Research Article |
Corresponding author: Hong Yu ( hongyu@ynu.edu.cn ) Academic editor: Nalin Wijayawardene
© 2024 Zhi-Qin Wang, Jing Zhao, Quan-Ying Dong, Yao Wang, Ying-Ling Lu, Run Luo, Hong Yu.
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:
Wang Z-Q, Zhao J, Dong Q-Y, Wang Y, Lu Y-L, Luo R, Yu H (2024) Multi-locus molecular phylogenetic analysis reveals two new species of Amphichorda (Bionectriaceae, Hypocreales). MycoKeys 106: 287-301. https://doi.org/10.3897/mycokeys.106.117205
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Amphichorda has been previously accepted as a member of the Cordycipitaceae and currently it is considered a member of the Bionectriaceae. The substrates of Amphichorda were complex and varied, being mainly animal faeces. This study reports two new species of Amphichorda from Yunnan Province in south-western China. Based on the five-gene (nrSSU, nrLSU, tef‐1α, rpb1 and rpb2) sequence and ITS data phylogenetic analysis, two new species, namely A. excrementa and A. kunmingensis, are proposed and a detailed description of the new species is provided. Amphichorda excrementa and A. kunmingensis were isolated from animal faeces in the park. The morphological characteristics of two novel species and seven known species in Amphichorda are also compared.
Coprophilous fungi, diversity, morphology, new taxa, taxonomy
Amphichorda Fr. was established to accommodate the type species A. felina (DC.) Fr., which was isolated from cat dung and previously classified in the genus Clavaria (
The taxonomic status of the type species has been controversial since the original description of the type species of the Amphichorda. Amphichorda felina was classified as Beauveria in 1980 (
During the surveys of entomopathogenic fungifrom two regions in Yunnan Province, China, the animal faeces were collected and three strains were isolated from the specimens. Based on morphological evidence together with the five-gene (nrSSU, nrLSU, tef‐1α, rpb1 and rpb2) sequence and ITS data analyses of some genera in Bionectriaceae, it was shown that the three strains belong to the genus Amphichorda. On the basis of its morphological characteristics and multi-locus molecular phylogenetic analyses, two new species were described. Furthermore, the morphological characteristics of two novel species and seven known species in Amphichorda were compared.
The specimens were collected in Kunming City, Yunnan Province, China in July 2019. In the field, it was placed in sterilised plastic pipes and brought to the laboratory for isolation. In order to obtain axenic cultures, part of the surface tissue of the specimen was cut off with a sterilised dissecting knife and then placed into a flask containing 10 ml of sterilised water and glass beads. Then the suspension was shaken for 10 min and diluted 50 times. Finally, the diluted suspension was applied on Petri dishes with potato dextrose agar (PDA: fresh potato 200 g/l, dextrose 20 g/l and agar 18 g/l) containing 0.1 g/l streptomycin and 0.05 g/l tetracycline. Then the Petri dish was placed in a room at 15 °C to allow it to grow, during which time the growing fungiwere transferred one by one to new Petri dishes. After isolation into pure cultures, they were transplanted to a PDA slant and stored at 4 °C. The specimens were deposited in the Yunnan Herbal Herbarium (YHH) of Yunnan University, China. The strain was deposited at the Yunnan Fungal Culture Collection (YFCC) of Yunnan University, China. The culture of the Amphichorda felina (CBS 250.34) was obtained from the culture collection (CBS) of the Westerdijk Fungal Biodiversity Institute (WI) in Utrecht, the Netherlands. The obtained strain CBS 250.34 was inoculated into PDA medium and re-cultured.
Colonies were incubated on PDA for three weeks in an incubator at 25 °C. The photograph was taken morphologically using a Canon 750 D camera (Canon Inc., Tokyo, Japan). The anamorphs (Conidiophores, Phialides and Conidia) in culture were observed using a light microscope (Olympus BX53). The growth rate of colonies was calculated using the method of
The genomic DNA was extracted from axenic living cultures using the Genomic DNA Purification Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The five-gene (nrSSU, nrLSU, tef‐1α, rpb1 and rpb2) and ITS were sequenced and the following primer pairs were used for PCR amplification. The nuclear ribosomal internal transcribed spacer region (ITS) was amplified with the primer pairs ITS4/ITS5 (
Based on the six-locus molecular, including ITS, nrSSU, nrLSU, tef‐1α, rpb1 and rpb2, phylogenetic analyses were performed using datasets retrieved from GenBank and those generated in this work. The DNA sequences newly generated have been submitted to GenBank. The sequences downloaded from the GenBank database were based on a previous study by
Species information and corresponding GenBank accession numbers of Amphichorda and close relative genera used in this study.
