New Xerophilic Species of Penicillium from Soil

Soil is one of the main reservoirs of fungi. The aim of this study was to study the richness of ascomycetes in a set of soil samples from Mexico and Spain. Fungi were isolated after 2% w/v phenol treatment of samples. In that way, several strains of the genus Penicillium were recovered. A phylogenetic analysis based on internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit 2 gene (rpb2) sequences showed that four of these strains had not been described before. Penicillium melanosporum produces monoverticillate conidiophores and brownish conidia covered by an ornate brown sheath. Penicillium michoacanense and Penicillium siccitolerans produce sclerotia, and their asexual morph is similar to species in the section Aspergilloides (despite all of them pertaining to section Lanata-Divaricata). P. michoacanense differs from P. siccitolerans in having thick-walled peridial cells (thin-walled in P. siccitolerans). Penicillium sexuale differs from Penicillium cryptum in the section Crypta because it does not produce an asexual morph. Its ascostromata have a peridium composed of thick-walled polygonal cells, and its ascospores are broadly lenticular with two equatorial ridges widely separated by a furrow. All four new species are xerophilic. Despite the genus Penicillium containing more than 480 known species, they are rarely reported as xerophilic.


Introduction
Soil is the natural reservoir of numerous organisms, such as algae, archaea, bacteria, fungi, protozoa, helminths, and arthropods, forming populations that are in a dynamic ecological balance. Many of these are responsible for degrading dead plants and animal remains to less complex molecules and contribute to the formation of humus and the maintenance of soil fertility [1]. Fungi grow in the space between soil particles, and nutrients necessary for their development are provided by organic matter and/or living roots. After early colonizers removed most of the soluble nutrients, fungi attack any remaining insoluble substrates by producing a broad spectrum of hydrolytic enzymes [2]. The products of enzymatic degradation are readily available, with various organisms competing for the same ecological niche. In addition, many of these fungi produce substances with antibiotic activity, which help them to compete more effectively for the scarcely available nutritional resources. Among them, species of the genus Penicillium are always present. The genus Penicillium was described by Link [3] in order to place three previously unknown fungi (Penicillium candidum, Penicillium glaucum, and Penicillium expansum, the latter corresponding to the type species of the genus). They typically produce brush-like structures (conidiophores) responsible for the formation of the asexual spores (conidia). At present, more than 480 species of Penicillium have been described [4,5] and more than 1300 records are available (Index of Fungi; http://www.indexfungorum.org/Names/Names.asp; 18 February 2021). Soil-borne Penicillium species are ubiquitous and present in a wide range of environmental conditions [4]. Several of them are extremophiles, meaning they can proliferate in low or high temperatures, in low pH values, and in high salt or sugar concentrations [6][7][8][9][10][11]. Only a few species have been reported as xerophilic, and consequently able to grow at a water activity (a w ) of 0.85 or below [12].
The aim of the present study was to resolve the taxonomic position and to identify several xerophilic isolates of Penicillium from soils in Spain and Mexico based on their morphology and a multilocus phylogenetic analysis (internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit 2 gene (rpb2)).

Sampling and Fungal Isolation
Soil samples were collected near the small villages of El Zapote (Michoacán state, Mexico) and Riaza (Castilla y León community, Spain). Riaza (41 • 16 59" N, 3 • 28 00" W) is at 1190 m above sea level (MASL), with a Mediterranean cold summer climate (according to the Köppen-Gieger climate classification), the average annual temperature is between 8 • C and 12 • C, the average annual rainfall is above 700 mm, and soils are based on metamorphic slate, quartzite, and schist, and covered with oaks (Quercus pyrenaica) trees. El Zapote (19 • 57 14.9" N, 101 • 38 34.8" W) is at 2100 MASL., with a dry-winter subtropical highland climate, the average annual temperature is 16-18 • C, the average annual rainfall is between 800 mm and 1000 mm, and soils are of extrusive volcanic origin, the vast majority of which are used to cultivate corn and sorghum. Samples from a horizon free from recognizable organic matter were placed into sterile plastic bags, which were then sealed. Once in the laboratory, the samples were stored at room temperature in the dark until they were processed. The methodology used for fungal isolation is described in Stchigel et al. [13]. Briefly, 1 g of soil was placed into a test tube, mixed with 5 mL 2% (w/v) phenol (Panreac, Barcelona, Spain) by shaking, and left to settle for 10 min. Then, the supernatant was discarded and the sediment was resuspended in 10 mL of sterilized water. The suspensions (1.6 mL) were poured into Petri dishes and mixed with 15 mL of molten (at 50-55 • C) sterile potato-carrot agar medium (PCA; potatoes, 20 g; carrot, 20 g, 1 L tap water). After jellification of the culture medium, Petri dishes were incubated at room temperature (22-25 • C) in the dark until 4-5 weeks. Cultures were examined periodically under a stereomicroscope and, when the formation of reproductive structures was observed, they were transferred to Petri dishes containing potato dextrose agar (PDA; Pronadisa, Madrid, Spain) [14] supplemented with L-chloramphenicol (100 mg/L), and incubated at 22-25 • C in the dark.

