Material and methods

Sampling and isolation

Rhizosphere soil samples were collected from agricultural fields in Pu’er and Baoshan cities, Yunnan Province, China. From each site, ten subsamples were collected from the 5–10 cm soil layer and combined into one composite sample. The composite samples were placed in sterile bags, transported to the laboratory, and stored at 4 °C prior to processing within one week.

For fungal isolation, 10 g of each soil sample was suspended in 90 mL sterile distilled water and shaken at 180 rpm for 30 min. The suspension was allowed to settle for 5 min, and 1 mL of the supernatant was serially diluted tenfold. Aliquots (100 μL) of 10−2 and 10−3 dilutions were spread onto Rose Bengal agar (RBA) supplemented with penicillin and streptomycin (50 μg/mL each) to inhibit bacterial growth (Martin 1950; Aldossari and Ishii 2021). Plates were incubated at 28 °C for 3–5 days. Emerging colonies were transferred to potato dextrose agar (PDA) and purified by repeated subculturing. A total of 32 Penicillium isolates were obtained, of which eight were selected for further taxonomic analyses.

Pure cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University (YMF), Kunming, China, and the ex-type cultures were additionally deposited in the Guangdong Microbial Culture Collection Center (GDMCC), Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China.

DNA extraction, amplification and sequencing

Genomic DNA was extracted from fresh mycelia following Ye et al. (2023) with minor modifications. Approximately 0.5 g of mycelium was scraped from PDA cultures and transferred to sterile tubes containing a steel bead. A total of 800 μL urea extraction buffer (7 M urea, 50 mM Tris-HCl, 1% SDS, 150 mM NaCl; pH 8.0) was added, and samples were homogenised for 5 min. The homogenate was centrifuged at 12,000 rpm for 5 min and the supernatant was collected. An equal volume of phenol–chloroform–isoamyl alcohol (25:24:1) was added and centrifuged under the same conditions. DNA was precipitated using an equal volume of isopropanol and one-tenth volume of 3 M sodium acetate, followed by incubation at –20 °C for 20 min. The DNA pellet was washed with 70% ethanol, air-dried, resuspended in 50 μL ddH2O, and stored at –20 °C.

Four loci were amplified, including the internal transcribed spacer (ITS), β-tubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2). Primer pairs ITS5/ITS4, Bt2a/Bt2b, CF1/Cmd6, and rpb2-5F2/rpb2-7R were used for ITS, BenA, CaM, and RPB2, respectively (White et al. 1990; Glass and Donaldson 1995; Hong et al. 2006; Sung et al. 2007; Peterson 2008). PCR reactions were performed in 25 μL volumes containing 1 μL genomic DNA, 1 μL of each primer (10 μM), 12.5 μL 2× MasterMix (Tiangen Biotech, Beijing, China), and 9.5 μL ddH2O. Amplifications were conducted using an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) following Visagie et al. (2014). PCR products were purified and sequenced in both directions using an ABI 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA). Newly generated sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and accession numbers are listed in Table 1.

