There are nine named species of buckeye butterflies (genus Junonia Hübner) in the Western Hemisphere. There is considerable geographic variation within Junonia species, and possible ongoing hybridization between species, suggesting that Junonia may be a ring species, but also making this a very difficult group to define taxonomically. We tried to determine whether two forms of Junonia from Argentina – conventionally referred to as Junonia genoveva hilaris C. & R. Felder, the light buckeye butterfly, and Junonia evarete flirtea (Fabricius), the dark buckeye butterfly – were genetically distinct species or simply colour forms of a single species using morphological characters, mitochondrial cytochrome oxidase I (COI) DNA barcodes, nuclear wingless (wg) locus DNA sequences, and anonymous nuclear Randomly Amplified Fingerprints (RAF). Phylogenetic analysis of COI identified two distinct mitochondrial haplotypes that differ by about 4% sequence divergence; one confined to light‐coloured Junonia specimens and one shared between some light‐coloured Junonia and all of dark‐coloured Junonia specimens. Analysis of nuclear wingless sequences revealed 32 alleles among 22 Junonia specimens and showed significant genetic differentiation between light‐coloured and dark‐coloured Junonia. Analysis of RAF genotypes suggests that there are actually three genetically distinct Junonia populations in Argentina: two with light wing coloration, and one with dark wing coloration. Genetic evidence of recent hybridization among these populations was also observed, consistent with the ring species hypothesis. Careful comparisons of morphological characters between Argentinian Junonia and Junonia species from elsewhere in South America suggests that the two light‐coloured populations correspond to J. genoveva and either a genetically disparate population of the same species or an undescribed cryptic Junonia species, The dark‐coloured population may correspond to J. wahlbergi Brévignon. Our data suggest that COI DNA barcodes by themselves are not very useful for studying Junonia taxonomy, population structure or evolution.

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Ecological and evolutionary theory has frequently been inspired by the diversity of colour patterns on the wings of butterflies. More recently, these varied patterns have also become model systems for studying the evolution of developmental mechanisms. A technique that will facilitate our understanding of butterfly colour-pattern development is germline transformation. Germline transformation permits functional tests of candidate gene products and of cis-regulatory regions, and provides a means of generating new colour-pattern mutants by insertional mutagenesis. We report the successful transformation of the African satyrid butterfly Bicyclus anynana with two different transposable element vectors, Hermes and piggyBac, each carrying EGFP coding sequences driven by the 3XP3 synthetic enhancer that drives gene expression in the eyes. Candidate lines identified by screening for EGFP in adult eyes were later confirmed by PCR amplification of a fragment of the EGFP coding sequence from genomic DNA. Flanking DNA surrounding the insertions was amplified by inverse PCR and sequenced. Transformation rates were 5% for piggyBac and 10.2% for Hermes. Ultimately, the new data generated by these techniques may permit an integrated understanding of the developmental genetics of colour-pattern formation and of the ecological and evolutionary processes in which these patterns play a role.

Species determination and definition in eukaryotes have traditionally been based on morphology, with little focus on genetic differentiation. Molecular methods allow for the independent assessment of morphology‐based taxonomic hypotheses. Three criteria used to define a full species for taxonomic purposes are morphological distinction, formation of a monophyletic lineage, and reproductive isolation. Junonia butterflies (Nymphalidae) are becoming an important experimental model system, but the taxonomy of many New World Junonia species is unclear. One of these species is J. coenia, which contains the subspecies J. coenia coenia, J. coenia grisea and J. coenia bergi. Previous studies suggest that J. coenia grisea may meet the criteria for full species status. Therefore, we evaluated the geographically isolated and rarely studied Bermuda buckeye butterfly J. coenia bergi to determine if it was similarly distinct. Physical examination of specimens and phylogenetic and population genetic analyses of mitochondrial cytochrome c oxidase subunit I, nuclear wingless, and complete mitochondrial genome sequences suggest that while J. coenia bergi is smaller in body size than many Junonia and has distinctive ventral hindwing colouration, it does not form a monophyletic lineage and shows indications of continued gene flow with North American mainland J. coenia coenia populations. Thus, J. coenia bergi does not meet the criteria for full species designation, but geographic isolation, morphological distinctiveness, and cultural importance suggest that it remain recognized as a subspecies of J. coenia. Similar analyses will be useful for addressing further taxonomic questions in Junonia and other taxa, especially where morphology‐based taxonomic determinations are ambiguous.

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