11 urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3 urn:lsid:zoobank.org:pub:A4FD9303-3F01-45D9-B438-FCCFE465D2F7 MycoKeys MC 1314-4057 1314-4049 Pensoft Publishers 10.3897/mycokeys.106.117205 117205 Research Article Hypocreales Sordariomycetes Phylogeny Taxonomy Asia Central Asia China Multi-locus molecular phylogenetic analysis reveals two new species of Amphichorda (Bionectriaceae, Hypocreales) Wang Zhi-Qin https://orcid.org/0000-0001-9022-3635 1 Writing - original draft Zhao Jing 1 Writing - review and editing Dong Quan-Ying 1 Data curation Wang Yao 1 Investigation Lu Ying-Ling 1 Visualization Luo Run 1 Visualization Yu Hong hongyu@ynu.edu.cn https://orcid.org/0000-0002-2149-5714 1 Writing - review and editing Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, Kunming, Yunnan, China Yunnan University Kunming China The International Joint Research Center for Sustainable Utilization of Cordyceps Bioresources in China and Southeast Asia, Yunnan University, Kunming, Yunnan, China Yunnan University Kunming China

Corresponding author: Hong Yu (hongyu@ynu.edu.cn, herbfish@163.com)

Academic editor: Nalin Wijayawardene

 2024 03 07 2024 106 287 301 053902AE-2A7B-5EAD-A453-C7DEBB768167 11 12 2023 06 05 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.

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 the National Natural Science Foundation of China (No. 31870017) 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

 Introduction

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 (Lamarck 1815; Fries 1825). At the present, seven species of the Amphichorda are now validly published (Zhang et al. 2017, 2021; Guerra-Mateo et al. 2023; Liu et al. 2023; Leão et al. 2024). The traditional phylogenetic placement of the genus Amphichorda was considered in the family Cordycipitaceae (Hypocreales). The Cordycipitaceae is the most complex group in the order Hypocreales because of its varied morphological characteristics and wide-ranging hosts and some genera present numerous taxonomical problems (Wang et al. 2020; Guerra-Mateo et al. 2023). In the studies of Zhang et al. (2017, 2021) and Liu et al. (2023) which report new species of the genus Amphichorda, the phylogenetic position of Amphichorda belongs to the Cordycipitaceae. However, Guerra-Mateo et al. (2023) conducted the phylogenetic analysis based on the nuclear ribosomal internal transcribed spacer region (ITS) and the nuclear ribosomal large subunit (nrLSU), considered Amphichorda to belong to the family Bionectriaceae and determined Amphichorda has close phylogenetic relationships with the genera Hapsidospora and Nigrosabulum. Leão et al. (2024) also proving the genus Amphichorda belongs to the family Bionectriaceae.

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 (Carmichael et al. 1980). However, early phylogenetic analyses showed that Beauveria felina was distant from other Beauveria species and that it was morphologically distinguished from other Beauveria species by the absence of elongate conidiogenous cells with apical denticulate rachis (Rehner et al. 2011; Zhang et al. 2017; Liu et al. 2023). The type strain of A. felina (= B. felina) seems to be unknown (Guerra-Mateo et al. 2023). Isaria cretacea J.F.H. Beyma type strain CBS 250.34 was considered to be the type strain of A. felina since I. cretacea was synonymised with A. felina (De Hoog 1972; Zhang et al. 2021; Guerra-Mateo et al. 2023). However, the criteria required for fungal epitypification were the substrate and geographic similarity (Guerra-Mateo et al. 2023). The substrate and geography were different between A. felina and the strain CBS 250.34, so this strain has not been designated as the epitype of A. felina (Lendemer 2020). Guerra-Mateo et al. (2023) proposed that the strain CBS 250.34 can be accepted as a reference to stabilise the nomenclature of A. felina, but should be avoided to indicate it as a type strain of A. felina (Zhang et al. 2017, 2021; Wang et al. 2020; Liu et al. 2023).

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.

 Materials and methods Fungal collection and isolation

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.

 Morphological observations

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 Liu and Hodge (2005) and it was categorised as: fast-growing (30–35 mm in diameter), moderately growing (20–30 mm in diameter) and slow-growing (< 20 mm in diameter).

