Morphological characteristics and phylogenetic evidence reveal two new species of Acremonium (Hypocreales, Sordariomycetes)

Abstract

Using chicken feathers as bait, Acremoniumglobosisporumsp. nov. and Acremoniumcurvumsp. nov. were collected from the soil of Yuncheng East Garden Wildlife Zoo and Zhengzhou Zoo in China. They were identified by combining the morphological characteristics and the two-locus DNA sequence (LSU and ITS) analyses. In the phylogenetic tree, both new species clustered into separate subclades, respectively. They were different from their allied species in their morphology. The description, illustrations, and phylogenetic tree of the two new species were provided.

Introduction

The genus Acremonium Link, established in 1929, with A.alternatum Link as the type species, is one of the largest and most complex genera of asexually typified. The morphological characteristics consist of hyphae septate, mostly tapered and lateral phialides, produced singly or in small groups, and unicellular conidia produced in mucoid heads or unconnected chains (Summerbell et al. 2011). Nowadays, Acremonium has 217 records in the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, retrieval on 30 Jun. 2022). The traditionally circumscribed Acremonium is polyphyletic, which explains why many Acremonium species were transferred to other genera and families (Yang et al. 2019). Thus, there are still many unidentified, suspect or misidentified taxa that require taxonomic investigation.

Due to the poor differentiation of asexual forms of the genus Acremonium, it is difficult to identify species only by morphological differences. To address this issue, there are many unidentified and suspicious species that require further phylogenetic analysis. To date, many isolates of Acremonium spp. lack the gene loci such as SSU, TEF 1-α and RPB2 (Table 1), therefore, phylogenetic analyses of this genus are generally performed based on the single locus sequences, especially LSU (Hyde et al. 2020).

Table 1.

Strains included in the present study.

