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. 2024 Mar 15;15:1329299. doi: 10.3389/fmicb.2024.1329299

Morphological and phylogenetic analyses reveal two new Penicillium species isolated from the ancient Great Wall loess in Beijing, China

Ruina Liang 1,2, Qiqi Yang 1, Ying Li 1, Guohua Yin 3, Guozhu Zhao 1,2,*
PMCID: PMC10978590  PMID: 38559343

Abstract

Introduction

Penicillium species exhibit a broad distribution in nature and play a crucial role in human and ecological environments.

Methods

Two Penicillium species isolated from the ancient Great Wall loess in the Mentougou District of Beijing, China, were identified and described as new species, namely, Penicillium acidogenicum and P. floccosum, based on morphological characteristics and phylogenetic analyses of multiple genes including ITS, BenA, CaM, and RPB2 genes.

Results

Phylogenetic analyses showed that both novel species formed a distinctive lineage and that they were most closely related to P. chrzaszczii and P. osmophilum, respectively.

Discussion

Penicillium acidogenicum is characterized by biverticillate conidiophores that produce globose conidia and is distinguished from similar species by its capacity to grow on CYA at 30°C. Penicillium floccosum is typically recognized by its restricted growth and floccose colony texture. The description of these two new species provided additional knowledge and new insights into the ecology and distribution of Penicillium.

Keywords: Aspergillaceae, DNA markers, phylogeny, taxonomy, new taxa

1. Introduction

Penicillium is widely distributed in various habitats, including soil, plants, air, and indoor settings, and various types of foods (Frisvad and Samson, 2004; Houbraken and Samson, 2011; Visagie et al., 2014a). Penicillium fungi, such as P. rubens for penicillin production, P. citrinum for synthesizing cholesterol-lowering drug mevastatin, P. camemberti and P. roqueforti for cheese production, and P. oxalicum with biocontrol potential, have significant economic value in antibiotic production, pharmaceutical synthesis, biocontrol, food processing, and food safety (Giraud et al., 2010; Houbraken et al., 2011a; Tsang et al., 2018; Steenwyk et al., 2019; Dumas et al., 2020; Yang et al., 2022). In addition, Penicillium can have some negative effects in some cases, such as producing a variety of mycotoxins that can cause food contamination and even threaten human health (Frisvad et al., 2004; Perrone and Susca, 2017; Stefanello et al., 2022).

Penicillium, established by Link (1809), derives its name from the Latin word penicillus, meaning small brush or paintbrush. The infrageneric classification system of Penicillium was mainly based on morphological characteristics in the past 100 years, whereas this phenotype-based sectional classification has been replaced by a system based on a multigene phylogeny in recent decades (Visagie et al., 2014a; Houbraken et al., 2016, 2020). Subgenera, sections, and series are usually transformed from well-supported clades based on DNA sequence analyses. Next-generation sequencing technology has made it possible to obtain a growing number of complete or nearly complete fungal genomes. Phylogenetic analysis based on whole genome sequences to determine the taxonomic position of Penicillium and its subordinate members is becoming an important trend in the future (Yang et al., 2016). Currently, 558 species of Penicillium were accepted (Wang et al., 2023) and were grouped into two subgenera, namely, Aspergilloides and Penicillium, 32 sections and 89 series (Houbraken et al., 2020). Species classified in the same section or series share many common features. For example, the series Canescentia and Atroveneta are closely related in phylogeny, but they can be distinguished by different extrolite profiles and colony textures. Therefore, defining a new species into a section or series could be highly predicted for their functional characteristics (Houbraken et al., 2020).

Penicillium section Citrina comprises a diverse range of species that exhibit a broad distribution and usually occur in soil habitats. Members of this group are distinguished by their symmetrically biverticillate conidiophores and relatively small, globose to subglobose conidia (Houbraken et al., 2011b; Visagie et al., 2014b; Visagie and Yilmaz, 2023). Furthermore, members of this section have a high potential to produce the mycotoxins citrinin (Houbraken et al., 2011b; Dutra-Silva et al., 2021; Yin et al., 2021). Currently, this section includes 47 species (Houbraken et al., 2020; Andrade et al., 2021; Nguyen et al., 2021; Ashtekar et al., 2022; Tan and Shivas, 2022; Visagie and Yilmaz, 2023). Penicillium sect. Osmophila was introduced by Houbraken et al. (2016) for species producing bi-, ter-, and quarter-verticillate conidiophores and demonstrating comparable growth rates on CYA when they were incubated at 15 and 25°C. This section currently only contains two species (Houbraken et al., 2020). Members of this section are isolated from soil, and no specific metabolites have been found (Houbraken et al., 2016). Due to the difficulty of delimiting the species within these sections solely based on phenotypic characteristics, a polyphasic approach incorporating morphological, extrolite, genetic data, and multigene phylogenetic analysis has been extensively employed for species identification (Visagie et al., 2014a).

