683 MycoKeys MycoKeys 116: 255-274 (2025) DOI: 10.3897/mycokeys.116.149376 Research Article Two new species of Penicillium (Eurotiales, Aspergillaceae) from China based on morphological and molecular analyses Rui-Na Liang’, Xiang-Hao Lin', Miao-Miao An', Guo-Zhu Zhao! 1 College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China 2 National Engineering Research Center of Tree Breeding and Ecological Restoration, Beijing Forestry University, Beijing 100083, China Corresponding author: Guo-Zhu Zhao (zhaogz@bjfu. edu. cn) OPEN Qaceess Academic editor: Ning Jiang Received: 11 February 2025 Accepted: 27 March 2025 Published: 25 April 2025 Citation: Liang R-N, Lin X-H, An M-M, Zhao G-Z (2025) Two new species of Penicillium (Eurotiales, Aspergillaceae) from China based on morphological and molecular analyses. MycoKeys 116: 255-274. https://doi. org/10.3897/mycokeys.116.149376 Copyright: © Rui-Na Liang et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Penicillium is a large and significant genus of fungi, exhibiting widespread distribution across diverse substrates. Ongoing taxonomic and nomenclatural revisions have led to an annual increase in the number of newly described species. This study described two new Penicillium species, i.e., P lentum and P tibetense, discovered in China. They have been identified and characterized through morphological examination and both single gene and multigene phylogenetic analyses. Based on these analyses, P lentum was classified within the section Brevicompacta, while P. tibetense was placed in the section Lanata-Divaricata. Both species exhibited the morphological features typical of their re- spective sections. Penicillium lentum is characterized by restricted growth with dense colonies on agar media and predominantly generates terverticillate conidiophores. Penicillium tibetense demonstrates rapid growth on media and has vigorous growth on CYA at 30 °C, producing biverticillate conidiophores. Comprehensive descriptions and detailed illustrations of these new species were presented. A morphological compari- son between the new species and their closely related taxa was provided. Key words: Aspergillaceae, DNA barcodes, section Brevicompacta, section Lanata-Divar- icata, taxonomy Introduction Penicillium is widely distributed across various substrates, primarily in soil, as well as in the atmosphere, food, plant tissues, and other environments. Several species possess considerable value for human applications in food produc- tion, biocontrol, and biotechnology. For instance, P sclerotiorum exhibits an- tagonistic activity against certain plant pathogens, demonstrating potential as a biocontrol agent (Jahan et al. 2024). The food industry utilizes P nalgiovense as starter cultures for dry-fermented sausages (Ludemann et al. 2010). The ca- pability of certain species to synthesize pigments has prompted the evaluation of these species for the production of highly stable and safe natural pigments (Morales-Oyervides et al. 2020). Nevertheless, mycotoxins generated by specif- ic species present a significant risk to human and animal health (Nielsen et al. 2017). Notably, patulin exhibits multiple toxicities, including genotoxicity and immunotoxicity, and is predominantly produced by P expansum and P griseo- fulvum (Bandoh et al. 2009; Puel et al. 2010; Tannous et al. 2014). 259 Rui-Na Liang et al.: Two new Penicillium species Link (1809) introduced the generic name Penicillium, which is classified in the family Aspergillaceae. Traditional taxonomy of Penicillium primarily relied on morphological characters, including colony diameter, texture, conidial color, and conidiophore branching patterns. However, the variability in morphology has pre- sented substantial challenges in accurately identifying novel species, frequently resulting in the erroneous classification of new isolates under known species (Visagie et al. 2016). Conversely, contemporary taxonomy adopts a polypha- sic strategy that incorporates morphological, extrolite, genetic, and multigene phylogenetic data (Visagie et al. 2014). Houbraken et al. (2020) delivered the most comprehensive update on the genus Penicillium based on a phylogenetic approach combined with phenotypic, physiologic, and extrolite data. This study recognized 483 species and introduced a novel series classification, which is deemed highly predictive of potential functional traits (Houbraken et al. 2020). Subsequently, Visagie et al. (2024b) applied GCPSR (Genealogical Concordance Phylogenetic Species Recognition) and phylogenetic analyses to reassess the list of Penicillium species published up to 31 December 2022, resulting in an updat- ed count of 535 species. An additional 100 species of this genus were described from 1 January 2023 to 31 December 2024 (Ansari et al. 2023; Crous et al. 2023; da Silva et al. 2023; Khuna et al. 2023; Li et al. 2023; Liu et al. 2023; Tan 2023; Tan and Shivas 2023, 2024; Tan et al. 2023, 2024a, 2024b; Wang et al. 2023; Zhang et al. 2023; Araujo et al. 2024; Crous et al. 2024; Liang et al. 2024; Lima et al. 2024; Nobrega et al. 2024; Song et al. 2024; Visagie et al. 2024a, 2024c; Zhang et al. 2024). The increase in species numbers in recent years indicates the possibility of numerous undiscovered Penicillium species, and their biodiversity, ecological functions, and potential for resource development warrant further investigation. During a comprehensive survey of Penicillium biodiversity in China, we found two isolates that could not be classified within existing species. In this paper, we compare these isolates with related species using multi-locus phylogenetic anal- yses