erio Camporesi 7 & Qiu-Ju Shang 1,2,3,4 & Ekachai Chukeatirote 4 & Ali H. Bahkali 8 & Kevin D. Hyde 1,2,3,8

Received: 19 November 2016 / Revised: 5 March 2017 / Accepted: 7 March 2017 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2017

Abstract Four new species and two new host records of Torula (Torulaceae, Pleosporales) are described and illustrated from herbaceous litter collected in Italy and Thailand. The new species possess colony, conidiophore and conidial characteristics that fit within the generic concept of Torula. Detailed morphological observations clearly demarcate four of these from extant species and are hence described as new (Torula chiangmaiensis sp. nov., Torula pluriseptata sp. nov., Torula chromolaenae sp. nov., Section editor: Marc Stadler This article is part of the “Special Issue in honour of the 70th birthday of Dr. Eric McKenzie” * Ali H. Bahkali [email protected] * Kevin D. Hyde [email protected] 1

Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China

2

World Agroforestry Centre, East and Central Asia, 132 Lanhei Road, Kunming 650201, People’s Republic of China

3

Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand

4

School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand

5

Department of Health Sciences, Faculty of Science, University of Mauritius, Reduit, Mauritius

6

Department of Botany, Goa University, No. 128/1-J, Azad Housing Society, Curca, P.O. Goa, Velha 403108, India

7

A.M.B. Gruppo Micologico Forlivese “Antonio Cicognani”, Via Roma, 18 Forlì, Italy

8

Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box: 2455, Riyadh 1145, Saudi Arabia

Torula mackenziei sp. nov.). Details of asexual morphs are described, and justifications for establishing these new species are provided. The nuclear are sequenced ribosomal RNA genes as well as protein coding genes to infer phylogenetic relationships and discuss phylogenetic affinities with morphologically similar species. Our morphological distinction is further supported by phylogenetic discrimination. In particular, phylogenies depict a close relationship of Torula chiangmaiensis and T. pluriseptata to T. hollandica, while T. chromolaenae and T. mackenziei constitute an independent phylogenetic lineage basal to T. herbarum and T. ficus. Torula ficus and T. masonii are also described and their phylogeny investigated as new host records from Bidens pilosa and Iris germanica, respectively. Keywords Asexual fungi . Dothideomycetes . Hyphomycetous . Multigene phylogeny

Introduction The family Torulaceae, first introduced by Corda (Sturm 1829), is known only by asexual characters of its erect micro- or macronematous conidiophores, with or without apical branches, and doliiform to ellipsoid or clavate, brown, smooth to verruculose, mono- to polyblastic, often cupulate conidiogenous cells and subcylindrical, phragmosporous, acrogenous, brown, dry, smooth to verrucose conidia, characteristically produced in branched chains (Crous et al. 2015; Su et al. 2016; Hyde et al. 2016). Catenate conidia or branched conidial chains are formed mostly by the terminal cell and sometimes even other cells of the conidia becoming fertile and producing newer conidia in chains (Ellis 1971; Crous et al. 2015). Crous et al. (2015) investigated phylogenetic relationships of the Torulaceae and provided DNA sequence data for four Torula species, and accepted

Mycol Progress

Dendryphion and Torula in Torulaceae within the order Pleosporales. Further taxonomic work by Su et al. (2016) introduced Neotorula and two new Dendryphion species, and Li et al. (2016) reported Sporidesmioides within Torulaceae. C u r r e n t l y, To r u l a , D en d r y p hi o n , N e o t o r ul a a n d Sporidesmioides are accommodated in Torulaceae (Su et al. 2016; Hyde et al. 2016; Li et al. 2016). Torula, typified by T. herbarum (Pers.) Link is characterized by terminal or lateral, monoblastic or polyblastic conidiogenous cells which have a basally thickened and heavily melanized wall, with the apex thin-walled and frequently collapsing and becoming coronate (Crane and Miller 2016). Additionally, Crane and Schoknecht (1977) reported aspects of conidiogenesis among Torula species observed using light and transmission electron microscopy. These morphological characters used to segregate Torula were also observed by Mason (1941), Hughes (1953), Subramanian (1971), Ellis (1971, 1976) and Bhat (2010). However, there is little information regarding the phylogenetic relationships of Torula prior to the study by Crous et al. (2015), who reported two new species. This paper introduces four new species and two new host records within the genus Torula collected in Italy and Thailand, and investigates their phylogenetic relationships based on DNA sequence analyses from nucleotides and protein genes.

Material and methods Isolation and identification The specimens were collected from herbaceous litter in Italy and Thailand during the period from 2014 to 2016. Samples were returned to the laboratory for examination and description of morphological characters. The specimens were observed under a Motic SMZ168 Series dissecting stereo microscope. The conidial structures were collected using a surgical needle and transferred into 10% lactoglycerol on a clean slide and examined under a Nikon Eclipse 80i compound microscope and photo-captured with a Canon 600D digital camera using differential interference contrast (DIC) microscopy. The morphological structures were photographed with a ZEISS SteREO Discovery.V8 stereo-microscope fitted with a ZEISS Axiocam ERc 5s microscope camera. Tarosoft (R) Image Frame Work program and Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems Inc., USA) were used for measurements and photographic plate drawing. Single spore isolations were carried out to obtain pure cultures as described in Chomnunti et al. (2014). Germinating conidia were transferred aseptically to potato dextrose agar (PDA) and malt extract agar (MEA) and grown at 16 to 30 °C in alternating day and night light. Colony characters were observed and recorded after 1 week and at weekly intervals thereafter.

