Abstract
Stingless bees serve as crucial pollinators and are increasingly recognized as models for investigating behavioral and genomic evolution in insects. In the genus Melipona, a major difference in heterochromatin organization defines two groups: Group I species (e.g., M. quadrifasciata) with < 50% of pericentromeric heterochromatin and Group II species (e.g., M. scutellaris) containing > 50% heterochromatin across their chromosomes. These differences are believed to correlate with genome size and transposable element (TE) content, offering a unique opportunity to explore how heterochromatin variation, TE dynamics, and chromosomal evolution interact in a phylogenetic context. We present pseudo-chromosome-level genome assemblies for M. quadrifasciata and M. scutellaris obtained through long-read sequencing and 3D chromosome conformation scaffolding. Comparative analyses reveal conserved synteny but marked divergence in structural variants and TE types. M. scutellaris exhibits an expansion of retrotransposons, particularly Gypsy/DIRS1 elements, concentrated in TE hotspots linked to chromosomal rearrangements and structural variants. This coincides with distinct methylation entropy patterns across the genome and an expansion of histone deacetylase orthologs. The increased proportion of retrotransposons in M. scutellaris is counterbalanced by more DNA transposons in M. quadrifasciata, resulting in genomes of similar overall sizes but with distinct heterochromatin distributions. Advancing our understanding of genome evolution in eusocial insects, we provide high-resolution genomic resources for two Melipona species that differ in heterochromatin content. Our results highlight the complex role of TEs in shaping genomes and underscore their influence on chromosomal and epigenetic innovation, providing compelling evidence that TE dynamics underlie the pronounced heterochromatic differences observed in Melipona.