Mycophagy of Attini Ants (Hymenoptera, Formicidae, Myrmicinae) with Agaricales Mushrooms (Basidiomycota, Agaricomycetes) at Riparian Zone in Southern Brazil

Attini ants (Hymenoptera, Formicidae, Myrmicinae) have great diversity in exploiting food resources. However, little is known about the mycophagy involving Agaricales fungi (Basidiomycota, Agaricomycetes). Moreover, these associations are of paramount importance in riparian zones, as the interaction among soil, fauna and flora is fundamental to the maintenance of these environments. Thus, the aim of this study was to describe cases of mycophagy between ants and fungi in order to understand how these associations occur in riparian zones. To this, collections were made between 2021-2022 in the valleys of the Rio Vacacaí, Rio dos Sinos and Rio Pardo in the state of Rio Grande do Sul, Brazil. The samples were analyzed for characters macro and microscopic and identified taxonomically. Mycophagy activities of six species of ants worker were cataloged, among them Acromyrmex niger , Acromyrmex versicolor , Tranopelta gilva , Tranopelta subterranea , Pheidole flavens and Mycetosoritis hartmanni , which included in their diet the mushrooms Agaricus rufoaurantiacus , Macrocybe titans , Agrocybe underwoodii , Dactylosporina steffenii , Lepiota micropholis and Neopaxillus echinospermus , respectively. The observed interactions demonstrate the dispersal of fungal spores through ant body structures, such as legs, antennae, thorax and abdomen; basidiomata as aliment source, such as pileus, lamellae and stipe; and generalized and specialized levels of predation on various fungi structures. Our results demonstrate unprecedented mycophagous relationships and show that Agaricales fungi can be considered a food source for Attini in riparian zones in Brazil south.


INTRODUCTION
The ant species Attini (Hymenoptera, Formicidae, Myrmicinae) is one of the most successful groups of Arthropods, both in terms of diversity and abundance. The group has a high social complexity and a great exploration ability in a variety of food niches (Houadria and Menzel, 2020).
Food from various parts of plants, such as leaves, seeds, fruits, and roots; also multiple kinds, such as carrion, manure, and others, are part of a wide variety of dietary habits of this group (Lanan, 2014). Many nutritional forms are used to some extent by ants, and consequently, some become specialists and others generalists in terms of their diet (Csata and Dussutour, 2019).
Little is known about the inclusion of fungi in ant diet, nor about the types of mycophagy habit involving the ants with Agaricales Fungi (Basidiomycota, Agaricomycetes) (Epps and Penick, 2018). Except for Euprenolepis ants, with reports of mycophagy in the Old World (Claridge and Trappe, 2005;Witte and Maschwitz, 2008;Von-Beeren et al. 2014), in addition to a select group of Attini endemic to the New World and known to cultivate fungi (de Fine et al. 2014). For these fungiculture ants, five types of agricultural systems are cataloged: lower agriculture, coral-fungus agriculture, yeast agriculture, generalized higher agriculture, and leafcutter agriculture (Branstetter et al. 2017).
In terms of fungal structures associated with ant feeding, gongylidia (swollen hyphae), hyphae tissues in general (pileus and stipe), spores, and mycelium are the most commonly reported (Epps and Penick, 2018). However, even with few studies on mycophagy in Hymenoptera, other Arthropod orders such as Acari, Coleoptera, Collembola, Diptera, Lepidoptera, and Orthoptera have already had important inferences regarding the inclusion of fungi in their diet (Schigel, 2012;Dash et al., 2018;Innocenti and Sabatini, 2018;Pollierer et al., 2020;Tozlu et al., 2022;Wakil et al., 2022). As nutritional importance of fungi, regarding the benefits of their consumption by arthropods, mainly minerals, vitamins and fats are listed (Biedermann and Vega, 2020).
In contrast, there are several reports of ants visiting the basidiomata of various fungi, mainly in relation to foraging and predation of larvae attached to the fungal tissues (Branstetter et al. 2017;Epps and Penick, 2018;Pereira et al. 2020). However, it is unclear whether the visit is limited to hunting larvae, or whether mycophagy also occurs. It is believed that many ants spread fungal spores Brazilian Journal of Animal and Environmental Research, Curitiba, v.5, n.4, p. 3935-3960, out./dez., 2022 even when there is no mycophagy because the spores can attach to the ants' body structures and travel with them short and long distances (Witte and Maschwitz, 2008;Von-Beeren et al. 2014;Epps and Penick, 2018).
Riparian zones are permanent protected areas where the presence of vegetation, mycota, and fauna play unique ecological interactions related to their conservation (Brasil, 2012). In this respect, little is known about the activity of ants in these areas, as well as the fungal community.
Nevertheless, nutrient cycling for fungi is an important process that takes place in all environments (White et al. 2018). The benefits cited for ants include supporting plant reproduction, improving the nutrient quality of the soil, and aerating the soil, among others (Tuma et al. 2020).
All points located in Rio Grande do Sul state, southern Brazil.

