Rev Fish Biol Fisheries (2009) 19:265–293
DOI 10.1007/s11160-009-9107-4
RESEARCH PAPER
Molecular identification methods of fish species:
reassessment and possible applications
Fabrice Teletchea
Received: 19 August 2008 / Accepted: 7 January 2009 / Published online: 28 January 2009
! Springer Science+Business Media B.V. 2009
Abstract Fish species identification is traditionally
based on external morphological features. Yet, in
many cases fishes and especially their diverse
developmental stages are difficult to identify by
morphological characters. DNA-based identification
methods offer an analytically powerful addition or
even an alternative. This work intends to provide an
updated and extensive overview on the PCR-methods
for fish species identification. Among the ten main
methods developed, three PCR-RFLP, PCR-FINS
and PCR-specific primers have been the most used.
Two other emerging methods, namely real-time PCR
and microarray technology, offer new potential for
quantification of DNA and simultaneous detection of
numerous species, respectively. Almost 500 species
have been targeted in the past decade, among which
the most studied belong to gadoids, scombroids, and
salmonids. The mitochondrial cytochrome b gene
was by far the most targeted DNA markers. The most
common applications belonged to the forensic, tax-
onomic, and ecological fields. At last, some key
problems, such as the degradation of DNA, the
reliability of sequences, and the use of scientific
names, likely to be encountered during the develop-
ment of molecular identification methods are
described. In conclusion, the tremendous advances
F. Teletchea (&)
UR AFPA, Nancy Université, MAN 34 Rue Sainte
Catherine, 54000 Nancy, France
e-mail: fabrice.teletchea@lsa-man.uhp-nancy.fr
in molecular biology in the past 10 years has
rendered possible the study of DNA from virtually
any substrates, offering new perspectives for the
development of various applications, which will
likely continue to increase in the future.
Keywords Mitochondrial DNA !
DNA barcoding ! Taxonomy ! PCR !
Food products ! Forensic
Introduction
Fish species identification is traditionally based on
external morphological features, including body
shape, pattern of colors, scale size and count, number
and relative position of fins, number and type of fin
rays, or various relative measurements of body parts
(Strauss and Bond 1990). Gill rakers are sometimes
counted to differentiate very similar species (e.g., Iff
At 2002). Otoliths, are also occasionally used,
especially when none of the former features are
available, e.g., to identify fossils or stomach contents
(Pierce and Boyle 1991; Granadeiro and Silva 2000).
Currently, some websites provide broad information
on fish morphology (www.fishbase.org), or more
specifically on otoliths (www.pescabase.org). Yet, in
some cases morphological features are of limited
value for identification and differentiation purposes,
even with whole specimens, because they can show
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either considerable intraspecific variations or small
differences between species. For instance it is very
difficult to differentiate the eight Barbus species of
the Iberian Peninsula based on morphological fea-
tures only (Callejas and Ochando 2001). Besides,
once most morphological features have been removed
during either digestion (e.g., in stomach contents) or
various processing (e.g., canning, filleting), the
identification becomes difficult or even impossible.
Furthermore, the identification of early life stages
(egg and larvae) is even more complicated than adult
identification (Strauss and Bond 1990). Taken all
together, these difficulties explained why researchers
have attempted to develop new methods for identi-
fying fish species without relying on morphological
features.
Traditional and official methods used in species
identification, including fish, are based chiefly on the
separation and characterization of specific proteins
using electrophoretic techniques, such as isoelectric
focusing: IEF (Rehbein 1990) and capillary electro-
phoresis: CE (Kvasnička 2005), high performance
liquid chromatography: HPLC (Hubalkova et al.
2007), or immunoassay systems, such as Enzyme-
Linked ImmunoSorbent Assay: ELISA (Asensio et al.
2008) (for a complete review see e.g., Mackie et al.
1999; Civera 2003; Moretti et al. 2003; Hubalkova
et al. 2007). Nowadays, different companies com-
mercialize ELISA diagnostic kits for various
applications, such as the authentication of species in
milk and cheese (Asensio et al. 2008). However, for
fish species identification, no immunological test kit
has yet been commercialized, probably because of the
potentially large number of species that might be
involved (Mackie et al. 1999). Although most of
these methods are of considerable value in certain
instances, they are not suitable for routine sample
analysis because proteins lose their biological activity
after animal death, and their presence and character-
istic depend on the cell types (Asensio 2007).
Besides, because most proteins are heat labile, they
become irreversibly denatured when the flesh is
cooked (e.g., in food products) and thus can no longer
be examined by techniques suitable for their native
states. It is, however, possible to solubilise some
heat-denatured proteins in denaturing solvents such
as 2% sodium dodecylsulphate or 8 M urea to obtain
species-specific profiles on electrophoresis (Etienne
et al. 2000). Antibodies used in immunoassay
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systems are also normally raised against undenatured
proteins and thus useful for fresh samples only; even
though some antibodies were developed against
thermostable proteins (Ansfield et al. 2000). This
results in that attention has now turned to DNA as a
source of information.
As an alternative to protein analysis, DNA-based
identification methods have been developed, chiefly
in the past decade (Teletchea et al. 2005). DNA
presents several advantages over protein analysis.
First, even though DNA might be altered by various
processing (canning, heating), it is more resistant and
thermostable than proteins are and it is still possible
to PCR (polymerase chain reaction) amplify small
DNA fragments (with sufficient information to allow
identification). Second, DNA could potentially be
retrieved from any substrate because it is present in
almost all cells of an organism. In addition, because
of the degeneracy of the genetic code and the
presence of many non-coding regions, DNA provides
much more information than proteins do. This results
in that the tremendous advances in molecular biol-
ogy, driven by the clinical arena, have now rendered
possible the identification of any species in virtually
any kind of organic substrate, such as muscle, fin, or
blood (Lockley and Bardsley 2000; Teletchea et al.
2005).
Focusing on the past decade (1997–2007), the
main goals of the present review are to provide: (a) a
brief description and comparison of the main methods
applied to fish species identification, (b) an assess-
ment of the species and DNA markers targeted, (c)
few examples of the possible applications, and
(d) a summary of the key problems that may be
encountered.
Description and comparison of methods
Most of the 153 studies reviewed here (Appendix)
have clearly focused on three among the ten methods
applied to fish species identification in the past
decade, i.e., PCR-RFLP, PCR-sequencing and PCR-
specific primers (Table 1). On the opposite, five
methods were barely used, and some seems even
abandoned today, namely PCR-SSCP, PCR-RAPD,
PCR-DGGE, PCR-ALFP, and cloning and sequenc-
ing. More recently, two new methods have emerged
in biology, which are real-time PCR and microarray
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Table 1 List and relative importance of the ten methods applied to fish species identification in the past decade (1997–2007)
Techniques Number References

