MycoKeys 33: 25-67 (20 | 8) A peer-reviewed open-access journal doi: 10.3897/mycokeys.33.23670 RESEARCH ARTICLE © MycoKkeys http://mycokeys.pensoft.net Launched to accelerate biodiversity research Identification of endophytic fungi from leaves of Pandanaceae based on their morphotypes and DNA sequence data from southern Thailand Saowaluck Tibpromma', Kevin D. Hyde'?, Jayarama D. Bhat?*, Peter E. Mortimer’, Jianchu Xu’, Itthayakorn Promputtha*, Mingkwan Doilom'”, Jun-Bo Yang’, Alvin M. C. Tang®, Samantha C. Karunarathna'? | Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, People’s Republic of China 2 Centre of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, Thailand 3 Formerly, Department of Botany, Goa University, Taleigdo, Goa, India 4 No. 128/1-], Azad Housing Society, Curca, Goa Velha, India § Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand 6 Environmental Science Research Centre, Faculty of Science, Chiang Mai University, 50200, Thailand 1 Germplasm Bank of Wild Species in Southwest China, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, China 8 Division of Applied Science, College of International Education, The Hong Kong Baptist University, Hong Kong SAR, China Corresponding author: Samantha C. Karunarathna (samanthakarunarathna@gmail.com) Academic editor: 7) Lumbsch | Received 16 January 2018 | Accepted 16 March 2018 | Published 28 March 2018 Citation: Tibpromma S, Hyde KD, Bhat JD, Mortimer PE, Xu J, Promputtha I, Doilom M, Jun-Bo Yang, Tang AMC, Karunarathna SC (2018) Identification of endophytic fungi from leaves of Pandanaceae based on their morphotypes and DNA sequence data from southern Thailand. MycoKeys 33: 25-67. https://doi.org/10.3897/mycokeys.33.23670 Abstract The authors established the taxonomic status of endophytic fungi associated with leaves of Pandanaceae collected from southern Thailand. Morphotypes were initially identified based on their characteristics in culture and species level identification was done based on both morphological characteristics and phyloge- netic analyses of DNA sequence data. Twenty-two isolates from healthy leaves were categorised into eight morphotypes. Appropriate universal primers were used to amplify specific gene regions and phylogenetic analyses were performed to identify these endophytes and established relationships with extant fungi. The authors identified both ascomycete and basidiomycete species, including one new genus, seven new species and nine known species. Morphological descriptions, colour plates and phylogenies are given for each taxon. Copyright S. Tibpromma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 26 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) Keywords Ascomycetes, Basidiomycota, biodiversity, phylogenetic analysis Introduction Endophytic fungi are beneficial to their host plants and have the ability to produce bioac- tive compounds that have applied uses (Fisher et al. 1994; Strobel et al. 2004; Gunatilaka 2006; Arnold et al. 2007; Saikkonen et al. 2010; Aly et al. 2010; Lin et al. 2010; Rajulu et al. 2011; Chowdhary et al. 2015). Research on endophytic fungi began approximately 30 years ago and has intensified over the past 20 years (Thomson et al. 1997; Arnold et al. 2000; Stone et al. 2000; Hyde and Soytong 2008; Lumyong et al. 2009). This rising interest in endophytic fungi dates back to Bills’ 1996 novel concept that mycelia sterilia isolates could be assigned to groups based on their degree of similarity in colony surface texture (Rodrigues 1994; Fisher et al. 1995; Lodge et al. 1996; Brown et al. 1998; Taylor et al. 1999; Umali et al. 1999; Frohlich et al. 2000). Lacap et al. (2003) used molecular data to demonstrate the reliability of Bill’s 1996 concept based on the cultural approach. Guo et al. (2000, 2003) found that morphological characteristics were insufficient to identify most endophyte isolates, especially when they do not sporulate and so DNA se- quence data were used for identification of these taxa. Although this has been followed by numerous authors using ITS sequence data analysis, the use of ITS alone is not accurate (Promputtha et al. 2005). Subsequent studies have shown that multi-gene analyses are needed to identify endophytes (Ko et al. 2011). Endophytic fungal strains have been isolated from many different plants includ- ing trees, vegetables, fruits, cereal grains and other crops (Rosenblueth and Martinez- Romero 2006). Dickinson (1976) published the first study of endophyte - leaf associa- tions. However, there has been less research on the endophytic fungi associated with the leaves of tropical plants (Promputtha et al. 2007). The high species diversity of en- dophytic fungi makes their study a pressing research area. Globally, endophytic fungi were estimated to comprise 7 % of the 1.5 million species of fungi (Hawksworth 2001; Chowdhary et al. 2015). The actual numbers may be far higher. Recently, Hawksworth and Lucking (2017) estimated that there are 2.2 to 3.8 million fungal taxa. Endophytes are expected to be numerous because their host-specificity will drive diversification and they can occupy several niches, including that of pathogens and saprobes (Zhou and Hyde 2001). Several studies have investigated the relationships between endophytes and saprotrophs and also between endophytes and pathogens (Petrini 1991; Yanna and Hyde 2002; Ghimire and Hyde 2004; Photita et al. 2004; Hyde et al. 2006). The authors have been investigating saprobic and endophytic fungi associated with Pandanaceae (Tibpromma et al. 2016a, b, c, 2017a, b) and, in this study, taxo- nomic details are presented regarding the endophytic fungi that were isolated. Pan- danaceae are monocotyledonous plants. Their associated endophytic fungi were first studied by McKenzie et al. (2002), with further research conducted by Thongkantha et al. (2008), Bungihan et al. (2011), Arifin (2013), Bungihan et al. (2013) and Es- kandarighadikolaii et al. (2015). Identification of endophytic fungi from leaves of Pandanaceae... 