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Research Article
Additions to Chaetothyriaceae (Chaetothyriales): Longihyalospora gen. nov. and Ceramothyrium longivolcaniforme, a new host record from decaying leaves of Ficus ampelas
expand article infoDanushka S. Tennakoon§|, Kasun M. Thambugala, Rajesh Jeewon#, Sinang Hongsanan¤, Chang-Hsin Kuo§, Kevin D. Hyde|
‡ Mae Fah Luang University, Chiang Rai, Thailand
§ National Chiayi University, Chiayi, Taiwan
| Kunming Institute of Botany, Chinese Academy of Science, Kunming, China
¶ Thammasat University, Pathumthani, Thailand
# University of Mauritius, Moka, Mauritius
¤ Shenzhen University, Shenzhen, China
Open Access

Abstract

A novel ascomycete genus, Longihyalospora, occurring on leaf litter of Ficus ampelas in Dahu Forest Area in Chiayi, Taiwan is described and illustrated. Longihyalospora is characterized by dark mycelium covering the upper leaf surface, elongate mycelial pellicle with ring of setae, pale brown to brown peridium, broadly obovoid, short pedicellate asci and hyaline, fusiform, elongated (tapering ends) and multi-septate ascospores with a thin mucilaginous sheath. Phylogenetic analyses of combined ITS, LSU and SSU sequence data revealed Longihyalospora as a distinct genus within the Chaetothyriaceae with high bootstrap support. Moreover, based on morphological similarities, Chaetothyrium vermisporum transferred to the new genus. In addition, Ceramothyrium longivolcaniforme is reported for the first time on Ficus ampelas. Newly added species are compared with other similar species and comprehensive descriptions and micrographs are provided.

Keywords

Moraceae, multi-gene phylogeny, mycelium pellicle, sooty mould, taxonomy

Introduction

The family Chaetothyriaceae was established by Hansford (1946) with the generic type Chaetothyrium Speg., and the members of this family are characterized by a loose network of dark mycelium over the substrate, ascomata produced beneath a mycelial pellicle, and forming beneath an external hyphal mat with or without setae (Batista and Ciferri 1962; von Arx and Müller 1975; Hughes 1976; Pereira et al. 2009; Chomnunti et al. 2012; Tian et al. 2014; Zeng et al. 2016). Due to some morphological similarities (i.e. bitunicate asci), Eriksson (1982) referred this family to the order Dothideales in Dothideomycetes, but subsequently, taxonomic studies have established its placement in Eurotiomycetes with support of molecular data (Chomnunti et al. 2012, 2014; Tian et al. 2014; Crous et al. 2015; Maharachchikumbura et al. 2018; Yang et al. 2018). Currently, 16 genera are accepted in Chaetothyriaceae, viz. Actinocymbe Höhn., Aphanophora Réblová & Unter., Beelia F. Stevens & R.W. Ryan, Camptophora Réblová & Unter., Ceramothyrium Bat. & H. Maia, Ceratocarpia Rolland, Chaetothyriomyces Pereira-Carvalho et al., Chaetothyrium Speg., Cyphellophoriella Crous & A.J. Sm., Euceramia Bat. & Cif., Microcallis Syd., Phaeosaccardinula P. Henn., Stanhughesia Constant., Treubiomyces Höhn., Vonarxia Bat. and Yatesula Syd. & P. Syd. (Wijayawardene et al. 2018).

During our survey of the taxonomy and diversity of leaf litter microfungi, two interesting fungal species were collected from Dahu forest, Chiayi in Taiwan. Morphological and multi-gene phylogenetic analyses were performed to establish their taxonomic placement.

Materials and methods

Sample collection, morphological studies and isolation

Decaying leaf litter samples of Ficus ampelas Burm.f. were collected from Dahu forest area in Chiayi, Taiwan and brought to the laboratory in plastic bags. The samples were incubated in plastic boxes at 25–30 °C for 3 days and examined following the methods described by Tian et al. (2014). Morphological observations were made using an Axioskop 2 Plus compound microscope and images were taken with an Axioskop 2 Plus compound microscope equipped with a Canon Axiocam 506 Color digital camera. Permanent slides were prepared by mounting fungal material in lactoglycerol and sealed by applying nail-polish around the margins of cover slips. All measurements were made with ZEN2 (blue edition) and images used for figures were processed with Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, USA).

