**3. Methods used to identify the interactions of bryophilic mushrooms**

The most used methods for the identification of bryophyta/Agaricomycetes associations are: optical and electronic microscopy, molecular and phylogenetic analyses, and *in vitro* culture experiments (**Figure 1**). Initially, it is necessary to identify the site of mushroom/bryophyte association, such as non-photosynthetic regions like rhizoids, or photosynthetic regions like the thalli or leaf structures. As for example, an optical microscopy analysis was used to identify the fungi *Chromocyphella muscicola* (Fr.) Donk in association with bryophytes, reporting this species usually known from the Northern Hemisphere in Brazil for the first time [21].

With the preparation of slides with KOH (5%), it is possible to visualize the structures of the mushrooms, especially the hyphae which are sometimes linked to the bryophyte cells. In the scanning electron microscope, slides were prepared as usually with reagents that can also be used to identify the association between Bryophytes and Agaricomycetes fungi [1]. As a differential, when analyzing the species *Sphagnum fuscum* Klinggräff with this methodology, it was possible to visualize the rudimentary appressoria that mechanically facilitated the entry into the cells of photosynthetic structures, belonging to the bryophilic species *Glomus mosseae* (T.H. Nicolson & Gerd.) Gerd. & Trappe [1]. An illustrative schematic of the step-by-step of these techniques is shown in **Figure 1A**.

The phylogenetic analyses can be made to detect the feeding and ecological habits using gene portions (ITS 1–2 and 5.8S rRNA) DNA extractions, sequencing and with subsequent bioinformatic analysis [19]. Analysis performed with *Mycena* sp. and *Galerina* sp. showed close evolutionary relationships with *Dicranum* sp. and *Hylocomium* sp. [19]. Key findings include that *Galerina* sp. showed a preference to associate with senescent, rather than photosynthetic tissues, and thus ancestral saprotrophic habit. On the other hand, *Mycena* sp. showed colonization in both tissues, and therefore ancestral parasitic habit [19]. In general, phylogenetics is performed in several steps: (I) Material preparation; (II) DNA extraction; (III) Sequencing; and (IV) Sequence analysis by bioinformatics [22]. This results in four advantages: an independent framework for clade construction; a well-supported statistical basis, as the sites of an alignment integrate matrices of different sizes; a low incidence of putative homeoplasies compared to morphological characters; and the implementation of evolutionary models applied independently to each base [23]. An illustrative schematization of these main steps is shown in **Figure 1B**.

*In vitro* culture experiments are performed to analyze the ecology of interactions and resistance of bryophytes. The bryophytic fungi are part of a diffuse group, often only detected by molecular analyses [24]. The basidioma emerges at specific periods, temperature and humidity, which can make it difficult to visualize between the photosynthesizing or non-photosynthesizing structures of bryophytes [25]. The mycelial phase is the most predominant fungal phase, and this structure can be visualized under microscope when associated to bryophytes. When growing the species *Atrichum androgynum* (Müll. Hal.) A. Jaeger in culture medium, an association with the fungi *Arthrobotrys oligospora* Fresen., was visualized which is known to capture nematodes [24]. This was only possible due to the growth of the fungi in

### **Figure 1.**

*Methodology frequently used for detection of Fungi-bryophyte associations (FBA). (A) Optical microscopy: association site, non-photosynthetic (1) or photosynthetic (2); slide cuts and use of reagents with cover by coverslip (3); microscope observation (4); FBA endophilic (5-A) or exophytic (5-B) structures. (B) Steps of molecular analyses of FBA: preparation of material for DNA extraction in bryophytes (1 and 2); preparation of material for DNA extraction in mushrooms (3 and 4); use of reagents for DNA extraction (5 and 6); sequencing of gene portions of interest (7); analysis of the sequences by bioinformatics techniques (8). (C) Visualization of FBA in culture medium: a mature bryophyte containing the capsule is isolated (1), and disposed in culture medium (2); the basidiomata (3), when lamellar region is cut and the structure is placed in culture medium (4); when the fungi is not visible, the hyphae grow in the culture medium starting from the bryophytes and can be isolated and cultured separately for species identification (5).*

culture medium, since it was not visible among the collected bryophytes [24]. Sometimes the structures of the fungi can be detected so that with the aid of microscopy, tweezers, and accessories the fungi can be isolated and grown separately in usual culture medium (like PDA). An illustration of the methodological steps mentioned above is shown in **Figure 1C**.