 DNA extraction, PCR and sequencing

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 (White et al. 1990). The nuclear ribosomal small and large subunit (nrSSU and nrLSU) were amplified with the primer pairs nrSSU-CoF/nrSSU-CoR and LR5/LR0R, respectively (Vilgalys and Hester 1990; Rehner and Samuels 1994; Wang et al. 2015a). The translation elongation factor 1α (tef‐1α) was amplified with the primers EF1α‐EF and EF1α‐ER (Bischof et al. 2006; Sung et al. 2007). The largest and second subunits of RNA polymerase II (rpb1 and rpb2) were amplified with the primers RPB1‐5′F/RPB1‐5′R and RPB2-5′F/RPB2-5′R, respectively (Bischof et al. 2006; Sung et al. 2007). The polymerase chain reaction (PCR) matrix was performed in a final volume of 50 µl and the detailed information was described by Wang et al. (2022). Amplification reactions were performed in the BIORAD T100TM thermal cycler (BIO-RAD Laboratories, Hercules, CA, United States). The PCR reactions followed the procedures of Wang et al. (2015b) and the PCR products were sequenced by the Beijing Genomics Institute (Chongqing, China).

 Phylogenetic analyses

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 Hou et al. (2023) and Leão et al. (2024). The taxonomic information and corresponding GenBank accession numbers used are provided in Table 1. Sequences were aligned with MEGA v.6.06 and used to remove poorly-aligned regions and for manual adjustment (Tamura et al. 2013). Six-locus molecular were concatenated together using Phylosuite v.1.2.2 (Zhang et al. 2020). The Maximum Likelihood (ML) tree was performed using IQ-tree v.2.1.3 and the Bayesian Inference (BI) tree was performed using MrBayes v.3.2.2 (Ronquist et al. 2012; Nguyen et al. 2015). The best-fitting likelihood model for BI and ML analyses was selected using ModelFinder (Kalyaanamoorthy et al. 2017). In the phylogenetic tree of Amphichorda and some other genera, the TN+F+I+G4 model was selected as the optimal model for the ML analyses, with 5000 ultrafast bootstraps (Hoang et al. 2017) in a single run. The GTR+F+I+G4 model was selected as the optimal model for the BI analysis and the four Markov Chain Monte Carlo chains run for 2 million generations from a random start tree with a sampling frequency of 100 generations, in which the initial 25% of sampled data were discarded as burn-in. Phylogenetic trees were visualised in FigTree v.1.4.3 and edited in Adobe Illustrator CS6. The values of ML bootstrap proportions (BP) (≥ 70%) and the BI posterior probability (PP) (≥ 0.70) are indicated at the nodes (BP/PP).

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
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

Boldface: data generated in this study; T: ex-type culture. A.E.G: A. E. Glenn personal collection; CBS: the culture collection of the Westerdijk Fungal Biodiversity Institute (WI); CGMCC: the China General Microbiological Culture Collection Center; COAD: the Laboratório de Micologia e Etiologia de Doenças Fúngicas de Plantas and Coleção Octávio Almeida Drummond; FMR: the culture collection of the Faculty of Medicine in Reus; KUMCC: the Kunming Culture Collection; LC: personal culture collection held in the lab of Dr Lei Cai; YFCC: the Yunnan Fungal Culture Collection (YFCC) of Yunnan University.

 Results Sequencing and phylogenetic analyses

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. 1). Our ML and BI analyses showed that two new species (i.e. A. excrementa and A. kunmingensis) and one known species were recognised. The new species, A. excrementa and A. kunmingensis, were well-supported by bootstrap proportions (BP = 90% and BP = 82%, respectively) and posterior probabilities (PP = 1.00 and PP = 0.96, respectively).

10.3897/mycokeys.106.117205.figure1 EFF94757-58A4-5C90-B454-7CA3D3A139FD

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.

https://binary.pensoft.net/fig/1084614 Taxonomy Fungi Hypocreales Bionectriaceae DBF78251-A66F-5FD7-AD9D-8DF84A7E84EE Amphichorda excrementa 851377 Hong Yu bis, Z.Q. Wang, Q.Y. Dong & Y. Wang sp. nov. Fig. 2 Etymology.

Refers to the excrement material from which this fungus was isolated.

