Research Article |
Corresponding author: Ting-Chi Wen ( tingchiwen@yahoo.com ) Academic editor: Marc Stadler
© 2018 Shi-Ke Huang, Rajesh Jeewon, Kevin D. Hyde, D. Jayarama Bhat, Putarak Chomnunti, Ting-Chi Wen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Huang S-K, Jeewon R, Hyde KD, Bhat DJ, Chomnunti P, Wen T-C (2018) Beta-tubulin and Actin gene phylogeny supports Phaeoacremonium ovale as a new species from freshwater habitats in China. MycoKeys 41: 1-15. https://doi.org/10.3897/mycokeys.41.27536
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A new species of Phaeoacremonium, P. ovale (Togniniaceae), was isolated during a diversity study of freshwater fungi from Yunnan Province in China. Morphological and cultural studies of the fungus were carried out and its sexual and asexual morphs (holomorph) are introduced herein. This species is characterised by peculiar long-necked, semi-immersed ascomata with oval to ellipsoid ascospores and ellipsoid to ovoid conidia. Phylogenetic analyses of a combined TUB and ACT gene dataset revealed that strains of P. ovale constitute a strongly supported independent lineage and are related to P. griseo-olivaceum and P. africanum. The number of nucleotide differences, across the genes analysed, also supports establishment of P. ovale as a new species.
1 new species, Togniniales , Sordariomycetes , Morphology, Phylogeny
Lignicolous freshwater fungi are important in nutrient recycling (
Phaeoacremonium (= Togninia), introduced by
Most species of Phaeoacremonium are plant or/and human pathogens and some have been recorded on arthropods or in soil (
Species of Togniniaceae have been reported to colonise substrates in different types of habitats and recent taxonomic studies have revealed additional new species (
Submerged dead wood was collected from Baoshan, Yunnan Province in China in October 2016, brought to the laboratory in zip lock plastic bags and treated in the laboratory following procedures detailed in
Total genomic DNA was extracted from mycelium using a Trelief Plant Genomic DNA Kit following the instructions of the manufacturer. The genomic DNA was amplified by using polymerase chain reaction (PCR) in a 25 μl reaction mixture. Partial regions of the beta-tubulin (TUB) and Actin (ACT) gene were amplified using the primer pairs T1 (
The quality of the amplified nucleotide sequences was checked and combined by SeqMan version 7.1.0 (44.1) and Finch TV version 1.4.0 (www.geospiza.com). Sequences used by
The phylogenetic analyses of combined gene regions (TUB and ACT) were performed using maximum-likelihood (ML) and Bayesian Inference (BI) methods. The best-fit model (GTR+G+I) was obtained using jModelTest 2.1.10 under the Akaike Information Criterion (AIC) calculations (
Strains and GenBank accession numbers of the isolates used in this study. Isolates from this study are marked with asterisk (*) and the type strains are in bold.
Species | Voucher/Culture | GenBank accession number | |
---|---|---|---|
TUB | ACT | ||
Phaeoacremonium africanum | CBS 120863 | EU128100 | EU128142 |
Phaeoacremonium album | CBS 142688 | KY906885 | KY906884 |
Phaeoacremonium alvesii | CBS 110034 | AY579301 | AY579234 |
Phaeoacremonium alvesii | CBS 729.97 | AY579302 | AY579235 |
