![handle is hein.journals/injlegame121 and id is 1 raw text is: Int J Legal Med (2007) 121: 1-8
DOI 10.1007/s00414-005-0056-8
Jeffrey D. Wells Diana W. Williams
Validation of a DNA-based method for identifying Chrysomyinae
(Diptera: Calliphoridae) used in a death investigation
Received: 30 June 2005 / Accepted: 27 September 2005 / Published online: 19 November 2005
© Springer-Verlag 2005
Abstract Many authors have proposed DNA-based meth-
ods for identifying an insect specimen associated with
human remains. However, almost no attempt has been
made to validate these methods using additional observa-
tions. We tested a protocol for identifying insects in the
blow fly subfamily Chrysomyinae (Diptera: Calliphoridae)
often found to be associated with a human corpse in
Canada or the USA. This method uses phylogenetic anal-
ysis of DNA sequence from a short segment of the mito-
chondrial gene for cytochrome oxidase one (COI). Test
chrysomyine COI sequences were obtained from 245
newly sequenced specimens and 51 specimens from the
published literature. Published sequences from representa-
tives of nonchrysomyine genera were also included to
check for the possibility of a false positive identification.
All of the chrysomyine test haplotypes were correctly
identified with strong statistical support, and there were no
false positives. This method appears to be an accurate and
robust technique for identifying chrysomyine species from
a death investigation in this geographic region. The far
northern species Protophormia atriceps was not evaluated;
therefore, caution is required in applying this method at
very high latitudes in North America.
Keywords Forensic entomology Mitochondrial DNA-
Cytochrome oxidase - Phylogeny - Species determination
Introduction
Correct identification of an insect specimen is usually a
crucial early step in a forensic entomological analysis [28].
J. D. Wells ( )
Department of Biology, West Virginia University,
Morgantown, WV 26506, USA
e-mail: jdwells@mail.wvu.edu
Fax: +1-304-2936363
D. W. Williams
US Army Investigation Laboratory,
Forest Park, GA 30297, USA
Although traditionally accomplished using anatomical
characters, this can be difficult or impossible for the
immature stages of many species [3]. Sperling et al. [29]
first proposed that such specimens should be identified
using mitochondrial DNA (mtDNA). The majority of
published insect mtDNA sequences cover some portion of
the genes for cytochrome oxidase subunits one and two
(COI + COII) [7]. Consequently, most authors have ad-
vocated the use of a region within COI + COII for forensic
insect identification (e.g., [8, 14, 20, 40]). However, some
closely related species could not be distinguished using the
COI + COII sequence [36, 42]. Protocols are available for
characterizing other mtDNA [37, 43] or nuclear [26, 30]
loci in forensically important Diptera, and some of these
may prove to be more useful than COI + COII for some
taxa.
A locus that works well for diagnosing species of one
taxonomic group may not work for a different group. The
use of mtDNA sequence data for species determination
requires an assumption of reciprocal mtDNA monophyly
when comparing a particular species with its closest rel-
ative in the data set [22, 24]. In other words, unless each
morphological species under consideration corresponds to
a single mtDNA lineage, it will not be possible to assign
at least some sample haplotypes to a single species
(Fig. 1).
Not surprisingly, carrion flies show intraspecific varia-
tion in mtDNA haplotypes [35], and geographic variation
is also possible [31]. Therefore, we believe that validation
of any molecular genetic species test for these insects
requires a population and geographic survey of the target
locus. Wells and Sperling [40] proposed a method for
identifying all forensically important species in the blow
fly subfamily Chrysomyinae (Diptera: Calliphoridae) like-
ly to be found in a human corpse in Canada or the USA.
This test relies on a short region of the COI sequence from
an evidence specimen and maximum parsimony phyloge-
netic analysis [33] to potentially associate a haplotype from
an unidentified specimen with a haplotype from an iden-
tified specimen. The current study aims to validate this
analytical method using newly generated and published but](ViewImageLocal?handle=hein.journals/injlegame121&div=1&collection=&method=preview&ext=.png&size=3)
What Is HeinOnline?
Contact us for annual subscription options:
Already a HeinOnline Subscriber?
profiles profiles most
Contact us for annual subscription options:
Already a HeinOnline Subscriber?
profiles profiles most
Contact us for annual subscription options:
Already a HeinOnline Subscriber?
profiles profiles most