Fissuroma (Aigialaceae: Pleosporales) appears to be hyperdiverse on Arecaceae: evidence from two new species from southern Thailand

ABSTRACT

Thailand and other tropical regions have high fungal diversity. Our investigation and examination of microfungi on palms (Arecaceae) revealed two new ascomycetous species of Fissuroma. Fissuroma arengae and F. wallichiae spp. nov. are introduced using morphological and phylogenetic evidence. The novel species have coriaceous ascomata, cylindrical-clavate asci and ascospores with a distinct and thin mucilaginous sheath. Fissuroma arengae is similar to F. wallichiae but can be distinguished by minor morphology, host substrate and gene base-pair differences. Phylogenetic analyses of combined LSU, ITS, SSU, tef1-α and rpb2 sequence data showed that these strains grouped within Fissuroma, further confirming this genus as monophyletic. The two new species are described and illustrated to support their taxonomic placement. Fissuroma appears to be a highly diverse genus often occurring on palms. It is likely that more research will result in numerous new taxa being discovered.

Keywords:
morphology; palm fungi; phylogeny; Thai fungi; two novel taxa

Introduction

To help understand the diversity of microfungi, we have been investigating the fungi on palms, Pandanaceae and grasses (Taylor & Hyde 2003; Hyde et al. 2007; Whitton et al. 2012; Thambugala et al. 2017; Goonasekara et al. 2018; Tibpromma et al. 2018). Our studies have constantly revealed new taxa, and would suggest that the estimated 2.2-3.8 million fungal species (Hawksworth & Lücking 2017) is certainly not an excessive number. In Thailand, the diversity has been shown to be extremely high with up to 96 % of species collected being new (Hyde et al. 2018). The high diversity of novel fungi on palms have been revealed in several studies (Hyde 1997; Fröhlich & Hyde 1999; 2000; Yanna et al. 2001; Pinnoi et al. 2006; Pinruan et al. 2007; Konta et al. 2017; Zhang et al. 2019). In one study, taxa on two adjacent palms of different genera had less than 6 % of overlapping species indicating the remarkable diversity likely to be harbored by different genera (Jones et al. 2014).

Aigialaceae was introduced by Suetrong et al. (2009) with three genera Aigialus, Ascocratera and Rimora. Aigialaceae members are characterized by carbonaceous ascomata without papilla, trabeculate pseudoparaphyses, cylindrical asci with apical rings and ascospores with a sheath or gelatinous appendages around the apical cells (Liu et al. 2011; Zhang et al. 2012; Hyde et al. 2013). Wijayawardene et al. (2018) accepted five genera in Aigialaceae, namely Aigialus, Ascocratera, Fissuroma, Neoastrosphaeriella and Rimora. Recently, Posidoniomyces was introduced by Vohník et al. (2019). Thus, six genera are currently accepted in Aigialaceae.

Fissuroma was established with F. maculans as the type species (Liu et al. 2011), and is characterized by black ascomata, opening with a slit-like ostiole, trabeculate pseudoparaphyses (sensuLiew et al. 2000), obclavate to cylindrical asci, and hyaline, fusiform, 1-septate ascospores (Liu et al. 2011; Phookamsak et al. 2015; Tennakoon et al. 2018). The asexual morph of Fissuroma is coelomycetous, being pleurophomopsis-like (Liu et al. 2011; Phookamsak et al. 2015). Currently, twenty Fissuroma species are accepted based on both morphology and phylogeny (Niranjan & Sarma 2018; Tennakoon et al. 2018; Wanasinghe et al. 2018; Zhang et al. 2020).

In this study, Fissuroma arengae and F. wallichiae are introduced as new species, from dead petioles of Arenga and Wallichia (Arecaceae), respectively. Their phylogenetic position was determined based on maximum likelihood, maximum parsimony and Bayesian inference of a combined LSU, ITS, SSU, tef1-α and rpf2 sequence dataset. Descriptions, illustrations and molecular data are provided to compare the new species with related taxa.

Materials and methods

Collection and isolation

Decayed rachides/petioles were collected from Thailand (Krabi and Phang-Nga Provinces) in 2014 on Arenga pinnata and Wallichia sp. Information on the environment, geographic location and host was recorded. The fungal taxa were identified based on morphological characteristics and phylogenetic analyses. Isolations and specimen examinations were conducted following the methods provided by Konta et al. (2016). Samples were taken to the laboratory in ziplock bags and morphological characteristics were observed using a Motic SMZ 168 series stereo-microscope. Free-hand sections were made using a razor blade and placed on a droplet of water on a glass slide. Morphological characters were observed and photo-micrographed using a Nikon ECLIPSE80i compound microscope with a fitted Canon 600D digital camera. Measurements were determined using an Image Framework program. Photo-plates were made by Adobe Photoshop CS6. Pure cultures were obtained using single ascospores isolation method (Chomnunti et al. 2014). Ascospore mass was transferred to a drop of sterile water on a flame-sterilized slide. The ascospore suspension was spread on a petri-dish containing malt extract agar (MEA) and incubated at 25-28 °C overnight. Germinating ascospores were transferred to fresh MEA dishes. Two palm samples were collected and three isolates were obtained from each sample. Holotype specimens and ex-type cultures were deposited in the herbarium of Mae Fah Luang University (MFLU) and Mae Fah Luang Culture Collection (MFLUCC) at Mae Fah Luang University, Chiang Rai, Thailand. Facesoffungi and Index Fungorum numbers were registered as outlined in Jayasiri et al. (2015) and Index Fungorum (2020).