Phaeoacremonium amstelodamense | CBS 110627 | AY579295 | AY579228 |
Phaeoacremonium amygdalinum | CBS 128570 | JN191307 | JN191303 |
Phaeoacremonium amygdalinum | CBS H-20507 | JN191305 | JN191301 |
Phaeoacremonium amygdalinum | CBS H-20508 | JN191306 | JN191302 |
Phaeoacremonium angustius | CBS 114992 | DQ173104 | DQ173127 |
Phaeoacremonium angustius | CBS 114991 | DQ173103 | DQ173126 |
Phaeoacremonium argentinense | CBS 777.83 | DQ173108 | DQ173135 |
Phaeoacremonium armeniacum | ICMP 17421 | EU596526 | EU595463 |
Phaeoacremonium aureum | CBS 142691 | KY906657 | KY906656 |
Phaeoacremonium australiense | CBS 113589 | AY579296 | AY579229 |
Phaeoacremonium australiense | CBS 113592 | AY579297 | AY579230 |
Phaeoacremonium austroafricanum | CBS 112949 | DQ173099 | DQ173122 |
Phaeoacremonium austroafricanum | CBS 114994 | DQ173102 | DQ173125 |
Phaeoacremonium austroafricanum | CBS 114993 | DQ173101 | DQ173124 |
Phaeoacremonium bibendum | CBS 142694 | KY906759 | KY906758 |
Phaeoacremonium canadens e | PARC327 | KF764651 | KF764499 |
Phaeoacremonium cf. mortoniae | ICMP 18088 | HM116767 | HM116773 |
Phaeoacremonium cinereum | CBS 123909 | FJ517161 | FJ517153 |
Phaeoacremonium cinereum | CBS H-20215 | FJ517160 | FJ517152 |
Phaeoacremonium cinereum | CBS H-20213 | FJ517158 | FJ517150 |
Phaeoacremonium croatiense | CBS 123037 | EU863482 | EU863514 |
Phaeoacremonium fraxinopennsylvanicum | CBS 101585 | AF246809 | DQ173137 |
Phaeoacremonium fraxinopennsylvanicum | CBS 110212 | DQ173109 | DQ173136 |
Phaeoacremonium fuscum | CBS 120856 | EU128098 | EU128141 |
Phaeoacremonium gamsii | CBS 142712 | KY906741 | KY906740 |
Phaeoacremonium geminum | CBS 142713 | KY906649 | KY906648 |
Phaeoacremonium globosum | ICMP 16988 | EU596525 | EU595466 |
Phaeoacremonium globosum | ICMP 17038 | EU596521 | EU595465 |
Phaeoacremonium globosum | ICMP 16987 | EU596527 | EU595459 |
Phaeoacremonium griseo-olivaceum | CBS 120857 | EU128097 | EU128139 |
Phaeoacremonium griseorubrum | CBS 111657 | AY579294 | AY579227 |
Phaeoacremonium griseorubrum | CBS 566.97 | AF246801 | AY579226 |
Phaeoacremonium hispanicum | CBS 123910 | FJ517164 | FJ517156 |
Phaeoacremonium hungaricum | CBS 123036 | EU863483 | EU863515 |
Phaeoacremonium inflatipes | CBS 391.71 | AF246805 | AY579259 |
Phaeoacremonium inflatipes | CBS 113273 | AY579323 | AY579260 |
Phaeoacremonium iranianum | CBS 101357 | DQ173097 | DQ173120 |
Phaeoacremonium iranianum | CBS 117114 | DQ173098 | DQ173121 |
Phaeoacremonium italicum | CBS 137763 | KJ534074 | KJ534046 |
Phaeoacremonium italicum | CBS 137764 | KJ534075 | KJ534047 |
Phaeoacremonium italicum | CBS H-21638 | KJ534076 | KJ534048 |
Phaeoacremonium junior | CBS 142697 | KY906709 | KY906708 |
Phaeoacremonium krajdenii | CBS 110118 | AY579324 | AY579261 |
Phaeoacremonium krajdenii | CBS 109479 | AY579330 | AY579267 |
Phaeoacremonium longicollarum | CBS 142699 | KY906689 | KY906688 |
Phaeoacremonium luteum | CBS 137497 | KF823800 | KF835406 |
Phaeoacremonium meliae | CBS 142710 | KY906825 | KY906824 |
Phaeoacremonium minimum | CBS 246.91 | AF246811 | AY735497 |
Phaeoacremonium minimum | CBS 100397 | AF246806 | AY735498 |
Phaeoacremonium mortoniae | CBS 211.97 | AF246810 | |
Phaeoacremonium nordesticola | CMM4312 | KY030807 | KY030803 |
Phaeoacremonium novae-zealandiae | CBS 110156 | DQ173110 | DQ173139 |
