Review
TRENDS in Biotechnology
Vol.23 No.7 July 2005
Food and forensic molecular
identification: update and challenges
Fabrice Teletchea, Celia Maudet and Catherine Hänni
Centre de Génétique Moléculaire et Cellulaire, CNRS UMR5534, UCB-Lyon I, 16, Rue Raphael Dubois, 69622 Villeurbanne Cedex,
France
The need for accurate and reliable methods for animal
species identification has steadily increased during past
decades, particularly with the recent food scares and the
overall crisis of biodiversity primarily resulting from the
huge ongoing illegal traffic of endangered species. A
relatively new biotechnological field, known as species
molecular identification, based on the amplification and
analysis of DNA, offers promising solutions. Indeed,
despite the fact that retrieval and analysis of DNA in
processed products is a real challenge, numerous
technically consistent methods are now available and
allow the detection of animal species in almost any
organic substrate. However, this field is currently facing
a turning point and should rely more on knowledge
primarily from three fundamental fields – paleogenetics,
molecular evolution and systematics.
Introduction
Recent food scares (e.g. BSE, avian flu, foot-and-mouth
disease, etc.), malpractices of some food producers,
religious reasons, food allergies and GMOS have tremendously reinforced public awareness regarding the composition of food products. However, because labels do not
provide sufficient guarantee about the true contents of a
product, it is necessary to identify and/or authenticate the
components of processed food, thus protecting both
consumers and producers from illegal substitutions [1].
In addition, trade of endangered species has contributed to severe depletion of biodiversity. Approximately
10–20% of all vertebrates and plant species are at risk of
extinction over the next few decades (IUCN; http://www.
redlist.org and CITES http://www.cites.org). Wildlife and
their products represent the third greatest illegal traffic
after drugs and arms [2] and one of the most serious
threats to the survival of animal populations is poaching.
Each year, millions of endangered animals are illegally
killed or captured for private zoo collections, hunt
trophies, ornamental objects (e.g. elephant ivory [3]),
human consumption (e.g. sea turtles and their eggs [4]) or
traditional medicine (e.g. tiger [5,6], rhinoceros [7]).
Food authentication and protection of biodiversity both
require reliable and accurate methods for determining,
without ambiguity, the animal species in a wide array
of degraded and processed substrates (Table 1). The
Corresponding author: Hänni, C. (hanni@univ-lyon1.fr).
Available online 31 May 2005
development of these methods should protect both
consumers and producers from frauds, and protect animal
species from over-exploitation or illegal trafficking. Molecular authentication or molecular traceability, which is
based on the PCR amplification of DNA, has been
developed in the past ten years and offers promising
solutions for these issues. Furthermore, this field will
probably experience tremendous development because:
(i) Most DNA methods developed have proved useful on
almost all organic substrates and will certainly
become the new legal standards for identification.
(ii) More regulations have introduced safety standards
into the chain of food production (e.g. European
Regulations such as the 2000/104/EC, which establishes that fish products can enter the commercial
circuit only if the commercial name, method of
production and capture area are clearly labelled or
the 2002/1774/EC, which bans the intra-species
recycling of animal by-products).
(iii) During the past decade, molecular identification tests
have only been developed for a few species but it is
likely that this number will steadily increase,
particularly among fish (Box 1).
Therefore, it is now crucial to reassess the different
molecular methods available for animal species identification, particularly in light of three fundamentals fields:
paleogenetics, molecular evolution and systematics. We
are convinced that these three fields could improve the
potential of analysing DNA in degraded substrates, help
to choose the most appropriate molecular markers and
highlight some common problems encountered in systematics that could result in erroneous identification.
Here we concentrate mainly on species identification and
not in the recognition of distinct populations of the same
species because the latter question requires different
concepts at some point.
