Bacterial and yeast isolates were obtained from all three media used (PCA, YPDA, and GYC). Following isolation and purification, a total of 25 yeast and 163 bacterial isolates was obtained across the different isolation media from 28 individuals of the seven Epipactis species studied (Appendix 1). Bacteria were recovered from all seven species (Table 1), representing 28 (80%) of the individuals examined, whereas yeasts were only found in E. helleborine, E. microphylla, E. muelleri, and E. palustris, covering nine (26%) individuals in total (Table 2). Colony counts on plates showing microbial growth ranged from one colony (i.e., for one and three individuals belonging to E. muelleri and E. neglecta, respectively) to over 300 colonies per plate (representing an “uncountable plate”) (Appendix 1). Highest microbial densities were observed in nectar samples from the species E. atrorubens and E. helleborine (on average >300 colony-forming units (CFUs) per plate for the different individuals and media tested). On the contrary, for E. muelleri and E. neglecta only a maximum of 6 and 7 CFUs per plate, respectively, was obtained. Intermediate counts (30–300 CFUs/plate) were obtained for the three other species, including E. microphylla, E. palustris, and E. purpurata (Appendix 1).
Using a 1% cut-off value, a total of 38 species-level OTUs was detected (Table 1). Although the rarefaction curve was tending to approach saturation, the Chao 2 estimator gave a predicted OTU richness of 60 (63%), indicating that our sampling detected a major part, but not all, of the total estimated species richness (Fig. 1). The recovered bacteria belonged to four major phyla, including Actinobacteria (6 OTUs), Bacteroidetes (1 OTU), Firmicutes (13 OTUs), and Proteobacteria (Alpha and Gamma subdivisions; 18 OTUs), the latter being the most frequent one (63.8% of isolates) (Table 1, Figs. 2 and 3). On the family level, the family of Enterobacteriaceae (Proteobacteria) was the most common one, representing 46.6% of all isolates, followed by the family of Microbacteriaceae (Actinobacteria) (14.7% of isolates) and Bacillaceae (Firmicutes) (13.5% of isolates) (Fig. 3). By far, the most common bacterial isolates obtained in this study represented OTUs corresponding to a nonspecified Enterobacteriaceae bacterium, namely OTU B23 (12.9% of isolates) and OTU B24 (17.1% of isolates), each occurring in three out of seven Epipactis species (Table 1). Other OTUs that were identified (>97.8% sequence identity with GenBank sequence) included members from the genera Acinetobacter, Bacillus, Curtobacterium, Dermacoccus, Enterobacter, Erwinia, Frigoribacterium, Leuconostoc, Microbacterium, Methylobacterium, Paenibacillus, Pectobacterium, Plesiomonas, Pseudomonas, Serratia, Sphingomonas, Staphylococcus, Tatumella, and Terrimonas (Table 1; Fig. 2). In contrast to bacteria, the diversity of yeasts was much lower (Table 2, Fig. 2), with a total of only 10 OTUs based on a 1% DNA dissimilarity cut-off value (Table 2). These belonged to two phyla, including Ascomycota (2 OTUs) and Basidiomycota (8 OTUs). The basidiomycetous yeast Cryptococcus, belonging to the family of Tremellaceae, was the most common yeast (OTU Y5 – OTU Y8), and was recovered from three species (E. microphylla, E. muelleri, and E. palustris), representing seven investigated plants (Table 2). All other yeast OTUs were only recovered from one or two individuals (Table 2).
Figure 2. Bayesian 50% majority consensus tree showing phylogenetic relationships between different large subunit and small subunit rRNA gene sequences from nectar-inhabiting bacteria (a) and yeasts (b) retrieved from seven Epipactis species and reference sequences of type strains found in GenBank. For ease of visualization, the dataset was limited to one representative sequence for each operational taxonomic unit (OTU) found in this study at a DNA dissimilarity cut-off value of 1%. Sequences are annotated by an abbreviation for the Epipactis species (EAT, E. atrorubens; EHE, E. helleborine; EMI, E. microphylla; ENE, E. neglecta; EPA, E. palustris; EPU, E. purpurata), the medium number (1, YPDA; 2, PCA; 3, GYC) from which the isolate was obtained, followed by an isolate number (see also Table 2). Branch support: Bayesian posterior probabilities (BPP).
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The total number of bacterial OTUs per nectar sample varied between 0 and 7 (one E. purpurata individual), with an average of 2.1 OTUs per sample. The total number of bacterial OTUs that could be associated with the plant species ranged from 2 (E. muelleri) to 12 (E. microphylla), whereas the average number of bacteria per plant species varied between 0.4 (E. muelleri) and 3.8 (E. purpurata) (Fig. 4A). The total number of yeast OTUs per nectar sample varied between 0 and 4 (one E. microphylla individual), with an average of 0.5. On the species level, the total number of yeast OTUs associated with the investigated Epipactis species varied between 0 (E. atrorubens, E. neglecta, and E. purpurata) and 8 (E. microphylla), with an average of 0–2 (E. microphylla) OTUs per species (Fig. 4B). Taken together, total OTU richness (i.e., the total number of bacterial and yeast OTUs per Epipactis species) varied between 4 (E. atrorubens) and 20 (E. microphylla). Finally, cluster analysis revealed that microbial communities of allogamous species differed from those of autogamous and facultatively autogamous species (Fig. 5).
Figure 4. Total (dark colors) and average (light colors) of bacterial operational taxonomic units (OTUs) based on a DNA dissimilarity cut-off value of 1%, encountered in the floral nectar of seven Epipactis species. Green bars refer to strictly allogamous species, orange–yellow bars to facultatively autogamous species, and red–pink bars to autogamous species. Orchid species: Epipactis atrorubens (Eatr), E. helleborine (Ehel), E. muelleri (Emue), E. microphylla (Emic), E. neglecta (Eneg), E. palustris (Epal), E. purpurata (Epur).
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Figure 5. Cluster analysis of microbial communities in the floral nectar of seven Epipactis species using the Sorensen (Bray-Curtis) distance measure and the Farthest neighbor-linkage method. Allogamous species are indicated in green circles, facultatively autogamous species in yellow triangles, and autogamous species in red squares. Orchid species: Epipactis atrorubens (Eatr), E. helleborine (Ehe), E. muelleri (Emu), E. microphylla (Emic), E. neglecta (Ene), E. palustris (Epal), E. purpurata (Epur) .
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