Corresponding author: Kurt Jordaens (
Academic editor: Z. T. Nagy
Forensic entomology uses the larval and pupal developmental stages of insects sampled on a corpse to estimate a minimum post-mortem interval (PMImin) of the corpse (
Currently, the most popular molecular method for organismal identification is DNA barcoding, which was promoted by
The monophyly of
Taxonomy of the subfamily
Genus/species | COI | COII | 16S | cyt |
ITS2 | 28S | ||
---|---|---|---|---|---|---|---|---|
251 bp | 350 bp | |||||||
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2(2) | 1(1) | 1(1) | ||||||
3(2) | 1(1) | 2(1) | 2(1) | |||||
5(2) | 1(1) | 10(6) | 2(1) | 2(2) | ||||
1(1) | ||||||||
1(1) | 11(2) | |||||||
1(1) | 2(2) | |||||||
1(1) | ||||||||
3(2) | 1(1) | 4(2) | ||||||
1(1) | ||||||||
9(2) | 2(2) | 1(1) | ||||||
6(2) | 1(1) | 5(1) | ||||||
79(11) | 28(7) | 66(31) | 20(3) | 2(2) | 42(3) | 4(2) | ||
9(7) | 3(3) | 7(1) | ||||||
1(1) | 1(1) | |||||||
1(1) | 1(1) | |||||||
7(4) | 1(1) | 14(2) | ||||||
2(2) | 1(1) | 1(1) | 2(1) | |||||
25(10) | 45(9) | 10(5) | 1(1) | 14(1) | 2(2) | |||
7(2) | 1(1) | 8(2) | ||||||
11(5) | 3(2) | 10(2) | ||||||
1(1) | ||||||||
7(6) | 6(2) | 1(1) | ||||||
7(1) | ||||||||
78(73) | 65(62) | 2(1) | 90(24) | 2(1) | ||||
3(3) | 1(1) | 1(1) | 4(1) | |||||
1(1) | 1(1) | |||||||
1(1) | 1(1) | 1(1) | ||||||
1(1) | 1(1) | 1(1) | ||||||
1(1) | 1(1) | 1(1) | ||||||
1(1) | ||||||||
1(1) | ||||||||
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48(20) | 30(9) | 15(2) | 15(2) | 17(10) | 36(2) | 38(2) | ||
17(7) | 1(1) | 2(2) | 1(1) | 1(1) | 4(2) | |||
2(2) | 1(1) | 1(1) | 1(1) | |||||
1(1) | ||||||||
1(1) | 1(1) | |||||||
1(1) | ||||||||
Total no. ind. | 339 | 194 | 95 | 39 | 32 | 263 | 66 | |
Total no. hapl. | 180 | 108 | 42 | 9 | 20 | 55 | 21 | |
Total no. spp. | 36 | 20 | 6 | 6 | 5 | 24 |
Because high COI sequence divergences are often indicating species level differentiation (e.g.
Sixty-one adult individuals of
Lateral (top) and dorsal (bottom) view of the male copulatory organs of
Color scoring of eleven external morphological characters of adult W European and N American
Character | W Europe and N America |
---|---|
calypters | white |
first spiraculum | white to yellow |
thoracic dorsum | metallic green-bluish to dark green |
scutellum | dark green |
legs | black |
abdomen | metallic green-bluish |
facial ridge | red-brown |
gena | black |
postgena | black |
first antennal segment | dark-brown to black |
second antennal segment | white-grey |
DNA was extracted from on one or two legs. The remaining parts of the vouchers are kept at the NICC (National Institute of Criminalistics and Criminology – Brussels, Belgium) as pinned material. Genomic DNA was extracted using the NucleoSpin Tissue kit (Macherey-Nagel). A fragment of 721 bp from the 5’-end of the COI gene, including the standard barcode region (
Each 25 µl PCR reaction was prepared using 1 × PCR buffer, 0.2 mM dNTPs, 0.4 μM of each primer, 2.0 mM MgCl2, 0.5 U of Taq DNA polymerase (Platinum®, Invitrogen), 2–4 µl DNA template (DNA was stored in 100 µl of elution buffer) and enough mQ-H2O to complete the total PCR reaction volume. The thermal cycler program consisted of an initial denaturation step of 4 min at 94 °C, followed by 30–40 cycles of 45–60 s at 94 °C, 30–60 s at a fragment depending annealing temperature and 90 s at 72 °C; with a final extension of 7 min at 72 °C. The annealing temperatures were 45 °C for COI and COII, 48 °C for 16S and cyt
Description of the
Marker | COI | COII | 16S | cyt |
ITS2 (without indels) | 28S (without indels) | |
---|---|---|---|---|---|---|---|
251 bp | 350 bp | ||||||
Fragment size (bp) | 655 | 472 | 251 | 350 | 512 | 380 (224) | 633 (592) |
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Total | |||||||
No of sequences | 50 | 30 | 15 | 15 | 17 | 36 | 37 |
No of haplotypes | 20 | 9 | 2 | 4 | 10 | 4 | 2 |
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No of sequences | 27 | 27 | 11 | 11 | 10 | 25 | 23 |
No of haplotypes | 14 | 7 | 1 | 3 | 7 | 1 | 2 |
Mean intra-NA distances (%) | 0.004 | 0.004 | - | 0.004 | 0.005 | - | 0.002 |
SE | 0.001 | 0.002 | - | 0.003 | 0.002 | - | 0.002 |
min. – max. | 0.002–0.008 | 0.002–0.006 | - | 0.003–0.006 | 0.002–0.008 | - | 0.002 |
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No of sequences | 23 | 3 | 4 | 4 | 7 | 11 | 14 |
No of haplotypes | 6 | 2 | 1 | 1 | 3 | 4(2) | 1 |
Mean intra-EU distances (%) | 0.003 | 0.002 | - | - | 0.002 | 0.002 | - |
SE | 0.001 | 0.002 | - | - | 0.007 | 0.002 | - |
min. – max. | 0.002–0.008 | 0.002 | - | - | 0.002–0.010 | 0.002 | - |
Mean p-distance between NA and EU | 0.04 | 0.037 | 0.004 | 0.006 | 0.053 | 0.001 | 0.001 |
SE | 0.007 | 0.008 | - | 0.003 | 0.009 | 0.001 | 0.001 |
min. – max. | 0.036–0.044 | 0.034–0.042 | 0.004 | 0.005–0.009 | 0.047–0.061 | 0–0.004 | 0–0.002 |
Other |
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Mean intraspecific p-distance | 0.005 | 0.014 | 0.028 | 0.014 | 0.003 | 0.008 | 0.003 |
SE | 0.009 | 0.014 | 0.009 | - | 0.002 | 0.005 | 0.004 |
min. – max. | 0–0.042 | 0–0.037 | 0.018–0.036 | 0.014 | 0.002–0.005 | 0.004–0.015 | 0–0.010 |
Mean interspecific p-distance | 0.066 | 0.046 | 0.038 | 0.023 | 0.079 | 0.085 | 0.007 |
SE | 0.005 | 0.005 | 0.006 | 0.004 | 0.007 | 0.011 | 0.002 |
min. – max. | 0.011–0.113 | 0.002–0.135 | 0.03–0.075 | 0.023–0.057 | 0.073–0.141 | 0.009–0.166 | 0–0.015 |
We did not detect morphological differences between N American and W European
Basic information of the different datasets can be found in
Neighbour-Joining tree (p-distances) of a 655 bp fragment of the mitochondrial cytochrome
Neighbour-Joining tree (p-distances) of a 472 bp fragment of the mitochondrial cytochrome
