Using eight universal primers and new designed 315 species-specific primers, we tried to retrieve COI sequences from 45 dried specimens of 36 butterfly species collected from 1959 to 1980. The eight universal primers were entirely failed in PCR amplification and sequencing of all specimens. In the other hand, the 315 primers, targeting fragments of 71–417 bp, generated various lengths of COI sequences ranged from 444 bp to 658 bp from all specimens. Among 284 primer pairs, 26 primer pairs designed for Limenitis camilla, Argynnis niobe, and Brenthis daphne were success to produce COI sequences of congeneric speices, Limenitis doerriesi, Argynnis nerippe, and Brenthis ino. It suggests that the species-specific primers can be applied for analyzing COI sequences of closely related species. Our study reveals that newly designed species-specific primers will be effective to retrieval of COI sequences of old butterfly specimens.

Bemisia tabaci (Hemiptera: Aleyrodidae) is one of the most important insect pests in the world. In the present study, the taxonomic status of B. tabaci and the number of species consisting of the B. tabaci complex were determined based on 1059 COI sequences of B. tabaci and 509 COI sequences of 153 hemipteran species. The genetic divergence within B. tabaci was conspicuously higher (onaverage,11.1%) than interspecific genetic divergence within the respective genera of the 153 hemipteran species (on average, 6.5%). This result indicates that B. tabaci is composed of multiple biological species that may belong to different genera or subfamilies. The phylogenetic tree constructed based on 212 COI sequences without duplications revealed that the B. tabaci complex is composed of a total of 31 putative species, including a new species, JpL. However, genetic divergences within five species, Asia III, Asia II 7, Mediterranean, New world, and Sub Saharan Africa 1, were higher than 3.5%, which has previously been used as a threshold of genetic boundary in distinguishing species of the B. tabaci complex. These results suggest the necessity to redefine the threshold for species boundaries to best distinguish the constituent species in the B.tabaci complex.