Species	Strains	LSU	ITS	SSU	TEF 1-α	RPB2
Acremoniumalcalophilum	CBS 114.92T	JX158443	DQ825967	JX158486	JX158399	JX158465
Acremoniumalternatum	CBS 407.66T	HQ231988	HE798150
Acremoniumalternatum	CBS 831.97	HQ231989
Acremoniumarthrinii	MFLU 18-1225T	MN036334		MN036335	MN038169
Acremoniumbehniae	CBS 146824T	MW175400	MW175360
Acremoniumbiseptum	CBS 750.69T	HQ231998
Acremoniumblochii	CBS 993.69	HQ232002	HE608636
Acremoniumborodinense	CBS 101148T	HQ232003	HE608635
Acremoniumbrachypenium	CBS 866.73T	HQ232004	AB540570
Acremoniumcamptosporum	CBS 756.69T	HQ232008		HQ232186
Acremoniumcavaraeanum	CBS 101149T	HF680202	HF680220
Acremoniumcavaraeanum	CBS 111656	HF680203	HF680221
Acremoniumcavaraeanum	CBS 758.69	HQ232012	HF680222
Acremoniumcerealis	CBS 207.65	HQ232013
Acremoniumcerealis	CBS 215.69	HQ232014
Acremoniumchiangraiense	MFLUCC 14-0397T	MN648329	MN648324
Acremoniumchrysogenum	CBS 144.62T	HQ232017		HQ232187
Acremoniumchrysogenum	CBS 401.65	MH870276	MH858636
Acremoniumcitrinum	CBS 384.96T	HF680217	HF680236
Acremoniumdimorphosporum	CBS 139050T	LN810506	LN810515
Acremoniumexiguum	CBS 587.73T	HQ232035
Acremoniumexuviarum	UAMH 9995T	HQ232036	AY882946
Acremoniumfelinum	CBS 147.81T	AB540488	AB540562
Acremoniumflavum	CBS 596.70T	HQ232037		HQ232191
Acremoniumflavum	CBS 316.72	MH872204	MH860487
Acremoniumfuci	CBS 112868T		AY632653
Acremoniumfuci	CBS 113889		AY632652
Acremoniumfusidioides	CBS 109069	HF680204	HF680223
Acremoniumfusidioides	CBS 991.69	HF680211	HF680230
Acremoniumfusidioides	CBS 840.68T	HQ232039	FN706542
Acremoniumhansfordii	CBS 390.73	HQ232043	AB540578
Acremoniumhennebertii	CBS 768.69T	HQ232044	HF680238
Acremoniuminflatum	CBS 212.69T	HQ232050
Acremoniummali	ACCC 39305T	MF993114	MF987658
Acremoniummoniliforme	CBS 139051T	LN810507	LN810516
Acremoniummoniliforme	FMR 10363	LN810508	LN810517
Acremoniumparvum	CBS 381.70A	HQ231986	HF680219
Acremoniumpersicinum	CBS 310.59T	HQ232077
Acremoniumpersicinum	CBS 101694	HQ232085
Acremoniumpinkertoniae	CBS 157.70T	HQ232089		HQ232202
Acremoniumpolychroma	CBS 181.27T	HQ232091	AB540567
Acremoniumpotronii	CBS 189.70	HQ232094
Acremoniumpseudozeylanicum	CBS 560.73T	HQ232101
Acremoniumpteridii	CBS 782.69T	HQ232102
Acremoniumpteridii	CBS 784.69	HQ232103
Acremoniumsclerotigenum	CBS 124.42T	HQ232126	FN706552	HQ232209
Acremoniumsclerotigenum	A101	KC987215	KC987139	KC987177	KC998961
Acremoniumsclerotigenum	A130	KC987242	KC987166	KC987204	KC998988
Acremonium sp.	E102	KC987248	KC987172	KC987210	KC998994	KC999030
Acremoniumspinosum	CBS 136.33T	HQ232137	HE608637	HQ232210
Acremoniumstroudii	CBS 138820T		KM225291
Acremoniumtumulicola	CBS 127532T	AB540478	AB540552
Acremoniumvariecolor	CBS 130360T	HE608651	HE608647
Acremoniumvariecolor	CBS 130361	HE608652	HE608648
Acremoniumverruculosum	CBS 989.69T	HQ232150
Acrophialophorahechuanensis	GZUIFR-H08-1T	MK926789	DQ185070	EU053286
Brunneomycesbrunnescens	CBS 559.73T	HQ231966	LN810520	HQ232184	LN810534
Brunneomyceshominis	UTHSC 06-415T	LN810509	KP131517		LN810535
Bryocentriabrongniartii	M139	EU940105		EU940052
Bryocentriabrongniartii	M190	EU940125		EU940052
Bryocentriametzgeriae	M140	EU940106
Bulbitheciumhyalosporum	CBS 318.91T	AF096187	HE608634
Cephalosporiumpurpurascens	CBS 149.62T	HQ232071
Cosmosporalavitskiae	CBS 530.68T	HQ231997
Emericellopsisalkalina	CBS 127350T	KC987247	KC987171	KC987209	KC998993	KC999029
Emericellopsisterricola	CBS 120.40T	U57082	U57676	U44112
Gliomastixroseogrisea	CBS 134.56T	HQ232121
Hapsidosporairregularis	ATCC 22087T	AF096192		AF096177
Kiflimoniumcurvulum	CBS 430.66T	HQ232026	HE608638	HQ232188
Lanatonectriaflavolanata	CBS 230.31	HQ232157
Leucosphaerinaarxii	CBS 737.84T	HE608662	HE608640
Nigrosabulumglobosum	ATCC 22102T	AF096195
Paracremoniumcontagium	CBS 110348T	HQ232118	KM231831		KM231966
Parasarocladiumbreve	CBS 150.62T	HQ232005
Parasarocladiumradiatum	CBS 142.62T	HQ232104		HQ232205
Pestalotiopsishawaiiensis	CBS 114491T	KM116239	KM199339		KM199514
Pestalotiopsisspathulata	CBS 356.86T	KM116236	KM199338		KM199513
Phialemoniumatrogriseum	CBS 604.67T	HQ231981	HE610367	FJ176825
Pseudoacremoniumsacchari	CBS 137990T	KJ869201	KJ869144
Sarcopodiumvanillae	CBS 100582	HQ232174	KM231780		KM231911
Sarocladiumbacillisporum	CBS 425.67T	HQ231992	HE608639	HQ232179
Sarocladiumbactrocephalum	CBS 749.69T	HQ231994	HG965006	HQ232180
Sarocladiumstrictum	CBS 346.70T	HQ232141	AY214439	HQ232211
Sarocladiumterricola	CBS 243.59T	HQ232046		HQ232196
Seliniapulchra	AR 2812	GQ505992	HM484859		HM484841
Trichotheciumcrotocinigenum	CBS 129.64T	HQ232018	AJ621773
Trichotheciumindicum	CBS 123.78T	AF096194		AF096179
Trichotheciumroseum	DAOM 208997	U69891		U69892
Trichotheciumsympodiale	ATCC 36477	U69889		U69890
Acremoniumcurvum	CGMCC 3.20954 = GZUIFR 22.035T	ON041050	ON041034	ON876754	ON494579	ON494583
Acremoniumglobosisporum	CGMCC 3.20955 = GZUIFR 22.036T	ON041051	ON041035	ON876755	ON494580	ON494584
Acremoniumglobosisporum	GZUIFR 22.037	ON041052	ON041036	ON876756	ON494581	ON494585
Acremoniumglobosisporum	GZUIFR 22.038	ON041053	ON041037	ON876757	ON494582	ON494586

Notes: “^T” stands for Ex-type strains.

