(a) Experimental procedures

We obtained potted Neoregelia compacta tank bromeliads from a nursery in Campinas, São Paulo, Brazil. To remove organisms and detritus, bromeliads were rinsed and air-dried. We obtained one-week-old tadpoles (Gosner's stage 25 [44]) of the bromeliad-specialist treefrog Ololygon alcatraz (Anura, Hylidae) from a Bd-free ex situ conservation breeding programme at Fundação Parque Zoológico de São Paulo, SP, Brazil. Using young, captive-bred amphibians ensured that initial host microbiomes were homogeneous and that microbiomes were actively assembling. For the Bd inoculum, we used the isolate CLFT 196 (Global Panzootic Lineage-2), which originated from a tadpole collected in São Paulo state, Brazil [45]. To obtain Bd zoospores for the inoculum, we added a growing liquid Bd culture to Petri plates containing 1% tryptone agar. Plates were incubated at 17°C for 9 days leading up to the experiment. We then added distilled water to the plates to form a zoospore suspension, collected the suspension and calculated the concentration of zoospores with a haemocytometer.

We used a 2 × 2 fully factorial experimental design with water treatment (natural or pre-sterilized) crossed with aquatic arthropod detritivores (present or absent). We used 12 replicate bromeliads for each of the four treatment combinations. We arranged the bromeliads in a blocked design, with 12 spatial blocks each containing one bromeliad from each treatment combination. The location of each bromeliad within each block was determined randomly. We collected water and arthropods for the experiment from wild bromeliads in a forest fragment in Campinas, São Paulo, Brazil (lat: −22.8316°, long: −46.9651°), containing vegetation representative of Brazil's Atlantic Forest. The water was collected with pipettes, filtered with 0.125-mm geological sieves and homogenized. For the pre-sterilized water treatment, we autoclaved the wild-collected water. We used the three most abundant aquatic detritivore species and selected the number of each species to be added to each bromeliad based on their relative abundances in the field. The three species were the microcrustacean Elpidium bromeliarum (Limnocytheridae; collector; n = 10), larvae of the mosquito Culex sp. (Culicidae; filter-feeder; n = 5) and larvae of the biting midge Forcipomyia (Phytohelea) sp. (Ceratopogonidae; collector; n = 4), which collectively feed on fine and coarse particulate matter, algae, microbes including fungi and bacteria, and smaller invertebrates throughout the water column [18,46,47]. Water and arthropods were stored at 20°C overnight prior to stocking bromeliads.

On day 0, we added the water treatment (100 ml of natural or pre-sterilized water), the arthropod treatment (present or absent), one randomly assigned O. alcatraz tadpole and a standardized zoospore inoculum (1.5 ml of the zoospore suspension at 3.22 × 10⁶ zoospores per ml) to each bromeliad. The experiment was performed under controlled laboratory conditions (20°C on a 12 h day/night cycle). We fed tadpoles every 3 d with dried and autoclaved Spirulina (10 mg). The Spirulina also served as a detritus source for the bromeliad community; the bromeliad species we used typically do not accumulate large fragments of leaf litter in the wild. On day 13, we quantified microeukaryotes (ciliates, flagellates and rotifers) in each bromeliad containing natural water. For each bromeliad, we collected 1 ml of water, fixed and stained the sample with glycerol and Rose Bengal, counted microeukaryotes in five 20-µl subsamples under a compound microscope and averaged the five counts. On day 17, we sampled environmental bacterial communities and Bd zoospore densities in each bromeliad by swabbing the submerged, inner leaf surfaces with a sterile swab. We also measured water pH (PCD-650, Oakton Instruments, Vernon Hills, IL, USA) and chlorophyll a concentration (Aquafluore, Turner Designs, San Jose, CA, USA) in each bromeliad. We then measured tadpole body mass as an estimate of sublethal fitness effects of Bd infection and euthanized tadpoles with an overdose of MS-222. Tadpoles and swabs were stored at −20°C until further processing.

Because bromeliads were not sterilized before the experiment and were held under open-air laboratory conditions during the experiment, we expected that bromeliads carried residual bacteria and that bromeliads would be colonized by ambient bacteria from the laboratory, but the differences between the natural and pre-sterilized water created divergent environmental bacterial reservoirs that were distinguishable throughout the experiment. Specifically, environmental bacterial communities in bromeliads containing natural water had higher phylogenetic bacterial diversity (figure 1a; electronic supplementary material, table S1), higher operational taxonomic unit (OTU) richness (table 1 and electronic supplementary material, table S1) and lower proportions of putatively Bd-inhibitory taxa (electronic supplementary material, table S1) than pre-sterilized water (see below). The composition of environmental bacterial communities also differed between natural and pre-sterilized water (figure 2; electronic supplementary material, table S1; see below).

Characteristics of the aquatic environment (a,b) and tadpole microbiomes (c,d) including average of Faith's phylogenetic bacterial diversity (a,c) and average logged Bd zoospore genomic equivalents detected (b,d). Error bars represent s.e. (Online version in colour.)

For bromeliad microecosystems containing tadpoles exposed to Batrachochytrium dendrobatidis, a water treatment (natural or pre-sterilized), and an arthropod treatment (present or absent) for 17 days: bacterial OTUs detected in the aquatic environment (total 1237 OTUs from 21 phyla) and in tadpole microbiomes (total 860 OTUs from 16 phyla). Nine environmental samples were excluded after rarefaction and six environmental samples were excluded due to contamination. Six tadpoles could not be recovered from bromeliads at the end of the experiment.

Bacterial community composition of the aquatic environment (enclosed by dashed line) and tadpole microbiomes, calculated using principal coordinates analysis based on unweighted UniFrac distances. Small circles indicate data points; large circles indicate group centroids. (Online version in colour.)