Genome-resolved metagenomics reveals a phylogenetically cohesive Acetilactobacillus-like species complex dominating stingless bee pot honey

Pot honey, the honey produced by stingless bees, is valued for its antimicrobial capacity, which may be influenced by its microbial content. While Lactobacillaceae species are commonly associated with honeybees and honey microbiomes, most studies have focused on Apis mellifera, leaving pot honey microbial diversity largely unexplored. We present the first pot honey shotgun metagenomic analysis from bee species Melipona beecheii and Scaptotrigona mexicana. We reconstructed 24 metagenome-assembled genomes (MAGs), 15 of which lacked close matches to any described species, showing[≤] 81% Average Nucleotide Identity (ANI) to available reference genomes. Phylogenetic analyses resolved these MAGs into four well-defined clades (intraclade ANI > 99%, interclade ANI[≤] 81%), consistent with four novel species within the family Lactobacillaceae. GTDB-Tk classification placed MAG clades 1 and 2 closest to Nicoliella, and clades 3 and 4 closest to Acetilactobacillus. We validated the presence of these lineages in honey by sequencing three isolates that clustered within MAG clade 2. Aminoacid similarity (AAI/cAAI) indicates the presence of two genus-level lineages: one occupying a transitional genomic space near Nicoliella, and a second representing an undescribed genus. The genomic similarity of our MAGs and isolates to those from pot honey or larval food in Malaysia, Brazil, and Australia suggests these taxa are closely associated with stingless bees and may contribute to honey properties. By reducing the genomic underrepresentation of evolutionarily divergent sister clades related to Nicoliella and Acetilactobacillus, our genome-resolved analyses reveal a globally distributed, phylogenetically cohesive Lactobacillaceae species complex dominating pot honey.