The type specimens were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand, and the Cryptogamic Herbarium, Kunming Institute of Botany Academia Sinica (HKAS), Yunnan, China. Ex-type living cultures are deposited in the Mae Fah Luang University Culture Collection (MFLUCC) and Kunming Institute of Botany Culture Collection (KUMCC). Faces of Fungi and MycoBank numbers are registered (Jayasiri et al. 2015; MycoBank 2016). New species status was justified following recommendations as outlined by Jeewon and Hyde (2016). DNA extraction, PCR amplification and sequencing Fungal mycelium was scraped off and transferred to a 1.5-mL microcentrifuge tube using a sterilized lancet for genomic DNA extraction. The Biospin Fungus Genomic DNA Extraction Kit (BSC14S1 (BioFlux®, China) was used to extract fungal genomic DNA, following the protocols in the manufacturer’s instructions. DNA amplification was performed by polymerase chain reaction (PCR) using the primer pairs as follows: LROR and LR5 (Vilgalys and Hester 1990) to amplify the partial ribosomal RNA for the nuclear large subunit (28S, LSU); NS1 and NS4 (White et al. 1990) to amplify the partial ribosomal RNA for the nuclear small subunit (18S, SSU); fRPB2-5F and fRPB2-7cR (Liu et al. 1999) to amplify the partial ribosomal RNA for the partial RNA polymerase second largest subunit (RPB2), and EF1-983F and EF1-2218R (Rehner 2001) to amplify the protein coding region for the translation elongation factor 1-alpha gene (TEF1-α). The final volume of the PCR reaction was 25 μl, containing 1 μl of DNA template, 1 μl of each forward and reward primer, 12.5 μl of 2×Power Taq PCR MasterMix and 9.5 μl of ddH2O. The PCR thermal cycling conditions for LSU, SSU and TEF1-α were as follows: initialization at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 50 s, elongation at 72 °C for 1 min, and a final extension at 72 °C for 10 min, followed by maintenance at 4 °C. The PCR thermal cycling program for RPB2 was processed by initialization at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C for 2 min, elongation at 72 °C for 90 s, and a final extension at 72 °C for 10 min. The PCR products were checked on 1% agarose gel electrophoresis stained with ethidium bromide. Purification and sequencing of the PCR products was carried out at Shanghai Majorbio Biopharm Technology Co., Ltd., China, using the same primers as described above. Sequence alignment and phylogenetic analyses Phylogenetic analyses were performed for single gene (LSU) as well as combined LSU, SSU and TEF1-α sequence data.

Mycol Progress

Sequences generated from this study were analysed using similar sequences obtained from GenBank and those derived from Tian et al. (2015), Ariyawansa et al. (2015) and Li et al. (2016) (Table 1), and analysed under different optimality criteria as outlined by Jeewon et al. (2002, 2003, 2013). The evolutionary model of nucleotide substitution for Bayesian analysis and maximum likelihood was selected independently for each locus using MrModeltest 2.2 (Nylander 2004). The sequence datasets were combined using BioEdit v.7.2.3 (Hall 1999). The single gene alignment was performed using MAFFT v.7 (Katoh and Standley 2013: http://mafft.cbrc.jp/alignment/server/) and manually aligned wherever necessary in MEGA version 6.0 (Tamura et al. 2013). Further analyses were performed using RAxML GUI v.0.9b2 (Stamatakis 2006, 2014; Stamatakis et al. 2008; Silvestro and Michalak 2010) with 1000 rapid bootstrap replicates using the GAMMAI model of nucleotide substitution. The best-scoring tree had final ML optimization likelihood of −24,926.710530. Bayesian an aly sis (BI) (Hu else nbe ck a nd R on qu ist 200 1; Huelsenbeck et al. 2001) was conducted with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001) to evaluate posterior probabilities (PP) (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002) by Markov chain Monte Carlo sampling (BMCMC). Six simultaneous Markov chains were run for one million generations, and trees were sampled every 100th generation and 10,000 trees were obtained. The first 2000 trees, representing the burn-in phase of the analyses, were discarded; the remaining 8000 trees were used for calculating posterior probabilities in the majority rule consensus tree (critical value for the topological convergence diagnostic is 0.01) (Cai et al. 2006). Bayesian posterior probabilities (BYPP) equal or greater than 0.90 are given below or above each node (Fig. 7). The phylograms were represented in TreeView (Page 1996) and drawn in Adobe/Microsoft PowerPoint and converted to jpeg files in Adobe Photoshop version CS5 (Adobe Systems Inc., USA). The new sequences were submitted to GenBank (Table 1). The alignment was deposited in TreeBASE (2016) under accession number 20535.

Results Taxonomy Torula pluriseptata J.F. Li, Phookamsak, Camporesi & K.D. Hyde, sp. nov. MycoBank number: MB 819534, Facesoffungi number: FOF 02709; Fig. 1. Etymology: Named after its multi-septate conidia. Holotype: MFLU 16–2814.

