|Goffredi et al - Supplemental Materials
General trends in bacteria associated with the commercial and artificial bromeliads. The bacterial community recovered from the commercially available species of Aechmea fasciata, with the highest tank pH, was uniquely dominated by Betaproteobacteria (48%) and Firmicutes (26%; Table 3, Figs. 7, S4). Only 4 bacterial families were shared among the commercial and natural bromeliads, regardless of pH. In addition to lack of exposure to the correct inoculum, commercial handling of bromeliads likely alters the chemical conditions within the tanks, possibly resulting in a dramatically different microbial community than observed naturally. For instance, the tank water of commercially grown plants is frequently removed or changed, resulting in a more neutral pH, less organic material for decomposition, and a disruption of deeper anoxic layers within the tank. Additionally, an artificial tank positioned in situ near bromeliad Amr1, for a duration of 12 mos, had a similar bacterial community to the plant host type, with the notable exception of many fewer Acidobacteria (3%) and greater abundance of Verrucomicrobia (20%; Table 3).
Phylogenetic identity of bacteria associated with bromeliads, regardless of pH. Members of the Bacteroidetes in this study were generally abundant within all bromeliad tanks (7-22% of the recovered ribotypes) and at least three groups, including the orders Bacteroidales, Sphingobacteriales, and Flavobacteriales were observed (Table 3, Fig. S2). While physiological potential cannot be determined exclusively from 16S rRNA similarity < 97%, many of the Bacteroidetes sequences recovered here grouped with organisms from other organic rich environments, including mangroves, wastewater, lake sediment, and phragmites rhizosphere, to name a few [2,3]. Ribotypes associated with bromeliad tanks clustered near taxa including Anaerophaga (family Marinilabiaceae) and members of the Chitinophagaceae (Fig. S2). The Chitinophagaceae, in particular, were present in all samples (including the artificial tank), although genus level differences were observed. The presence of the Chitinophagaceae is consistent with the idea that chitin, the main component of arthropod exoskeletons, is thought to be readily available within the bromeliad environment as insects become trapped and likely degraded by resident bacteria. Bacteria attached to chitin particles within the tank water were often visualized via fluorescence microscopy (Fig. S3). Surprisingly, Bacteroidetes were not recovered from the commercially available bromeliad, A. fasciata (Table 3).
Verrucomicrobia, typically observed in eutrophic or heavily polluted habitats, were also common (3-11% of recovered ribotypes) in the bromeliad tank habitat, including many previously recognized from organic rich environments, including sphagnum peat bogs and phragmites wetland sediments (Fig. S4). Notably, Verrucomicrobia comprised a much larger proportion (~20%) of the recovered diversity from the artificial tank, however, ribotypes were not associated with subdivision 3, as were the residents of the natural tanks. Additionally, Planctomycetes were also commonly recovered, especially from acidic bromeliad tanks, comprising, in one case, up to 15% of the recovered diversity, as compared to soil and the less acidic bromeliads (representing only 0-3%, Table 3, Fig. S4). Planctomycetes are now known to be ubiquitous inhabitants of soils, and all members currently in pure culture are chemoheterotrophic . This group may, thus, also play an important role in the breakdown of organic material within bromeliad tanks.
Supplemental Figure legends
Figure S1. Bromeliad species used in this study. (A) Aechmea mariae-reginae (‘Amr’), approx. 145 cm across. (B) A. nudicaulis (‘An’), approx. 15-20 cm across.
Figure S2. Phylogenetic relationships among Bacteroidetes, Verrucomicrobia, and Planctomycetes associated with Costa Rican bromeliads, an artificial tank, and a nearby soil sample, relative to selected cultured and environmental sequences in public databases, based on sequence divergence within the 16S rRNA gene. A neighbor- joining tree with Kimura two-parameter distances is shown with Zea mays (X86563) used as an outgroup (not shown). The symbols at the nodes represent bootstrap values from parsimony methods obtained from 100 replicate samplings (open symbol = 70-90%, closed symbol = 90+% bootstrap support).
Figure S3. Fluorescence in situ hybridization (FISH) microscopy showing a variety of bacteria (red) recovered from bromeliad tank debris in association with chitin (green). Cells were hybridized with a general bacterial probe (Eub338) labelled with Cy3 while chitin was labeled with a fluorescein conjugated bacterial chitin binding protein (P5211S, New England Biolabs). Scale bars = 10 µm. Samples for FISH, initially preserved in paraformaldehyde, were rinsed twice with 1X phosphate-buffered saline (PBS), transferred to 70% ethanol, and stored at -20°C. Hybridization and wash steps were carried out according to , with 50 g ml-1 Eub338_Cy3 probe and 10 g ml-1 chitin-binding probe. Samples were imaged on a Nikon Eclipse E80i fluorescence microscope
Figure S4. Phylogenetic relationships among Firmicutes associated with Costa Rican bromeliads, a commercially available bromeliad, and a nearby soil sample, relative to selected cultured and environmental sequences in public databases, based on sequence divergence within the 16S rRNA gene. A neighbor- joining tree with Kimura two-parameter distances is shown with Flavobacterium psychrophilum, (AF090991) used as an outgroup (not shown). The symbols at the nodes represent bootstrap values from parsimony methods obtained from 100 replicate samplings (open symbol = 70-90%, closed symbol = 90+% bootstrap support). +/- designations in brackets following ‘Bio325’ strains indicate their respective abilities to degrade pectin, cellulose (via -glucosidase), and chitin, in that order.
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