DNA extraction and amplification (PCR)

Genomic DNA was extracted from fungal mycelium using the Biospin Fungus Genomic DNA extraction Kit (BioFlux, P.R. China) following the manufacturer’s protocol. The partial nucleotide genes were subjected to PCR amplification and sequencing of the large subunit (28S, LSU) (Vilgalys & Hester 1990), the internal transcribed spacer (ITS) (White et al. 1990), the small subunit (18S, SSU) (White et al. 1990), the translation elongation factor 1-alpha (tef1-α) (Rehner 2001; Rehner & Buckley 2005) and the RNA polymerase II second largest subunit (rpb2) (Liu et al. 1999). For primers and conditions see Table 1.

Table 1
Details of genes/loci with PCR primers and PCR conditions.

Genes/loci	PCR primers (forward/reverse)	PCR conditions
LSU, ITS, SSU, tef1-α	LR5/LR0R, ITS5/ITS4, NS4/NS1, EF1-983F/EF1-2218R	a; 95 °C: 30 s, 55 °C: 50 s, 72°C: 30 s (35 cycles); c
rpb2	fRPB2-5f/fRPB2-7cR	b; 95 °C: 1 min, 54 °C: 2 min, 72 °C: 1.5 min (35 cycles); c

• ^a
  Initiation step of 95 °C: 3 min. ^b Initiation step of 95 °C: 5 min. ^c Final elongation step of 72 °C: 10 min and final hold at 4 °C.

The total volume of PCR mixtures for amplification were 25 μl containing 8.5 μl ddH₂O, 12.5 μl 2× Easy Taq PCR SuperMix (mixture of Easy Taq TM DNA Polymerase, dNTPs and optimized buffer (Beijing Trans Gen Biotech Co., Beijing, P.R. China), 2 μl of DNA template, 1 μl of each forward and reverse primers (10 pM). The quality of PCR products was checked on 1 % agarose gel electrophoresis stained with 4S green nucleic acid (Life Science Products & Services, Shanghai, P.R. China). Purification and sequencing of PCR products were carried out by Sangon Biotech Co., Shanghai, P.R. China. The resulting fragments were sequenced in both forward and reverse directions. The DNA sequences generated were analysed. Consensus sequences were computed using SeqMan software. The new sequences generated in this study were deposited in GenBank (Tab. 2).

Table 2
GenBank accession numbers of sequences used in phylogenetic analysis.

Species	Strains	GenBank accession numbers					References
		LSU	ITS	SSU	tef1-α	rpb2
Aigialus grandis T	BCC 18419	GU479774	-	GU479738	GU479838	GU479813	Suetrong et al. 2009
A. grandisT	BCC 20000	GU479775	-	GU479739	GU479839	GU479814	Suetrong et al. 2009
A. mangrovis	BCC 33563	GU479776	-	GU479741	GU479840	GU479815	Suetrong et al. 2009
A. parvus	BCC 32558	GU479779	-	GU479743	GU479843	GU479818	Suetrong et al. 2009
A. parvus	BCC 18403	GU479778	-	GU479744	GU479842	GU479817	Suetrong et al. 2009
A. rhizophorae	BCC 33572	GU479780	-	GU479745	GU479844	GU479819	Suetrong et al. 2009
A. rhizophorae	BCC 33573	GU479781	-	GU479746	GU479845	GU479820	Suetrong et al. 2009
Ascocratera manglicola T	JK 5262C	GU301799	-	GU296136	-	GU371763	Schoch et al. 2009
A. manglicolaT	HHUF 30032	GU479783	-	GU479748	GU479847	GU479822	Suetrong et al. 2009
A. manglicolaT	BCC 09270	GU479782	-	GU479747	GU479846	GU479821	Suetrong et al. 2009
Astrosphaeriella fusispora T, E	MFLUCC 10-0555	KT955462	-	KT955443	KT955425	KT955413	Phookamsak et al. 2015
A. neofusisporaE	MFLUCC 11-0161	KT955463	-	KT955444	KT955426	KT955418	Phookamsak et al. 2015
Fissuroma aggregatum	KT 984	AB524591	-	AB524450	AB539105	AB539092	Tanaka et al. 2009
F. aggregatum	KT 767	AB524590	-	AB524449	-	-	Tanaka et al. 2009
F. arengae E	MFLUCC 15-0325A	MN726232	MN726238	MN726244	MN937554	-	This study
F. arengae	MFLUCC 15-0325B	MN726233	MN726239	MN726245	MN937555	-	This study
F. arengae	MFLUCC 15-0325C	MN726234	MN726240	MN726246	MN937556	-v	This study
F. bambusaeE	MFLUCC 11-0160	KT955468	-	KT955448	KT955430	KT955417	Phookamsak et al. 2015
F. bambusae	MFLUCC 11-0198	KT955469	-	KT955449	KT955431	KT955449	Phookamsak et al. 2015
F. calamiE	MFLUCC 13-0836	MF588993	-	MF588983	MF588975	-	Wanasinghe et al. 2018
F. caryotae	MFLUCC 16-1383	MN712335	MN735992	MN699322	MN744228	-	Zhang et al. 2020
F. caryotaeE	MFLU 17-1253	MF588996	-	MF588986	MF588979	-	Wanasinghe et al. 2018
F. maculansT, E	MFLUCC 10-0886	JN846724	JN846710	JN846734	-	-	Liu et al. 2011
F. maculans	MFLUCC 10-0887	JN846725	JN846712	JN846736	-	-	Phookamsak et al. 2015
F. maculans	MFLUCC 10-0888	JN846726	JN846713	JN846737	-	-	Phookamsak et al. 2015
F. maculans	MFLUCC 11-0023	JN846728	JN846714	JN846738	-	-	Phookamsak et al. 2015
F. neoaggregatumE	MFLUCC 10-0554	KT955470	-	KT955450	KT955432	KT955412	Phookamsak et al. 2015
F. neoaggregatum	MFLUCC 13-0227	KT955471	-	KT955451	KT955433	KT955407	Phookamsak et al. 2015
F. palmae E	MFLU 19-0820	MN712336	-	-	MN744229	-	Zhang et al. 2020
F. taiwanenseE	FU30861	MG189605	-	MG189607	MG252072	-	Tennakoon et al. 2018
F. taiwanense	FU30862	MG189606	-	MG189608	MG252073	-	Tennakoon et al. 2018
F. thailandicum	MFLUCC 11-0189	KT955472	-	KT955452	KT955434	KT955423	Phookamsak et al. 2015
F. thailandicumE	MFLUCC 11-0206	KT955473	-	KT955453	KT955435	KT955415	Phookamsak et al. 2015
F. wallichiae	MFLUCC 15-0315A	MN726235	MN726241	MN726247	MN953045	-	This study
F. wallichiae E	MFLUCC 15-0315B	MN726236	MN726242	MN726248	MN953046	MN915108	This study
F. wallichiae	MFLUCC 15-0315C	MN726237	MN726243	MN726249	MN953047	-	This study
Neoastrosphaeriella aquatica E	MFLUCC 18-0209	MK138829	MK138710	MK138789	-	-	Bao et al. 2019
N. krabiensisT	MFLUCC 11-0022	JN846727	JN846711	JN846735	-	-	Liu et al. 2011
N. krabiensisT, E	MFLUCC 11-0025	JN846729	-	JN846739	-	-	Liu et al. 2011
N. sribooniensisE	MFLUCC 13-0834	MF588997	-	MF588987	MF588977	-	Wanasinghe et al. 2018
Posidoniomyces atricolor T, E	BRK-21	MK656107	MK656111	MK656115	-	-	Vohník et al. 2019
P. atricolorE	BRK-97	MK656110	MK656114	MK656118	-	MK656935	Vohník et al. 2019
Rimora mangrovei T	JK 5246A	GU301868	-	GU296193	-	GU371759	Schoch et al. 2009
R. mangroveiT	JK 5437B	GU479798	-	GU479765	-	-	Schoch et al. 2009