Saprobic on a dead aerial branch of Clematis vitalba Linn. (Ranunculaceae). Sexual morph: Undetermined. Asexual morph: Colonies effuse on host, black, powdery. Mycelium partly immersed, composed of septate, branched, smooth, pale brown hyphae. Conidiophores (2.8–)3–4.3 μm long × 2.5– 3 μm diam (x = 3.8 × 2.8 μm, n = 10), macronematous, mononematous, solitary, erect, pale brown, verruculose, thick-walled, consisting of 1–2 cells or reduced to conidiogenous cells, subcylindrical to subglobose, arising from prostrate hypha. Conidiogenous cells (3–)3.2–3.5 μm long × 3.8–4.6 μm diam (x = 3.4 × 4.1 μm, n = 10), polyblastic, terminal, dark brown to black, smooth to minutely verruculose, thick-walled, doliiform to ellipsoid. Conidia (14–)23.5–36(−55) μm long × (3.3–)3.6–4.4 μm wide (x = 30.5 × 4.1 μm, n = 20) solitary to catenate, acrogenous, simple, phragmosporous, dark brown, with apical cell pale brown, minutely verruculose, 3–10-septate, rounded at both ends, composed of subglobose cells, slightly constricted at some septa, chiefly subcylindrical. Conidial secession schizolytic. Cultural characteristics: Conidia germinating on PDA within 14 h and germ tubes produced from the tip cell. Colonies growing on PDA, reaching 5 cm in 10 days at 30 °C, mycelium partly immersed to superficial, slightly effuse, hairy, with regular edge, pale to medium brown; sexual or asexual spores not formed within 60 days. Material examined: ITALY, Forlì-Cesena [FC], Trivella di Predappio, on a dead aerial branch of Clematis vitalba Linn. (Ranunculaceae), 19 February 2014, Erio Camporesi, IT 1729 (MFLU 16–2814, holotype), ex-type living culture MFLUCC 14–0437, KUMCC 16–0034; (isotype in HKAS, under the code of HKAS 96294). Notes: Torula pluriseptata is distinct from the other species treated here. Torula pluriseptata resembles T. hollandica in having subglobose cells and catenate conidia (Crous et al. 2015), but differs in the number of septa (3–10-septate vs. 2–4-septate), slender conidia (23.5–36 × 3.6–4.4 μm vs. 13– 26 × 6–7 μm) and smaller conidiogenous cells (3.2–3.5 × 3.8– 4.6 μm vs. 6–7 × 6–7 μm). Torula chiangmaiensis J.F. Li, Phookamsak & K.D. Hyde, sp. nov. MycoBank number: MB 819535, Facesoffungi number: FOF 02710; Fig. 2. Etymology: Named after the location from which it was collected, Chiang Mai, Thailand. Holotype: MFLU 16–2815. Saprobic on a branch of dead herbaceous plant. Sexual morph: Undetermined. Asexual morph: Colonies effuse on host, black. Mycelium partly immersed to superficial on the substrate, composed of septate, branched, smooth, light brown hyphae. Conidiophores 8–12.6(−16.1) μm long × 4.5–5.2 μm wide (x = 11.8 × 4.9 μm, n = 20), macronematous, mononematous, solitary, erect, subhyaline to paler brown,

Mycol Progress Table 1

Taxa used in the phylogenetic analysis and their corresponding GenBank numbers

Species

Arthopyrenia salicis Arthopyrenia salicis Biatriospora mackinnonii Biatriospora mackinnonii Biatriospora marina Byssosphaeria salebrosa Dendryphion aquatic Dendryphion europaeum Dendryphion europaeum Dendryphion namum Dendryphion namum Dendryphion submersum Elongatopedicellata lignicola Herpotrichia macrotricha Hysterium angustatum Melanomma pulvis-pyrius Monotosporella tuberculata Neooccultibambusa chiangraiensis Neoroussoella bambusae Neotorula aquatic Neotorula submerse Nigrograna mackinnonii Occultibambusa bambusae Occultibambusa bambusae Occultibambusa fusispora Occultibambusa pustule Paradictyoarthrinium diffractum Paradictyoarthrinium diffractum Paradictyoarthrinium tectonicola Paradictyoarthrinium tectonicola Pleomassaria siparia Prosthemium stellar Roussoella angustior Roussoella chiangraina Roussoella hysterioides Roussoella intermedia Roussoella magnatum Roussoella neopustulans Roussoella nitidula Roussoella nitidula Roussoella scabrispora Roussoella siamensis Roussoella thailandica Roussoella pustulans Roussoellopsis macrospora Roussoellopsis tosaensis Seriascoma didymospora Seriascoma didymospora Sporidesmium australiense Sporidesmioides thailandica Sporidesmioides thailandica Torula chiangmaiensis Torula chromolaenae Torula ficus Torula ficus Torula ficus Torula herbarum Torula herbarum Torula hollandica Torula mackenziei

Culture collection

CBS 368.94 CBMAI1330 CBS 110022 CBS 674.75 CY 1228 SMH 2387 MFLUCC 15–0271 CPC 23231 CPC 22943 HKAS 84010 HKAS 84012 MFLUCC15–0271 MFLUCC 15–0642 GKM 196 N CBS 236.34 CBS 124080 CBS 256.84 MFLUCC 12–0584 MFLUCC 11–0124 MFLUCC 15–0342 HKAS 92660 E5202H MFLUCC 13–0855 MFLUCC 11–0394 MFLUCC 11–0127 MFLUCC 11–0502 MFLUCC12–0557 MFLUCC 13–0466 MFLUCC 12–0556 MFLUCC 13–0465 CBS 279.74 CBS 126964 MFLUCC 15–0186 MFLUCC 10–0556 CBS 125434 CBS170.96 MFLUCC 15–0185 MFLUCC 11–0609 MFLUCC 11–0182 MFLUCC 11–0634 MFLUCC 11–0624 MFLUCC 11–0149 MFLUCC 11–0621 KT 1709 MFLUCC 12–0005 KT1659 MFLUCC 11–0179 MFLUCC 11–0194 HKUCC 10833 MFLUCC 13–0840 KUMCC 16–0012 KUMCC 16–0039 KUMCC 16–0036 KUMCC 16–0035 KUMCC 16–0038 CBS 595.96 CBS 111855 CBS 379.58 CBS 220.69 MFLUCC 13–0839

GenBank accession number

References

LSU

SSU

TEF1-α

RPB2

AY779288 JN903536 GQ387614 GQ387613 GQ925848 GU385162 KU500573 KJ869202 KJ869203 KU500575 KU500574 KU500572 KX421368 GU385176 FJ161180 GU456323 GU301851 KU764699 KJ474839 KU500576 KX789217 KJ605422 KU863112 KU863113 KU863114 KU863115 KP744497 KP744498 KP744499 KP744500 DQ678078 AB553781 KT281979 KJ474840 AB524622 KF443382 KT281980 KJ474841 KJ474843 KJ474842 KJ474844 KJ474845 KJ474846 AB524623 KJ474847 AB524625 KU863116 KU863117 DQ408554 KX437757 KX437758 KY197856 KY197860 KY197858 KY197859 KF443385 KF443386 KF443383 KF443384 KY197861

