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Abstract The relationships between biodiversity-ecosystem functioning (BEF) for microbial communities are poorly understood despite the important roles of microbes acting in natural ecosystems. Dilution-to-extinction (DTE), a method to manipulate microbial diversity, helps to fill the knowledge gap of microbial BEF relationships and has recently become more popular with the development of high-throughput sequencing techniques. However, the pattern of community assembly processes in DTE experiments is less explored and blocks our further understanding of BEF relationships in DTE studies. Here, a microcosm study and a meta-analysis of DTE studies were carried out to explore the dominant community assembly processes and their potential effect on exploring BEF relationships. While stochastic processes were dominant at low dilution levels due to the high number of rare species, the deterministic processes became stronger at a higher dilution level because the microbial copiotrophs were selected during the regrowth phase and rare species were lost. From the view of microbial functional performances, specialized functions, commonly carried by rare species, are more likely to be impaired in DTE experiments while the broad functions seem to be less impacted due to the good performance of copiotrophs. Our study indicated that shifts in the prokaryotic community and its assembly processes induced by dilutions result in more complex BEF relationships in DTE experiments. Specialized microbial functions could be better used for defining BEF. Our findings may be helpful for future studies to design, explore, and interpret microbial BEF relationships using DTE.

ABSTRACT With the development and reduced costs of high-throughput sequencing technology, environmental dark matter, such as novel metagenome-assembled genomes (MAGs) and viruses, is now being discovered easily. However, due to read length limitations, MAGs and viromes often suffer from genome discontinuity and deficiencies in key functional elements. Here, by applying long-read sequencing technology to sediment samples from a Tibetan saline lake, we comprehensively analyzed the performance of high-fidelity (HiFi) reads and the possibility of integration with short-read next-generation sequencing (NGS) data. In total, 207 full-length nonredundant 16S rRNA gene sequences and 19 full-length nonredundant 18S rRNA genes were directly obtained from HiFi reads, which greatly surpassed the retrieval performance of NGS technology. We carried out a cross-sectional comparison among multiple assembly strategies, referred to as ‘NGS’, ‘Hybrid (NGS+HiFi)’, and ‘HiFi’. Two MAGs and 29 viruses with circular genomes were reconstructed using HiFi reads alone, indicating the great power of the ‘HiFi’ approach to assemble high-quality microbial genomes. Among the 3 strategies, the ‘Hybrid’ approach produced the highest number of medium/high-quality MAGs and viral genomes, while the ratio of MAGs containing 16S rRNA genes was significantly improved in the ‘HiFi’ assembly results. Overall, our study provides a practical metagenomic resolution for analyzing complex environmental samples by taking advantage of both the short-read and HiFi long-read sequencing methods to extract the maximum amount of information, including data on prokaryotes, eukaryotes, and viruses, via the ‘Hybrid’ approach. IMPORTANCE To expand the understanding of microbial dark matter in the environment, we did the first comparative evaluation of multiple assembly strategies based on high-throughput short-read and HiFi data from lake sediments metagenomic sequencing. The results demonstrated great improvement of the ‘Hybrid’ assembly method (short-read next-generation sequencing data plus HiFi data) in the recovery of medium/high-quality MAGs and viral genomes. Further analysis showed that HiFi data is important to retrieve the complete circular prokaryotic and viral genomes. Meanwhile, hundreds of full-length 16S/18S rRNA genes were assembled directly from HiFi data, which facilitated the species composition studies of complex environmental samples, especially for understanding micro-eukaryotes. Therefore, the application of the latest HiFi long-read sequencing could greatly improve the metagenomic assembly integrity and promote environmental microbiome research.

The Asian clam Corbicula fluminea is a keystone zoobenthos in freshwater ecosystems. However, its associated microbiome is not well understood. We investigated the bacterial communities of this clam and its surrounding environment, including sediment and water simultaneously, in a large lake by means of 16S rRNA gene sequencing. Approximately two-thirds of the bacterial operational taxonomic units (OTUs) associated with clams were observed in the surrounding environment and mostly from particle-associated samples. The associated bacterial communities were site specific and more similar to environmental bacteria from the same site than those at other sites, suggesting a local environmental influence on host bacteria. However, the significant differences in bacterial diversities and compositions between the clam and the environment also indicated strong host selection pressure on bacteria from the surrounding environment. Bacteria affiliated with Firmicutes, Spirochaetes, Tenericutes, Bacteroidetes, Epsilonbacteraeota, Patescibacteria, and Fusobacteria were found to be significantly enriched in the clams in comparison to their local environment. Oligotyping analyses of the core-associated bacterial OTUs also demonstrated that most of the core OTUs had lower relative abundances and occurrence frequencies in environmental samples. The core bacterial OTUs were found to play an important role in maintaining the stability of the bacterial community network. These core bacteria included the two most abundant taxa Romboutsia and Paraclostridium with the potential function of fermenting polysaccharides for assisting host clams in food digestion. Overall, we demonstrate that clam-associated bacteria were spatially dynamic and site specific, which were mainly structured both by local environments and host selection. [ABSTRACT FROM AUTHOR]