Penicillium gercinae and Penicillium stangiae (Eurotiomycetes, Ascomycota), two new species from soil in Brazil

Two new Penicillium species are reported from soil of an agroforestry system and an Atlantic Rainforest fragment in Pernambuco, Northeast Brazil. Using a polyphasic approach, combining partial β -tubulin ( BenA ), calmodulin ( CaM ), internal transcribed spacer (ITS), and RNA polymerase II second largest subunit ( RPB2 ) gene sequences and morphological characters, Penicillium gercinae sp. nov. (section Ramigena , series Georgiensia ) , and Penicillium stangiae sp. nov. (section Lanata-Divaricata , series Dalearum ) are described. Descriptions based on morphological features are provided. Penicillium gercinae is phylogenetically closely related to P. georgiense ; however, P. gercinae does not grow on Czapek yeast extract (CYA) and has monoverticillate and biverticillate conidiophores. Penicillium stangiae is phylogenetically closely related to P. penarojense and P. vanderhammenii . As diagnostic features, P. stangiae had slower growth on CYA (28-30 mm in 7 days) and on DG18 (6-7 mm in 7 days) and lacked the production of acid on creatine agar. This research contributes to the expansion of knowledge on the taxonomy and diversity of Penicillium species in Brazil.


Introduction
Penicillium species can occur in a wide range of habitats and it is considered one of the most common genus of fungi able to grow on culture media (Visagie et al. 2016).In the last decade, species of this genus have been described from specific habitats or substrates (Houbraken et al. 2016; Penicillium has been extensively revised owing to new taxonomic insights (mainly based on molecular data) and the introduction of the single-name nomenclature system in fungi (Houbraken & Samson 2011;Visagie et al. 2014;Houbraken et al. 2020).In the most recent overview of Eurotiales, Penicillium species were allocated on 2 subgenera, 32 sections, and 89 series (Houbraken et al. 2020).
Penicillium subgenus Aspergilloides comprises species with conidiophores in which at least a portion of the stipes terminate in a vesicular swelling (Houbraken et al. 2011), with 19 sections, including Ramigena and Lanata-Divaricata.According to Houbraken et al. (2020), section Ramigena comprises two series, Georgiensia and Ramigena, and includes species in which monoverticillate conidiophores are evident; however, divaricate branching at various levels without an arrangement pattern is consistently observed.Section Lanata-Divaricata comprises five series, Dalearum, Janthinella, Oxalica, Rolfsiorum, and Simplicissima, and includes fast-growing species with large, spreading colonies (Houbraken et al. 2020).The species on this section form mostly strongly divaricate conidiophores, with terminal, subterminal and intercalate metulae.In the latter, there are intercalary monoverticillate conidiophores.In addition, the terminal cluster often consists of prolongation of the main axis (Houbraken et al. 2020).
The present describes two new Penicillium species from soils with different land use management systems in Brazil.

Study area
Soil samples were obtained from an Atlantic Rainforest fragment in Paudalho city, Pernambuco, Brazil (7°5712″S, 35°0626″W) (Fig. 1A).The vegetation is classified as dense ombrophilous forest, characterized by evergreen forest with a canopy of up to 15 m with emergent trees reaching 40 m in height, and dense shrub vegetation comprising arborescent ferns, bromeliads, and palms.Climbing plants, epiphytes (bromeliad and orchids), and ferns are also abundant (Alves et al. 2021).Soil samples were also collected from an agroforestry system (AFS) in the same city (7°5727″S, 35°0624″W) (Fig. 1B).This system is going through an agroecological transition process since 2013, Penicillium gercinae and Penicillium stangiae (Eurotiomycetes, Ascomycota), two new species from soil in Brazil with an unproductive sugarcane monoculture being replaced with fruit trees and crops (Alves et al. 2021).

Isolation
Soil samples were stored in plastic bags and processed as described by Barbosa et al. (2016).At the laboratory, 25 g of each soil sample was suspended in 225 mL of sterile distilled water and 1 mL of this suspension was transferred to a test tube containing 9 mL of sterile distilled water.This procedure was repeated until a 10 -³ dilution was achieved.An 1 mL aliquot of this final dilution was poured on Petri dishes with Sabouraud Agar (AS) (40 g L -1 dextrose, 10 g L -1 peptone, and 15 g L -1 agar, pH 5.5) with chloramphenicol (0.017 g mL -1 ) and Bengal rose (0.05 g L -1 ).Each dilution was conducted in triplicate.Petri dishes were incubated at 27 ± 2 °C for 10 days.After incubation, the colonies were purified until single colonies were obtained (Alves et al. 2021).Among the 100 fungal strains obtained from the soil, 25 belonged to Penicillium (Alves et al. 2021), and two of those were new species.The strains of the new species were deposited at the Micoteca URM Profa.Maria Auxiliadora Cavalcanti culture collection and the holotypes (slide preparation) at the Herbário Pe.Camille Torrend, both at the Federal University of Pernambuco, Recife, Brazil (Barbosa et al. 2020).

