DNA barcodes are very useful for species identification especially when identification by traditional morphological characters is difficult. However, the short mitochondrial and chloroplast barcodes currently in use often fail to distinguish between closely related species, are prone to lateral transfer, and provide inadequate phylogenetic resolution, particularly at deeper nodes. The deficiencies of short barcode identifiers are similar to the deficiencies of the short year identifiers that caused the Y2K problem in computer science. The resolution of the Y2K problem was to increase the size of the year identifiers. The performance of conventional mitochondrial COI barcodes for phylogenetics was compared with the performance of complete mitochondrial genomes and nuclear ribosomal RNA repeats obtained by genome skimming for a set of caddisfly taxa (Insect Order Trichoptera). The analysis focused on Trichoptera Family Hydropsychidae, the net-spinning caddisflies, which demonstrates many of the frustrating limitations of current barcodes. To conduct phylogenetic comparisons, complete mitochondrial genomes (15 kb each) and nuclear ribosomal repeats (9 kb each) from six caddisfly species were sequenced, assembled, and are reported for the first time. These sequences were analyzed in comparison with eight previously published trichopteran mitochondrial genomes and two triochopteran rRNA repeats, plus outgroup sequences from sister clade Lepidoptera (butterflies and moths). COI trees were not well-resolved, had low bootstrap support, and differed in topology from prior phylogenetic analyses of the Trichoptera. Phylogenetic trees based on mitochondrial genomes or rRNA repeats were well-resolved with high bootstrap support and were largely congruent with each other. Because they are easily sequenced by genome skimming, provide robust phylogenetic resolution at various phylogenetic depths, can better distinguish between closely related species, and (in the case of mitochondrial genomes), are backwards compatible with existing mitochondrial barcodes, it is proposed that mitochondrial genomes and rRNA repeats be used as next generation DNA barcodes.

Decades of research on the highly modified wings of Drosophila melanogaster has suggested that insect wings are divided into two Anterior-Posterior (A-P) compartments separated by an axis of symmetry. This axis of symmetry is created by a developmental organizer that establishes symmetrical patterns of gene expression that in turn pattern the A-P axis of the wing. Butterflies possess more typical insect wings and butterfly wing colour patterns provide many landmarks for studies of wing structure and development. Using eyespot colour pattern variation in Vanessa butterflies, here we show an additional A-P axis of symmetry running between wing sectors 3 and 4. Boundaries of Drosophila mitotic clones suggest the existence of a previously undetected Far-Posterior (F-P) compartment boundary that coincides with this additional A-P axis. A similar compartment boundary is evident in butterfly mosaic gynandromorphs. We suggest that this additional compartment boundary and its associated developmental organizer create an axis of wing colour pattern symmetry and a gene expression-based combinatorial code, permitting each insect wing compartment to acquire a unique identity and allowing for the individuation of butterfly eyespots.

Up to 9 described species ofJunoniabutterflies occur in the Americas, but authorities disagree due to species similarities, geographical and seasonal variability, and possible hybridization. In dispute is whether CaribbeanJunoniaare conspecific with South American species.Cytochrome oxidase I(COI) barcodes,wingless(wg) sequences, and Randomly Amplified Fingerprints (RAF) were studied to revealJunoniapopulation structure in French Guiana, Guadeloupe, Martinique, and Argentina. Phylogenetic analysis ofCOIrecovered 2 haplotype groups, but mostJunoniaspecies can have either haplotype, soCOIbarcodes are ambiguous. Analysis of nuclearwinglessalleles revealed geographic patterns but did not identifyJunoniaspecies. Nuclear RAF genotyping distinguished 11 populations ofJunoniaarranged into 3 clusters. Gene flow occurs within clusters but is limited between clusters. One cluster included all Argentinian samples. Two clusters included samples from French Guiana, Martinique, and Guadeloupe and appear to be divided by larval host plant use (Lamiales versus Scrophulariales). ManyJunoniataxa were distributed across populations, possibly reflecting patterns of genetic exchange. We had difficulty distinguishing between the Caribbean formsJ. zonalisandJ. neildi, but we demonstrate that CaribbeanJunoniaare genetically distinct from South AmericanJ. evareteandJ. genoveva, supporting the taxonomic hypothesis that they are heterospecific.