Microbial community distribution patterns, environmental drivers, microbial community species interactions, and community building mechanisms are the focus of microbial ecology research. In this study, we analyzed the dynamics of EBC and their environmental impact factors from July to November, and revealed the structural changes, co-occurrence pattern characteristics, and community assembly processes of epiphytic biofilm bacterial communities.
The EBC diversity of the Caohai submerged plants in November was higher than that in July (Xia et al., 2020). The change in the bacterial community at the phylum level on the surface of macroalgae is driven by the season (Lachnit et al., 2011). In this study, the diversity of EBC showed an increasing trend from July to November when analyzing its characteristics. The differences in epiphyte community composition between growing seasons were mainly due to changes in host growth status and surrounding environmental factors (Xia et al., 2020). Diatom species diversity and composition exhibit large seasonal or spatial variation, which may be related to the life history, leaf morphology, and characteristics of the host plant (He et al., 2020). This difference is closely related to the development of epiphytic biofilms. Planktonic bacteria increase the diversity and abundance of EBC through adsorption, adhesion, and biofilm formation (Kimkes and Heinemann, 2020).
Biological interactions are considered the main drivers of microbial assembly processes (Ju and Zhang, 2015). Identifying microbial interactions with keystone species is essential for a better understanding of microbial community diversity and function (Ren et al., 2015). In freshwater ecosystems, the interactions between epiphytic bacterial taxa are largely unknown. Network analysis is an effective tool for describing microbial interactions at different taxonomic levels (Manoeli et al., 2014). Construction of correlation-based species–species co-occurrence networks that can reflect important details of ecological processes such as community cooperation, habitat filtering, and historical effects, as well as reflecting mathematical associations between different bacterial populations (Zhao et al., 2016). From summer to winter, the planktonic bacterial co-occurrence network evolved from complex to simple, and the benthic bacterial community had strong seasonal stability (Jiao et al., 2020). Our study shows that the number of ecological connections (links) between EBC nodes and network connectivity and complexity gradually increased from July to November.
The differences in networks between the time periods may be because the bacterial community composition differs between seasons; bacterial diversity is higher in November, and higher biodiversity promotes interactions between bacterial communities (Sun et al., 2021a), thus increasing their co-occurrence patterns (Widder et al., 2014). Simultaneously, bacterial diversity and abundance increase, and bacteria utilize quorum sensing (Qs) to further develop a complex and well-structured community lifestyle by activating or repressing target genes (Haque et al., 2019). Intercellular communication promotes the development of biofilms and enhances interactions between bacterial communities (Guzmán-Soto et al., 2021). Finally, EBC forms a dense community with stronger interactions between bacterial members (e.g., metabolic linkage and horizontal gene transfer) (Broszat and Grohmann, 2014). The dynamics of EBC are influenced by the host plant, the period of plant growth (Li et al., 2021), differentiated morphology or metabolic profile (Hempel et al., 2009), plant leaf size and certain traits of leaves (e.g., enzyme activity and secondary metabolites) (Buchan et al., 2014), and plant characteristics (He et al., 2020). Host plants influence the bacterial community and thus, the co-occurrence pattern of the network. Furthermore, temperature is often considered to be one of the key factors influencing seasonal variation in bacterial symbiosis patterns (Ren et al., 2019). Water temperature simultaneously influences various other environmental factors (e.g., DO, Chl a, and metabolic rate), thus exerting a strong control on the biodiversity patterns in aquatic ecosystems (Wang et al., 2019).
Increased complexity can also lead to increased ecological community stability (Herren and McMahon, 2018; Mougi and Kondoh, 2012). The community structure may be more stable in November than in other months. We also determined the topological role of each OTU in a microbial network composed of all biofilm samples using network analysis. Prediction of Keystone OTUs in the EBC network is based on network scores (Pi and Zi), and these keystone species play an essential role in maintaining the complexity and stability of the EBC network (Banerjee et al., 2018; Herren and McMahon, 2018). More keystone species can lead to more stable and organized microbial networks (Banerjee et al., 2018; Qi et al., 2019). The influence of key taxa on community structure in co-occurrence networks is not governed by their relative abundance, but rather by their strong ecological associations (Banerjee et al., 2018; Shi et al., 2016). October and November had the most complex co-occurrence pattern and had more keystone species compared to other months, indicating a gradual stabilization of the EBC structure. The change from relatively scattered bacterial communities to the formation of mature and stable bacterial communities from July to November may be a developmental process of biofilms from formation to maturity.