In the present study, two new species of Acremonium were identified in a survey of keratinolytic fungi from China, which were enriched by the baiting technique. We provided a description, illustrations, and phylogenetic tree for the two new species.

Materials and methods

Fungal isolation and morphology

Soil samples were collected from Yuncheng East Garden Wildlife Zoo (35°6'26"N, 111°4'24"E) (three isolates), Yuncheng City, Shanxi Province and Zhengzhou Zoo (34°47'20"N, 113°40'41"E) (one isolate), Zhengzhou City, Henan Province, China by Yu-Lian Ren on July 2021. We collected 3–10 cm below the soil surface, placed the samples in sterile Ziploc plastic bags (Kaixin Biotechnology, Guizhou, China), and transported them to the laboratory (Zhang et al. 2019a, b). Then, they were treated and isolated according to the baiting method (using chicken feathers as bait: a method specifically designed for isolating keratinophilic microbes) of Zhang et al. (2020a, b; 2021). We washed the chicken feathers, sterilized them in an autoclave for 30 minutes at 121 °C, and dried them in an oven at 50 °C. The sterile and dried chicken feathers were mixed with soil samples and then wet with sterile distilled water and cultured at darkroom temperature for 1 month (Li et al. 2022).

Then, the 2 g samples were weighed in a conical flask with glass beads containing 20 mL sterile water and mixed evenly by eddy shock for 10 min. Next, 1 mL samples were mixed evenly in 9 mL sterile water in a sterile environment and diluted to 10^-3. Then, 1 mL 10^-3 samples were put into a sterile petri dish, and SDA medium containing 50 mg/L penicillin and 50 mg/L streptomycin was added and mixed. The target strains were isolated. The purified strains were transferred to PDA, OA, and MEA plates for dark culture at 25 °C for 7 days. Microscopic features were examined by making direct wet mounts with 25% lactic acid on PDA, with a light microscope.

The cultures were placed to slowly dry at 50 °C to produce the dried holotype. The dried holotype was deposited in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS), while ex-type living culture was stored in PDA test tubes which were deposited in the China General Microbiological Culture Collection Center (CGMCC), and the Institute of Fungus Resources, Guizhou University, Guiyang City, Guizhou, China (GZUIFR).

DNA extraction, PCR amplification, and sequencing

We used a 5% chelex-100 solution for total genomic DNA extraction. ITS1/ITS4 (White et al. 1990), LROR/LR7 (Vilgalys and Hester 1990), EF1-983F/ EF1-2218R (Rehner and Buckley 2005), fRPB2-5f/ fRPB2-7cR (Liu et al. 1999), and NS1 and NS4 (White et al. 1990) primers were used for amplification of the internal transcribed spacers (ITS), the 28S nrRNA locus (LSU), translation elongation factor 1-alpha gene region (TEF 1-α), RNA polymerase II second largest subunit gene (RPB2), and small subunit rDNA (SSU), respectively. Purification and sequencing were performed by Quintarabio (Wuhan, China). The new sequences were submitted to GenBank (Table 1).

Phylogenetic analyses

The ITS and LSU sequences of Acremonium were downloaded from GenBank (Table 1). Two strains of Pestalotiopsisspathulata (CBS 356.86) and P.hawaiiensis (CBS 114491) were chosen as the outgroup taxa. The TBtools were used for name simplification and renaming (Chen et al. 2020). Sequences were aligned by MAFFT v7.037 (Katoh and Standley 2013). Multi-locus was concatenated by PhyloSuite v1.16 (Zhang et al. 2020a).

Bayesian inference (BI) and maximum likelihood (ML) methods were used in the analysis. For BI analysis was conducted with MrBayes v3.2 (Ronquist et al. 2012) and Markov chain Monte Carlo (MCMC) simulations; ML analysis was performed using IQ-TREE v1.6.11 (Nguyen et al. 2015), as outlined in Li et al (2022). All analyses were performed in PhyloSuite V1.16 (Zhang et al. 2020b).

Results

Phylogeny

Based on a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the LSU sequences, our isolates were identified as belonging to the genus Acremonium. To further determine the phylogenetic position of these strains, we performed a multi-locus phylogenetic analysis. The dataset was composed of LSU (1–430 bp) and ITS (431–1005 bp) gene, comprising a total of 1005 characters (including gaps). The best-fit partition model for ML analysis and BI analysis is shown in Table 2. The results showed that the CGMCC 3.20955, GZUIFR 22.037, and GZUIFR 22.038 are still grouped in the Pinkertoniae-clade (Fig. 1). The CGMCC 3.20954 is still grouped in the Chrysogenum-clade (Fig. 1).

