Small reserves as hotspots for Fungi preservation in Brazil

Small conservation areas have been created in many countries usually to protect plants and animals, but no priorities are deserved in protecting fungi. The creation of preservation areas is meeting a new problem: there are only small remaining areas, since exploration is destroying large forests. Applying the technique of the transects in three different areas in Brazil (Pampa and Cerrado biomes and Amazonian Forest), diversity and ecological data were collected for Agaricomycetes fungi, mainly order Agaricales, and compared to the fungi already known and their conservation status to understand how fungi are protected indirectly with the creation of small protected areas. The samples were collected in differents permanent protected areas (APP – permanent protected areas) in river margins in southern Brazil (RPAS), Saint Hilaire Forest in the Universidade de Goiás (MSH - ca. 20 ha) in Goiânia/GO and in the Universidade Federal do Amazonas Protected Forest (Manaus/AM - MUFAM), all located in Brazil. In MUFAM, 140 specimens were collected in 2014 and 2018 resulting in 47 species. In MSH 86 specimens were collected in 2019 and 2020 with 31 species identified. In RPAS 278 new species records for the Brazilian Pampa biome were cataloged, 23 new records for the state of Rio Grande do Sul and 4 new registrations for Brazil. This demonstrates the importance of small forest fragments to fungi preservation and maintenance, being efforts in that direction important in future studies of new conservation areas implementation.


Introduction
Even though small reserves are extremely common around the world, studies on their conservational value are disproportionally uncommon in scientific works, which usually focus on defining their value relative to one large reserve, to one or two taxa or ecosystem types (Volenec & Dobson, 2019). Despite many efforts being done in Europe and North America to preserve fungi biodiversity, few initiatives are under course in South America, except for creation of preservation areas but without referring to fungi species. Most European countries have generated red lists for fungi but this is not true for countries in Latin America (Senn-Irlet et al., 2007).
The lack of knowledge is one of the major problems relating to fungi preservation, especially in South America, where countries with high biodiversity numbers like Brazil and Colombia, which have incomplete fungal species lists, are still beginning to study the complex ecological role of this group (Putzke & Putzke 2017;Putzke et al. 2020Putzke et al. , 2021. But it is also a problem to all the world since only 56 species of fungi have their status evaluated globally by the UCN red list (Hawksworth et al. 2017).
Habitat reduction and pollution are considered the most important causes of worldwide loss of biodiversity, being the creation of small reserves also of huge importance (Serengil et al., 2011;Avigliano et al., 2019). Agaricales fungi are generally annual and popularly known as mushrooms. They began to be studied by specialists in Brazil mostly in the XIX century (Fidalgo, 1985). The list counts for 1.011 species (Putzke, 1994) increased to ca. 1800 in 2018 (Putzke & Putzke, 2018). The studies done so far are essentially taxonomic, and rarely ecological/conservational or involving relationships between the fungi and the forest and rarely on preservation of fungi.
There are also citations of introduced fungi in this country and preoccupations are related to the fact that those species Research, Society andDevelopment, v. 11, n. 10, e103111032395, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i10.32395 could contact native plants to form mycorrhiza, competing with the native fungi species. One recent example of such invasion is cited in South America were Amanita muscaria (L.) Lam., usually associated with pine plantations is now found associated with a native oak species of Colombia (Vargas et al. 2019). The impact of these exotic species to this exclusive forest and over other native fungi needs to be better studied.
There are six terrestrial biomes recognized in Brazil: Pampa, Cerrado, Amazônia, Caatinga, Mata Atlântica and Pantanal. The first three are among the less studied in relation to their biodiversity. The Brazilian Pampa biome covers an average area of 176,496 km² and is characterized by being restricted only to Rio Grande do Sul State (southern Brazil), Uruguay and parts of Argentina. The biome is formed by the phytogeographic regions Contact Savana-Steppe, Steppe, Deciduous Seasonal Forest, Semi-deciduous Seasonal Forest and Pioneer Vegetation Areas (IBGE, 2012). The forest formations are basically found along the watercourses in the region, and must host an unknown biodiversity since they correspond to the last remnants of natural forest (Metzger, 2003).
The Cerrado is considered the largest hotspot in the Western Hemisphere, and the most biodiverse tropical savanna in the world, with an approximate extension of 2 million km2, with about 99.3% of its area is in Brazil (Sawyer et al., 2016). The cerrado is formed by a complex vegetation, including forest, grassland, and savannah formations, with a high number of endemic species (Ribeiro & Walter, 2008). The Atlantic Forest has the largest number of fungi records (3,017 species), followed by Amazonia (1,050), Caatinga (999), Cerrado (638) and Pampa (84) and Pantanal (35) (Maia et al. 2015). This shows that the 3 biomes selected for this study are poorly surveyed. In a recently published work on fungi in Brazil (BFG et al. 2021), 1461 species of fungi were reported for the state of Rio Grande do Sul, 867 species for the Amazon and 189 species for Goiás, characterizing Goiás as the seventh Brazilian state with lower occurrence of Fungi in the country. The states of Rio Grande do Sul and Amazonas stand out as the third and fourth, respectively, with more species of fungi in Brazil, however, in Rio Grande do Sul, most of the work was carried out in the Atlantic Forest biome, highlighting the importance of carrying out work in the Pampa biome (Silva et al., 2020).
Many initiatives to preserve the biodiversity in Brazil have been done by universities, many of them creating and maintaining forests inside their campus or creating and managing small areas outside. This is the case of forest inside the Universidade Federal do Amazonas (northern Amazonian Forest), in the Universidade Federal de Goiás (UFG -Middle West region -Bosque Auguste Saint Hilaire of the UFG has 27 hectares). It is important also the forest relicts in the river margins, called Permanent Protected Areas and that sometimes are small forest but its fungi composition is still not well studied. In order to better understand the importance of small areas in preserving fungi, specially Agaricomycetes, this work is proposed, dealing with the taxonomy and distribution of this group in the small natural reserves of Brazil, choosing areas in Southern, Central and northern Brazil.

