To our knowledge, this is the first study ever conducted to evaluate the geographic and environmental specificity of the epiphytic bacterial communities of red macroalgae G. lemaneiformis cultivated at three different geographic locations by using next generation sequencing. Previous studies focused on spatial and temporal diversity of the epiphytic bacterial communities from macroalgae14,18,21. Few reports demonstrated that differences in the epiphytic bacterial communities composition on the surface of macroalgae from different geographic locations are likely due to environmental selections18,32. Environmental selection can produce substantial biogeographic patterns in the global microbe population33. This suggests that there are selective mechanisms in place, which determine the assemblage of species that exist in one environment or the other13. In order to understand this phenomenon, we investigated the epiphytic bacterial communities composition and their correlation at spatial level and as well as the role of different environmental factors such as nitrogen and phosphorous in shaping epiphytic bacterial communities composition. In our study, the similarity among the epiphytic bacterial communities were higher in G. lemaneiformis collected from three different sampling sites which are 300 m apart i.e. NR: 17.5%-66.92% similarity, NA: 12.5%-64.77% similarity, and LJ: 50%-75.07% similarity when compared with the G. lemaneiformis collected from NA and LJ which are 500 km apart exhibiting only 7.5% similarity. In addition to that we also found that the similarity of the epiphytic bacterial communities among different sampling sites in any of the three locations was much higher than that between the two most distant locations (NA and LJ, 500 km apart). This correlation fits in the distance-decay of similarity model, in which the decrease in similarity of microbial communities is related to the increase of geographic distance28 and our findings are consistent with previous study conducted by Roth-Schulze et al.1 Variations in similarity of epiphytic bacterial communities correlated by distance have also been observed in marine, soil, sediment and plant-associated ecosystems1.
We found considerable variations of epiphytic bacterial communities composition of G. lemaneiformis among three different geographic locations. Briefly, the epiphytic bacterial communities of G. lemaneiformis from NR, NA, and LJ were mainly composed of Flavobacteriaceae, Saprospiraceae, Muribaculaceae (Bacteroidetes), Hyphomonadaceae, Sphingomonadaceae, Rhodobacteraceae (Alphaproteobacteria), Cellvibrionaceae, Thiohalorhabdaceae, Nitrincolaceae, Thiotrichaceae, Halomonadaceae (Grammaproteobacteria), Ruminococcaceae, Lachnospiraceae (Firmicutes), Microtrichaceae (Actinobacteria), Trueperaceae (Deinococcus-Thermus) and Rare taxa (S. Fig. 1 & S. Tab. 3). These observations are consistent with previously demonstrated studies of marine algal-associated bacterial communities. For example, the epiphytic bacterial communities on the surface of the red alga Delisea pulchra was found to be comprised of Rhodobacteraceae, Sphingomonadaceae, Flavobacteriaceae, Planctomycetaceae and unclassified Grammaproteobacteria34, whereas the green alga U. australis hosts Alphaproteobacteria, Bacteroidetes, Planctomycetes and unclassified Grammaproteobacteria13. Fucus vesiculosus has been found to be associated with a high proportion of Alphaproteobacteria, Bacteroidetes, Verrucomicrobia, and Cyanobacteria in summer, while Grammaproteobacteria in winter35. The core microbial communities were often defined as the suite of members shared among microbial communities from similar habitats36. Discovering core microbial communities is important for understanding the stable and consistent components across complex microbial assemblages. Our study revealed significant differences of the core epiphytic bacterial communities across the three different geographic locations (S. Fig. 2). For instance, G. lemaneiformis at NR had Cyclobacteriaceae, Pseudomonadaceae, Sphingobacteriaceae, Xanthobacteraceae, Burkholderiaceae, Beijerinckiaceae, Nocardiaceae, Rhizobiaceae and Micrococcaceae as the dominant bacterial family, while at NA and LJ Veillonellaceae, Bifidobacteriaceae, Coriobacteriaceae, Porticoccaceae, Bacillaceae and Sandaracinaceae, Staphylococcaceae, Corynebacteriaceae, Blastocatellaceae, RhizobialeslncertaeSedis, Flammeovirgaceae, Shewanellaceae were found dominate bacteria respectively.