Species | Strain | ITS | nrSSU | nrLSU | tef‐1α | rpb1 | rpb2 |
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Alloacremonium humicola | CBS 613.82 | NR_189433 | – | NG_229089 | OQ470786 | – | OQ453888 |
Alloacremonium ferrugineum | CBS 102877 | NR_189432 | – | NG_228721 | OQ470785 | – | OQ453887 |
Amphichorda cavernicola | CGMCC3.19571 | MK329056 | – | MK328961 | MK335997 | – | – |
Amphichorda cavernicola | LC12481 | MK329057 | – | MK328962 | MK335998 | – | – |
Amphichorda cavernicola | LC12553 | MK329059 | – | MK328964 | MK336000 | – | – |
Amphichorda cavernicola | LC12560 | MK329061 | – | MK328966 | MK336002 | – | – |
Amphichorda coprophila | CBS 247.82 T | MH861494 | – | MH873238 | OQ954487 | – | – |
Amphichorda coprophila | CBS 424.88 | OQ942929 | – | OQ943166 | OQ954488 | – | – |
Amphichorda excrementa | YFCC AECCS848T | - | OR913433 | OR913439 | OR917446 | OR917451 | OR917443 |
Amphichorda felina | CBS 250.34 | MH855498 | – | OQ943167 | OQ954490 | – | – |
Amphichorda felina | CBS 250.34 | - | OR913436 | OR913440 | OR917447 | OR917450 | OR917444 |
Amphichorda felina | CBS 648.66 | OQ942930 | – | MH870575 | OQ954491 | – | – |
Amphichorda guana | CGMCC3.17908T | KU746665 | KY883262 | KU746711 | KX855211 | KY883202 | KY883228 |
Amphichorda guana | CGMCC3.17909 | KU746666 | KY883263 | KU746712 | KX855212 | KY883203 | – |
Amphichorda kunmingensis | YFCC AKYYH8414T | - | OR913435 | OR913438 | OR917448 | OR917452 | – |
Amphichorda kunmingensis | YFCC AKYYH8487 | - | OR913434 | OR913437 | OR917449 | OR917453 | OR917445 |
Amphichorda littoralis | FMR 17952 | OQ942925 | – | OQ943162 | OQ954483 | – | – |
Amphichorda littoralis | FMR 19404T | OQ942924 | – | OQ943161 | OQ954482 | – | – |
Amphichorda littoralis | FMR 19611 | OQ942926 | – | OQ943163 | OQ954484 | – | – |
Amphichorda monjolensis | COAD 3124 | OQ288256 | – | OQ288260 | OR454090 | – | OQ405040 |
Amphichorda monjolensis | COAD 3125 | OQ288257 | – | – | – | – | OQ405041 |
Amphichorda monjolensis | COAD 3120 | OQ288258 | – | – | – | – | OQ405042 |
Amphichorda yunnanensis | KUMCC 21-0414 | ON426823 | – | – | OR025977 | OR022016 | OR022041 |
Amphichorda yunnanensis | KUMCC 21-0415 | ON426824 | – | – | OR025976 | OR022015 | OR022040 |
Amphichorda yunnanensis | KUMCC 21-0416T | - | – | – | OR025975 | OR022014 | OR022039 |
Bulbithecium ammophilae | CBS 178.78 | NR_189437 | – | NG_242039 | OQ470793 | – | OQ453895 |
Bulbithecium arxii | CBS 737.84 | NR_145040 | – | HQ232159 | OQ470794 | – | OQ451834 |
Bulbithecium borodinense | CBS 101148 | OQ429506 | – | HQ232003 | – | – | – |
Bulbithecium ellipsoideum | CBS 993.69 | NR_189438 | – | NG_242040 | OQ470796 | – | OQ453896 |
Bulbithecium hyalosporum | CBS:318.91 | MH862256 | AF096172 | OQ055419 | OQ470797 | – | OQ453897 |
Bulbithecium pinkertoniae | CBS 157.70 | NR_159611 | NG_062816 | NG_058554 | OQ470799 | – | OQ453898 |
Bulbithecium spinosum | CBS 136.33 | OQ429512 | NG_062819 | NG_056971 | OQ470802 | – | OQ453899 |
Bulbithecium truncatum | CBS 113718 | NR_189439 | – | NG_242041 | OQ470803 | – | OQ453900 |
Claviceps purpurea | SA cp11 | - | EF469122 | EF469075 | EF469058 | EF469087 | EF469105 |
Geosmithia lavendula | CBS 344.48 | MH856380 | – | MH867927 | – | – | – |