Phaeoacremonium novae-zealandiae | CBS 110157 | DQ173111 | DQ173140 |
Phaeoacremonium occidentale | ICMP 17037 | EU596524 | EU595460 |
Phaeoacremonium oleae | CBS 142704 | KY906937 | KY906936 |
*Phaeoacremonium ovale | KUMCC 17-0145 | MH395327 | MH395325 |
*Phaeoacremonium ovale | KUMCC 18-0018 | MH395328 | MH395326 |
Phaeoacremonium pallidum | CBS 120862 | EU128103 | EU128144 |
Phaeoacremonium parasiticum | CBS 860.73 | AF246803 | AY579253 |
Phaeoacremonium parasiticum | CBS 113585 | AY579307 | AY579241 |
Phaeoacremonium parasiticum | CBS 514.82 | AY579306 | AY579240 |
Phaeoacremonium paululum | CBS 142705 | KY906881 | KY906880 |
Phaeoacremonium pravum | CBS 142686 | KY084246 | KY084248 |
Phaeoacremonium proliferatum | CBS 142706 | KY906903 | KY906902 |
Phaeoacremonium prunicola | CBS 120858 | EU128095 | EU128137 |
Phaeoacremonium prunicola | CBS 120858 | EU128096 | EU128138 |
Phaeoacremonium pseudopanacis | CPC 28694 | KY173609 | KY173569 |
Phaeoacremonium roseum | PARC273 | KF764658 | KF764506 |
Phaeoacremonium rosicola | CBS 142708 | KY906831 | KY906830 |
Phaeoacremonium rubrigenum | CBS 498.94 | AF246802 | AY579238 |
Phaeoacremonium rubrigenum | CBS 112046 | AY579305 | AY579239 |
Phaeoacremonium santali | CBS 137498 | KF823797 | KF835403 |
Phaeoacremonium scolyti | CBS 113597 | AF246800 | AY579224 |
Phaeoacremonium scolyti | CBS 113593 | AY579293 | AY579225 |
Phaeoacremonium scolyti | CBS 112585 | AY579292 | AY579223 |
Phaeoacremonium sicilianum | CBS 123034 | EU863488 | EU863520 |
Phaeoacremonium sicilianum | CBS 123035 | EU863489 | EU863521 |
Phaeoacremonium sp. | KMU 8592 | AB986584 | AB986583 |
Phaeoacremonium spadicum | CBS 142711 | KY906839 | KY906838 |
Phaeoacremonium sphinctrophorum | CBS 337.90 | DQ173113 | DQ173142 |
Phaeoacremonium sphinctrophorum | CBS 694.88 | DQ173114 | DQ173143 |
Phaeoacremonium subulatum | CBS 113584 | AY579298 | AY579231 |
Phaeoacremonium subulatum | CBS 113587 | AY579299 | AY579232 |
Phaeoacremonium tardicrescens | CBS 110573 | AY579300 | AY579233 |
Phaeoacremonium tectonae | MFLUCC 13-0707 | KT285563 | KT285555 |
Phaeoacremonium tectonae | MFLUCC 14-1131 | KT285570 | KT285562 |
Phaeoacremonium theobromatis | CBS 111586 | DQ173106 | DQ173132 |
Phaeoacremonium tuscanicum | CBS 123033 | EU863458 | EU863490 |
Phaeoacremonium venezuelense | CBS 651.85 | AY579320 | AY579256 |
Phaeoacremonium venezuelense | CBS 110119 | AY579318 | AY579254 |
Phaeoacremonium venezuelense | CBS 113595 | AY579319 | AY579255 |
Phaeoacremonium vibratile | CBS 117115 | DQ649063 | DQ649064 |
Phaeoacremonium viticola | CBS 113065 | DQ173105 | DQ173128 |
Phaeoacremonium viticola | CBS 101737 | AF246817 | DQ173129 |
Pleurostomophora richardsiae | CBS 270.33 | AY579334 | AY579271 |
Wuestneia molokaiensis | CBS 114877 | AY579335 | AY579272 |
The combined TUB and ACT sequence dataset comprised 98 strains of Phaeoacremonium. The tree was rooted with Pleurostoma richardsiae (CBS 270.33) and Wuestineaia molokaiensis (CBS 114877). The alignment comprised 947 total characters including gaps (TUB: 646bp; ACT: 301bp). ML and BI analyses yielded trees which were topologically congruent in terms of species groupings. RAxML analysis yielded a best scoring tree with a final optimisation likelihood value of -15310.399369 (Fig.
