Strong agreement in absolute marine prokaryote abundance from epifluorescence microscopy and internal-standard-corrected metagenomics

Abstract Quantifying microbial cell abundance in natural environments with taxonomic resolution remains a central challenge in microbial ecology. While epifluorescence microscopy reliably enumerates free-living cells, it lacks taxonomic specificity and is less suitable for quantifying cells in sediments, soils, and on surfaces. Furthermore, while most omics approaches profile microbial communities, they typically provide only relative abundances. Recent advances in quantitative metagenomics using single-copy marker genes and genomic internal standards enable taxon-specific abundance estimates in absolute units (e.g. haploid genome equivalents L-1). Here, we directly compared SYBR Green–based epifluorescence cell counts with spike-in metagenomic estimates derived from bacterial recA and archaeal radA gene abundances in seawater samples collected monthly at five depths (5 to 890 m) from the San Pedro Ocean Time-series (SPOT) during 2023–2024. Across all depths, quantitative metagenomic estimates of prokaryotic abundance closely matched SYBR Green–based counts (slope = 1.05; Pearson’s r = 0.85). In addition to validating total abundance, quantitative metagenomics provided high-resolution, taxon-specific estimates across depths and sampling dates. The strong agreement between microscopy cell counts and metagenomic estimates from unfractionated SPOT samples suggests that most prokaryotes were free-living rather than particle associated. These results validate quantitative metagenomics as a robust approach for estimating absolute prokaryotic abundance, establishing a framework for taxonomically resolved quantification in microbial ecology and ecosystem modeling.