Isolates (for Ceramothyrium longivolcaniforme Zeng, T.C. Wen & K.D. Hyde) were obtained from single ascospores following the methods described in Chomnunti et al. (2014). Germinated ascospores were transferred to potato dextrose agar (PDA) and incubated at 25 °C in normal light. Subsequent sub culturing was done carefully to ensure no contaminants are used to generate DNA sequence data. Culture characteristics were observed after two weeks. Type specimens were deposited in the Mae Fah Luang University Herbarium (MFLU) and living cultures were deposited in Mae Fah Luang University Culture Collection (MFLUCC). Faces of Fungi and Index Fungorum numbers were provided as in Jayasiri et al. (2015) and Index Fungorum (2019).

DNA extraction and PCR amplification

Fresh mycelia were scraped (for Ceramothyrium longivolcaniforme) using a sterile scalpel from pure cultures growing on PDA medium at 25 °C and kept in a 1.5 ml micro-centrifuge tube and used as starting material for DNA extraction. When fungi failed to germinate in a culture medium, DNA was extracted directly from ascomycete fruiting bodies (for Longihyalospora ampeli) by following a modified protocol of Zeng et al. (2018) protocol: 15–20 fruiting bodies (> 500 µm diam., 10 fruiting bodies) were removed from the host substrate using a sterilized needle and transferred to a drop of sterile water, placed in a sterile Eppendorf tube (1.5 mL) under aseptic conditions.

The genomic DNA was extracted using a DNA extraction kit (E.Z.N.A Fungal DNA Mini Kit, D3390-02, Omega Bio-Tek) following the manufacturer’s protocol. The DNA product was kept at 4 °C for DNA amplification and maintained at -20 °C for long-term storage. DNA was amplified by Polymerase Chain Reaction (PCR) for three genes, the large subunit (28S, LSU), small subunit (18S, SSU) and internal transcribed spacers (ITS1-5.8S-ITS2). The LSU gene was amplified by using the primers LR0R and LR5 (Vilgalys and Hester 1990; Rehner and Samuels 1994); SSU gene was amplified using the primers NS1 and NS4 (White et al. 1990); nuclear ITS was amplified by using the primers ITS5 and ITS4 (White et al. 1990). The amplification reactions were performed in 25µl of total reaction that contained 9.5 µl of sterilized water, 12.5 µl of 2×Power Taq PCR MasterMix (Tri-I Biotech, Taipei, Taiwan), 1 μl of each forward and reverse primers and 1 μl of DNA template. PCR thermal cycle program for ITS, LSU and SSU were as detailed by Tian et al. (2016). The PCR products were analyzed by 1.5% agarose gels containing the Safeview DNA stain (GeneMark, Taipei, Taiwan) to confirm the expected molecular weight of a single amplification product. PCR products were purified and sequenced with primers mentioned above by Tri-I Biotech, Taipei, Taiwan. Nucleotide sequences were deposited in GenBank (Table 1).

Table 1.

GenBank and culture collection accession numbers of species included in the present phylogenetic study. The newly generated sequences are shown in bold.

Species Strain/Voucher no. GenBank accession no.
ITS LSU SSU
Aphanophora eugeniae CBS 124105 FJ839617 FJ839652
Brycekendrickomyces acaciae CBS 124104 MH863350 MH874874
Camptophora hylomeconis IFRDCC 2661 MF285228 MF285230
C. hylomeconis CBS 113311 EU035415 KC455295
Capronia fungicola CBS 614.96 KY484990 FJ358224 FJ225722
C. mansonii CBS 101.67 AF050247 MH870591 AF346422
Ceramothyrium aquaticum LC306299 LC360299 LC360296
C. carniolicum AFTOL-ID 1063 EF413628 EF413627
C. carniolicum CBS 175.95 KC978733 KC455251 KC455294
C. exiguum LC306297 LC360297 LC360295
C. ficus MFLUCC 15-0228 KT588601 KT588599
C. ficus MFLUCC 15-0229 KT588602 KT588600
C. longivolcaniforme MFLU 16-1306 KP324929 KP324931
C. longivolcaniforme MFLUCC 19-0252 MN219715 MN238770 MN238773
C. melastoma CPC 19837 KC005771 KC005793
C. menglunense MFLU 16-1874 KX524148 KX524146
C. phuquocense LC306298 LC360298 LC360294
C. podocarpi CPC 19826 KC005773 KC005795
C. thailandicum MFLUCC 10-0008 KP324928 HQ895835
C. thailandicum MFLU 13-0632 HQ895838 KP324930
Chaetothyrium agathis MFLUCC 12-0113 KP744437 KP744480
C. brischoficola MFLUCC 10-0012 HQ895839 HQ895836
Cladophialophora minourae CBS 556.83 AY251087 FJ358235 FJ225734
C. emmonsii CBS 640.96 KX822192 KC809995 KX822192
Cyphellophoriella pruni CPC 25120 KR611878
Leptoxyphium fumago CBS 123.26 MH854862 GU214430 GU214535
L. madagascariense CBS 124766 MH863407 GQ303308
Longihyalospora ampeli MFLU 19-0824 MN219716 MN238771 MN238774
L. ampeli MFLU 19-0825 MN219717 MN238772 MN238775
Knufia cryptophialidica DAOM 216555 JN040500 EF137364
K. cryptophialidica DAOM 216553 JN040504 EF137363
K. perforans CBS 885.95 MH862564 MH874191
K. perforans CBS 726.95 KC978746 KC978741 KC978739
Minimelanolocus asiaticus MFLUCC 15-0237 KR215604 KR215610 KR215615
M. melanicus MFLUCC 15-0415 KR215608 KR215613 KR215618
Phaeosaccardinula dendrocalami IFRDCC 2663 KF667243 KF667246
P. dendrocalami IFRDCC 2649 KF667242 KF667245
P. ficus MFLUCC 10-0009 HQ895840 HQ895837
P. multiseptata IFRDCC 2639 KF667241 KF667244
Trichomerium deniqulatum MFLUCC 10-0884 JX313654 JX313660
T. follicola MFLUCC 10-0058 JX313653 JX313659
T. gleosporum MFLUCC 10-0087 JX313656 JX313662
Vonarxia vagans CBS 123533 FJ839636 FJ839672 KC455310
V. vagans CPC 15152 FJ839637 FJ839673

