2.1.1. Solvent-Extracted Pigment

*Chlorociboria aeruginosa* UAMH 11657 (isolated from a decaying hardwood log in Haliburton, ON, Canada), *Chlorociboria aeruginascens* UAMH 7615 (isolated in Lake District, UK), and Scytalidium *cuboideum* (Sacc. and Ellis) Sigler and Kang UAMH 11517 (isolated from *Quercus* sp. in Memphis, TN, USA) were used to inoculate petri dishes containing 2% malt extract agar (MEA) (20 g of bacteriological malt extract (VWR, Radnor, PA, USA), 15 g of agar (VWR), 1 l of deionized water) amended with sterile white rotted wood chips from either *Acer saccharum* or *Populus grandidentata*, following the protocol set by Robinson et al. [53]. Cultures were harvested once plates were completely pigmented, with times ranging from four (*Scytalidium cuboideum*) to twelve weeks (*Chlorociboria* spp.). Plates were opened and left to dry for 48 h, then ground using a blender (Oster Precise Blend, Boca Raton, FL, USA) until reaching a maximum size of ~5 mm. The resulting powder and 45 mL of dichloromethane (DCM) (VWR, Radnor, PA, USA) were combined in a 250 mL Erlenmeyer flask with a 2 mm × 5 mm VWR Spinbar magnetic stir bar. The flask was closed with a rubber cap and a stirred at 220 rpm for 30 min on a VWR Dylastir stir plate. The resulting solution was then filtered through VWR 415 Whatman Filter Paper to remove the wood chip particles. The extract was collected in a borosilicate glass vial (Ace glass, Vineland, NJ, USA) and sealed with non-evaporative polyseal-cone-lined caps.

## 2.1.2. Pigments from Liquid Media

Liquid media were prepared following methods in Weber et al. [54]. Sterilized and cooled 150 mL mason jars containing 50 mL of 2% malt broth (20 g of VWR bacteriological malt extract, 1 l of deionized water) were inoculated with active fungal cultures of either *C. aeruginosa* or *S. cuboideum* using one plug of approximately 2 mm in diameter. Jars were then incubated at room temperature (21 ◦C) for 28 days on an open shelf.

Pigment from liquid media was tested in three ways. First, liquid media were used directly in zebrafish assays. Second, liquid media were autoclaved at 121 ◦C for 30 min. Finally, media from fungal liquid malt cultures were also cleaned independently using Strata SPE 2 g/12 mL columns (Phenomenex). The column was conditioned by adding 4 mL of HPLC acetonitrile (CAN) solvent to remove trapped air and activate the SPE particles, before the solvent was removed and 4 mL HPLC grade water was added to maximize the sorbent interaction with target analytes. For all species, liquid media culture was filtered through 415 Whatman filter paper (VWR) twice before 10 mL was loaded onto activated column, where a visible band of pigment was formed. Contaminants were removed from the column through the addition of 10 mL 50% acetonitrile (ACN) in HPLC grade water. Pigment was eluted using 2 to 4 mL of 100% of HPLC-grade chloroform (EMD Millipore, Burlington, MA, USA). About 10 mL of the pigment mixture sample was used to achieve less than 0.5 mL of each purified pigment in two to three hours. This method was used to obtain pigment with a reduced amount of contaminants to reduce and identify potential effects of the extracts on the zebrafish embryos.