and morphological character assessments. As a result, the isolates are de- scribed as species new to science. This study is expected to offer new perspec- tives on the diversity, function, ecology, and distribution of Penicillium members. Materials and methods Isolates Soil samples were collected from the rhizosphere of plants in the Kangyu Tun- nel, Tibet, China, while indoor dust samples were sourced from Beijing Forestry University, Beijing, China. To isolate the fungus, the samples were suspended in sterile water at a ratio of 1:10, vortexed to ensure homogeneity, and then dilut- ed to 10* concentrations. Each of 100 uL from 10%, 10°, and 10% dilutions was spread on potato dextrose agar (PDA) and Martin medium with 50 ppm peni- cillin and 50 ppm streptomycin. The cultures were incubated at 25 °C for 5-7 days. Individual colonies were then picked from the plates and transferred to fresh PDA plates until pure cultures were obtained. Type specimens, preserved as dry cultures, were deposited in the Fungarium (HMAS), Institute of Microbi- ology, Chinese Academy of Sciences, while ex-type strains, maintained as living cultures, were stored at the China General Microbiological Culture Collection Centre (CGMCC). MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 256 Rui-Na Liang et al.: Two new Penicillium species Morphological studies Morphological observations of colonies were conducted under strictly standard- ized conditions, encompassing media preparation, inoculation technique, incuba- tion parameters, and description methods (Visagie et al. 2014). Colony characters and diameters were recorded from cultures grown on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), yeast extract sucrose agar (YES), dichloran 18% glycerol agar (DG18), and creatine sucrose agar (CREA) at 25 °C for 7 days. Additional CYA plates were incubated at 30 and 37 °C. Color names and codes adhered to the book “Color Standards and Color Nomenclature” (Ridgway 1912). Ehrlich reaction was employed to assess the production of indole metabolites; a violet ring observed within ten min was deemed a positive result, while other color changes were interpreted as negative (Lund 1995; Houbraken et al. 2016). For light microscopic observations, slides were prepared from cultures grown on MEA, and phenol glycerin solution was used as mounting fluid, with cotton blue staining if necessary. In addition, a field emission scanning electron microscope (Hitachi SU8010, Japan) was employed to examine microstructural characteris- tics. Agar blocks (3-4 mm x 3-4 mm) were fixed in 2.5% v/v glutaraldehyde at 4 °C for 8-12 hr, then washed three times for 10 min each with 0.1M phosphate buffer. Dehydration was performed with a gradient of ethanol (30, 50, 70, 95, and 100% v/v) for 10-20 min per step, followed by replacement with tert-butanol and ultimate vacuum freeze-dried and gold-sprayed for observation (Wei et al. 2024). DNA extraction, sequencing, and phylogenetic analyses Colonies were cultivated on MEA plates for 5-7 days, and DNA extraction was conducted using the E.Z.N.A.® Fungal DNA Mini Kit (Omega Bio-Tek, Inc., Unit- ed States). The internal transcribed spacer (ITS), beta-tubulin (BenA), calmod- ulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes were amplified using primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), CMD5/CMD6 (Hong et al. 2006), and RPB2-5F/RPB2- 7CR (Liu et al. 1999), respectively. Polymerase chain reaction (PCR) amplifi- cation followed Visagie et al. (2014). Sequencing reactions were performed by Sangon Biotech (Shanghai) Company Limited, China. DNAMAN software (Lynnon Biosoft) was used for the assembly and trimming of the Sanger chro- matograms. Sequences were submitted to GenBank (www.ncbi.nIm.nih.gov). Sequence similarity searches were conducted using the mega BLAST pro- gram of basic local alignment search tool (BLAST) within the NCBI core nu- cleotide database (core_nt). Comprehensive sequence datasets were com- piled containing newly generated sequences alongside reference sequences sourced from GenBank (Table 1). Sequence alignments were performed us- ing the ClustalW algorithm and subsequently manually edited using MEGA 11 (Tamura et al. 2021). The resulting multiple sequence alignments have been deposited in TreeBASE (submission number: 31847) (www.treebase. org). Phylogenetic trees were constructed based on the ITS, BenA, CaM, and RPB2 genes as well as the concatenated sequences of the latter three genes. Phylogenetic analyses were conducted using both maximum likelihood (ML) and Bayesian Inference (BI). ML phylogenies were performed using |Qtree v. 1.6.12 (Nguyen et al. 2015), including 1000 standard non-parametric boot- MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 257 Rui-Na Liang et al.: Two new Penicillium species Table 1. Strains of Penicillium used for phylogenetic analyses. Species P. abidjanum P alagoense P. amphipolaria P annulatum P araracuaraense P astrolabium P ausonanum P austrosinense P. bialowiezense P bissettii P. brasilianum P. brevicompactum P. buchwaldii P camponoti P cataractarum P coffeatum P. daleae P echinulonalgiovense P excelsum P expansum P. fengjieense P fennelliae P flaviroseum P fructuariae-cellae P. globosum P griseoflavum P griseopurpureum P guaibinense P. guangxiense P hainanense P infrabuccalum P jianfenglingense P jinyunshanicum P. kongii P laevigatum P. lentum P. mariae-crucis P. marykayhuntiae P neocrassum P. newtonturnerae P. ochrochloron Strain Substrate and origin CBS 246.67" Soil, lvory Coast URM 8086' Leaves of Miconia sp., Brazil CBS 140997' Soil, Antarctica CBS 135126° Air sample, South Africa CBS 