Morphologic analysis
The strains were inoculated at three points on culture plates containing Czapek yeast extract agar (CYA), CYA supplemented with 5 % NaCl (CYAS), yeast extract sucrose agar (YES), creatine agar (CREA), Czapek agar (CZ), dichloran 18 % glycerol agar (DG18), malt extract agar (MEA, Oxoid), and oatmeal agar (OA) (Samson et al. 2010).All strains were incubated at 25 °C for 7 days.Additional CYA and MEA plates were incubated at 15 °C, 30 °C, and 37 °C.Colony diameters were measured after 7 days of incubation, and colony characteristics (presence of soluble pigments, exudates, obverse and reverse colors, and color of mycelium) were recorded, Rayner (1970).Microscopic observations were made using a microscope Nikon Eclipse Ni equipped with a photo camera Nikon Digital Sight DSFi2.Images were captured using software NIS-elements software (Nikon) and converted to format Tagged Image File Format (tiff) to preserve high definition graphics for color raster images.These observations were performed for colonies grown on MEA, and the presence of a sexual stage was investigated in CYA, MEA and OA cultures maintained at 25 °C for at least 40 days.Lactic acid (80 %) was used for mounting, and 96 % ethanol was used to remove excess conidia (Barbosa et al. 2018).

Phylogenetic analyses
DNA extraction was performed using the DNA Wizard Genomic DNA Purification Kit, following the manufacturer's recommendations.The β-tubulin (BenA), calmodulin (CaM), internal transcribed spacer (ITS), and RNA polymerase II second largest subunit (RPB2) gene regions were amplified using the primers Bt2a and Bt2b, CMD5 and CMD6, ITS1 and ITS4, and RPB2-5F and RPB2-7CR, respectively (Houbraken et al. 2019), with PCR conditions as described by Visagie et al. (2014).The PCR products were purified using the Exosap illustrative enzyme ExoProStar™ 1-Step (GE Healthcare Life Sciences, Little Chalfont, UK) and sequenced at the LABCEN/CCB sequencing platform at the UFPE (Recife, Brazil) using the same primers.The electropherograms were analyzed using Sequencher 4.7 (Gene Codes, Ann Arbor, MI, USA), from which the consensus nucleotide sequences were obtained and exported as FASTA files.

DNA sequence alignment and phylogenetic analyses
The sequences obtained were aligned with sequences of Penicillium section Ramigena and section Lanata-Divaricata available in the NCBI database, through alignment in the MAFFT v.7 online interface (Katoh & Standley 2013) (http:// mafft.cbrc.jp/alignment/server/)and manual adjustment.For section Lanata-Divaricata, P. glabrum and P. oxalicum were used as outgroups; for section Ramigena, Penicillium sections Charlesia and Eremophila were used as outgroups (Visagie et al. 2016).The generated DNA sequences were deposited in GenBank under the accession numbers listed in Table 1.The phylogenies from the BenA, CaM, ITS, and RPB2 sequences were analyzed both separately and combined.MrBayes 3.2.6 (Ronquist et al. 2012) was used to generate phylogenies based on Bayesian inference with 5000000 generations, a sampling frequency of 1000 generations, and a burn-in fraction of 25 %.The best-fit models of nucleotide substitution, implementing the Bayesian information criterion, were estimated separately for each gene region using MrModeltest 2.3 (Nylander 2004).Phylogenetic reconstructions using maximum likelihood analysis were performed on the CIPRES Science Gateway portal (Miller et al. 2010) using RAxML-HPC2 on XSEDE (8.2.10) with default parameters (Stamatakis 2014).Phylogenetic trees were plotted using FigTree 1.4.3 (Rambaut 2009).The phylogenetic relationship between the strains and the accepted species of sections Ramigena and Lanata-Divaricata were determined by analysis of single and concatenated sequence datasets of four loci (BenA, CaM, ITS, and RPB2).
Strains URM 6020 and URM 8347 of this species formed a well-supported clade with P. vanderhammenii and P. penarojense in the BenA (1 pp, 100 % bs), ITS (0.99 pp, 89 % bs) (Fig. 4) and the combined phylogeny (1 pp, 98 % bs) (Fig. 5).Analysis of CaM and RPB2 sequences could not resolve the phylogenetic position of Penicillium stangiae sp.nov. in the same clade with P. vanderhammenii and P. penarojense.In the CaM phylogeny (1 pp, 100 % bs), URM 6020 and URM 8347 took a basal position to a large clade that also included P. vanderhammenii and P. penarojense (Fig. 4) and, in the RPB2 phylogeny (1 pp, 99 %), URM 8347 took a basal position to a large clade that included P. vanderhammenii and P. penarojense (Fig. 4).An overview of the length of each dataset and the most optimal substitution model for each dataset is presented in Table 2.

