Brazilian Atlantic Forest and Pampa Biomes in the spotlight: an overview of Aspergillus , Penicillium , and Talaromyces ( Eurotiales ) species and the description of Penicillium nordestinense sp. nov.

The knowledge of the geographical distribution of fungi is essential to promote the understanding of global and national fungal diversity patterns. In this study, we provide a literature-based checklist of Aspergillus , Penicillium , and Talaromyces species recorded in the biomes of the Brazilian Atlantic Forest and the Brazilian Pampa. Only Penicillium digitatum was reported for the Pampa biome. For the Atlantic Forest, a total of 169 species (68 Aspergillus , 79 Penicillium


Introduction
The Atlantic Forest is the second largest tropical forest in South America, and one of the most diverse on the planet, containing over 2,960 tree species (Zwiener et al. 2020), 2,645 tetrapoda species (Figueiredo et al. 2020), and 1,401 species of social insects (Feitosa et al. 2020).This rainforest harbors a high level of endemic species of the global fauna and flora (Myers et al. 2000;Grelle et al. 2021).The Atlantic Forest is a biome that, which currently covers approximately 15 % of the Brazilian territory, and is a place for approximately 72 % of the Brazilian population (SOS Mata Atlântica 2017).The Atlantic Forest is considered a global conservation hotspot because of its highincidence of endemic species, and high rate of habitat loss because of anthropogenic pressure (solely 12.4 % of the forest that originally existed still subsists) (CIB 2000;Myers et al. 2000;SOS Mata Atlântica 2021).
The degradation of the Atlantic Forest is one of the most alarming conservation challenges in the world.The forest destruction for agriculture and biofuels, charcoal market, deforestation for cattle ranching, and wild animal hunting has resulted in the elimination of several species and has potentially contributed to the erosion of the genetic diversity of species (Brown & Brown 1992;Ferrer-Paris et al. 2019;Wilson et al. 2021).It is logical to suppose that several species have already been extinct before their discovery and several others will be extinct soon (Morellato & Haddad 2000).Owing to its high level of endemism and diversity in numerous groups of organisms, the Atlantic Forest has been recognized as a top priority for conservation in South America (Klumpp et al. 1996;Myers et al. 2000;Marques et al. 2020).
The Pampa biome covers an area of approximately 700,000 km2, extending across the territories of Argentina, Brazil, Uruguay, and Paraguay (IBGE 2004).The Brazilian Pampa is the second smallest biome in the country, occupying an area of 176,496 km² (2.10 % of the national territory), present solely in the state of Rio Grande do Sul.The Pampas are in the southern temperate zone, with well-defined seasons (Roesch et al. 2009), having a subtropical predominantly rainy climate, with no systematic dry period and negative temperatures in winter (IBGE 2019).The dense forest in this biome is not very expressive, and predominantly consists of herbaceous and shrub type vegetation (Roesch et al. 2009).In the Brazilian Pampa, 23 % of its territory is composed of pastures (BRASIL 2010), facilitating the implementation of agricultural activities.Compared to other Brazilian biomes, the Pampa has a high percentage of agricultural land use and it has a small number of conservation units (Ferreira et al. 2012).Human activities like cultivation of exotic crops for commercial purposes and changes in the ecosystem for agricultural activities have aggravated the deforestation of Brazilian Pampa (Sulzbacher et al. 2018).It is estimated that approximately 48.70 % of this biome has already been modified by human activity, leaving approximately 5 % of native forest (BRASIL 2010).
Fungi are crucial in ecosystems, acting as decomposers of organic material, sometimes pathogens, and mutualistic partners of approximately all terrestrial multicellular organisms (Heilmann-Clausen et al. 2015).For example, lichenized fungi mediate most primary production and nitrogen fixation in desert and polar ecosystems (Honegger 2012); mycorrhizal fungi are important for nutrient cycling, for mineral weathering and carbon storage in forest ecosystems (Courty et al. 2010); fungal endophytes are mutualistic and prevent damage against biotic and abiotic factors (Bamisile et al. 2018).Despite these, the importance of fungi in the maintenance and quality of life on the planet is rarely considered in the conservation actions (Heilmann-Clausen et al. 2015).In Brazil, the knowledge of the fungal biodiversity has been a way to strengthen the importance of the protection and conservation of biomes (Sette et al. 2013).Biodiversity checklists are an important step in providing relevant information for the conservation planning and service of the environment (Piepenbring et al. 2020).A checklist of fungal species in tropical forests is particularly important for the comparison of regions, enabling identification of threatened species and their habitats, and providing data for ecological/ biogeographical predictive modeling of species in local and global conservation strategies (Barbosa et al. 2020).