10.3897/mycokeys.106.117205.figure2 0C7AD6A2-AB31-5F27-ABB6-F53AB53DE09D

Morphology of Amphichorda excrementaA, B colony character on PDA medium after 30 d (A obverse B reverse) C–F conidiophores, conidiogenous cells and conidia. Scale bars: 2 cm (A, B); 10 µm (C–F).

https://binary.pensoft.net/fig/1084615 Type.

China, Yunnan Province, Kunming City, Changchongshan Country Park, 11 July 2019, Hong Yu and Yao Wang (YHH AECCS200777, holotype; YFCC AECCS848, ex-type).

 Description.

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.

 Substrate.

Animal faeces.

 Distribution.

China.

 Commentary.

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).

 Fungi Hypocreales Bionectriaceae D064A027-4CEB-5360-85A4-0286C61AC039 Amphichorda felina 562082 (DC.) Fr., Syst. orb. veg. (Lundae) 1: 170 (1825). Fig. 3 Description.

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.

10.3897/mycokeys.106.117205.figure3 D27B3FB5-8F03-5CC9-A23D-244C5EC66DAA

Morphology of Amphichorda felinaA, B colony character on PDA medium after 30 d (A obverse B reverse) C–K conidiophores, conidiogenous cells and conidia. Scale bars: 2 cm (A, B); 10 µm (C–F, I, K); 5 µm (G–H, J).

https://binary.pensoft.net/fig/1084616 Substrate.

Pupa of Anaitis efformata, rabbit dung, mouldy leaves, porcupine dung, cat dung.

 Distribution.

Argentina, Britain, France, Germany.

 Commentary.

Guerra-Mateo et al. (2023) proposed that the strain CBS 250.34 can be accepted as a reference to stabilise the nomenclature of Amphichorda felina and thus the genus Amphichorda, but should be avoided to indicate it as a type strain. In this study, the strain, CBS 250.34, was available in the CBS culture collection and morphological observations were made. Its morphology was generally consistent with those described by De Hoog (1972), with one difference being that this study extended the phialides (1.5–8.5 × 1.8–2.9 µm) and conidia size range of this species (2.5–4.7 × 2–3.5 µm).

 Fungi Hypocreales Bionectriaceae 8BB759EA-C045-52A6-9308-3D269DFD9A6B Amphichorda kunmingensis 851378 Hong Yu bis, Z.Q. Wang, Q.Y. Dong & Y. Wang sp. nov. Fig. 4 Etymology.

Named from the location Kunming City where the species was collected.

 Type.

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).

10.3897/mycokeys.106.117205.figure4 1791BA94-B230-54B8-AE7E-7439FCAA092D

Morphology of Amphichorda kunmingensisA, B colony character on PDA medium after 30 d (A obverse B reverse) C–K conidiogenous cells and conidia. Scale bars: 2 cm (A, B); 20 µm (C–E, H); 10 µm (F–G, I–K).

https://binary.pensoft.net/fig/1084617 Description.

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.

 Substrate.

Animal faeces.

 Distribution.

China.

 Other material examined.

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).

 Commentary.

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 2). Amphichorda kunmingensis differs from A. excrementa in its usually curved and longer phialides (6.1–17.5 × 1.4–2.9 μm vs. 4.1–13.9 × 1.3–2.1 μm) and larger conidia (2.3–4.2 × 1.6–3.0 μm vs. 1.7–3.0 × 1.2–2.5 μm). Amphichorda kunmingensis differs from A. yunnanensis in the shape of its phialides and narrower conidia.

Geographical location, hosts/substrates and asexual morphology of Amphichorda.

Species Country Host/Substrate Conidiophores Phialides (μm) Conidia (μm) References
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 Zhang et al. (2021)
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 Guerra-Mateo et al. (2023)
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 De Hoog (1972); In this study
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 Zhang et al. (2017)
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 Guerra-Mateo et al. (2023)
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 Leão et al. (2024)
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 Liu et al. (2023)
Discussion

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 2). They were similar to those of Beauveria and all species of Amphichorda do not have the elongate conidiogenous cells with apical denticulate rachis that are characteristic of Beauveria.