Phylogenetic analysis

Phylogenetic analyses were performed based on a combined ITS, LSU and SSU DNA sequence data. Newly generated sequences were subjected to a standard BLAST search of GenBank to aid in phylogenetic taxon sampling. Other sequences used in the analyses (Table 1) were obtained from GenBank based on recently published data (Zeng et al. 2016; Maharachchikumbura et al. 2018; Yang et al. 2018). The multiple alignments were made with MAFFT v. 7 at the web server (http://mafft.cbrc.jp/alignment/server), using default settings (Katoh and Standley 2013). The alignment was refined manually with BioEdit v. 7.0.5.2 (Hall 1999) where necessary. The tree topologies obtained from a single gene sequence data were compared prior to the combined gene analysis for checking the incongruence in overall topology of the phylogenetic tree.

Maximum likelihood trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008; Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using GTRGAMMA model with 1,000 bootstrap replicates. Maximum parsimony analysis (MP) was performed in PAUP v. 4.0b10 (Swofford 2002), with the heuristic search option and 1,000 random replicates. Maxtrees was set to 1,000 and branches of zero length were collapsed and all multiple parsimonious trees were saved. Descriptive tree statistics for parsimony (Tree Length [TL], Consistency Index [CI], Retention Index [RI], Relative Consistency Index [RC] and Homoplasy Index [HI] were calculated.

A Bayesian analysis (GTR+I+G model) was conducted with MrBayes v. 3.1.2 (Huelsenbeck and Ronqvist 2001) to evaluate posterior probabilities (PP) (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo sampling (BMCMC). Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every 100th generation, thus 10,000 trees were obtained. The suitable burn-in phases were determined by inspecting likelihoods and parameters in Tracer version 1.6 (Rambaut et al. 2014). Based on the tracer analysis, the first 1,000 trees representing 10% were discarded as the burn-in phase in the analysis. The remaining trees were used to calculate posterior probabilities in the majority rule consensus tree (critical value for the topological convergence diagnostic set to 0.01). Phylograms were visualized with FigTree v1.4.0 (Rambaut 2012) and annotated in Microsoft Power Point (2010). The final alignment and trees were deposited in TreeBASE, submission ID: 24826.

Results

Phylogenetic analysis

The combined dataset of ITS, LSU and SSU sequences comprised 2531 characters, of which 1492 characters are constant, 801 characters are parsimony-informative, while 238 variable characters are parsimony-uninformative in the maximum parsimony (MP) analysis (TL = 3011, CI = 0.515, RI = 0.698, RC = 0.360, HI = 0.485). LSU contains 900 total characters (constant = 645, informative = 217, uninformative = 38), ITS contains 759 total characters (constant = 332, informative = 364, uninformative = 63) and SSU contains 872 characters (constant = 515, informative = 220, uninformative = 137). The RAxML analysis of the combined dataset yielded a best scoring tree (Figure 1) with a final ML optimization likelihood value of -17222.496803. The matrix had 1040 distinct alignment patterns, with 37.84 % of undetermined characters or gaps. All analyses (ML, MP and BYPP) gave similar results and in agreement with previous studies based on multi-gene analyses (Zeng et al. 2016; Maharachchikumbura et al. 2018).