Methodology
There were chosen two small reserves (one in Amazonia and other in central Brazil) and Permanent Protected Areas near rivers in Southern Brazil. The areas are described below:

Sampling in the Amazonas (Northern Brazil):
The area of the UFAM campus is 6.7 million meters square, with a perimeter of 16.9km of land. This area is the third largest natural fragment in an urban area in the world and the first in the country, which contributed for the creation of the Environmental Protection Area -APA UFAM. The APA was created in 2012, totaling 759.15ha, encompassing fragments of the Amazonian Forest, now totally isolated (Caldas, 2016). The field survey was done from February 19 to 24 2018, in the forest preserved inside the campus of the Universidade Federal do Amazonas (UFAM). The samples were collected using the "walking methodology" (Filgueiras et al., 1994), searching for Agaricomycetes fungi. The samples were studied in the Mycology Laboratory of the UFAM and all collections dehydrated and deposited in the JP herbarium.

Sampling in Goiás (Center Brazil):
Samples were collected during the rainy seasons 2018/2019 and 2020/2021 in the Bosque Auguste Saint Hilaire of the Universidade Federal de Goiás (UFG -Campus II), located in Goiânia city, the capital of Goiás state in the coordinates 16º 36'11" S and 49º15'39" W, at 695 m alt. and has ca. 20 ha. One of the few areas with primitive vegetation in the municipality of Goiânia, it is located on Campus II of the UFG. It is a remnant of the semideciduous forest of the Cerrado biome plant formation. The site, which is protected by a screen and has ecological trails, and is used for the development of Environmental Education activities, visitation of students of different levels and development of numerous scientific researches. The samples were also collected using the walking methodology and were dehydrated and deposited in the Herbarium of the UFG.

Sampling in Rio Grande do Sul (Southern Brazil):
The collections in the Pampa biome were carried out in the state of Rio Grande do Sul, southern Brazil, in all seasons of the year 2019. 10 collection points were selected in a longitudinal gradient between the municipalities of São Gabriel and Porto Alegre (the capital), in which riparian vegetation occurs, with an approximate distance of 20 km from one point to another. In all three areas the samples were collected using the walking methodology. All the mushrooms found were collected and taken to the local laboratories for identification and samples were dehydrated and deposited in the herbarium housed in each university. All samples from these three collecting areas photographed following the Macrofunge Image Capture Protocol (Bittencourt et al., 2022) and the identification was made using microscopes and usual morphological/anatomical techniques, as well as the specific bibliography available for the area (Putzke & Putzke, 2018).

Manaus -MUFAM
There were collected 140 specimens, being identified 41 species (29.3%) ( Table 1). The list is incomplete mostly because of the poorly known large genera like Marasmius, Marasmiellus and Mycena abundant in the mentioned forest. The 12 taxa (29.2%) not identified to species level are not cited to the country or represent new species to science. The Agaricales fungi have received little attention in the Amazon region, especially in relation to basic taxonomic surveys. The works of Singer (1973;1976;1989), Singer and Araujo (1979), Araújo (1984) and Singer & Aguiar (1986) demonstrate that diversity is very large and adapted to regional conditions, but since then few studies have dedicated greater attention to this giant Brazilian region (despite disperse in other surrounding countries). In the beginning of this century, basic agaricology has returned to be the subject of studies, especially from the surveys carried out by Souza and Aguiar (2004) involving the Agaricales of the Walter Egler Reserve, where 39 species are mentioned, but only 6 have been identified to the specific level, being many specimens identified with a cf., aff. or sp.. Souza and Aguiar (2007) describe Marasmius species to the same area. Oliveira et al. (2009) neotypify Marasmius amazonicus Henn. Since then, few surveys have been carried out, leaving the immense agaricine flora of the state of Amazonas largely ignored.