• Notes: Newly generated sequences are in bold; ^T denotes the type species of the genus; ^E denotes the ex-type culture.

Phylogenetic analyses

The sequences generated in this study were subjected to BLAST search in GenBank to identify closely related sequences. Sequence data retrieved from GenBank and recent publications were used as references (Tennakoon et al. 2018; Wanasinghe et al. 2018; Zhang et al. 2020). Sequences of the LSU, ITS, SSU, tef1-α and rpb2 were analysed individually and in combination. A total of 44 taxa were used for the phylogenetic analyses. Astrosphaeriella fusispora (MFLUCC 10-0555) and A. neofusispora (MFLUCC 11-0161) were selected as the outgroup taxa. Absent sequence data (i.e. ITS, tef1-α, rpb2 sequence data) in the alignments were treated as missing data. Sequence alignments were carried out with MAFFT v.6.864b (Katoh & Standley 2013) and were manually improved where necessary. The single gene datasets were combined using Mega7 (Kumar et al. 2016). Data were converted from fasta to nexus and PHYLIP format with Alignment Transformation Environment online, https://sing.ei.uvigo.es/ALTER/ (Glez-Peña et al. 2010). The tree topologies obtained from single gene sequence data were compared prior to the combined gene analysis for checking the incongruence in the overall topology of the phylogenetic tree.

Maximum likelihood (ML) analysis was performed using RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008; Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) with GTRGAMMA model and set as 1,000 bootstrap replicates. Bayesian analysis was performed at CIPRES using Bayesian analysis on XSEDE (v.3.2.6) as part of the “MrBayes on XSEDE” tool (Huelsenbeck & Ronquist 2001; Miller et al. 2010). GTR+I+G model was selected by using MrModelTest 2.2 (Nylander 2004) under the Akaike information criterion (AIC) as the best-fit models of the combined dataset for maximum likelihood and Bayesian analysis (Nylander 2004). Bayesian posterior probabilities (BYPP) were determined by Markov chain Monte Carlo sampling (MCMC) in MrBayes on XSEDE v.3.2.6. Six simultaneous Markov chains were run for 815,000 generations and trees were sampled every 1,000^th generation. MCMC heated chain was set with a “temperature” value of 0.20. All sampled topologies beneath the asymptote (25 %) were discarded as part of a burn-in procedure; the remaining trees (816) were used for calculating posterior probabilities in the majority rule consensus tree. Maximum parsimony (MP) analysis was carried out with PAUP v 4.0b10 (Swofford 2002). Statistical supports for branches of the most parsimonious tree were estimated using maximum parsimony bootstrap analysis with 1,000 bootstrap replicates (Felsenstein 1985). All characters were unordered and of equal weight, and gaps were treated as missing data. Descriptive tree statistics for parsimony (tree length [TL], consistency index [CI], retention index [RI], relative consistency index [RC] and homoplasy index [HI]) were calculated for trees generated under different optimality criteria. Bootstrap support values for ML, MP and BYPP are given near to each node (Fig. 1).