Geosmithia pallidum | CBS 260.33 | OQ429599 | – | OQ055509 | OQ470909 | – | OQ453998 |
Hapsidospora chrysogena | CBS 144.62 | NR_189452 | NG_062810 | HQ232017 | OQ470953 | – | OQ454043 |
Hapsidospora flava | CBS 596.70 | NR_189453 | NG_062812 | NG_056983 | OQ470957 | – | OQ454047 |
Hapsidospora globosa | CBS 512.70 | NR_160124 | – | NG_064081 | OQ470963 | – | OQ454053 |
Hapsidospora inversa | CBS 517.70 | NR_189454 | – | OQ055565 | OQ470967 | – | OQ454057 |
Hapsidospora irregularis | CBS 510.70 | NR_160123 | – | MH871595 | OQ470968 | – | OQ454058 |
Hapsidospora stercoraria | CBS 516.70 | OQ429662 | – | OQ055568 | OQ470970 | – | OQ454060 |
Hapsidospora variabilis | CBS 100549 | NR_189456 | – | NG_229091 | OQ470971 | – | OQ454061 |
Myriogenospora atramentosa | AEG 96-32 | - | AY489701 | AY489733 | AY489628 | AY489665 | DQ522455 |
Ovicillium subglobosum | CBS 101963 | NR_154335 | – | NG_069329 | OQ471085 | – | OQ454170 |
Ovicillium attenuatum | CBS 399.86 | NR_154333 | – | NG_229092 | OQ471083 | – | OQ454168 |
Proxiovicillium blochii | CBS 427.93 | - | HQ232182 | HQ232001 | – | – | – |
Proxiovicillium lepidopterorum | CBS 101239 | NR_189482 | – | NG_242070 | OQ471145 | OQ454214 | |
Proliferophialis apiculata | CBS 303.64 | NR_189480 | – | NG_242064 | OQ471122 | – | OQ454207 |
Proliferophialis apiculata | CBS 397.78 | OQ429798 | – | OQ055694 | – | – | OQ454209 |
Stilbocrea macrostoma | CBS 141849 | OQ429874 | – | OQ430123 | – | – | OQ454273 |
Stilbocrea walteri | CBS 144627 | NR_160063 | – | NG_242075 | – | – | – |
Waltergamsia parva | CBS 381.70A | NR_163808 | – | NG_242083 | OQ471279 | – | OQ454346 |
Waltergamsia pilosa | CBS 124.70 | NR_163809 | – | OQ430199 | OQ471282 | – | OQ454349 |
The phylogenetic tree was inferred using 54 strains of 12 genera from Bionectriaceae and Clavicipitaceae, including Alloacremonium, Amphichorda, Bulbithecium, Claviceps, Geosmithia, Hapsidospora, Myriogenospora, Ovicillium, Proxiovicillium, Proliferophialis, Stilbocrea and Waltergamsia. Two strains (Claviceps purpurea SA cp11 and Myriogenospora atramentosa AEG 96-32) of Clavicipitaceae were selected as the outgroup. The final length of the six-locus molecular sequence concatenated dataset was 5,798 bp, including 766 bp for ITS, 1,391 bp for nrSSU, 859 bp for nrLSU, 850 bp for tef‐1α, 781 bp for rpb1 and 1,151 bp for rpb2. Phylogenetic trees from the BI and ML analyses exhibited similar topologies that had ten recognised, statistically well‐supported clades in Bionectriaceae. The four strains were clustered in the genus Amphichorda based on the phylogenetic analyses of the combined dataset (Fig.
Phylogenetic tree of Amphichorda and close relative genera was constructed, based on Maximum Likelihood (ML) and Bayesian Inference (BI) analysis using six-locus molecular (ITS, nrSSU, nrLSU, tef‐1α, rpb1and rpb2) sequences. The values of ML bootstrap proportions (BP) (≥ 70%) and the BI posterior probability (PP) (≥ 0.70) are indicated at the nodes (BP/PP). The new taxa were highlighted in bold.
China, Yunnan Province, Kunming City, Changchongshan Country Park, 11 July 2019, Hong Yu and Yao Wang (YHH AECCS200777, holotype; YFCC AECCS848, ex-type).
