Investigation of the Taxonomic Relationships Among Closely Related Butterflies of the Gelon Group in the Genus Graphium Based on Nucleotide Sequences of Two Mitochondrial Genes

It has been hypothesized that the butterfly Graphium weiskei goodenovii should be reassigned the name Graphium kosii based on certain wing characteristics. However, this suspicion remains unconfirmed, perhaps due to the extreme difficulty of obtaining the former butterfly species for examination. In the present study, we performed DNA sequence analysis on Graphium weiskei goodenovii, Graphium kosii gigantor and Graphium kosii kosii to study this unclarified taxonomic matter; there has been no previous DNA analysis of G. weiskei goodenovii and G. kosii kosii. The DNA of the G. weiskei goodenovii sample we acquired was in very poor condition, so we performed a specialized PCR involving a series of PCRs that amplify four brief COI regions (approximately 180 to 240 bp), to obtain a full barcode sequence. Analysis of the sequences obtained, including phylogenetic trees constructed, produced two results. First, the DNA sequence identity analysis among the traditional gelon group taxa, i.e., G. weiskei goodenovii, Graphium weiskei weiskei, Graphium kosii gigantor, and Graphium kosii kosii, supported the traditional concept that G. weiskei goodenovii and Graphium weiskei weiskei were the same species. Specifically, the percentage difference between G. weiskei weiskei and G. weiskei goodenovii was 1.06%, whereas the differences between G. weiskei goodenovii and the two above subspecies of G. kosii, as well as those between G. weiskei weiskei and those two subspecies, were between 2% and 3%. Second, the percentage differences in ND5 sequences between G. weiskei weiskei and those two kosii subspecies were unexpectedly small, at 1.04% each. In addition, the COI phylogenetic trees did not clearly support that G. weiskei and G. kosii are different species. Therefore, these results indicate the taxonomic classification that G. weiskei and G. kosii are different species may not be definitive. Further studies, including cross-fertilization experiments, may produce new insights into this taxonomic matter.