|Other Abstract||Seagrass meadow is one of the most valuable coastal ecosystems, serving significant ecological functions in coastal protection, carbon sequestration, fish nursery, and water purification. As the only angiosperm that can live in seawater, seagrass host abundant and diverse communities of microorganisms. These microbes fundamentally influence the seagrass physiology and health, and also regulate the biogeochemical dynamics of entire seagrass meadows. Under the scenario of intensified anthropogenic activities and global warming, a systematic understanding of the direct or indirect effects of microbial activities on seagrass health and growth is still lacking. Although the biogeographical distribution of bacteria associated with seagrass has been well depicted, knowledge of ecologically important archaea and microeukaryotes on how to interact with seagrass remain scarce.
In this study, three typical temperate seagrass meadows (Zostera japonica) distributed in northern China (Dongying, Weihai, and Dalian) were investigated. With amplicon sequencing, metagenomics, qPCR, and microcosm approaches, we aimed to reveal the followings: (1) the effects of season variations, habitat heterogeneity, and seagrass colonization on the distributions of three-domain microorganisms at the community level; (2) the dynamic of functional microbial guilds involved in nitrogen and sulfur cycles with seagrass growth, and their metabolic differences between vegetated and bare areas at the gene level; (3) the hypothesis that “warming threatens seagrass health by broking the balance of benthic sulfur-cycling microorganisms”, and then verified by a microcosm experiment. The main findings are listed as follows:
(1) The microeukaryotic assemblages associated with surficial sediments of Z. japonica were mainly composed of diatoms (21.8%) and dinoflagellates (20.4%). Ammonia-oxidizing archaea (Ca. Nitrosopumilus and Ca. Nitrocosmicus) was the dominant archaeal group (58.1%). Sulfur-cycling taxa Sulfurovum, Woeseia, and Sva0081 were frequently detected (18.8%) in benthic bacterial communities. The network analysis showed a relatively high co-occurrence between archaea and eukaryotes, indicating their close interkingdom interactions. In contrast with seagrass-vegetated effects, the α-diversity of the three-domain microorganisms was more affected by habitat heterogeneity and seasonal variations. A possible reason is that environmental factors changed evidently on a large spatial scale, which masked the effects of seagrass vegetation. The microeukaryotic community exhibited higher α-diversity estimators in spring and summer than those in autumn and winter. For the bacterial and archaeal assemblages, however, α-diversity estimators were higher in summer and autumn than those in spring and winter. The highest sensitivity of microbial diversity to environmental shifts was bacteria, followed by microeukaryotes, and archaea the last.
(2) Community structures of the three-domain microorganisms were significantly affected by both seagrass colonization and seasonal changes, and the effect of the former (R > 0.08, P < 0.001) appears to be greater than the latter (R > 0.07, P < 0.011) within a given meadows. It indicated that the rhizosphere effect was a major driving force for the microbial distribution. Compared with bare areas, higher contents of SO42−, total organic carbon (TOC), and total organic nitrogen (TON) were detected in the seagrass-vegetated sediments where enriched several functional groups, such as sulfate-reducing bacteria (SRB), diazotrophic bacteria, fiber-degrading microorganisms (Bathyarchaeota and MBG-D), methanogenic archaea (Methanolobus), etc. However, seagrass-vegetated sediments had lower diversity of microalgae and pathogens, which may be suppressed by algicidal bacteria inhabiting in seagrass surface and metabolites secreted by seagrass. Furthermore, seagrass meadows possessed distinct microbial taxa in different seasons, which were mainly driven by DO, nutrients (SO42−, NH4+, NO3−), and heavy metals (Cr, Cd, and As).
(3) Shotgun sequencing revealed that δ- and γ-proteobacteria were the most prevalent SRB members, while γ- and α-proteobacteria were the major sulfur oxidizers. Sulfide oxidizing gene fccA dominated in the sulfur-cycling gene pool. Thiosulfate reduction genes were enriched in seagrass-vegetated sediments, and genes encoding dissimilatory sulfate reductase, tetrathionate reductase, and sulfur-oxidizing protein were detected with higher abundance in the bare sediments. Temperature and DO were the major influential environmental factors on the diversty and the relative abundance of sulfur-cycling genes. The main participants in nitrogen cycling were composed of γ- and α-proteobacteria. Gene hao encoding hydroxylamine dehydrogenase and gene narI/narV and nrfA encoding nitrate reductase were the most abundant in the nitrogen-cycling gene pool of Z. japonica system. The relative abundances of genes hao and nifK were more frequently detected in the vegetated areas, and more abundant nosZ and nirS genes were found in unvegetated sediments. The distribution of denitrifying genes (nrfA, narGH, nosZ, nirS) presented lower abundances in the winter but higher in the summer, whereas an opposite pattern was found in genes nifDHK that related to nitrogen fixation. Therefore, these nitrogen fixators may play a key role in seagrass overwintering by providing nitrogen nutrients. Random forest analysis showed that the temperature and NH4+ were the crucial environmental drivers on the diversity and abundance of nitrogen-cycling genes. SRB was a key group coupling the transformations of nitrogen and sulfur elements. In addition, genes involved in heavy metal resistance, single-carbon (C1) metabolic pathways, and inorganic phosphate transportation were highly detected in seagrass ecosystems.
(4) The microcosm assays validated that the temperature plays a key role in shaping benthic bacterial community structure in seagrass sediments. The qPCR results showed that bacteria, SRB, and sulfur-oxidizing bacteria (SOB) had consistent patterns in response to warming, and their abundances increased firstly and then decreased from 26°C to 35°C. In addition, SRB:SOB ratio increased with warming, indicating that SOB is more readily to be suppressed by high temperature than SRB. The imbalance between SRB and SOB might lead to the accumulation of sulfide, which is harmful to seagrass health. These findings basically verified our assumption.
In conclusion, we comprehensively investigated the dynamics of diversity, abundance, and functions of bacteria, archaea, and microeukaryotes among the three seagrass meadows across four seasons. The complex network relationships among the three-domain microorganisms and the regulation mechanisms of environmental drivers (such as nutrient concentrations, heavy metals, and temperature) were unveiled at large, and we proposed a microbial-mediated frame of "warming-sulfur cycling-seagrass degradation". These findings improved our understanding of the interactions between three-domain microorganisms and seagrass, and provided new insights into the health monitoring and ecological restoration of seagrass meadows.|