Maximum likelihood phylogenetic tree generated from analysis of a combined TUB and ACT sequences dataset for 98 taxa of Togniniaceae. Pleurostoma richardsiae (CBS 270.33) and Wuestineaia molokaiensis (CBS 114877) are the outgroup taxa. ML support values greater than 70% (BSML, left) and Bayesian posterior probabilities greater than 0.90 (BYPP, right) are indicated above the nodes. The strain numbers are noted after the species names. Ex-type strains are indicated in bold. Isolates from this study are indicated in red.
CHINA, Yunnan Province, Baoshan, stream along the roadside; saprobic on dead wood, 21 December 2016; Huang S.K. (KUN HKAS99550, holotype; MFLU MFLU18-1076, isotype); ex-type living culture (KUMCC 17-0145; KUMCC 18-0018). GenBank no. (ITS: MH399732, TUB: MH395327, ACT: MH395325; ITS: MH399733, TUB: MH395328, ACT: MH395326)
The name ovale refers to the oval shaped ascospores.
Sexual morph: Ascomata 225–300 μm (n = 5), on wood, perithecial, solitary, semi-immersed, unilocular, subglobose to globose, black, ostiolate, with ostiolar neck erumpent through bark of host when mature. Neck 445–645 × 35–45 μm (x̄ = 530 × 40 μm, n = 5), centrally ostiolate, contorted, lined with hyaline periphyses. Peridium 17–40 μm diam., membranous, composed of dark brown to hyaline cells of textura angularis. Hamathecium composed of 2–6 μm wide, hyaline, septate paraphyses, slightly constricted at septa and gradually narrowed towards apex. Asci 11–20 × 3–6 μm (x̄ = 15.5 × 5 μm, n = 30), 8-spored, unitunicate, clavate, with short pedicel, apically rounded. Ascospores 3–5 × 1.5–3 μm (x̄ = 3.5 × 2 μm, n = 50), bi-seriate, hyaline, oval to ellipsoid, aseptate, smooth-walled, rounded at the ends. Asexual morph: Mycelium on culture, partly superficial, composed of septate, branched, hyaline, rarely verrucose, hyphae 1.5–3 μm diam., rarely with adelophialides. Conidiophores usually arising from hyaline hyphae, mononematous, unbranched, occasionally constricted at basal septum, hyaline. Phialides 8–15 × 2–4 μm (x̄ = 9.5 × 3 μm, n = 20), terminal, monophialidic, elongate-ampulliform and attenuated at base. Conidia 2.5–6 × 1–2.5 µm (x̄ = 4 × 2 μm, n = 30), hyaline, ellipsoid to ovoid, aseptate.
Phaeoacremonium ovale (HKAS99550, holotype). a Substrate b, c Ascoma on host d Squashed neck e Ascoma in vertical section fPeridiumg Asci surrounded by paraphyses h Asci i Septate paraphyses j–m Asci with ascospores n Germinating ascospores. Note: Fig. i stained in Congo red reagent, fig l stained in Melzer’s reagent. Scale bars: 500 µm (c); 200 µm (d); 100 µm (e); 50 µm (f, i); 30 µm (n); 20 µm (g–h); 10 µm (j–m)
Ascospore germinating on PDA within 1 week at 23°C, germ tubes produced from ends. Colonies growing on PDA, reaching 2 cm diam. and sporulating after 30 days. Colonies semi-immersed to superficial, irregular in shape, flat, slightly raised, with undulate edge, slightly rough on surface, cottony to fairly fluffy, colony from above, greyish-brown (5F3–5,
Phaeoacremonium is currently accommodated in the monogeneric family Togniniaceae (
In this study, we introduce a novel taxon of Phaeoacremonium from dead wood collected in a stream in the Yunnan Province, China and describe its sexual and asexual morph. Examination of morphological characters reveal that our species is sufficiently distinct from extant species to establish it as a new species. Analyses of the combined DNA sequence dataset from partial TUB and ACT genes also support that this taxon is a Phaeoacremonium species and phylogenetically distinct from other species (Fig.
This work was supported by the National Natural Science Foundation of China (No. 31760014), the Science and Technology Foundation of Guizhou Province (No. [2016]2863 & [2017]5788). Dr. Qi Zhao is acknowledged for his support and thanks to Dr. Sajeewa Maharachchikumbura for his valuable help in phylogenetic analyses. Shaun Pennycook is thanked for checking and correcting the Latin name.