Ecological niche theory and neutrality theory are used to explain the process of microbial community construction (Bahram et al., 2016; Sloan et al., 2006). The ecological niche theory (deterministic process) emphasizes the importance of determinism and proposes that ecological selection is a unique ecological process that affects species homogeneity and abundance (Chave, 2004). Neutral theory (stochastic processes) emphasizes that neutral, often stochastic processes can largely regulate the assembly and dynamics of community structure (Vanwonterghem et al., 2014). Several studies suggest that deterministic processes play a major role in community construction (Chen et al., 2021; Gad et al., 2020; Jiao et al., 2020; Zhou et al., 2021), while others have reported the dominant role of stochastic processes (Chen et al., 2019; Sun et al., 2021b; Wang et al., 2020). In the present study, the EBC was dominated by deterministic processes. Compared to planktonic and benthic bacterial communities, epiphytic biofilms have many specific physicochemical properties, such as vegetative properties that can shape bacterial communities to exhibit "host specificity"(Hempel et al., 2009), which may lead to more complex assembly mechanisms.
After the formation of microcolonies of epiphytic bacteria, cells continue to proliferate and aggregate (Haagensen et al., 2015). The synthesis of extracellular polymers (EPS), which form a "closed microenvironment,” can reduce the diffusion rate of bacteria (stochastic process) (Seymour et al., 2017). Simultaneously, complex interactions between algae and bacteria attached to the biofilm may increase biological selection (deterministic process) (Foster et al., 2011).
Our results show that the deterministic processes dominate the construction of the EBC, and the ratio of deterministic to stochastic processes varies among months, with the relative proportion of deterministic processes gradually increasing and the relative proportion of stochastic processes decreasing. Deterministic processes include selection imposed by environmental filtering and biological interactions, and stochastic processes include diffusion, random birth/death, and drift (Burns et al., 2016; Xue et al., 2018). From July to November, the complexity of the bacterial co-occurrence network increased, indicating that the interaction between species was enhanced (Wu et al., 2019), and that the deterministic process may be gradually enhanced, with the alpha diversity of bacteria also showing an increasing trend. The November EBC has higher biodiversity, higher network complexity, and more stable communities which are less susceptible to random processes (diffusion and drift). These results suggest that deterministic processes may play an important role in maintaining the diversity and function of epiphytic bacteria in aquatic ecosystems. Concurrently, the increase in temperature enhanced the metabolic dynamics and molecular irregular movement of the bacterial community, increasing the randomness of the colonization and extinction of bacterial community members (Ren et al., 2017). The bacterial community may be more affected by randomness in summer, which is consistent with our results. However, (He et al., 2020) showed that random processes played a dominant role in community construction and were significantly correlated with alpha diversity, and random processes may be beneficial for the maintenance of epiphytic bacterial diversity. Therefore, in different lakes, the process of biofilm bacterial community construction of submerged plants may not be consistent and may be related to lake nutrient status, temperature, and even host plants. Subsequently, it is necessary to carry out studies on biofilm bacterial communities in different lakes to clarify the construction mechanism and influencing factors of biofilm bacterial communities.
Co-occurrence networks allow for a more in-depth analysis of ecological processes of microbial community construction, such as neutral processes and species selection (Faust and Raes, 2012; Layeghifard et al., 2017). In this study, random and deterministic processes jointly regulated the dynamic changes in EBC. With the gradual strengthening of the dominant role of deterministic processes, the co-occurrence model had a larger network scale, and the relationship between species became closer. Deterministic processes may be beneficial in shaping the complex co-occurrence patterns of EBC.