Riparian Vegetation in Southern Brazil -RPAS
The riparian vegetation of the Brazilian Pampa Biome has been scarcely studied in terms of its agaricobiota, with forest formations remaining rare in a predominantly herbaceous/shrub ecosystem. Studying these areas may allow establishing new parameters for the distribution of Fungi. In this sense, 10 riparian forest formations were studied along a 200 km (east to west) transect in the center of Rio Grande do Sul state -Southern Brazil, in seasonal expeditions in 2019 to collect and identify the agaricoid fungi species. 278 new species records for the Brazilian Pampa biome were cataloged, 23 new records for the state of Rio Grande do Sul and 4 new registrations for Brazil (Table 2). Among the families found, there was a higher incidence of Marasmiaceae (77 species), followed by Agaricaceae (45 species), Tricholomataceae (30 species) and Mycenaceae (30 species), covering a total of approximately 65% of the collected species (Table 2).

Saint Hilaire Forest in the Universidade de Goiás (MSH) in Goiânia municipality -Goiás State
From the 86 samples collected, 31 were identified to species level and 51 to genus, the remaining only to family rank ( Table 1). The unidentified species were probably new citations or new species to science. Identified species related to the place of occurrence. Table 1 -List of species collected in the forest preserved inside the campus of the Universidade Federal de Manaus (UFAM) and Saint Hilaire Forest in the Universidade de Goiás (MSH) in Goiânia municipality -Goiás State.   4 2,4,5,7,9,10 Source: Authors (2022).

Discussion
The In the studies presented here, the organisms of the represented about 30% of the cataloged species, a lower number than other works, but it is still the family with the highest incidence. There are species that are characterized by their greater resistance to drier environments, since they show reactivation, the ability to stop their growth in times of drought and grow again later when the environment presents a certain humidity (Singer 1986). This character may explain the high rate of species of this family in studies on taxonomy and diversity.
In MUFAM and MSH, the family with the highest occurrence was Agaricaceae, family that has a considerable rate of specimens in the Brazilian mycota, with 85 genera and about 1340 species distributed throughout the world (Kirk et al., 2008).
This family is characterized by hosting a variety of edible individuals, including some species of the genus Agaricus, popularly known as champignons (Wartchow, 2018). Rosa & Capelari (2009)  The Agaricaceae has a worldwide distribution, with a range of representatives in tropical and temperate regions, as well as in arctic-alpine and desert areas. However, the diversity of individuals in the family has a certain disparity in the occurrence from one region to another (Vellinga 2004), which requires more studies on the specimens of this family found in the Brazilian biomes, to elucidate more information on ecology and diversity of this family in the area.
The Southeast, Northeast and South regions of Brazil lead numbers of research on Agaricales in Brazil, on the other hand, the North and Midwest regions have a reduced number of research on this group of fungi (Carvalho et al., 2022).
Agaricales studies for the Cerrado are rare, highlighting some works at specific level. Calaça et al. (2020) presented the first In addition, specimens of Agaricus, Cantharellus, Flammulina, Gymnopilus, Macrolepiota, Oudemansiella and Pleurotus, known for their edibility, were also identified in the study areas, highlighting the possible biotechnological application and cultivation of the mycota found in the three biomes studied.
Among the collected species, ca. 50% were identified only to the genus level, which requires further studies to confirm new occurrences or the registration of new species to science. Furthermore, from the list, 10% represents new confirmed occurrences for the state of Rio Grande do Sul or Brazil and 99% are cited for the first time for the Pampa biome.

Conclusion or Final Considerations
The high number of species mentioned for the first time for the Pampa and Cerrado biomes, mainly, can be explained because there are only a few studies on the Agaricales fungi in these regions of Brazil. Also, areas with a reasonable number of studies, such as the Amazon biome, presented new occurrences not yet reported to science, showing the need for current studies on Agarics in these areas. Therefore, due to the high number of specimens collected in this work, there are indications that the biomes may have a greater diversity of Agaricales fungi, which requires further studies to estimate the richness and diversity of these organisms in the areas. This corroborates the idea that small areas can resume a great fungal diversity, deserving preservation at any coasts.