DNA Extraction, Amplification, and Sequencing
Total DNA was extracted directly from colonies on MEA after 7-10 days incubation at 25 • C in the dark, following the Fast DNA kit protocol (Bio 101, Inc., Vista, CA, USA) with the homogenization step repeated three times with a FastPrep FP120 instrument (Thermo Savant, Holbrook, NY, USA). After each homogenization, the sample was kept in ice for 10 min. DNA was quantified with GeneQuant pro (Amersham Pharmacia Biotech, Cambridge, England) [23]. Extracted DNA was used to amplify the internal transcribed spacer (ITS) (ITS5/ITS4 primers) [24], a fragment of the beta-tubulin (BenA) (T10/Bt2b primers) [25], a fragment of the calmodulin (CaM) (Cmd5/Cmd6 primers) [26] and a fragment of the RNA polymerase II subunit 2 gene (rpb2) (RPB2-5F/RPB2-7cR primers) [27]. The PCR amplifications were made in a total volume of 25 µL containing 5 µL 10x PCR Buffer (Invitrogen, CA, USA), 0.2 µM dNTPs, 0.5 µM of each primer, 1 U Taq DNA polymerase, and 1-10 ng nuclear DNA. PCR conditions for ITS, BenA, CaM, and rpb2 were set as follows: initial denaturation at 95 • C for 5 min, followed by 35 cycles of denaturation, annealing and extension, and a final extension step at 72 • C for 10 min. For ITS amplification, the 35 cycles were 45 s at 95 • C, 45 s at 53 • C, and 2 min at 72 • C; for the BenA region, 30 s at 95 • C, 1 min at 55 • C, and 90 s at 72 • C; for CaM region, 30 s at 94 • C, 1 min at 55 • C, and 90 s at 72 • C; and for the rpb2 region, 45 s at 95 • C, 1 min at 56 • C, and 90 s at 72 • C. Single-band PCR products were purified from agarose gels and sequenced at Macrogen Europe, which uses large-scale sequencing developed by "Applied Biosystem," which works using the Sanger method (Macrogen Inc., Madrid, Spain). Sequence assembly and editing were carried out using SeqMan software v. 7.0 (DNAStar Lasergene, Madison, WI, USA). GenBank accession numbers for the newly generated sequences in this study and others corresponding to reference or ex-type strains are listed in Supplementary Table S1.

Phylogenetic Analysis
The sequences of the four loci generated in this study were compared with those of the National Center for Biotechnology Information using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE= BlastSearch&LINK_LOC=blasthome; 10 July 2019). To determine the phylogenetic relationship of all isolates, a combination of ITS-BenA-CaM-rpb2 was built to distinguish among other species of Penicillium belong to the sections Alfrediorum, Crypta, Lanata-Divaricata, Lasseniorum, Oxalica, and Torulomyces ( Figure 1). Penicillium toxicarium NRRL 6172, Penicillium restrictum NRRL 1748, and Penicillium corylophilum CBS 330.79 (section Exilicaulis) were selected as outgroups. The sequence alignments and the maximum-likelihood (ML) and Bayesian inference (BI) phylogenetic analyses were performed as was described by Valenzuela-Lopez et al. [28]. The final matrices used for phylogenetic analyses were deposited in TreeBASE (www.treebase.org; accession number: 25066; 12 July 2019).