113149° Leaf litter, Colombia CBS 122427° Grapes, Portugal CBS 148237' Sediment of freshwater stream, Spain CGMCC 3.18797' Acidic soil, China CBS 227.28" Soil under conifers, Poland CBS 140972' Soil from spruce forest, Canada CBS253:55! Herbarium exsiccata, Brazil NRRL 28139 Stroma of a wood decay fungus, USA CV1492 Unknown, South Africa CBS 257.29" Unknown, Belgium CBS 116980 Wheat, United Kingdom CBS 116935 Wheat, United Kingdom CBS 116929 Wheat flour, Denmark CBS:11,7181" Hordeum vulgare, Denmark CBS 140982' Carpenter ants, Canada CBS 140974' Fallen nuts of Carya cordiformis, Canada CGMCC 3.25152" Soil, China CBS 211.28" Soil under conifer, Poland CBS 328.597 Unknown, Japan DTO 357-D7" Brazil nut shell, Brazil ITAL 7804 Flowers, Brazil CBS 325.48" Malus sylvestris, USA CGMCC 3.25157" Soil, China CBS 711.68" Soil, Congo CGMCC 3.18805" Acidic soil, China CBS 145110° Dried fruit of Vitis vinifera, Italy CBS 144639° Acidic soil, China CGMCC 3.18799" Acidic soil, China CBS 406.65" Soil under Pinus sp., United Kingdom CCDCA 11512" Soil, Brazil CBS 144526° Soil, China CGMCC 3.18798" Acidic soil, China CBS 140983' Camponotus pennsylvanicus, Canada CGMCC 3.18802' Acidic soil, China CGMCC 3.25162" Soil, China AS3.15329' leaf sample of Cotoneaster sp., China CGMCC 3.18801' Acidic soil, China CGMCC 3.28596" Indoor dust, Beijing, China = B24 CBS 271:83' Secale cereale, Spain BRIP 74934a‘™ Soil, Australia CBS 122428° Grapes, Madeira BRIP 74909a' Soil, Australia CBS 357.48" Copper sulphate solution, USA DTO 189-A6 Unknown, Japan MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 GenBank accession numbers ITS GU981582 MK804503 KT887872 JX091426 GU981597 DQ645804 LR655808 KY495007 EU587315 KT887845 GU981577 AY484917 JX091398 AY484912 JAS ISLES JX313156 JX313152 JX313164 KT887855 KT887847 0Q870815 GU981583 GU981587 KR815341 KT749963 AY373912 0Q870765 JX313169 KY495032 MK039434 KY495014 KY495011 KF296408 MH674389 KY494986 KY495009 KT887856 KY495016 0Q870766 KC427191 KY495015 PQ643282 GU981593 OR271913 DQ645805 OP903478 GU981604 KC346347 BenA GU981650 MK802333 KT887833 JX091514 GU981642 DQ645793 LR655809 KY495116 AY674439 KT887806 GU981629 DQ645795 JX091533 AY674437 JX313181 JX313174 JX313170 MN969374 KT887816 KT887808 ORO51121 GU981649 GU981631 KP691061 KT749959 AY674400 ORO51156 MN969382 KY495141 KU554679 KY495123 KY495120 KF296467 MH674391 KY495095 KY495118 KT887817 KY495125 ORO51157 KC427171 KY495124 PQ519854 GU981630 OR269446 DQ645794 OP921964 GU981672 KC346324 CaM MN969234 MK802336 KT887794 JX141545 MN969237 DQ645808 LR655810 MN969328 AY484828 KT887767 MN969239 AY484825 JX141574 AY484813 JX313147 JX313140 JX313136 JX313148 KT887777 KT887769 ORO51298 MN969251 KX961269 KR815342 KT749962 DQ911134 ORO51333 JX3813751 MN969329 MK045337 MN969330 MN969331 MN969261 MH674393 MN969332 MN969333 KT887778 MN969334 ORO051334 KC427151 MN969335 PQ519855 MN969275 DQ645809 OP921962 MN969280 KC346341 RPB2 JN121469 MK802338 MN969177 KF296410 KF296414 JN406634 LR655811 KY495061 JN406604 MN969178 KF296420 JN406594 JN406637 MN969179 MN969180 ORO51466 KF296427 KX961301 MN969166 JF417427 ORO51489 JN406536 KY495083 KY495067 KY495064 KF296431 KY495045 KY495062 MN969181 KY495069 ORO51490 KY495068 PQ519856 KF296439 OR269440 JN406633 OP921963 KF296445 KC346318 258 Rui-Na Liang et al.: Two new Penicillium species Species P olsonii P onobense P panissanguineum P. paraherquei P. pauciramulum P. pedernalense P. penarojense P. piscarium P pulvillorum P. rolfsii P. roodeplaatense P rotoruae P rubriannulatum P. salamii P. simplicissimum P. singorense P. skrjabinii P. soliforme P spathulatum P. spinuliferum P stangiae P. stolkiae P. subfuscum P. subrubescens P. subrutilans P. svalbardense P tail P tanzanicum P terrarumae P tularense P. vanderhammenii P vasconiae P vickeryae P viridissimum P wotroi P tibetense P. yuyongnianii P zonatum Strain CBS 232.60" CBS 174.81 CBS 140989° CBS 338.59" CGMCC 3.25164! CBS 140770° CBS 113178° CBS 362.48" CBS 280.39" CBS 275.83 CBS 368.48" DTO 444-C8 CBS 145838" CGMCC 3.18804" CBS 135391° CBS 372.48" CBS 138214° CBS 439.75" CGMCC 3.18806" NNO72390 NNO72399 CBS 117192" CBS 144483' URM 8347' CBS 315.67" CBS 147455' CBS 132785' CGMCC 3.25174 CBS 122416 CGMCC 3.25176 CBS 140968° CBS 131811' CS23-08 CBS 430.69" CBS 431.69 CBS 126216' CBS 339.79" BRIP 72552a" CGMCC 3.18796" CBS 118171' CGMCC 3.28597" = XZ5-3 CGMCC 3.25187" CBS 992.72" Substrate and origin Musa, France Soil, andosol, Spain Soil near termite mound, Tanzania Soil, Japan Soil, associated with nest of Formicidae, China Litopenaeus vannamei, Ecuador Leaf litter, Colombia Cod-liver oil emulsion, Germany Acidic soil, United Kingdom Rye grain, Spain Fruit of Ananas sativus, USA Soil, South Africa Pinus radiata timber stake in ground contact, New Zealand Acidic soil, China Salami, Italy Secale cereale, Spain House dust, Thailand Soil, Russia Acidic soil, China Acidic soil, China Acidic soil, China Mouldy chestnut (Castanea sp.), France Acidic soil, associated with Litchi chinensis, China Soil, Brazil Soil, South Africa Soil, South Africa Soil of Helianthus tuberosus field, Finland Soil, China Glacial ice, Svalbard Soil, China Soil near termite mound, Tanzania Soil contaminated by heavy metals, China Unknown, China Soil under Pinus ponderosa and Quercus kelloggii, USA Soil under Pinus ponderosa and Quercus kelloggii, USA Leaf litter, Colombia Soil, Spain Soil, Australia Acidic soil, China Leaf litter, Colombia Rhizosphere soil, Tibet, China Soil, China Soil, USA MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 GenBank accession numbers ITS EU587341 GU981575 KT887862 AF178511 0Q870726 KU255398 GU981570 GU981600 AF178517 