Phoridae are a family of necrophagous flies commonly found in indoor death scene. They account for approximately 19.7% of the entomofauna in human cadavers in Korea. Additionally, this taxon is an indicator of indoor hygiene, and these flies appear in environments where access by other necrophagous insects is difficult, such as enclosed rooms. Thus, they are likely to be used as forensic evidence. Despite their importance in forensic investigations and environmental hygiene, detailed studies on the taxonomy and molecular barcoding for this family are scarce, including in Korea. Because accurate taxonomic information regarding necrophagous insects collected from a death-related scene is essential during medicolegal investigations, molecular barcoding data could be useful as well as reliable. In this paper, full-length nucleotide sequences of genes coding for the cytochrome c oxidase subunit I (COI) in 79 Phoridae larvae collected from 20 medicolegal autopsy cases in Korea were phylogenetically analyzed by comparing their sequences to the foreign barcoding data of Phoridae. Six mitochondrial haplogroups were identified, which two of them matched to foreign Phoridae fly species haplotypes,
Necrophagous insect species are utilized as estimators of minimum postmortem intervals (mPMIs) in the medicolegal entomologic practice [
Lateral view of female
Because most samples recovered from medicolegal autopsies are in immature stages, the exact taxonomic information at the species level is rarely available unless the collected samples are reared to adulthood or have been molecularly barcoded. Because of the paucity of expert taxonomists and DNA barcoding data, taxonomic information regarding the collected Phoridae samples is rarely available. Moreover, it is not yet known how many species of the genus
Experimental samples were obtained during autopsies or samples that had been stored from 2 to 4 years below -20°C in 70% ethanol that were used. A total of 79 samples were collected from 20 human cadavers in Seoul, Incheon, and Gyeonggi provinces in Korea. Seasonal changes had no effect on the presence of Phoridae, which occurred from February or April to December in 2015–2017. The samples were identified as Phoridae based on the morphological identification by observing the characteristic inferiorly directed posterior spiracles [
Samples in 70% ethanol were dried on a paper towel for 5 min. After the larvae were chopped using cutter and forceps, DNA extraction was performed according to the manufacturer’s instructions using an Exgene Tissue Mini Kit (GeneAll Biotechnology Co., Ltd., Seoul).
Primers were designed to amplify the entire length of the COI gene (Table
Primer information.
Primer name | Sequences | Binding locations |
---|---|---|
F1 | 5 | tRNA-Cys |
R1 | 5 | 653-678 on COI |
F2 | 5 | 163-182 on COI |
R2 | 5 | 1035-1055 on COI |
F3 | 5 | 740-759 on COI |
R3 | 5 | 26-46 on COII |
F3-2 | 5 | 812-838 on COI |
R3-2 | 5 | 24-49 on COII |
Three primer pairs were used to amplify three different regions with overlaps to cover the complete COI gene. Primer pair F3-2/R3-2 was designed for samples for which amplification failure was observed using the primer pair F3/R3. The nucleotide sequence of the
Three contigs of the COI gene based on the amplicons generated by the three primer pairs were assembled to construct the full-length COI gene sequence using ChromasPro 2.1.5 software (Technelysium Pty Ltd., South Brisbane, Australia). Only COI gene regions were selected and aligned for further analysis. A neighbor-joining phylogenetic tree was constructed using MEGA X software [
List of COI gene sequences for Phoridae flies obtained in this study.