The species of Amphichorda has an extremely wide distribution, including Argentina, Canada, China, France, Germany, Great Britain, Spain (Table 2). Amongst the Amphichorda species, A. felina, A. cavernicola, A. guana and A. monjolensis were found in caves, especially A. felina, which was widely distributed in caves (Vanderwolf et al. 2013; Zhang et al. 2017, 2021; Vanderwolf et al. 2018; Leão et al. 2024). Amphichorda littoralis was found in Mediterranean coast sediments at 20 m depth (Guerra-Mateo et al. 2023). In contrast to the particular ecology of caves and the sea, A. coprophila was isolated from rabbit, chipmunk and porcupine dung and A. yunnanensis was isolated from the wing surfaces of Rhinolophus affinis (Guerra-Mateo et al. 2023; Liu et al. 2023). Amphichorda excrementa and A. kunmingensis were isolated from animal faeces in the Park. The substrates of Amphichorda were complex and varied, being mainly animal faeces, i.e. bird, cat, bat, chipmunk, rabbit and porcupine dung, but they have also been isolated in the pupa of Anaitis efformata, mouldy leaves, plant debris, sediments, fragments of floating rubber tyres, wing surfaces of Rhinolophus and soil. Most species of the genus Amphichorda have been isolated on animal faeces and are quite unique to their parasitic environments. This is unique to the biological characteristics and ecological habits for the genus Amphichorda.

Coprophilous fungi, particularly coprophilous ascomycetes, will be a rich source of antibiotics and other biologically important secondary metabolites (Bills et al. 2013). Species of the genus Amphichorda tend to have special physiological and metabolic characteristics due to the uniqueness of their growth environment. Additionally, some of their species have been reported to have high application value, such as A. felina, which was a well-known producer of insecticidal cyclodepsipeptide and cyclosporin C (Langenfeld et al. 2011; Chung et al. 2013; Xu et al. 2018). Furthermore, the study by Liang et al. (2021) successfully established a genetic transformation system in A. guana strain LC5815, which facilitated the development of bioactive secondary metabolites in fungi. Two new species of the genus Amphichorda, described in the present study, were isolated from animal faeces and may have good potential for natural product research.

 Additional information Conflict of interest

The authors have declared that no competing interests exist.

 Ethical statement

No ethical statement was reported.

 Funding

This work was supported by the National Natural Science Foundation of China (No. 31870017).

 Author contributions

Data curation: QYD. Investigation: YW. Visualization: RL, YLL. Writing - original draft: ZQW. Writing - review and editing: JZ, HY.

 Author ORCIDs

Zhi-Qin Wang https://orcid.org/0000-0001-9022-3635

Hong Yu https://orcid.org/0000-0002-2149-5714

 Data availability

All of the data that support the findings of this study are available in the main text.

