Freshwater ecosystems play an important role in providing habitats for a variety of unique species, supporting key ecosystem functions, and providing essential ecosystem services (Cid et al., 2021). However, aquatic biodiversity in freshwater ecosystems has faced significant threats and dramatic declines globally due to increasing anthropogenic disturbances, such as hydrological alteration, habitat fragmentation, overexploitation, and water pollution (Vörösmarty et al., 2010; Burlakova et al., 2011; Geist, 2011; Reis et al., 2019). Hydrological connectivity is vital to sustaining hydrological and ecological processes between the river and adjacent waterbodies such as secondary channels, lakes, and wetlands (Amoros and Bornette, 2002). First, hydrological connectivity can have a profound effect on aquatic microbial communities through pH, organic substrate availability, and nutrient concentrations (Argiroff et al., 2016). Then, a high degree of hydrological connectivity supports a high level of species richness (Heino et al., 2014), including fish, macroinvertebrates, and zooplankton (Leigh and Sheldon, 2009; Shen and Liu, 2021; Zheng et al., 2022) because of the high dispersal rate. The rivers and lakes that were connected can be understood as a continuum, particularly in studies of aquatic microbial ecology (Tang et al., 2020). At present, there is a knowledge gap concerning the relationships between microbial communities in lakes and their input rivers in the river–lake continuum. The extent to which input rivers influence microbial communities in their downstream lakes has yet to be thoroughly investigated. This limited our understanding of aquatic ecosystem structure and function, preventing effective predictions of lake ecosystem responses to environmental change.

Benthos macroinvertebrates serve as excellent indicators of aquatic ecosystem structure and function because they contribute to biogeochemical cycling such as carbon, nitrogen, and sulfur (Dos Santos et al., 2011; Elgueta et al., 2021). For example, a high Tubificidae and Chironomus larvae density can influence the release of nitrogen and sulfur, which can accelerate the decomposition rate of organic detritus, as well as regulate the exchange of matter between sediments and water (Fukuhara and Sakamoto, 1987). Neotrypaea californiensis, another globally distributed macrofauna, transports oxygenated and anoxic water in sediments, causing oxic-anoxic oscillations (Liu et al., 2012). In spite of the direct impact macroinvertebrates have on sediment biogeochemical cycling, it has been suggested macroinvertebrates contribute more to sediment processes through their diverse direct and indirect interaction with sediment microbes (Grandy et al., 2016; Petersen and Osvatic, 2018). Caddisflies created niches enriched with denitrifiers to engineer the stream microbiome through low-oxygen niche creation (Bertagnolli et al., 2023). The presence of Naidid worms increased the relative abundance of Betaproteobacteria and decreased the relative abundance of Chlorobi in the surface sediment (Zeng et al., 2014). In addition to facilitating the vertical and horizontal distribution of the elements along with the sediment, various physical activities of macrozoobenthos disrupt natural sediment architecture and affect benthic microbial assemblage by reshuffling substratum during various physical activities (Adámek and Maršálek, 2012; Chakraborty et al., 2022).

In the past few decades, researchers have been interested in quantifying the influence of macrozoobenthos on sediment microbes, yet most studies on microbial responses to invertebrates have focused on single species of invertebrates, such as earthworms, isopods, or millipedes (Maraun and Scheu, 1996; Dempsey et al., 2011; Crowther et al., 2015). Researchers studying the relationship between macroinvertebrate-driven changes in microbial community composition and functions have found that macroinvertebrates can significantly impact microbial biomass and have contrasting effects on fungal and bacterial communities (Chang et al., 2017). However, in natural environments, multiple macroinvertebrate taxa are simultaneously influencing microbial communities through different pathways. Therefore, accurately measuring microbial responses to diverse macroinvertebrates at the community level is essential for comprehending the role of macrozoobenthos in shaping soil microbial communities.

