Three New Species of Penicillium from East and Northeast China

Penicillium species are ubiquitous in the environment and are of substantial importance, especially in industrial and medical aspects. During our investigation of the biodiversity of Penicillium, three new species were discovered in soil samples collected from East and Northeast China. They were determined as new to science based on morphological comparisons and phylogenetic analyses, and were found to belong to the subgenus Penicillium section Robsamsonia and subgenus Aspergilloides sections Aspergilloides and Citrina. Descriptions and illustrations of these species are provided, and their geographic distributions are also discussed.


Introduction
Species of Penicillium Link are ubiquitous.Some of them are of industrial and medical importance.A range of efficient plant-polysaccharide-degrading enzymes are secreted by Penicillium species, such as P. oxalicum Currie & Thom, and are very useful for sustainable bioproduction [1].Penicillium sumatraense Svilv.has the potential to be adopted in algal biorefinery processes for biofuel production because of its arsenal of degrading enzymes [2].Enzymes from Penicillium species are also crucial for the enhanced saccharification of agroindustrial wastes, which can be used to build a bio-based economy [3].In the medicinal field, the best-known antibiotic penicillin was produced by P. chrysogenum Thom [4].And more than 280 compounds have been reported from this genus, exhibiting lots of bioactive effects, e.g., antimicrobial, anticancer, antiviral, and antioxidant effects [5].On the other hand, P. digitatum (Pers.)Sacc. is not only a major source of postharvest decay in citrus fruits worldwide [6] but is also a rare human pathogen that can cause fatal pneumonia in immunocompromised hosts [7,8].
The genus Penicillium was established in 1809, and P. expansum Link was designated as the type species.It is the most speciose in the order Eurotiales.In a monography published in 2014, 354 species were accepted in this genus [9], and 483 species were recognized in one that was published in 2020 [10].By the end of 2022, 64 species had been further added to this group [11].In the last year, 54 new species were described, and 43 of them were discovered in Southwestern China [12].This leads to the species number of the genus over 600 at this moment.In China, more than 170 Penicillium species have been recorded, of which 91 were originally described from this country [12].
During an investigation into the biodiversity of Penicillium, three new species were discovered from the soil samples collected in East and Northeast China.Their descriptions and illustrations are provided here.

DNA Extraction, PCR Amplification, and Sequencing
DNA was extracted from the cultures grown on PDA for 7 days using the Plant Genomic DNA Kit (DP305, TIANGEN Biotech, Beijing, China).Polymerase chain reaction (PCR) amplifications of the internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) gene regions were conducted using the routine methods [9].The products were purified and subjected to sequencing on an ABI 3730 DNA Sequencer (Applied Biosystems, Foster, CA, USA).Although the ITS region, the proposed universal DNA barcode for fungi, is helpful to classify a Penicillium species at the section or series level, it is not sufficient to distinguish them at species level.However, ITS sequences are still provided here, as they might be beneficial to other researchers.

Phylogenetic Analyses
The forward and reverse sequences newly generated in this study were assembled using Seqman v. 7.1.0(DNASTAR Inc., Madison, WI, USA).The assembled sequences were deposited in GenBank.The sequences used for phylogenetic analyses are listed in Tables 1-3.Sequences of each of the three single gene datasets (BenA, CaM and RPB2) and the concatenated ones were aligned using MAFFT v. 7.221 [19], then manually edited and concatenated in BioEdit v. 7.1.10 [20] and MEGA v. 11.0.13[21].Maximum likelihood (ML) analyses were conducted using RAxML-HPC2 [22] on XSEDE 8.2.12 on CIPRES Science Gateway v. 3.3 [23] with the default GTRCAT model and bootstrap (BP) iteration setting.Bayesian inference (BI) analyses were performed with MrBayes v. 3.2.7 [24].Appropriate nucleotide substitution models and parameters were determined using Modeltest v. 3.7 [25].Four MCMC chains were run for at least 1 million generations, and posterior probability (PP) values were estimated with the remaining 75% of trees after a burn-in phase.The consensus trees were viewed in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 28 December 2023)).