Species | Strain | Substrate/Host and locality | GenBank accession number
ITS | BenA | CaM | RPB2
P. abidjanum | CBS 246.67T | Savannah soil, Ivory Coast | GU981582 | GU981650 | MN969234 | JN121469
P. amphipolaria | DAOMC 250551T | Soil, Quartermain Mountains, Antarctic Dry Valleys, Antarctica | KT887872 | KT887833 | KT887794 | MN969177
P. annulatum | CBS 135126T | Air, Stellenbosch, South Africa | JX091426 | JX091514 | JX141545 | KF296410
P. ausonanum | FMR 16948T | Fluvial sediment, Spain | LR655808 | LR655809 | LR655810 | LR655811
P. austrosinense | CBS 144505T | Acidic soil, Hainan, China | KY495007 | KY495116 | MN969328 | KY495061
P. bissettii | CBS 140972T | Picea forest soil, Québec, Canada | KT887845 | KT887806 | KT887767 | MN969178
P. boreae | CBS 111717T | Unknown source, Unknown country | AF481122 | JN617715 | AF481138 | MN969107
P. brefeldianum | CBS 235.81T | Human alimentary tract, Unknown country | AF033435 | GU981623 | EU021683 | KF296421
P. camponoti | CBS 140982T | Carpenter ant (Camponotus pennsylvanicus), New Brunswick, Canada | KT887855 | KT887816 | KT887777 | MN969179
P. caperatum | CBS 443.75T | Soil, NSW, Australia | KC411761 | GU981660 | MN969242 | KF296422
P. coeruleum | CBS 141.45T | Unknown source, Unknown country | GU981606 | GU981655 | MN969247 | KF296425
P. coffeatum | CGMCC 3.25152T | Soil, China | OQ870815 | OR051121 | OR051298 | OR051466
P. cremeogriseum | CBS 223.66T | Forest soil, Kiev, Ukraine | GU981586 | GU981624 | MN969250 | KF296426
P. curticaule | CBS 135127T | Soil, Malmesbury, South Africa | FJ231021 | JX091526 | JX141536 | KF296417
P. daleae | CBS 211.28T | Conifer soil, Poland | GU981583 | GU981649 | MN969251 | KF296427
P. donggangicum | AS 3.15900T | Tidal-flat soil, Liaoning, China | MW946996 | MZ004914 | MZ004918 | MW979253
P. ehrlichii | CBS 324.48T | Unknown substrate, Poland | GU981578 | GU981652 | MN969253 | KF296428
P. elleniae | CBS 118135T | Forest leaf litter, Araracuara, Colombia | GU981612 | GU981663 | MN969254 | KF296429
P. excelsum | DTO 357-D7T | Bertholletia excelsa nut shell, Amazon, Brazil | KR815341 | KP691061 | KR815342 | MN969166
P. flaviroseum | CBS 144479T | Acidic soil, Hainan, China | KY495032 | KY495141 | MN969329 | KY495083
P. fructuariae-cellae | CBS 145110T | Corvina withered grapes, Verona, Italy | MK039434 | KU554679 | MK045337 | MK520927
P. glaucoroseum | CBS 138908T | Soil, Virginia, USA | MN431390 | MN969383 | MN969257 | MN969119
P. griseopurpureum | CBS 406.65T | Pinus soil, Lancashire, UK | KF296408 | KF296467 | MN969261 | KF296431
P. guaibinense | CCDCA 11512T | Soil, China | MH674389 | MH674391 | MH674393 | –
P. hainanense | CBS 144527T | Acidic soil, Hainan, China | KY495009 | KY495118 | MN969333 | KY495062
P. janthinellum | CBS 340.48T | Soil, Nicaragua | GU981585 | GU981625 | MN969268 | JN121497
P. javanicum | CBS 341.48T | Camellia sinensis root, Java, Indonesia | GU981613 | GU981657 | MN969269 | JN121498
P. jianfenglingense | CBS 144640T | Acidic soil, Hainan, China | KY495016 | KY495125 | MN969334 | KY495069
P. koreense | KACC 47721T | Soil, South Korea | KJ801939 | KM000846 | MN969317 | MN969159
P. laevigatum | NN 072364T | Acidic soil, Hainan, China | KY495015 | KY495124 | MN969335 | KY495068
P. limosum | CBS 339.97T | Marine sediment, Nagasaki, Japan | GU981568 | GU981621 | MN969271 | KF296433
P. lineolatum | CBS 188.77T | Copse soil, Japan | GU981579 | GU981620 | MN969272 | KF296434
P. ludwigii | CBS 417.68T | Polished Oryza sativa seed, Japan | KF296409 | KF296468 | MN969273 | KF296435
P. longlingense | YMF 1.11137T | Soil, Yunnan, China | PZ279979 | PZ306492 | PZ306501 | PZ306510