Dongting Lake is the first large lake in the area downstream of the Yangtze River basin, characterized by a clear seasonal rhythm consisting of a dry season and a flood season with a highly fluctuating water level (Liang et al., 2018). As a representative river-connected lake, Dongting Lake receives water from four primary tributaries (Xiangjiang River, Zishui River, Yuanjiang River, and Lishui River) and has three outfalls (Songzi, Taiping, and Ouchi). Inflow water is regulated by Dongting Lake, which discharges into the Yangtze River. Thus, Dongting Lake is an inland lake with a flood channel and has a complex river–lake relationship with its inflow rivers. It is an ideal site at which to identify the effects of tributary rivers on the microbial communities in the main lake. Rivers and lakes can only generally be understood as a continuum because rivers change constantly as they move from headwaters to downstream lakes (Ylla et al., 2013). Previous studies have found that the discharge from the upstream tributaries in the lake basin and the water recharge via the connection to the Yangtze River outside the basin would influence the nutrient concentration of water and sediment in Dongting Lake directly (Tian et al., 2017). Meanwhile, microorganisms travel from one habitat to another along with particulate organic matter as a result of river and lake connectivity (Tang et al., 2020). Thus, a reorganization of microbial communities is created with the influences of environmental factors. It is crucial to take into account the entire network of interconnected water bodies (such as rivers and lakes) in order to comprehensively grasp the principles governing the assembly of microbial communities in aquatic ecosystems.

While the temporal and spatial variations of microbial community composition in Dongting Lake and its upstream tributaries have been examined recently, little is known about what and how the biotic and abiotic variables influenced the microbial community under the river–lake continuum. In this study, the Dongting Lake watershed worked as a typical river-connected lake ecosystem to investigate the microbial diversity, community composition, and functional types in both tributary river and lake systems simultaneously. We hypothesized that (i) the main lake has higher species diversity than the tributary rivers because of more unique species from various tributary rivers; (ii) biotic (macrozoobenthos) and abiotic factors both help drive the microbial community pattern in sediment; and (iii) the functional types would be influenced with the replacement of unique OTUs. This study is the first attempt to explore the effects of macrozoobenthos on microbial communities at the community level in a river–lake continuum. Therefore, it makes a fundamental contribution to the mechanism understanding of a protective microbial ecology in the river–lake continuum.

This study comprehensively investigated the sediment environment characters and microbial community composition, diversity, and function types of bacteria and fungi in Dongting Lake (D) and its tributary rivers, including Xiangjiang River (X), and pooling from three outfalls (P). The results revealed significant differences in sediment properties and zoobenthos community compositions among the main lake and tributary rivers, indicating distinct biotic and abiotic environmental characteristics. The main lake had higher nutrient contents than tributary rivers. In detail, the contents of TP, NO³⁻-N, and NH⁴⁺-N were higher in D than those in X, while OM was higher in D than that in P. In a previous study, water inputs from rivers can alter the dissolved oxygen, conductivity, turbidity, and nutrient levels, among other variables, in lakes (Weigelhofer et al., 2015; Castillo, 2020). Lake sediments may permanently bury 70% of nutrient-enriched particulate nutrients derived from tributaries, rather than releasing them into the environment (Klump et al., 1997). For example, nutrient inputs such as phosphorus and nitrogen in Lake Erie originated from the Maumee River (Stow et al., 2015). Thus, sediment nutrient in lakes was considered to be higher than that in tributary rivers. It was typically found that river mainstems have higher concentrations of nitrate than lakes and backwaters, where depletion of nitrates is common (Houser, 2016). In addition, lower turbulence in lakes would further promote the enrichment of nutrient elements and reduce their release effect to overlying water compared to tributary rivers. Meanwhile, connections between rivers and lakes were crucial to determining macrozoobenthos assemblages (Pan et al., 2011). For example, tributaries serve as the main richness source for the Oligochaeta assemblage (Annelida) of the Neotropical dammed river (Ragonha et al., 2014). The present study showed that macrozoobenthos communities in Dongting Lake and its tributary rivers were composed of Mollusca, Arthropoda, and Annelida. Arthropoda and Mollusca were the dominant phyla in D, where tributary river P was dominated by Mollusca, and tributary river X was dominated by Arthropoda and Annelida. The Benthos macroinvertebrate community in the main Dongting Lake was identified to be higher than that in its tributary rivers under the action of species communication in the river–lake continuum (Roesti et al., 2015). Aquatic environment characteristics, including water physical and chemical variables (e.g., dissolved oxygen, conductivity, alkalinity, and temperature), land use, and landscape characteristics, are essential to understanding macroinvertebrate community distribution and diversity (Melo, 2009; Rezende et al., 2014). Environmental heterogeneity (e.g., nutrient levels and composition, hydrological conditions) between the main lake and tributary rivers in the present study drove the community differences in benthic macrofauna (Mao et al., 2023). Water velocity also affects benthic taxa through their respiration and feeding strategies (Hamid et al., 2021). Chironomids (Arthropoda) and Oligochaetes (Annelida) are generally tolerant of high nutrient concentrations and low oxygen levels and can outcompete sensitive taxa such as Mollusca (Brauns et al., 2007).

