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Most living species of New World marsupials are classified in the family Didelphidae, with 98 species and 18
genera currently recognized. We sequenced fragments of two mitochondrial genes of didelphid marsupials from the Atlantic
Forest of eastern South America, a biodiversity hotspot. We evaluated sequence divergences within and among species
and contrasted the efficiency of cytochrome c oxidase subunit I (COI) with cytochrome b (CytB) in species-level diagnosis.
The average intraspecific genetic divergence of COI and CytB was 2.0% and 1.9%, respectively; which was
about five times lower than the comparison among species of the same genus (11.2 and 10.8%). In both genes, divergence
levels among closely related species are usually higher than within species. The barcoding gap is similar in COI and CytB,
indicating that either gene can be used in molecular diagnoses of didelphid species. DNA barcodes are a welcome addition
to traditional taxonomic methods when viewed as additional diagnostic characters in the context of integrative taxonomy.