DNA from food and forensic samples
Fresh food products or forensic samples without processing are suitable for many types of molecular or protein
analyses (traditional biochemical approaches based on
proteins used either electrophoretic, chromatographic or
immunological techniques; reviewed in [8]). Unfortunately, because most foodstuffs and forensic samples are
processed, DNA is usually altered. However, several
research fields had already worked with such DNA:
ancient DNA studies or paleogenetics (studying DNA
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Vol.23 No.7 July 2005
Table 1. Examples of forensic or food substrates for molecular analysis
Samples analyzed
Food products
Canned products
Species targeted
Extraction methods
Fragment targeted
Fragment size (bp)
Refs
Tuna
Cyt b
171
[58]
Canned products
Sardine
Chloroform, methanol,
water
Chelexa, phenol, chloroform,
isoamyl alcohol
Guanidium thiocyanate
(GUSCN)
Dneasy tissue kitb
152
[59]
145–313
[60]
Meat-and-bone meal in
compound feeds
Blood or meat meal, pet
food, baby food
‘Mortara’ salami
Goat cheese
Foie gras
Beef, sheep, pig, chicken
Ruminant, avian, fish and
pig
Goose
Beef
Goose and mule duck
Caviar
Sturgeon species
Genomic prep kitc
Dneasy tissue kitb
Phenol, chloroform, isoamyl
alcohol
Not indicated
Whale
Cyt b
Lys
tRNA
, ATPase
12s rRNA, tRNAval,
16S rDNA
Cyt b
D-loop
5S rDNA
104–290
[61]
350
413
250–1000
[62]
[63]
[56]
Cyt b, 16S, 12S
Not indicated
[55]
Not indicated
D-loop
155–378
[64]
Human
Chelexa, QIAmpb
128–250
[65]
Chinese alligator
Tiger
Phenol/chloroform
Chelex1, phenol, chloroform
Microsatelittes,
Amelogenin
Cyt b
Cyt b
180
165
[66]
[6]
African elephant
QIAamp kitb
70–251
[3]
Faeces
Tiger
510
[67]
Muscle, blood, eggs, skin
Soup, dried fin, cartilage
pills
Sea turtles
Shark
Guanidium thiocyanate
(GUSCN)
Phenol-chloroform; Dneasyb
Phenol-chloroform; Dneasyb
12S, cyt b, microsatellites
Cyt b
Cyt b
Cyt b
875–876
155–188
[4]
[68]
Forensic products
Dried, salted and unfrozen
strips of meat
Formalin fixed paraffin
embded tissues
Skin, tanned hide, scales
Pills and plasters made with
tiger’s bone
Elephant tusk (Ivory)
a
BioRad (http://www.bio-rad.com).
Applied Biosystems (http://www.appliedbiosystems.com).
Amersham Biosciences (http://www1.amershambiosciences.com).
b
c
from fossil bones or ancient organic remains), human
forensics (studying DNA from hairs, saliva, blood etc. from
crime scenes), non-invasive ecological studies (studying
DNA from animal faeces or hairs found in the field) and
more recently, food authentication. Taken together, these
fields have demonstrated that, (i) despite being altered,
DNA is more resistant and thermostable than proteins
are and it is still possible to PCR amplify small DNA
fragments (with sufficient information to allow identification) and (ii) DNA could potentially be retrieved from
any substrate because it is present in almost all cells of an
organism. In addition, molecular evolution and phylogenetics have shown that, because of the degeneracy of the
genetic code and the presence of many non-coding regions,
DNA provides much more information than proteins
do. However, in processed products, DNA is altered and
displays several particular features that must be taken
into account.
Substrates, DNA quality and contaminations
Short DNA fragments. First, during production processes,
food products might be subject to thermal treatments
(cooking, pasteurization, etc.), high pressure, pH modifications, irradiation, drying and so on. For example, many
food products are heated up to 1008C for 10–60 min and
are exposed to a pH!4. Consequently, molecular
Box 1. Review of species identified
To evaluate the species for which at least one method is currently
available, we attempted to collate comprehensively, but not exhaustively, all studies published in the past decade. It seemed that almost
all of these studies (w100 reviewed) had focused on a few species
belonging to three main groups of vertebrates: mammals (36%),
actinopterygian fishes (34%) and birds (20%).
† Within mammals, more than one-third (41%) of the studies only
dealt with one or all of the same four livestock species (cattle, pig,
sheep and goat). The remaining mammal species studied are usually
endangered ones, such as seven species of animals for bushmeat [69],
rhinoceroses [7] or tigers [5].
† Within birds, two-thirds (60%) only dealt with chicken and/or
turkey, few dealt with other birds such as goose and duck [70] or
ostrich [71].