The comprehensive analysis of sediment microbial community composition, diversity, and distribution patterns across Dongting Lake and its tributary rivers provides valuable insights into the ecological dynamics of aquatic microbes in the river–lake continuum. The majority of microbes generally existed in both the lake and its tributary rivers under the exchanges of water and sediment in the river–lake continuum, with more than 50% sharing OTUs in bacterial and fungal communities. It is evident that the microbes and suspended matter entering the aquatic ecosystem from runoff are mixed with the microbial population inhabiting river and lake sediments. However, bacterial and fungal community dissimilarities were significantly different among the main lake and its tributary rivers, with the main effect of species replacement processes. On the one hand, Xiangjiang River consisted of more unique OTUs with the main lake compared to the pooling from three outfalls, which suggested a higher similarity of microbial community between the main Dongting Lake and the three outfalls. The three outfalls served as important exchange channels between Dongting Lake and Yangtze River, providing more than 30% of the runoff volume and 70% of the sediment load to Dongting Lake (Yu et al., 2018; Tian et al., 2021). The Xiangjiang River drains an area of 94,000 km² and mainly derives its water from rainfall, flowing northward to Dongting Lake from the South Mountain Range (Xie et al., 2021). Microorganisms are transported together with particulate organic matter from one habitat to another, leading to microbial community coalescence due to the connection between the river and the lake (Bourhane et al., 2022). The dissimilarity of microbial communities between the Xiangjiang River and the main Dongting Lake was higher than that between the pooling from three outfalls and the main lake, due to the differences in hydrology conditions. On the other hand, the bacterial community had lower species richness in the Xiangjiang River than that in the main lake and the three outfalls, while the fungal community displayed reversed results. The dissimilarity of fungal communities between Dongting Lake and its tributary rivers was higher than that for the bacterial community. There are several possible explanations for this result. First, there are a variety of differences among bacteria and fungi characteristics, including growth rate and dispersal capacity, which may affect the assembly of benthic microbial communities and their biogeographic patterns in lake sediments (Zhao et al., 2022). A large proportion of fungal spores spread over short distances, which reduced the fungi exchanges among different habitats. The fungal community finally showed higher diversity in the Xiangjiang River than in the main lake and the pooling from three outfalls. Then, benthic bacterial communities may be better able to utilize simple compounds in the sediments, which will allow them to establish diversity more rapidly than fungal communities (Santonja et al., 2018). In other words, bacteria would be more tolerant to environmental disturbances compared to fungi.

Biotic and abiotic factors in sediment habitat drive the contrast diversity patterns and processes of the microbial community among Dongting Lake and its tributary rivers by regulating the community’s unique species. Lakes and rivers were typically subjected to different environmental conditions, such as flow velocity, water residence time, organic matter quantity and quality, and nutrient content, which can affect the compositions and functions of the microbial community. PH, TP, and ORP were found to be the important abiotic variables for predicting the bacterial community structure, yet, pH, TP, and OP were key regulators for fungal community structure. Sediment pH dominated the biogeographic pattern of benthic bacterial and fungal communities in main lakes and rivers, in agreement with previous studies (Zhao et al., 2022). Sediment pH serves as an indicator of sediment condition as well as a key factor in the biogeographic distribution of benthic microbes (Hou et al., 2020). As most microbes have an intracellular pH close to neutral and their optimum pH range is narrow, bacteria grow slowly to enhance stress resistance during soil pH fluctuations. In stressful environments, slow-growing species might not reach a high biomass but may remain healthy (Jousset et al., 2017). Thus, pH would regulate the microbial community structure by affecting the relative abundance of bacterial and fungal unique species. Similarly, Mollusca was a vital biotic variable for predicting sediment bacterial and fungal community structure by affecting the relative abundance of unique species. The bioturbation and feeding habits of macroinvertebrates might have a significant impact on the structure of microbial communities (Li et al., 2020). On one hand, Mollusca has been found to affect the microbial community diversity and the abundance of microbes that transform nitrogen. For example, the presence of Mollusca resulted in a sharp decrease in the phyla Gemmatimonadetes, Actinobacteria, Acidobacteria, Plantomycetes, Chloroflexi, Firmicutes, Crenarcheota, and Verrucomicrobia (Black et al., 2017). A similar result was found in this study that Mollusca would decrease the unique phyla Actinobacteria, Acidobacteria, Plantomycetes, and Gemmatimonadetes in the bacterial community and Ascomycota in the fungal community. On the other hand, the existence of multiple species may be maintained by biotic interactions (e.g., competition), which prevent microbes from finely partitioning niche axes with small population sizes under limited sediment resource conditions. Additionally, Mollusca, including Mytilus edulis, Pecten maximus, Ostrea edulis, Modiolus modiolus, and Mya arenaria, feed on suspensions and actively filter and retain particles in the surrounding water, including bacteria that are free-living and encapsulated in particles (Mudadu et al., 2021; Kijewska et al., 2023). In contrast, Annelida can influence the relative abundance of unique species to regulate the bacterial community structures in sediment. The phylum Annelida consists of segmented worms, including earthworms, lugworms, ragworms, and leeches, which primarily inhabit freshwater environments as either free-living or parasitic organisms (Wang et al., 2021). Their main ecological role is to rework sediment habitats. As some of the most sensitive organisms in sediment, bacteria, especially unique species in different sections of the river–lake continuum, were positively affected when the abundance of Annelida changed, which was opposite to the effects observed with Mollusca.