Neighbour-Joining tree (p-distances) of a 512 bp fragment of the mitochondrial cytochrome
The mean p-distances between
For the 251 bp dataset, all eleven NA specimens had the same haplotype with a p-distance of 0.004 to the EU haplotype (four specimens) (
On the one hand, our results show that the mean p-distance of other intrageneric interspecific comparisons (COI: 5–6.8%, COII: 4.8-5.9%, cyt
North American and W European
Our findings may have important implications for the use of
Intraspecific mtDNA divergence in other
Although there is no doubt that COI is a useful tool for the identification of forensically important
So far, the forensically important species within the
In conclusion, we observed substantial differentiation between N American and W European
We wish to thank Françoise Hubrecht for her support, Knut Rognes for his help with the literature, and Sofie Vanpoucke (NICC) for her help in making the pictures of the genitalia. We thank Jens Amendt, Richard Zehner and Benoît Vincent for providing part of the W European
Neighbour-Joining tree (p-distances) of a 350 bp (
Neighbour-Joining tree (p-distances) of a 404 bp (229 bp without indels) fragment of the nuclear internal transcribed spacer 2 (ITS2). Bootstrap values ≥ 70% are shown at the nodes. N gives the number of specimens of that haplotype. EU =
Neighbour-Joining tree (p-distances) of a 633 bp fragment of the nuclear 28S gene. Bootstrap values ≥ 70% are shown at the nodes. N gives the number of specimens of that haplotype. EU =
Sampling localities, voucher numbers and GenBank numbers of the
Species | continent/country | country/state | city/county | latitude/longitude | voucher no. | GenBank accession no. | |||||
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COI | COII | 16S | cyt |
ITS2 | 28S | ||||||
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Europe | Belgium | Andrimont |
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NICC 0323 |
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Andrimont |
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NICC 0324 |
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Andrimont |
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NICC 0325 |
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Andrimont |
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NICC 0326 |
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Andrimont |
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NICC 0327 |
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Andrimont |
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NICC 0328 |
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Andrimont |
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NICC 0329 |
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Andrimont |
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NICC 0331 |
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Andrimont |
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NICC 0332 |
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Andrimont |
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NICC 0334 |
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Andrimont |
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NICC 0336 |
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Auderghem |
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NICC 0032 |
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Custine |
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NICC 0314 |
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Genk |
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NICC 0038 |
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Genk |
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NICC 0355 |
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Hastière |
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NICC 0027 |
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Laeken |
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NICC 0044 |
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Liège |
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NICC 0048 |
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Liège |
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NICC 0638 |
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Liège |
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NICC 0640 |
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Liège |
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NICC 0641 |
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Messancy |
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NICC 0317 |
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Schaerbeek |
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NICC 0035 |
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Schoonaarde |
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NICC 0359 |
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Schoonaarde |
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NICC 0360 |
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Steendorp |
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NICC 0054 |
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Toernich |
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NICC 0024 |
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France | Sarreguemines |
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NICC 0295 |
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Sarreguemines |
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NICC 0296 |
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Germany | Frankfurt |
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NICC 0301 |
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Frankfurt |