AY538333 – GQ387553 GQ387552 GQ925835 – KU500580 – – KU500582 KU500581 KU500579 KX421369 – GU397359 GU456302 – KU712458 – KU500583 – – KU872116 KU872117 – KU872118 – KP753960 – KP753961 DQ678027 AB553650 – – AB524481 KF443390 – KU872122 – – – KU872125 – AB524482 KJ739608 AB524484 KU872119 KU872120 – KX437759 KX437760 KY197863 KY197867 KY197865 KY197866 KF443387 KF443391 KF443388 KF443389 KY197868

KF443404 – – – GU479848 GU327748 – – – AY383601 – – – GU327755 FJ161096 GU456265 GU349006 – KJ474848 – – – – – – – – – – – DQ677923 – – KJ474849 AB539115 – – KJ474850 KJ474852 KJ474851 KJ474853 KJ474854 – AB539116 KJ474855 AB539117 – – – KX437766 KX437767 KY197876 KY197880 KY197878 KY197879 KF443402 KF443403 KF443400 KF443401 KY197881

KF443397 – – – GU479823 – – – – – – – – – – GU456350 – – KJ474856 – – – KU940170 KU940171 KU940172 – KX437765 KX437764 – KX437763 DQ677976 – – KJ474857 AB539102 – – – KJ474859 KJ474858 KJ474860 KJ474861 – AB539103 KJ474862 AB539104 KU940173 KU940174 DQ435080 KX437761 KX437762 – KY197873 KY197871 KY197872 KF443395 KF443396 GU456362 KF443393 KY197874

Ahmed et al. 2014 Passarini et al. 2013 Ahmed et al. 2014 Suetrong et al. 2009 Mugambi and Huhndorf 2009 Su et al. 2016 Crous et al. 2014 Su et al. 2016

Ariyawansa et al. 2015 Mugambi and Huhndorf 2009 Boehm et al. 2009 Zhang et al. 2009 Schoch et al. 2009 Doilom et al. 2016 Liu et al. 2014 Su et al. 2016 Hyde et al. 2016 Shaw et al. 2015 Dai et al. 2016

Liu et al. 2015

Tanaka et al. 2010 Wohlbach et al. 2011 Ariyawansa et al. 2015 Liu et al. 2014 Ahmed et al. 2014 Ariyawansa et al. 2015 Liu et al. 2014

Tanaka et al. 2009 Liu et al. 2014 Tanaka et al. 2009 Dai et al. 2016 Shenoy et al. 2006 Li et al. 2016 In this study

Crous et al. 2015

In this study

Mycol Progress Table 1 (continued) Species

Torula masonii Torula pluriseptata Torula masonii Torula sp. Versicolorisporium triseptatum

Culture collection

KUMCC 16–0033 MFLUCC 14–0437 CBS245.57 CBS245.57 JCM 14775

GenBank accession number

References

LSU

SSU

TEF1-α

RPB2

KY197857 KY197855 KR873289 KR873290 AB330081

KY197864 KY197862 – – –

KY197877 KY197875 – – –

KY197870 KY197869 – – –

Crous et al. 2015 Hatakeyama et al. 2008

The newly generated sequences are indicated in underlined bold font, while the type strains are in black bold font CBS Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CBMAI Brazilian Collection of Environmental and Industrial Microorganisms, Campinas State University, Brazil; CMW Tree Pathology Co-operative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; CPC Collection of Pedro Crous housed at CBS; HKAS Cryptogamic Herbarium, Kunming Institute of Botany Academia Sinica (HKAS), Yunnan, China; HKUCC University of Hong Kong Culture Collection, Department of Ecology and Biodiversity, Hong Kong, China; JCM Japan Collection of Microorganisms, Japan; KUMCC Kunming Institute of Botany Culture Collection, Chinese Science Academy, Kunming, China; MFLUCC Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; GKM G.K. Mugambi; JK J. Kohlmeyer; KT K. Tanaka; SMH S.M. Huhndorf

smooth to minutely verruculose, thick-walled, consisting of 2–3 cells, without apical branches, with ampulliform cells, arising from prostrate hypha. Conidiogenous cells 3.4–

Fig. 1 Torula pluriseptata (MFLU 16–2814, holotype) a Colonies on dead branch of Clematis vitalba. b Mass of conidia. c Conidiophores with conidiogenous cell. d Budding on conidia. e–h, j–l Conidia. i Conidia in catenated chains. m Germinated conidia. Scale bars (a) 500 μm, (b) 10 μm, (c–m) 5 μm

6.5 μm long × 4.8–7.6 μm diam (x = 5.3 × 6.8 μm, n = 20), polyblastic, terminal, dark brown to black, smooth to minutely verruculose, thick-walled, ellipsoid to coronal. Conidia

Mycol Progress Fig. 2 Torula chiangmaiensis (MFLU 16–2815, holotype) a Colonies on branch of dead herbaceous plant. b Hyphae. c Conidia in mass. d–f Conidiophores with conidiogenous cells. g–h Immature conidia. i–n Conidia. Scale bars (a) 200 μm, (b) 20 μm, (c, k–n) 10 μm, (d–h) 5 μm, (i–j) 7 μm