† Within fishes, the number of species studied is much higher than
in the two previous groups. This was expected, because the number of
exploited species is far higher than either in mammals or birds. Indeed
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O1200 species are fished (http://www.fishbase.org/search.cfm) and
w220 are farmed (both numbers include shellfish) [72]. Nevertheless,
as with the two previous groups, we observed a strong bias towards
several species, such as tuna (20%), salmon (16%) and sturgeon (6%).
By contrast, the most valuable fish have hardly been studied, such as
sardines [73,59] and cod [74], which are the first (22 472 563 metric
tons in 2002) and second (8 392 479 metric tons in 2002) most
important groups ‘harvested’ worldwide (http://www.fao.org),
respectively.
† Finally, we found several studies (10%) that focused on other
groups, such as clam species [75] or sea turtles [4].
Consequently, despite the relatively high number of studies
published, the number of species for which a method of detection
is currently available is still small, probably less than 100 animal
species overall. Thus it seems likely that in the future the number
of species studied will increase significantly, particularly within
the fish.
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TRENDS in Biotechnology
Box 2. Example of simultaneous detection of mixed species
in foodstuff
DNA was first extracted from a pet food can labelled ‘meat flavour’
(Figure I, [76]) and was amplified using trans-vertebrates (enabling
the amplification of all vertebrate species) or ‘universal primers’
designed by Kocher et al. [26]. The amplified DNA (a 380 bp portion
of the Cytochrome b gene) was then cloned using a commercial
cloning kit (TOPO TA cloning, Invitrogen; http://www.invitrogen.
com/) to separate each molecule of DNA, and 30 clones were
sequenced using a direct sequencing method. Sequences obtained
were compared to those in the sequence bank GenBank (http://www.
ncbi.nlm.nih.gov/Genbank/index.html). A mix of five species
sequences was observed: seven clones of beef (Bos taurus), four
clones of pig (Sus scrofa), five clones of duck (Cairina moschata),
seven clones of chicken (Gallus gallus), and seven clones of sea trout
(Salmo trutta). Today, this approach is the only one (with DNA chips)
that enables broad identification of animal species used in a food
product, even if, to our knowledge, it has rarely been used.
Canned pet food
DNA isolation
PCR amplification using trans-vertebrates primers
Cloning and sequencing of 30 clones
TRENDS in Biotechnology
Figure I.
identification methods from such highly degraded substrates should be based on the analysis of very short DNA
fragments, preferably between 100–200 base pairs
(e.g. researchers were not able to amplify fragments
longer that 200 bp from canned tunas [9] or in processed
animal meals [10,11]).
Low amounts of DNA. DNA is not only degraded but is
also present in small quantities that considerably
reduce the number of DNA fragments with suitable
size for molecular analysis. Thus, paleogenetics and
non-invasive molecular studies have shown that
increasing the number of PCR cycles, up to 45 or 55
cycles, is often required to get enough amplified DNA
for subsequent analysis. However, DNA from other
components, fraudulently or accidentally included, and
from minority constituents, could be present in very
small quantities. Species detection methods should
therefore be very specific to provide reliable results.
Contaminations. Because PCR is such a sensitive
method, sometimes a single exogenous DNA molecule
could be preferentially amplified instead of the degraded
one (reviewed in [12]). Thus, food and forensic samples
should be manipulated (before PCR) in a dedicated
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Vol.23 No.7 July 2005
361
laboratory [physically isolated from those where unaltered DNA (from fresh samples) and amplified DNA are
handled] and appropriate negative controls (extraction
and PCR blanks) should be processed on every run.
PCR inhibitors. Many food or forensic constituent
products could be co-purified with the target DNA and
are known to be inhibitors of DNA amplification. These
include organic and phenolic compounds, polysaccharides
(or products of the Maillard reaction), glycogen, fats, milk
proteins, collagen, iron, cobalt or fulvic acids (reviewed in
[13] and [14]). Other more widespread inhibitors include
bacterial cells, non-target DNA and exogenous contaminants [13]. Therefore, the absence of amplification in food
or forensic samples (i.e. a negative readout) could be
explained by the inhibition of the PCR amplification
rather than by an insufficient amount or the absence of
the target DNA. Thus, to detect a species in a food product,
DNA isolation methods should allow removal of inhibitors
(e.g. [15]). Despite the numerous protocols already
described (examples are given in Table 1) to date, no
general extraction method has proved useful with all the
different matrices encountered [16]. Therefore, each new
substrate requires the development of new extraction and
amplification methods or the adaptation of existing ones.
