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

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.


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based on morphological characteristics in the past 100 years, whereas this phenotype-based sectional classification has been replaced by a system based on a multigene phylogeny in recent decades (Visagie et al., 2014a;Houbraken et al., 2016Houbraken et al., , 2020)).Subgenera, sections, and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 quarterverticillate 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.

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).

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).

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).

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 TreeBASE 2 with the submission number 30834.

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 3.25421 T and CGMCC 3.25422 T ).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.

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).

3.3.2
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).

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.

FIGURE 1 ML
FIGURE 1 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 ML
FIGURE 2 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.

FIGURE 3 ML
FIGURE 3 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 ML
FIGURE 4 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.

TABLE 1
Strains of the genus Penicillium used for phylogenetic analyses.

TABLE 2
Length and substitution models for each dataset used in phylogeny.

TABLE 3
Morphological features for new species and their closely related species.