(5.4–)25.5–70(−86.5) μm long × 5.6–7.8 μm diam (x = 59.6 × 6.6 μm, n = 20) solitary to catenate, acrogenous, phragmosporous, light brown to greyish-brown, smooth to minutely verruculose, 4–12-septate, composed of moniliform cells, slightly constricted at some septa, chiefly subcylindrical. Conidial secession schizolytic. Cultural characteristics: Conidia germinating on PDA within 14 h and germ tubes produced from terminal cells. Colonies growing on PDA, reaching 5 cm in 21 days at 30 °C, mycelium partly immersed to superficial, slightly effuse, hairy, with regular edge, maroon to yellowish-brown; sexual morph not formed within 60 days. Material examined: THAILAND, Chiang Mai, Mae Taeng (Mushroom Research Center), on a branch of dead herbaceous plant, 12 March 2016, Junfu Li, H-MRC45 (MFLU 16–2815, holotype), ex-type living culture KUMCC 16–0039, MFLUCC; (isotype in HKAS, under the code HKAS 96295). Notes: Torula chiangmaiensis described herein as a new species, resembles Bahusaganda ambrosiae in having conidia composed of moniliform cells and coronate conidiogenous cells (Crane and Miller 2015). However, conidia in Torula chiangmaiensis are catenate, with less septa and smaller (4–12septate, 25.5–70 × 5.6–7.8 μm vs. 6–15-septate, 55–110 × 8–

13 μm). Phylogenetic analyses support T. chiangmaiensis as a new species in Torula as well. Torula masonii P.W. Crous, IMA Fungus 6 (1): 192 (2015). MycoBank number: MB812806, Facesoffungi number: FOF 02711; Fig. 3. Saprobic on a dead aerial stem of Iris germanica Linn. (Iridaceae). Sexual morph: Undetermined. Asexual morph: Colonies effuse on host, black, powdery. Mycelium immersed on the substrate, composed of septate, branched, smooth, subhyaline hyphae. Conidiophores 7.6–20 μm long × 3.5– 5.4 μm wide (x = 16.8 × 4.5 μm, n = 20), macronematous, mononematous, solitary, straight to flexuous, subhyaline to paler brown, smooth to minutely verruculose, thick-walled, consisting of 2–3 cells, without apical branches, subcylindrical, arising from prostrate hypha. Conidiogenous cells 6.4–7.2 μm long × 4.8–6.6 μm diam (x = 6.6 × 5.3 μm, n = 20), polyblastic, terminal, dark brown to black, smooth to minutely verruculose, thick-walled, ellipsoid to coronate. Conidia 15.6–25.4 μm long × 6.8–11.5 μm wide (x = 21.6 × 9.5 μm, n = 20) catenated, acrogenous, phragmosporous, greyish-brown, smooth to minutely verruculose, 2–5-septate, often with a dark coronal

Mycol Progress Fig. 3 Torula masonii (MFLU 16–2816) a Colonies on dead branch of Iris germanica. b–c, d–f Conidiophores with conidiogenous cell. g–h Immature conidia. j–k Conidial budding. i, l–t Conidia. u Germinated conidia. Scale bars (a) 200 μm, (u) 20 μm, (b–t) 5 μm

conidiogenous cell at apex, chiefly subcylindrical. Conidial secession schizolytic. Cultural characteristics: Conidia germinating on PDA within 14 h and germ tubes produced from the apex. Colonies growing on PDA, reaching 5 cm in 15 days at 30 °C, mycelium partly immersed to superficial, slightly effuse, hairy, with regular edge, black; sexual morph not formed within 60 days. Material examined: ITALY, Forlì-Cesena [FC], Monte Cavallo-Meldola, on a dead aerial stem of Iris germanica Linn. (Iridaceae), 16 March 2016, Erio Camporesi, IT2888 (the re-examined specimens deposited in MFLU 16–2816; HKAS 96296), living culture KUMCC 16–0033, MFLUCC. Notes: Torula masonii was introduced by Crous et al. (2015) based on the fungus sporulating in culture which was collected from Brassica sp. in UK. In this study, we re-examined T. masonii on a dead aerial stem of Iris germanica from Italy. The study showed that T. masonii is also characterized by conidia having a dark terminal coronate cell which is conidiogenous. Torula ficus P.W. Crous, IMA Fungus 6 (1): 192 (2015). MycoBank number: MB812804, Facesoffungi number: FOF 02712; Fig. 4. Saprobic on a dead branch of Chromolaena odorata Linn. (Asteraceae). Sexual morph: Undetermined. Asexual morph:

Colonies effuse on host, black, powdery. Mycelium partly immersed to superficial on the substrate, composed of septate, branched, smooth, hyaline hyphae. Conidiophores 9.4– 12.5 μm long × 3.7–4.5 μm wide (x = 10.2 × 3.9 μm, n = 20), macronematous, mononematous, solitary, erect, paler brown to brown, smooth to distinctly verrucose, thick-walled, consisting of 1–2 cells, without apical branches, ampulliform, arising from hypha. Conidiogenous cells 5.4–8.5 μm long × 5– 7.4 μm wide (x = 6.7 × 5.6 μm, n = 20), polyblastic, terminal, dark brown to black, smooth to minutely verruculose, thickwalled, ellipsoid to coronal. Conidia 12–20 μm long × 4.6– 6.6 μm diam (x = 16.3 × 5.4 μm, n = 20) catenate, acrogenous, phragmosporous, light brown to greyish-brown, smooth to distinctly verrucose, 2–4-septate, rounded and paler coloured at apex, slightly constricted at some septa, chiefly subcylindrical. Conidial secession schizolytic. Cultural characteristics: Conidia germinating on PDA within 14 h and germ tubes produced from the apex. Colonies growing on PDA, reaching 5 cm in 10 days at 30 °C, mycelium partly immersed to superficial, slightly effuse, cottony, with regular edge, greyish-brown to brown; sexual morph not formed within 60 days. Material examined: THAILAND, Chiang Rai, Doi Pui, on a dead branch of Chromolaena odorata Linn. (Asteraceae),