During a survey of Penicillium diversity in China, two strains isolated from the ancient Great Wall loess at Qingshui Town, Mentougou District, Beijing, were identified as two new species by multiphase classification. In this study, we provided the morphology of these new species and conducted the phylogenetic analyses using the internal transcribed spacer rDNA area (ITS), partial β-tubulin (BenA), calmodulin (CaM), and the RNA polymerase II second largest subunit (RPB2) genes and compared them with closely related species. The description of these two novel species is expected to enrich our comprehension of Penicillium ecology and distribution.

2. Materials and methods

2.1. Sampling and isolation

Soil samples were collected from loess at the base of the ancient Great Wall (Hongshui Kou section) (39°59′16”N, 115°28′54″E) in Mentougou District, Beijing, China. Cultures were isolated from the soil using the dilution plate method. Initially, 10 g of soil sample was thoroughly mixed with 90 mL of sterile water to prepare a soil suspension. This suspension was then serially diluted to 10−2, 10−3, and 10−4 concentrations. Subsequently, 100 μL of each diluted suspension was plated on potato glucose agar (PDA) with penicillin (50 ppm) and streptomycin (50 ppm) (Lin, 2010). All plates were incubated at 25°C. Type specimens (dry cultures) were deposited in the Fungarium (HMAS), Institute of Microbiology, Chinese Academy of Sciences. Ex-type strains (living cultures) were deposited in the China General Microbiological Culture Collection Centre (CGMCC).

2.2. Morphology

Colony characters were observed for strains 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). The cultures were incubated at 25°C for 7 days, with extra CYA plates incubated at 30 and 37°C, which are useful for species distinction. Culture media preparation, inoculation technique, and incubation conditions followed the methods described by Visagie et al. (2014a). Color names and codes referred to the Color standards and color nomenclature (Ridgway, 1912). For microscopic observations, slides were made from colonies that have been growing on MEA for 7 days, using phenol glycerin solution as mounting fluid or staining with cotton blue. The isolates were tested for indole metabolite production using the Ehrlich reagent and a filter paper method (Lund, 1995). A violet ring observed within 10 min was considered a positive reaction, while any other color was defined as a negative response (Houbraken et al., 2016).

2.3. Observation of scanning electron microscope

Strains were grown for 5–7 days on MEA or PDA, and the conidiophores were mature. Agar blocks (4 × 4 mm) with conidial structures were cut with a blade before transferring to sterile Petri dishes. They were initially fixed with 2.5% glutaraldehyde at room temperature for 2 h, followed by an overnight incubation at 4°C. Subsequently, a gradient dehydration process involved varying ethanol concentrations (30, 50, 70, 95, and 100%), before a transition to tert-butanol (Zhang et al., 2016; Mukherjee et al., 2022). Finally, the samples were freeze-dried, sprayed with gold, and observed using FESEM (Hitachi SU8010, Japan).

2.4. DNA extraction, sequencing, and phylogenetic analysis

Strains were grown on PDA for 7 days and DNA was extracted using the E.Z.N.A.® Fungal DNA Mini Kit (Omega Bio-Tek, Inc., United States), involving fungal tissue disruption and lysis, isopropanol precipitation of DNA, precipitation of proteins, and DNA elution. Primers, PCR amplification, and DNA sequencing methods used for the ITS, BenA, CaM, and RPB2 genes were based on the description of Visagie et al. (2014a). The newly generated sequences were submitted to GenBank.1

Sequence datasets were established using newly generated sequences and reference-type sequences retrieved from GenBank. All datasets were aligned using MEGA 11 implementing the Align by ClustalW option (Tamura et al., 2021). Datasets were analyzed using maximum likelihood (ML) and Bayesian inference (BI). ML analyses were performed within IQtree v. 1.6.12 (Nguyen et al., 2015) and tested by standard non-parametric bootstrap analyses for 1,000 replications (Visagie et al., 2021). The best model for ML was determined using ModelFinder (Kalyaanamoorthy et al., 2017), a fast model-selection method implemented in IQtree. Bayesian inference (BI) analyses were conducted using MrBayes v. 3.2.7 (Ronquist et al., 2012), with a sampling frequency of 100 and the exclusion of the initial 25% of trees as burn-in. The sequences used for phylogenetic analyses in this study are listed in Table 1. Gene sequence alignment datasets were stored in TreeBASE2 with the submission number 30834.

Table 1.

Strains of the genus Penicillium used for phylogenetic analyses.

