New data from South American hotspots uncover a greater diversity in Gracilinanus (Didelphimorphia: Didelphidae) mouse opossums

The genus Gracilinanus ranges from savannas to dense forests in South America, yet its systematics have never been thoroughly investigated across its wide distributional range. We assessed Gracilinanus phylogenetic relationships, species boundaries, and geographical limits using mtDNA sequences. Our analysis confirmed the distinctiveness of the six recognized species (G. aceramarcae, G. agilis, G. emiliae, G. marica, G. microtarsus, and G. peruanus), with a mean p-distance for interspecific nucleotide sequence divergences ranging from 13–16.2% and robust phylogenetic support (BPP > 0.95; BS > 75%). Refined species delimitation approaches (GMYC, PTP, ASAP) revealed potential cryptic diversity, suggesting up to 20 candidate species. Three geographically structured and divergent lineages (4.1–4.8% sequence divergence) were identified within G. agilis, extending its Cerrado range. Within G. emiliae, we found divergence values ranging from 4.7–5.7% and the first known record for the northeastern Atlantic Forest. Three divergent clades were recovered within G. microtarsus (9.0–9.8% sequence divergence), including a new lineage for the northern Atlantic Forest. For G. peruanus, we found two divergent lineages (7.2%) and the first documented occurrence for Amazonian lowland forest. This comprehensive sampling revealed greater genetic diversity in Gracilinanus, extending its geographic limits. Here we propose nine putative new species, emphasizing a hidden diversity that warrants formal description and further increases the taxonomic diversity of this genus. These newly identified lineages underscore the urgency of inventorying and conserving the threatened ecosystems of the Cerrado and Atlantic Forest hotspots.