www.nature.com/scientificreports OPEN Distribution, Interaction and Functional Profles of Epiphytic Bacterial Communities from the Rocky Intertidal Seaweeds, South Africa Ramganesh Selvarajan1*, Timothy Sibanda2, Siddarthan Venkatachalam3, Henry J. O. Ogola1,4, Chinedu Christopher Obieze 5 & Titus A. Msagati6 Interrelations between epiphytic bacteria and macroalgae are multifaceted and complicated, though little is known about the community structure, interaction and functions of those epiphytic bacteria. This study comprehensively characterized the epiphytic bacterial communities associated with eight diferent common seaweeds collected from a rocky intertidal zone on the Indian Ocean at Cape Vidal, South Africa. High-throughput sequencing analyses indicated that seaweed-associated bacterial communities were dominated by the phyla Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, Planctomycetes, Actinobacteria and Verrucomicrobia. Energy-dispersive X-ray (EDX) analysis showed the presence of elemental composition in the surface of examined seaweeds, in varying concentrations. Cluster analysis showed that bacterial communities of brown seaweeds (SW2 and SW4) were closely resembled those of green seaweeds (SW1) and red seaweeds (SW7) while those of brown seaweeds formed a separate branch. Predicted functional capabilities of epiphytic bacteria using PICRUSt analysis revealed abundance of genes related to metabolic and biosynthetic activities. Further important identifed functional interactions included genes for bacterial chemotaxis, which could be responsible for the observed association and network of elemental-microbes interaction. The study concludes that the diversity of epiphytic bacteria on seaweed surfaces is greatly infuenced by algal organic exudates as well as elemental deposits on their surfaces, which triggers chemotaxis responses from epiphytic bacteria with the requisite genes to metabolise those substrates. Microorganisms are found associated with a wide variety of hosts including animate and inanimate1. In some cases, microorganisms are increasingly recognized to play a signifcant role in host health and metabolism2,3. For example, in terrestrial ecosystem, plants are highly dependent on their associated microbes to support their development and protection under stressors4. Similarly, in marine ecosystems, the surface of marine organisms like macroalgae (seaweeds) ofen represents a highly active association between hosts and microbes5. Tese microbes play crucial roles such as supplying the seaweeds with vitamins6 and fatty acids7, as well as regulating growth of the seaweed8. In addition, some are producers of antimicrobials9 while others are involved in nutrient biogeochemical cycling and hence support growth and adaptation and acclimation to environmental stress10. Seaweed-associated epiphytic microbial communities are extremely complex, and can best be described as cos- mopolitan, constituting of bacteria, fungi, archaea, viruses diatoms and protozoa8. However, their biodiversity and functional associations remain largely unexplored and poorly understood. 1Department of Environmental Sciences, College of Agricultural and Environmental Sciences, UNISA, Johannesburg, South Africa. 2Department of Biological Sciences, University of Namibia, Mandume Ndemufayo Ave, Pionierspark, Windhoek, Namibia. 3Arctic Division, National Centre for Polar and Ocean Research, Vasco-da-Gama, Goa, India. 4Centre for Research, Innovation and Technology, Jaramogi Oginga Odinga University of Science and Technology, Bondo, Kenya. 5Department of Microbiology, University of Port Harcourt, Port Harcourt, Nigeria. 6Nanotechnology and Water Sustainability Research Unit, College of Science, Engineering and Technology, University of South Africa- Science Campus, Florida, South Africa. *email: [email protected] SCIENTIFIC REPORTS | (2019) 9:19835 | https://doi.org/10.1038/s41598-019-56269-2 1 www.nature.com/scientificreports/ www.nature.com/scientificreports SW1 SW2 SW3 SW4 SW5 SW6 SW7 SW8 OTUs 431 1038 504 316 737 460 120 746 Dominance_D 0.034 0.052 0.121 0.147 0.025 0.045 0.431 0.127 Simpson_1-D 0.967 0.948 0.879 0.853 0.975 0.955 0.569 0.873 Shannon_H 4.502 4.065 3.075 3.024 4.847 3.983 1.544 3.647 Evenness_e^H/S 0.209 0.0561 0.043 0.065 0.1728 0.117 0.039 0.051 Chao-1 525.01 523.13 611.85 480.19 997.01 722.62 83.11 1072.71 ACE 220.07 179.33 381.61 173.42 444.1 307.72 40.38 445.39 Table 1. Estimated OTU richness and diversity indices of the eight diferent seaweeds collected from intertidal zones of Mission Rocks, South Africa. Earlier, studies investigated the bacterial communities of seaweeds using culture dependent analysis7,11–13. In one such study, Beleneva and Zhukova14 reported