Species Strain Substrate and location GenBank accession numbers
ITS BenA CaM RPB2
P. acidogenicum CGMCC3.25421T = CC-1 Soil, Beijing, China OR512884 OR531524 OR538539 OR538541
P. allii-sativi DTO 149-A8T Bulbs of Allium sativum (garlic), Mendoza, Argentina JX997021 JX996891 JX996232 JX996627
P. anatolicum CBS 479.66T Soil, Turkey AF033425 JN606849 JN606571 JN606593
P. argentinense CBS 130371T Soil, Valdes Peninsula, prov. Chubet, Argentina JN831361 JN606815 JN606549 MN969105
P. atrofulvum CBS 109.66T Soil, Katanga near Kipushi, Zaire JN617663 JN606677 JN606387 JN606620
P. aurantiacobrunneum CBS 126228T Air sample, Cake factory, Give, Denmark JN617670 JN606702 MN969238 MN969106
P. cairnsense CBS 124325T Soil, Atherton Tableland, Australia JN617669 JN606693 MN969240 MN969108
P. carneum CBS 112297T Moldy rye bread, Denmark HQ442338 AY674386 HQ442322 JN406642
P. christenseniae CBS 126236T Soil in the native forest near the base of the aerial tram, Costa Rica JN617674 JN606680 MN969243 JN606624
P. chrysogenum CBS 306.48T Cheese, Storrs, Connecticut AF033465 JF909955 JX996273 JN121487
P. chrzaszczii CBS 217.28T Woodland soil, Puszcza Bialowieska Forest, Poland GU944603 JN606758 MN969244 JN606628
P. citrinum CBS 139.45T Unknown AF033422 GU944545 MN969245 JF417416
P. confertum CBS 171.87T Cheek pouches of Dipodomys spectabilis, Arizona, United States JX997081 AY674373 JX996963 JX996708
P. copticola CBS 127355T Tortilla, United States JN617685 JN606817 JN606553 JN606599
P. cosmopolitanum CBS 126995T Heathland soil, Eersel, Cartierheide, The Netherlands JN617691 JN606733 MN969249 MN969113
P. decaturense CBS 117509T Old resupinate fungus, Ramsey Lake State Park, Decatur, United States GU944604 GU944604 MN969252 JN606621
P. desertorum DTO 148-I6T Cool desert soil under Oryzopsis hymenoides, Wyoming, United States JX997011 JX996818 JX996937 JX996682
P. dipodomys CBS 110412T Cheek pouches of Dipodomys spectabilis, Arizona, United States MN431359 AY495991 JX996950 JF909932
P. dokdoense JMRC:SF:013606T Soil, Dokdo Island in the East Sea of Korea MG906868 MH243037 MH243031 n.a.
P. egyptiacum CBS 244.32T Soil, Cairo, Egypt AF033467 KU896810 JX996969 JN406598
P. euglaucum CBS 323.71T Soil, Argentina JN617699 JN606856 JN606564 JN606594
P. expansum CBS 325.48T Fruit of Malus sylvestris, United States AY373912 AY674400 DQ911134 JF417427
P. flavigenum CBS 419.89T Flour, Lyngby, Denmark JX997105 AY495993 JX996281 JN406551
P. floccosum CGMCC3.25422T = CC-2 Soil, Beijing, China OR512914 OR594640 OR594641 OR538540
P. gallaicum CBS 167.81T Air, Madrid, Spain JN617690 JN606837 JN606548 JN606609
P. godlewskii CBS 215.28T Soil under pine, Bialowieska, Poland JN617692 JN606768 MN969258 JN606626
P. goetzii CBS 285.73T Soil, Calgary, Canada JX997091 KU896815 JX996971 JX996716
P. gorlenkoanum CBS 408.69T Soil, Syria GU944581 GU944520 MN969259 JN606601
P. halotolerans DTO 148-H9T Salt marsh, Egypt JX997005 JX996816 JX996935 JX996680
P. hetheringtonii CBS 122392T Soil of beach, Land’s End Garden, Treasure Island, United States GU944558 GU944538 MN969263 JN606606
P. kewense CBS 344.61T Unknown, Great Britain AF033466 KU896816 JX996973 JF417428
P. lanosocoeruleum CBS 215.30T Culture contaminant, United States JX997110 KU896817 JX996967 JX996723
P. manginii CBS 253.31T Unknown GU944599 JN606651 MN969274 JN606618
P. mediterraneum FMR 15188T Herbivore dung, Balearic Islands, Spain LT899784 LT898291 LT899768 LT899802
P. miczynskii CBS 220.28T Soil under conifer, Tatra mountains, Poland GU944600 JN606706 MN969277 JN606623