Figure 1.
Maximum-likelihood (ML) phylogenetic tree of Penicillium section Alfrediorum, Crypta, Lanata-Divaricata, Lasseniorum, Oxalica, and Torulomyces inferred from the combined internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit 2 gene (rpb2) loci. Support in nodes is indicated above thick branches and is represented by posterior probabilities (Bayesian inference (BI) analysis) of 0.95 and higher and/or bootstrap values (ML analysis) of 70% and higher. Some branches were shortened; these are indicated by two diagonal lines with the number of times a branch was shortened. Fully supported branched (100% BS/1 PP) are indicated in bold. T =ex-type strains. Alignment length 2249 bp. The sequences not generated by us were retrieved from EMBL/GenBank and are indicated in Supplementary Table S1. Maximum-likelihood (ML) phylogenetic tree of Penicillium section Alfrediorum, Crypta, Lanata-Divaricata, Lasseniorum, Oxalica, and Torulomyces inferred from the combined internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit 2 gene (rpb2) loci. Support in nodes is indicated above thick branches and is represented by posterior probabilities (Bayesian inference (BI) analysis) of 0.95 and higher and/or bootstrap values (ML analysis) of 70% and higher. Some branches were shortened; these are indicated by two diagonal lines with the number of times a branch was shortened. Fully supported branched (100% BS/1 PP) are indicated in bold. T = ex-type strains. Alignment length 2249 bp. The sequences not generated by us were retrieved from EMBL/GenBank and are indicated in Supplementary Table S1.

Molecular Phylogeny
The Blast search gave the following results: strain FMR  We carried out individual and combined phylogenetic analyses with ITS, BenA, CaM, and rpb2 sequences to resolve the taxonomical position of our strains using the sequences of type strains of the accepted species of Penicillium into the sections Lanata-Divaricata and Torulomyces. A concatenated dataset from 98 sequences contained a total of 2249 characters including gaps (575 of them for ITS, 444 for BenA, 476 for CaM, and 755 for rpb2), from which 966 were parsimony informative (114 for ITS, 257 for BenA, 310 for CaM, and 285 for rpb2). The sequence datasets did not show conflict in the tree topologies for the 70% reciprocal bootstrap trees, which allowed us to combine the four genes for the multi-locus analysis. The ML analysis showed similar tree topology and was congruent with that obtained in the Bayesian inference analysis. The phylogenetic tree ( Figure 1) was divided into six main clades representing the sections Alfrediorum, Crypta (100% BS/1 PP), Lanata-Divaricata (100% BS/1 PP), Lasseniorum, Oxalica (100% BS/1 PP), and Torulomyces (100% BS/1 PP). Three of our strains were placed into the section Lanata-Divaricata clade-FMR 17,424, which formed a sister branch of a terminal clade; FMR 17,381, which formed in another terminal clade containing P. meloforme CBS 445.74 (100% BS/1 PP); and FMR 17,612, which formed a terminal clade (100% BS/1 PP) with P. brefeldianum CBS 235.81 and P. limosum CBS 339.97. On the other hand, strain FMR 17,380 was located in the section Crypta, into a terminal clade (100% BS/1 PP), together with P. cryptum.

Taxonomy
Because FMR 17,424 forms a sister branch distant from the nearest terminal clade composed by FMR 17,381 and P. meloforme, and because FMR 17,381 differs phylogenetically and phenotypically from mentioned species, both strains are proposed as two new species of section Lanata-Divaricata as follows.
Notes: Penicillium meloforme and Penicillium siccitolerans sp. nov., which form a wellsupported terminal clade in our tree (Figure 1), are the species most phylogenetically related to P. melanosporum. Penicillium melanosporum differs from P. meloforme, because the former produces an asexual morph and lacks a sexual morph, while the second one forms a sexual morph and the asexual morph is only produced on MY70FG. Penicillium siccitolerans differs from P. melanosporum by the production of sclerotia. Penicillium melanosporum also produces shorter stipes than those of P. meloforme (15-70 × 2-3 µm versus 150-500 × 2-3 µm) [29], and bigger conidia (4-5 µm diameters) than those of P. meloforme (2-3 × 1-2.5 µm) and of P. siccitolerans (2-3 × 1-2.5 µm). Moreover, P. melanosporum differs from P. meloforme and P. siccitolerans by the production of a mucilaginous brown to dark brown exopigment surrounding the conidia, and because the last of the conidia produced remains attached to the phialide. Nevertheless, P. melanosporum and P. siccitolerans are capable to grow on CYA at 40 • C, while P. meloforme does not grow at 37 • C [29].
Etymology: From Latin siccus-, dry, and -tolerans, tolerance, due to the ability of this fungus to grow at a low water activity.
Culture characteristics (14 days at 25 °C): Colonies on CYA reaching 4-5 mm diameters, slightly raised, velvety, margins regular, white (4A1), exudates absent, sporulation absent; reverse yellowish-white (4A2), soluble pigment absent. On MEA reaching 21-22 mm diameters, slightly raised, velvety, margins regular, white (3A1), sporulation absent, Notes: Penicillium sexuale differs significantly from P. cryptum [31], the phylogenetically nearest species (see Figure 1), by a very a late production of ascospores into the ascostromata (after 2 months growing on PDA; after two weeks in P. cryptum), and because does not produce an asexual morph in any of the culture media tested.