Nevertheless, several ancient DNA studies have shown
that the use of the PTB (N-phenecylthiazolium bromide)
allows cleaveage of cross-links between proteins and DNA
(Maillard reaction) and thus enhances the subsequent
PCR reaction by permitting the movement of the Taq
polymerase on the DNA molecule [17,18]. Moreover, it is
also possible to neutralize some of these inhibitors by the
addition of bovine serum albumin (BSA) or spermidine to
the PCR reagents [19,20].
Chemical modifications and PCR artefacts
These are probably the least known features of DNA in
food and forensic applications but paleogenetic studies
showed that environment (acidity, UV light, moisture,
etc.) could induce chemical modifications of the DNA
molecule. In this case, modified DNA molecules might
display breaks or artifactual mutations (reviewed in [21]).
Consequently, absence of DNA degradation from a food
product, particularly for extensively processed ones,
should be checked before using methods based on few
variable sites. These DNA modifications can indeed
produce misidentification of species because the DNA
sequence obtained is slightly different from the reference
one. Although these chemical degradations have never
been studied in food products, Ram et al. [22] found
inexplicable variations in DNA extracted from canned
tuna; this variability could be because of chemical
modifications of the DNA molecule studied.
Paleogenetic and non-invasive studies have also shown
that PCR amplification starting from tiny amounts of
altered DNA could induce artifactual results (e.g. allelic
drop-out in microsatellite analysis [23] or chimeric
molecules produced by jumping PCR [24]).
Mix of individuals and species
DNA extracted from a food product is a mix of DNA of
many origins: bacterial, vegetable, animal and fungi.
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Furthermore, several individuals of the same species
(sometimes from different lineages) could be used in a food
product. However, unfortunately almost none of the
available methods allow the simultaneous identification
of a wide array of species (or individuals) mixed in food
products. This simultaneous detection of several species is
certainly one of the greatest challenges in the field, but
still remains unresolved. Two methods have the potential
to offer convincing and reliable solutions – cloning and
sequencing (Box 2) or DNA chips. DNA chips (also known
as DNA macroarray or DNA microarray) allow the
examination of complex mixtures of PCR products and,
potentially, the identification of hundreds or thousands of
species simultaneously. Such methods have already been
used in numerous fields such as in ecology (e.g. for the
simultaneous detection of five marine fish pathogens [25])
but not extensively for species identification in food and
forensic samples (F. Teletchea et al., unpublished data).
Despite all of these technical constraints and difficulties, it has been shown that DNA could be retrieved and
analysed with sufficient size and information to allow the
correct identification of species from a wide array of
substrates by using different extraction protocols (Table 1,
Box 3).
DNA markers for species identification
DNA retrieved from food and forensic products displays
several specific features that complicate its detection.
These characteristics underline why, for species identification in these kinds of products, the mitochondrial DNA
genome (mtDNA) is preferentially targeted compared to
nuclear DNA. Indeed, because there are several copies of
mtDNA inside a cell (w1000!the copies of nuclear DNA)
it is more likely to amplify a fragment within this genome
rather than within the nuclear genome. Besides, this
small circular genome (w16 kb in most vertebrate species)
displays maternal inheritance in most animal species, is
haploid, and does not undergo recombination – characteristics that make its study easier and more straightforward. Lastly, mtDNA generally evolves much faster than
nuclear DNA and thus enables even closely related species
to be differentiated and identified.
Vol.23 No.7 July 2005
More precisely, a suitable DNA marker for identification at the species level should be sufficiently variable
between species (particularly between the closest ones)
and display either low or no intra-specific variations
across the geographic distribution area. In addition, this
marker should be widely studied for a large number of
species to enable comparison of the nucleotide sequence
from an unknown sample with reference sequences in a
database. The gene encoding cytochrome b satisfied most
of these criteria and is by far the most studied gene for
phylogeny. It is certainly used in more than half of the
phylogenetic studies published in the past decade (more
than 50 000 sequences available in GenBank in 2004).