GU981601 JN617705 OR819195 MN315103 KY495029 HG514431 GU981588 KJ775674 GU981576 KY495038 KY495019 KY495022 JX313165 KY495040 MW648590 AF033444 MT949907 KC346350 0Q870816 GU981603 0Q870778 KT887841 MN431397 0Q870751 AF033487 JX313167 GU981574 GU981599 OP903479 KY495004 GU981591 PQ643284 0Q870820 GU981581 BenA AY674445 GU981627 KT887823 KF296465 ORO51111 KU255396 GU981646 GU981668 GU981670 GU981671 GU981667 OR820176 MN315104 KY495138 HG514437 GU981632 KJ775167 GU981626 KY495147 KY495128 KY495131 MN969400 KY495149 MW646388 JN617717 MT957412 KC346327 ORO51137 DQ486644 ORO51170 KT887802 KX650295 ORO51141 KC427175 AY674433 GU981647 GU981653 OP921966 KY495113 GU981637 PQ519857 ORO51175 GU981651 CaM DQ658165 MN969281 KT887784 MN969285 ORO51288 MN969322 MN969287 MN969288 MN969289 KC346336 MN969294 OR820180 MN315102 MN969336 HG514432 MN969297 KJ775403 MN969299 MN969337 KY494959 KY494962 JX313149 MN969338 MW646390 AF481135 MT957454 KC346330 ORO51314 KC346338 ORO051347 KT887763 MN969323 ORO51318 JX313135 JX313134 MN969308 MN969309 MN969339 MN969313 PQ519858 ORO51352 MN969315 RPB2 JN121464 KF296447 MN969182 KF296449 ORO51457 MN969184 KF296450 KF296451 KF296452 KF296423 KF296455 OR820186 MT240842 KY495080 MN969160 JN121507 MN969138 EU427252 KY495047 KY495072 KY495074 JN406636 KY495090 MW646392 JN121488 MT957480 KC346306 ORO51479 KF296457 ORO51496 MN969183 MN969185 ORO51481 JN121516 KF296458 MN969144 OP921965 KY495059 KF296460 PQ519859 ORO51499 KF296461 259 Rui-Na Liang et al.: Two new Penicillium species strap replicates with the best partition scheme and substitution model se- lected using ModelFinder (Kalyaanamoorthy et al. 2017). BI phylogenies were run in MrBayes v. 3.2.7 (Ronquist et al. 2012). Best fit models were selected according to the Akaike information criterion (AIC) using MrModeltest v. 2.4 (Nylander 2004). Posterior probabilities (PP) were estimated using Markov Chain Monte Carlo (MCMC) sampling, set to run for 1,000,000 generations with the average standard deviation of split frequencies less than 0.01 as the stopping criterion. In cases where this threshold was not achieved, the run was continued until the condition was met. Additionally, the initial 25% of the generated trees were discarded as burn-in. Results Morphology Two novel species, Penicillium lentum and P tibetense, were introduced within the sections Brevicompacta and Lanata-Divaricata, respectively, based on com- prehensive phylogenetic analyses. General morphological characteristics and ecological information for the species included in these sections are provided in Table 2. Both newly described species exhibited morphological traits con- sistent with their respective sections. Specifically, R lentum displayed limited growth with dense colonies on agar media and primarily produced terverticil- late conidiophores. In contrast, P. tibetense demonstrated rapid growth on agar media, particularly exhibiting robust development on CYA at 30 °C, and predom- inantly formed biverticillate conidiophores. The morphological features of the new species and their closely related species are summarized in Table 3. Table 2. Morphological and ecological data pertaining to the sections of the new species in this study. Section Morphology Ecology References Brevicompacta Colonies restricted (occasionally moderately fast), Mainly soil and foods, (Houbraken and Samson 2011; texture velutinous; conidiophores terverticillate or mul- | also on plant leaves and | Frisvad et al. 2013; Wang and Wang tiramulate branched with wide stipes, smooth-walled. rotting wood. 2013; Houbraken et al. 2020) Lanata-Divaricata Colonies grow rapidly, occasionally moderately fast; | Commonly foundin soil,| | (Houbraken and Samson 2011; conidiophores monoverticillate, biverticillate or divari- | also on rotting leaf litter Houbraken et al. 2020) cate, occasionally terverticillate. and vegetable. Table 3. Morphological features of new species and their closely related taxa. Specie Growth rates (mm) Conidiophores _Cleistothecia Conidia CYA | CYA30°C | CYA37°C| _ branching /sclerotia Size Shape _|_ Roughening P lentum 7-10 | Nogrowth | Nogrowth | Terverticillate, Absent O25 (55 Broadly Smooth sometimes 2.5 um ellipsoidal biverticillate P tularense? | n.a. n.a. n.a. Asymmetric and | Cleistothecia | 2.2-2.6 um | Globose to Smooth divaricate subglobose P tibetense 42-50) 42-52 21-27 Biverticillate Absent 1.5-3 um | Globose to | Finely rough subglobose P excelsum® | 35-50 n.a. 8-22 Biverticillate, Absent 4-5x Ellipsoidal Smooth sometimes 2-3.2 um terverticillate *Description based on Paden (1971), "Description based on Taniwaki et al. (2016). MycoKkeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 Acid production on CREA Absent na. Absent Absent 260 Rui-Na Liang et al.: Two new Penicillium species Phylogenetic analyses A BLAST search revealed that strain CGMCC 3.28596 is most closely related to Penicillium tularense (Identities: ITS: 97.52%, BenA: 81.13%, CaM: 84.91%, RPB2: 91.00%) within section Brevicompacta, and strain CGMCC 3.28597 exhibits the highest similarity to P excelsum (Identities: ITS: 98.64%, BenA: 94.37%, CaM: 89.66%, RPB2: 94.84%) within section Lanata-Divaricata. Section Brevicompacta The analyses of the concatenated dataset (BenA, CaM, and RPB2) comprised 20 predominantly ex-type strains, each with a total sequence length of 1876 bp (BenA: 469 bp, CaM: 512 bp, RPB2: 895 bp). Phylogenetic analyses divided section Brevicompacta into four distinct clades, with the new species Penicil- lium lentum forming a robustly supported clade alongside P. tularense (100% bs, 1.00 pp) (Fig. 1). In the phylogenetic analyses of individual genes, the