In 2015, the Brazilian fungal diversity was estimated to have approximately 5,719 species, distributed over 1,246 genera of the 102 orders, with the Atlantic Forest having the largest number of records (approximately 3,017 species) and Pampa with 84 species (Maia et al. 2015).However, this estimation was mainly based on the data made available by mycologists in the database of the Flora do Brasil -Algas, Fungos e Plantas project (http://floradobrasil.jbrj.gov.br/),making the Brazilian fungal diversity still underestimated.For example, endophytic and bee-related fungi were not considered by Maia et al. (2015).A few other lists of fungi from Brazil have been recently published, revealing a remarkable fungal diversity, which is still found in the country (e.g., Bezerra et al. 2019;Barbosa et al. 2020;Chikowski et al. 2020).
Following the extensive overview of Aspergillus, Penicillium and Talaromyces species in the Caatinga forest published by Barbosa et al. (2020), this study aimed to summarize the records of Aspergillus, Penicillium, and Talaromyces species/names in the Atlantic Forest and Pampas biome by presenting a current list of valid species names, their substrate and distribution.In addition, we present the description of Penicillium nordestinense sp.nov.from and creatine sucrose agar (CREA).Media preparation, inoculation, incubation, and microscope preparations were conducted as described by Samson et al. (2010).Additional CYA and MEA plates were incubated at 15, 30, and 37 °C.Colony diameters were measured after 7 days of incubation, and colony characteristics were recorded.Microscopic observations were made from colonies grown on MEA, and the presence of a sexual stage was investigated in cultures incubated on CYA, MEA, and OA, and the cultures were incubated for at least 40 days at 25 °C.Lactic acid (60 %) was used as a mounting fluid, and 96 % ethanol was used to remove excess conidia.Color names and alphanumeric codes used in descriptions refer to Rayner (1970).Features of the novel species were compared to their closest relatives based on notes provided in previous studies.
Genomic DNA extractions were made from 7-day-old colonies grown on MEA using the Promega DNA isolation kit (Wizard Genomic DNA Purification Kit).A polymerase chain reaction (PCR) amplification of the ITS barcode (ITS1, 5.8S rDNA, and ITS2), tubulin (BenA), calmodulin (CaM), and RNA polymerase II, the second largest subunit (RPB2) genes, was performed using the methods described by Visagie et al. (2014).PCR products were purified using the Exonuclease/ Alkaline Phosphatase mix (Cellco Biotec.), and sequenced on the sequencing platform at UFPE (Recife, Brazil) using the same primer pairs.For phylogenetic analysis, sequence datasets were generated by combining the newly generated sequences with reference (ex-type) sequences from previous studies deposited in the nucleotide database at NCBI (GenBank).The sequences were aligned using MAFFT v.7 (Katoh & Standley 2013), and manually optimized using MEGA v. 6.06 (Tamura et al. 2013).Initially, the positioning of the new species in section Lanata-Divaricata was analyzed using a concatenated dataset with BenA and CaM sequences.After this initial analysis, more comprehensive ITS, BenA, CaM, and RPB2 sequence datasets for series Janthinella were generated and analyzed.The combined datasets for section Lanata-Divaricata and series Janthinella were made by concatenating the individual alignments using Mesquite v. 3.04 (Maddison & Maddison 2016).Phylogenetic trees were constructed by maximum likelihood analyses (ML) using RAxML-HPC v. 8.2.8 (Stamatakis 2014) BlackBox with 1,000 rapid bootstrap inferences via the CIPRES science gateway (http://www.phylo.org/)(Miller et al. 2012), adopting default parameters.Bayesian inference (BI) analysis was performed in MrBayes 3.2.2(Ronquist et al. 2012).In the Bayesian analyses, every 1,000 generations were sampled, and the first 25 % of the samples were discarded.The most suitable substitution model was determined separately for each gene region using jModelTest v. 2.1.7(Posada 2008).Trees were visualized in FigTree v. 1.1.2(Rambaut 2016), and edited in Adobe Illustrator v. 5.1.BI posterior probabilities (pp) and bootstrap (bs) values were labelled at the nodes.Branches with full support in BI and ML analyses were thickened.Values below 0.95 pp and 70 % bs support were not shown, or they were indicated with a hyphen.