Collection and taxonomic identification of specimens
The Rapid Survey method used for tree vegetation (Walter and Guarino, 2006) was adapted to collect basidiomata with signs of mycophagy. The fungal material with the ants was collected under license from SISBIO n°78538-1. For the mushrooms, collections were performed according to Putzke and Putzke (2017) in Putzke and Putzke (2017) and data available on the Index Fungorum platform (http://www.indexfungorum.org/). The ants found in basidiomata were stored in tubes with 70% alcohol. The keys of Fisher and Cover (2007) and Baccaro et al. (2015) were used for identification, as well as the data available on the AntWiki platform (http://www.antwiki.org/). Specimens were analyzed according to their macromorphological and micromorphological characteristics using the Olympus CH-2 optical microscope. All collected specimens (mushrooms and ants) were stored in the Laboratório de Taxonomia de Fungos of Universidade Federal do Pampa, São Gabriel campus, Rio Grande do Sul state, Brazil.

Statistical analyzes of the data
ANOVA test was conducted under the model of variables with the "predation location" factor and predation levels for the "amount" factor, conducted in the form of data percentage (Sabin and Stafford, 1990). Comparisons among the means of spore quantity, as well as of each individual were conducted under the LSMeans model (Lenth and Lenth, 2018), applying the Test-Tukey at 5% significance using the Estat v.2.0 program (Estat, 1994).

RESULTS AND DISCUSSION
For the Attini six species of workers ants were cataloged involved in mycophagy activities: Acromyrmex niger, Acromyrmex versicolor, Tranopelta gilva, Tranopelta subterranea, Pheidole flavens and Mycetosoritis hartmanni. The ants were associated with six species of Agaricales: Agaricus rufoaurantiacus, Macrocybe titans, Agrocybe underwoodii, Dactylosporina steffenii, Lepiota micropholis and Neopaxillus echinospermus respectively. After the identification of the specimens, two taxonomic keys were prepared, one for the identification of the mushrooms and the other for identifying the ants, both involved in mycophagy.

Agaricus rufoaurantiacus Heinem and Acromyrmex niger (Smith, 1858)
The fungus has a pileus 2 -3 cm in diameter, convex and umbonate. Pileus pale cream with orange-brown scales (  in the Atlantic Forest biome, the species has no cataloged occurrence, as well as for Brazil. There are only records of occurrence for North and Central America (Fowler, 1988;Julian and Fewell, 2004), and a single occurrence in South America for Guyana (Fernández and Sendoya, 2004), being considered a species native to the Neo-Arctic region.
The ant has a broad, rectangular head, without the presence of tubercles and with poorly

Mycophagic associations between ants and mushrooms
About 70 individuals of Acromyrmex niger showed associations with Agaricus rufoaurantiacus on the soil surface during the daytime at point A. At the site, 7 basidiomata were found in the area and 4 contained signs of mycophagy. The main parts of the basidiomata involved in predation were the pileus surface, lamellae and stipe. Mycophagous associations have already been described for Acromyrmex niger with other Agaricales fungi, including Leucoagaricus gongylophorus, where besides preying on all parts of the basidiome, it is also associated with its cultivation as a fungiculture (Aylward et al. 2013). For Agaricus rufoaurantiacus, this is the first record of association with this ant species. However, in the study by Wartchow et al. (2008), unidentified nests of leafcutter ants were found near basidiomata of this mushroom in the Atlantic Forest biome in Pernambuco.

About 20 individuals of Acromyrmex versicolor showed mycophagy associations with
Macrocybe titans on the soil surface at point C at night. In the area, two basidiomata of Macrocybe titans were found there, and only 1 showed signs of predation. In the mushroom, only the surface of the pileus showed signs of mycophagy, including a small portion of the lamellar margin region.
For Acromyrmex versicolor, the title of fungiculture is frequent and connected to the survival of their nests (Clark and Fewell, 2014). Also, Leucocoprinus sp. has already been reported in fungiculture association with this ant species (Bizarria et al. 2022 al. 1998; Mueller and Mata, 2001). Nevertheless, this is the first report of mycophagy between the two species of this study.
Approximately 7 individuals of Tranopelta gilva were found during the day at point B in mycophagy activity with Agrocybe underwoodii on the soil surface. A total of 5 basidiomata of Agrocybe underwoodii were found in the region and 3 specimens showed signs of mycophagy, only the bordon of the lamellae showed signs of predation. Although Tranopelta gilva is cited as a fungiculture ant (Fernandez, 2003), there are no known cases in the literature with Agaricales fungi.
For Agrocybe underwoodii, this is the first report of mycophagy associated with ants. However, there are citations for the genus from Russia (Rayner et al. 1985) and Malaysia (Fung and Tan, 2022) with unidentified fire ants.

Approximately 2 individuals of Mycetosoritis hartmanni showed mycophagous activity with
Neopaxillus echinospermus on the soil surface at point A during the day. In the area, 3 basidiomata of Neopaxillus echinospermus were found and 1 showed signs of mycophagy. The surface part of the pileus and a small part of the lamellar margin showed signs of predation. For Mycetosoritis hartmanni, mycophagy associations with fungal gardens are recorded in the literature, including Escovopsis and Trichoderma (Sanchez-Peña, 2010;Solomon et al. 2011), but both belong to the Ascomycota. For Agaricales fungi, the ant has a description of association with Lepiota (Batra and Batra, 1967). For Neopaxillus echinospermus, the report of mycophagous association is unpublished.

