The objective of this study was to build a DNA barcode reference library for the true butterflies of Peninsula Malaysia and assess the value of attaching subspecies names to DNA barcode records. A new DNA barcode library was constructed with butterflies from the Museum of Zoology, University of Malaya collection. The library was analysed in conjunction with publicly available DNA barcodes from other Asia-Pacific localities to test the ability of the DNA barcodes to discriminate species and subspecies. Analyses confirmed the capacity of the new DNA barcode reference library to distinguish the vast majority of species (92%) and revealed that most subspecies possessed unique DNA barcodes (84%). In some cases conspecific subspecies exhibited genetic distances between their DNA barcodes that are typically seen between species, and these were often taxa that have previously been regarded as full species. Subspecies designations as shorthand for geographically and morphologically differentiated groups provide a useful heuristic for assessing how such groups correlate with clustering patterns of DNA barcodes, especially as the number of DNA barcodes per species in reference libraries increases. Our study demonstrates the value in attaching subspecies names to DNA barcode records as they can reveal a history of taxonomic concepts and expose important units of biodiversity.
The Neotropical butterfly Dryas iulia has been collected from several locations in Thailand and Malaysia since 2007, and has been observed breeding in the wild, using introduced Passiflora foetida as a larval host plant. The butterfly is bred by a butterfly house in Phuket, Thailand, for release at weddings and Buddhist ceremonies, and we hypothesized that this butterfly house was the source of wild, Thai individuals. We compared wing patterns and COI barcodes from two, wild Thai populations with individuals obtained from this butterfly house. All Thai individuals resemble the subspecies D. iulia modesta, and barcodes from wild and captive Thai specimens were identical. This unique, Thai barcode was not found in any of the 30 specimens sampled from the wild in the species' native range, but is most similar to specimens from Costa Rica, where many exporting butterfly farms are located. These data implicate the butterfly house as the source of Thailand's wild D. iulia populations, which are currently so widespread that eradication efforts are unlikely to be successful.
Sex ratios are subject to distortion by a range of inherited parasites. Although it has been predicted that the presence of these elements will result in spatial and temporal variation in host sex ratio, testing of this hypothesis has been constrained by availability of historical data. We here determine spatial and temporal variation in sex ratio in a interaction between a butterfly and male-killing Wolbachia bacteria by assaying infection presence in museum specimens, and from this inferring infection prevalence and phenotype in historical populations. Comparison of contemporary and museum samples revealed profound change in four of five populations examined. Two populations become extremely female biased, associated with spread of the male-killer bacterium. One evolved from extremely female biased to a sex ratio near parity, resulting from the infection losing male-killing activity. The final population fluctuated widely in sex ratio, associated with varying frequency of the male killer. We conclude that asynchronous invasion and decline of sex-ratio distorters combines with the evolution of host suppressors to produce a rapidly changing mosaic of sex ratio. As a consequence, the reproductive ecology of the host species is likely to be fundamentally altered over short time scales. Further, the study demonstrates the utility of museum specimens as "silent witnesses" of evolutionary change.