The phylogeny recovered herein also agrees with previously established ones in that Ceramothyrium is within the Chaetothyriales (Zeng et al. 2016; Maharachchikumbura et al. 2018; Yang et al. 2018). Our new collection (MFLUCC19-0252) grouped in a well-supported clade (80% ML, 100% MP and 0.92 BYPP) with other Ceramothyrium species (Figure 1). In particular, it shows a close affinity to Ceramothyrium longivolcaniforme (holotype, MFLU16-1306). MFLU 19-0824 and MFLU 19-0825 constitute in a strongly supported subclade and is phylogenetically distinct from other genera in family (77% ML, 65% MP, 0.99 BYPP) (Figure 1).

Figure 1. 

RAxML tree based on a combined dataset of ITS, LSU and SSU partial sequences of 45 taxa. Bootstrap support values for maximum likelihood (ML and, maximum parsimony (MP) values higher than 60 % and Bayesian posterior probabilities (BYPP) greater than 0.90 are given above each branch respectively. The new isolates are in red. Ex-type strains are in bold. The tree is rooted by Leptoxyphium fumago (CBS 123.26) and L. madagascariense (CBS 124766).

Taxonomy

Ceramothyrium longivolcaniforme X.Y. Zeng, T.C. Wen & K.D. Hyde, Phytotaxa 267(1): 54 (2016)

Figure 2

Description

Epiphytic on decaying leaves of Ficus ampelas Burm.f. Covering the upper leaf surface with dark mycelium without penetrating host tissues. Mycelial pellicle elongate, subiculum-like, comprising hyphae that are mostly narrow, 3.5–4.5 μm wide (x- = 3.8 μm, n= 20), brownish, slightly constricted at the septa, dense, radiating outward, anastomosing at the tips with cells of the hyphal network. Sexual morph: Ascomata 130–180 μm high, 200–250 μm diam. (x- = 155 × 220 µm, n = 10) in diameter, superficial, solitary, pale brown, globose to subglobose, coriaceous, somewhat flattened when dry, covered by a mycelial pellicle, with a circumferential space filled with sparse mycelium around the mature ascomata. Peridium 18–25 μm wide (x- = 23.5 μm, n= 20), light brown, with compressed, hyaline, inner cells of textura angularis and light brown outer cells of textura angularis. Asci (62–)70–90 × 30–60 μm (x- = 81 × 44 µm, n = 20), 8-spored, bitunicate, broadly obovoid, short pedicellate, apically rounded, with well-developed ocular chamber. Ascospores 30–45(–47) × 8–16 μm (x- = 36 × 12 µm, n = 30), crowded or overlapping, irregularly triseriate, hyaline, oblong to ellipsoid, muriform, with 7 transversal septa and 6 longitudinal septa, slightly constricted at the septa, smooth-walled, surrounded by a mucilaginous sheath. Asexual morph: Not observed.

Culture characteristics

Colonies on PDA reaching 3 mm diameter after 2 weeks at 25–30 °C, slow growing, spreading, with folded, velvety, wavy margin, consist of dark mycelium, colony color from above: olivaceous green; colony color from below: dark brown to black, not producing pigments in PDA.

Material examined

Taiwan, Chiayi, Fanlu Township area, Dahu forest, decaying leaves of Ficus ampelas Burm.f (Moraceae), 20 June 2018, D.S. Tennakoon, H10 (MFLU19-0823), living culture (MFLUCC19-0252).

Notes

In this study, a sample of Ceramothyrium longivolcaniforme was collected from dead leaves of Ficus ampelas (Moraceae) in Taiwan. The new collection shares a close phylogenetic relationship with Ceramothyrium longivolcaniforme (MFLU16-1306) (Figure 1). The morphology of our collection (MFLUCC19-0252) fits with the type material of Ceramothyrium longivolcaniforme (MFLU16-1306) in having elongate mycelial pellicle, broadly obovoid, short pedicellate asci and hyaline, oblong to ellipsoid, muriform ascospores with a mucilaginous sheath (Zeng et al. 2016). However, the ascospores are slightly larger (30–45 × 8–16 μm) than MFLU16-1306 (28–37 × 7–13 μm) (Table 2). Ceramothyrium longivolcaniforme has been previously reported from Thailand on unidentified sp. (not F. ampelas) and thus, we provide the new host record of Ceramothyrium longivolcaniforme on Ficus ampelas (Moraceae). Remarkably, this is the first Ceramothyrium species collected from Taiwan.

Figure 2. 