P. mononematosum CBS 172.87T Heavily molded seed, Amaranthus, Arizona, United States JX997082 AY495997 JX996964 JX996709
P. nalgiovense CBS 352.48T Ellischauer cheese, Czechoslovakia AY371617 KU896811 JX996974 JX996719
P. neomiczynskii CBS 126231T Soil, New Zealand JN617671 JN606705 MN969278 MN969128
P. nothofagi CBS 130383T Soil under Nothofagus, Chile JN617712 JN606732 JN606507 MN969129
P. osmophilum NRRL 5922T Arable soil, Netherlands EU427295 MN969391 KU896846 JN121518
P. pancosmium CBS 276.75T Old basidioma of Armillaria mellea, on hardwood log; Meech Lake, Gatineau Park, Gatineau County, Quebec, Canada JN617660 JN606790 MN969284 MN969130
P. paneum CBS 101032T Moldy rye bread, Denmark HQ442346 AY674387 HQ442331 KU904361
P. pasqualense CBS 126330T Soil, Easter Island, Chile JN617676 JN606673 MN969286 JN606617
P. paxilli CBS 360.48T Optical instrument, Barro Colorado Island, Panama GU944577 JN606844 JN606566 JN606610
P. persicinum CBS 111235T Soil, Qinghai, China JX997072 JF909951 JX996954 JN406644
P. psychrosexuale CBS 128137T Wooden crate in cold-store of apples covered by Fibulorhizoctonia psychrophile, the Netherlands HQ442345 HQ442356 HQ442330 KU904362
P. quebecense CBS 101623T Air in sawmill, Quebec, Canada JN617661 JN606700 JN606509 JN606622
P. raphiae CBS 126234T Soil under Raphia, Las Alturas, Costa Rica JN617673 JN606657 MN969292 JN606619
P. restrictum CBS 367.48T Soil, Honduras AF033457 KJ834486 KP016803 JN121506
P. roqueforti CBS 221.30T French Roquefort cheese, United States KC411724 MN969397 MN969295 JN406588
P. roseopurpureum CBS 226.29T Unknown, Belgium GU944605 JN606838 JN606556 JN606613
P. rubens CBS 129667T Unknown JX997057 JF909949 JX996263 JX996658
P. samsonianum CBS 138919T Grassland along the banks of Qumar River, Qinghai, China KJ668590 KJ668582 KJ668586 KT698899
P. sanguifluum CBS 127032T Soil, Norway JN617681 JN606819 JN606555 MN969135
P. shearii CBS 290.48T Soil, Tela, Honduras GU944606 JN606840 EU644068 JN121482
P. sinaicum CBS 279.82T Marine sludge, Egypt JX997090 KU896818 JX996970 JN406587
P. sizovae CBS 413.69T Soil, Syria GU944588 GU944535 MN969298 JN606603
P. steckii CBS 260.55T Cotton fabric treated with copper naphthenate, Colorado Island, Panama GU944597 GU944522 MN969300 JN606602
P. sucrivorum DTO 183-E5T Mite inside infructescence of Protea repens, South Africa JX140872 JX141015 JX141506 MN969140
P. sumatrense CBS 281.36T Soil, Toba Heath, Sumatra, Indonesia GU944578 JN606639 MN969301 EF198541
P. tardochrysogenum CBS 132200T Soil, McMurdo Dry Valley, Antarctica JX997028 JX996898 JX996239 JX996634
P. terrigenum CBS 127354T Soil, Hawaii, United States JN617684 JN606810 JN606583 JN606600
P. tropicoides CBS 122410T Soil of rainforest, Thailand GU944584 GU944531 MN969303 JN606608
P. tropicum CBS 112584T Soil under Coffea arabica, Karnataka GU944582 GU944532 MN969304 JN606607
P. ubiquetum CBS 126437T Soil, Wilson Botanical Garden, Costa Rica JN617680 JN606800 MN969306 MN969142
P. vancouverense CBS 126323T Soil under maple tree, Vancouver, British Columbia, Canada JN617675 JN606663 MN969307 MN969143
P. vanluykii CBS 131539T Lechuguilla Cave, Carlsbad, United States JX997007 JX996879 JX996220 JX996615
P. waksmanii CBS 230.28T Woodland soil, Puszcza Bialowieska Forest, Poland GU944602 JN606779 MN969310 JN606627
P. wellingtonense CBS 130375T Soil, Wellington, New Zealand JN617713 JN606670 MN969311 JN606616
P. westlingii CBS 231.28T Soil under conifer, Denga Goolina, Poznan, Poland GU944601 JN606718 MN969312 JN606625
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3. Results