Vibrio spp. as the single most abundant bacteria on the surfaces of brown and red macroalgal species collected from Peter the Great Bay in the Sea of Japan. Other identifed bacterial strains included Sulftobacter spp., Halomonas spp., Acinetobacter spp., Planococcus spp., Arthrobacter spp., and Agromyces spp. Similarly, Wang et al.15 reported Halomonas and Vibrio as the major bacterial groups isolated from the surfaces of four diferent seaweed in China. Tey observed closely-knit genetic relationships among the isolates, which also displayed host species specifcity. Host specifcity observations as well as temporal and spatial variations were further confrmed by culture-independent studies of Egan et al.16. Other researchers studied bacterial communities in green (Chlorophyta), brown (Phaeophyta) and red (Rhodophyta) macroalgae using community fngerprinting methods such as denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP)17–19. Most community analysis revealed that members of Alphaproteobacteria and Gammaproteobacteria were the dominant bacterial-seaweed associated phyla. Other frequently encountered phyla include the Bacteroidetes, Actinobacteria, Planctomycetes and Chlorofexi19–22. Te advent of culture independent techniques has seen a great deal of research efort being focused on exploring microbial communities associated with seaweeds in order to understand the ecological basis of seaweed-microbe interactions8. Some research fndings seem to insinuate that diferent macroalgal species in the same ecological niche are associated with specifc bacterial communities19,20, while others suggest that macroalgal species can have similar bacterial communities even if they are located in diferent ecological niches14,15. In addition to spatial variabil- ity, epiphytic bacterial community compositions are also driven by diferences in seasonal, environmental and physiological characteristics including elemental composition and nutrient availability23. Te occurrence and relative abundance of epiphytic bacterial species may also vary depending on their specifc interactions with abiotic factors. In the last two decades, advances in next generation sequencing technologies have now made it feasible to characterize the core seaweed-associated epiphytic bacterial communities from diferent ecological niches. Up until now, the diversity of epiphytic bacterial communities has most likely been underestimated due to the lim- itations of the conventional methods8,16. Consequently, there is a need for in-depth analysis to characterize the epiphytic bacterial community compositions as well as to understand the epiphytic bacterial functions and their interactions with abiotic gradients. In this study, we used next-generation sequencing analysis to provide in-depth insights into the diversity of seaweed-associated epiphytic bacterial communities of eight diferent seaweed spe- cies that are commonly found in the rocky intertidal zone at Cape Vidal, South Africa. Additionally, prediction of functional abilities and bacteria-seaweed interactions using co-network analysis allowed us to infer diferences and relationships across seaweed epiphytes. To the best of our knowledge, this is the frst study to investigate the seaweed associated epiphytic bacterial communities using next generation sequencing technology in South Africa. Results Scanning electron microscopy of collected seaweeds revealed variations in surface morphology between the sea- weed species (S. Fig. 1). Results of energy-dispersive X-ray (EDX) analysis showed the presence of ten diferent chemical elements in the cell wall at varying concentrations dependent on seaweed type. Among the identifed ten elements, the most abundant was carbon, with higher level detected in Hypnea rosea (SW3: 71.01%), followed by Sargassum incisifolium (SW2: 64.14%). Comparatively, red seaweeds (SW6 & SW7) had higher levels of calcium than other seaweeds, while potassium was higher in green and brown seaweeds than red seaweeds. Sulphur con- tent ranged between 0.36–4.45%, with a magnitude in the order of SW1≫SW5 > SW8 > SW6 > SW3 > SW7 > S W2 > SW4. Te detailed elemental compositions in the seaweeds are given in S. Table 1. Epiphytic bacterial diversity. Afer fltering out low quality and chimeric sequences, a total of 243,488 quality reads were obtained from the eight diferent seaweed samples, which were then used for downstream analysis. Overall, 4,352 OTUs were predicted across all the samples based on the Silva rRNA gene database at the cut-of level of 97%. Among the eight seaweed samples, brown seaweed SW2 (1,038 OTUs) harboured the highest number of unique OTUs, while the lowest number of OTUs were recorded in red seaweed SW7 (120 OTUs). In contrast, sample SW8