Ceramothyrium longivolcaniforme (MFLU19-0823, new host record). a, b Appearance of colony (black spots) on host leaf c mycelial pellicle d vertical section through ascoma e section of peridium f–i asci j–m ascospores n ascospore stained in Indian ink showing mucilaginous sheath o germinating ascospore p, q colony from above and below. Scale bars: 50 µm (d), 10 µm (e), 20 µm (f–i), 10 µm (j–o).

Table 2.

Comparison of ascospore characters among species of Ceramothyrium.

Species Numbers of septa Host /Locality Size (μm) References
C. anacardii 3 33–50 × 7–9.5 Batista and Maia (1956)
C. aurantii 3–6 18.9–27 × 5.4–8 Batista and Maia (1956)
C. biseptatum 2 Macaranga tanarius/ Philippines 14–16 × 4.5–5.5 Batista and Ciferri (1962)
C. boedijnii 3 Theobroma cacao/ Papua New Guinea 15–20 × 5–7 Batista and Ciferri (1962)
C. calycanthi 6–10 Calycanthus sp./ Georgia 24.5–37 × 6.5–9.5 Batista and Ciferri (1962)
C. carniolicum 3 Pyrola rotundifolia/ Sweden 18–20 × 4–5.5 Eriksson (1992)
C. cinereum 7 35–42 × 7–9 Batista and Maia (1956)
C. citricola 3–4 Citrus aurantium/ Brazil 14–30 × 2.5–11 Mendes et al. (1998)
C. coffeanum 3 Coffea robusta/ New Guinea 12–16 × 4–6 Batista and Ciferri (1962)
C. cordiae 3 Cordia rufescens/ Brazil 10–13.5 × 4–5.4 Eriksson (1992)
C. europaeum 3 Pogonophora schomburgkiana/ Brazil 16–20 × 4–5.5 Eriksson (1992)
C. globosum 6–9 transversal 50–58 × 5–6 Batista and Maia (1956)
C. griseolum 4–6 Aleurites moluccana/ Brazil 19–25 × 4–5 Eriksson (1992)
C. gustaviae 3–5 Gustavia augusta/ Brazil 22–25 × 3.7–5 Eriksson (1992)
C. gymnopogonis 2 Alyxia scandens/ Samoa 15 × 5 Dingley et al. (1981)
C. jambosae Eugenia malaccensis/ Brazil Eriksson 1992
C. linnaeae 3–4 Lycopodium annotinum/ Sweden 12–18 × 3–5 Constantinescu et al. 1989
C. longivolcaniforme (MFLU 16-1306) 7 transversal Unidentified/ Thailand 28–37 × 7–13 Zeng et al. (2016)
6 longitudinal
C. longivolcaniforme (MFLU 19-0823) 7 transversal Ficus ampelas / Taiwan 30–45 × 8–16 This study (New host record)
6 longitudinal
C. lycopodii 7 Lycopodium annotinum/ Sweden 45 × 4 Constantinescu et al. (1989)
C. martinii 5–7 20–27 × 7–9 Barr (1993)
C. moravicum 2–3 10–14 × 3–5 Petrak (1961)
C. paiveae 1–4 Paivaea langsdorffii/ Brazil 12.5–22 × 3.7–6 Mendes et al. (1998)
C. paraense 3–7 Anacardium sp./ Brazil 20–30 × 3.5–4 Mendes et al. 1998
C. parenchymaticum 5–7 Didymopanax morototoni/ Cuba 30–40 × 8–10 Batista and Ciferri 1962
C. peltatum 6–9 28–32 × 4.5–6.5 Batista and Maia (1956)
C. philodendri 1–7 Philodendron imbe/ Brazil 17.5–32.5 × 5–7.5 Mendes et al. (1998)
C. thailandicum 7–9 transversal Lagerstroemia sp./ Thailand 24.7–35.5 × 5.7–8.7 Chomnunti et al. (2012)

Longihyalospora Tennakoon, C.H Kuo & K. D Hyde, gen. nov.

Etymology

Referring to the long, hyaline ascospores.

Description

Epiphytic on the upper surface decaying leaves, appearing as small black dots. Covering the upper leaf surface with dark mycelium without penetrating host tissues. Mycelial pellicle elongate, subiculum-like, comprising hyphae that are mostly narrow, dense, dark brown. Mycelial setae broad, dark brown, scattered, discrete, arranged as a ring around the pellicle, unbranched, formed on dense, dark hyphae. Sexual morph: Ascomata superficial, solitary, dark brown to black, globose to subglobose, coriaceous, uni-locular, somewhat flattened when dry, covered by a mycelial pellicle. Peridium pale brown to brown, with compressed, hyaline, inner cells of textura angularis and dark brown outer cells of textura angularis, fusing and indistinguishable from the host tissues. Asci 8-spored, bitunicate, broadly obovoid, slightly stalked, apically rounded, with a well-developed ocular chamber. Ascospores overlapping, irregularly triseriate, hyaline, fusiform, elongated, multi-septate, slightly constricted at the septa, tapering to the ends, smooth-walled, surrounded by a thin mucilaginous sheath. Asexual morph: Not observed.