27 The objectives of the present study were to establish the endophytic fungal com- munity on selected Pandanaceae collected in southern Thailand. The authors isolated 22 endophytic isolates and sorted them in morphotypes and identified the taxa based on DNA sequence analyses. Both ascomycete and basidiomycete genera were identi- fied, including one new genus, seven new species and nine known species. The recom- mendations of Jeewon and Hyde (2016) were followed when introducing the new species based on molecular data. Materials and methods Sample collection and fungal isolation Healthy mature leaves of Pandanus and Freycinetia species (Pandanaceae, Figure 1) were collected from Chumphon (10°57'38.2"N 99°29'21.8"E) and Ranong (9°55'15.9"N, 98°38'30.7"E) provinces of southern Thailand during the rainy season (December) of 2016. Leaves with physical damage or showing signs of pathogenic infection were excluded from the study. In total, more than 100 healthy leaves were placed in Ziploc plastic bags, preserved with ice and transported to the laboratory. Leaves were ran- domly cut into 0.5 cm size pieces (10 pieces/leaf) using a hole puncher under aseptic conditions. These sections were soaked in 95 % ethanol for 1 minute, then in 3 % so- dium hypochlorite solution for 3 minutes and finally in 95 % ethanol for 30 seconds. All samples were rinsed with sterile distilled water and dried on sterile tissue paper. Leaf sections were placed in Malt Extract Agar (MEA), Potato Dextrose Agar (PDA) and Water Agar (WA). They were incubated at room temperature (25-30 °C) for 1-3 days. If hyphal tips of any fungal colony appeared during incubation, the colony was transferred to new PDA plates and incubated to obtain pure cultures. Cultures and identification The above methods resulted in 22 isolates which were separated into morphotypes based on visual assessment of the similarity of the cultures (Bills 1996; Umali et al. 1999; Frohlich et al. 2000; Lacap et al. 2003). All of these cultures were grown on Po- tato Dextrose Agar (PDA). Growth rate measurements are shown in Table 1 with col- ony colour defined with the Methuen Handbook of Colour (Kornerup and Wanscher 1967). New taxa were examined in pure culture, allowing photographs, records of morphological characteristics and descriptions to be recorded. Herbarium specimens were prepared from cultures that were dried in silica gel. The holotypes were deposited in the Mae Fah Luang University Herbarium (Herb. MFLU), Chiang Rai, Thailand and in the Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China. The ex-types cultures were deposited in the Mae Fah Luang University Culture Col- lection (MFLUCC) with duplicates deposited in the BIOTEC Culture Collection 28 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) i ES ¥ : fi DE S Sati oh A N 1 A \- C My } Pr a 5 folte SARS ns, | x aL Ss Bat Ky — ¥ [wd Figure |. Habitats of the host plants: a, b Pandanus spp. €, d Freycinetia spp. Laboratory (BCC) and the Kunming Institute of Botany Culture (KMUCC). New taxa were registered in Facesoftungi (FoF) (Jayasiri et al. 2015) and MycoBank (Crous et al. 2004). DNA extraction, PCR amplification, and sequencing Genomic DNA was extracted from pure fungal cultures using Biospin Fungal Genom- ic DNA extraction Kit-BSC14S1 (BioFlux, PR. China). Polymerase chain reaction (PCR) was used to amplify partial gene regions of Internal Transcribed Spacers (ITS), 28S ribosomal RNA (LSU), 18S ribosomal RNA (SSU), RNA polymerase II second largest subunit (RPB2), 8-tubulin (Tub2), Actin (ACT), Glyceraldehyde-3- Phosphate Dehydrogenase (GADPH), Chitin synthase 1 (CHS-1) and Translation Elongation Factor l-alpha (TEF1) using primers as shown in Table 1. The total volume of PCR mixtures for amplifications were 25 pl containing 8.5 pl ddH,O, 12.5 pl 2x Easy Taq PCR Super Mix (mixture of Easy Taq TM DNA Polymerase, dNTPs and optimised buffer (Beijing Trans Gen Biotech Co., Chaoyang District, Beijing, PR China), 2 ul of DNA template, 1 pl of each forward and reverse primers (10 pM). The quality of PCR Identification of endophytic fungi from leaves of Pandanaceae... 29 Table |. Details of genes/loci with PCR primers and protocols. Gene/Loci PCR primers (Forward/Reverse) References ESU LROR/LRS5 Vilgalys and Hester 1990 ITS ITSS/ITS4 White et al. 1990 SSU NS1/NS4 White et al. 1990 983F/2218 Rehner 2001 TEF1 728F/986R Carbone and Kohn 1999 RPB2 fRPB2-5f/fFRPB2—7cR Liu et al. 1999 } BT2a/BT 2b Glass and Donaldson 1995 6-tubulin > - ; TAT O’Donnell and Cigelnik 1997 Actin 512F/783R Carbone and Kohn 1999 GHS-1 79F/354R Carbone and Kohn 1999 1 2 Myll . 2002 GADPH Gpd1/Gpd yllys et al. 200 GDF/GDR Templeton et al. 1992 products was checked on 1 % agarose gel electrophoresis stained with 4S green nucleic acid (Life Science Products & Services, Cat. No: A616694). Purification and sequenc- ing of PCR products were carried out by Sangon Biotech Co., Shanghai, China. Phylogenetic analysis The sequence data generated during this study were the subject of BLAST searches in the nucleotide database of GenBank (www http://blast.ncbi.nlm.nih.gov/) to deter- mine their most probable closely related taxa. Sequence data were retrieved from Gen- Bank based on recent publications. Raw forward and reverse sequences were assem- bled using Geneious Pro.v4.8.5. Sequence alignments were carried out with MAFFT v.6.864b (Katoh and Standley 2016) and alignments were manually improved where necessary. Ihe sequence datasets were combined using BioEdit v.7.2.5 (Hall 2004). Maximum Likelihood (ML) and Bayesian Inference (BI) analyses were performed for the sequence dataset. The phylogenetic trees were configured in FigureTree v. 1.4 (Rambaut and Drummond 2008) and edited using Microsoft Office PowerPoint 2007 and Adobe Illustrator CS3 (Adobe Systems Inc., USA). Results and discussion Identification of morphotypes Twenty-two fungal isolates from Pandanus and Freycinetia species were recovered and these mycelia sterilia were separated into eight morphotypes based on the similarity of their culture characteristics, as summarised in Table 2 (Bills 1996; Umali et al. 1999; Frohlich et al. 2000; Lacap et al. 2003). 30 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) Table 2. Culture characteristics of the 22 strains (8 morphotypes) of mycelia sterilia on PDA. Size (cm) of colony i Edge Isolate code PEOS | Pandanus sp. | 4.6 >A Circular 4A1 4A2 Aerial Undulate 1 Pandanus sp. é Circular Aerial Entire Pandanus sp. Circular Flat Entire Pandanus sp. : 5 Irregular Aerial Undulate Pandanus sp. : ; Irregular 4A3 Aerial Undulate Irregular 5B2 5A2 Aerial Undulate Irregular 4A1 4A3 Aerial Undulate Irregular 5D4 5C4 Flat Undulate Circular | 6A1/6D3 |6A1/6F5| Aerial Undulate Circular 5F4 5F7 Aerial Curled Freycinetia sp. Freycinetia sp. Freycinetia sp. Pandanus sp. Pandanus sp. Freycinetia sp. Irregular Flat Filamentous F 1 5B2 5D5 5 PE25 | Pandanussp.| >A Aerial Entire y PE26 | Pandanus sp. | 3.1 Aerial Undulate PE52 | Pandanus sp. | 1.2 Aerial Undulate 5 PE35 | Pandanus sp. | 1.1 Aerial | Filamentous Aerial | Curled Aerial | Curled 6 Flat__| Undulate Aerial | Undulate FE41 | Freycinetia sp.