Genetic variation in neotropical butterflies is associated with sampling scale, species distributions, and historical forest dynamics.

Previous studies of butterfly diversification in the Neotropics have focused on Amazonia and the tropical Andes, while southern regions of the continent have received little attention. To address the gap in knowledge about the Lepidoptera of temperate South America, we analysed over 3000 specimens representing nearly 500 species from Argentina for a segment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Representing 42% of the country's butterfly fauna, collections targeted species from the Atlantic and Andean forests, and biodiversity hotspots that were previously connected but are now isolated. We assessed COI effectiveness for species discrimination and identification and how its performance was affected by geographic distances and taxon coverage. COI data also allowed to study patterns of genetic variation across Argentina, particularly between populations in the Atlantic and Andean forests. Our results show that COI discriminates species well, but that identification success is reduced on average by ~20% as spatial and taxonomic coverage rises. We also found that levels of genetic variation are associated with species' spatial distribution type, a pattern which might reflect differences in their dispersal and colonization abilities. In particular, intraspecific distance between populations in the Atlantic and Andean forests was significantly higher in species with disjunct distributions than in those with a continuous range. All splits between lineages in these forests dated to the Pleistocene, but divergence dates varied considerably, suggesting that historical connections between the Atlantic and Andean forests have differentially affected their shared butterfly fauna. Our study supports the fact that large-scale assessments of mitochondrial DNA variation are a powerful tool for evolutionary studies.

Barcoding the butterflies of southern South America: Species delimitation efficacy, cryptic diversity and geographic patterns of divergence.

Because the tropical regions of America harbor the highest concentration of butterfly species, its fauna has attracted considerable attention. Much less is known about the butterflies of southern South America, particularly Argentina, where over 1,200 species occur. To advance understanding of this fauna, we assembled a DNA barcode reference library for 417 butterfly species of Argentina, focusing on the Atlantic Forest, a biodiversity hotspot. We tested the efficacy of this library for specimen identification, used it to assess the frequency of cryptic species, and examined geographic patterns of genetic variation, making this study the first large-scale genetic assessment of the butterflies of southern South America. The average sequence divergence to the nearest neighbor (i.e. minimum interspecific distance) was 6.91%, ten times larger than the mean distance to the furthest conspecific (0.69%), with a clear barcode gap present in all but four of the species represented by two or more specimens. As a consequence, the DNA barcode library was extremely effective in the discrimination of these species, allowing a correct identification in more than 95% of the cases. Singletons (i.e. species represented by a single sequence) were also distinguishable in the gene trees since they all had unique DNA barcodes, divergent from those of the closest non-conspecific. The clustering algorithms implemented recognized from 416 to 444 barcode clusters, suggesting that the actual diversity of butterflies in Argentina is 3%-9% higher than currently recognized. Furthermore, our survey added three new records of butterflies for the country (Eurema agave, Mithras hannelore, Melanis hillapana). In summary, this study not only supported the utility of DNA barcoding for the identification of the butterfly species of Argentina, but also highlighted several cases of both deep intraspecific and shallow interspecific divergence that should be studied in more detail.