Mycol Progress Fig. 4 Torula ficus (MFLU 16–2817) a Colonies on dead branch of Chromolaena odorata. b Conidiophores. c, f–i Budding on conidia. j–s Conidia. Scale bars (a) 200 μm, (c) 10 μm, (b, d–s) 5 μm

17 July 2015, Ausana Mapook, DP 59 (the re-examined specimens deposited in MFLU 16–2817, HKAS 96297), living culture KUMCC 16–0035, MFLUCC. Notes: Torula ficus, collected from Ficus in Europe, was reported by Crous et al. (2015) based on a sporulating culture. Torula ficus is morphologically similar to T. herbarum, but distinct as it has longer and wider conidia (Crous et al. 2015). In our study, we re-examine T. ficus which was collected from Chromolaena odorata in Thailand. This is a new record from this host as well as from a tropical region. Torula chromolaenae J.F. Li, Phookamsak, A. Mapook & K.D. Hyde, sp. nov. MycoBank number: MB 819536, Facesoffungi number: FOF 02713; Fig. 5. Etymology: Named after the host genus from which it was collected, Chromolaena. Holotype: MFLU 16–2819. Saprobic on a dead branch of Chromolaena odorata Linn. (Asteraceae). Sexual morph: Undetermined. Asexual morph: Colonies discrete on host, black, powdery. Mycelium immersed on the substrate, composed of septate, branched, smooth, light

brown hyphae. Conidiophores 5–6.3 μm long × 3.5–4.6 μm diam (x = 5.8 × 4 μm, n = 10), macronematous, mononematous, solitary, erect, light brown, verruculose, thick-walled, consisting of one cell, without apical branches, ellipsoid to subglobose, arising from hypha. Conidiogenous cells (3.5–)4.5–5.1 μm long × 4.8–5.6 μm wide (x = 4.7 × 5.4 μm, n = 10), polyblastic, terminal, light brown to brown, paler at apex, smooth to minutely verruculose, thickwalled, doliiform. Conidia 12.1–16.5 μm long × (3.6–)4.1– 5 μm wide (x = 14.5 × 4.3 μm, n = 20) catenated, acrogenous, simple, phragmosporous, light brown to brown, smooth to minutely verruculose, 2–3-septate, rounded at both ends, often paler at apex, slightly constricted at some septa, chiefly subcylindrical. Conidial secession schizolytic. Cultural characteristics: Conidia germinating on PDA within 14 h and germ tubes produced from the apex. Colonies growing on PDA, reaching 5 cm in 14 days at 30 °C, mycelium partly immersed to superficial, slightly effuse, cottony, with regular edge, greyish-white to grey; sexual morph not formed within 60 days. Material examined: THAILAND, Chiang Mai, Mae Taeng (Mushroom Research Center), on a dead branch of

Mycol Progress Fig. 5 Torula chromolaenae (MFLU 16–2819, holotype) a Colonies on dead branch of Chromolaena odorata. b, l Conidia in catenated chain. c, k Branched chains of conidia. d–h Conidiophores with conidiogenous cells. i–j Budding on conidia. m Conidiophores. n–p Conidia. q Germinated conidia. Scale bars (a) 500 μm, (q) 10 μm, (b–p) 5 μm

Chromolaena odorata (Asteraceae), 26 December 2015, Ausana Mapook, MRC1 (MFLU 16–2819, holotype), extype living culture KUMCC 16–0036, MFLUCC; (isotype in HKAS, under the code of HKAS 96299). Notes: Torula chromolaenae, described herein as a new species, is distinct from other species in having smaller and doliiform conidiogenous cells and 2–3-septate conidia. Torula mackenziei J.F. Li, Phookamsak & K.D. Hyde, sp. nov. MycoBank number: MB 819537, Facesoffungi number: FOF 02714; Fig. 6. Etymology: Named in honour of the mycologist Dr. E.H.C. McKenzie, in recognition of his contribution to the study of hyphomycetes. Holotype: MFLU 16–2820. Saprobic on dead branch of Bidens pilosa Linn. (Asteraceae). Sexual morph: Undetermined. Asexual morph: Colonies sporadic on host, black, powdery. Mycelium immersed on the substrate, composed of septate, branched, smooth, light brown hyphae. Conidiophores 3–4.3 μm long × 3.4–3.7 μm wide (x = 3.8 × 3.5 μm, n = 10), macronematous, mononematous, solitary, erect, light brown, minutely verruculose, thick-walled, consisting of 1–2 cells or reduced to conidiogenous cells, without apical branches, ellipsoid to subglobose, arising from hypha. Conidiogenous cells (3.6–)4.3–4.7 μm long × 4–4.8 μm wide (x = 4.5 × 4.5 μm, n = 10), polyblastic, terminal, dark brown to black, paler at apex, smooth to minutely verruculose, thick-

walled, globose to ellipsoid. Conidia 9.4–18.5 μm long × (3.8–)4.4–4.7 μm wide (x = 13.8 × 4.5 μm, n = 20) catenated, acrogenous, phragmosporous, light brown to greyish-brown, smooth to minutely verruculose, 2–3-septate, rounded at both ends, often paler at apex, composed of moniliform cells, slightly constricted at some septa, chiefly subcylindrical. Conidial secession schizolytic. Cultural characteristics: Conidia germinating on PDA within 14 h and germ tubes produced from the apex. Colonies growing on PDA, reaching 5 cm in 10 days at 30 °C, mycelium partly immersed to superficial, slightly effuse, cottony, with regular edge, white to grey; sexual morph not formed within 60 days. Material examined: THAILAND, Chiang Rai, Doi Mae Salong, on a dead branch of Bidens pilosa Linn. (Asteraceae), 29 September 2013, Junfu Li, H-2 (MFLU 16–2820, holotype), ex-type living culture MFLUCC 13–0839, KUMCC; (isotype in HKAS, under the code of HKAS 96300). Notes: Torula mackenziei is distinct from other Torula species in having greyish-brown conidia composed of moniliform cells and 2–3 septa. Phylogenetic analyses of combined LSU, SSU, TEF1-α and RPB2 DNA sequence data The combined LSU, SSU, TEF1-α and RPB2 dataset comprises 65 taxa, with Hysterium angustatum as the outgroup taxon. Bayesian inference (BI) and maximum likelihood