|_ >A Flat Filamentous 7 Aerial | Entire 8 FE101 | Freycinetiasp.| _ A Completely covering plate, Cladosporium angustiherbarum CPC 17814 ~ Cladosporium phlei CBS 358.69 [Cladosporium herbarum UTHSC DI-13-220 Cladosporium herbarum CBS 121621 [_ Cladosporium herbaroides CBS 121626 er Cladosporium macrocarpum UTHSC DI-13-191 Cladosporium macrocarpum CBS 121623 [— Cladosporium echinulatum CBS 123191 ~ Cladosporium versiforme CPC 19053 ~ Cladosporium variabile CBS 121636 ~~ Cladosporium iridis CBS 138.40 [_ Cladosporium spinulosum CBS 119907 00- Cladosporium ossifragi CBS 842.91 Cladosporium soldanellae CPC 13153 Cladosporium scabrellum CBS 126358 ee Cladosporium pseudochalastosporoides CPC 17823 CREPE AES SET ETT ITI 39 Figure 7. Phylogram generated from maximum likelihood analysis based on ITS, TEF1 and Actin se- quenced data. Maximum likelihood bootstrap is given above/below the nodes. The newly generated se- quences in red bold. ‘The tree is rooted with Cercospora beticola. 40 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) Figure 8. Cladosporium endophyticum (MFLU 18-0005, holotype). a Colony on MEA media b Myce- lium masses Ce Conidia and conidiogenous cells f, g Conidia h Conidia and conidiogenous cells. Scale bars: 5 um (b=h), 10 um (h). brown, subglobose to globose with verrucose, less often short-ovoid conidia, narrower at both ends (Zalar et al. 2007), while C. endophyticum has globose to ovoid, hyaline to pale-olivaceous conidia with rounded ends. Here, the authors introduce the new species C. endophyticum and provide an updated phylogenetic tree for the genus Cladosporium. Pleosporales Luttr. ex M.E. Barr, 1987 Massarinaceae Munk. Remarks. The family Massarinaceae was introduced by Munk (1956) under Pleospo- rales together with Cucurbitariaceae and Didymosphaeriaceae. Later, Barr (1987) seg- regated Massarinaceae under Lophiostomataceae based on morphology, while based on multigene phylogenetic analysis Schoch et al. (2009) also showed Massarinaceae is a distinct family in order Pleosporales. Recently, Zhang et al. (2009, 2012) recognised Massarinaceae as a distinct lineage based on both morphology and molecular phylog- eny. In this study, a new species of endophytic Massarina, based on morphological and phylogenetic support, is introduced from Pandanus sp. in Thailand. Massarina pandanicola Tibpromma & K.D. Hyde, sp. nov. MycoBank number: MB823839 Facesoffungi number: FoF03904 Figure 10 Etymology. name referring to the host genus of the plant on which the fungus was first discovered (Pandanus). 98 Identification of endophytic fungi from leaves of Pandanaceae... Corynespora endiandrae CBS 138902 | Helminthosporium dalbergiae H 4628 10 Ss 99 ~ Helminthosporium magnisporum H 4627 Helminthosporium hispanicum L109 = Helminthosporium velutinum H 4626 r4 Massarina pandanicola MFLUCC 17-0596 Massarina cisti CBS 266.62 99 100 81 | Massarina eburnea CBS 473.64 Massarina eburnea JCM 14422 Stagonospora paspali CBS 331.37 |_100_f Stagonospora paludosa CBS 135088 Stagonospora pseudocaricis CBS 135132 100 | Pseudodidymosphaeria spartii MFLUCC 13-0273 32 Pseudodidymosphaeria spartii MFLUCC 14-1212 Pseudodidymosphaeria spartii CBS 183.58 ~_ Suttonomyces clematidis MFLUCC 14-0240 100 100 75 | Periconia sp. H 4151 ~ Periconia sp. H 4600 Periconia digitata KT 644 100_{~ Dictyosporium digitatum KT 2660 100 87 94 Dictyosporium tetrasporum KT 2865 Dictyocheirospora pseudomusae KH 412 Katumotoa bambusicola KT 1517a 100 0.04 95 Lentithecium fluviatile CBS 122367 100__| Macrodiplodiopsis desmazieri CPC 24648 Macrodiplodiopsis desmazieri CBS 125026 | Camarographium koreanum CBS 117159 ~ Pseudochaetosphaeronema larense CBS 640.73 io [| Aquastroma magniostiolata KT 2485 o[ Multiseptospora thailandica MFLUCC 11-0183 100 Parabambusicola bambusina H 4321 Parabambusicola bambusina KH 139 100 [| Falciformispora senegalensis CBS 196.79 a= L% {~ Falciformispora lignatilis BCC 21118 100 ~_ Falciformispora tompkinsii CBS 200.79 100 [ Halomassarina thalassiae BCC 17054 96 Halomassarina thalassiae JK 5262D 100 | Trematosphaeria grisea CBS 332.50 ~ Trematosphaeria pertusa CBS 122368 Asteromassaria pulchra CBS 124082 91 | ~ Inflatispora pseudostromatica YZ-2011 89 ~ Monodictys capensis HR 1 Bactrodesmium cubense CBS 680.96 65 97 Helicascus nypae BCC 36752 99 84 ~~ Morosphaeria ramunculicola BCC 18405 Morosphaeria velataspora BCC 17059 ~ Aquilomyces rebunensis KT 732 100 | Sulcatispora sp. KT 1607 100 ~ Sulcatispora sp. KT 2982 100 { Bambusicola massarinia MFLUCC 11-0389 ~ Bambusicola splendida MFLUCC 11-0439 1° ___| Pseudoasteromassaria fagi KT 2966 Pseudoasteromassaria fagi KT 3432 97 s4[. Matsushimamyces bohaniensis CBEC001 100 { Latorua grootfonteinensis CBS 369.72 ~ Latorua caligans CBS 576.65 100 | Polyschema congolensis CBS 542 92 84 Polyschema terricola CBS 301.65 78 67} [611 Polyschema larviformis CBS 463.88 ~ Polyschema sclerotigenum UTHSC DI14-305 ~ Matsushimamyces venustum CBS 140212 95 [~~ Pseudoxylomyces elegans KT 2887 Longipedicellata aptrootii MFLUCC 10-0297 Fuscostagonospora sasae KT 1467 [| Montagnula aloes CPC 19671 —— Alternaria alternata AFT 69| Didymosphaeria rubi-ulmifolii MFLUCC 14-0024 99 Neokalmusia brevispora KT 1466 _ Deniquelata barringtoniae MFLUCC 11-0422 Pleospora herbarum CBS 191.86 4] Massarinaceae Periconiaceae Dictyosporiaceae Macrodiplodiopsidaceae Parabambusicolaceae Trematosphaeriaceae Morosphaeriaceae Sulcatisporaceae Bambusicolaceae Latoruaceae Longipedicellataceae Didymosphaeriaceae Figure 9. Phylogram generated from maximum likelihood analysis based on ITS, TEF1, SSU, LSU and RPB2 sequenced data. Maximum likelihood bootstrap values are given above/below the nodes. The newly generated sequences in red bold. The tree is rooted with Alternaria alternata and Pleospora herbarum. Holotype. MFLU 18-0004 Description. Colonies on PDA attaining 9 cm diam. in 4 weeks at room temperature, slow growing, white to yellow-white. Mycelium superficial and immersed composed of septate, branched, 2.5—7 um wide, sub-hyaline, with smooth and thick-walled hyphae. Sexual morph Undetermined. Asexual morph Conidiophores 12-25 um high, 8-14 um diam. (X = 15.12 x 10.45 um, n = 10), enteroblastic, phialidic, cylindrical or sub-cylindri- cal, sub-hyaline. Conidia 3-5 x 1-3 pm (X = 4.34 x 1.75 um, n = 30), cylindrical, hyaline, smooth and thin-walled, aseptate, rounded ends, guttulate, without sheet or appendages. 42 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) Figure 10. Massarina pandanicola (MFLU 18-0004, holotype). a Colony on MEA media b Mycelium masses C=g Conidia and conidiogenous cells h Conidia. Scale bars: 20 um (b), 2 um (c=g), 5 um (h). Culture characteristics. Colonies on PDA (Figure 2, PE52), superficial, white to yellow-white, irregular, undulate with smooth and raised on surface media; reverse yellow-white. Generative hyphae simple-septate, branched, sub-hyaline, with guttulate cells, thin-walled. Sporulating in culture within 3 months (Figure 10). Material examined. THAILAND, Chumphon, Pathio District, on healthy leaves of Pandanus sp. (Pandanaceae), 1 December 2016, S. Tibpromma PE52 (MFLU 18- 0004, holotype); HKAS100854, paratype, ex-type living cultures, MFLUCC 17- 0596 = KUMCC 17-0293. Genbank numbers. ITS=MG646958, LSU=MG646947, SSU=MG646979, TEF1=MG646986. Notes. The genus Massarina has been known as a phylogenetically diverse group in the order Pleosporales based on molecular data (Liew et al. 2002) and most members in Massarina except for the type species (M. eburnea) are morphologically variable. The taxon, Massarina pandanicola collected from Pandanus sp. in Thailand is introduced here as a new species with both morphology and phylogeny support. The morphology of the taxon showed similar conidia with Massarina eburnean (Tanaka et al. 2015), but based on phy- logenetic analysis of combined ITS, LSU, SSU and TEF1 gene sequence data, the new taxon MV. pandanicola is well-separated from other species in Massarina (Figure 9) with high bootstrap support (79 % in ML). This is the first record of Massarina from Pandanus sp. Pleosporaceae Nitschke Remarks. ‘The family Pleosporaceae was introduced by Nitschke (1869) and is the larg- est family of the order Pleosporales (Hyde et al. 2013; Ariyawansa et al. 2015b; Liu et al. 2017). Members of this family can be endophytes, aquatic or terrestrial saprobes, plant Identification of endophytic fungi from leaves of Pandanaceae... 43 pathogens or opportunistic animal pathogens (Sivanesan 1984; Carter and Boudreaux 2004). A backbone tree for Pleosporaceae was provided by Ariyawansa et al. (2015a). In this study, Alternaria burnsii is reported from a Pandanus sp. host in Thailand. Alternaria burnsii Uppal, Patel & Kamat, Indian J. Agric. Sci. 8: 49 (1938) Culture characteristics. Colonies on PDA (Figure 2, PE26), superficial, white-orange to cream, circular, entire edge, smooth, flossy, velvety and raised on surface media; reverse yellow-white at the margin and yellow-brown in centre. Not sporulating in culture. GenBank numbers. [TS=MG646973, LSU=MG646952, TEF1=MG646987. Notes. Alternaria burnsii was introduced by Uppal et al. (1938) from India on Cum- nium cyminum. This species has a close phylogenetic relationship with Alternaria tomato and A. jacinthicola (Woudenberg et al. 2015). Results from phylogenetic analysis show that the authors’ collection belongs to Al/ternaria burnsii with a relatively high bootstrap support (89% in ML) (Figure 11). Nucleotides across the ITS regions of Alternaria burnsii CBS 108.27 and the isolates were compared and the authors noted that they are identical. Alternaria gaisen CBS 632.93 3) Alternaria alternata CBS 175.52 6069 Al/ternaria alternata CBS 174.52 1 Alternaria alternata CBS 118814 Alternaria alternata MFLUCC 14-1184 Alternaria alternata CBS 916.96 ~ Alternaria longipes CBS 540.94 89 Alternaria burnsii MFLUCC17-0582 Alternaria burnsii CBS 108.27 Alternaria perpunctulata CBS 115267 Alternaria perpunctulata EGS 51-130 Alternaria alternantherae CBS 124392 | Alternaria pseudorostrata CBS 119411 Sect. Alternaria Sect. Alternantherae oe 0099 Alternaria sp. CBS 108.27 oy) Alternaria porri CBS 116698 ~_ Alternaria macrospora CBS 117228 100 Alternaria saponariae CBS 116492 2 i Alternaria gypsophilae CBS 107.41 Alternaria vaccariicola CBS 118714 100 Alternaria eureka CBS 193.86 100 ~~ Alternaria leptinellae CBS 477.90 92 100; ~~ Alternaria planifunda CBS 537.83 — Alternaria proteae CBS 475.90 00 ii Alternaria cheiranthi CBS 190384 99 ~_ Alternaria sp. CBS 115.44 96 Alternaria aspera CBS 115269 Alternaria obovoideum CBS101229 Alternaria cucurbitae CBS 483.81 sf Alternaria leucanthemi CBS 421.65 I : I 100 Alternaria panax CBS 482.81 | Sect. Porri Sect. Gypsophilae Sect. Eureka Sect. Embellisiodes Sect. Cheiranthus Sect. Pseudoulocladium Sect. Ulocladioides Sect. Teretispora 81 ~~ Alternaria photistica CBS 212.86 Sect. Panax eS Alternaria phragmospora CBS 274.70 __100_| Alternaria chlamydospora CBS 491.72 Sect. Phragmosporae Alternaria mouchaccae CBS 119671 100 | Alternaria embellisia CBS 339.71 _|— Alternaria chlamydospora CBS 491.72 Sect. Embellisia ~ Alternaria telluster CBS 583.83 Alternaria frumenti EGS 44-001 69 Alternaria infectoria CBS 210.86 Alternaria hordeicola EGS 50-184 Alternaria ethzedia CBS 197.86 Alternaria triticina EGS 17-061 © Alternaria metachromatica EGS 38-132 | Alternaria breviramosa CBS 121331 3 Alternaria obclavata CBS 124120 9 Alternaria malorum CBS 173.80 Sect. Infectoriae Sect. Chalastospora ~ Alternaria malorum CPC 15567 Pleospora herbarum 0.02 Figure | 1. Phylogram generated from maximum likelihood analysis based on ITS, TEF1, LSU and RPB2 sequence data. Maximum likelihood bootstrap values are given above/below the nodes. The newly generated sequences are in red bold. The tree is rooted with Pleospora herbarum. 44 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) Sordariomycetes O.E. Erikss. & Winka Diaporthales Nannf. Diaporthaceae Hohn. ex Wehm. Remarks. The family Diaporthaceae was introduced by von Hohnel (1917) and was placed in the order Diaporthales. This family comprised two Diaporthe genera (Phomopsis and Mazzantia) (Wehmeyer 1975; Castlebury et al. 2002). Later, Dia- porthaceae was given the synonym Valsaceae (Barr 1978). Based on DNA sequence data, some other genera have been placed in Diaporthaceae (Dai et al. 2014; Voglmayr and Jaklitsch 2014). Recently, Maharachchikumbura et al. (2015) and Senanayake et al. (2017) listed further genera that belong to Diaporthaceae. In this study, a new and a known species of Diaporthe from Pandanaceae hosts in Thailand is reported. Diaporthe pandanicola Tibpromma & K.D. Hyde, sp. nov. MycoBank number: MB823840 Facesoffungi number: FoF03905 Figure 13 Etymology. Name referring to the host genus on which the fungus was first discovered (Pandanus). Holotype. MFLU 18-0006 Culture characteristics. Colonies on PDA (Figure 2, PE77), superficial, white, circu- lar with entire edge, smooth and raised on surface media, flossy and velvety; reverse yellow- white, 9 cm diam. in 10 days. Generative hyphae simple-septate, branched, sub-hyaline, cells with guttules, thin-walled, 1.5—7 um wide. Not sporulating in culture (Figure 13). Material examined. THAILAND, Chumphon, Pathio District, on healthy leaves of Pandanus sp. (Pandanaceae), 1 December 2016, S. Tibpromma PE77 (MFLU 18- 0006, holotype); HKAS100858, paratype, ex-type living cultures, MFLUCC 17- 0607 = KUMCC 17-0297. GenBank numbers. [TS=MG646974, 8-tubulins=MG646930, ACT=MG646930. Notes. Diaporthe species are plant pathogens, endophytes or saprobes (Carroll 1986; Garcia-Reyne et al. 2011; Udayanga et al. 2011, 2012, 2014, Hyde et al. 2014). Here, a new species Diaporthe pandanicola is introduced based on phylogeny support. Based on phylogenetic analysis, the new species was well-separated from closely related species of Diaporthe (61% in ML, 0.97 in PP). However, this isolate is an endophytic fungus and did not sporulate in culture during 5 months (Figure 13). Diaporthe siamensis Udayanga, X.Z. Liu & K.D. Hyde, 2012 Culture characteristics. Colonies on PDA (Figure 2, PE37), superficial, white to yellow- white, irregular, curled and raised on media surface, flossy; under surfaceyellow-white. Identification of endophytic fungi from leaves of Pandanaceae... 45 | Diaporthe pascoei BRIP 54847 | Diaporthe litchicola BRIP 54900 87/1[ Diaporthe pseudomangiferae CBS 101339 ~ Diaporthe pseudomangiferae CBS 388.89 Diaporthe eugeniae CBS 444.82 at Diaporthe pandanicola MFLUCC 17-0607 Ea: a 6140.97 go/1 | ———~ Diaporthe pterocarpicola MFLUCC 10-0580 68/1 =~ Diaperthe arecae CBS 161.64 67/0.99 | Diaporthe perseae CBS 151.73 ~ Diaporthe arecae CBS 535.75 ~ Diaporthe arengae CBS 114979 74, Diaporthe musigena CBS 129519 ~ Diaporthe fraxini-angustifoliae BRIP 54781 99/1 | Diaporthe aseana MFLUCC 12-0299 wu pa ~ Diaporthe tectonigena MFLUCC 12-0767 ~ Diaporthe hongkongensis CBS 115448 100/1__[ Diaporthe siamensis MFLUCC 10-0573 60/-- ~_ Diaporthe siamensis MFLUCC 17-0591 100/1_|Diaporthe biguttulata ZJUD47 Diaporthe biguttulata ZJUD48 1001 [| —~—~—~—S——C Ditaporrthe discoidispora ZJUD89 ~ Diaporthe discoidispora ZJUD87 100/1_ | Diaporthe citriasiana ZJUD30 Diaporthe citriasiana ZJUD8 1 69/0.97| 64/097 100/1 Figure 12. Phylogram generated from maximum likelihood analysis based on ITS, TEF1 and $-tubulin sequenced data. Maximum likelihood (left) and Bayesian inference (right) bootstrap values are given above/ below the nodes. The newly generated sequences are in red bold. The tree is rooted with Diaporthe ambigua. Figure 13. Diaporthe pandanicola (MFLU 18-0006, holotype). a-c Mycelia masses. Scale bars: 5 um (a=c). GenBanknumbers. [TS=MG646975, TEF1=MG646989, 8-tubulin=MG646925, ACT=MG646940. Notes. In the phylogenetic analysis, the authors’ collection grouped with Dia- porthe siamensis MFLUCC 10-0573 with high statistical values of 100% in ML and 1.00 in PP. Diaporthe siamensis is an endophytic fungus collected from a Pandanaceae host in Thailand. 46 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) [ Colletotrichum henenanse CGMCC 3.17354 | Colletotrichum ti ICMP 4832 [ Colletotrichum aotearoa ICMP 18537 4 Colletotrichum syzygicola MFLUCC 10-0624 ~ Colletotrichum cordylinicola CMP 18579 Colletotrichum psidii ICMP 19120 66/0'99 | Colletotrichum temperatum Coll883 “7 ~ Colletotrichum camelliae CGMCC 3.14925 ./099 Colletotrichum clidemiae ICMP 18658 2x Colletotrichum wuxiense CGMCC 3.17894 Ln ‘ Colletotrichum kahawae ICMP 17816 -/1 are Colletotrichum rhexiae Coll1026 Colletotrichum fructivorum Coll1414 Colletotrichum jiangxiense CGMCC 3.17363 90/096 Colletotrichum xanthorrhoeae ICMP 17903 100/1 | Colletotrichum grossum CAUG7 89/0.94 [~ Colletotrichum theobromicola ICMP. 18649 -Colletotrichum grevilleae CBS 132879 Colletotrichum proteae CBS 132882 | Colletotrichum queenslandicum ICMP 1778 -/980°~ Colletotrichum pandanicola MFLUCC 17-0571 ~ Colletotrichum tropicale ICMP 18653 | Colletotrichum musae ICMP 19119 | Colletotrichum hebeiense MFLUCC 13-0726 ~ Colletotrichum conoides CAUGI7 -Colletotrichum aenigma ICMP 18608 x _-/0L8 Colletotrichum viniferum GZAAS5.08601 4% 95/I sO. =s Gloeosporioides complex Colletotrichum aeschynomenes ICMP 17673 4/ Colletotrichum alienum ICMP 12071 40.84 Colletotrichum nupharicola ICMP 18187 ‘i i A Colletotrichum fructicola MFLUCC 17-0555 , Colletotrichum fructicola MFLUCC 17-0613 | Colletotrichum fructicola 1CMP 18581 1-88” Colletotrichum siamense ICMP 18578 7 ~ Colletotrichum asianum ICMP 18580 ale Colletotrichum salsolae ICMP 19051 y Colletotrichum gloeosporioides CBS 112999 Colletotrichum endophytica MFLUCC 13-0418 Colletotrichum alatae ICMP 17919 Colletotrichum horii ICMP 10492 Colletotrichum truncatum CBS 151. Dd oo = = 0.03 Figure 14. Phylogram generated from maximum likelihood analysis based on combined ITS, Actin, 6-tubulin, GADPH and CHS-1 sequenced data. Maximum likelihood (left) and Bayesian inference (right) bootstrap values are given above/below the nodes. The newly generated sequences are in red text. The tree is rooted with Colletotrichum truncatum. Glomerellales Chadef. ex Réblova et al. Glomerellaceae Locq. ex Seifert & W. Gams, in Zhang et al. (2007) Remarks. The family Glomerellaceae was introduced by Locquin (1984), but was in- validly published. To date, most Glomerellaceae have been recorded to be pathogens (Maharachchikumbura et al. 2016b). Earlier studies reported that the position of the family Glomerellaceae was not stable (Zhang et al. 2006; Kirk et al. 2001; Kirk et al. 2008). Réblova et al. (2011) resolved the placement of Glomerellaceae by using phy- logenetic analysis of combined ITS, LSU, SSU and RPB2 sequence data. Recently, the family Glomerellaceae was established based on the genus Glomerella (Zhang et al. 2006), which had been given a synonym under its asexual morph Colletotrichum (Ma- harachchikumbura et al. 2015). Recently, Jayawardena et al. (2016) provided notes Identification of endophytic fungi from leaves of Pandanaceae... 47 on currently accepted species of Colletotrichum. In this study, the authors introduce a new endophytic Colletotrichum species and report a known species of endophytic Colletotrichum from gloeosporioides species complex based on morphology and phy- logenetic analysis. Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde, 2009 Culture characteristics. Colonies on PDA (Figure 2, PE84, 88), superficial, white to olivaceous in the beginning and later become olivaceous to dark-olivaceous, circu- lar, entire edge, smooth, dense and raised on surface media; reverse dark-olivaceous. Sporulating in culture after 1 month. GenBanknumbers. MFLUCC17-0613ITS=MG646968, 8-tubulin=MG646927, GAPDH=MG646932, CHS-1=MG646937, ACT=MG646939. MFLUCC_ 17- 0555 ITS=MG646969, §-tubulins=MG646928, GADPH=MG646933, CHS- 1=MG646936, ACT=MG646944. Notes. The gloeosporioides species complex is mainly plant pathogens (Weir et al. 2012) and some species are endophytes (Liu et al. 2015). Colletotrichum fructicola has a wide host range (Weir et al. 2012) and was originally reported from coffee berries in Thailand (Prihastuti et al. 2009). In this study, the authors followed Jayawardena et al. (2016) and identify the collection as Colletotrichum fructicola which was isolated from a Pandanaceae host. Based on phylogenetic analysis, this taxon grouped with Colletotri- chum fructicola with 90 % in ML and 1.00 in PP. The ITS, 8-tubulin, GAPDH, CHS- 1 and ACT DNA nucleotide comparison showed that the taxon and other strains of Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde have 100% similarity. Colletotrichum pandanicola Tibpromma & K.D. Hyde, sp. nov. MycoBank number: MB823841 Facesoffungi number: FoF03906 Figure 15 Etymology. name referring to the host genus (Freycinetia). Holotype. MFLU 18-0003 Description. Colonies on PDA attaining 9 cm diam. in 7 days at room tempera- ture, dark-grey. Sexual morph Undetermined. Asexual morph Conidiophores hyaline, smooth-walled, cylindrical to slightly inflated. Conidia 9-18 um high, 4-8 um diam. ( X = 13.39 x 5.35 um, n = 20), hyaline, cylindrical with rounded ends tapering slightly towards the base, smooth, septate, guttulate. Culture characteristics. Colonies on PDA (Figure 2, PEO9), superficial, white in the beginning and later becoming dark-grey, circular, entire edge, smooth, flossy, velvety and raised on surface media; reverse dark. Sporulating in culture after 1 month. 48 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) Figure 15. Colletotrichum pandanicola (MFLU 18-0003, holotype). a Colony on PDA media b Conidia and conidiogenous cells c=g Conidia on PDA culture. Scale bars: 5 um (b), 2 um (c=). Material examined. THAILAND, Chumphon, Pathio District, on healthy leaves of Pandanus sp. (Pandanaceae), 1 December 2016, S. Tibpromma PE09 (MFLU 18-0003, holotype); GZAAS 16-0145, paratype, ex-type living cultures, MFLUCC 17-0571. GenBank numbers. ITS=MG646967, §8-tubulin=MG646926, GAPDH= MG646931, CHS-1=MG646935, ACT=MG646938. Notes. Colletotrichum pandanicola is introduced here as a new species in the gloe- osporioides species complex based on morphological and phylogenetic data. The phy- logenetic analysis shows that this new taxon is well-separated from other known Colle- totrichum species (Figure 14). The authors also compared nucleotides of $-tubulin, GAPDH, CHS-1 and ACT and found that there are differences between Colletotri- chum tropicale and this new species (8-tubulin 7 bp, GAPDH 11 bp, CHS-1 7 bp and ACT 3 bp). Magnaporthaceae P.F. Cannon Remarks. The family Magnaporthaceae was introduced by Cannon (1994) and was placed as a family within the class Sordariomycetes (Kirk et al. 2001; Lumbsch and Huhndorf 2007). According to Thongkantha et al. (2009), the placement of the taxa Magnaporthaceae has long been problematic due to a lack of convincing morphological Identification of endophytic fungi from leaves of Pandanaceae... 49 9 ! Mycoleptodiscus terrestris CMU Cib179 By ~~ Mycoleptodiscus terrestris CBS 231.53 100 100 _| Mycoleptodiscus indicus VUAMH 10746 Mycoleptodiscus indicus UAMH 8520 ~ Mycoleptodiscus endophytica MFUCC 17-0545 90 Buergenerula spartinae ATCC 22848 67| 8 Magnaporthe poae M47 76| Magnaporthe salvinii M21 ~ Slopeiomyces cylindrosporus CBS 609.75 ~ Bussabanomyces longisporus CBS 125232 99[ Deightoniella cibiessia CPC18916 ; ’ U Pseudopyricularia kyllingae HYKB202-1-2 | Pyriculariaceae ~ Pyricularia borealis CBS 461.65 100 { Ophioceras dolichostomum CBS 114926 . - Ophioceras dolichostomum HKUCC10113 Ophioceraceae —___ Ophioceras leptosporum CBS 894.70 AFTOL-ID 172 Magnaporthaceae 100 Thyridium vestitun 0.07 Figure 16. Phylogram generated from maximum likelihood analysis based on combined ITS, LSU, SSU and TEF1 sequenced data. Maximum parsimony bootstrap values are given above/below the nodes. The newly generated sequences are in red bold. ‘The tree is rooted with Thyridium vestitum. characteristics and inconclusive molecular data. Thongkantha et al. (2009) established a new order, Magnaporthales, to accommodate Magnaporthaceae, based on a combi- nation of morphological characteristics and the phylogenetic analysis of combined sequence data. Maharachchikumbura et al. (2015) provided an updated outline of the family Magnaporthaceae with 20 genera, which included both sexual and asexual morphs. In this study, Mycoleptodiscus endophyticus is introduced as a new species. Mycoleptodiscus endophyticus Tibpromma & K.D. Hyde, sp. nov. MycoBank number: MB823842 Facesoffungi number: FoF03907 Figure 17 Etymology. Named after its original habitat as an endophytic fungus. Holotype. MFLU 18-0001 Culture characteristics. Colonies on PDA (Figure 2, FE101), superficial, dark oli- vaceous with circular rings with filiform edge and rough and raised on media surface; reverse dark olivaceous. Mycelium composed of branched, pale-brown to dark-brown, thick-walled, guttulate, hyphae, with cells sub-globose to ovoid in shape. Not sporulat- ing in culture. Material examined. THAILAND, Ranong, Muang, on healthy leaves of Freyci- netia sp. (Pandanaceae), 3 December 2016, S. Tibpromma FE101 (MFLU 18-0001, holotype); HKAS100847, paratype, ex-type living cultures, MFLUCC 17-0545 = KUMCC 17-0263. GenBank numbers. LSU=MG646946, SSU=MG646978, TEF1=MG6469835. Notes. Mycoleptodiscus Ostaz. (1968) belongs to Magnaporthaceae, Magna- porthales. Since 1968, there have been 17 records of Mycoleptodiscus in Index Fun- 50 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) masses d—f Vegetative hyphae in culture. Scale bars: 10 um (bd), 5 pm (e, f). gorum. Most of these species were described without molecular data. In this study, a new species Mycoleptodiscus endophyticus is introduced, based on culture charac- teristics and phylogenetic analysis (100 % in ML). Mycoleptodiscus endophyticus was found as an endophytic fungus on leaves of Freycinetia sp; Mycoleptodiscus freycinetiae Whitton, K.D. Hyde & McKenzie was found as a saprobic fungus on the same host but there was no molecular data available to confirm this identification. The authors were unable to compare the morphological differences between the new taxon and Mycoleptodiscus freycinetiae, because only culture characteristics are presented here for this new taxon (Fig. 17). Sporocadaceae Corda, 1842 Remarks. Sporocadaceae was introduced by Corda (1842) with Pestalotiopsis-like asex- ual morphs and confirmed by Senanayake et al. (2015). Members of Sporocadaceae are saprobes, endophytes or foliar pathogens in tropical and temperate regions (Jeewon et al. 2004; Tanaka et al. 2011). Pestalotiopsis can be found as saprobes or pathogens worldwide (Jeewon et al. 2002, 2003; Maharachchikumbura et al. 2011, 2012, 2013, 2014a, b, 2016a, c). Recently, Chen et al. (2017) provided updates for this genus based on morphology and phylogeny. In this study, two known species of Pestalotiopsis from Pandanaceae hosts were isolated. Identification of endophytic fungi from leaves of Pandanaceae... Pestalotiopsis \s Pestalotiopsis jesteri CBS109350 | Pestalotiopsis portugalica CBS 393.48 | Pestalotiopsis novae-hollandiae CBS 130973 | Pestalotiopsis inflexa MFLUCC 12-0270 100! Pestalotiopsis intermedia MFLUCC 12-0259 ?| — Pestalotiopsis linearis MFLUCC 12-0271 82, Pestalotiopsis chamaeropis CBS 186.71 | Pestalotiopsis australis CBS 114193 Pestalotiopsis unicolor MFLUCC 12-0276 9s) Pestalotiopsis scoparia CBS 176.25 Pestalotiopsis monochaeta CBS 144.97 /Pestalotiopsis sequoiae MFLUCC 13-0399 Pestalotiopsis hollandica CBS 265 33 Pestalotiopsis brassicae CBS 170.26 | Pestalotiopsis italiana MFLUCC 12-0657 ~ Pestalotiopsis verruculosa MFLUCC 12-0274 \00/ Pestalotiopsis lushanensis LC8183 ~ Pestalotiopsis clavata MFLUCC 12-0268 | estalotiopsis parva CBS 265.37 Pestalotiopsis grevilleae CBS 114127 Pestalotiopsis knightiae CBS 114138 Pestalotiopsis biciliata CBS 236.38 $3 [\4' Pestalotiopsis biciliata CBS 124463 Pestalotiopsis biciliata CBS 790.68 ~ Pestalotiopsis brachiata LC2988 | Pestalotiopsis australasiae CBS 114126 73 Pestalotiopsis telopeae CBS 114161 88 , Pestalotiopsis oryzae CBS 353.69 4,| Pestalotiopsis rhodomyrtus HGUP 4230 00, Pestalotiopsis trachicarpicola OP068 Pestalotiopsis kenyana CBS 442.67 ~ Pestalotiopsis photinicola GZCC 16-0028 ‘| Pestalotiopsis dracontomelon MFUCC 10-0149 a ~~ Pestalotiopsis rosea MFLUCC 12-0258 ~_ Pestalotiopsis digitalis |CMP 5434 | Pestalotiopsis jinchanghensis LC8191 ~ Pestalotiopsis colombiensis CBS 118553 Pestalotiopsis shorea MFLUCC 12-0314 Pestalotiopsis subshorea HGUP 4118 40 9¢ Pestalotiopsis papuana CBS 331.96 Pestalotiopsis adusta MFLUCC 10-0146 70|| Pestalotiopsis licualacola HGUP 4057 Pestalotiopsis humus CBS 336.97 Pestalotiopsis diploclisiae CBS 115587 Pestalotiopsis racaenea HGUP 4037 Pestalotiopsis malayana CBS 102220 | Pestalotiopsis furcata MFLUCC 12-0054 Se = In S S 1/00. af 84 99 100 [ Pestalotiopsis jiangxiensis LC4399 Pestalotiopsis jiangxiensis MFLUCC 17-0567 100 Pestalotiopsis microspora MFLUCC 17-0619 Pestalotiopsis microspora DPX3-1 78 0.08 estalotiop Pestalotiopsis longiappendiculata LC3013 Pestalotiopsis camelliae MFLUCC 12-0277 ~ Pestalotiopsis yanglingensis LC3375 Pestalotiopsis hawaiiensis CBS 114491 Pestalotiopsis arengae CBS 331.92 Pestalotiopsis arceuthobii CBS 434.65 97 Pestalotiopsis spathulata CBS 356.86 sis diversiseta MFLUCC 12-0287 D096 Figure 18. Phylogram generated from maximum likelihood analysis based on the combination of ITS, 6-tubulin and TEF1 sequenced data. Maximum parsimony bootstrap is given above/below the nodes. The newly generated sequences are in red bold. The tree is rooted with Seiridium camelliae. Pestalotiopsis jiangxiensis F. Liu & L. Cai, 2017 Culture characteristics. Colonies on PDA (Figure 2, PEO5), superficial, white at the margin with yellow-white in the centre, with circular to undulate at the edge and raised 52 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) and dense aerial mycelia on surface; reverse yellow-white. Sporulating in culture after 2 months. GenBank numbers. ITS=MG646966, ACT=MG646942, GAPDH=MG646934, 8-tubulin=MG646929. Notes. The authors’ collection from Pandanaceae host in Thailand was identified as Pestalotiopsis jiangxiensis. This taxon grouped with Pestalotiopsis jiangxiensis LC4399 which is collected from Eurya sp., with high bootstrap support of 100% in ML. Pestalotiopsis microspora (Speg.) G.C. Zhao & N. Li, 1995 Culture characteristics. Colonies on PDA (Figure 2, PE92), superficial, white to yellow-white, edge irregular, flossy and velvety; under surface yellow-white to yellow. Sporulating in culture after 2 months. GenBank numbers. ITS=MG646965, ACT=MG646943. Notes. Pestalotiopsis microspora was isolated from a Pandanaceae host in ‘Thailand. This strain clusters with Pestalotiopsis microspora DPX3-1 with a strong bootstrap support. Saccharomycetes Debaryomycetaceae Kurtzman & M. Suzuki Remarks. Debaryomycetaceae was introduced by Kurtzman and Suzuki in 2010 and was typified by Debaryomyces Klécker. Meyerozyma belongs to family Debaryomyceta- ceae and was detailed in Kurtzman and Suzuki (2010). In this study, Meyerozyma carib- bica was found on a Pandanaceae host as an endophytic fungus. Species identification was confirmed by DNA sequence data. Meyerozyma caribbica (Vaughan-Mart., Kurtzman, S.A. Mey. & E.B. O'Neill) Kurtzman & M. Suzuki, Mycoscience 51(1): 8 (2010) Culture characteristics. Colonies on PDA (Figure 2, PE75, 98), superficial, white to yellow-white, rings with irregular, undulate edge and curled, raised on the surface media; reverse yellow-white to yellow at the margin and dark-brown at the centre. Sporulating in culture after 2 months. GenBank numbers. MFLUCC 17-0556 ITS=MG646971, LSU=MG646950, SSU=MG646977. MFLUCC 17-0606 ITS=MG646972, LSU=MG646951, SSU=MG646980. Notes. Meyerozyma caribbica collected in this study is represented by two endo- phytic isolates from Pandanaceae. Phylogenetic analysis also supported the identifica- tion of this sample as Meyerozyma caribbica. Identification of endophytic fungi from leaves of Pandanaceae... 53 Candida sake Y-1622 Candida palmioleophila Y-17323 99 65 Candida glaebosa Y-6949 94 FL Candida pseudoglaebosa Y-17911 '— Candida saitoana Y-17316 Candida fluviatilis Y-7711 99 —Scheffersomyces stipitis Y-7124 91 '— Scheffersomyces segobiensis Y-11571 Scheffersomyces shehatae var. insectosa Y-12854 100 |Scheffersomyces shehatae var. shehatae Y-17029 Scheffersomyces shehatae var. lignosa Y-12856 Scheffersomyces ergastensis Y-17652 Candida iS Scheffersomyces 98 71 |_______Scheffersomyces coipomoensis Y-17651 66>—Lodderomyces sojae Y-17909 I | '-Lodderomyces tropicalis Y-12968 Lodderomyces neerlandica Y-27T057 Lodderomyces maltosa Y-17677 Lodderomyces elongisporus YB-4239 Lodderomyces 96 | agbeaiecree Lodderomyces albicans Y-12983 L Lodderomyces dubliniensis Y-17841 odderomyces viswanathii Y-6660 Lodderomyces lodderae Y-17317 _Lodderonwvees parapsilosis Y-12969 99 _,—Spathaspora passalidarum Y-27907 Spathaspora jeffriesii Y-27738 74, —Spathaspora lyxosophila Y-17539 (_______________ Spathaspora insectamans Y-7786 Millerozyma acaciae Y-7117 91 Spathaspora Scheffersomyces etchellsii Y-7121 Millerocyma 100 -Scheffersomyces spartinae JCM 10741 }t———___— Schwanniomyces Bacueopoleorpits YB-4229 Debaryomyces nepalensis Y-7108 Debaryomyces prosopidis Y-27369 74| Debaryomyces coudertii Y-7425 Debaryomyces hansenii var. fabryi Y-17914 Debaryomyces psychrophila Y-17665 Scheffersomyces Debaryomyces Yamadazyma triangularis Y-5714 98 Yamadazyma friedrichii Y-17653 100 8-—— Yamadazyma buinensis Y-11706 Yamadazyma membranifaciens Y-2089 99 Yamadazyma atmosphaerica Y-17642 Yamadazyma atlantica Y-17759 a insectorum Y-7787 60 Yamadazyma mexicana Y-11818 7 Yamadazyma conglobata Y-1504 7 '— Yamadazyma aaseri YB-3897 Vere Cayo > Yamadazyma scolyti Y-5512 67| tamadazyma tenuis Y-1498 -— Yamadazyma diddensiae Y-7589 98 “_ Yamadazyma dendronema Y-7781 100 -—— Yamadazyma nakazawae var. nakazawae Y-7903 ¢— Yamadazyma nakazawae var. akitaensis Y-7904 L Yamadazyma philogaea Y-7813 Candida glucosophila Y-17781 Meyerozyma guilliermondii Y-2075 ‘| 95_-—Meyerozyma caribbica MFLUCC 17-0556 Meyerozyma caribbica MFLUCC 17-0606 Meyeroryma Meyerozyma caribbica Y-27274 _L Priceom iyces fermenticarens Y-17321 92 Priceomyces media Y-7122 Priceomyces castillae Y-7501 — Debaryomyces carsonii YB-4275 | Debaryomyces robertsiae Y-6670 Debaryomyces udenii Y-17354 Schwanniomyces castellii Y-7423 + Candida fragi Y-17910 I 99 Priceomyces Debaryomyces Y 91 Candida quercitrusa Y-5392 “LCandi da natalensis Y-17680 -—— Candida sophiaereginae Y-17668 gcandida zeylanoides Y-1774 Candida santamariae var santamariae JCM 1816 Candida santamariae var. santamariae Y-6656 Candida oleophila Y-2317 i Candida boleticola Y-17080 Candida schatavii Y-17078 {_________________ Babjeviella inositovora Y-12698 Babjeviella a a Schizosaccharomyces pombe Y-1279 I Candida 0.02 Figure 19. Phylogram generated from maximum likelihood analysis based on combined LSU and SSU sequence data. Maximum parsimony bootstrap is given above/below the nodes. The newly generated sequences are in red text. The tree is rooted with Schizosaccharomyces pombe. Conclusion In this study on fungal endophytes found on leaves of Pandanaceae, it was found that the taxa belonged to both Ascomycota and Basidiomycota. The majority of the taxa were Ascomycota, as found in most previous endophytic studies (Crozier et al. 2006; Selim et al. 2017). In classical mycology, most endophytic fungi were described based on their morphological features (Barseghyan and Wasser 2010). However, there are difficulties in identifying ascomycetes to the species level based only on morphological features (Lu et al. 2012), because they have only a small set of morphological charac- teristics and exhibit homoplasy (Barseghyan and Wasser 2010). The 22 endophytic fungal strains found in this study were chiefly identified using their microscopic characteristics and DNA sequence data and holotype materials in the 54 Saowaluck Tibpromma et al. / MycoKeys 33: 25-67 (2018) form of dried cultures. Future studies are however needed to recollect the taxa which are sporulating to describe sexual and asexual characteristics (sensu Lacap et al. 2003). In this study, 22 endophytes were isolated and sorted into eight morphotype based on colony characteristics. The authors, however, subjected all isolates to phylogenetic analysis and found they belong to 16 different taxa. The taxa were sorted roughly into morphotypes, but they did not reflect the actual species. Several isolates of this study did not sporulate, but are introduced as new species because DNA sequence com- parison and multi-gene phylogenetic analyses provided sufficient evidence to show that they are distinct taxa (Jeewon and Hyde 2016). The new taxa are, however, code compliant, as they are provided with MycoBank numbers, full descriptions, colour photographss and illustrations. The species composition of endophytic microorganisms is likely to depend on the plant age, genotype, sampled tissue, host type and season of isolation (Rosen- blueth and Martinez-Romero 2006). Promputtha et al. (2007) showed that endo- phytic species can change their ecological strategies and adopt a saprotrophic life- style. However, it was found that for the cultures of some endophytic fungal species, mycelia are the only visible morphological structures. According to these conclu- sions, the authors agree with Petrini (1991), Yanna and Hyde (2002), Ghimire and Hyde (2004) and Hyde et al. (2006) regarding the relationships between fungal endophytes and saprobic fungi. However, the use of next-generation sequencing (NGS) (Shendure and Ji 2008) is another option for identification of fungal species that cannot be cultured in vitro and has now become popular. These methods have also been applied to large-scale culture-independent molecular biological methods (Zoll et al. 2016). Future developments in technology are likely to produce further novel methods that mycologists could apply to the field of taxonomy (e.g. Hawks- worth and Lucking 2017). Acknowledgements We would like to thank Molecular Biology Experimental Centre at Kunming Institute of Botany for their help with sequencing work. Saowaluck Tibpromma thanks the Mushroom Research Foundation (MRF), Chiang Rai, Thailand for financial support. Kevin D. Hyde thanks Mae Fah Luang University for the grant “Biodiversity, phylog- eny and role of fungal endophytes of Pandanaceae” (Grant number: 592010200112) and the Chinese Academy of Sciences (project number 2013T2S0030), for the award of Visiting Professorship for Senior International Scientists, at Kunming Institute of Botany and Chiang Mai University. Andrew Stevenson, Fiona Worthy, Sajeewa Maha- rachchikumbura, Danushka Sandaruwan, Chada Norphanphoun, Asha Dissanayake, Ruvishika Jayawardena and Kasun Thambugala are thanked for their help and valu- able suggestions. Samantha C. Karunarathna thanks Yunnan Provincial Department of Human Resources and Social Security funded postdoctoral project (number 179122) and National Science Foundation of China (NSFC) project code 31750110478. Pe- Identification of endophytic fungi from leaves of Pandanaceae... 55 ter E. Mortimer thanks the National Science Foundation of China (NSFC) project codes 41761144055 and 41771063. 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