PCR-RLFP 71 [1, 3, 4, 6, 8, 9, 21, 23, 24, 26, 28–31, 35, 36, 38, 39, 44, 46–48, 50, 52, 54, 57–66, 70, 73,
76–78, 80, 84–88, 91, 92, 96, 100, 104–108, 112, 113, 120, 123–125, 127, 130, 133, 134, 139,
145–147, 150, 151]
PCR-sequencing 43 [2, 12, 15, 17, 18, 40, 43, 45, 46, 48, 51, 53, 56, 63–67, 70, 71, 73–75, 79, 81, 82, 94, 102,
104, 107, 115, 116, 118, 124, 126, 129, 132, 136, 137, 140, 142, 148, 149]
PCR-specific 23 [5, 7, 9, 11, 14, 27, 33, 34, 40, 50, 68, 69, 89, 95, 97, 98, 100, 117, 122, 127, 128, 138, 141]
primers
PCR-SSCP 9 [10, 32, 37, 39, 109–112, 144]
Real-time PCR 9 [25, 41, 49, 55, 70, 90, 135, 138, 143]
PCR-RAPD 5 [19, 20, 42, 83, 121]
PCR-DGGE 5 [39, 45, 47, 48, 153]
PCR-AFLP 3 [93, 152, 153]
Cloning and 2 [45, 72]
sequencing
Microarray 1 [13]
Number, number of studies using this method (the number is superior to the 153 studies reviewed here because studies can develop
more than one method)
References, numbers correspond to studies listed in the Appendix

technology. The first method seems to be more and
more used, while only one study has applied micro-
array technology to fish species identification in the
past decade (Babola et al. 2004). A brief description
and comparison of all these methods are provided
below.
Descriptions of the methods
analyzed. Lin and Hwang (2007) used this technique
to identify eight species of scombroids. Two sets of
primers were designed to amplify 126 and 146 bp of
the mitochondrial cytochrome b gene, and five
restriction enzymes were determined to analyze the
short length fragments. The method was successfully
applied to the authentication of species in 18
commercial canned tuna.

