11urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3MycoKeysMC1314-40571314-4049Pensoft Publishers10.3897/mycokeys.61.4705647056Research ArticleAscomycotaChaetothyrialesTaxonomyAsiaAdditions to Chaetothyriaceae (Chaetothyriales): Longihyalospora gen. nov. and Ceramothyriumlongivolcaniforme, a new host record from decaying leaves of FicusampelasTennakoonDanushka S.https://orcid.org/0000-0003-2306-1255123ThambugalaKasun M.https://orcid.org/0000-0002-6210-05044JeewonRajeshhttps://orcid.org/0000-0002-8563-957X5HongsananSinanghttps://orcid.org/0000-0003-0550-31526KuoChang-Hsinchkuo@mail.ncyu.edu.twhttps://orcid.org/0000-0001-9011-65301HydeKevin D.23Department of Plant Medicine, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, TaiwanMae Fah Luang UniversityChiang RaiThailandCenter of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, ThailandNational Chiayi UniversityChiayiTaiwanKey Laboratory for Plant Biodiversity and Biogeography of East Asia (KLPB), Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, ChinaKunming Institute of Botany, Chinese Academy of ScienceKunmingChinaIndustrial Science and Management (International Program), Faculty of Science and Technology, Thammasat University (Rangsit Center), Klong Luang, Pathumthani 12121, ThailandThammasat UniversityPathumthaniThailandDepartment of Health Sciences, Faculty of Science, University of Mauritius, Reduit, MauritiusUniversity of MauritiusMokaMauritiusShenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518000, ChinaShenzhen UniversityShenzhenChina
Corresponding author: Chang-Hsin Kuo (chkuo@mail.ncyu.edu.tw)
Academic editor: A. Miller
2019161220196191109F9BCAADB-E9D7-539B-8A84-A3DCDB6D317235921900410201919112019Danushka S. Tennakoon, Kasun M. Thambugala, Rajesh Jeewon, Sinang Hongsanan, Chang-Hsin Kuo, Kevin D. HydeThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
A novel ascomycete genus, Longihyalospora, occurring on leaf litter of Ficusampelas 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, Chaetothyriumvermisporum transferred to the new genus. In addition, Ceramothyriumlongivolcaniforme is reported for the first time on Ficusampelas. Newly added species are compared with other similar species and comprehensive descriptions and micrographs are provided.
Tennakoon DS, Thambugala KM, Jeewon R, Hongsanan S, Kuo C-H, Hyde KD (2019) Additions to Chaetothyriaceae (Chaetothyriales): Longihyalospora gen. nov. and Ceramothyrium longivolcaniforme, a new host record from decaying leaves of Ficus ampelas. MycoKeys 61: 91–109. https://doi.org/10.3897/mycokeys.61.47056
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 methodsSample collection, morphological studies and isolation
Decaying leaf litter samples of Ficusampelas 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 Ceramothyriumlongivolcaniforme 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 Ceramothyriumlongivolcaniforme) 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 Longihyalosporaampeli) 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).
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
Aphanophoraeugeniae
CBS 124105
FJ839617
FJ839652
–
Brycekendrickomycesacaciae
CBS 124104
MH863350
MH874874
–
Camptophorahylomeconis
IFRDCC 2661
MF285228
MF285230
–
C.hylomeconis
CBS 113311
EU035415
–
KC455295
Caproniafungicola
CBS 614.96
KY484990
FJ358224
FJ225722
C.mansonii
CBS 101.67
AF050247
MH870591
AF346422
Ceramothyriumaquaticum
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
–
Chaetothyriumagathis
MFLUCC 12-0113
KP744437
KP744480
–
C.brischoficola
MFLUCC 10-0012
HQ895839
HQ895836
–
Cladophialophoraminourae
CBS 556.83
AY251087
FJ358235
FJ225734
C.emmonsii
CBS 640.96
KX822192
KC809995
KX822192
Cyphellophoriellapruni
CPC 25120
KR611878
–
–
Leptoxyphiumfumago
CBS 123.26
MH854862
GU214430
GU214535
L.madagascariense
CBS 124766
MH863407
GQ303308
–
Longihyalosporaampeli
MFLU 19-0824
MN219716
MN238771
MN238774
L.ampeli
MFLU 19-0825
MN219717
MN238772
MN238775
Knufiacryptophialidica
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
Minimelanolocusasiaticus
MFLUCC 15-0237
KR215604
KR215610
KR215615
M.melanicus
MFLUCC 15-0415
KR215608
KR215613
KR215618
Phaeosaccardinuladendrocalami
IFRDCC 2663
KF667243
KF667246
–
P.dendrocalami
IFRDCC 2649
KF667242
KF667245
–
P.ficus
MFLUCC 10-0009
HQ895840
HQ895837
–
P.multiseptata
IFRDCC 2639
KF667241
KF667244
–
Trichomeriumdeniqulatum
MFLUCC 10-0884
JX313654
JX313660
–
T.follicola
MFLUCC 10-0058
JX313653
JX313659
–
T.gleosporum
MFLUCC 10-0087
JX313656
JX313662
–
Vonarxiavagans
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.
ResultsPhylogenetic 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 Ceramothyriumlongivolcaniforme (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).
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 Leptoxyphiumfumago (CBS 123.26) and L.madagascariense (CBS 124766).