Sexual morph : Undetermined. Asexual morph: Colonies on PDA attaining a diameter of 42–44 mm after a month at 25 °C, white to cream, with high mycelial density, cottony, with a yellow margin, reverse pale yellow. Hyphae branched, smooth-walled, septate, hyaline, 0.6–1.3 µm wide. Cultures readily produced phialides and conidia after 3 weeks on potato dextrose agar at room temperature. Conidiophores arising laterally from hyphae, cylindrical, straight or slightly curved, hyaline and occasionally branched. Phialides arising laterally from aerial hyphae, occasionally solitary, mostly in whorls of 2–3 on lateral branches from the mycelia, basal portion cylindrical or flask-shaped, usually curved, 4.1–13.9 × 1.3–2.1 µm, tapering abruptly towards the apex, have a distinctly thin neck. Conidia 1.7–3.0 × 1.2–2.5 µm, one-celled, smooth-walled, hyaline, globose to elliptical, single. Chlamydospores not observed.
Animal faeces.
China.
Phylogenetic analyses showed that Amphichorda excrementa formed a separate clade with statistical support from the BI posterior probabilities (PP = 1.00) and the ML bootstrap proportions (BP = 90%) and was closely related to A. felina, A. yunnanensis and A. monjolensis. However, A. excrementa can be distinguished from three species by morphological differences. The phialides of A. excrementa were longer (4.1–13.9 × 1.3–2.1 µm) than those of A. felina (1.5–8.5 × 1.8–2.9 µm) and the conidia were smaller than those of A. felina (1.7–3.0 × 1.2–2.5 µm vs. 2.5–4.7 × 2–3.5 µm). The phialides of A. excrementa were longer (4.1–13.9 × 1.3–2.1 µm) than those of A. yunnanensis (4–12 × 1–4 µm) and the conidia were smaller than those of A. felina (1.7–3.0 × 1.2–2.5 µm vs. 2–5 × 2–4 µm). The conidia of A. monjolensis were longer than those of A. excrementa (2.8–3.7 × 1.8–2.9 µm vs. 1.7–3.0 × 1.2–2.5 µm).
The morphological description of this study is based on the specimen, CBS 250.34. Sexual morph: Undetermined. Asexual morph: Colonies on PDA attaining a diameter of 36–38 mm after a month at 25 °C, white to creamy-white, hard texture, felt-like, reverse black-brown, many conidia assemble to form powder. Hyphae branched, smooth-walled, septate, hyaline, 1.2–2.4 µm wide. Phialides arising laterally from aerial hyphae, erect or irregularly curved, 1.5–4.1 × 1.8–2.9 µm. Conidia 2.9–4.7 × 2.4–3.5 µm, one-celled, smooth-walled, hyaline, broadly ellipsoid or subglobose, single or aggregated into spheres. Chlamydospores not observed.
Pupa of Anaitis efformata, rabbit dung, mouldy leaves, porcupine dung, cat dung.
Argentina, Britain, France, Germany.
Named from the location Kunming City where the species was collected.
China, Yunnan Province, Kunming City, Wild Duck Lake Forest Park, 16 July 2019, Hong Yu and Yao Wang (YHH AKYYH200704, holotype; YFCC AKYYH8414, ex-type).
Sexual morph : Undetermined. Asexual morph: Colonies on PDA attaining a diameter of 52–54 mm after a month at 25 °C, white to pale grey, with low mycelial density, lanose. Hyphae hyaline, branched, smooth-walled, septate, 0.7–1.9 µm wide. Cultures readily produced phialides and conidia after 3 weeks on potato dextrose agar at room temperature. Phialides arising laterally from aerial hyphae, solitary, occasionally in simple whorls on lateral branches from the mycelia, basal portion cylindrical or fusiform, straight or irregularly bent, 6.1–17.5 × 1.4–2.9 µm. Conidia 2.3–4.2 × 1.6–3.0 µm, one-celled, smooth-walled, hyaline, globose to elliptical, single or aggregating in small heads at the apex of conidiogenous cells. Chlamydospores not observed.
Animal faeces.
China.
China, Yunnan Province, Kunming City, Wild Duck Lake Forest Park, 16 July 2019, Hong Yu and Yao Wang (YHH AKYYH200776, paratype; YFCC AKYYH8487, ex-paratype).
Three species of Amphichorda were from China and A. yunnanensis was distributed in Yuxi City, Yunnan Province. The two new species in this study were from Kunming City, Yunnan Province. According to the phylogenetic tree, the new species, A. kunmingensis, forms a separate branch in Amphichorda and is sister to A. guana. However, it differs from A. guana by its smaller conidia. Although A. kunmingensis, A. excrementa and A. yunnanensis were all collected from Yunnan, their morphology was quite different (see Table
Geographical location, hosts/substrates and asexual morphology of Amphichorda.
