HUMAnN2 is a next-generation method for species-level functional profiling of metagenomes and metatranscriptomes. It improves upon previous methods by incorporating a tiered search strategy that enables faster, more accurate, and species-resolved functional profiling. HUMAnN2 identifies known microbial species, aligns reads to their pangenomes, performs translated search on unclassified reads, and quantifies gene families and pathways. It is 3x faster than pure translated search and produces more accurate gene family profiles (89% vs. 67%). HUMAnN2 is applied to clinal variation in marine metabolism, ecological contribution patterns among human microbiome pathways, variation in species’ genomic vs. transcriptional contributions, and strain profiling. It introduces "contributional diversity" to explain patterns of ecological assembly across different microbial community types. HUMAnN2's tiered search strategy is more accurate and efficient than existing methods. It is compared to HUMAnN1, COGNIZER, MEGAN, and ShotMAP, and is found to be more accurate and faster. HUMAnN2 is also accurate for metatranscriptomes and robust to communities with species lacking reference genomes. It quantifies community-encoded functions and stratifies their abundances according to who performs them. These data can be explored using traditional diversity measures to define "contributional diversity," which reflects the distribution of functions across species. HUMAnN2 is applied to environmental microbial communities, demonstrating its applicability to marine metagenomes. It quantifies KEGG Orthogroup (KO) abundance in a dataset of 45 marine metagenomes from the Red Sea. HUMAnN2 identifies high-variance KOs not detected by HUMAnN1 and shows that KO abundances are often associated with sample temperature. It also profiles strain-level functional variation in well-covered community species, identifying subspecies-level clades from metagenomes based on presence/absence of functions observed across sequenced isolate genomes. HUMAnN2 can profile paired metagenomes and metatranscriptomes to compare microbial community functional potential and activity. It is applied to the Inflammatory Bowel Disease Multi-omics Database (IBDMDB) to analyze core pathways. HUMAnN2's stratified profiles confirm Methanobrevibacter smithii as a consistent contributor to these pathways, resulting in low within- and between-subject distributional diversity. HUMAnN2 is a new approach for functional profiling of meta'omically sequenced microbial communities. It introduces a novel tiered search algorithm that provides exceptionally accurate profiles for characterized members of microbial communities, with fallback to translated search for uncharacterized members. These tiers operate jointly in far less time than traditional pure translated search. Moreover, tiered search provides taxonomic stratification of microbial functions at the species-level, thus quantifying the community abundance of functions whileHUMAnN2 is a next-generation method for species-level functional profiling of metagenomes and metatranscriptomes. It improves upon previous methods by incorporating a tiered search strategy that enables faster, more accurate, and species-resolved functional profiling. HUMAnN2 identifies known microbial species, aligns reads to their pangenomes, performs translated search on unclassified reads, and quantifies gene families and pathways. It is 3x faster than pure translated search and produces more accurate gene family profiles (89% vs. 67%). HUMAnN2 is applied to clinal variation in marine metabolism, ecological contribution patterns among human microbiome pathways, variation in species’ genomic vs. transcriptional contributions, and strain profiling. It introduces "contributional diversity" to explain patterns of ecological assembly across different microbial community types. HUMAnN2's tiered search strategy is more accurate and efficient than existing methods. It is compared to HUMAnN1, COGNIZER, MEGAN, and ShotMAP, and is found to be more accurate and faster. HUMAnN2 is also accurate for metatranscriptomes and robust to communities with species lacking reference genomes. It quantifies community-encoded functions and stratifies their abundances according to who performs them. These data can be explored using traditional diversity measures to define "contributional diversity," which reflects the distribution of functions across species. HUMAnN2 is applied to environmental microbial communities, demonstrating its applicability to marine metagenomes. It quantifies KEGG Orthogroup (KO) abundance in a dataset of 45 marine metagenomes from the Red Sea. HUMAnN2 identifies high-variance KOs not detected by HUMAnN1 and shows that KO abundances are often associated with sample temperature. It also profiles strain-level functional variation in well-covered community species, identifying subspecies-level clades from metagenomes based on presence/absence of functions observed across sequenced isolate genomes. HUMAnN2 can profile paired metagenomes and metatranscriptomes to compare microbial community functional potential and activity. It is applied to the Inflammatory Bowel Disease Multi-omics Database (IBDMDB) to analyze core pathways. HUMAnN2's stratified profiles confirm Methanobrevibacter smithii as a consistent contributor to these pathways, resulting in low within- and between-subject distributional diversity. HUMAnN2 is a new approach for functional profiling of meta'omically sequenced microbial communities. It introduces a novel tiered search algorithm that provides exceptionally accurate profiles for characterized members of microbial communities, with fallback to translated search for uncharacterized members. These tiers operate jointly in far less time than traditional pure translated search. Moreover, tiered search provides taxonomic stratification of microbial functions at the species-level, thus quantifying the community abundance of functions while