Discussion
Xerophilic fungi are those able to grow at a water activity (a w ) of 0.85 or below [12]. The order Eurotiales, which contains the genus Penicillium, includes several xerophilic genera. Among them, one of the best characterized is Aspergillus. This genus has many species of xerophilic habit, mostly foodborne but also inhabitants of soil, able to grow at a w of up to 0.67 [32,33]. Other eurotialean xerophilic genera are Monascus [34][35][36], Talaromyces [10], and Xerochrysium [37]. However, the most xerophilic of the fungal taxa is Xeromyces bisporus, able to grow at a w between 0.61-0.62 [38].
There are a large number of published studies on xerophilic species and on the physiological mechanisms involved for the genus Aspergillus, but conversely very few studies on species of Penicillium. Our findings strongly suggest that more studies are needed for a better understanding of the diversity of extremophilic species of Penicillium, and the mechanisms involved in their adaptation to extreme environments.
Species of Penicillium living in soils, caves, and buildings, and causing food spoilage, such as P. brevicompactum, P. chrysogenum, P. cinnamopurpureum, P. implicatum, and P. janczewskii grow at a minimum a w of 0.78; Penicillium corylophilum, P. fellutanum, P. viridicatum, and P. verrucosum develop at a w as low 0.80; and Penicillium aurantiogriseum, P. citrinum, P. expansum, P. griseofulvum, and P. restricum grow at a w of 0.81-0.82 [12,[39][40][41][42][43][44]. As these species grow at a w lower than 0.85, all of them must be considered as xerophilic [43]. Very recently, Penicillium apimei, P. meliponae, and P. mellis have been described in honey produced by stingless bees in Brazil [10]. Despite these species were isolated from a sugar-rich substrate, with a w usually lower than 0.60, and being included in other recent taxonomic studies on Penicillium species, the ability to grow at low water activity was not tested.
In the present study, the multigene-based phylogeny (using ITS, BenA, CaM, and rpb2 sequences) allowed us initially to describe four new species of Penicillium isolated from soil samples collected from Spain and Mexico-P. melanosporum, P. michoacanense, P. siccitolerans of the section Lanata-Divaricata, and P. sexuale, of the section Crypta. All four Penicillium spp. were able to grow at a w of 0.76 (on MY70FG culture medium) formed colonies, thus demonstrating they were xerophilic. The asexual morph of P. melanosporum resembles those of P. brunneoconidiatum and P. tsitsikammaense [45] of the section Aspergilloides. However, the strongly ornamented dark brown conidia were only observed in the latter, produced by the phialides in P. melanosporum, all conidia being equally ornamented in the other two species. The asexual morph of P. michoacanense and P. siccitolerans is also reminiscent of the species of the genus within the section Aspergilloides [45] than those of the section Lanata-Divaricata. P. michoacanense and P. siccitolerans produce sclerotia, but these are made up of polygonal cells that are thick-walled in the former and thin-walled in latter. Finally, P. sexuale differs from P. cryptum because it does not form the typical asexual morph consisting in (mostly) conidiophores with solitary phialides, presenting only a sexual morph.

Conclusions
In the present study, we report the isolation of four new xerophilic species of the genus Penicillium, all of them isolated from soil samples. Three of these species were placed in the section Lanata-Divaricata (P. melanosporum, P. michoacanense, and P. siccitolerans), and the fourth in section Crypta (P. sexuale). Our study shows that there are metabolic groups still to be explored within the genus Penicillium, and that known species need to be characterized physiologically in depth, such as species of the genus Aspergillus.