3.1. Isolates and identification

Isolations resulted in six fungal isolates obtained from the ancient Great Wall loess, with two suspect new species CC-1(CGMCC 3.25421T) and CC-2(CGMCC 3.25422T). Through sequencing of ITS, BenA, CaM, and RPB2 genes, CC-1(CGMCC 3.25421T) generated gene fragments with sizes of 549 bp, 459 bp, 534 bp, and 1,070 bp, and CC-2(CGMCC 3.25422T) with sizes of 541 bp, 453 bp, 520 bp, and 1,053 bp, respectively. The blast results of ITS, BenA, CaM, and RPB2 genes showed that CC-1 was closely related to P. chrzaszczii (Identities: ITS: 99.62%, BenA: 95.62%, CaM: 96.21%, RPB2: 97.27%), while CC-2 was closely related to P. osmophilum (Identities: ITS: 98.34%, BenA: 96.22%, CaM: 95.31%, RPB2: 96.97%). Preliminary identification based on blast results of sufficient gene and morphological characteristics, strain CC-1 was designated as a member of Penicillium section Citrina and strain CC-2 was a member of section Osmophila, but neither strain could be identified as any known species, so further phylogenetic analyses were performed.

3.2. Phylogenetic analyses

Phylogenetic analyses of the Penicillium sections Citrina, Chrysogena, Osmophila, and Roquefortorum were conducted using the ITS, BenA, CaM, and RPB2 genes, along with a concatenation of the latter three genes, to determine the phylogenetic position of the new species (CGMCC 3.25421T and CGMCC 3.25422T). A total of 43 ex-(neo) type strains were involved in the analyses of the individual and combined datasets of section Citrina, while 26 ex-(neo) type strains were involved in the analyses of the sections Chrysogena, Osmophila, and Roquefortorum. A summary of the length and substitution models for each dataset is provided in Table 2.

Table 2.

Length and substitution models for each dataset used in phylogeny.

Dataset
Section Citrina Section Osmophila and Roquefortorum and Chrysogena
ITS dataset Length (bp) 572 588
Substitution model (BI) GTR + I + G GTR + G
Substitution model (ML) TIM2 + F + I + G4 TIM2e + I
BenA dataset Length (bp) 459 437
Substitution model (BI) GTR + G HKY + I + G
Substitution model (ML) TIM2e + G4 K2P + G4
CaM dataset Length (bp) 611 505
Substitution model (BI) GTR + I + G K80 + G
Substitution model (ML) TIM2e + I + G4 K2P + G4
RPB2 dataset Length (bp) 914 960
Substitution model (BI) GTR + I + G SYM + G
Substitution model (ML) TNe + I + G4 TNe + G4
Concatenated dataset (BenA-CaM-RPB2) Length (bp) 1984 1902
Substitution model (BI) GTR + I + G SYM + G
Substitution model (ML) TIM2e + I + G4 TNe + G4
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3.2.1. Section Citrina

Phylogenetic analyses based on concatenated datasets (BenA-CaM-RPB2) divided section Citrina into nine clades (Figure 1), which is consistent with the study by Houbraken et al. (2020). Penicillium acidogenicum (CC-1 = CGMCC3.25421T) was introduced as a new species, comprising a well-supported distinct clade related to species P. chrzaszczii in series Westlingiorum (95% bs, 1.00 pp) (Figure 1). The phylogenetic analyses of single-gene revealed that the CaM phylogeny better resolved the relationship between these branches in section Citrina compared to the ITS, BenA, and RPB2 phylogenies. ITS has poor discriminatory ability in this section. BenA, CaM, and RPB2 can easily distinguish the new species, but BenA cannot reliably distinguish among P. decaturense, P. pancosmium, and P. ubiquetum (Figure 2).

Figure 1.

Figure 1

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ML phylogenetic tree for concatenated datasets (BenA-CaM-RPB2) of Penicillium acidogenicum and Penicillium section Citrina. Penicillium restrictum was chosen as the outgroup. Bootstrap values (bs) above 70% or posterior probability (pp) above 0.95 are shown at nodes. The branches with more than 95% bs and 1.00 pp values are thickened. Names in red text indicate strains that belong to the new species in this study. *Indicates bs = 100% or pp = 1.00, T = ex-type strain.

Figure 2.

Figure 2

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ML phylogenetic tree for individual gene dataset of ITS, BenA, CaM, and RPB2 of Penicillium acidogenicum and Penicillium section Citrina. Penicillium restrictum was chosen as the outgroup. Bootstrap values (bs) above 70% or posterior probability (pp) above 0.95 are shown at nodes. The branches with more than 95% bs and 1.00 pp values are thickened. Names in red text indicate strains that belong to the new species in this study. *Indicates bs = 100% or pp = 1.00, T = ex-type strain.

3.2.2. Section Chrysogena, Osmophila, and Roquefortorum

Phylogenetic analyses revealed a new species in section Osmophila and described it as Penicillium floccosum (CC-2 = CGMCC3.25422T). This species was grouped in a clade as P. osmophilum with robust support (100% bs, 1.00 pp) (Figure 3). In the single-gene phylogenies, P. floccosum and P. osmophilum formed a clade with a high degree of support, except for ITS (Figure 4). Compared with ITS, BenA, CaM, and RPB2 can easily distinguish all species of these sections.