Figure 1
Bayesian inference tree based on a combined dataset of LSU, ITS, SSU, tef1-α and rpb2 partial sequences. Bootstrap support values for maximum likelihood (ML), maximum parsimony (MP) higher than 50 % and Bayesian posterior probabilities (BYPP) greater than 0.90 are given above each branch respectively. Branches with 100 % ML, 100 % MP and 1.00 BYPP are shown with a red dot. Novel taxa are in blue. Ex-type strains are in bold. The tree is rooted to Astrosphaeriella fusispora (MFLUCC 10-0555) and A. neofusispora (MFLUCC 11-0161) (Astrosphaeriellaceae).

The phylogenetic trees were configured in FigTree v1.4.0 (Rambaut 2012) and edited using Microsoft Office PowerPoint 2010 and Adobe Photoshop CS6 (Adobe Systems, USA). The alignments and respective phylogenetic trees were deposited in TreeBASE, submission ID: 25410 (http://www.treebase.org/).

Results

Phylogenetic analyses

The combined multigene dataset comprised 44 taxa from seven genera (Aigialus, Ascocratera, Fissuroma, Neoastrosphaeriella, Posidoniomyces, Rimora (Aigialaceae), and Astrosphaeriella (Astrosphaeriellaceae) in Pleosporales (Tab. 2). The RAxML analysis of the combined dataset yielded the best-scoring tree (Fig. 1) with a final ML optimization likelihood value of -18651.882128. The alignment comprised a total of 5,088 characters including gaps. The matrix had 1,359 distinct alignment patterns, with 36.76 % undetermined characters or gaps, 3,872 constant characters and 207 parsimony-uninformative characters. Estimated base frequencies were: A = 0.249050, C = 0.245449, G = 0.278730, T = 0.226771; substitution rates AC = 1.424415, AG = 3.977296, AT = 0.940998, CG = 1.292848, CT = 9.900102, GT = 1.000000; gamma distribution shape parameter α = 0.179440. Tree-Length = 0.781448. Maximum parsimony analysis of the remaining 1,009 parsimony-informative characters resulted in 1,000 trees with TL = 2261, CI = 0.677, RI = 0.843, RC = 0.571, HI = 0.323. Bayesian posterior probabilities from MCMC were evaluated with a final average standard deviation of the split frequency of 0.009698. The Bayesian analysis resulted in a tree with similar topology and clades as the ML and MP trees. The bootstrap values for ML and MP greater than 50 % and BYPP more than 0.90 are given at the nodes. Phylogenetic analyses of combined LSU, ITS, SSU, tef1-α and rpb2, showed that the two novel species of Fissuroma clustered in a single clade within Aigialaceae.

Fissuroma arengae Konta & K.D. Hyde., sp. nov.

Index Fungorum number: IF557045, Facesoffungi number: FoF06903, Fig. 2

Etymology: Epithet refers to host genus, Arenga.

Holotype: MFLU 15-0300.

Figure 2
Fissuroma arengae (MFLU 15-0300, holotype). A, B: The forest in Phang-nga Province. C: Palm samples (Arenga pinnata, Arecaceae). D: Appearance of ascomata on host. E: Close-up of ascoma. F: Vertical cut of ascoma. G: Vertical section of ascoma. H: Section of peridium. I: Close-up of ostiole. J-M: Asci and ascospores with pseudoparaphyses. N: Pseudoparaphyses. O-R: Ascospores. S, T: Germinated ascospores. U: Colony on MEA. bars: D = 1,000 μm, E, F = 500 μm, G = 200 μm, H-N = 50 μm, O-T = 20 μm.

Saprobic on dead rachis of Arenga pinnata. Sexual morph: Ascomata 510-970 μm long, in vertical section 175-350 μm high, 425-870 μm diam., dark brown, coriaceous, solitary, scattered, gregarious, hemispherical, semi-immersed, immersed beneath host epidermis, appearing as raised areas. Ostioles central, apapillate, with carbonaceous, thin, slit-like opening. Peridium 40-76 μm wide at sides, 8-23 μm wide at base, dark brown to black, thick-walled, of textura prismatica, poorly developed at the base, thick at sides towards the apex. Hamathecium composed of dense, 1.1-2.7 μm wide, trabeculate, hyaline pseudoparaphyses, anastomosing at the apex, embedded in a gelatinous matrix. Asci 110-170 × 20-30 μm (x̅ = 133 × 24 μm, n = 30), 8-spored, bitunicate, fissitunicate, cylindrical to obclavate, short pedicellate, narrow and rounded apex, with a small ocular chamber. Ascospores 40-55 × 10-16 μm (x̅ = 48 × 14 μm, n = 30), overlapping, 1-2-seriate, fusiform, hyaline, smooth-walled, tapering to pointed apices, 1-septate, septum near median, constricted at the septum, straight to curved, surrounded by a thin distinctive sheath, 1.5-3.8 μm wide (x̅ = 2.6 μm, n = 30), smooth-walled, with two large guttules near septum, and two minute guttules towards ends of ascospores. Asexual morph: Undetermined. Appressoria: not formed. Distribution: Thailand.

Culture characteristics: Colonies on MEA reaching 20 mm diam. after two weeks at 25-30 °C, colonies medium dense, circular, convex, surface slightly rough with edge entire, effuse, velvety to hairy, margin well-defined, colony from above, white to cream at the margin, brown at the center, not producing pigments in culture.