 References AraújoJPMLebertBMVermeulenSBrachmannAOhmRAEvansHCde BekkerC (2022) Masters of the manipulator: Two new hypocrealean genera, Niveomyces (Cordycipitaceae) and Torrubiellomyces (Ophiocordycipitaceae), parasitic on the zombie ant fungus Ophiocordyceps camponoti-floridani.Persoonia49(1): 171–194. https://doi.org/10.3767/persoonia.2022.49.05 BelyagoubiLBelyagoubi-BenhammouNJuradoVDupontJLacosteSDjebbahFOunadjelaFZBenaissaSHabiSAbdelouahidDESaiz-JimenezC (2018) Antimicrobial activities of culturable microorganisms (Actinomycetes and Fungi) isolated from Chaabe Cave, Algeria.International Journal of Speleology47(2): 189–199. https://doi.org/10.5038/1827-806X.47.2.2148 BillsGFGloerJBAnZ (2013) Coprophilous fungi: Antibiotic discovery and functions in an underexplored arena of microbial defensive mutualism.Current Opinion in Microbiology16(5): 549–565. https://doi.org/10.1016/j.mib.2013.08.001 BischofJFRehnerSAHumberRA (2006) Metarhizium frigidum sp. nov.: A cryptic species of M. anisopliae and a member of the M. favoviride Complex.Mycologia98(5): 737–745. https://doi.org/10.1080/15572536.2006.11832645 CarmichaelJWKendrickWBConnersILSiglerL (Eds) (1980) Genera of Hyphomycetes. University of Alberta Press. ChungYMEl-ShazlyMChuangDWHwangTLAsaiTOshimaYAshourMLWuYCChangFR (2013) Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, induces the production of anti-inflammatory cyclodepsipeptides from Beauveria felina.Journal of Natural Products76(7): 1260–1266. https://doi.org/10.1021/np400143j De HoogGS (1972) The Genera Beauveria, Isaria, Tritirachium and Acrodontium gen. nov.Studies in Mycology1: 1–41. FriesEM (1825) Systema orbis vegetabilis: Primas Lineas Novae Constructionis Periclitatur; E Typographia Academica. Lunde, Norway 1: 170. Guerra-MateoDGenéJBaulinVCano-LiraJF (2023) Phylogeny and Taxonomy of the Genus Amphichorda (Bionectriaceae): An Update on Beauveria-like Strains and Description of a Novel Species from Marine Sediments.Diversity15(7): 795. https://doi.org/10.3390/d15070795 HoangDTChernomorOvon HaeselerAMinhBQVinhLS (2017) UFBoot2: Improving the ultrafast bootstrap approximation.Molecular Biology and Evolution35(2): 518–522. https://doi.org/10.1093/molbev/msx281 HouLWGiraldoAGroenewaldJZRämäTSummerbellRCHuangGZCaiLCrousPW (2023) Redisposition of acremonium-like fungiin Hypocreales.Studies in Mycology105(1): 23–203. https://doi.org/10.3114/sim.2023.105.02 KalyaanamoorthySMinhBQWongTKFvon HaeselerAJermiinLS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates.Nature Methods14(6): 587–589. https://doi.org/10.1038/nmeth.4285 LamarckJB (1815) Flore Françoise ou Descriptions Succinctes de Toutes les Plantes Qui Croissent Naturellement en France, Disposées Selon Une Nouvelle Méthode D’Analyse, et Précédées Par un Exposé des Principes Élémentaires de la Botanique.Libraire Chez Desray, Paris, vol. 6, 30 pp. https://doi.org/10.5962/bhl.title.112968 LangenfeldABlondAGueyeSHersonPNayBDupontJPradoS (2011) Insecticidal cyclodepsipeptides from Beauveria felina.Journal of Natural Products74(4): 825–830. https://doi.org/10.1021/np100890n LeãoAFCondéTODutraYLGRosadoAWCGrazziottiPHNevesSCFragaLMSKasuyaMCMPereiraOL (2024) Amphichorda monjolensis sp. nov., a new fungal species isolated from a Brazilian limestone cave, with an update on acremonium-like species in Bionectriaceae. Brazilian Journal of Microbiology. https://doi.org/10.1007/s42770-024-01289-y LendemerJC (2020) Epitypes are forever: Best Practices for an Increasingly Misused Nomenclatural Action.Taxon69(5): 849–850. https://doi.org/10.1002/tax.12289 LiangMLiWQiDChenCCaiLYinB. (2021) Establishment of a genetic transformation system in guanophilic fungus Amphichorda guana. Journal of Fungi 7: 138. https://doi.org/10.3390/jof7020138 LiuMHodgeKT (2005) Hypocrella zhongdongii sp. nov., the teleomorph of Aschersonia incrassata.Mycological Research109: 818–824. https://doi.org/10.1016/10.1017/S095375620500290X LiuXFTibprommaSHughesACChethanaKWTWijayawardeneNNDaiDQDuTYElgorbanAMStephensonSLSuwannarachNXuJCLuLXuRFMaharachchikumburaSSNZhaoCLBhatDJSunYMKarunarathnaSCMortimerPE (2023) Culturable mycota