Type species

Longihyalospora ampeli Tennakoon, C.H Kuo & K. D Hyde.

Longihyalospora ampeli Tennakoon, C.H Kuo & K.D. Hyde, sp. nov.

Figure 3

Etymology

Species name based on the host Ficus ampelas, from which it was collected.

Holotype

MFLU 19-0824

Description

Epiphytic on the upper surface decaying leaves, appearing as small black dots. Covering the upper leaf surface with dark mycelium without penetrating host tissues. Mycelial pellicle (190–) 200–250 (–258) µm diam., elongate, subiculum-like, comprising hyphae that are mostly narrow, 1–2 μm wide (x- = 1.5 μm, n= 20), dense, dark brown. Mycelial setae (197–) 200–225 (–231) µm long, at base 10–12 µm wide, at apex 2–3 µm wide, dark brown, scattered, discrete, arranged as a ring around the pellicle, unbranched, formed on dense, dark hyphae. Sexual morph: Ascomata 55–90 μm high, 150–200 μm diam. (x- = 76 × 168 µm, n = 10) in diameter, superficial, solitary, dark brown to black, globose to subglobose, coriaceous, uni-locular, somewhat flattened when dry, covered by a mycelial pellicle. Peridium 18–25 μm wide (x- = 23.5 μm, n= 20), pale brown to brown, with compressed, hyaline, inner cells of textura angularis and dark brown outer cells of textura angularis. Asci (82–) 90–115 (–120) × 52–62 μm (x- = 106 × 57 µm, n = 20), 8-spored, bitunicate, broadly obovoid, slightly stalked, apically rounded, with well-developed ocular chamber. Ascospores (74–) 76–98(–105) × 10–12 μm (x- =84 × 10.8 µm, n = 30), overlapping, irregularly triseriate hyaline, elongate fusiform, (6–) 8–11 (–12) septa, slightly constricted at the middle septum, tapering to the ends, smooth-walled, surrounded by a 3.5–5 µm wide mucilaginous sheath. Asexual morph: Not observed.

Material examined

Taiwan, Chiayi, Fanlu Township area, Dahu forest, decaying leaves of Ficus ampelas (Moraceae), 20 June 2018, D.S. Tennakoon, H50B1 (MFLU 19-0824, holotype), H50B2 (MFLU19-0825, isotype).

Notes

Longihyalospora is described herein as a new monotypic genus in Chaetothyriaceae. Longihyalospora differs from other genera in Chaetothyriaceae by a combination of a dark mycelium covering the upper leaf surface, an elongate mycelial pellicle, ring of setae around the pellicle, pale brown to brown peridium with hyaline inner layers, broadly obovoid, short pedicellate asci and hyaline, elongate fusiform and 8–11-septate ascospores, with tapering ends and a thin mucilaginous sheath. In our phylogenetic analyses, Longihyalospora ampeli species constitutes a strongly supported sub clade, which is nested independently from other genera in Chaetothyriaceae (Figure 1).

Figure 3. 

Longihyalospora ampeli (MFLU 19-0824, holotype). a Host leaf b appearance of colony (black spots) on leaf c ring of setae around the pellicle d mycelial pellicle with setae e mycelial pellicle cells f, g vertical section through ascoma h section of peridium i–m asci n–r ascospores s ascospore stained in Indian ink showing a mucilaginous sheath. Scale bars: 100 µm (c), 75 µm (d), 20 µm (e, f), 50 µm (g), 10 µm (h), 50 µm (i–m), 20 µm (n–s).

Longihyalospora vermisporum (Hansf.) Tennakoon, C.H. Kuo & K.D. Hyde, comb. nov.

Chaetothyrium vermisporum Hansf., Mycol. Pap. 15: 151 (1946). Morphological description: See Hansford (1946), Hofmann and Piepenbring (2006).

Recorded hosts

Canthium sp. (Rubiaceae) Hansford no. 1327; Hugonia platysepalae (Linaceae) Hansford no. 1384; Ventilago africana (Rhamnaceae), Hansford no. 2930 (Hansford, 1946).

Known distribution

Uganda (Hansford, 1946), Panama (Hofmann and Piepenbring 2006).