Metabolic pathway predictions are essential for describing the metabolic activity of microbes (Liu et al., 2021). The microbial functional types were significantly different among Dongting Lake and its tributary rivers with the changes in microbial communities. First, the functional dissimilarity among lakes and rivers was strengthened in unique species compared to all species in bacterial and fungal communities. The result indicated that the unique species replacement in specific habitats would help to enhance some specific function types as an adaptation strategy in varying environments. Then, the results of our PICRUSt2 prediction approach showed that changes in unique species generally affected the metabolism functions of bacterial and fungal communities, which is consistent with previous studies (Yang et al., 2022). The main difference for bacterial functional types is that amino acid metabolism and lipid metabolism were enriched in the Xiangjiang River, and the metabolism of cofactors and vitamins was enriched in the main Dongting Lake. In addition, the fungal function types of carbohydrate metabolism and metabolism of cofactors and vitamins were also enriched in tributary rivers. The pentose phosphate pathway is an alternative to glycolysis, allowing microorganisms to metabolize a greater variety of carbohydrates without oxygen (Wamelink et al., 2008). A higher abundance of pentose phosphate pathway in the Xiangjiang River meant that microbes might have a stronger potential to mineralize a greater diversity of organic carbon substrate than those in the main Dongting Lake. Pyridoxal 5′-phosphate is a B6 vitamer, which regulates several metabolic processes, including several metabolic reactions, such as amino acid biosynthesis and degradation, iron metabolism, nucleotide utilization, cofactor biosynthesis, and biofilm formation (Kuo et al., 2023). The lipid IVA biosynthesis is one of the main conserved structures in diverse Gram-negative pathogens, which is the most toxic part (Amin et al., 2023).

Although the main Dongting Lake and its tributary rivers are parts of a lake–river continuum system, there are significant differences in the biotic and abiotic environments of their sediment habitats. The value of pH was found to be the most important abiotic variable for predicting the bacterial and fungal community structures, and the abundance of Mollusca was found to be the most important biotic variable. The bacterial and fungal community compositional dissimilarities among Dongting Lake, Xiangjiang River, and pooling from three outfalls were dominated by species replacement processes. In particular, the biotic and abiotic factors affected the number and the relative abundance of bacterial and fungal unique OTUs and resulted in the changes in community composition for these unique OTUs. Mollusca, pH, TP, NO₃-N, and NH₄-N were negatively related to the relative abundance of Actinobacteria, Acidobacteria, Gemmatimonadetes, Planctomycetes, and Ascomycota, while Annelida and ORP were positively related to the relative abundance of Actinobacteria and Gemmatimonadetes. Finally, the functional dissimilarity between lakes and rivers was strengthened in unique species compared to all species in bacterial and fungal communities, which helped to improve the habitat environment in main Dongting Lake and promote the process of microbial growth. Therefore, the results of this study provided a theoretical reference for further understanding of microbial community structure, function, and the key influencing factors in lake–river continuum sediments.