Multi-locus molecular phylogenetic analysis reveals two new species of Amphichorda (Bionectriaceae, Hypocreales)

Amphichorda has been previously accepted as a member of the Cordycipitaceae and currently it is considered a member of the Bionectriaceae. The substrates of Amphichor-da 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 (nr SSU , nr LSU , 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. kun-mingensis were isolated from animal faeces in the park. The morphological characteristics of two novel species and seven known species in Amphichorda are also compared.


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(Zhang et al. , 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. (2017Zhang et al. ( , 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(Zhang et al. , 2021;;Wang et al. 2020;Liu et al. 2023).
During the surveys of entomopathogenic fungi from 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.

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 fungi were 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 Gen-Bank 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.2.1.3and 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).

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).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, 0.6-1.3µm 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.
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 × 1.8-2.9µm) and conidia size range of this species (2.5-4.7 × 2-3.5 µm).Etymology.Named from the location Kunming City where the species was collected.
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 × 1.4-2.9μm vs. 4.1-13.9× 1.3-2.1 μm) and larger conidia (2.3-4.2 × 1.6-3.0μm 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.

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. 2017Zhang et al. , 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.

Table 1 .
Species information and corresponding GenBank accession numbers of Amphichorda and close relative genera used in this study.
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.