Notes

Chaetothyrium vermisporum was introduced by Hansford (1946) which was collected from Uganda based on morphological characteristics. Subsequently, it has been collected from Panama by Hofmann and Piepenbring (2006). After in-depth morphological investigations, we found that Chaetothyrium vermisporum shares some similar morphology with Longihyalospora ampeli by having mycelial pellicle with ring of setae, pale brown to brown peridium and hyaline, fusiform, elongated and multi-septate ascospores (Hansford (1946). However, Chaetothyrium vermisporum can be distinguished from Longihyalospora ampeli by having hyaline surface mycelium, smaller asci (60 × 30 µm) and ascospores (35–50 × 5–6 µm) without a mucilaginous sheath, whereas Longihyalospora ampeli has dark brown mycelium, larger asci (90–115 × 52–62 µm) and ascospores (76–98 × 10–12 μm) with mucilaginous sheath. Therefore, we synonymized Chaetothyrium vermisporum under Longihyalospora based on high morphological similarities. Fresh collections with molecular data are needed to clarify the phylogenetic affinity of Longihyalospora vermisporum.

Additionally, we compared our collection with Chaetothyrium guaraniticum Speg. (type species of Chaetothyrium). Longihyalospora ampeli can be distinguished from Chaetothyrium guaraniticum by many morphological characters, viz. C. guaraniticum has 1-septate shorter ascospores (10–14 × 4–5 µm) and lacks a mucilaginous sheath (Spegazzini 1888), whereas L. ampeli has multi-septate (8–11), longer (84 × 10.8 µm) ascospores with a mucilaginous sheath. Further collections are needed to resolve the phylogenetic position and relationships between members of Chaetothyrium and Longihyalospora species.

Discussion

Sooty molds are an interesting group of fungi in tropical and temperate regions in worldwide (Chomnunti et al. 2014; Hongsanan et al. 2015; Farr and Rossman 2019; Kwon et al. 2019). Their morphology has been well-studied but their phylogenetic relationships are poorly understood due to the difficulty of obtaining good-quality DNA samples (Chomnunti et al. 2011, 2014; Zeng et al. 2016; Zeng et al. 2019). Currently, seven sooty mold forming families have been reported, viz. Antennulariellaceae Woron., Capnodiaceae Höhn., Euantennariaceae S. Hughes & Corlett ex S. Hughes, Metacapnodiaceae S. Hughes & Corlett (Dothideomycetes) and Chaetothyriaceae Hansf. ex M.E. Barr, Coccodiniaceae Höhn. ex O.E. Erikss., and Trichomeriaceae Chomnunti & K.D. Hyde (Eurotiomycetes) (Reynolds 1998; Winka et al. 1998; Hughes and Seifert 2012; Hyde et al. 2013; Chomnunti et al. 2014; Hongsanan et al. 2016).

Chaetothyriaceae species are widespread in tropical and temperate regions (Hofmann and Piepenbring 2006; Chomnunti et al. 2011, 2014; Hongsanan et al. 2015; Zeng et al. 2016; Maharachchikumbura et al. 2018; Yang et al. 2018; Farr and Rossman 2019). Wijayawardene et al. (2018) accepted 16 genera in Chaetothyriaceae, but currently only seven genera (Aphanophora, Camptophora, Ceramothyrium, Chaetothyrium, Cyphellophoriella, Phaeosaccardinula and Vonarxia) have DNA sequence data. The main morphological differences of Chaetothyriaceae genera are mentioned in Table 3.

Table 3.

Synopsis of sexual morphs of Chaetothyriaceae genera discussed in this study.

Genus name Ascomata or mycelium setose/glabrous Asci Ascospores References
Shape Number of spores/ascus Shape Color Septation Sheath
Actinocymbe Höhn. Glabrous straight to sickle shape 8 club shaped hyaline to light brown 9 Verma and Kamal (1987)
Beelia F. Stevens & R.W. Ryan Glabrous broadly ellipsoidal 8 cylindrical hyaline 5 yes Li et al. (2011)
Camptophora Réblová & Unter. Glabrous long-ellipsoid to obovoid 8 obovoid to pyriform hyaline 1–3 or muriform no Yang et al. (2018)
Ceramothyrium Bat. & H. Maia Glabrous clavate or pyriform 8 oblong to ellipsoid or cylindrical clavate hyaline 3–10 or muriform yes Zeng et al. (2016), Chomnunti et al. (2012)
Ceratocarpia Rolland Glabrous clavate to broadly clavate 8 ellipsoid to fusiform light brown muriform no Tian et al. (2014)
Chaetothyrium Speg. Setose broadly ovoid or oblong 8 oblong to ellipsoidal or obovoid hyaline 4–7 or muriform no Chomnunti et al. (2012), Liu et al. (2015)
Chaetothyriomyces Pereira-Carv et al. Glabrous broadly clavate 16 elliptical hyaline 1 no Pereira et al. (2009)
Euceramia Bat. & Cif. Glabrous ellipsoid to pyriform 8 clavate-fusoid hyaline 4–5 no Batista and Ciferri (1962)
Longihyalospora Tennakoon, C.H. Kuo & K.D. Hyde Setose broadly obovoid 8 fusiform and elongated hyaline 8–11 yes This study
Microcallis Syd. Glabrous clavate 8 oblong to clavate hyaline 1 no Sydow (1926), Chomnunti et al. (2011)
Phaeosaccardinula Henn. Glabrous obovoid to oval 4–6 oblongellipsoid to reniform hyaline or pale brown muriform yes Yang et al. (2014), Maharachchikumbura et al. (2018)
Treubiomyces Höhn. setose clavate 8 oblong to clavate hyaline muriform no Höhnel (1909), Pohlad (1989)
Yatesula Syd. & P. Syd. Glabrous clavate 4–8 oblong to clavate brownish yellow 3–4 or muriform no Ellis and Everhart, (1893), Sydow and Sydow (1917)