This gene also displayed both (i) conserved regions
allowing the determination of primers such as ‘universal’
primers published by Kocher et al. [26], which could be
used to amplify a wide range of vertebrate species (see
Box 2), and (ii) regions with a high level of variability,
which allows the evolutionary studies of even closely
related species. Nevertheless, in the cases of identification
of breeds, geographic origins, or individual assignments,
markers should be different and those showing an
important intra-specific variability will be very useful
(e.g. D-loop [27]; microsatellites [28,29]; or coding region
[30]). Thus, in some cases, a strong haplotypic structure
within a species can allow allocation of an individual to a
particular geographic population. The large number of
reconstructed phylogeography using mtDNA genes clearly
illustrated that infraspecific information can be used to
improve identification and potentially to identify geographic origins of new invasive species.
Thus, as expected, among approximately one hundred
molecular identification studies (collated from the literature comprehensively but not exhaustively in the past
decade) almost half of them targeted the cytochrome b
(44%), then the 12S (11%), 16S (8%) and D-loop (8%).
Lastly, about fifteen other markers have occasionally been
used, mostly located within mtDNA [e.g. cytochrome c
oxidase II (COX2)]. Besides, despite the technical problem
of analyzing DNA in these highly degraded samples (as
mentioned above), few studies targeted nuclear markers
such as microsatelittes (Table 1).
Box 3. Examples of food or forensic molecular identification
Three molecular identification methods today represent O90% of all
studies published: PCR-RFLP, Species-specific PCR and PCR-FINS.
PCR-RFLP and species-specific PCR successes could be explained by
the short development time necessary to design molecular markers,
low cost and straightforward results.
PCR-RFLP (restriction fragment length polymorphism). For example,
Hold et al. [77] differentiated 36 fish species by digesting a 464 bp
cytochrome b fragment with six different restriction enzymes. They
showed that this method was still useful with processed products (1008C
during 15 min) and also when several species were mixed.
Species-specific PCR. Wand and Fang [5] analyzed tiger DNA in
meat, faeces and dried skin and therefore showed that this protected
animal was still illegally traded. Moreover, using a multiplex of
species-specific primers Dalmasso et al. [61] proposed to identify the
presence of the most used vertebrate groups in feedstuff products
(ruminant, poultry, fish and pork).
PCR-FINS (forensically informative nucleotide sequencing) is the
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most direct means for obtaining information from PCR products,
(i.e. by sequencing). Compared to the two previous methods, this
method presents two main advantages: (i) it is not too sensitive to
intraspecific variations; and (ii) by using cloning (Box 2) it is possible to
detect several species in the same product. Jérôme et al. [59] used
direct sequencing of a 103-bp diagnostic sequence to differentiate 14
sardine species used in food preparation. This method was successfully applied to 45 out of 47 commercial canned sardine and sardinetype products tested.
New methods such as real time PCR (see [78] for a detailed
presentation) and DNA chips have not been developed significantly
for species identification to date. This low interest is particularly
because of longer developing times and costs compared with the
previous methods. However, in our laboratory, we have developed a
DNA chip that allows the simultaneous detection of numerous
vertebrate species in processed food and forensic samples
(F. Teletchea et al., unpublished data).
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TRENDS in Biotechnology
Caution with mtDNA: the Numts
The vast majority of identification methods developed in
the past decade were based on mitochondrial sequences.
However, we would like to emphasize that mitochondrial
sequences could be obtained from a nuclear copy of the
mtDNA rather than from endogenous mitochondrial DNA.
Indeed, translocated pieces of mitochondrial DNA (known
as Numts) are sometimes integrated into the nuclear
genome. Thus, during analysis, these Numts might be
amplified inadvertently by the PCR in addition to or even
instead of the authentic target mtDNA and therefore led
to erroneous results [31]. For example, while studying the
snapping shrimp genus Alpheus, Williams and Knowlton
[32] found multiple copies of the COX1 gene in at least ten
species. They observed that within a single animal,
differences between the real mtDNA and pseudogene
sequences ranged from 0.2–18.8%. Thalmann et al. [31]
found similar results within gorillas. Bensassson et al. [33]
provided a detailed census of Numts in eukaryotic groups
(they found Numts in 82 different species) and described
methods for detecting them (e.g. checking for unique
changes and odd substitution patterns). However, current
practices do not preclude inadvertent analysis of Numts
and much caution should be exercised when using
mitochondrial sequences for identification purposes –
explicit measures need to be taken to authenticate
mtDNA sequences [31].