Species | Country | Host/Substrate | Conidiophores | Phialides (μm) | Conidia (μm) | References |
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Amphichorda cavernicola | China | Bird faeces; soil; plant debris; animal faeces; bat guano | Cylindrical, straight or slightly curved, occasionally branched | Fusiform or ellipsoidal, straight or irregularly bent, 4.5–8.0 × 2.0–3.0 | Broadly ellipsoidal to subglobose, 2.5–4.0 × 2.0–3.5 |
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A. coprophila | Canada; England | Chipmunk, rabbit and porcupine dung | Straight or flexuous, unbranched, bearing lateral or terminal conidiogenous cells, arranged singly or in whorls | Flask-shaped, usually with a strongly bent neck, 6–10 × 2–2.5 | Subglobose to somewhat ellipsoidal, 3.5–5.5 × 2–3 |
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A. excrementa | China | Animal faeces | Cylindrical, straight or slightly curved, occasionally branched | Occasionally solitary, mostly in whorls of 2–3, basal portion cylindrical or flask-shaped, usually curved, 4.1–13.9 × 1.3–2.1 | Globose to elliptical 1.7–3.0 × 1.2–2.5 | In this study |
A. felina | Britain, Germany, Argentina, France | Pupa of Anaitis efformata; rabbit dung; mouldy leaves; porcupine dung; cat dung | Straight | Solitarily or in small groups, consisting of a swollen, flask-shaped or curved, occasionally elongate basal part, 1.5–8.5 × 1.8–2.9 | Subglobose, ellipsoidal or ovoidal, sometimes with a pointed base, 2.5–4.7 × 2–3.5 |
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A. guana | China | Bat guano | Straight or slightly curved | Fusiform or ellipsoidal, straight or irregularly bent, 7–10 × 2–3 | Broadly ellipsoid to subglobose, 4.5–5.5 × 3.5–5 |
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A. kunmingensis | China | Animal faeces | - | Solitary, occasionally in simple whorls, basal portion cylindrical or fusiform, straight or irregularly bent, 6.1–17.5 × 1.4–2.9 | Globose to elliptical 2.3–4.2 × 1.6–3.0 | In this study |
A. littoralis | Spain | Sediments; fragment of floating rubber tire | Straight or flexuous, commonly unbranched, bearing lateral or terminal conidiogenous cells, arranged singly or in whorls of 2–4 | Flask-shaped, usually with a strongly bent neck, 6–10 (–11.5) × 1.5–2 | Subglobose, 3–4 × 2.5–3 |
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A. monjolensis | Brazil | on PDA plate consumed by an insect | Cylindrical, bearing one or more conidiogenous cells, straight or slightly bent, solitary or synnematous, sometimes branched | Flask-shaped, straight or irregularly bent, 3.1–6.1 × 2.7–5.1 | Holoblastic, 2.8–3.7×1.8–2.9 |
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A. yunnanensis | China | Wing surfaces of Rhinolophus | Cylindrical, straight or slightly curved, branched | Monoblastic to polyblastic, ampulliform to flask-shaped, 4–12 × 1–4 | Globose to oval, slightly ellipsoid, 2–5 × 2–4 |
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The phylogenetic analyses, based on the five-gene (nrSSU, nrLSU, tef‐1α, rpb1 and rpb2) sequence and ITS data were conducted and Amphichorda excrementa and A. kunmingensis were introduced. The morphological characteristics of the new species are similar to those of other Amphichorda species. Its conidiophores straight or slightly curved; phialides solitary, simple whorls or several whorls, straight or irregularly bent, usually curved, tapering abruptly towards the apex; conidia solitary or clumped, one-celled, shape variable (Table
The species of Amphichorda has an extremely wide distribution, including Argentina, Canada, China, France, Germany, Great Britain, Spain (Table
Coprophilous fungi, particularly coprophilous ascomycetes, will be a rich source of antibiotics and other biologically important secondary metabolites (
The authors have declared that no competing interests exist.
No ethical statement was reported.
This work was supported by the National Natural Science Foundation of China (No. 31870017).
Data curation: QYD. Investigation: YW. Visualization: RL, YLL. Writing - original draft: ZQW. Writing - review and editing: JZ, HY.
Zhi-Qin Wang https://orcid.org/0000-0001-9022-3635
All of the data that support the findings of this study are available in the main text.