on bats in central and southern Yunnan Province, China.Mycosphere14(1): 497–662. https://doi.org/10.5943/mycosphere/14/1/7 NguyenLTSchmidtHAvon HaeselerAMinhBQ (2015) IQ-TREE: A fast and efective stochastic algorithm for estimating maximum likelihood phylogenies.Molecular Biology and Evolution32(1): 268–274. https://doi.org/10.1093/molbev/msu300 RehnerSASamuelsGJ (1994) Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences.Mycological Research98(6): 625–634. https://doi.org/10.1016/S0953-7562(09)80409-7 RehnerSAMinnisAMSungGHLuangsa-ardJJDevottoLHumberRA (2011) Phylogeny and systematics of the anamorphic, entomopathogenic genus Beauveria.Mycologia103(5): 1055–1073. https://doi.org/10.3852/10-302 RonquistFTeslenkoMvan der MarkPAyresDLDarlingAHöhnaSLargetBLiuLSuchardMAHuelsenbeckJP (2012) MrBayes 3.2: Efcient Bayesian phylogenetic inference and model choice across a large model space.Systematic Biology61(3): 539–542. https://doi.org/10.1093/sysbio/sys029 SungGHHywel-JonesNLSungJMLuangsa-ArdJJShresthaBSpataforaJW (2007) Phylogenetic classifcation of Cordyceps and the clavicipitaceous fungi.Studies in Mycology57: 5–59. https://doi.org/10.3114/sim.2007.57.01 TamuraKStecherGPetersonDFilipskiAKumarS (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0.Molecular Biology and Evolution30(12): 2725–2729. https://doi.org/10.1093/molbev/mst197 VanderwolfKJMallochDMcalpineDFForbesGJ (2013) A world review of fungi, yeasts, and slime molds in caves.International Journal of Speleology42(1): 77–96. https://doi.org/10.5038/1827-806X.42.1.9 VilgalysRHesterM (1990) Rapid genetic identifcation and mapping of enzymatically amplifed ribosomal DNA from several Cryptococcus species.Journal of Bacteriology172(8): 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 WangYBYuHDaiYDWuCKZengWBYuanFLiangZQ (2015a) Polycephalomyces agaricus, a new hyperparasite of Ophiocordyceps sp. infecting melolonthid larvae in southwestern China.Mycological Progress14(9): 70. https://doi.org/10.1007/s11557-015-1090-7 WangYBYuHDaiYDChenZHZengWBYuanFLiangZQ (2015b) Polycephalomyces yunnanensis (Hypocreales), a new species of Polycephalomyces parasitizing Ophiocordyceps nutans and stink bugs (Hemipteran adults).Phytotaxa208: 34–44. https://doi.org/10.11646/phytotaxa.208.1.3 WangYBWangYFanQDuanDEZhangGDDaiRQDaiYDZengWBChenZHLiDDTangDXXuZHSunTNguyenTTTranNLDaoVMZhangCMHuangLDLiuYJZhangXMYangDRSanjuanTLiuXZYangZLYuH (2020) Multigene phylogeny of the family Cordycipitaceae (Hypocreales): New taxa and the new systematic position of the Chinese cordycipitoid fungus Paecilomyces hepiali.Fungal Diversity103(1): 1–46. https://doi.org/10.1007/s13225-020-00457-3 WangZQWangYDongQYFanQDaoVMYuH (2022) Morphological and Phylogenetic Characterization Reveals Five New Species of Samsoniella (Cordycipitaceae, Hypocreales).Journal of Fungi (Basel, Switzerland)8(7): 747. https://doi.org/10.3390/jof8070747 WhiteTJBrunsTDLeeSBTaylorJW (1990) Amplifcation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: InnisMAGelfandDHSninskyJJWhiteTJ (Eds) PCR protocols: a guide to methods and applications.Academic, New York, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 XuLLiYBigginsJBBowmanBRVerdineGLGloerJBAlspaughJABillsGF (2018) Identifcation of cyclosporin C from Amphichorda felina using a Cryptococcus neoformans differential temperature sensitivity assay.Applied Microbiology and Biotechnology102(5): 2337–2350. https://doi.org/10.1007/s00253-018-8792-0 ZhangZFLiuFZhouXLiuXZLiuSJCaiL (2017) Culturable mycobiota from Karst caves in China, with descriptions of 20 new species.Persoonia39(1): 1–31. https://doi.org/10.3767/persoonia.2017.39.01 ZhangDGaoFLJakovlićIZouHZhangJLiWXWangGT (2020) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies.Molecular Ecology Resources20(1): 348–355. https://doi.org/10.1111/1755-0998.13096 ZhangZFZhouSYEurwilaichitrLIngsriswangSRazaMChenQZhaoPLiuFCaiL (2021) Culturable mycobiota from Karst caves in China II, with descriptions of 33 new species.Fungal Diversity106(1): 29–136. https://doi.org/10.1007/s13225-020-00453-7

Zhi-Qin Wang and Jing Zhao contributed equally to this work