Microbial communities have been widely characterized using metabarcoding of environmental DNA, which is derived from living (including dormant and active forms) and dead cells. Despite advances in discriminating living and dead cells, methodologies for further distinguishing the dormant from active members are lacking. Propidium monoazide (PMA) is a photoreactive dye that can selectively cross-link to the DNA of broken cells. By taking advantage of PMA, we have proposed an new approach combined DNA, RNA and PMA-DNA based Illumina sequencing to discriminate dormant phylotypes from active and dead forms in nature. We applied this approach to characterize the diversity and community structure of bacteria, archaea, and microeukaryotes in marine water column and sediments. We found that 1) considerable proportions of OTU or ASV richness detected in the bulk DNA pools were attributed to the dormant and dead microbes, indicating that the “active” microbial richness was largely overestimated in many DNA-based studies; 2) the spatial and temporal patterns of microbial richness and assemblage structure of active fractions were generally different from those those based solely on metabarcoding of 16S and 18S rRNA genes; and 3) the main environmental drivers identified varied among these fractions, and those based on the total rDNA. Taken together, our study demonstrates that the dead and dormant are non-negligible in the marine total DNA pools, they should therefore be discriminated from the active ones, for example, by using the triple metabarcoding approach, for a better understanding of the persistence and resilience of microbial diversity in marine ecosystems.

microbial community structure,metabarcoding analysis,Eco-physiology,Microbial diversity