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NICC 0302 |
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Frankfurt |
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NICC 0303 |
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Frankfurt |
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NICC 0304 |
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Frankfurt |
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NICC 0305 |
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Frankfurt |
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NICC 0306 |
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Frankfurt |
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NICC 0307 |
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USA | Indiana | Rensselaer Co. |
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NICC 0275 |
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Rensselaer Co. |
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NICC 0276 |
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Rensselaer Co. |
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NICC 0277 |
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Rensselaer Co. |
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NICC 0278 |
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Rensselaer Co. |
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NICC 0279 |
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Texas | Brazos |
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NICC 0265 |
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Brazos |
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NICC 0266 |
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Brazos |
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NICC 0267 |
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Brazos |
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NICC 0268 |
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Brazos |
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NICC 0269 |
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Virginia | Pr. Williams Co. |
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NICC 0260 |
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Pr. Williams Co. |
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NICC 0261 |
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Pr. Williams Co. |
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NICC 0262 |
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Pr. Williams Co. |
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NICC 0263 |
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Pr. Williams Co. |
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NICC 0264 |
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Washington | Snohomish Co. |
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NICC 0270 |
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Snohomish Co. |
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NICC 0271 |
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Snohomish Co. |
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NICC 0272 |
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Snohomish Co. |
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NICC 0273 |
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Snohomish Co. |
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NICC 0274 |
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Wyoming | Park Co. |
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NICC 0255 |
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Park Co. |
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NICC 0256 |
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Park Co. |
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NICC 0257 |
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Park Co. |
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NICC 0258 |
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Park Co. |
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NICC 0259 |
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Europe | Belgium | Andrimont |
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NICC 0030 |
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Andrimont |
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NICC 0095 |
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Andrimont |
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NICC 0096 |
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Andrimont |
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NICC 0336 |
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Andrimont |
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NICC 0337 |
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Andrimont |
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NICC 0338 |
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Andrimont |
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NICC 0339 |
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Andrimont |
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NICC 0340 |
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Andrimont |
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NICC 0341 |
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Andrimont |
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NICC 0342 |
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Auderghem |
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NICC 0033 |
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Auderghem |
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NICC 0358 |
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Text file with the alignments for all the gene fragments studied. (doi: