Phytotaxa 449 (2): 149–163 ISSN 1179-3155 (print edition) https://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2020 Magnolia Press Article ISSN 1179-3163 (online edition)

https://doi.org/10.11646/phytotaxa.449.2.3

Saprobic in Thailand: Phaeoseptum hydei sp. nov., a new terrestrial ascomycete in Phaeoseptaceae

DHANUSHKA N. WANASINGHE1,2,3,4,8, PETER E. MORTIMER2,9, CHANOKNED SENWANNA1,5,10 & RATCHADAWAN CHEEWANGKOON1,6,7,11* 1 Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand 2 Key Laboratory for Plant Biodiversity and Biogeography of East Asia (KLPB), Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan China. 3 World Agroforestry, East and Central Asia, 132 Lanhei Road, Kunming 650201, Yunnan, China. 4 Center for Mountain Futures, Kunming Institute of Botany, Honghe County 654400, Yunnan, China. 5 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, Thailand 6 Innovative Agriculture Research Center, Faculty of Agriculture, Chiang Mai University, 50200, Thailand. 7 Center of Excellence in Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, Thailand, 50200. 8 [email protected]; https://orcid.org/0000-0003-1759-3933 9 [email protected]; https://orcid.org/0000-0003-3188-9327 10 [email protected]; https://orcid.org/0000-0002-1008-4514 11 [email protected]; https://orcid.org/0000-0001-8576-3696 *Corresponding author

Abstract

During a survey of saprobic microfungi in Thailand, a dothideomycetous was found on a dead twig of Delonix regia, on the Chiang Mai University campus. This fungus is characterized by fully immersed ascomata under a small blackened pseudoclypeus, pseudoparenchymatous peridium, cellular pseudoparaphyses, cylindrical-clavate asci with a distinct pedicel, overlapping 3–4-seriate, pale to dark brown, broadly fusoid, 7–9-transversally septate ascospores with a vertical septum in nearly all median cells. Multigene phylogenetic analyses, using partial sequences from the 28S nrRNA gene (LSU), 18S nrRNA gene (SSU), internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) of the nrDNA operon and the translation elongation factor 1-alpha region (TEF) demonstrated a monophyletic affiliation of the new strain, accommodating the species of Phaeoseptum in the family Phaeoseptaceae. With further morphological and phylogenetic investigations, we justify the new fungus as a novel species, Phaeoseptum hydei in Phaeoseptaceae. Detailed descriptions and illustrations are provided for Phaeoseptum hydei and this novel species compared with the remaining species found in the . An updated checklist of microfungi recorded on Delonix regia from around the world is also provided.

Keywords: 32-spored asci, Delonix regia, microfungi,

Introduction

Pleosporales is one of the most species-rich orders in , including 88 accepted families and ~580 genera (Hongsanan et al. 2020). Members of Pleosporales are cosmopolitan and adapted to various habitats, sometimes with extreme environmental conditions (Wanasinghe et al. 2018a). Life modes of pleosporalean members can be recognized as epiphytes, endophytes, or parasites of living leaves or stems, hyperparasites on fungi or insects, lichenised, or as saprobes of dead plant stems, leaves, bark or dung (Zhang et al. 2012, Hyde et al. 2013, Ariyawansa et al. 2019). Phaeoseptaceae is one of these families of Pleosporales, which has recently been established. Hyde et al. (2018) introduced this family based on the genus Phaeoseptum, which had previously been placed in Halotthiaceae (Zhang et al. 2013). Hyde et al. (2018) also accommodated Lignosphaeria, Neolophiostoma, Decaisnella formosa and Thyridaria macrostomoides in Phaeoseptaceae. Most recently, Liu et al. (2019) introduced Pleopunctum to accommodate two hyphomycetous species, P. ellipticum and P. pseudoellipticum in Phaeoseptaceae and excluded Neolophiostoma from the family. After all of these amendments, the family is currently comprised of 11 species viz. Decaisnella formosa, Lignosphaeria fusispora, L. thailandica, Phaeoseptum aquaticum, P. carolshearerianum, P. mali, P. manglicola, P.

Accepted by Sajeewa Maharachchikumbura: 12 May 2020; published: 18 Jun. 2020 149 terricola, Pleopunctum ellipsoideum, P. pseudoellipsoideum and Thyridaria macrostomoides. This study is a continuity of our past studies on saprobic Dothideomycetes in Thailand (Wanasinghe et al. 2016, 2017a, b, 2018b). The first aim of the current study was to identify the new fungus, by sequencing three non-translated loci (ITS, LSU, SSU) and one protein-coding region (TEF). Secondly, we wanted to investigate the molecular phylogenetic relationships of the new strain with close alliances, using ML, MP and BI analyses in both individual and concatenated gene alignments. A third aim was to provide an up to date, global list of microfungi recorded from Delonix regia.

Materials and methods

Isolates and specimens

Fresh fungal material was collected on a dead twig of Delonix regia (Fabaceae) from Chiang Mai University campus, Chiang Mai Province, Thailand, during the dry season (February). The specimen was brought to the laboratory in a paper bag and examined with a Motic SMZ 168 Series microscope. Single ascospore isolation was carried out following the method described in Chomnunti et al. (2014). Germinated spores were individually transferred to Potato Dextrose Agar (PDA) plates and grown at 25 °C in the daylight. Isolates including accession numbers of gene sequences are listed in Table 1. Isolates listed as MFLUCC are those maintained in the collection of the Culture Collection of Mae Fah Luang University, Chiang Rai, Thailand. Specimens have been deposited in the Mae Fah Luang University (MFLU) fungarium, Chiang Rai, Thailand. Faces of Fungi and Index Fungorum numbers are provided as outlined in Jayasiri et al. (2015) and Index Fungorum (2020).

Morphological observations

Hand sections of the ascomata were mounted in tap water and the following characteristics were measured and evaluated: ascomata diameter, height, colour and shape; width of peridium; length and width (at the widest point) of asci and ascospores. Images were captured with a Canon 550D digital camera fitted to a Nikon ECLIPSE 80i compound microscope. Measurements were made with the Tarosoft (R) Image Frame Work program and images used for figures processed with Adobe Photoshop CS5 Extended version 10.0 software (Adobe Systems, USA).

DNA extraction, PCR amplifications and sequencing

Mycelia for DNA extraction from the isolate was grown on PDA for 3–4 weeks at 25 °C and total genomic DNA was extracted from the scraped axenic mycelium with a sterile scalpel. Mycelium was ground to a fine powder with liquid nitrogen and DNA extracted using the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, P.R. China) following the instructions of the manufacturer. DNA to be used as template for PCR were stored at 4 °C for use in regular work and duplicated at -20 °C for long-term storage. DNA sequence data was obtained from the partial sequences of four gene regions, the ITS, LSU, SSU and TEF. Nuclear ITS was amplified using the primers ITS5 and ITS4 (White et al. 1990). LSU was amplified using the primers LR0R and LR5 (Vilgalys & Hester 1990, Rehner & Samuels 1994). SSU was amplified using the primers NS1 and NS4 (White et al. 1990), TEF was amplified using primers EF1-983F and EF1-2218R (Liu et al. 1999, Rehner & Buckley 2005). Polymerase chain reaction (PCR) was carried out in a volume of 25 μl which contained 12.5 μl of 2 × Power Taq PCR MasterMix (Bioteke Co., China), 1 μl of each primer (10 μM), 1 μl genomic DNA and 9.5 μl deionized water. The reaction was then allowed to run for 35 cycles. The PCR profile was as follows: initial denaturation 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 50 s, elongation at 72 °C for 90 s, and final extension at 72 °C for 10 min. The amplified PCR fragments were sent to a commercial sequencing provider (BGI, Ltd Shenzhen, P.R. China). The nucleotide sequence data acquired were deposited in GenBank (Table 1).

150 • Phytotaxa 449 (2) © 2020 Magnolia Press WANASINGHE ET AL. TABLE 1. Taxa used in the phylogenetic analysis and their GenBank numbers. The newly generated sequences are indicated in bold. GenBank Accession no Species Strain no LSU SSU ITS TEF Alfoldia vorosii CBS 145501T MK589354 MK589346 JN859336 MK599320 Amorocoelophoma cassiae MFLUCC 17-2283T NG_066307 NG_065775 NR_163330 MK360041 Angustimassarina acerina MFLUCC 14-0505T KP888637 NG_063573 NR_138406 KR075168 Angustimassarina populi MFLUCC 13-0034T KP888642 NG_061204 KP899137 KR075164 Angustimassarina quercicola MFLUCC 14-0506T KP888638 NG_063574 KP899133 KR075169 Crassiclypeus aquaticus CBS 143643T LC312530 LC312472 LC312501 LC312559 Decaisnella formosa BCC 25616 GQ925846 GQ925833 - GU479851 Decaisnella formosa BCC 25617 GQ925847 GQ925834 - GU479850 Flabellascoma cycadicola CBS 143644T LC312531 LC312473 LC312502 LC312560 Forliomyces uniseptata MFLUCC 15-0765T NG_059659 NG_061234 NR_154006 KU727897 Gloniopsis praelonga CBS 112415 FJ161173 FJ161134 - FJ161090 Guttulispora crataegi MFLUCC 13-0442T KP888639 KP899125 KP899134 KR075161 Halotthia posidoniae BBH 22481 GU479786 GU479752 - - Hysterium angustatum MFLUCC 16-0623 FJ161180 GU397359 - FJ161096 Leptoparies palmarum CBS 143653T LC312543 LC312485 LC312514 LC312572 Lignosphaeria fusispora MFLUCC 11-0377T KP888646 - NR_164233 - Mauritiana rhizophorae BCC 28866 GU371824 GU371832 - GU371817 Misturatosphaeria aurantiacinotata GKM 1238T NG_059927 - - GU327761 Phaeoseptum aquaticum CBS 123113T JN644072 - KY940803 - Phaeoseptum carolshearerianum NFCCI-4221T MK307813 MK307816 MK307810 MK309874 Phaeoseptum carolshearerianum NFCCI-4384 MK307815 MK307818 MK307812 MK309876 Phaeoseptum hydei MFLUCC 17-0801T MT240623 MT240624 MT240622 MT241506 Phaeoseptum mali MFLUCC 17-2108T MK625197 - MK659580 MK647990 Phaeoseptum manglicola NFCCI-4666T MK307814 MK307817 MK307811 MK309875 Phaeoseptum terricola MFLUCC 10-0102T MH105779 MH105780 MH105778 MH105781 Platystomum crataegi MFLUCC 14-0925T KT026109 KT026113 KT026117 KT026121 Pleopunctum ellipsoideum MFLUCC 19-0390T MK804517 MK804514 MK804512 MK828510 Pleopunctum pseudoellipsoideum MFLUCC 19-0391T MK804518 - MK804513 MK828511 Pseudoaurantiascoma kenyense GKM 1195T NG_059928 - - GU327767 Pseudolophiostoma cornisporum CBS 143654T LC312544 LC312486 LC312515 LC312573 Ramusculicola thailandica MFLUCC 13-0284T KP888647 KP899131 KP899141 KR075167 Sporormurispora atraphaxidis MFLUCC 17-0742T NG_059880 NG_061296 NR_157546 - Sulcosporium thailandicum MFLUCC 12-0004T KT426563 KT426564 MG520958 - Teichospora melanommoides CBS 140733T KU601585 - NR_154632 KU601610 Teichospora pusilla CBS 140731T KU601586 - NR_154633 KU601605 Teichospora rubriostiolata CBS 140734T KU601590 - NR_154634 KU601609 Thyridaria macrostomoides GKM 1033 GU385190 - - GU327776 Thyridaria macrostomoides GKM 1159 GU385185 - - GU327778 Thyridaria macrostomoides GKM 224N GU385191 - - GU327777 Vaginatispora appendiculata MFLUCC 16-0314T KU743218 KU743219 KU743217 KU743220 Westerdykella ornata CBS 379.55 GU301880 GU296208 AY943045 GU349021

SAPROBIC DOTHIDEOMYCETES IN THAILAND Phytotaxa 449 (2) © 2020 Magnolia Press • 151 Molecular phylogenetic analyses

Sequencing and sequence alignment

Sequences generated from different primers of the four genes were analysed with other sequences retrieved from GenBank (Table 1). Sequences with high similarity indices were determined from a BLAST search to find the closest matches with taxa in Pleosporales, and from recently published data (Dayarathne et al. 2020). Loci were aligned with the online version of MAFFT v. 7 (Katoh et al. 2019) after which the alignments were manually checked and improved where necessary using BioEdit v. 7.0.5.2 (Hall 1999). Ambiguous regions were excluded from the analyses and gaps were treated as missing data.

Phylogenetic analyses

The phylogenetic methods used in this study included maximum-likelihood (ML), maximum parsimony (MP) and Bayesian criteria (BI). ML analyses for both single-locus and the final concatenated alignment were performed with RAxML-HPC2 on XSEDE v. 8.2.10 (Stamatakis 2014) using a GTR+GAMMA substitution model with 1000 bootstrap iterations. For the BI analyses, the optimal substitution model for combined dataset was determined to be GTR+I+G using MrModeltest software v. 2.2. (Nylander 2004). The BI analysis was computed with MrBayes v. 3.2.6 (Ronquist et al. 2012) with six simultaneous Markov Chain Monte Carlo chains from random trees over 2 M generations (trees were sampled every 100th generation), ending the run automatically when standard deviation of split frequencies dropped below 0.01. Both RAxML and Bayesian analyses were run on the CIPRES Science Gateway portal (Miller et al. 2012). MP analysis was conducted with PAUP v. 4.0b10 (Swofford 2002), inferring trees with the heuristic search option with TBR branch swapping and 1000 random sequence additions. The robustness of equally parsimonious trees was evaluated by 1000 bootstrap replications. Alignment gaps were treated as missing characters in the analysis, where they occurred in relatively conserved regions. Tree scores, including consistency index, retention index, rescaled consistency index and homoplasy index (CI, RI, RC and HI) were also calculated for all the trees generated under different conditions as measures of homoplasy in the data. The Kishino-Hasegawa tests (Kishino & Hasegawa 1989) were performed in order to determine whether trees were significantly different. Phylograms were visualized with FigTree v1.4.0 program (Rambaut 2012) and reorganized in Microsoft power point (2016). The finalized alignment and tree were deposited in TreeBASE, submission ID: S25995 (http://purl.org/ phylo/treebase/phylows/study/TB2: S25995).

Results

Phylogenetic analysis

The concatenated alignment (LSU, SSU, ITS and TEF) contained of 42 strains with 3277 characters representing six families (Pleosporales) including the new taxon proposed in this study. The strains of Hysterobrevium angustatum (MFLUCC 16-0623) and Gloniopsis praelonga (CBS 112415) in the Hysteriales were used as the out-group taxa. The RAxML analysis of the combined dataset yielded a best scoring tree (Fig. 1) with a final ML optimization likelihood value of -19799.686753. The matrix had 1200 distinct alignment patterns, with 24.53 % of undetermined characters or gaps. Parameters for the GTR+I+G model of the combined loci were as follows: Estimated base frequencies; A = 0.239914, C = 0.257396, G = 0.275859, T = 0.226831; substitution rates AC = 1.071858, AG = 2.431144, AT = 1.448298, CG = 1.104719, CT = 6.926814, GT = 1.000; proportion of invariable sites I = 0.484902; gamma distribution shape parameter α = 0.549328. The bootstrap support values (ML-BS) were mapped on the tree as the first value (Fig. 1; ML-BS >70 % shown). The MP analyses generated the maximum of 11 equally most parsimonious trees, the first of which is shown in Fig. 1 (Length = 3127, CI = 0.484, RI = 0.679, RC = 0.329, HI = 0.516), and the bootstrap support values (MP-BS) were mapped on the tree as the second value (MP-BS >70 % shown). From the analysed characters, 2235 were constant, 224 were variable and parsimony-uninformative and 818 were parsimony-informative. MrModelTest recommended that the Bayesian analysis should use dirichlet base frequencies and the GTR+I+G model. The BI analysis ran 170000 generations before the average standard deviation for split frequencies reached below 0.01

152 • Phytotaxa 449 (2) © 2020 Magnolia Press WANASINGHE ET AL. (0.009019). The alignment contained a total of 1201 unique site patterns. After discarding the first 20 % of generations, 1361 trees remained from which 50 % consensus trees and posterior probabilities (PP) were calculated (Fig. 1; third value: PP >0.95 shown). The ML phylogeny (Fig. 1) showed the same terminal family clades as those presented in the MP and BI phylogeny.

FIGURE 1. RAxML tree based on analysis of a combined dataset of LSU, SSU, ITS and TEF partial sequences. Bootstrap support values for ML and MP higher than 60 % and Bayesian posterior probabilities (BYPP) for BI greater than 0.95 are defined above the internal branches respectively. The ex-type strains are in bold; the new isolate is in blue. The tree is rooted to Hysterobrevium angustatum (MFLUCC 16-0623) and Gloniopsis praelonga (CBS 112415) in the Hysteriales.

SAPROBIC DOTHIDEOMYCETES IN THAILAND Phytotaxa 449 (2) © 2020 Magnolia Press • 153 To assess tree outputs, we divided the Phaeoseptaceae taxa in the cladogram into five clades (A–E). Each clade clustered separately from the rest with strong bootstrap supports for the all three formats of analyses. Our new isolate, Phaeoseptum hydei (MFLUCC 17-0801) clustered in Clade A with P. aquaticum (CBS 123113), P. carolshearerianum (NFCCI-4221, NFCCI-4384), P. mali (MFLUCC 17-2108), P. manglicola (NFCCI-4666) and P. terricola (MFLUCC 10-0102) with 100 % ML, MP and 1.00 PP statistical support (Fig. 1). Two representative strains of Decaisnella formosa (BCC 25616 and 25617) grouped in Clade B with 100 % ML, MP and 1.00 PP statistical support (Fig. 1). Three strains designated as ‘Thyridaria macrostomoides’ (GKM 1033, GKM 224N, GKM 1159) clustered in Clade C with 70 % ML, 95 % MP and 0.98 PP statistical support (Fig. 1). Clade D comprises Lignosphaeria fusispora (MFLUCC 11-0377) and L. thailandica (MFLUCC 11-0376) while Pleopunctum ellipsoideum (MFLUCC 19-0390) and P. pseudoellipsoideum (MFLUCC 19-0391) strains are basal (Clade E) and all these taxa constitute strongly supported monophyletic lineages (100 % ML /100 % MP /1.00 PP).

Taxonomy

Phaeoseptum hydei Wanas., Senwanna & Mortimer, sp. nov.

Index Fungorum Number: IF557397, Facesoffungi Number: FoF 07860; FIGURE 2. Etymology: Named in honour of British mycologist Kevin David Hyde, who celebrates his sixty-fifth birthday and for his innumerable contributions to ascomycete .

Saprobic on dead twigs in terrestrial habitats. Sexual morph: Ascomata 350–500 μm high, 300–380 μm diam. (x̅ = 411.4 × 323.6 μm, n = 10), scattered to gregarious, fully immersed under a small blackened pseudoclypeus, appearing as black, elongated regions on host surface; ascomata depressed spherical, laterally flattened. Pseudoclypeus composed of host cells with black deposits. Peridium 8–20(–40 at apex) μm wide, pseudoparenchymatous, of thin- walled cells, at apex comprising isodiametric angular cells that are more pigmented outwardly, at sides with flattened hyaline cells, at base of angular pigmented cells. Hamathecium comprising 1.5–2.5 μm (n = 20), wide septate, cellular pseudoparaphyses, situated between and above the asci, embedded in a gelatinous matrix. Asci 110–180 × 20–30 μm (x̅ = 141.5 × 36.5 μm, n = 30), 8-spored, bitunicate, fissitunicate, cylindrical-clavate, with a distinct pedicel (30–45 μm long; x̅ = 35.9 μm, n = 30), apically rounded with a minute ocular chamber. Ascospores 20–25 × 6–7 μm (x̅ = 22.3 × 6.4 μm, n = 50), uniseriate at base and overlapping 3–4-seriate at apex, pale to dark brown, broadly fusoid with broadly rounded ends, slightly curved, 7–9-transversally septate, with a vertical septum in nearly all median cells, not constricted at the septa, the septa partly pale brown, having at maturity a thickened and heavily pigmented appearance, wall smooth, without sheath or appendages. Asexual morph: Undetermined. Culture characteristics: Colonies on PDA reaching 4 cm diam. after 30 days at 25°C, circulate in shape, smooth margin, from above dark brown, dense, flattened, umbonate, fairly fluffy, reverse greenish grey. Hyphae septate branched, hyaline, thin, smooth-walled. Known distribution: Thailand, on dead twigs of Delonix regia (Boj. ex Hook.) Raf. Material examined: Thailand, Chiang Mai, Mueang Chiang Mai District, Suthep, Chiang Mai University, 18.794368N, 98.959904E, on a dead twig of Delonix regia (Fabaceae), 15 February 2017, Chanokned Senwanna DHA17-1 (MFLU 17-0660, holotype), ex-type living culture, MFLUCC 17-0801. Notes: Phaeoseptum hydei was found on a dead twig of Delonix regia from Chiang Mai, Thailand. Its morphology comparable with the type species of the genus Phaeoseptum, P. aquaticum, and other species in Phaeoseptum in having fully immersed ascomata under a small blackened pseudoclypeus, cylindrical-clavate asci with a distinct pedicel and broadly fusoid, brown ascospores with multi-transverse septa. The phylogenetic analyses of a combined sequence data-set (LSU, SSU, ITS and TEF) positions our novel species, Phaeoseptum hydei (MFLUCC 17-0801) with other remaining taxa in Phaeoseptum (Clade A) with strong statistical support (100 % ML, MP and 1.00 PP, Fig. 1). The phylogeny shows Phaeoseptum hydei is basal to Phaeoseptum species with close affinity to P. terricola (MFLUCC 10-0102). Morphologically, Phaeoseptum terricola differs from P. hydei in having 8-spored and comparatively small asci (64–90 × 13–17.5 μm) whereas P. hydei has 32-spored asci that are larger (110–180 × 20–30 μm). Phaeoseptum terricola also has a relatively smaller (137–214 high × 155–224 diam.) ascomata compared with P. hydei (350–500 high × 300–380 diam.). The 32-spored asci is a unique characteristic for all sampled fruiting bodies of the holotype and this feature has not seen any of the other species in Phaeoseptum (Table 2).

154 • Phytotaxa 449 (2) © 2020 Magnolia Press WANASINGHE ET AL. . (2016) . (2020) . (2020) et al et al et al . (2013) . (2018) et al et al Hyde Zhang Dayarathne Phukhamsakda Dayarathne Terrestrial This study Freshwater Estuarine Terrestrial Estuarine , Delonix regia Robinia pseudoacacia marina Avicennia Malus halliana marina Avicennia Suaeda monoica m) Host Habitat Reference μ 3–4-seriate 1–3-seriate 1–2-seriate 2-seriate 2–3-seriate m) ( Ascospore μ 110–180 × 20–30110–180 32-spored 20–25 × 6–7 135–190 × 19–258-spored 7–9 transverse septa 30.5–38 × 9.5–12 103–195 × 18–238-spored 9–13(–16) transverse septa 29–37.5 × 8.5–12.5 85–190 × 19–328-spored 8–10 transverse septa 27–38 × 8–13 102–212 × 17–27.58-spored 27–36 × 7.5–13 transverse septa 11–14 64–90 × 13–17.58-spored 9–13 transverse septa 19–25 × 5–7 transverse septa 9–10(–11) Unknown wood Terrestrial m) Asci ( μ species with morphological features discussed in this study. 350–500 high, 300–380 diam. 300–400 high, 400–600 diam. 290–620 high, 295–400 diam. 320–375 high, 320–360 diam. 210–420 high, 170–374 diam. 137–214 high, 155–224 diam. Phaeoseptum Synopsis of 2-seriate Species Ascomata size ( P. carolshearerianum P. P. mali manglicola P. terricola P. P. aquaticum P. Phaeoseptum hydei TABLE 2. TABLE

SAPROBIC DOTHIDEOMYCETES IN THAILAND Phytotaxa 449 (2) © 2020 Magnolia Press • 155 FIGURE 2. Phaeoseptum hydei (MFLU 17-0660, holotype). a, b. Appearance of ascomata on host substrate. c. Section of ascoma. d. Peridium. e. Pseudoparaphyses. f–j. Asci. k–n. Ascospores. o. Colony on PDA. Scale bars: b = 500 μm, c = 100 μm, d, e, k–n = 10 μm, f–j = 20 μm.

Discussion

Phaeoseptaceae is a family comprised of a heterogenous group of taxa that are monophyletic with 100 ML/ 96 MP and /1.00 BYPP bootstrap support (Fig. 1). The species in this family have diversified habitats, different types of asexual morphs and diverged morphological characteristics. The habitat of the taxa can be freshwater (Phaeoseptum aquaticum), marine (Decaisnella formosa) or terrestrial environments (Lignosphaeria spp., P. carolshearerianum, P. hydei, P. mali, P. manglicola, P. terricola, Pleopunctum spp.) (Wahab and Jones 2003, Suetrong et al. 2009, Zhang et al. 2013, Thambugala et al. 2015, Liu et al. 2019, Phukhamsakda et al. 2019). The asexual morph can be varying from hyaline coelomycetous (Lignosphaeria thailandica) to pigmented hyphomycetous (Pleopunctum ellipticum) (Thambugala et al. 2015, Liu et al. 2019). The asexual morph of Phaeoseptum is yet to be discovered. Within the family, ascomata are located under a blackened clypeus/pseudoclypeus, pseudoparenchymatous peridium with a thick apex, cellular pseudoparaphyses and having cylindrical asci with a long pedicel are shared characteristics. But the ascospore arrangement inside an ascus and the features of ascospores are dissimilar among species in this group. The spore arrangement can be uniseriate (Decaisnella formosa), biseriate (Phaeoseptum carolshearerianum) or 3–4-seriate (P. hydei). Spore colour is hyaline, pale brown to dark brown and the septations can be vary from three (Lignosphaeria fusispora) to 16 (Phaeoseptum aquaticum).

156 • Phytotaxa 449 (2) © 2020 Magnolia Press WANASINGHE ET AL. The morphology-based taxonomic information and phylogenetic sequencing data of Decaisnella species and Thyridaria macrostomoides are not linked appropriately. Such scenarios create insufficiently complete understandings of the natural classification in these genera. Broader taxon sampling, the gathering of accurate, morphology-based taxonomic information and phylogenetic sequencing data are all needed to clarify taxa into their appropriate taxonomic ranks. One exciting finding herein is the 32-spored asci of Phaeoseptum hydei. Thirty-two-spored asci is not a common feature in ascomycetes and to the best of our knowledge, out of the 20 classes in Ascomycota (Wijayawardene et al. 2020), only species from five classes are known to have 32-spore baring asci in their life cycles. They are Candelariella vitellina (Candelariales) in Candelariomycetes (Castello and Nimis 1994), Pycnidiophora and Westerdykella species (Pleosporales) in Dothideomycetes (Zhang et al. 2012), Neoprotoparmelia nigra (Lecanorales) in Lecanoromycetes (Dos Santos et al. 2019), Streptotheca psychrophila (Thelebolales) in (Bergman & Shanor 1957) and Lichenoverruculina sigmatospora (Amphisphaeriales) (Spegazzini 1889, Etayo and Rosato 2008), Podospora pleiospora, Schizothecium alloeochaetum, S. dakotense, S. simile (Sordariales) in Sordariomycetes (Doveri 2004, Cai et al. 2005). Among ascomycetous fungi, using the number of spores in an ascus should be further evaluated prior to establish as a diagnostic feature to differentiate species or generic level classification. Delonix regia (Hook.) Raf. (Poinciana regia Boj. ex Hook, Gul mohar) is an ornamental tree (10–18 m) commonly known as a ‘Flamboyant’ with fern-like bipinnately compound leaves and attractive red peacock flowers (El-Gizawy et al. 2018). Flamboyants originate from Madagascar, but it is now almost extinct there. However, this has widely used in reforestation programs, due to its high ornamental value and nitrogen fixation (Lorenzi et al. 2003). This exotic species is well adapted to tropical climates (Zuffo et al. 2016), and has been used in the afforestation programs in most tropical and subtropical areas of the world including Bermuda, Brazil, Burkina Faso, China, Cyprus, Egypt, Eritrea, Ethiopia, India, Jamaica, Kenya, Mexico, Niger, Nigeria, Puerto Rico, Singapore, South Africa, Sri Lanka, Sudan, Tanzania, Thailand, Uganda and USA.

TABLE 3. Checklist of microfungi on Delonix regia (Most were extracted form, Farr & Rossman, 2020). Species Order Family Country Reference Arthoniomycetes Arthonia sp. Arthoniales Arthoniaceae Cuba Urtiaga (1986) Dothideomycetes Diplodia sp. Botryosphaeriales Botryosphaeriaceae USA Alfieri et al. (1984) Dothiorella sp. Venezuela Urtiaga (1986) Sphaeropsis sp. USA Alfieri et al. (1984) Cercospora delonicis J.M. Yen 1964 Capnodiales Mycosphaerellaceae Singapore Yen (1964) (=Pseudocercospora delonicis*) Pseudocercospora delonicis (J.M. Yen) J.M. Singapore Crous & Braun (2003) Yen 1980 Phoma sp. Pleosporales Didymellaceae Zimbabwe Whiteside (1966) Didymosphaeria mulleri muelleri Mukerji & S. Didymosphaeriaceae India Pande & Rao (1998) Kapoor 1969 Hendersonia sp. Zimbabwe Whiteside (1966) Phaeoseptum hydei Wanas., Senwanna & Phaeoseptaceae Thailand This study Mortimer 2020 Alternaria tenuis Nees 1816 (=Alternaria Pleosporaceae India Pande & Rao (1998) alternata*) Helicomyces sp. Tubeufiales Tubeufiaceae Cuba Arnold (1986) Helicosporium sp. Cuba Arnold (1986) Dothideomycetes genera incertae sedis Pseudorobillarda sojae Uecker & Kulik 1986 - - Thailand Plaingam et al. (2005) (=Stauronematopsis sojae*) Lecanoromycetes ...continued on the next page

SAPROBIC DOTHIDEOMYCETES IN THAILAND Phytotaxa 449 (2) © 2020 Magnolia Press • 157 TABLE 3. (Continued) Species Order Family Country Reference Phaeographis sp. Graphidales Graphidaceae Cuba Urtiaga (1986) Physcia sp. Caliciales Physciaceae Venezuela Urtiaga (1986) Leotiomycetes Erysiphe quercicola S. Takam. & U. Braun 2007 Erysiphales Erysiphaceae Brazil Dallagnol et al. (2012) Phyllactinia corylea var. subspiralis E.S. India Pande & Rao (1998) Salmon 1906 (=Phyllactinia dalbergiae*) Phyllosticta sp. USA Alfieri et al. (1984) Phomopsis gymnocladi Byzova 1968 Phacidiales Phacidiaceae China Chi et al. (2007) Phomopsis sp. USA Alfieri et al. (1984) cinereal Pers. 1801 Triblidiales Sclerotiniaceae China Zhang (2006) Sordariomycetes Beltrania rhombica Penz. 1882 Amphisphaeriales Beltraniaceae New Caledonia Huguenin (1966) Dinemasporium sp. Chaetosphaeriales Chaetosphaeriaceae India Pande & Rao (1998) Tympanopsis acanthostroma (Mont.) E. Müll. Coronophorales Scortechiniaceae Puerto Rico, Virgin Stevenson (1975) & Arx 1955 Islands Delonicicola siamense R.H. Perera, Delonicicolales Delonicicolaceae Thailand Perera et al. (2018) Maharachch. & K.D. Hyde 2017 Colletotrichum sp. Glomerellales Glomerellaceae USA Alfieri et al. (1984) Acremonium alternatum Link 1809 Hypocreales Bionectriaceae India Gams (1975) Cylindrocarpon gracile Bugnic. 1939 New Caledonia Huguenin (1966) (=Calonectria brassicae*) Calonectria indusiata (Seaver) Crous 2002 Nectriaceae Germany Crous (2002) Fusarium oxysporum Schltdl. 1824 Hong Kong Lu et al. (2000), Zhuang (2001) Fusarium sp. USA, Zimbabwe Whiteside (1966), Aoki et al. (2018), Na et al. (2018) Neocosmospora sp. United States Sandoval-Denis et al. (2019) Cercosporella theae Petch 1917 (=Calonectria India Spaulding (1961) indusiata*) Cirrenalia nigrospora Somrith., Chatmala & Microascales Halosphaeriaceae Thailand Somrithipol et al. (2002) E.B.G. Jones 2002 Parascedosporium putredinis (Corda) Lackner Microascales Microascaceae Thailand Perera et al. (2018) & de Hoog 2011 Diatrypella leguminacearum M.S. Patil 1985 Xylariales Diatrypaceae India Patil (1985), Pande (2008) Diatrypella verruciformis (Ehrh.) Nitschke 1867 Bermuda Vizioli (1923) Camillea broomeana (Berk. & M.A. Curtis) Graphostromataceae Sierra Leone Laessoe et al. (1989) Læssøe, J.D. Rogers & Whalley 1989 Theissenia pyrenocrata (Theiss.) Maubl. 1914 Hypoxylaceae Sierra Leone Deighton (1936) Ustulina deusta (Hoffm.) Lind 1913 Xylariaceae Malawi, Tanzania Spaulding (1961) (=Kretzschmaria deusta*) * Current name

Micro-fungi on Delonix regia have been studied for nearly a century, with specimens being collected from around the world (Table 4). Herbert Hice Whetzel (1877–1944), an American plant pathologist and mycologist, started to report microfungi from this host by examining Diatrypella verruciformis from the fallen pods in March 1922. However, despite the long history of study, there are relatively few taxa that have been described from this host, totaling less than 40 (Table 4). Delonix regia grows well in areas with both high and scanty rainfall, that also suits for a rapid fungal growth, reproduction and speciation. Therefore, robust and updated fungal classification with more fresh sampling on this host should facilitate a proper taxonomic understanding between the host and its dependents. So far, four species

158 • Phytotaxa 449 (2) © 2020 Magnolia Press WANASINGHE ET AL. have reported from Delonix regia in Thailand viz. Cirrenalia nigrospora, Delonicicola siamense, Parascedosporium putredinis and Pseudorobillarda sojae (Table 3). In this study, we introduce another micro fungal species, Phaeoseptum hydei on this host from Thailand. This study also provides a checklist of microfungi on Delonix regia and highlights this as an ideal, but still undervalued, host to conduct diverse research on micro-fungal occurrences.

Acknowledgments

Peter E. Mortimer thanks the National Science Foundation of China and the Chinese Academy of Sciences for financial support under the following grants: 41761144055. Dhanushka Wanasinghe would like to thank CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2019PC0008) and the 64th batch of China Postdoctoral Science Foundation (grant no.: Y913083271). This research work was partially supported by Chiang Mai University. Danushka S. Tennakoon is thanked for his invaluable assistance.

References

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