Species concepts
The literature about species concepts might be more
extensive than that about any other subject in evolutionary biology and therefore it is obviously out of the scope of
the present review to discuss the validity of all these
concepts (see [34]). Rather, we illustrate the three main
problems usually encountered in systematics that could
result in erroneous molecular identification methods if
current practices do not take them into account.
Complexes of cryptic species, closely related species and
introgression
The same scientific names could refer to highly divergent
molecular groups. While studying the mitochondrial
sequence variation (927 pb fragment of the ATPase and
COX3 genes) within and between tuna species (Thunnus
spp.), Takeyama et al., [35] found two divergent groups
(diverging by w4,5%) within the northern bluefin tuna,
Thunnus thynnus, (i.e. T. t. thunnus, living in the Atlantic
ocean and T. t. orientalis, in the Pacific ocean). Thus, they
showed that a re-evaluation of previous restriction
methods was required to avoid inconsistent profiles and
erroneous tuna identification. This result has considerable
consequences as Thunnus thynnus is one of the most
highly prized fish worldwide and Atlantic populations are
on the red list. Similarly, Ludt et al. [36] found two distinct
groups of red deer diverging by 5–6% (complete cytochrome b gene) and concluded that the Cervus elaphus
species is clearly subdivided giving two valid species
Cervus canadensis (occurring in Asia and North America)
and Cervus elaphus (inhabiting Europe). Hird et al. [37]
also found that the sequences of the partial cytochrome b
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Vol.23 No.7 July 2005
363
gene (447 bp) differed by 3% between the English and USA
breeds of turkey (Meleagris gallopavo).
Two valid species could be genetically closely related.
While studying the complete cytochrome b gene variability within the bear family, Talbot and Shields [38] found
that brown bears (Ursus arctos) inhabiting the islands of
southeastern Alaska seemed to be more closely related to
polar bears (Ursus maritimus) than to any other brown
bear populations. Similar conclusions were obtained
between the Greenland cod Gadus ogac and the Pacific
cod Gadus macrocephalus, in which sequences of partial
cytochrome b gene (401 bp) and COX1 (495 bp) were
identical [39]. The authors concluded that these two
species are in fact the same and should be synonymized.
Hybridization between closely related species (introgression) can occur if these species share overlapping
habitats or sometimes through human intervention
(e.g. during captive breeding). Within the group of the
Bovini (comprising cattle-like species) several hybrids
have been described (e.g. [40–42]). Similarly, several fish
species show interspecific mitochondrial introgression
(charr [43,44]; sturgeon [45]). These introgressions could
sometimes disturb molecular identification of closely
related species [46].
For all these reasons, more caution should be taken in
this area. First with the use of scientific names, indeed
most authors were quite elusive about the species they
studied, for example neither the name of the author of
authority who identified the species (e.g. Gadus morhua
Linnaeus, 1758) nor geographical distribution and biological information were indicated; and sometimes only
common names were given (e.g. [47,48]). Second, researchers should be reminded that scientific names could refer to
different contents according to the progress of science [49]
and that discrepancies could exits between taxonomical
conclusions obtained from morphological and molecular
characters even in well known groups, as shown here.
Therefore identification methods cannot only rely on one
or several sequences per species as was the case in the
methods reviewed here. All the problems highlighted here
are particularly critical within fish, where there are
numerous examples of such unclear species boundaries
and species complexes [50]. In addition, because this
group is likely to become the most studied one in the next
decades, it is likely that these kinds of situations will also
become more and more common in the molecular identification field (for a detailed review of species already
analyzed see Box 1).
Recommendations
Whatever the species, a thorough analysis should be made
each time a new group is to be studied (ideally with
taxonomists of each group) and several samples from the
full distribution range should be taken into consideration
to validate the method [51,52]. Voucher specimens should
ideally be preserved, and submitters of sequences should
conform to the ICZN (International Commission on
Zoological Nomenclature; http://www.iczn.org/) rules of
nomenclature [51].
Finally, because sequences from international banks
are usually taken as reference, we would like to emphasize
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Box 4. What is DNA taxonomy, and why is it so
controversial?