Mycol Progress Fig. 6 Torula mackenziei (MFLU 16–2820, holotype) a Colonies on dead branch of Bidens pilosa. b, l Conidia in catenated chain. c, k Branched chains of conidia. d–h Conidiophores with conidiogenous cells. i–j Immature conidia. m Conidiophores. n–q Conidia. Scale bars (a) 100 μm, (b–q) 5 μm

(ML) analyses of the combined dataset resulted in phylogenetic reconstructions with largely similar topologies, and the best-scoring RAxML tree is shown in Fig.7. Most of the core genera of Torulaceae (Crous et al. 2015) and Roussoellaceae (Liu et al. 2014) in Pleosporales (Wijayawardene et al. 2014) are included in our phylogenetic analysis (Fig. 7), and they are represented with well-supported clades. The new taxa cluster with relatively moderate support (≥ 50% ML, 0.90 PP) with other species of Torula within Torulaceae (Pleosporales, Dothideomycetes).

Discussion Torula is introduced here to represent the asexual genus of the ascomycete family Torulaceae within the order Pleosporales. The most recent taxonomic work on this genus is that of Crane and Miller (2015), who presented a generic key to differentiate Torula from seven allied hyphomycete genera. DNA sequence data were not available from Crane and Miller (2016), and DNA-based phylogenies are rather scarce among this genus. A recent intergeneric revision was provided by Crous et al. (2015), who investigated phylogenetic relationships of the Torulaceae and accepted Torula and Dendryphion within this

family in Pleosporales based on 28S rDNA sequence analysis. The latest DNA-based phylogenetic study on the Torulaceae was conducted by Su et al. (2016) and Li et al. (2016), who revisited the family and included four genera: Torula, Dendryphion, Neotorula and Sporidesmioides. During our fungal diversity studies in Italy and Thailand, we collected a few species with unusual morphologies from herbaceous plant litter. Close microscopic examination revealed that these species belonged to the genus Torula, and interestingly, four of the taxa could not be ascribed to any known taxa within Torula according to recent classification provided by Crous et al. (2015) and Crane and Miller (2016). We also note morphological differences among extant species, and hence four new species, viz. Torula chiangmaiensis sp. nov., T. pluriseptata sp. nov., T. chromolaenae sp. nov., and T. mackenziei sp. nov., as well as two new host records (T. masonii and T. ficus) are described and illustrated in this study. Our molecular phylogeny based on a concatenated dataset also provides further evidence to support our newly collected taxa as belonging to the genus Torula with high support (Fig. 7). To justify our new fungal species, we follow the recommendations as outlined by Jeewon and Hyde (2016). In particular, we note, despite morphological similarities, some remarkable

Mycol Progress Fig. 7 Phylogenetic construction using RAxML-based analysis of a combined LSU, SSU, TEF1-α and RPB2 dataset. Bootstrap support values for maximum likelihood (ML, black) equal to or greater than 50% and Bayesian posterior probabilities (PP, red) equal to or greater than 0.90 are shown above the nodes. The tree is rooted to Hysterium angustatum. The type strains are in black bold and the newly generated sequences are indicated in blue bold

differences that segregate our new taxa from existing ones. For instance, T. chromolaenae and T. mackenziei resemble T. herbarum, which is the type species having solitary to catenate, septate, brownish and round-ended conidia, mononematous conidiophores and polyblastic conidiogenous cells. However, T. chromolaenae is unique, as it is the only species characterized by relatively smaller and doliiform conidiogenous cells and commonly two-septate conidia (4.5– 5.1 μm long × 4.8–5.6 μm diam vs. 5–8 μm long × 6–8 μm diam), in contrast to already described species such as T. herbarum and T. ficus, which have predominantly 3–4-septate conidia and larger conidiogenous cells. Torula mackenziei can also be differentiated from T. herbarum, as the former possesses conidia composed mainly of moniliform cells, and smaller (9.4– 18.5 μm long × 4.4–4.7 μm diam vs. 16–18 μm long × 6–7 μm

diam) (Fig. 6, Table 2). Interestingly, molecular phylogeny generated here corroborates our morphologically based assumption that T. chromolaenae and T. mackenziei could be related to T. h e r b a r u m ( F i g . 7 ) . O u r p h y l o g r a m i n d i c a t e s T. chromolaenae and T. mackenziei as sister taxa to T. herbarum and T. ficus, but their phylogenetic distinctiveness is fully supported, as the two new taxa constitute an independent lineage with high statistical support. ITS pairwise sequence comparison also reveals some distinct nucleotide differences between the two new species. We note that there are 28 notable nucleotide differences among the 535 analysed nucleotides between T. chromolaenae and T. mackenziei. Our phylogeny also reveals that Torula pluriseptata and T. chiangmaiensis are related to T. hollandica, as these three species are positioned together in a well-supported subclade. However, T. pluriseptata and

3.4–6.5 × 4.8–7.6 μm, polyblastic, smooth to minutely verruculose, ellipsoid to coronal, dark brown to black. (3.5–)4.5–5.1 × 4.8–5.6 μm, polyblastic, smooth to minutely verruculose, doliiform, light brown to brown, paler at apex. 5.4–8.5 × 5–7.4 μm, polyblastic, smooth to minutely verruculose, ellipsoid to coronal, dark brown to black. 5–8 × 6–8 μm, mono- to polyblastic, verruculose at apex, doliiform to ellipsoid, brown 6–7 × 6–7 μm, mono- to polyblastic, verruculose, doliiform, brown.