Taxonomy
The new species P. nordestinense is described below and the phylogenetically closely related species P. setosum is validated.
MycoBank MB 842080 Etymology: "nordestinense", referring to the region Northeast of Brazil (Nordeste, in Portuguese).In this region is the state of Pernambuco, where the type species was isolated.
Notes: Penicillium setosum is herewith validated, as the original description did not state that the holotype was preserved as a metabolically inactive culture.

Discussion
Tropical regions are typically the most diverse in plants and animals species (Brown 2014;Raven et al. 2020), as well as a broad variety of interactions between them (Brown 2014).However, regarding fungi, there is a deficit of data in the literature that estimate the diversity of fungi in the tropics (Hawksworth 2001;Hawksworth & Lücking 2017).Our knowledge about fungal diversity and their roles in tropical ecosystems is still incomplete (Aime & Brearley 2012).Species checklists are essential tools to provide information for ecological studies, biodiversity assessments, conservation reports, and public politics related to biodiversity conservation.A few initiatives are helpful aids for compiling lists of fungal species, such as the MycoBank (https://www.mycobank.org/),Index Fungorum The soil is the most frequently reported source of Aspergillus, Penicillium, and Talaromyces in the Atlantic Forest, which was also reported by Barbosa et al. (2020) in a recent checklist of fungi in the Caatinga forest in Brazil.Other substrates with significant occurrence of species were water and leaf litter.Fungi are present and prominent in all soils, and their fungal communities may have an extreme species richness (Taylor & Sinsabaugh 2015).Those fungi are dispersed through mycelial growth and more rapidly over larger distances via spores, which are dispersed principally by wind (Taylor & Sinsabaugh 2015), but also through insects and other animals (Magyar et al. 2016).Owing to their ability to produce a wide variety of extracellular enzymes, fungi are able to break down all types of organic matter and decompose soil components, thereby regulating the balance of carbon and nutrients (Žifčáková et al. 2016).The fungal diversity can be affected by soil and plant properties, providing evidence for strong links among soil fungal diversity and plant and soil properties (Yang et al. 2017).Most fungi consume living or dead plant materials as their primary nutrients source, and a large number of fungi display some degree of specialization toward their living or dead plant substrates.Thus, plant community composition plays a dominant role in determining the fungal community in an environment (Taylor & Sinsabaugh 2015).
In Brazil, the state of Pernambuco has been historically highlighted in mycological studies because of the existence of the former Institute of Mycology, of the University of Recife (currently the Departamento de Micologia at the Universidade Federal de Pernambuco), which was founded by Augusto Chaves Batista (Bezerra et al. 2017).The mycological effort in this region explains the reason for the highest number of fungal records because since 1954, several fungal surveys were developed, and their focus was mainly on the soil (Barbosa et al. 2020).
Aspergillus had the highest species richness in the Atlantic Forest.Species of Aspergillus are found across the world and might be more dominant in regions with tropical and subtropical climates (Klich 2002).A few species, owing to the production of important metabolites such antibiotics, mycotoxins, enzymes, organic acids, and phenolic compounds, are used in several biotechnological processes (Dagenais & Keller 2009).Aspergillus species belonging to sections Nidulantes and Nigri were the most frequently reported.Aspergillus nidulans is the most common section Nidulantes species reported in the Atlantic Forest, and produces, like other section Nidulantes species, typical biseriate conidiophores with pale brown pigmented stipes, and when present, the ascomata embedded in masses of Hülle cells (Chen et al. 2016).A total of 11 species in this section are reported in our checklist.Members of Aspergillus section Nidulantes are widely distributed in nature and may play significant roles in decomposition processes (Raper & Fennell 1965).The section Nigri comprises several species important to agricultural and food production, human and other animal health, and occur frequently throughout the world.The taxonomy of section Nigri is confusing and complex because of subtle differences between the species.A few species, such as A. carbonarius, A. japonicus, and A. aculeatus, can be easily recognized using morphologic criteria; however, species related to the A. niger (series Nigri) are difficult to distinguish using morphologic criteria (Samson et al. 2004).Thus, the polyphasic taxonomy has been necessary for accurate identification, as well as description of novel species, for Aspergillus, as well as Penicillium and Talaromyces.