Material examination: THAILAND, Phang-Nga Province, on dead rachis and leaflet of Arenga pinnata (Wurmb) Merr. (Arecaceae), 5 December 2014, Sirinapa Konta, PHR07f (MFLU 15-0300, holotype); ex-type living culture, MFLUCC 15-0325A; ibid. MFLUCC 15-0325B and MFLUCC 15-0325C

Notes: Fissuroma arengae is similar to F. maculans in having ascomata with slit-like ostioles and hyaline, fusiform, didymosporous ascospores. However, F. arengae has larger asci (110-170 × 20-30 μm vs. 65-125 × 10-17 μm) and ascospores (40-55 × 10-16 μm vs. 29-38 × 4-8 μm). In addition, the ascospores of F. arengae are smooth whereas those of F. maculans are verruculose (Liu et al. 2011). In phylogenetic analyses, F. arengae formed a separate branch as a sister clade to F. maculans with high support (93 % ML, 99 % MP, 0.99 BYPP, Fig. 1). Comparison of the LSU, SSU and ITS genes of F. arengae and F. maculans showed minimal nucleotide differences; 2/841 bp (0.24 %) in LSU, 1/992 bp (0.1 %) in SSU and 3/530 bp (0.57 %) in ITS (Tab. 3). Thus, a comparison of base pairs of LSU, SSU and ITS cannot separate all species of Fissuroma (Tab. 3).

Table 3
A comparison of the nucleotide polymorphisms of Fissuroma arengae (MFLUCC 15-0325) for all species in Fissuroma.

Species	Strain number	LSU	ITS	SSU	tef1-α	References
F. aggregatum	KT984	2.4 % (21/856 bp)	-	1.0 % (10/988 bp)	6.87 % (65/945 bp)	Tanaka et al. 2009
F. bambusae	MFLUCC 11-0160	4.34 % (35/805 bp)	-	1.24 % (10/808 bp)	7.82 % (68/869 bp)	Phookamsak et al. 2015
F. calami	MFLUCC 13-0836	0.87 % (7/807 bp)	-	0.66 % (7/1057 bp)	5.5 % (48/872 bp)	Wanasinghe et al. 2018
F. caryotae	MFLU 17-1253, MFLUCC 16-1383 (for ITS)	0.84 % (7/830 bp)	9.67 % (54/558 bp)	0.57 % (6/1057 bp)	4.13 % (36/872 bp)	Wanasinghe et al. 2018; Zhang et al. 2020
F. maculans	MFLUCC 10-0886	0.24 % (2/841 bp)	0.57 % (3/530 bp)	0.1 % (1/992 bp)	-	Liu et al. 2011
F. neoaggregatum	MFLUCC 10-0554	4.15 % (33/795 bp)	-	1.56 % (15/964 bp)	7.22 % (63/872 bp)	Phookamsak et al. 2015
F. palmae	MFLU 19-0820	1.64 % (14/853 bp)	-	-	4.62 % (30/648 bp)	Zhang et al. 2020
F. thailandicum	MFLUCC 11-0206	4.12 % (348/825 bp)	-	1.23 % (12/937 bp)	8.57 % (66/872 bp)	Phookamsak et al. 2015
F. wallichiae	MFLUCC 15-0315	0.22 % (2/901 bp)	2.11 % (13/615 bp)	0 % (0/1049 bp)	1.48 % (15/1014 bp)	This study
F. taiwanense	FU30861	1.1 % (10/903 bp)	-	1.1 % (11/1028 bp)	3.36 % (30/892 bp)	Tennakoon et al. 2018

• Notes: ‘-’ do not have sequence; ‘0’ no base pair similarity; base pair differences included gaps.

Fissuroma wallichiae Konta & K.D. Hyde., sp. nov.

Index Fungorum number: IF557046, Facesoffungi number: FoF06904, Fig. 3

Etymology: Epithet refers to host genus, Wallichia.

Holotype: MFLU 15-0290.

Figure 3
Fissuroma wallichiae (MFLU 15-0290, holotype). A: The forest in Krabi Province. B: Palm samples (Wallichia sp., Arecaceae). C, D: Appearance of ascomata on host. E: Vertical cut of ascoma. F: Vertical section of ascoma. G: Close-up of ostiole. H: Section of peridium. I-L: Asci and ascospores with pseudoparaphyses. M: Pseudoparaphyses. N-S: Ascospores. T, U: Germinated ascospores. V: Colony on MEA. bars: B = 3 cm, C, D = 1,000 μm, E = 500 μm, F = 100 μm, G, H, N-U = 20 μm, I-M = 50 μm.

Saprobic on dead petioles of Wallichia sp. Sexual morph: Ascomata 360-870 μm long, in vertical section 170-330 μm high, 400-650 μm diam., dark brown, coriaceous, solitary, scattered, gregarious, hemispherical, semi-immersed, immersed beneath host epidermis, appearing as raised areas. Ostioles central, apapillate, with carbonaceous, thin, slit-like opening. Peridium 30-95 μm wide at sides, 19-75 μm wide at base, dark brown to black, thick-walled, of textura prismatica, poorly developed at the base, thick at sides towards the apex. Hamathecium composed of dense, 1-2.4 μm wide, trabeculate, hyaline pseudoparaphyses, anastomosing at the apex, embedded in a gelatinous matrix. Asci 105-150 × 16-30 μm (x̅ = 123 × 24 μm, n = 30), 8-spored, bitunicate, fissitunicate, cylindrical to obclavate, short pedicellate, narrow and rounded apex, with a small ocular chamber. Ascospores 40-55 × 6-20 μm (x̅ = 48 × 10 μm, n = 30), overlapping, 1-3-seriate, fusiform, hyaline, smooth-walled, tapering to pointed apices, 1-septate, septum nearly median, constricted at the septum, straight to curved, surrounded by a thin distinctive sheath, 1.2-4.1 μm wide (x̅ = 2.5 μm, n = 30), smooth-walled, with two large guttules near septum, and two minute guttules towards ends of ascospores. Asexual morph: Undetermined. Appressoria: not formed. Distribution: Thailand.