Table 2.

The best-fit substitution models are used in multi-locus phylogenetic construction.

	LSU	ITS
ML analysis	TN+F+R5	GTR+F+R4
BI analysis	GTR+F+I+G4	GTR+F+I+G4

Figure 1.

Phylogenetic tree of the genus Acremonium constructed from LSU and ITS. Bayesian posterior probability (≥ 0.95) and ML bootstrap values (≥ 70%) are indicated along branches (BPP/ML).

Taxonomy

. Acremonium globosisporum

Xin Li, Y.F. Han & Z.Q. Liang sp. nov.

35BF27DA-FC43-57D8-989A-49581DCE8C51

843765

Fig. 2

Figure 2.

Morphology of Acremoniumglobosisporum sp. nov. a–f colony on PDA, OA and MEA after 7 d at 25 °C (upper surface and lower surface) g–j conidia are borne on the phialides k–l winding hyphae and inflate hyphae. Scale bars: 4 mm (a–f); 10 μm (g–l).

Type.

Yuncheng East Garden Wildlife Zoo, Yuncheng City, Shanxi Province, China N35°6'26", E111°4'24", isolated from green belt soil, July 2021, Yu-Lian Ren (dried holotype culture HMAS 351939, ex-holotype culture CGMCC 3.20955 = GZUIFR 22.036). ITS sequences, GenBank ON041035; LSU sequences, GenBank ON041051; SSU sequences, GenBank ON876755; TEF 1-α sequences, GenBank ON494580; RPB2 sequences, GenBank ON494584.

Description.

Colonies on PDA and OA at 25 °C attaining 11–13 mm and 9–11 mm diam respectively after 7 d, white, flat or raised, velvety to slightly cottony. On MEA at 25 °C, reaching 8–10 mm after 7 d, white to yellowish white, raised, slimy. Hyphae hyaline, septate, sometimes winding and inflate, 1.5–11.0 µm wide. Sporulation abundant. Phialides are mostly borne singly, hyaline, erect to slightly curved, sometimes forming a collarette, 9.0–22.0 µm long, tapering from 1.5–3.5 µm near the base to 0.5–1.5 µm. Conidia cohering in long chains, with minutely truncate ends, up to 27.5 µm long, globose or subglobose, 2.5–4.5 × 2.5–4.5 µm (x– ± SD = 3.4 ± 0.77 × 3.6 ± 0.52, n = 50) diam. Chlamydospores and teleomorph stage were not observed.

Etymology.

globosisporum. A reference to the global conidia.

Additional specimens examined.

Yuncheng East Garden Wildlife Zoo, Yuncheng City, Shanxi Province, China N35°6'26", E111°4'24", isolated from green belt soil, July 2021, Yu-Lian Ren, GZUIFR 22.037, ITS, LSU, SSU, TEF 1-α, RPB2 sequences GenBank ON041036, ON041052, ON876756, ON494581, ON494585; GZUIFR 22.038, ITS, LSU, SSU, TEF 1-α, RPB2 sequences GenBank ON041037, ON041053, ON876757, ON494582, ON494586.

Known distribution.

Yuncheng City, Shanxi Province, China.

Notes.

The phylogeny results showed that the CGMCC 3.20955, GZUIFR 22.037 and GZUIFR 22.038 still nested in the Pinkertoniae-clade. The morphological characteristics of Acremoniumglobosisporum were similar to other species of the Pinkertoniae-clade in that phialides were erect on the hyphae; sporulation was abundant, and conidia were subglobose (Ito et al. 2000). However, Acremoniumglobosisporum hyphae were sometimes winding and inflated, with conidia cohering in long chains, unlike other species.

. Acremonium curvum

Xin Li, Y.F. Han & Z.Q. Liang sp. nov.

7DC71370-B32D-59C2-B927-D407C2A66239

843766

Fig. 3

Figure 3.

Morphology of Acremoniumcurvum sp. nov. a–f colony on PDA, OA and MEA after 7 d at 25 °C (upper surface and lower surface) g, I, j conidia are borne on the phialides h Winding hyphae. Scale bars: 4 mm (a–f); 10 μm (g–j).

Type.