new species, together with P tularense, consistently formed a well-supported clade, mostly with high support values (>97% bs, 1.00 pp), except for ITS (Fig. 2). ser. Brevicompacta P. astrolabium CBS 122427" P. salamii CBS 135391" P. olsonii CBS 232.60" ser. Olsoniorum ser. Buchwaldiorum xjxp P. tularense CBS 430.69" P. tularense CBS 431.69 | P. lentum CGMCC 3.28596" ser. Tularensia P. expansum CBS 325.48" _ 7 /% 0.10 Figure 1. ML tree based on the concatenated data set (BenA, CaM, and RPB2) of section Brevicompacta. Penicillium ex- pansum CBS 325.48" was designated as the outgroup. Nodes display bootstrap values (bs) exceeding 70% or posterior probabilities (pp) greater than 0.95. Branches with bs of 95% or higher and pp of 1.00 are depicted in bold. The strain described as the new species P lentum is indicated with blue text. * Indicates bs = 100% or pp = 1.00, ' = ex-type strain. MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 261 Rui-Na Liang et al.: Two new Penicillium species «ja P. tularense CBS 430.69! P. tularense CBS 431.69 P. lentum CGMCC 3.28596" P. spathulatum CBS 117192? P. roodeplaatense DTYO 444-C8 P. buchwaldii CBS 117181" P. buchwaldii CBS 116980 9910.9) P. buchwaldii CBS 116935 P. buchwaldii CBS 116929 P. salamii CBS 135391? P. olsonii CBS 232.60' P. astrolabium CBS 122427' P. brevicompactum CV 1492 P. brevicompactum NRRL 28139 P. brevicompactum CBS 257.29° | P. kongii AS3.15329" P. neocrassum CBS 122428' P. expansum CBS 325.48" 0.02 P. kongii AS3.15329° 90/-¢ P. brevicompactum CV 1492 P. brevicompactum NRRL 28139 CaM P. brevicompactum CBS 257.29" P. neocrassum CBS 1224287 P. fennelliae CBS 711.68" P. bialowiezense CBS 227.28! P. salamii CBS 135391" P. olsonii CBS 232.60' P. astrolabium CBS 122427" «ep P. tularense CBS 430.697 P. tularense CBS 431.69 P. buchwaldii CBS 116935 /0.99 P. buchwaldii CBS 116929 P. buchwaldii CBS 117181" P. buchwaldii CBS 116980 P. spathulatum CBS 117192" P. roodeplaatense DTO 444-C8 P. expansum CBS 325.48" 0.05 P. bialowiezense CBS 227.28! P. fennelliae CBS 711.68" P. lentum CGMCC 3.28596" P. brevicompactum NRRL 28139 P. brevicompactum CBS 257.29" P. brevicompactum CV 1492 P. kongii AS3.15329" P. neocrassum CBS 122428" P. bialowiezense CBS 227.28" P. fennelliae CBS 711.68" P. salamii CBS 135391" P. olsonii CBS 232.60 P. astrolabium CBS 122427' P. buchwaldii CBS 117181 «ep P. buchwaldii CBS 116980 P. buchwaldii CBS 116935 P. buchwaldii CBS 116929 P. spathulatum CBS 117192? P. roodeplaatense DTO 444-C8 «/*{ P. tularense CBS 430.69! P. tularense CBS 431.69 P. lentum CGMCC 3.28596" P. expansum CBS 325.48" 0.05 P. buchwaldii CBS 117181! P. spathulatum CBS 117192" P. roodeplaatense DTO 444-C8 P. lentum CGMCC 3.28596" P. tularense CBS 430.69' P. neocrassum CBS 122428" P. brevicompactum CBS 257.29" P. fennelliae CBS 711.68" P. bialowiezense CBS 227.28" P. salamii CBS 135391" P. astrolabium CBS 122427' P. olsonii CBS 232.60° P. expansum 325.48" 97/%* RPB2 0.10 Figure 2. ML trees for section Brevicompacta based on ITS, BenA, CaM, and RPB2. Penicillium expansum CBS 325.48° was designated as the outgroup. Nodes display bootstrap values (bs) exceeding 70% or posterior probabilities (pp) greater than 0.95. Branches with bs of 95% or higher and pp of 1.00 are depicted in bold. The strain described as the new species P lentum is indicated with blue text. * Indicates bs = 100% or pp = 1.00, ' = ex-type strain. MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 Section Lanata-Divaricata In this section, we selected the series Simplicissima, Dalearum, and Rolfsiorum, comprising 71 predominantly ex-type strains, for phylogenetic analyses based on the concatenated dataset totaling 1876 bp (BenA: 504 bp, CaM: 617 bp, RPB2: 755 bp). The resulting phylogenies revealed that Penicillium tibetense is closely related to P. excelsum (64% bs, 0.97 pp; not depicted in Fig. 3). However, the significant evolutionary divergence observed supports the recognition of 262 Rui-Na Liang et al.: Two new Penicillium species P. taii CGMCC 3.25176" 71/0.99— P. yuyongnianii CGMCC 3.25187" 81/* P. globosum CBS 144639" ats q P. cataractarum CBS 140974? 1 P. newtonturnerae BRIP 74909a! P. guangxiense CBS 144526" pk P. griseoflavum CGMCC 3.18799" P.mariae-crucis CBS 271.83" P. echinulonalgiovense CBS 328.59! gg#-— P. jinvunshanicum CGMCC 3.251627 P. laevigatum CGMCC 3.18801" P. spinuliferum CBS 144483" 90/*— fied 96/*L P. vickeryae BRIP 72552a' P. araracuarense CBS 113149! */% P. wotroi CBS 118171' P. onobense CBS 174.81" 99g P. paraherquei CBS 338.59" -/0.97 96% P. brasilianum CBS 253.55" P. skrjabinii CBS 439.75" P. alagoense URM 8086! «jp— P. infrabuccalum CBS 140983! P. pedernalense CBS 140770! P. fengjieense CGMCC 3.25157" +) P. panissanguineum CBS 140989" Bo/* P. subfuscum CBS 147455" P. simplicissimum CBS 372.48" P. tanzanicum CBS 140968" «yx P. pauciramulum CGMCC 3.25164" 86/0.98 P. ausonanum CBS 148237" 8sL_ P. viridissimum CGMCC 3.18796! P. amphipolaria CBS 140997" =i 86/.- P. austrosinense CGMCC 3.18797" -/0.99 99/0.98[L. P. guaibinense CCDCA 11512! P. marykayhuntiae BRIP 74934a! P. singorense CBS 138214" o7/* P. penarojense CBS 113178" : P. vanderhammenii CBS 126216! 20. 