Hence, these findings lead to conclusion that these epiphytic bacteria on the surface of different macroalgae may be vital to hosts. For example, cross-kingdom chemical signals derived from members of Halomonas (Halomonadaceae), Roseobacter (Rhodobacteraceae) and Sulfitobacter (Flavobacteriaceae) are beneficial for the thallus development of Ulva mutabilis (Chlorophyta)37. Few studies reveal that Dimethylsulfoniopropionate (DMSP) plays a key role in the macroalgal-bacterial interactions38. Alphaproteobacteria, which are morphologically and metabolically extremely diverse14, have a critical role in the assimilation of DMSP in the oceans and contribute significantly to the global sulphur cycling39. Furthermore, DMSP, usually produced by macroalgae including Ulva sp., attract some bacteria38. It’s worth mentioning that the members of family Hyphomonadaceae are widely dispersed in marine environment and play an important role in mineralizing dissolved organic matters in oligotrophic waters18. From our finding we assume that the ability of G. lemaneiformis to grow normally in three different oligotrophic waters may partly benefit from the mineralization of these microorganisms on its surface. In addition, previous study performed by Holmström et al.40,41 had suggested multiple bioactive compounds produced by the genus Pseudoalteromonas present on the surface of Ulva lactuca might play an important role in the chemical defense against biofouling in the marine environment.
Studies on holobionts had shown either host-specificity of epiphytic bacterial communities within different species, or geographic differences of epiphytic bacterial communities between different locations1,16,18,21,32. For instance, the epiphytic bacterial communities on the surface of different host species were taxonomically and functionally distinct, and this distinction was not due to the phylogeny of host, but was due to physicochemical properties of host16. As Lachnit et al.42 suggested that the difference in the epiphytic bacterial communities on different algae host are due to the physiochemical properties of macroalgae surface which allow the settlement and colonization of specific bacteria. In terms of geographic diversity, Roth-Schulze et al.1 demonstrated that the same algal-genus across different regions can harbor different microbial communities at the taxonomic and functional level, which could be due to local geographic conditions and host specificity.
To explore the specificity of the epiphytic bacterial communities associated with G. lemaneiformis and its correlation with different environmental factors. Our study demonstrates that the concentrations of TN, TP, NO3-N and DIN at NR were significantly higher as compare to NA and LJ (p < 0.05), while the concentration of NO2-N at NR was significantly lower as compare to LJ (p < 0.05) and NA (p > 0.05) besides having similar environmental conditions i.e. temperature, pH, salinity, dissolved oxygen, electrical conductance, and total dissolved solid. It has been reported that changes in environmental conditions, such as nutrient concentration, nutrient ratio and temperature, can affect the physicochemical properties of macroalgae43. Subsequently, Van Alstyne44 suggested that Ulva lactuca and Ulva obscura grown in high nitrogen concentration have higher DMSP content than that in low nitrogen concentration. It suggests that different environmental conditions can affect the content of algal-associated compounds. There are increasing evidences which demonstrate that the compounds associated with algal surface can mediate epiphytic bacterial colonization, abundance and communities composition of macroalgae45–47. The above studies also show that the differences in the epiphytic bacterial communities composition of macroalgae led by the changes of environmental factors is attributed to the variation of physicochemical properties on the surface of macroalgae. Therefore, we speculate that the variation of the epiphytic bacterial communities of G. lemaneiformis is probably related to the differences of algal-associated compounds caused by environmental conditions.
Notably, there were considerable changes of the epiphytic bacterial communities on the surface of G. lemaneiformis from three different geographic locations regardless of the taxonomic level (Figure 5, S. Fig. 1 and S. Tab. 3). At the level of phyla (Figure 5), the most predominant phylum associated with G. lemaneiformis at NR was Bacteroidetes, while Proteobacteria at NA and LJ. Our findings are similar with previous study of surface-associated bacterial communities on macroalgae, conducted by Burke et al.13, which reveal that epiphytic bacteria of U. australis are dominated by Proteobacteria- and Bacteroidetes13. While at family level (S. Tab. 4), the relative abundance of Muribaculaceae, Ruminococcaceae and Lachnospiraceae at NR was significantly higher (p < 0.05) than that at NA and LJ, and no significant differences (p > 0.05) was observed between NA and LJ. The above results showed that there were significant differences of the epiphytic bacterial communities composition on the surface of G. lemaneiformis between NR and NA and also between NR and LJ, but no significant differences were observed between NA and LJ which is contrary to previous reports which reveal that the algal epiphytic bacterial communities varies from different locations1,32. The possible explanation for that might be due to secretion of certain compounds on the surface of G. lemaneiformis which regulate the epiphytic bacteria communities composition.
