Estimation of the postmortem interval (PMI) is an essential component in investigations of unnatural death (1). Before a corpse begins to decompose, the PMI can be estimated by observation of early postmortem changes, such as body cooling, muscle rigidity, and postmortem lividity. These early postmortem changes are the results of physical or chemical consequences, which do not alter the overall consistency of the corpse. However, because the early postmortem changes are disrupted by late postmortem changes, such as putrefaction and autolysis, which gradually degrade the corpse, estimation of the PMI in a decomposing body has been a difficult task in forensic practice (1). Tiny insects gathering around the corpse can provide an alternative clue because the growth rates and the occurrence of insects are dependent on the ambient temperature and the stages of decomposition, respectively (2). In most developed countries, forensic entomology, which applies insect data to the forensic investigation, is now a major approach used to estimate PMI after decomposition occurs.

Because different insect species have different growth rates and different geographic or climatic distributions, accurate species identification is critical in the practice of forensic entomology (3-6). However, species identification of insects of forensic importance, especially flies, is a very complicated and difficult process, in which the small anatomic identification keys are observed with a stereomicroscope (7). Moreover, most insect specimens recovered from the scene are immature stages, such as eggs, larvae, or pupae, which are in many instances indistinguishable among different species (8). For accurate and convenient species identification, DNA-based methods have been developed, usually analyzing the mitochondrial cytochrome c oxidase I (COI) gene or the ribosomal internal transcribed spacer 2 (ITS 2) (9-11). Because DNA sequences are divergent among different species, species identification is possible with the DNA sequence data. Currently, COI gene sequences are used as a main molecular identification tool for insects (12-14).

The subfamily Luciliinae is one of the major groups in the Family Calliphoridae (7). However, there have been limited studies about COI sequences of Korean Luciliinae fly species (15, 16). Furthermore, full-length sequences of COI genes of the Korean Luciliinae fly species have not yet been determined. The current study is the first to determine the full-length sequences of COI genes of Korean Luciliinae flies, which the authors believe, are the most important necrophagous fly species, from late spring to early fall, in Korea.

Species-level paraphyly or even polyphyly in mitochondrial DNA, is not uncommon in the Animal Kingdom. According to Funk and Omland (28), at least 702 arthropod species are polyphyletic (26.5%). For these polyphylies, several explanations have been offered, which consist of inadequate sequence data, inaccurate taxonomy, interspecific hybridization or introgression, and incomplete sorting. In another study by Cognato (29), the intraspecific percent sequence distance of the COI genes in a lice species, Columbicola macrourae, was the highest (26.0%) among 26 insect species. However, this extraordinary intraspecific percent distance may be due to parasitic inhabitation of the louse (30). According to Cognato (29), intraspecific percent distances of the COI genes in 8 fly species (Order: Diptera) range from 0.04 to 3.5%. Therefore, intraspecific percent distances in H. ligurriens, 0 to 8.6% (in the Results section), are quite unusual among fly species. Although Wells et al. (19) insisted that validity of the genus Hemipyrellia is highly questionable, in our analysis, all sequences of H. ligurriens, except for two Taiwanese sequences, show distances within 3.6%. Further investigations, by other taxonomists, into the Taiwanese H. ligurriens formerly identified by Chen et al. (17), could ascertain the level of applicability, substantiate findings, and answer questions regarding the unusual COI haplotype.

L. illustris and L. caesar, which showed overlaps between intra- and interspecific sequence distances, are closely related sister species. Wells et al. (19) also noted a paraphyly between these two species and indicated, because L. caesar does not exist in the New World, identification of L. illustris in North America would be possible. Despite this paraphyly, because mixed clades occurred only in European strains, Asian strains are possibly distinguishable with COI sequence analyses. However, more sequence collection from various Asian and European regions would be helpful to prevent possible misidentification and to reinforce the validity of paraphyletic European sequences. Harvey et al. (20) insisted that an alternative gene choice, perhaps nuclear markers like ITS regions, may resolve COI para/polyphylies in some Calliphoridae taxa, especially when it is caused by incomplete lineage sorting. Regarding L. caesar and illustris, alternative gene analyses, including nuclear genes, may be applicable.

In conclusion, the Korean Luciliinae fly species are identifiable with COI sequence analyses. However, because of the inconsistent COI haplotypes of H. ligurriens, L. caesar, and L. illustris in a worldwide-scale comparison, more individuals from various geographic regions and alternative nuclear genes, for these three species, should be investigated.