P. longlingense | YMF 1.11153 | Soil, Yunnan, China | PZ279981 | PZ306494 | PZ306503 | PZ306512
P. longlingense | YMF 1.11156 | Soil, Yunnan, China | PZ279980 | PZ306493 | PZ306502 | PZ306511
P. longlingense | YMF 1.11658 | Soil, Yunnan, China | PZ344665 | PZ347309 | PZ357595 | PZ330332
P. longyangense | YMF 1.10573T | Soil, Yunnan, China | PZ279982 | PZ306495 | PZ306504 | PZ306513
P. longyangense | YMF 1.11657 | Soil, Yunnan, China | PZ344664 | PZ347308 | PZ357594 | PZ330331
P. malacosphaerulum | CBS 135120T | Soil, Malmesbury, South Africa | FJ231026 | JX091524 | JX141542 | KF296438
P. melanosporum | CBS 146938T | Soil, Spain | LR655192 | LR655196 | LR655200 | LR655204
P. meloforme | CBS 445.74T | Soil, Papua New Guinea | KC411762 | GU981656 | MN969276 | KF296440
P. michoacanense | FMR 17612T | Soil, Mexico | LR655194 | LR655198 | LR655202 | LR655206
P. nordestinense | URM 8423T | Pollen from Melipona scutellaris nests, Brazil | OV265270 | OV265324 | OV265272 | OM927721
P. ochrochloron | CBS 357.48T | Copper sulphate solution, Washington, USA | GU981604 | GU981672 | MN969280 | KF296445
P. ortum | CBS 135669T | Forest soil, Kiev, Ukraine | JX091427 | JX091520 | JX141551 | KF296443
P. penarojense | CBS 113178T | Forest leaf litter, Peña Roja, Colombia | GU981570 | GU981646 | MN969287 | KF296450
P. piscarium | CBS 362.48T | Cod-liver oil emulsion, Norway | GU981600 | GU981668 | MN969288 | KF296451
P. pulvillorum | CBS 280.39T | Acidic soil, UK | AF178517 | GU981670 | MN969289 | KF296452
P. pulvillorum | CBS 275.83 | Rye grain, Spain | GU981601 | GU981671 | KC346336 | KF296423
P. raperi | CBS 281.58T | Soil, Bedford, UK | AF033433 | GU981622 | MN969291 | KF296453
P. reticulisporum | CBS 122.68T | Soil, Japan | AF033437 | MN969394 | MN969293 | KF296454
P. rolfsii | CBS 368.48T | Unknown source, Unknown country | JN617705 | GU981667 | MN969294 | KF296455
P. rotoruae | CBS 145838T | Pinus radiata timber, New Zealand | MN315103 | MN315104 | MN315102 | MT240842
P. rubriannulatum | NN 072456T | Acidic soil, Hainan, China | KY495029 | KY495138 | MN969336 | KY495080
P. siccitolerans | FMR 17381T | Soil, Spain | LR655193 | LR655197 | LR655201 | LR655205
P. singorense | CBS 138214T | House dust, Songkhla, Thailand | KJ775674 | KJ775167 | KJ775403 | MN969138
P. soli | KUMCC 18-0202T | Soil, China | MT152337 | MT161681 | MT178249 | MT384372
P. soliforme | CBS 144482T | Acidic soil, Hainan, China | KY495038 | KY495147 | MN969337 | KY495047
P. stangiae | URM 8347T | Soil, Brazil | MW648590 | MW646388 | MW646390 | MW646392
P. subrubescens | CBS 132785T | Helianthus tuberosus soil, Helsinki, Finland | KC346350 | KC346327 | KC346330 | KC346306
P. svalbardense | CBS 122416T | Glacial ice, Svalbard, Norway | GU981603 | DQ486644 | KC346338 | KF296457
P. terrarumae | CBS 131811T | Heavy-metal-contaminated soil, Guizhou, China | MN431397 | KX650295 | MN969323 | MN969185
P. tibetense | CGMCC 3.28597T | Rhizosphere soil, Tibet, China | PQ643284 | PQ519857 | PQ519858 | PQ519859
P. vanderhammenii | CBS 126216T | Forest leaf litter, Araracuara, Colombia | GU981574 | GU981647 | MN969308 | KF296458
P. vasconiae | CBS 339.79T | Acid-washed brown soil, Spain | GU981599 | GU981653 | MN969309 | MN969144
P. viridissimum | CBS 144484T | Acidic soil, Hainan, China | KY495004 | KY495113 | MN969339 | KY495059
P. yunnanense | CBS 144485T | Acidic soil, Yunnan, China | KY494990 | KY495099 | MN969340 | KY495048
P. zhenyuanense | YMF 1.11149T | Soil, Yunnan, China | PZ344666 | PZ347310 | PZ357596 | PZ330333
P. zhenyuanense | YMF 1.11656 | Soil, Yunnan, China | PZ344667 | PZ347311 | PZ357597 | PZ330334
P. zonatum | CBS 992.72T | Coastal marsh soil, North Carolina, USA | GU981581 | GU981651 | MN969315 | KF296461