Penicillium is one of the most common and diverse fungal genera, and according to the last list of accepted species, includes 483 species (Houbraken et al. 2020).Several Penicillium species are economically important as sources of antibiotics, organic acids, enzymes, pharmaceuticals, and several other metabolites; however, a few of them cause food spoilage, produce mycotoxins, and cause human and other animal diseases (Pitt 1994;Frisvad et al. 2004).Members of the Penicillium section, Lanata-Divaricata and Sclerotiorum are abundant in the Brazilian Atlantic Forest.Penicillium section Sclerotiorum was introduced by Houbraken & Samson (2011), and most of the species in this group share the production of yellow to orange mycelia and have a reverse orange or reddish colony and bright-colored sclerotia (Visagie et al. 2013).Recently, P. barbosae and P. limae isolated from sugarcane cultivation soils in Atlantic Forest in northeastern Brazil were described as new in the section Sclerotiorum, series Adametziorum (Ramos et al. 2021).The novel species, P. nordestinense, described in this study belongs to section Lanata-Divaricata, series Janthinella.Species of this section are common in soil (Diao et al. 2018) and frequently found on rotting leaf litter (Houbraken et al. 2011).Species identification in this section is difficult, and a DNA-based approach using BenA sequencing, if needed supplemented with a CaM sequence, is suggested for reliable results (Visagie et al. 2015).Previous studies reported the occurrence of section Lanata-Divaricata species as endophytes (P.alagoense, Crous et al. 2019a;P. brasilianum, P. cluniae and P. echinulonalgiovense, Freire et al. 2020) and in honey, bee pollen, and nests of M. scutellaris (Barbosa et al. 2018); however, the ecological significance remains unknown.
The  (Sun et al. 2020).In our checklist, we found species belonging to sections Helici, Islandici, Talaromyces, and Trachyspermi.Species classified in section Talaromyces were commonly found occurring in the Atlantic Forest.This is the largest section of the genus Talaromyces, and it includes 72 species (Houbraken et al. 2020).Members of this section exhibit a great diversity in morphological characters and were isolated from a diverse range of substrates (e.g., Jiang et al. 2018;Barbosa et al. 2018;Wei et al. 2021).Although few members of Talaromyces were reported as opportunistic human pathogens, such T. marneffei, most of them are producers of anticancer, antibacterial, antifungal, antiproliferative, and antioxidative compounds; several enzymes; and natural pigments (Narikawa et al. 2000;Bladt et al. 2013;Frisvad et al. 2013;Maeda et al. 2013;Zhai et al. 2016;Kumari et al. 2018;Lian et al. 2018;Nicoletti et al. 2018;Xu et al. 2018).
The correct identification and description of new species must follow standardized methodologies to make species identifications across laboratories more consistent (e.g., Samson et al. 2010;Houbraken et al. 2011;Visagie et al. 2014;Yilmaz et al. 2014;Frisvad et al. 2019).All guidelines were based on approaches, including morphology, multigene phylogenies, physiology, and extrolite data.Currently, few studies in Brazilian Atlantic Forest have adopted this approach for species identification (e.g., Fungaro et al. 2017;Barbosa et al. 2018;Barros-Correia et al. 2020) and previous studies that did not use polyphasic methods could have misidentified species in very related and complex groups.We also reinforced the importance of preserving strains in a public reference fungal culture collection (e.g., URM culture collection in Brazil (Micoteca URM Profa.Maria Auxiliadora Cavalcanti -https://www.ufpe.br/micoteca)(Barbosa et al. 2020).
Although the Atlantic Forest is one of the world's top biodiversity hotspots (Rezende et al. 2018) with a diversified mosaic of habitats that harbors several species, there is a gap between species estimates and the number of species discovered.Only 19 names (5 Aspergillus, 10 Penicillium, and 4 Talaromyces) are described on and typified with specimens from the Atlantic Forest.Most of these were isolated from soil and substrates related to stingless bees.This fact is considerably insufficient, considering that we have investigated literature published over 65 years (1955 to 2020).The question "how many Aspergillus, Penicillium, and Talaromyces species there are in Brazil?" is indisputably important to mention here through the vast importance and applicability of these fungal species.Currently, only a few research groups focus on studying the diversity of these fungi in Brazil because of several technical limitations, e.g., several groups are still unable to apply modern identification techniques.In addition, there are very few taxonomy experts and enthusiasts studying Aspergillus, Penicillium, and Talaromyces taxonomy in Brazil (R.N.Barbosa -personal communication).This can be seen, for example, in the scarcity of studies in the Pampa biome.Among the six studies that were analyzed, only one isolate was identified at the species level.