Culture characteristics: Colonies on MEA reaching 10-20 mm diameter after 2 weeks at 25-30 °C, colonies medium dense, circular, convex, surface slightly rough with edge entire, effuse, velvety to hairy, margin well-defined, colony from above, white to cream at the margin, brown to dark greenish at the center, producing brown pigments in the agar.

Material examination: THAILAND, Krabi Province, on dead petioles of Wallichia sp. (Arecaceae), 3 December 2014, Sirinapa Konta, KBC03b (MFLU 15-0290, holotype); ex-type living culture, MFLUCC 15-0315B; ibid. MFLUCC 15-0315A and MFLUCC 15-0315C.

Notes: Phylogenetically, F. wallichiae is closely related to F. taiwanense with statical support (53 % ML, 91 % MP, 0.94 BYPP, Fig. 1). Fissuroma wallichiae resembles F. taiwanense in having ascomata with slit-like ostioles and hyaline, fusiform, 1-septate ascospores. F. wallichiae differs from F. taiwanense by having larger ascomata (in vertical section part), with dark brown to black-walled peridium, thin a mucilaginous sheath without club-shaped appendages at ends of ascospores (Tennakoon et al. 2018). Comparison of the LSU, SSU and tef1-α nucleotides of F. wallichiae and F. taiwanense showed a high difference in nucleotide bases; 12/904 bp (1.32 %) in LSU, 11/1029 bp (1.06 %) in SSU and 34/892 bp (3.81 %) in tef1-α (Tab. 4). In addition, F. wallichiae was found on a palm in Thailand while F. taiwanense was found on Hedychium coronarium (Zingiberaceae) in Taiwan. We also compared base pairs difference of F. wallichiae to all species of Fissuroma that are available in GenBank (Tab. 4).

Table 4
A comparison of the nucleotide polymorphisms of Fissuroma wallichiae (MFLUCC 15-0315) for all species in Fissuroma.

Species	Strain number	LSU	SSU	tef1-α	ITS	rpb2	References
F. aggregatum	KT984	2.79 % (24/861 bp)	1.1% (10/988 bp)	7.13 % (67/939 bp)	-	7.83 % (84/1072 bp)	Tanaka et al. 2009
F. arengae	MFLUCC 15-325	0.22 % (2/901 bp)	0 % (0/1049 bp)	1.48 % (15/1014 bp)	2.11 % (13/615 bp)	-	This study
F. bambusae	MFLUCC 11-0160	4.59 % (37/805 bp)	1.24 % (10/808 bp)	7.94 % (69/869 bp)	-	8.4 % (69/819 bp)	Phookamsak et al. 2015
F. calami	MFLUCC 13-0836	1.11 % (9/809 bp)	0.86 % (9/1036 bp)	6.42 % (56/872 bp)	-	-	Wanasinghe et al. 2018
F. caryotae	MFLU 17-1253, MFLUCC 16-1383 (for ITS)	1.08 % (9/829 bp)	0.76 % (8/1055 bp)	4.58 % (40/872 bp)	9.13 % (51/558 bp)	-	Wanasinghe et al. 2018; Zhang et al. 2020
F. maculans	MFLUCC 10-0886	0.49 % (4/809 bp)	0.1 % (1/992 bp)	-	2.63 % (14/531 bp)	-	Liu et al. 2011
F. neoaggregatum	MFLUCC 11-0206	4.4 % (35/795 bp)	1.35 % (13/963 bp)	7.56 % (66/872 bp)	-	9.27 % (76/819 bp)	Phookamsak et al. 2015
F. palmae	MFLU 19-0820	1.64 % (14/851 bp)	-	4.62 % (30/648 bp)	-	-	Zhang et al. 2020
F. taiwanense	FU30861	1.32 % (12/904 bp)	1.06 % (11/1029 bp)	3.81 % (34/892 bp)	-	-	Tennakoon et al. 2018
F. thailandicum	MFLUCC 11-0206	4.36 % (36/824 bp)	1.17 % (11/937 bp)	7.91 % (69/872 bp)	-	8.66 % (71/819 bp)	Phookamsak et al. 2015

• Notes: ‘-’ do not have sequence; ‘0’ no base pair similarity; base pair differences included gaps.

Discussion

Liu et al. (2011) introduced Fissuroma with F. maculans as type species and F. aggregatum. Subsequently, another seven species were introduced based on morphology and molecular data (Phookamsak et al. 2015; Wanasinghe et al. 2018; Tennakoon et al. 2018; Zhang et al. 2020). Fissuroma kavachabeejae and F. microsporum were introduced using morphology (Niranjan & Sarma 2018). Most recently, F. palmae was described by Zhang et al. (2020). Fissuroma species have been recorded on two host families (Arecaceae, Poaceae) of which eight from twenty species were found on Arecaceae (F. calami, F. caryotae, F. fissuristoma, F. kavachabeejae, F. maculans, F. microsporum, F. palmae) and this supports that Fissuroma is hyperdiverse on palms (Liu et al. 2011; Phookamsak et al. 2015; Niranjan & Sarma 2018; Wanasinghe et al. 2018; Zhang et al. 2020). All known species have been recorded from Asian countries (Brunei, China, India, Japan, Taiwan, Thailand), and only from terrestrial habitats.