92/* P. stangiae URM 8347" P. jianfenglingense CGMCC 3.18802" 92/* +e P. rubriannulatum CGMCC 3.18804" = P. griseopurpureum CBS 406.65" lb oe P. daleae CBS 211.28" P. abidjanum CBS 246.67" P. zonatum CBS 992.72! «xe P. camponoti CBS 140982" P. subrutilans CGMCC 3.25174! «te P. terrarumae CBS 131811' P. terrarumae CS23-08 P. piscarium CBS 362.48! P. rolfsii CBS 368.48" xx) P. ochrochloron CBS 357.48" +/* P. ochrochloron DTO 189-A6 -/0.97. | uli P. rotoruae CBS 145838' 86/* P. svalbardense CBS 122416" -/0.99—t— P. flaviroseum CGMCC 3.18805! P. subrubescens CBS 132785" «ep P. pulvillorum CBS 275.83 P. pulvillorum CBS 280.39! xpy P. soliforme NNO72390 =f P. soliforme NNO72399 P. soliforme CGMCC 3.18806" — P. annulatum CBS 135126" «je, P. excelsum DTO 357-D7' 98/% P. excelsum ITAL 7804 -/0.95 -/0.97 P. tibetense CGMCC 3.28597" P. bissettii CBS 140972" 82/0.99 P. fructuariae-cellae CBS 145110! P. hainanense CGMCC 3.18798" P. vasconiae CBS 339.79" P. coffeatum CGMCC 3.25152! P. stolkiae CBS 315.67! % /%* % /* * /' 0.05 Figure 3. ML tree based on the concatenated data set (BenA, CaM, and RPB2) of section Lanata-Divaricata (series Sim- plicissima, Dalearum, and Rolfsiorum). Penicillium stolkiae CBS 315.67' was designated as the outgroup. Nodes display bootstrap values (bs) exceeding 70% or posterior probabilities (pp) greater than 0.95. Branches with bs of 95% or higher and pp of 1.00 are depicted in bold. The strain described as the new species P tibetense is indicated with blue text. * In- dicates bs = 100% or pp = 1.00, ' = ex-type strain. MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 263 Rui-Na Liang et al.: Two new Penicillium species P tibetense as a distinct species (Fig. 3). Phylogenetic analyses of individual genes within series Rolfsiorum demonstrated generally weak clustering sup- port, with variations observed among the ITS, BenA, CaM, and RPB2 datasets. Furthermore, P ochrochloron and P rotoruae share identical ITS sequences, making them indistinguishable through ITS phylogeny alone (Fig. 4). P. tibetense C@MCC 3.28597! P. fructuariae-cellae CBS 145110" P. hainanense CGMCC 3.18798" P. bissettii CBS 140972" gg/xpP. excelsum DTO 357-D7" P. excelsum ITAL 7804 P. vasconiae CBS 339.797 P. pulvillorum CBS 275.83 P. pulvillorum CBS 280.39" P. ochrochloron CBS 357.48" P. rotoruae CBS 145838" P. ochrochloron DTO 189-A6 P. svalbardense CBS 122416° P. terrarumae CBS 1318117 P. terrarumae CS23-08 P. camponotum CBS 140982? P. subrutilans CGMCC 3.25174" P. flaviroseum CGMCC 3.18805" P. subrubescens CBS 132785" P. soliforme CGMCC 3.18806! P. soliforme NNO72390 P. soliforme NNO72399 P. annulatum CBS 135126' P. rolfsii CBS 368.48" P. piscarium CBS 362.48" P. coffeatum CGMCC 3.25152" P. stolkiae CBS 315.67" ITS 0.01 «sep P. terrarumae CBS 1318117 P. terrarumae CS23-08' P. svalbardense CBS 122416" CaM aj P. ochrochloron CBS 357.48" P. ochrochloron DTO 189-A6 P. rotoruae CBS 1458387 P. subrubescens CBS 132785" «xp P. camponotum CBS 1409821 P. subrutilans CGMCC 3.25174" P. rolfsii CBS 368.48" P. annulatum CBS 135126' «yep P. pulvillorum CBS 280.39° P. pulvillorum CBS 275.83 P. flaviroseum CGMCC 3.18805" xy P. excelsum DTO 357-D7* P. excelsum ITAL 7804 P. tibetense CGMCC 3.28597" _ P. piscarium CBS 362.487 P. soliforme CGMCC 3.18806" P. soliforme NNO72390 */I P_ soliforme NNO72399 P. bissettii CBS 140972 P. fructuariae-cellae CBS 145110 P. vasconiae CBS 339.791 P. coffeatum CGMCC 3.25152? P. stolkiae CBS 315.67° 0.05 BenA P. hainanense CGMCC 3.18798" «ep P. camponotum CBS 140982! P. subrutilans CGMCC 3.25174" P. subrubescens CBS 132785" P. flaviroseum CGMCC 3.18805" «yap P. terrarumae CBS 131811" P. terrarumae CS23-08 P. piscarium CBS 362.48" */*| P. ochrochloron CBS 357.48" P. ochrochloron DTO 189-A6 P. rotoruae CBS 145838? P. svalbardense CBS 122416' «41 P. pulvillorum CBS 280.39! P. pulvillorum CBS 275.83 P. rolfsii CBS 368.48" + /0.99| P. soliforme NNO72390 ,| eld ' P. soliforme NNO72399 7 P. soliforme CGMCC 3.18806" «sat P. excelsum DTO 357-D7" P. excelsum ITAL 7804 P. tibetense C@MCC 3.28597" 10.95 P. annulatum CBS 135126° P. bissettii CBS 140972" P. fructuariae-cellae CBS 145110° P. vasconiae CBS 339.79' P. hainanense CGMCC 3.18798" P. coffeatum CGMCC 3.25152! P. stolkiae CBS 315.67° -/0.99 0.05 gg/#| P. ochrochloron CBS 357.48" P. ochrochloron DTO 189-A6 P. rotoruae CBS 145838" P. svalbardense CBS 122416' P. flaviroseum CGMCC 3.18805" P. subrubescens CBS 132785! «yep P. terrarumae CBS 1318117 P. terrarumae CS23-08 «x P. camponotum CBS 140982' P. subrutilans CGMCC 3.25174" P. rolfsii CBS 368.48" P. piscarium CBS 362.48" P. soliforme NNO72390 P. soliforme NNO72399 P. soliforme CGMCC 3.18806" P. tibetense CG@MCC 3.28597" P. excelsum DTO 357-D7"™ «ep P. pulvillorum CBS 280.39 P. pulvillorum CBS 275.83 P. annulatum CBS 135126' P. bissettii CBS 140972' P. hainanense CGMCC 3.18798" P. vasconiae CBS 339.79" P. coffeatum CGMCC 3.25152" P. stolkiae CBS 315.67" RPB2 0.99 0.05 Figure 4. ML trees for section Lanata-Divaricata series Rolfsiorum based on ITS, BenA, CaM, and RPB2. Penicillium stolki- ae CBS 315.67' was designated as the outgroup. Nodes display bootstrap values (bs) exceeding 70% or posterior proba- bilities (pp) greater than 0.95. Branches with bs of 95% or higher and pp of 1.00 are depicted in bold. The strain described as the new species P tibetense is indicated with blue text. * Indicates bs = 100% or pp = 1.00, ' = ex-type strain. MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 264 Rui-Na Liang et al.: Two new Penicillium species Taxonomy Penicillium lentum R.N. Liang & G.Z. Zhao, sp. nov. MycoBank No: 857346 Fig. 5 Infrageneric classification. Subgenus Penicillium, section Brevicompacta, se- ries Tularensia. Etymology. The specific epithet “lentum” is derived from lentus (Latin), re- flecting the slow growth rate characteristic of this species. Type. CHINA * Beijing, Haidian District, Beijing Forestry University, 40°0'20'N, 116°20'51"E, from indoor dust, 1 February 2024, collected by G.Z. Zhao, B24 (holotype HMAS 353385, dried culture; culture ex-type CGMCC 3.28596). Colony diameter after 7 d (mm). CYA 7-10; CYA 30 °C, 37 °C no