Figure 3.

Figure 3

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ML phylogenetic tree for concatenated datasets (BenA-CaM-RPB2) of Penicillium floccosum and Penicillium sections Chrysogena, Osmophila, and Roquefortorum. Penicillium expansum was chosen as the outgroup. Bootstrap values (bs) above 70% or posterior probability (pp) above 0.95 are shown at nodes. The branches with more than 95% bs and 1.00 pp values are thickened. Names in red text indicate strains that belong to the new species in this study. *Indicates bs = 100% or pp = 1.00, T = ex-type strain.

Figure 4.

Figure 4

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ML phylogenetic tree for individual gene dataset of ITS, BenA, CaM, and RPB2 of Penicillium floccosum and Penicillium sections Chrysogena, Osmophila, and Roquefortorum. Penicillium expansum was chosen as the outgroup. Bootstrap values (bs) above 70% or posterior probability (pp) above 0.95 are shown at nodes. The branches with more than 95% bs and 1.00 pp values are thickened. Names in red text indicate strains that belong to the new species in this study. *Indicates bs = 100% or pp = 1.00, T = ex-type strain.

3.3. Taxonomy

3.3.1. Penicillium acidogenicum R. N. Liang and G. Z. Zhao, sp. nov.

Figure 5

Figure 5.

Figure 5

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Penicillium acidogenicum CGMCC 3.25421. (A) Colonies on medium for 7 days (left to right, first row: CYA, YES, DG18, and MEA obverse; second row: CYA, YES, DG18 reverse, and CREA obverse); (B) SEM micrograph of cleistothecia; (C–E) Conidiophores; (F) Conidia; (G,H) SEM micrograph of conidiophores; (I) SEM micrograph of conidia. Scale bars: B = 50 μm, C–H = 10 μm, I = 5 μm.

MycoBank number: 850530.

Infrageneric classification: subgenus Aspergilloides, section Citrina, series Westlingiorum.

Etymology: “acidogenicum” refers to the acid-producing characteristics of colonies grown on CREA.

Type: CHINA. Beijing, Mentougou District, Qingshui Town, from the ancient Great Wall loess, 27 August 2022, collected by G. Z. Zhao, CC-1 (holotype HMAS 352643, dried culture; culture ex-type CGMCC 3.25421).

Colony diameter after 7 days (mm): CYA 18–21; MEA 20–23; YES 24–29; DG18 16–19; CREA: 10–13; CYA 30°C 14–16; CYA 37°C no growth.

Colony characteristics (7 days): CYA at 25°C: Colonies moderately deep, sulcate, slightly elevated at the center; margins entire; mycelium white; texture floccose and funicolose; sporulation moderate, conidia light grayish vinaceous (R. Pl. VIII); exudate clear; reverse light orange-yellow (R. Pl. LXXI); soluble pigment absent. CYA at 30°C: Colonies moderately deep, sulcate, slightly elevated at the center; margins entire; mycelium white; texture floccose and funicolose; sporulation moderate, conidia light grayish vinaceous (R. Pl. XXXII) to gray; exudate clear; reverse orange-pink (R. Pl. LII); soluble pigment absent. MEA at 25°C: Colonies moderately deep, radially sulcate; margins entire; mycelium white; texture floccose; sporulation sparse, conidia livid pink (R. Pl. IV); exudate clear; reverse cadmium orange (R. Pl. XLVIII); soluble pigment absent. YES at 25°C: Colonies moderately deep, sulcate, elevated at the center; margins entire; mycelium white; texture floccose; sporulation sparse to absent; exudate absent; reverse orange (R. Pl. L); soluble pigment absent. DG18 at 25°C: Colonies moderately deep, sulcate, raised at the center; margins entire; mycelium white; texture floccose; sporulation sparse to absent; exudate absent; reverse pale orange-yellow (R. Pl. LXX); soluble pigment absent. CREA at 25°C: weak growth, moderate acid production. Ehrlich reaction negative.

Micromorphology: Cleistothecia produced on CYA and MEA, 80–130 μm diam; conidiophores biverticillate; stipes smooth-walled, 80–235 × 2.5–3.5 μm; metulae divergent, 2–4 per stipe, 8–13 × 1.5–3 μm; phialides ampulliform, 4–8 per metula, 5.5–7.5 × 2–2.5 μm; conidia globose, finely rough, 2–3 μm diam.