Results
To determine the phylogenetic positions of the new species, single-gene datasets (BenA, CaM and RPB2) and concatenated ones were compiled and analyzed for Penicillium subgen.Penicillium sect.Robsamsonia ser.Robsamsonia, subgen.Aspergilloides sect.Aspergilloides ser.Glabra, and sect.Citrina ser.Sumatraensia.Detailed characteristics of the datasets are listed in Table 4. Phylogenies of Penicillium subgen.Penicillium sect.Robsamsonia ser.Robsamsonia are given in Figure 1.In the BenA and concatenated phylogenies (Figure 1A,D), the strain HLJ59-03 was closely related to P. coprobium with strong support (MLBP = 100% or BIPP = 1.00).Although HLJ59-03 was also a sister taxon of P. coprobium in the ML tree based on RPB2 sequences (Figure 1C), the statistical support between them was very weak.HLJ59-03 was absent in the CaM phylogeny (Figure 1B) because of the failure of PCR amplification.Phylogenies of Penicillium subgen.Aspergilloides sect.Aspergilloides ser.Glabra are shown in Figure 2. In the RPB2 and concatenated phylogenies (Figure 2C,D), strain SHL01-03 clustered with P. glabrum receiving strong support (MLBP = 100% or BIPP = 1.00).In contrast, SHL01-03 was a sister taxon of P. frequentans with weak support in the ML tree of BenA sequences (Figure 2A) and as an independent lineage in the CaM phylogeny (Figure 2B).Phylogenies of Penicillium subgen.Aspergilloides sect.Citrina ser.Sumatraensia were depicted in Figure 3. Strain SHL06-18 was shown as an independent lineage in the BenA and CaM phylogenies (Figure 3A,B), while it was clustered with P. sumatraense in the RPB2 analysis and the concatenated ML tree (Figure 3C,D).C, 7 days: Colonies nearly circular, plain, protuberant and funiculose at centers, slightly concentrically sulcate; margins narrow, entire; mycelia white; texture velutinous; sporulation dense; conidia en masse dull green; soluble pigments absent; exudates absent; reverse yellow brown, white at margins.
Notes: This species is closely related to P. frequentans (Figure 2A) and P. glabrum phylogenetically (Figure 2C,D).It differs from P. frequentans in 7 bp for BenA, 5 bp for CaM and 22 bp for RPB2; and from P. glabrum in 10 bp for BenA, 8 bp for CaM and 10 bp for RPB2.Morphologically, it differs from the above two species in being able to grow on CYA at 37 °C and having smooth-walled and slightly larger conidia (3.0-3.5 vs. 2.5-3.0 µm) [28,29].Their morphological distinctions are listed in Table 6.Notes: This species is closely related to P. frequentans (Figure 2A) and P. glabrum phylogenetically (Figure 2C,D).It differs from P. frequentans in 7 bp for BenA, 5 bp for CaM and 22 bp for RPB2; and from P. glabrum in 10 bp for BenA, 8 bp for CaM and 10 bp for RPB2.Morphologically, it differs from the above two species in being able to grow on CYA at 37 • C and having smooth-walled and slightly larger conidia (3.0-3.5 vs. 2.5-3.0 µm) [28,29].Their morphological distinctions are listed in Table 6.C, 7 days: Colonies nearly circular, slightly protuberant at centers, radially sulcate; margins wide, entire; mycelia white; texture velutinous; sporulation moderately dense; conidia en masse bluish green to dull green; soluble pigments absent; exudates abundant, clear; reverse carneous or flesh-colored.

Table 4 .
Detailed characteristics of the datasets.

of Seq. Length of Alignment (bp) No. of Variable Sites No. of Parsimony- Informative Sites Model for BI
Abbreviations of models: SYM+G (symmetrical model with Gamma distribution); TrNef+I+G (equal-frequency Tamura-Nei model with invariant sites and Gamma distribution).

Table 7 .
Morphological and molecular comparisons of new species and their closely related species in the series Sumatraensia.