What is DNA barcoding?
Herbert et al. [79] recently proposed a DNA-based barcoding system
for all animal species, similar in practice to a supermarket barcode.
According to the proponents of this system, the basic procedures of
DNA barcoding would be straightforward [80]. A tissue sample is
taken from a collected individual and DNA is extracted from this. This
DNA serves as the reference sample from which one or several gene
regions are, as a first approximation, an identification tag for the
species from which the respective individual was derived. This
sequence is then made available via appropriate references, against
which sequences from sampled individuals can be compared (http://
www.barcodinglife.com/). Put simply, given the long history of use
of molecular markers for molecular identification of species Moritz
and Cicero [46] considered that there is nothing fundamentally new
in this DNA barcoding concept, except increased scale and proposed
standardization.
Why is it so controversial?
However, the main problem with the DNA barcoding initiative is that
the proponents of it propose not only to use DNA for identifying
species (the ‘DNA barcode’, which seems to be generally accepted)
but also for defining species, and therefore they want to give DNA a
central and mandatory role in taxonomy (the so-called ‘DNA
taxonomy’). Indeed they think that this is the only way to overcome
the current impediment of taxonomy and particularly they consider
that (i) it is impossible to describe biological diversity with traditional
approaches [81], (ii) the current system depends heavily on
specialists, whose knowledge is frequently lost when they retire
[80], and (iii) species identification based on morphological characters has several significant limitations [79].
Such remarks have already received outright condemnation,
chiefly from taxonomists, on both the theoretical (e.g. [49,82,83])
and practical level (e.g. [46,84]). Among the numerous criticisms, the
main one is that DNA sequences, as morphological characters, are
only data and could not serve to define a species on their own. In
fact, what defines a species is an intractable debate that cannot be
resolved satisfactorily using part of a single gene. The circumscription of a species will therefore always remain an opinion based on all
data available [49,84]. Finally, molecular identification, using
mtDNA, can be misled by Numts, introgression (as we have
illustrated here) but also by incomplete lineage sorting, retention
of polymorphism or absence of polymorphism (see [85] for a more
complete review).
For all these reasons, most taxonomists consider that relegating
taxonomy, rich in theory and knowledge, to a high-tech service
industry would decidedly be a setback for science. They argued that,
instead of discarding more than 250 years of knowledge, new
generations of taxonomists must be trained in initiatives such as the
Partnerships for Enhancing Expertise in Taxonomy, PEET program
([86]; http://web.nhm.ku.edu/peet/).
that they could contain false sequences. For example,
Forster [53] found that half of all published studies of
human mtDNA sequences contain mistakes, not to
mention Numts (as described above). Therefore, Harris
[54] proposed several solutions to check the quality of the
published sequences and the ‘simplest’ is to re-sequence
them. Another solution would be to use as many reference
sequences as possible resulting from different studies
(when available) and never base a method on only one
reference sequence. Consequently, even though there is a
great need for molecular identification in numerous fields,
protection of the biodiversity, food traceability and also
ecology [46], much more caution should be taken in
developing molecular identification methods and more
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Vol.23 No.7 July 2005
generally in implementing the recent call for a ‘DNA
taxonomy’ (Box 4).
Conclusion
Despite these technical and conceptual challenges, molecular species identification in food products and forensic
samples is likely to increase exponentially. Indeed,
numerous reliable methods now exist for identifying
species in almost all kinds of substrates (Table 1 and
Box 3) and some approaches have already produced
significant and interesting results, for example in gourmet
food (such as species identification in caviar [55] or in foie
gras [56]) and on forensic samples made from endangered
species (e.g. tiger [5] or rhinoceros [7]). Molecular
identification has already proven useful in court [4,57]
and some methods are currently used in industry [1,76].
Nevertheless, to become more accurate and reliable, this
biotechnological field will have to take into account the
technical and conceptual knowledge of its nearest fundamental fields.
Acknowledgements
We thank our team for constructive discussions, Brian B. Rudkin and
Vincent Laudet for critical reading of the article, and CNRS UCB-Lyon1,
Région Rhône-Alpes and Ministère de l’Education Nationale, de
l’Enseignement Supérieur et de la Recherche for financial support.
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