Experimental design and animal husbandry
Metamorphic toads (Bufonidae) collected from Arizona, California, Louisiana, Ohio, and Tennessee, USA, (see Table S1 for specific collection locations) were shipped overnight to Tampa, Florida. Although the experiment was designed to cross six host populations with six Bd strains (a 6x6 design), we could not collect enough Quebec (QC) hosts and the experiment was reduced to a 5x6 design. Breeding times varied across collection locations and so we used a randomized block design in which identical protocols were employed to minimize variation (see Supplemental Methods and Table S1 for details). To eliminate any existing Bd infections from field-collected animals, we subjected all animals to a 10-day 30° C Bd clearance treatment (Supplemental Methods) (Chatfield & Richards-Zawacki 2011; McMahon et al.2014). Toads were then maintained individually in containers at 18.5° C for a 7-10 day acclimation period (Raffel et al.2013). We fed toads ad libitum with vitamin- and mineral-dusted crickets and provided a fresh container and bedding twice per week (Supplemental Methods).
We crossed 5 closely related North American toad species (HostAZ, HostCA, HostLA, HostOH, HostTN) with 6 Bd strains (BdAZ, BdCA, BdLA, BdOH, BdTN, BdQC; Table S2) and one sham control (artificial spring water; ASW) for a total of 35 experimental treatment combinations. Strains were pulled from cryopreservation from the Bd isolate cryopreservation library of Dr. Joyce Longcore. The number of replicates per treatment varied between 6 and 18 because of variation in the availability of toads across the populations (Table S1). An experimental unit was an individual toad. Each toad population was split into seven treatments (6 Bd strains and one ASW sham). Mortality was monitored daily. All toads were weighed before the Bd exposure, and again at death or at 70 d post-exposure. At the end of the experiment, all remaining toads were swabbed for Bd load, euthanized with phosphate-buffered benzocaine, and stored at -20° C.