growth; MEA 6-9: YES 9-13:DG18 7-1'1;CREA-.3.5-5. Colony characteristics (7 d). CYA at 25 °C: Colonies deep, raised at center, margins low, narrow, irregular; mycelium white; texture velutinous, floccose ar- eas present; sporulation moderate to good, conidia antique green (R. Pl. VI); exudate clear; reverse capucine buff (R. PI. Ill); soluble pigment absent. MEA at 25 °C: Colonies deep, raised at center, margins low, narrow, entire; myceli- um white; texture velutinous, floccose areas present; sporulation moderate to good, conidia celandine green (R. Pl. XLVII) to deep turtle green (R. Pl. XXXII); exudate clear; reverse light orange-yellow (R. PI. Ill); soluble pigment absent. YES at 25 °C: Colonies deep, radially and concentrically sulcate, raised at cen- ter, margins low, narrow, entire; mycelium white; texture velutinous and fascicu- late; sporulation good to strong, conidia glaucous-green (R. Pl. XXXIII); exudate absent; reverse cinnamon (R. Pl. XXIX); soluble pigment absent. DG18 at 25 °C: Colonies low, plane, margins low, wide, entire; mycelium white; texture veluti- nous and fasciculate; sporulation good, conidia bluish gray-green (R. PI. XLII); exudate absent; reverse antimony yellow (R. Pl. XV); soluble pigment absent. CREA at 25 °C: Weak growth, no acid production. Ehrlich reaction negative. Micromorphology. Conidiophores biverticillate to terverticillate; stipes smooth-walled, 70-236.5 x 2.5-4.5 um; rami two when present, 6.5-18 x 2-4 um; metulae divergent, 2-4 per branch/ramus, 4.0-13.0 x 2.5-4.5 um; phialides ampulliform, 3-8 per metula, 4.5—8.0 x 2-3 um; conidia broadly ellip- soidal, smooth-walled, 2-3 x 1.5-2.5 um. Notes. Penicillium lentum belongs to section Brevicompacta and is most closely related to P. tularense (Fig. 1). Penicillium tularense produces light brown to pale tan cleistothecia, which are not found in the new species (Paden 1971). Additionally, P lentum has broadly ellipsoidal conidia, while P tularense produces globose to subglobose conidia (Table 3). Penicillium tibetense R.N. Liang & G.Z. Zhao, sp. nov. MycoBank No: 857347 Fig. 6 Infrageneric classification. Subgenus Aspergilloides, section Lanata-Divarica- ta, series Rolfsiorum. MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 265 Rui-Na Liang et al.: Two new Penicillium species Figure 5. Penicillium lentum CGMCC 3.28596. A Colonies on medium at 25 °C for 7d (left to right, top row: CYA, YES, DG18, MEA obverse; second row: CYA reverse, YES reverse, DG18 reverse, CREA obverse) B-E conidiophores F conidia G-I SEM micrograph of conidiophores J SEM micrograph of conidia. Scale bars: 10 um (B-1); 2 um (J). wk Etymology. The specific epithet “tibetense” denotes the geographical origin of the species, indicating its discovery in Tibet. Type. CHINA * Tibet, Changdu City, Basu County, Kangyu Tunnel, 30°33'53'N, 96°15'25'E, from rhizosphere soil of grasses, 19 July 2023, collected by X.W. Peng, XZ5-3 (holotype HMAS 353386, dried culture; culture ex-type CGMCC 3.28597). MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 266 Rui-Na Liang et al.: Two new Penicillium species Figure 6. Penicillium tibetense CGMCC 3.28597. A Colonies on medium at 25 °C for 7d (left to right, top row: CYA, YES, DG18, MEA obverse; second row: CYA reverse, YES reverse, DG18 reverse, CREA obverse) B-D conidiophores E conidia F, G SEM micrograph of conidiophores H SEM micrograph of conidia. Scale bars: 10 um (B-G); 2 um (H). Colony diameter after 7 d (mm). CYA 42-50; CYA 30 °C 42-52; CYA 37 °C 21-27; MEA 48-52; YES 46-52; DG18 20-26; CREA 24-26. Colony characteristics (7 d). CYA at 25 °C: Colonies low to moderately deep, radially sulcate, margins low, narrow, entire; mycelium white; texture floccose; MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 267 Rui-Na Liang et al.: Two new Penicillium species sporulation moderate, conidia livid pink (R. Pl. XXVII); exudate clear; reverse light purple-drab (R. Pl. XLV) to avellaneous (R. Pl. XL); soluble pigment ab- sent. CYA at 30 °C: Colonies low to moderately deep, radially sulcate, margins low, narrow, entire; mycelium white; texture floccose; sporulation moderate, conidia livid pink (R. Pl. XXVII); exudate clear; reverse brownish vinaceous (R. Pl. XXXIX); soluble pigment absent. CYA at 37 °C: Colonies moderately deep, radially sulcate, margins low, narrow, entire; mycelium white; texture floccose; sporulation sparse, conidia livid pink (R. Pl. XXVII); exudate clear; reverse light buff (R. Pl. XV); soluble pigment absent. MEA at 25 °C: Colonies low to moderately deep, radially sulcate, margins low, narrow, entire; mycelium white; texture floccose; sporulation sparse to moderate, conidia pale brown- ish vinaceous (R. Pl. XXXIX); exudate clear; reverse antimony yellow (R. PI. XV); soluble pigment absent. YES at 25 °C: Colonies moderately deep, ran- domly sulcate, margins low, wide, entire; mycelium white; texture floccose; sporulation moderate, conidia antique green (R. Pl. VI); exudate clear; reverse antimony yellow (R. Pl. XV); soluble pigment absent. DG18 at 25 °C: Colo- nies low, radially sulcate, margins low, wide, entire; mycelium white; texture floccose; sporulation sparse, conidia ecru-drab (R. Pl. XLVI); exudate absent; reverse orange-pink (R. PI. Il); soluble pigment absent. CREA at 25 °C: Strong growth, no acid production. Ehrlich reaction negative. Micromorphology. Conidiophores biverticillate; stipes finely rough-walled, 27-364.5 x 2-3 um; metulae appressed to divergent, 2-4 per stipe, 8-15 x 1.5-3 um; phialides ampulliform to cylindrical, 2-6 per metula, 5-10.5 x 1.5- 3 um; conidia globose to subglobose, finely rough-walled, 1.5-3 um