Batista and Maia (1956) established the genus Ceramothyrium and designated Ceramothyrium paiveae Bat. & H. Maia as the type species, which has been collected from Brazil. Ceramothyrium species are characterized by a mycelial pellicle that covers the ascomata with a circumferential space around the maturing ascomata, lack of setae and hyaline, transversely pluriseptate ascospores (Batista and Maia 1956; Chomnunti et al. 2012; Tsurumi et al. 2018). Most Ceramothyrium species have been collected from terrestrial habitats and their asexual morph has been recorded as Stanhughesia Constant. (Chomnunti et al. 2012; Réblová et al. 2013; Wijayawardene et al. 2017; Tsurumi et al. 2018). Ceramothyrium species seem to have a diverse distribution since they have been recorded from both temperate and tropical countries (i.e. Brazil, Canada, Georgia, Indonesia, Thailand, Panama, Philippines, South Africa, Sweden, Vietnam) (Hofmann and Piepenbring 2006; Chomnunti et al. 2012; Crous et al. 2012; Zeng et al. 2016; Tsurumi et al. 2018; Farr and Rossman 2019). Host-specificity of the taxa in this group has not yet been proven, since they have been recorded from various plant families (i.e. Arecaceae, Anacardiaceae, Ericaceae, Lycopodiaceae, Lythraceae, Melastomataceae, Podocarpaceae, Rubiaceae) (Batista and Maia 1956; Chomnunti et al. 2012; Hongsanan et al. 2015; Farr and Rossman 2019). Combined phylogenetic analyses with a larger taxon sampling provide a better resolution of interspecific relationships of Ceramothyrium within Chaetothyriaceae (Chomnunti et al. 2014; Zeng et al. 2016; Maharachchikumbura et al. 2018; Yang et al. 2018).

Recent studies have revealed that Ceramothyrium is a species rich genus. For instance, in the last few years, numerous Ceramothyrium species have been described. Ceramothyrium longivolcaniforme, C. menglunense were introduced by Zeng et al. (2016) and Hyde et al. (2016) respectively. Yen et al. (2018) introduced three Ceramothyrium species, viz. C. aquaticum, C. phuquocense and C. exiguum. Currently, there are 41 Ceramothyrium epithets in Index Fungorum (2019).

Most previous Chaetothyriaceae studies have been based on brief descriptions with line drawings and without DNA sequence data (i.e. Actinocymbe, Beelia, Ceratocarpia, Chaetothyriomyces, Euceramia, Microcallis, Stanhughesia, Treubiomyces and Yatesula). Therefore, it is essential to focus on DNA sequence data to clarify the phylogenetic affinity of above genera in Chaetothyriaceae in future studies. Thus, it is necessary to collect more fungi similar to Chaetothyriaceae in different geographic regions and hosts, isolate them into cultures, describe their morphology, analyze their DNA sequences and investigate their phylogenetic relationships for a better identification and classification.

Acknowledgments

We thank the Department of Plant Medicine, National Chiayi University (NCYU) for providing facilities for DNA molecular experiment. We also thank Mae Fah Luang University grant number 56101020032 for supporting studies on Dothideomycetes. We also extend our gratitude to Dr. Shaun Pennycook for checking species’ names. The authors would like to thank N.I de Silva, Wilawan Punyaboon, Chada Norphanphoun and Dr. Samantha Karunarathne for their valuable suggestions and help. K.D. Hyde thanks Chiang Mai University for the award of Visiting Professorship. R. Jeewon thanks the University of Mauritius for research support.

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