Notes: Phylogenetic analyses clustered Penicillium acidogenicum within a sister clade alongside 10 species, including P. chrzaszczii, P. godlewskii, P. waksmanii, P. outeniquaense, P. ubiquetum, P. pancosmium, P. decaturense, P. cosmopolitanum, P. westlingii, and P. nothofagi. The new species P. acidogenicum is phylogenetically most closely related to P. chrzaszczii, P. outeniquaense, P. waksmanii, and P. godlewskii. However, the former (colony 14–16 mm diam) can grow on CYA at 30°C, and the latter four cannot grow at 30°C. Penicillium acidogenicum can produce acid on CREA which is easily distinguished from the non-acid-producing character of closely related species (Table 3). The growth of P. acidogenicum is more restricted on DG18 (colony 16–19 mm) than P. chrzaszczii (20–27 mm), P. outeniquaense (20–21 mm), and P. waksmanii (16–27 mm) (Houbraken et al., 2011b; Visagie and Yilmaz, 2023).

Table 3.

Morphological features for new species and their closely related species.

Species Growth rates (in mm) Conidiophores branching Cleistothecia/sclerotia Conidia References
CYA CYA 30°C CYA 37°C MEA Shape Roughening
P. chrzaszczii 25–33 No growth No growth 21–28 Symmetrically biverticillate Absent Globose to subglobose Finely rough Houbraken et al. (2011b)
P. outeniquaense 25–27 Germination No growth 18–19 Biverticillate/mono- or terverticillate rare Absent Globose Finely rough Visagie and Yilmaz (2023)
P. godlewskii 15–25 No growth No growth 12–20 Symmetrically biverticillate Absent Globose to subglobose Finely rough Houbraken et al. (2011b)
P. waksmanii (20–) 25–32 No growth No growth 18–24(−30) Symmetrically biverticillate Absent Globose to subglobose Finely rough Houbraken et al. (2011b)
P. acidogenicum 18–21 14–16 No growth 20–23 Biverticillate Cleistothecia Globose Finely rough This study
P. osmophilum 14–26 n.a. n.a. 35 (2 weeks) Biverticillate-divaricate/monoverticillate rare Cleistothecia Broadly ellipsoidal or subglobose Smooth Stolk and Veenbaas-Rijks (1974), Pitt (1979), and Houbraken et al. (2016)
P. floccosum 24–27 10–13 No growth 24–27 Terverticillate Absent Globose Smooth This study
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3.3.2. Penicillium floccosum R. N. Liang and G. Z. Zhao, sp. nov.

Figure 6

Figure 6.

Figure 6

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Penicillium floccosum CGMCC 3.25422. (A) Colonies on medium for 7 days (left to right, first row: CYA, YES, DG18, and MEA obverse; second row: CYA, YES, DG18 reverse, and CREA obverse); (B–D) Conidiophores; (E) Conidia; (F,G) SEM micrograph of conidiophores; (H) SEM micrograph of conidia. Scale bars: B–G = 10 μm, H = 5 μm.

MycoBank number: 850534.

Infrageneric classification: subgenus Penicillium, section Osmophila, series Osmophila.

Etymology: “floccosum” refers to its floccose colony texture.

Type: CHINA. Beijing, Mentougou District, Qingshui Town, from the ancient Great Wall loess, 27 August 2022, collected by G. Z. Zhao, CC-2 (holotype HMAS 352644, dried culture; culture ex-type CGMCC 3.25422).

Colony diameter after 7 days (mm): CYA 24–27; MEA 24–27; YES 31–34; DG18 18–19; CREA: 5–8; CYA 30°C 10–13; CYA 37°C no growth.

Colony characteristics (7 days): CYA at 25°C: Colonies moderately deep, radially sulcate, elevated at the margin; margins entire; mycelium white; texture floccose; sporulation moderate, conidia dark greenish glaucous (R. Pl. CXXXV); exudate clear; reverse peach red (R. Pl. XXXVII); soluble pigment absent. CYA at 30°C: Colonies moderately deep, radially sulcate, elevated at the center; margins entire; mycelium white; texture floccose; sporulation absent; exudate absent; reverse peach red (R. Pl. XXXVII); soluble pigment absent. MEA at 25°C: Colonies moderately deep, radially sulcate, elevated at the center; margins entire; mycelium white to pale yellow; texture floccose; sporulation sparse, conidia tea green (R. Pl. CXXII); exudate absent; reverse orange to cadmium orange (R. Pl. L); soluble pigment absent. YES at 25°C: Colonies moderately deep, radially sulcate, elevated at center; margins entire; mycelium white; texture floccose; sporulation sparse, conidia cadet gray (R. Pl. CLXXXV) to calamine blue (R. Pl. CLXXI); exudate absent; reverse cadmium yellow (R. Pl. LXVIII) to cadmium orange (R. Pl. L); soluble pigment absent. DG18 at 25°C: Colonies moderately deep, radially sulcate, raised at the center; margins entire; mycelium white; texture floccose; sporulation absent; exudate absent; reverse light orange–yellow (R. Pl. LXXI); soluble pigment absent. CREA at 25°C: weak growth, no acid production. Ehrlich reaction negative.

