DNA sequence alignment and Phylogenetic analysis
DNA sequences of Penicillium and Acaulium strains determined in this study is deposited in GenBank and accession numbers are listed in Table 1. CaM and TUB sequences could not be obtained for all strains. A dataset of partial RPB2 sequence (RPB1: 1–816) for 77 strains, including our isolates and type or reference strains of related species and outgroup, was used for a preliminary phylogenetic analysis. The general time reversible (GTR) model with gamma distributed (+G) and invariant sites (+I) was the most suitable model for ML and BI. Aspergillus niger NRRL 326NT was selected as outgroup. The phylogenetic tree was constructed and bootstrap values for likelihood
(ML analysis) (≥ 50%) and branch support values for posterior probability (PP) (≥
0.95) are illustrated. Phylogeny of 38 Penicillium strains which isolated from herbivore dung based on RPB2 sequences revealed that these strains belong to six sections, 12 known species (Fig. 1). Twenty-three Penicillium strains isolated from chinchilla and belong to sect. Fasciculata, sect. Citrina and sect. Lanata-divaricata, respectively. Fifteen Penicillium strains isolated from marmot and belong to sect. Chrysogena, sect. Citrina, sect. Fasciculata, sect. Ramigena and sect. Roquefortorum, respectively. Multiple sequence alignment and multi-gene phylogentic analysis were further carried out by testing TUB, ITS, CAM, RPB2 and a detailed phenotypic study (Figs. 2, 3). The strains isolated from chinchilla dung could be identified as five species, P. polonicum, P. aurantiogriseum, P. steckii, P. fructuariae-cellae, P. oxalicum. However, the strains isolated from marmot dung could be identified as eight species, P. polonicum, P. echinulatum, P. allii-sativi, P. dipodomyis, P. paneum, P. roqueforti, P. steckii, P. citrinum. Besides, P. polonicum and P. steckii could be isolated from chinchilla and marmot.
In addition, BLAST searches of GenBank using the ITS sequences of four Acaulium strains isolated from the healthy marmot revealed that CGMCC 3.20206T (MZ157171), CGMCC 3.20207 (MZ157173) and CGMCC 3.20208 (MZ157172) showed 93% similarity to Acaulium acremonium CBS 290.38 (MH855966) (Table 1). The three Acaulium strains had 40 variable positions, including 6 transitions, 2 transversions and 32 indels in the ITS regions. The isolate TBS429 showed 99% similarity to Acaulium album CBS 539.85 (NR_159559). The dataset of combined 4 loci (ITS: 1–434; LSU: 435–1186; TEF: 1187–1658; TUB: 1659–1866) comprised 1,866 characters. The phylogenetic tree was constructed based on 20 taxa including Graphium penicillioides CBS 102632T as outgroup. The terminal branch support value and additional internal branch support value of the Bayesian posterior probabilities (PP) (≥ 95%) and Bootstrap value (BP) (≥ 50%) are indicated on the nodes. The species belonging to Acaulium clustered in a separate clade with a high bootstrap value. Acaulium stericum clustered with Acaulium pannemaniae CBS 145025T, with Bayesian posterior probability and bootstrap support of 99% and 100% (Fig. 4). Moreover, morphological comparisons showed differences between these species. Therefore we assigned the new species in Acaulium. Coupled with analysis of the polymorphisms in the ITS and TUB regions and morphological characters, three strains were designated as a new species A. stericum.
Taxonomy
Acaulium stericum L. Su, H. Zhu & C. Qin, sp. nov. (Fig. 5).
MycoBank No.: MB 840126
Etymology: The specific epithet ‘stericum’, indicating the type strain isolated from the dung of animal.
Colonies on PDA reaching 8 mm diameter after 15 days at 25 °C, slow growing, raised centrally, with irregular margin, white. On SDA reaching 10 mm diameter, slow growing, raised centrally, aerial mycelium absent or sparse, white to cream. On CMA reaching 21 mm diameter, moderately growing, planar, white, margin discrete. On OA reaching 30 mm diameter, planar, white, raised centrally. Hyphae hyaline to subhyaline, smooth-walled, 2–5 μm (x̅ = 3.3 μm) wide. Conidiophores often arising from the substratum or from the aerial mycelium, branched or unbranched, septate, smooth, cylindrical, 3–16 × 2–6 μm (x̅ = 8.0 × 4.0 μm). Conidiogenous cells percurrent in conidiophores or produced on hyphae in laterally, flask-shaped, subhyaline and smooth-walled, 6–20 × 2–6 μm (x̅ = 11.7 × 3.6 μm). Conidia formed in slimy heads at the apex of the phialides, ellipsoidal to fusiform, with a truncate base and rounded or bluntly pointed apex, smooth, 3–8 × 2–5 μm (x̅ = 5.7 × 3.3 μm). Chlamydospores formed short chains by schizolytic secession intercalary or laterally, irregular, thick- walled. Ascomata not observed.
Typification: CHINA, Beijing, Fangshan District, in the North Center for Experimental Animal Resources, Institute of Medical Laboratory of Animal Science, Chinese Academy of Medical Sciences, 116°13¢ E, 39°48¢ N, 58 m above sea level, from fresh fecal samples of healthy Marmota monax, 30 July 2020, collected and isolated by Lei Su, HMAS 249146 (holotype), ex-type culture CGMCC 3.20206.
Other cultures examined: CHINA. Beijing: Daxing, E 116°13’, N 39°48’, 58 m above sea level, isolated as the fresh fecal samples of healthy marmot, 30 July 2020, Lei Su. CGMCC 3.20207, CGMCC 3.20208.
Notes: The phylogenetic analysis shows that the ex-type culture of Acaulium stericum grouped with statistical support with species of A. pannemaniae. However, Compared to A. pannemaniae, A. stericum produces the short conidiophores. Besides, the dimensions of conidiogenous cells and conidia, and the irregular chlamydospores for two species are apparent different (Fig. 5).