Morphological characters of F. arengae and F. wallichiae fit well with the description of the generic type of Fissuroma. Our phylogenetic analysis also supported the new taxa placement within Fissuroma. A comparison of nucleotide base pairs in Tables 3 and 4 which showed that the protein-coding gene regions (tef1-α, rpb2) have a high percentage of base pair differences. A comparison of LSU and ITS nucleotides revealed a high percentage of base pair differences for some species, while SSU showed few differences (< 1.6 %) among species (Tabs. 3, 4). The phylogeny recovered herein also agrees with that previously established for Fissuroma species in Aigialaceae (Tennakoon et al. 2018; Wanasinghe et al. 2018; Zhang et al. 2020). However, the phylogenetic analysis of combined LSU, ITS and SSU sequence data could not separate species of Fissuroma well. Thus, for species-level identification of Fissuroma it is better to use protein-coding genes such as tef1-α and rpb2; rpb2 sequences are required to confirm the monophyly of these species.

Acknowledgements

KD Hyde thanks the Thailand Research Funds for the grant “Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (RDG6130001)”. Sirinapa Konta is grateful to Saowaluck Tibpromma, SC Karunarathna, EHC McKenzie, Zhang Shengnan, Ishani Goonasekara, Sirilak Radbouchoom, Ausana Mapook, Paul Kirk and Shaun Pennycook for their valuable suggestions. Mingkwan Doilom thanks the 64th batch of China Postdoctoral Science Foundation (Y913082271) and the 5th batch of Postdoctoral Orientation Training Personnel in Yunnan Province (Y934283261).