(3.6–)4.3–4.7 × 4–4.8 μm, polyblastic, smooth to minutely verruculose, globose to ellipsoid, dark brown to black. 6.4–7.2× 4.8–6.6 μm, polyblastic, dark, smooth to minutely verruculose, ellipsoid to coronate, brown to black. (3–)3.2–3.5 × 3.8–4.6 μm, polyblastic, smooth to minutely verruculose, doliiform to ellipsoid, dark brown to black.

8–12.6(−16.1) × 4.5–5.2 μm, macronematous, mononematous, consisting of 2–3 ampulliform cells, subhyaline to paler brown.

5–6.3 × 3.5–4.6 μm, macronematous, mononematous, consisting of an ellipsoid cell, light brown.

9.4–12.5 × 3.7–4.5 μm, macronematous, mononematous, consisting of 1–2 ampulliform cells, paler brown to brown.

Reduced to conidiogenous cells, or with one supporting cell, mononematous, brown.

Reduced to conidiogenous cells.

3–4.3 × 3.4–3.7 μm, macronematous, mononematous, consisting of 1–2 ellipsoid cells or reduced to conidiogenous cells, light brown. 7.6–20 × 3.5–5.4 μm macronematous, mononematous, consisting of 2–3 cells, subcylindrical, subhyaline to paler brown.

(2.8–)3–4.3 × 2.5–3 μm, macronematous, mononematous, consisting of 1–2 subglobose cells or reduced to 1-celled conidiogenous cells, pale brown

Torula chiangmaiensis

Torula chromolaenae

Torula ficus

Torula herbarum

Torula hollandica

Torula mackenziei

Torula pluriseptata

Torula masonii

Conidiogenous cells

Conidiophores

Morphological differences among taxa in Torula

Species name

Table 2

Crous et al. 2015

(15–)16–24 × 6–7 μm, 3–4-septate, composed of subglobose cells, brown, apex pale brown. 13–26 × 6–7 μm, predominantly 4-septate, in branched chains, composed of subglobose cells, brown, apex pale brown, branching cell in conidial chain darker brown than other cells. 9.4–18.5 × (3.8–)4.4–4.7 μm, 2–3-septate, composed moniliform cells, rounded at both ends, catenated, light brown to greyish-brown, often paler at apex. 15.6–25.4 μm long × 6.8–11.5 μm wide, 2–5-septate, often with a dark coronal conidiogenous cell at apex, greyish-brown. (14–)23.5–36(−55) × (3.3–)3.6–4.4 μm, 3–10-septate, composed of subglobose cells, dark brown.

This study

Crous et al. 2015 In this study

This study

Crous et al. 2015

Crous et al. 2015 This study

This study

This study

Reference

12–20 × 4.6–6.6 μm, 2–4-septate, rounded and paler coloured at apex, light brown to greyish-brown.

12.1–16.5 × (3.6–)4.1–5 μm, 2–3-septate, rounded at both ends, catenated, light brown to brown, often paler at apex,

(25.5–)43.6–70(−86.5) × 5.6–7.8 μm, 4–12-septate, composed of moniliform cells, light brown to greyish-brown.

Conidia

Mycol Progress

Mycol Progress

T. chiangmaiensis are different in having smaller conidiogenous cells (3.2–3.5 × 3.8–4.6 μm, 3.4–6.5 × 4.8–7.6 μm vs 6–7 × 6– 7 μm) and larger conidia (23.5–36 × 3.6–4.4 μm, 43.6–70 (−86.5 × 5.6–7.8 μm vs 13–26 × 6–7 μm), and conidiophores are absent in T. hollandica (Table 2). Despite a close phylogenetic relatedness among these three species, sequence analyses indicate that they can be treated as three distinct taxa (Fig. 7). Across the ITS sequences, there are 15 notable nucleotide differences among the 528 analysed nucleotides between T. pluriseptata and T. chiangmaiensis, and these align with the recommendations of Jeewon and Hyde (2016) to establish new species. This study also reveals new host records for Torula ficus and T masonii. While they have previously been collected from host species Ficus religiosa (Cuba) and Brassica (UK), respectively, we recovered T. ficus from Chromolaena odorata, an herbaceous host (in Thailand), whereas T. masonii was collected from dead aerial stem of Iris germanica (in Italy). Another peculiar morphological observation noted herein is a characteristic dark corona cell at the apex among T. masonii, but no such detail has been described in Crous et al. (2015). Could this be a phylogenetic indicator of T. masonii, as our phylogeny supports its distinctiveness from all other species analysed? Acknowledgements We are grateful to the Mushroom Research Foundation and Thailand Research Fund (TRF: BRG5280002) for supporting this research, and for MFLU grant number 567110754 to support Hyphomycetes studies. Prof. Kevin D. Hyde thanks the Chinese Academy of Sciences (project no. 2013T2S0030) for the award of Visiting Professorship for Senior International Scientists at Kunming Institute of Botany. Dr. Rungtiwa Phookamsak expresses sincere appreciation for the CAS President’s International Fellowship for Postdoctoral Researchers, project number 2017 PB0072, for financial support. Ausana Mapook is grateful for financial support provided by Research and Researchers for Industries (RRI) PHD57I0012 under the Thailand Research Fund. Jun-Fu Li thanks Dr. Shaun Pennycook, Dhanushka Wanasinghe, Sirinapa Konta, Qing Tian, Dr. Mingkwan Doilom and Zonglong Luo for their assistance and valuable suggestions. Prof. Darbhe J. Bhat and Dr. Jeewon Rajesh would like to thank Mae Fah Luang University for the opportunities provided to them as visiting professors to the Center of Excellence in Fungal Research. The authors extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its Prolific Research Group funding (PRG1436-09).

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