Levels of interaction and predation of ants with the mushrooms
The present study reports the interaction of six ant species in mycophagous activities with six Agaricales fungi. Our results describe novel associations with ants already cited in literature as fungiculture and not cited. For the ants, the report is similar once that of the activities mycophagy involved fungi with and without a description of the application in Attini fungiculture. The interactions involving these associations are summarized in Figure 7. In general, it could be seen that the ants not only eat but also carry parts of the fungi to their nests. Several parts of the basidiomata were associated with predatory activities, such as the surface of the pileus, the lamellae, and the stipe. According to Von-Beeren et al. (2014), ant harvesting performed directly on basidiomata affects spore dispersal and has a significant positive impact on the local fungal community, as does ant harvesting of seeds, which affects the plant community (Nelsen et al. 2018). Under this premise, the ant structures that contained the most fungal spores followed in descending order: legs, antennae, head, thorax, and abdomen (Figure 7 A).
Thus, the data presented help in questions relevant to the known Attini mycophagy, describing predation standards and reported forms of association.
Studies show that certain ants visit the basidiomata to capture larvae of arthropods in general and that many are predatory in the basidiome itself (Witte and Maschwitz, 2008). Ants are also known to collect fungi to grow them in their nests (Epps and Penick, 2018). However, the main point of contention is whether or not ants benefit nutritionally from fungal structures, such as the surface of the pileus, lamellae and stipe. According to de Moura and Okura (2022) Acromyrmex niger and Acromyrmex versicolor had a high percentage of predation compared to the other species of this study (Figure 7 C). According to Clark and Fewell (2014), Acromyrmex has a collective foraging pattern that is fast and efficient. This is evidence of optimization of mycophagous strategy, as they were also the species that benefited most from the diverse structures of basidiomata. According to Wartchow et al. (2008), the species of this genus tend to be generalists in terms of feeding, which would explain the diversity of structures integrated into mycophagy.
Therefore, it can be concluded that mycophagy is generalized in these species.
For Tranopelta gilva and Tranopelta subterranea there are descriptions of generalist dietary habits (Rayner et al. 1985), particularly in relation to the diversity of decomposing organic matter (Fernandez, 2003). For the samples of this study, specifications related to mycophagy were cataloged, the mycophagy occurred preferably in lamellae region of the basidiomata (Figure 7 C).
These data are consistent with the reports of Fung and Tan (2022), in which more than 20 species of edible mushroom were implicated in predation by fire ants in lamellae region, including individuals of Tranopeltra. Thus, is possible to infer that occur a specialization of mycophagy on the lamellae regions involving these ants.
Pheidole flavens has a diet that is classified as generalist (Wilson, 2003), Attini ants are known for their generalist diet (Lanan, 2014). However, few data are available on the diet of fungi, and these data refer only to a selected group of fungiculture ants (Witte and Maschwitz, 2008). In a controlled laboratory study with individuals of Aphaenogaster, a generalist ant group known for its carnivorous habits, Epps and Penick (2018) infer that not only was prey (larvae) collected in basidiomata, but that workers also collected pieces of mushroom free of prey, chewed the same, and deposited them in their nests. In the nest, the workers continued the process of macerating the basidiome pieces and distributing them all over the colony. In our study, only worker ants were identified, but our observations and sampling were limited to the soil surface.
Further studies could elucidate the mycophagous associations of these species and their internal interactions within the colony, as well as possible cultivator groups that have not yet been described.
The species this study inhabit a very important ecosystem, there are many unknown interactions in riparian zones (Vidon et al. 2019). As previously described in the literature, fungi in mycorrhizal associations integrate a nutritional mycelial network, and among the species studied, Dactylosporina steffenii forms pseudorriza (Putzke and Putzke, 2017). In this sense, the diversity of ants active in these zones is underestimated, and they could play an important role in the interaction plants-fungi-soil. Another important point to study is the spread and maintenance of the fungal network that plays an active role in the nutrient cycling of these environments. In this case, the dispersal of spores over short or long distances by ants could be an important issue for the local fungal community.

CONCLUSION
From the findings related to the ants, it can be deduced that mycophagous Attini is an underestimated group that contradicts the idea that they use basidiomata only as hunting grounds or act only as disseminators of fungal spores. Each ant showed peculiarities in relation to the associated fungi, preferences structures and the levels of predation in basidiomata. In the case of Agaricales fungi, our results revealed new interactions that will serve as a basis for further studies, especially in relation to nest dynamics associated with mycophagy and fungiculture.
However, it is possible to conclude that these interactions in the riparian zones studied may favor either the predator or the fungus grower, the prey or the culture. In addition, the ability of ants to exploit a temporally limited resource such as basidiomata may be related to their success in food diversity. Our study reveals a diverse social potential of mycophagous, as specificities related to food resource exploitation were cataloged. It can be concluded that Attini-Mycophagous integrate an important group in the riparian zones of southern Brazil.