*2.1. Laboratory Rearing*

Data on fungal pathogens present in laboratory rearing of Lepidoptera were obtained as a by-product of ecological experiments performed at the University of Tartu, Estonia, in 2014-2017. Four lepidopteran species—*Ematurga atomaria* L., *Cabera pusaria* L., *Hypomecis punctinalis* Scopoli (Geometridae), and *Orthosia gothica* L. (Noctuidae)—were subjected to identical experimental design ([13–15], and unpublished). Specifically, in order to record growth rates, developmental periods, and final weights, we reared the larvae from eggs to pupae singly in 50 mL plastic vials at different temperatures and weighed them periodically. Within each combination of year and species, the insects were reared simultaneously under common garden design, while the timing of experiments with different species did not coincide due to natural phenological differences of the insects. The data on the fifth species, *Chiasmia clathrata* L. (Geometridae), were obtained from a technically similar study in which growth parameters of selection lines were compared (Välimäki et al., unpublished). The larvae were fed with leaves of host plants collected in the field in Tartu, or surroundings of the town. The larvae of *E. atomaria* were fed on *Trifolium repens* L. (Fabaceae), *Vaccinium myrtillus* L. (Ericaceae) and *Salix alba* L. (Salicaceae); *C. pusaria* on *Alnus glutinosa* (L.) Gaertn. (Betulaceae); *H. punctinalis* on *Betula pendula* Roth. (Betulaceae), *Tilia cordata* Mill. (Tiliaceae), and *Quercus robur* L. (Fagaceae); *O. gothica* on *B. pendula*; and *C. clathrata* on *Lathyrus pratensis* L. (Fabaceae).

The larvae were allowed to pupate in moist *Sphagnum* moss, known for its antiseptic properties. This should have minimized the insects' risk of being infected during the pupal period, allowing us to focus on infections acquired during the larval stage. The pupae were kept overwinter in thermoregulated chambers at about 0 ◦C. In spring, adult moths were allowed to eclose at room temperature. The pupae that failed to eclose and eventually died were inspected for visual signs of fungal infection.

## *2.2. Field Experiment*

To record the community of insect-associated fungi in near natural conditions, we reared larvae of *C. pusaria* on living wild host plants in mixed forest fragments in the surroundings of Tartu (between 58◦26 N, 26◦30 E and 58◦24 N, 26◦39 E), Estonia, in the course of 2 years. Newly hatched larvae were enclosed in 50 × 30 cm<sup>2</sup> polyester bags (*N* = 81), in which they remained until pupation. Ten larvae per bag were placed on 3 different host plants of the moth—grey alder (*Alnus incana* (L.) Moench), silver birch (*Betula pendula* Roth.), and downy birch (*B. pubescens* Ehrh.)—in the first half of July 2016 and 2017 and were checked weekly for pupation. After pupation, the insects were placed individually into Empera 124 N polystyrene vials with sterilized peat moss (*Sphagnum* sp.). The moss was sterilized by keeping it at 100 ◦C for 4 h. Vials were sterilized with 10% NaOCl. Pupae overwintered in thermoregulated chambers at 2 ◦C for 3 months. In January, adult moths were allowed to eclose at 24 ◦C and 12:12 h of light/darkness cycle. Insects that died during the pupal period were inspected for visual signs of fungal infection.

#### *2.3. Identification of Fungi and Their Host Ranges*

To identify the fungi and preserve these as pure living cultures, we inoculated visible fungal material (only anamorphs were encountered) to Petri dishes with 2% malt extract agar (Oxoid, Cambridge, UK) supplemented with antibiotics (1% of streptomycin and tetracycline). After a few weeks of growth in culture, the fungi were identified on the basis of morphological traits using keys provided by Domsch et al. [16], Samson et al. [12], and Seifert et al. [17]. A culture isolate representing each morphotype was subjected to DNA extraction to confirm the identification.

The procedures of growing the mycelium, extracting DNA, conducting PCR, and sequencing followed the protocols described by Põldmaa et al. [18]. Ribosomal DNA full ITS and partial LSU sequences were obtained from 91 fungal isolates. The sequences along with their metadata were uploaded in PlutoF, a data managemen<sup>t</sup> and publishing platform [19], and made available via UNITE database [20]. The ITS rDNA sequences were incorporated in the UNITE species hypotheses (SH), which served as the basis for species identification, by choosing an appropriate distance threshold value in each case (Table 1). The Basic Local Alignment Search Tool (blastn) at National Centre for Biotechnology Information [21] was used to check for similar sequences not ye<sup>t</sup> incorporated in the UNITE database.

**Table 1.** Species of fungi isolated from lepidopteran hosts (the field experiment data in brackets). UNITE species hypothesis (SH) codes are presented to facilitate communication on detected fungi [22].


\* DOI = digital object identifier, SH DOI is displayed here as a short code; \*\* insect hosts abbreviated as EA—*Ematurga atomaria*, CP—*Cabera pusaria*, HP—*Hypomecis punctinalis*, CC—*Chiasmia clathrata*, OP—*Orthosia gothica*, UP—unidentified pupa. 5

All pupae infected with a fungus were deposited at the TU fungarium (accession numbers TU133001-133196) and representative isolates at the TFC culture collection (TFC202234- 202344) in the Natural History Museum and Botanical Garden, University of Tartu.