Zhengzhou Zoo, Zhengzhou City, Henan Province, China N34°47'20", E113°40'41", isolated from green belt soil, July 2021, Yu-Lian Ren (dried holotype culture HMAS 351938, ex-holotype culture CGMCC 3.20954 = GZUIFR 22.035). ITS sequences, GenBank ON041034, LSU sequences, GenBank ON041050; SSU sequences, GenBank ON876754; TEF 1-α sequences, GenBank ON494579; RPB2 sequences, GenBank ON494583.

Description.

Colonies on PDA and OA at 25 °C attaining 11–14 mm and 7–9 mm diam respectively after 7 d, white, flat, radially folded or rugose. On MEA at 25 °C, reaching 6–8 mm after 7 d, white to yellowish-white, slimy. Hyphae hyaline, septate, sometimes winding, 1.5–2.5 µm wide. Sporulation abundant. Phialides are mostly borne singly, curved, slightly inflated at the base, tapered at the tip, up to 38.0 µm long. tapering from 1.5–3.5 µm near the base to 0.5–1.5 µm. Conidia cohering together on the top of phialides, one-celled, solitary, or several fascicled, ovoid or subglobose, 3.0–7.0 × 2.5–3.5 µm (x– ± SD = 4.1 ± 1.18 ×3.2 ± 0.77, n = 50) diam. Chlamydospores and teleomorph stage were not observed.

Etymology.

curvum. Referring to the curved Phialides.

Known distribution.

Henan Province, China.

Notes.

Based on the multi-locus analysis we found that Acremoniumcurvum had close phylogenetic affinities to other taxa of the Chrysogenum-clade. Morphologically, A.curvum was similar to other taxa of the Chrysogenum-clade in having simple or rarely branched conidiophores, slightly inflated at the base and tapered at tip phialides, and ovoid to subglobose conidia (Yang et al. 2019). Conidia of Hapsidosporairregularis and A.curvum had several fascicled at the tips of the conidiophores (Malloch and Cain 1970). However, A.curvum was differentiated by having mostly curved phialides and the conidia were several fascicled at the tips of the phialides.

Discussion

In the present study, four strains of Acremonium fungi were isolated from soil in the Shanxi and Henan Province, China. Two-locus (LSU and ITS) phylogenetic analyses in combination with morphological characteristics were used for identification. As a result, two new species of A.curvum (one isolate) and A.globosisporum (three isolates) were proposed.

With the development of biotechnology, a growing number of studies have combined morphological and phylogenetic features to distinguish between species. This provides the basis for more precise species naming. Generally, the fungal ITS marker includes considerably more sequence variability, and consequently provides high interspecific resolution, and also some degree of intraspecific variability (Nilsson et al. 2008). Therefore, ITS has been widely used in studies of fungal inter- and intraspecific relationships (Dai et al. 2020; Szczepańska et al. 2021). There are numerous ITS sequences stored in public databases, which are incomparable to other molecular markers (Zhang et al. 2022). In addition, according to Vu et al. (2019), combining ITS and LSU can improve the accuracy of fungal species discrimination with high generality. They think that fungi commonly present in clinical, environmental, or economically relevant communities can often be identified to species level by their ITS and LSU barcodes (Vu et al. 2019).

Although, Yang et al. (2019) used a multi-locus phylogenetic analysis in introducing the new species Acremoniumarthrinii, lacking loci such as SSU, TEF 1-α and RPB2 (Table 1) in the isolates of Acremonium spp. were relatively serious, so it is not difficult to find that the strains with SSU and TEF 1-α in this analysis were not yet 50% or even 30% of the total number of strains. Therefore, although we sequenced these above loci in the new isolates, they were not included in the phylogenetic analysis. In the future, the phylogeny relationships of Acremonium members will undoubtedly vary and become clearer with the increase of the number and type of molecular used.

In recent years, Acremonium spp. has been reported to cause immunocompetent and immunocompromised individual diseases, such as brain abscess (Anis et al. 2021), fungal keratitis (Liu et al. 2021), fungal osteomyelitis (Jalan et al. 2021), and fungal maxillary sinusitis (Durbec et al. 2011). In the present study, all strains were isolated by a method specifically designed for the isolation of keratinophilic microbes. Therefore, more studies are necessary to confirm whether A.curvum and A.globosisporum are opportunistic infectious pathogens that infect the skin and cause skin infection, as well as their potential application in the degradation of keratin-rich matrices.

Citation

Li X, Zhang Z-Y, Ren Y-L, Chen W-H, Liang J-D, Pan J-M, Huang J-Z, Liang Z-Q, Han Y-F (2022) Morphological characteristics and phylogenetic evidence reveal two new species of Acremonium (Hypocreales, Sordariomycetes). MycoKeys 91: 85–96. https://doi.org/10.3897/mycokeys.91.86257