References

• Bao DF, Luo ZL, Jeewon R, Nalumpang S, Su HY, Hyde KD. 2019. Neoastrosphaeriella aquatica sp. nov. (Aigialaceae), a new species from freshwater habitats in southern Thailand. Phytotaxa 391: 197-206.
• Chomnunti P, Hongsanan S, Aguirre-Hudson B, et al 2014. The sooty moulds. Fungal Diversity 66: 1-36.
• Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791.
• Fröhlich J, Hyde KD. 1999. Biodiversity of palm fungi in the tropics: are global fungal diversity estimates realistic. Biodiversity & Conservation 8: 977-1004.
• Fröhlich J, Hyde KD. 2000. Palm microfungi. Fungal Diversity Press 3: 1-375.
• Glez-Peña D, Gómez-Blanco D, Reboiro-Jato M, Fdez-Riverola F, Posada D. 2010. ALTER: program-oriented conversion of DNA and protein alignments. Nucleic Acids Research 38: 14-18.
• Goonasekara ID, Jayawardene RS, Saichana N, Hyde KD. 2018. Checklist of microfungi on grasses in Thailand (excluding bambusicolous fungi). Asian Journal of Mycology 1: 88-105.
• Hawksworth DL, Lücking R. 2017. Fungal diversity revisited: 2.2 to 3.8 million species. The Fungal Kingdom 4: 79-95.
• Huelsenbeck JP, Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755.
• Hyde KD, Bussaban B, Paulus B, et al 2007. Diversity of saprobic microfungi. Biodiversity and Conservation 16: 7-35.
• Hyde KD, Jones EBG, Liu JK, et al 2013. Families of Dothideomycetes. Fungal Diversity 63: 1-313.
• Hyde KD, Norphanphoun C, Chen J, et al 2018. Thailand’s amazing diversity: up to 96 % of fungi in northern Thailand may be novel. Fungal Diversity 93: 215-239.
• Hyde KD. 1997. Diversity of ascomycetes on palms in the tropics. Biodiversity of Tropical Microfungi 1: 141-156.
• Index Fungorum. 2020. Index Fungorum. http://www.indexfungorum.org/names/Names.asp 30 Oct. 2019.
  » http://www.indexfungorum.org/names/Names.asp
• Jayasiri SC, Hyde KD, Ariyawansa HA, et al 2015. The Faces of Fungi database: fungal names linked with morphology, phylogeny and human impacts. Fungal Diversity 74: 3-18.
• Jones EG, Hyde KD, Pang KL. 2014. Freshwater fungi: and fungal-like organisms. Berlim, Boston, Walter de Gruyter GmbH & Co KG.
• Katoh K, Standley K. 2013. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30: 772-780.
• Konta S, Hongsanan S, Eungwanichayapant PD, et al 2017. Leptosporella (Leptosporellaceae fam. nov.) and Linocarpon and Neolinocarpon (Linocarpaceae fam. nov.) are accommodated in Chaetosphaeriales. Mycosphere 8: 1943-1974.
• Konta S, Hongsanan S, Tibpromma S, et al 2016. An advance in the endophyte story: Oxydothidaceae fam. nov. with six new species of Oxydothis Mycosphere 7: 1425-1446.
• Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.
• Liew EC, Aptroot A, Hyde KD. 2000. Phylogenetic significance of the pseudoparaphyses in Loculoascomycete taxonomy. Molecular Phylogenetics and Evolution 16: 392-402.
• Liu JK, Phookamsak R, Jones EBG, et al 2011. Astrosphaeriella is polyphyletic, with species in Fissuroma gen. nov., and Neoastrosphaeriella gen. nov. Fungal Diversity 51: 135-154.
• Liu YJ, Whelen S, Hall BD. 1999. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799-1808.
• Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees. SC10 Workshop on Gateway Computing Environments (GCE10). https://www.phylo.org/sub_sections/portal/sc2010_paper.pdf
  » https://www.phylo.org/sub_sections/portal/sc2010_paper.pdf
• Niranjan M, Sarma VV. 2018. New Ascomycetous fungi in the family Aigialaceae from Andaman Islands, India. Current Research in Environmental & Applied Mycology (Journal of Fungal Biology) 8: 351-359.
• Nylander JAA. 2004. MrModeltest 2.0. Program distributed by the author. Evolutionary Biology Centre. Uppsala, Uppsala University.
• Phookamsak R, Norphanphoun C, Tanaka K, et al 2015. Towards a natural classification of Astrosphaeriella-like species; introducing Astrosphaeriellaceae and Pseudoastrosphaeriellaceae fam. nov. and Astrosphaeriellopsis, gen. nov. Fungal Diversity 74: 143-197.
• Pinnoi A, Lumyong S, Hyde KD, Jones EBG. 2006. Biodiversity of fungi on the palm Eleiodoxa conferta in Sirindhorn peat swamp forest, Narathiwat, Thailand. Fungal Diversity 22: 205-218.
• Pinruan U, Hyde KD, Lumyong S, McKenzie EHC, Jones EBG. 2007. Occurrence of fungi on tissues of the peat swamp palm Licuala longicalycata Fungal Diversity 25: 157-173.
• Rambaut A. 2012. FigTree version 1.4.0. http://tree.bio.ed.ac.uk/software/figtree/. 1 Nov. 2019.
• Rehner S. 2001. Primers for Elongation Factor 1-α (EF1-α). http://ocid.NACSE.ORG/research/deephyphae/EF1primer. pdf
  » http://ocid.NACSE.ORG/research/deephyphae/EF1primer. pdf
• Rehner SA, Buckley E. 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97: 84-98.
• Schoch CL, Crous PW, Groenewald JZ, et al 2009. A class-wide phylogenetic assessment of Dothideomycetes. Studies in Mycology 64: 1-15.
• Stamatakis A, Hoover P, Rougemont J. 2008. A rapid bootstrap algorithm for the RAxML web servers. Systematic Biology 57: 758-771.
• Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313.
• Suetrong S, Schoch CL, Spatafora JW, et al 2009. Molecular systematics of the marine Dothideomycetes. Studies in Mycology 64: 155-173.
• Swofford DL. 2002. PAUP: phylogenetic analysis using parsimony, version 4.0 b10. Sunderland, MA, Sinauer Associates.
• Tanaka K, Hirayama K, Yonezawa H, et al 2009. Molecular taxonomy of bambusicolous fungi: Tetraplosphaeriaceae, a new pleosporalean family with Tetraploa-like anamorphs. Studies in Mycology 64: 175-209.
• Taylor JE, Hyde KD. 2003. Microfungi of tropical and temperate palms. Fungal Diversity Press 12: 121-459.
• Tennakoon DS, Phookamsak R, Kuo CH, Goh TK, Jeewon R, Hyde KD. 2018. Morphological and phylogenetic evidence reveal Fissuroma taiwanense sp. nov. (Aigialaceae, Pleosporales) from Hedychium coronarium Phytotaxa 338: 265-275.
• Thambugala KM, Wanasinghe DN, Phillips AJL, et al 2017. Mycosphere notes 1-50: Grass (Poaceae) inhabiting Dothideomycetes. Mycosphere 8: 697-796.
• Tibpromma S, Hyde KD, McKenzie EH, et al 2018. Fungal diversity notes 840-928: micro-fungi associated with Pandanaceae. Fungal Diversity 93: 1-160.
• Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238-4246.
• Vohník M, Borovec O, Kolaříková Z, Sudová R, Réblová M. 2019. Extensive sampling and high-throughput sequencing reveal Posidoniomyces atricolor gen. et sp. nov. (Aigialaceae, Pleosporales) as the dominant root mycobiont of the dominant Mediterranean seagrass Posidonia oceanica MycoKeys 55: 59-86.
• Wanasinghe DN, Jeewon R, Jones EBG, Boonmee S, Kaewchai S. 2018. Novel palmicolous taxa within Pleosporales: Multigene phylogeny and taxonomic circumscription. Mycological Progress 17: 571-590.
• White TJ, Bruns T, Lee SJWT, Taylor JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications 18: 315-322.
• Whitton SR, McKenzie EHC, Hyde KD. 2012. Fungi associated with Pandanaceae. Dordrecht, Springer.
• Wijayawardene NN, Hyde KD, Lumbsch HT, et al 2018. Outline of ascomycota: 2017. Fungal Diversity 88: 167-263.
• Yanna Ho WH, Hyde KD. 2001. Fungal communities on decaying palm fronds in Australia, Brunei, and Hong Kong. Mycological Research 105: 1458-1471.
• Zhang SN, Hyde KD, Jones EBG, Cheewangkoon R, Liu JK. 2020. Additions to Fissuroma and Neoastrosphaeriella (Aigialaceae, Pleosporales) from palms. Mycosphere 11: 269-284.
• Zhang SN, Hyde KD, Jones EG, Jeewon R, Cheewangkoon R, Liu JK . 2019. Striatiguttulaceae, a new pleosporalean family to accommodate Longicorpus and Striatiguttula gen. nov. from palms. MycoKeys 49: 99-129.
• Zhang Y, Crous PW, Schoch CL, Hyde KD. 2012. Pleosporales. Fungal Diversity 53: 1-221.

Publication Dates

• Publication in this collection
  03 Aug 2020
• Date of issue
  Apr-Jun 2020

History

• Received
  24 Jan 2020
• Accepted
  30 Mar 2020