Epiphytic on decaying leaves of Ficusampelas 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 Ficusampelas Burm.f (Moraceae), 20 June 2018, D.S. Tennakoon, H10 (MFLU19-0823), living culture (MFLUCC19-0252).
Notes.
In this study, a sample of Ceramothyriumlongivolcaniforme was collected from dead leaves of Ficusampelas (Moraceae) in Taiwan. The new collection shares a close phylogenetic relationship with Ceramothyriumlongivolcaniforme (MFLU16-1306) (Figure 1). The morphology of our collection (MFLUCC19-0252) fits with the type material of Ceramothyriumlongivolcaniforme (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). Ceramothyriumlongivolcaniforme has been previously reported from Thailand on unidentified sp. (not F.ampelas) and thus, we provide the new host record of Ceramothyriumlongivolcaniforme on Ficusampelas (Moraceae). Remarkably, this is the first Ceramothyrium species collected from Taiwan.
Ceramothyriumlongivolcaniforme (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).
https://binary.pensoft.net/fig/365506
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
Macarangatanarius/ Philippines
14–16 × 4.5–5.5
Batista and Ciferri (1962)
C.boedijnii
3
Theobromacacao/ 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
Pyrolarotundifolia/ Sweden
18–20 × 4–5.5
Eriksson (1992)
C.cinereum
7
–
35–42 × 7–9
Batista and Maia (1956)
C.citricola
3–4
Citrusaurantium/ Brazil
14–30 × 2.5–11
Mendes et al. (1998)
C.coffeanum
3
Coffearobusta/ New Guinea
12–16 × 4–6
Batista and Ciferri (1962)
C.cordiae
3
Cordiarufescens/ Brazil
10–13.5 × 4–5.4
Eriksson (1992)
C.europaeum
3
Pogonophoraschomburgkiana/ 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
Aleuritesmoluccana/ Brazil
19–25 × 4–5
Eriksson (1992)
C.gustaviae
3–5
Gustaviaaugusta/ Brazil
22–25 × 3.7–5
Eriksson (1992)
C.gymnopogonis
2
Alyxiascandens/ Samoa
15 × 5
Dingley et al. (1981)
C.jambosae
–
Eugeniamalaccensis/ Brazil
–
Eriksson 1992
C.linnaeae
3–4
Lycopodiumannotinum/ 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
Ficusampelas / Taiwan
30–45 × 8–16
This study (New host record)
6 longitudinal
C.lycopodii
7
Lycopodiumannotinum/ 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
Paivaealangsdorffii/ 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
Didymopanaxmorototoni/ 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
Philodendronimbe/ 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)
AnimaliaChaetothyrialesChaetothyriaceaeBA6AF1D7-4875-50A6-A448-7A5329BE7CFBLongihyalosporaIndex Fungorum number: IF 556715Facesoffungi number: FoF06136Tennakoon, C.H Kuo & K. D Hydegen. 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.
Longihyalosporaampeli Tennakoon, C.H Kuo & K. D Hyde.
AnimaliaChaetothyrialesChaetothyriaceaeF066F419-72D8-5847-AB64-0D4E5AC2B474LongihyalosporaampeliIndex Fungorum number: IF 556716Facesoffungi number: FoF06137Tennakoon, C.H Kuo & K.D. Hydesp. nov.Figure 3Etymology.
Species name based on the host Ficusampelas, 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.
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, Longihyalosporaampeli species constitutes a strongly supported sub clade, which is nested independently from other genera in Chaetothyriaceae (Figure 1).
Longihyalosporaampeli (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).
https://binary.pensoft.net/fig/365507AnimaliaChaetothyrialesChaetothyriaceaeC47738F9-D32D-5CD5-996C-89B275C7FD0BLongihyalosporavermisporumIndex Fungorum number: IF 556717Facesoffungi number: FoF01679(Hansf.) Tennakoon, C.H. Kuo & K.D. Hydecomb. nov.≡ Chaetothyriumvermisporum Hansf., Mycol. Pap. 15: 151 (1946). Morphological description: See Hansford (1946), Hofmann and Piepenbring (2006). Recorded hosts.
Uganda (Hansford, 1946), Panama (Hofmann and Piepenbring 2006).
Notes.
Chaetothyriumvermisporum 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 Chaetothyriumvermisporum shares some similar morphology with Longihyalosporaampeli by having mycelial pellicle with ring of setae, pale brown to brown peridium and hyaline, fusiform, elongated and multi-septate ascospores (Hansford (1946). However, Chaetothyriumvermisporum can be distinguished from Longihyalosporaampeli by having hyaline surface mycelium, smaller asci (60 × 30 µm) and ascospores (35–50 × 5–6 µm) without a mucilaginous sheath, whereas Longihyalosporaampeli has dark brown mycelium, larger asci (90–115 × 52–62 µm) and ascospores (76–98 × 10–12 μm) with mucilaginous sheath. Therefore, we synonymized Chaetothyriumvermisporum under Longihyalospora based on high morphological similarities. Fresh collections with molecular data are needed to clarify the phylogenetic affinity of Longihyalosporavermisporum.
Additionally, we compared our collection with Chaetothyriumguaraniticum Speg. (type species of Chaetothyrium). Longihyalosporaampeli can be distinguished from Chaetothyriumguaraniticum 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.
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 Ceramothyriumpaiveae 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. Ceramothyriumlongivolcaniforme, 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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