Micromorphology: Conidiophores terverticillate; stipes smooth to nearly smooth-walled, 70–300 × 2–4 μm; rami 11–21 × 1.5–3 μm; metulae divergent, 2–4 per branch/ramus, 8–12 × 1.5–2.5 μm; phialides ampulliform to cylindrical, 2–6 per metula, 6–7.5 × 1.5–2.5 μm; conidia globose, smooth, 2–3.5 μm diam.

Notes: Penicillium floccosum is classified in the section Osmophila and phylogenetically closely related to P. osmophilum. However, P. osmophilum produces ascomata, a feature lacking in the new species. Penicillium floccosum produces globose conidia and is distinguished from P. osmophilum which produces pear-shaped to ellipsoidal, occasionally subglobose conidia (Table 3; Stolk and Veenbaas-Rijks, 1974).

4. Discussion

The delimitation of Penicillium species currently relies on a polyphasic approach, typically including morphological characteristics, extrolites data, and multigene phylogenetic analyses. DNA sequence markers used for identification and phylogeny include ITS, BenA, CaM, and RPB2 genes (Houbraken et al., 2020). The ITS region is widely recognized as a universal barcode for fungi (Schoch et al., 2012). Nevertheless, in Penicillium, ITS is inadequate to distinguish between all closely related species, and secondary markers including BenA, CaM, and RPB2 genes are often needed to identify isolates to species accurately (Visagie et al., 2014a). BenA offers accurate identification of Penicillium species as do CaM and RPB2 (Visagie et al., 2016). Furthermore, RPB2 contains almost no introns, making it robust and easy to align for phylogenies (Vetrovsky et al., 2016). However, the RPB2 gene is usually difficult to amplify, probably because the RPB2 gene sequence varies significantly among different fungal species, and thus, the universal primers contain some degenerate bases that reduce the specificity of PCR amplifications (Liu et al., 1999; Diao et al., 2019). To enhance the all-inclusiveness of the RPB2 database, it is possible to design primers with higher specificity targeting Penicillium and reduce the generation of non-specific amplification products. On the other hand, cloning of the amplification products and sequencing of the recombinant plasmids can be performed to obtain the target gene sequence (Zhou and Gomez-Sanchez, 2000).

In this study, we introduced two new species Penicillium acidogenicum and P. floccosum, belonging to sections Citrina and Osmophila, respectively, based on a polyphasic approach. The morphological characteristics of the new species and their closely related species are summarized in Table 3. Section Citrina members are frequently found in soil and have also been recovered from indoor environments and food products (Samson et al., 2010; Houbraken et al., 2011b). This shows that members of the group have a relatively wide range of habitats. Accurate species identification is crucial for section Citrina strains. Some members of this group produce mycotoxins citrinin, which is widely recognized as a harmful contaminant in food and feed (Houbraken et al., 2010; Gao et al., 2013). For example, Schmidt-Heydt et al. (2019) performed whole genome sequencing on P. citrinum DSM 1997 and revealed the biosynthesis gene cluster for citrinin. Section Osmophila currently includes three species, P. osmophilum, P. samsonianum, and the new species P. floccosum identified in this study. Species in this group have restricted colony growth with smooth-walled conidiophores and conidia (Stolk and Veenbaas-Rijks, 1974; Houbraken et al., 2016) and are mostly isolated from soil, while P. osmophilum is also isolated from the roots of the plant Colobanthus quitensis (Hereme et al., 2020).

Soil fungi, distinguished by their abundance and diversity, play a fundamental role in the ecosystem. A few Ascomycota taxa including Penicillium species dominate the soil fungal communities (Egidi et al., 2019). The discovery of two new species from the ancient Great Wall loess in Beijing predicts that there may still be a large number of undescribed species in special soil habitats. Therefore, using a polyphasic approach to study Penicillium from the soil will enrich the species diversity of the genus and provide more ideas and insights for us to understand the function of fungi in the ecosystem.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

Author contributions

RL: Data curation, Formal analysis, Investigation, Visualization, Writing – original draft. QY: Investigation, Visualization, Writing – original draft. YL: Investigation, Visualization, Writing – original draft. GZ: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Writing – review & editing. GY: Conceptualization, Methodology, Resources, Supervision, Validation, Writing – review & editing.

Funding Statement

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Survey project on alien invasive species and grassland pests in the Mentougou District (2022HXFWSWXY038), the Fundamental Research Funds for the Central Universities at Beijing Forestry University (2021ZY61), and the University–Industry Collaborative Education Program (202102083002).

Footnotes

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

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Associated Data

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Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

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