Phylogenetic analysis

Sequences of ITS, BenA, CaM, and RPB2 obtained in this study were initially compared with reference sequences in GenBank using BLAST to determine their closely related species. Reference strains were selected from recent taxonomic studies of Penicillium section Lanata-Divaricata. A total of 70 ingroup strains representing 64 species of series Janthinella, Dalearum and Rolfsiorum were included. Penicillium boreae S.W. Peterson & Sigler CBS 111717T (section Stolkia) was chosen as the outgroup.

Sequences for each locus were aligned using ClustalX v1.83 and manually edited in BioEdit v7.0. The individual alignments were concatenated into a combined dataset, and ambiguously aligned regions were excluded. Maximum likelihood (ML) analysis was conducted using IQ-TREE 2 (Minh et al. 2020), with the concatenated dataset partitioned by locus and the best-fit substitution models and partitioning scheme selected using ModelFinder under the MFP+MERGE option (Kalyaanamoorthy et al. 2017). Branch support was assessed using 1,000 ultrafast bootstrap replicates. Bayesian inference (BI) analysis was performed in MrBayes v3.1.2 under the GTR+G substitution model (Ronquist and Huelsenbeck 2003). Four independent runs with four Markov chains were executed for 5,000,000 generations, sampling every 500 generations. The first 25% of trees were discarded as burn-in. The average standard deviation of split frequencies fell below 0.01, indicating convergence among the independent runs. Nodes with bootstrap support (BS) ≥ 75% and posterior probabilities (PP) ≥ 0.90 were considered statistically supported. The final phylogeny was inferred from the ML analysis and visualized using TreeView.

Morphological characterisation

For morphological observation, a small amount of conidial suspension or mycelial fragments was taken from the margin of actively growing colonies on potato dextrose agar (PDA) using a sterile inoculation needle. Point inoculations were performed at three equidistant points (forming an equilateral triangle) on the surface of the following media: malt extract agar (MEA), Czapek yeast autolysate agar (CYA), and yeast extract-sucrose agar (YES). Cultures were incubated in the dark at 25 °C for 7 days. Colony characteristics, colony diameters, and pigment production were recorded for each strain on MEA, CYA, and YES.

Slide cultures were prepared following Cai et al. (2009) to observe micromorphological structures. Microscopic features were observed using an Olympus BX51 microscope (Tokyo, Japan) equipped with differential interference contrast (DIC) optics. Photomicrographs were captured using an Olympus DP10 digital camera operated with Olympus DP Controller software (Version 3.1.1208). Thirty individual structures were randomly measured, and average morphological dimensions were calculated.