Furthermore, in order to have better explanation of our findings, RDA48 and Pearson correlation analysis30 are employed. Interestingly, the epiphytic bacterial communities on the surface of G. lemaneiformis at NR exhibited a positive correlation with TN, TP, NO3-N and DIN, and a negative correlation with NO2-N (Figure 6 and 7). For example, clinically important bacterial genus, Escherichia-Shigella, was found on the surface of G. lemaneiformis at NR2 and was extremely significant correlated with NO3-N, DIN, TP and TN (Figure 7). Escherichia-Shigella, an enteric pathogen, being released from waste water treatment plant49 and can secrete toxins to the surrounding environment50. The correlation between the abundance of Escherichia-Shigella and environmental factors could be of ecological/health concerns51. However, the epiphytic bacterial communities on the surface of G. lemaneiformis at NA and LJ showed a positive correlation with NO2-N, and a negative correlation with TN, TP, NO3-N and DIN. The above results lead to make conclusion that the differences of epiphytic bacterial communities associated with G. lemaneiformis from different geographic locations is because of environmental factor rather than different geographical locations, which is consistent with previous study18. In that study Asparagopsis-associated bacterial communities have been observed to be modulated by environmental conditions. Moreover, Roth-Schulze et al.1 suggested that most of the Ulva-associated bacterial communities are horizontally derived from the environment as macroalgae U. australis isolated from distinct geographic locations have been observed to share only two low-abundance OTUs. We observed different findings that G. lemaneiformis from NA and LJ (500 km away from each other) only share 7.5% similarity. It is surprisingly interesting that environmental factors rather than geographic different locations caused changes in the epiphytic bacterial communities of G. lemaneiformis, which is contrary to what was previously reported by Roth-Schulze et al.,1. Therefore, we hypothesize that the epiphytic bacterial communities of G. lemaneiformis is regulated by nitrogen and phosphorus (environmental factors) rather than geographical locations.
Replicate samples from the same Gracilaria species found at the same location showed certain variability in the epiphytic bacterial communities composition (17.5%-66.92%, 12.5%-64.77%, 50%-75.07% Bray-Curtis similarity for NR, NA and LJ, respectively) which is similar with previous studies which have shown high level of intraspecies variability of epiphytic bacterial communities associated with macroalgae1,15. This could be due to the depth level of sequencing14, or as reported in lottery model in which random variation can be seen in the recruitment of the epiphytic bacterial communities on algae surface22 which was in actual developed for the coexistence of reef fish species share the same niche. Briefly this hypothesis asserts that recruitment of species having same tropic abilities in any of the ecosystem is stochastic fashion i.e. who so ever gets there first wins the space, but they must share similar ecologies52,53. Furthermore, in order to draw a clear hypothesis regarding the environmental factor and their role in the selection of the epiphytic bacterial communities of macroalgae, we suggest a study for the comparative epiphytic bacterial communities composition of other algae in the presence of similar environmental factors in future.
Moreover, it is worth noting that there were considerable changes in the composition of epiphytic bacterial communities on G. lemaneiformis at three different geographic locations, however the functional composition of epiphytic bacterial communities remained similar. There is an emerging consensus that the bacterial community composition on macroalgae is mainly driven by functional genes rather than taxonomic composition15,17. In our study, the functional capabilities of epiphytic bacterial communities on G. lemaneiformis at different locations were similar, which indicate similar functions of these bacterial communities at different locations. As regard the fundamental factor that causing the differences in the composition of bacterial communities was microenvironment established by the physiological and biochemical properties of the algal host3,17.
We found bacterial genes associated with these amino acids including glycine, alanine, arginine, proline, glutamic, and aspartic acids which majorly contribute in algal proteins54 (S. Tab. 4). This could be an explanation for higher percentage of bacterial genes assigned to amino acid metabolism in our study (Figure 8). In addition, abundant functional genes related to carbohydrate metabolism found which are believed to be involved in the mineralization of dissolved organic matter under oligotrophic environment of coastal water (Figure 8). Similar to our finding, Selvarajan et al.17 reported higher abundance of bacterial functional genes associated with carbohydrate metabolism in all seaweeds at intertidal zones of Mission Rocks, Cape Vidal, Leven Point, South Africa. The limitation of current study is that all the inferences are based on predicted functional characters by PICRUSt annotation. It is not a complete substitute for metagenomic research, so there could be inherent inaccuracies in interpreting functional biogeography in certain ecosystem.