diam. Notes. Penicillium tibetense is classified in section Lanata-Divaricata and exhibits a close phylogenetic relationship to P excelsum (Fig. 3). This novel species generates globose to subglobose, finely rough-walled conidia that dis- tinguish it from P. excelsum (Table 3). Additionally, PR. tibetense demonstrates more robust growth on CYA at 37 °C compared to P. excelsum (21-27 mm vs. 8-22 mm) (Taniwaki et al. 2016). Discussion Penicillium, a ubiquitous and diverse fungal genus, plays pivotal roles in natural ecosystems while maintaining substantial economic importance and signifi- cant relevance to human affairs. The recent rapid increase in newly described species within this genus suggests that numerous taxa remain undiscovered. Given the extensive biotechnological applications of Penicillium species, ac- curate taxonomic identification is paramount, necessitating comprehensive species delineation through polyphasic approaches. In the present study, we introduced two novel species: one belonging to section Brevicompacta and the other to section Lanata-Divaricata. Section Brevicompacta currently comprises 15 species distributed across four series (Visagie et al. 2024a, 2024b) and is represented in our findings by the newly described P lentum sp. nov. This species, classified within series Tularensia, is characterized by predominantly terverticillate conidiophores and demonstrates a close relationship with other members of section Brevicom- pacta (Table 2). Section Lanata-Divaricata is characterized by its remarkable species diversity and rapid colony growth, primarily comprising soil-inhabiting MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 268 Rui-Na Liang et al.: Two new Penicillium species species, with over 90 taxa currently recognized across five series (Houbraken et al. 2020; Visagie et al. 2024b). The newly identified Penicillium tibetense as- signed to series Rolfsiorum exhibits characteristic rapidly expanding colonies and produces biverticillate conidiophores, consistent with the morphological features typical of this series. Members of section Lanata-Divaricata are eco- logically significant as decomposers of organic matter (Lichtner et al. 2022), with notable biotechnological potential exemplified by P subrubescens, which has demonstrated efficient inulinase production (Mansouri et al. 2013). Phylogenetic analyses of section Brevicompacta demonstrated that our strain P lentum formed a well-supported clade with its closest relative, P. tu- larense (Fig. 1), a relationship corroborated by shared morphological charac- teristics such as conidiophore branching patterns and growth rates. However, our strain could be clearly distinguished from P. tularense based on distinct phenotypic features, including the presence or absence of cleistothecia and differences in conidial morphology (Table 3). The phylogenetic relationships within section Lanata-Divaricata remain unresolved, primarily due to the poor support values in certain clades (Houbraken et al. 2020), exemplified by a clade comprising P camponoti, P. piscarium, P rolfsii, PR subrutilans, and P. terrar'umae (Fig. 3), which highlights the persistent challenges in resolving certain taxo- nomic groups even with multigene phylogenetic approaches. To address these limitations, we recommend expanding the taxonomic sam- pling to include strains from diverse geographical origins and ecological niches. This strategy would not only generate additional reference sequences but also facilitate the discovery of novel species and the detection of infraspecific vari- ation (Visagie and Houbraken 2020). Furthermore, sequencing additional gene regions represents a promising approach to enhance phylogenetic resolution (Visagie et al. 2021). The rapid development of high-throughput sequencing technologies has resulted in an accelerated increase in genomic data availabil- ity (Kapli et al. 2020), positioning phylogenomics as an essential tool in modern fungal taxonomy and systematics. By leveraging genome-scale data, phyloge- nomics is poised to overcome the limitations of single gene or multigene anal- yses, providing robust statistical support for clade resolution and enabling the reconstruction of a highly resolved fungal tree of life (Burki et al. 2020; Zhou and May 2023). These advancements underscore the transformative potential of phylogenomics in addressing long-standing taxonomic challenges and refin- ing our understanding of fungal evolutionary relationships. Acknowledgements We express our sincere gratitude to Professor Xia-Wei Peng for her invaluable assistance in the collection of soil samples from Tibet. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. MycoKeys 116: 255-274 (2025), DOI: 10.3897/mycokeys.116.149376 269 Rui-Na Liang et al.: Two new Penicillium species Funding This work was funded by the National Natural Science Foundation of China (No. 31570019, 31093440) and the Survey Project of Alien Invasive Species and Grassland Pest in Mentougou District (2022HXFWSWXY038). Author contributions Rui-Na Liang: Formal analysis, investigation, data curation, writing — original draft prepa- ration, visualization; Xiang-Hao Lin and Miao-Miao An: Investigation, visualization; Guo- Zhu Zhao: Conceptualization, methodology, validation, resources, writing - review and editing, supervision, funding acquisition. All authors have read and agreed to the pub- lished version of the manuscript. Data availability All of the data that support the findings of this study are available in the main text. All sequences generated in this study have been submitted to GenBank. 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