Fungi are the second most species-rich organism group after insects (Purvis & Hector 2000).In this way, it is challenging to conduct fungal inventory, as they are still scarce when compared to other organisms such as plants and animals.Although the fungal diversity has been extensively investigated, and the development of molecular phylogeny has revealed an unexpected fungal diversity with an increased number of novel taxa, it is still necessary to increase and incentivize the development of researches including collection and preservation of specimens in fungal herbaria and culture collections.In addition, the survey and correct use of fungal diversity is relevant to several industry segments (e.g., agriculture, pharmacology, food, and biotechnology).

Conclusions
This checklist indicates that the number of mycological studies for prospective Aspergillus, Penicillium, and Talaromyces diversity in Brazilian Atlantic Forest and Pampa biomes is still scarce and extremely necessary.The soil is the most cited substrate for these species, the conditions of the soil environment are optimal for their growth, mainly because of the abundance of leaf litter and other compounds.Despite the degraded state of the Atlantic Forest and Pampa, the biological potential of several of them still lacks a basic understanding, highlighting the importance of conducting inventories.To date, few species are described from these biomes, and this reflects the importance of increasing the number of mycologists, in particular, specialists in taxonomy.Our data provide a framework to study the biogeography of Aspergillus, Penicillium, and Talaromyces species in tropical environments, and contribute to a better understanding of Brazilian fungal diversity.
Brazilian Atlantic Forest and Pampa Biomes in spotlight: an overview of Aspergillus, Penicillium, and Talaromyces (Eurotiales) species and the description of Penicillium nordestinense sp.nov.

Figure 1 .
Figure 1.Geographical location of the Brazilian Atlantic Forest and Pampa biomes.

Figure 3 .Figure 4 .
Figure 3. Phylogenetic position of Penicillium section Lanata-Divaricata strains and the new species P. nordestinense based on a combined dataset containing ITS, BenA, CaM and RPB2 sequences.The new species is highlighted.Values below 0.95 pp and 70 % are not shown and indicated with a hyphen.
genus Talaromyces was introduced by Benjamin (1955) to accommodate a group of sexually reproducing Penicillium species.In 2011, Samson et al. re-evaluated the genus and transferred most species of Penicillium belonging to the subgenus Biverticillium to Talaromyces.In the monographic treatment of the genus by Yilmaz et al. (2014), 88 species were accepted and these were classified in seven sections: Bacillispori, Helici, Islandici, Purpurei, Subinflati, Talaromyces, and Trachyspermi.Six years later, a new section named Tenues was proposed published between 1955 and 2020 reported members of Eurotiales, and were included in this checklist.In total, 169 species (68 Aspergillus, 79 Penicillium, and 22 Talaromyces) have been recorded in the Atlantic Forest.Penicillium digitatum was the only reported species for the Pampa biome.In Aspergillus, the reported species are distributed over 17 sections, with section Nidulantes and Nigri species being the most common.In Penicillium, species classified in 16 sections are listed, they mainly belong to sections Lanata-Divaricata and Sclerotiorum.Regarding the genus Talaromyces, four sections have been recorded, with Talaromyces being the most common.According to our survey, 19 species were typified in previous studies with material from the Atlantic Forest biome: A. barbosae, A. bezerrae, A. brasiliensis, A. labruscus, A. recifensis, P. apimei, P. barbosae, P. brasilianum, P. fernandes, P. guaibinense, P. limae, P. meliponae, P. mellis, P. reconvexovelosoi, P. restingae, T. brasiliensis, T. mycothecae, T. Pernambucoensis, and T. pigmentosus.In addition, Penicillium nordestinense is described here as a new species, see "Taxonomy" section.Regarding the distribution of records by Brazilian states, Pernambuco has the highest number of records (43 Aspergillus spp., 67 Penicillium spp., 20 Talaromyces spp.), followed by São Paulo (27 Aspergillus spp., 29 Penicillium spp., 11 Talaromyces spp.), and Rio de Janeiro (22 Aspergillus spp., 27 Penicillium spp., 7 Talaromyces spp.).