Haplogroup name | Sample name | Sequence coverage on COI | Length (nucleotide) | Location | GenBank | Reference |
---|---|---|---|---|---|---|
sca1 | 1-2, 1-3, 7-2, 10-3, 11-1, 12-1, 13-1, 13-2, 13-4, 14-1, 14-2, 14-3, 14-4, 15-4, 17-2, 17-3, 17-4, 19-2, 20-2 | All | 1,539 bp | South Korea | MT396274, MT396280, MT396287, MT296299, MT296304, MT396306, MT396320, MT396340-MT396343, MT396346, MT 396350-MT396351, MT396353-MT396354, MT396362-MT396364 | Newly sequenced |
sca2 | 3-3 | All | 1,539 bp | South Korea | MT396277 | Newly sequenced |
sca3 | 7-3, 8-1, 8-2, 8-4, 10-1, 10-4, 18-1, 18-2 | All | 1,539 bp | South Korea | MT396275-MT396276, MT396279, MT396290, MT396310, MT396313, MT396358-MT396359 | Newly sequenced |
sca4 | 1-1, 3-1, 3-2, 4-1, 4-2, 4-4, 5-3, 5-4, 11-2, 11-3, 11-4 | All | 1,539 bp | South Korea | MT396271-MT396272, MT396278, MT396282, MT396286, MT396291, MT396293, MT396295, MT396301, MT396307, MT396309 | Newly sequenced |
sca5 | 1-4, 2-1, 2-3, 2-4, 3-4, 4-3, 5-1, 7-4 8-3, 9-2, 10-2, 13-4, 15-1, 15-2, 15-3, 17-1, 19-1, 19-3, 19-4 | All | 1,539 bp | South Korea | MT396273, MT396292, MT396294, MT396298, MT396300, MT396302, MT396312, MT396314, MT396317-MT396319, MT396344-MT396345, MT396347, MT396349, MT396352, MT396361, MT396366-MT396367 | Newly sequenced |
sca6 | 2-2, 9-1, 9-3, 9-4, 12-2, 12-3, 12-4, 16-1, 16-2, 16-3, 16-4, 18-3, 18-4 | All | 1,539 bp | South Korea | MT396284-MT396285, MT396288, MT396303, MT396305, MT396308, MT396311, MT396348, MT396355-MT396357, MT396360, MT396365 | Newly sequenced |
Spi | 5-2, 6-1, 6-2, 6-3, 6-4, 7-1, 20-1, 20-3 | All | 1,539 bp | South Korea | MT396281, MT396283, MT396289, MT396296, MT396297, MT 396315, MT396316, MT396321 | Newly sequenced |
The schematic redrawing of a phylogenetic tree constructed using nucleotide sequences from 79 Korean Phoridae larvae revealed seven groups, i.e., sca1, sca2, sca3, sca4, sca5, sca6, and spi. Six groups—sca1 to sca6—clustered together away from the spi group with distances ranging from 11.64–12.38%. Previously published nucleotide sequences NC023794 (
A schematic of a neighbor-joining phylogenetic tree using the sequences of the COI gene from 79 Phoridae larvae and two reference nucleotide sequences (NC023794 M
Percent distances of full-length COI gene sequences among seven Phoridae fly groups.
sca1 | |||||||
---|---|---|---|---|---|---|---|
sca2 | |||||||
sca3 | |||||||
sca4 | |||||||
sca5 | |||||||
sca6 | |||||||
Spi | |||||||
sca1 | sca2 | sca3 | sca4 | sca5 | sca6 | Spi |
Intragroup percent distances of full-length COI gene sequences in seven Phoridae fly groups.
Group name | Intragroup differences |
---|---|
sca1 | 0.001709928 |
sca2 | n/c |
sca3 | 0.000997865 |
sca4 | 0.001228661 |
sca5 | 0.005312176 |
sca6 | 0.003115576 |
Spi | 0.001137102 |
The number of base differences per site calculated by averaging over all sequence pairs between groups is shown. This analysis involved 79 nucleotide sequences. Codon positions included are 1st+2nd+3rd+noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1539 positions in the final dataset.
There are no strict criteria that is determining the conspecificity or heterospecificity based on nucleotide sequence distances. Furthermore, nucleotide sequence distances between 1% and 2% were empirically considered as a gray zone, whereas when the distances >2%, the samples were generally accepted as being different species [
The nucleotide sequences of the COI genes from 79 necrophagous Phoridae flies recovered from human cadavers were analyzed. Based on this analysis, we confirmed the presence of COI gene sequences corresponding to those of two previously known species,
The data is full-length nucleotide sequences of the cytochrome c oxidase subunit I (COI) genes of necrophagous Phoridae in South Korea. These can be found at NCBI Genbank, and there is no restrictions on data access.
The authors declare that they have no conflicts of interest.
We would like to thank Editage (
The original phylogeny before schematic redrawing is available as a supplementary figure: “See Supplementary Figure 1 in the Supplementary Material for comprehensive image analysis.” The detailed percent distances table between species and reference genes (NC023794 and MN832848) are available as Supplementary Tables 1 and 2.