Discussion
Several new species of Penicillium were described in Brazil recently, some of them isolated from soil (Crous et al. 2014;Taniwaki et al. 2015;Crous et al. 2019;Ramos et al. 2021).We introduce P. gercinae in the section Ramigena and P. stangiae in the section Lanata-Divaricata, both species from soil in the Atlantic Forest environment.
The new species P. gercinae belongs to the series Georgiensia and produces monoverticillate and biverticillate conidiophores, indicating a relationship with other taxa of the small section Ramigena.A sexual state was not observed in the new species described in the present study.P. gercinae is phylogenetically close to P. georgiense, but it does not grow on CYA nor produce acid on CREA, and has globose conidia.P. georgiense is no longer a sole, basal species in section Charlesia clade (Sun et al. 2021), being actually supported in clade of section Ramigena (Houbraken et al. 2020) and, until now, form the series Georgiensia with P. gercinae.
Penicillium stangiae belongs to this series Dalearum in the section Lanata-Divaricata, and produces monoverticillate, biverticillate, and divaricate conidiophores, colonies in shades of green, smooth walled, globose to subglobose conidia.Sexual morphs were not observed in cultures.Several features observed in new species are in compliance with the characteristics features for this series.As in Houbraken et al. (2020), the series Dalearum has species with moderately fast growing colonies, conidial color en masse dull green, bluish green to gray-green; conidiophores monoverticillate, biverticillate, and divaricate, occasionally terverticillate, conidia variable shaped, and variable growth at 37 °C.
The present study is the first report of a new species in section Ramigena, found in the soil of an agroforestry system in Northeast Brazil.Most species in section Ramigena have been isolated from organic substrates (soil, leaf litter, and wood) (Tab.1).It is noteworthy that agroecological transition areas contain microorganisms that favor the use of carbon sources in the soil, thus making this nutrient available to plants during the agroecological transitional process, promoting soil fertility (Hecht 2018).In the present study, we report for the first time, in the soil of the Atlantic Rainforest fragment in Northeast Brazil, a new species in section Lanata-Divaricata series Dalearum.Species in this section have an affinity for acidic soils and are acidtolerant (Diao et al. 2018;Alves et al. 2021).In response to phosphorus deficiency in soil and recalcitrant lignin-rich organic matter, fungi can release organic acids and enzymes for nutrient acquisition, developing the rhizosphere and enzymatic processes that promote tolerance to low pH in plants (Deepa et al. 2010;Fujii 2014).The results of the current study contribute to the taxonomic knowledge of Penicillium at soil of different land use.

Figure 1 .
Figure 1.Locations of the soil samples collected in the Atlantic Rainforest (A) and in the agroforestry system (B) for isolation of Penicillium specimens.

Figure 3 .Figure 4 .
Figure 3. Phylogenetic position of the new Penicillium species in the series Georgiensia (section Ramigena) based on a combined dataset containing β-tubulin (BenA), calmodulin (CaM), internal transcribed spacer (ITS), and RNA polymerase II second largest subunit (RPB2) sequences.The new species P. gercinae sp.nov.(URM 8383 and URM 8348 T) are highlighted in bold.T = Type.

Table 1 .
Sequences of strains obtained by NCBI (National Center for Biotechnology Information) used for phylogenetic analyses of Penicillium section Lanata-Divaricata with emphasis on the series Dalearum and section Ramigena.

Table 2 .
Length (bp), variable positions (pb and %) in the alignments, and nucleotide substitution models used in phylogenetic analyses.