PCR-RFLP PCR-sequencing

PCR-RFLP (Restriction Fragment Length Polymor-
phism) is a method in which an amplified fragment is
cut by endonucleases, which recognized specific
restriction sites, resulting in few smaller fragments
of different sizes. The different fragments are then
separated by agarose gel electrophoresis. This
method is simple, robust, and much easier to perform
and less costly than PCR-sequencing, explaining why
it is the most popular. Besides, a single pair of
primers can produce a fragment that can be used for
the identification of multiple species with judicious
choice of restriction enzymes. The main disadvan-
tages are that incomplete digestion may occasionally
occur and intraspecific variations could delete or
create additional restriction sites (Lockley and Bards-
ley 2000). Besides, its dependence on restriction
enzymes requires previous knowledge of the sample
The sequencing option has been advocated under the
name FINS: forensically informative nucleotide
sequencing (Bartlett and Davidson 1992). The DNA
sequence is obtained by sequencing the purified PCR
fragment using the same primers. Sequencing was
traditionally time consuming, technically demanding,
and required good data handling capacity; yet this is
clearly the method that produces the largest amount
of information (Lockley and Bardsley 2000; Asensio
2007). Today both prices and time have significantly
been reduced, which explained why this method
has become the preferred one. For instance, Maretto
et al. (2007) sequenced a 430 bp fragment of the
mitochondrial 16S rRNA gene to identify four
economically important species belonging to the
Gadiformes. They identified three SNPs (single
nucleotide polymorphism) that allows unambiguous

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discrimination between the four species. Balitzki-
Korte et al. (2005) used a new sequencing method-
ology, called pyrosequencing, which is well suited for
the sequencing of short stretches located next to the
sequencing primer. This technique is based on the
detection of pyrophosphate (PPi) that is released
from dNTPs during the DNA synthesis. Based on a
cascade of enzymatic reactions, visible light is
generated and detected. The amount of light is
directly related to the number of incorporated
nucleotides (Ronaghi 2001). Balitzki-Korte et al.
(2005) demonstrated that the detection by pyrose-
quencing of only 20 nucleotides following the
sequencing primer, within a 149-bp fragment of the
mitochondrial 12S rDNA gene, was sufficient to
identify the biological origin of samples by align-
ment with a reference sequence database.
PCR-specific primers
Detailed sequence information has become available
for many species and consequently phylogenetically
information single base polymorphisms may be iden-
tified that enable species-specific primers to be
designed. Under suitable stringent reaction conditions,
such primers generate a fragment, visualized by
agarose gel electrophoresis, only in the presence of
DNA from a given species. This procedure is applica-
ble only when some previous knowledge of the
material analyzed is available and identification is
made on a small set of putative species. Besides,
appropriate controls should be included to preclude the
possibility of false positive or negative results being
obtained (the lack of amplified fragment on the gel may
be due to technical problems rather than due to the
absence of the target DNA). Multiplex PCR is a variant
of PCR-specific primers, which enables simultaneous
amplification of many targets of interest by using more
than one pair of primers in one reaction tube. Michelini
et al. (2007) developed a one-step triplex PCR-based
assay to discriminate between three tuna species,
yellowfin tuna Thunnus albacares, bigeye tuna Thun-
nus obesus, and skipjack tuna Katsuwonus pelamis.
The species of origin of the DNA was indicated by the
distinctive size of the PCR product, i.e., 246 bp for
yellowfin tuna, 262 bp for bigeye tuna and 113 bp for
skipjack tuna. Multipex PCR has the potential to
produce considerable savings of time and effort within
the laboratory without comprising test utility.
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PCR-SSCP
PCR-SSCP (Single Strand Conformation Polymor-
phism) is a technique based on the relationship
between the electrophoretic mobility of a single-
stranded DNA and its folded conformations, which in
turn reflects the nucleotide sequence. The amplified
product is denatured to a single-stranded form
(ssDNA) and electrophoresed on a non-denaturing
polyacrilamide gel (PAGE). The differential migra-
tion of the ssDNA (in theory two bands of ssDNA
have to be expected, one per strand) originates in the
difference in DNA sequence between samples. PCR-
SSCP is fast and easy to perform. Besides, because
even single base changes in a sequence are likely to
result in different conformations, highly close-species
may be accurately discriminated, even using very
short fragments. Yet, this technique displays three
major disadvantages: (a) the necessity to run refer-
ences and samples side by side on the same gel
(because SSCP profiles are highly dependent on the
conditions of native PAGE), (b) intra-species varia-
tion may result in different conformations leading to
wrong identification, and (c) sometimes more than
two bands are visible, differing in intensity. The
reason for this phenomenon may be that the ssDNA
exists in several states of conformation depending on
the electrophoretic conditions. Weder et al. (2001)
studied the interest of a SSCP method, originally
developed to identify tuna and bonito species, for
discriminating other fish and animal species. Two to
four strong ssDNA bands were obtained for several
fish species (e.g., blue ling, carp), while other fish
species resulted in either weak (cod, spined dogfish)
or no ssDNA bands (halibut, herring). They demon-
strated that increasing the stringency of PCR
conditions caused a more pronounced difference
between strong and weak ssDNA bands. Besides,
they also found that inter-laboratory reproducibility
(using different chemicals, such as PCR kits, gel
rehydratation buffer) of the method was good.
Real-time PCR
Real-time PCR (also known as quantitative PCR,
Real-time quantitative PCR, or qPCR) is a method of
simultaneous DNA amplification and quantification.
In real-time PCR a fluorescent reporter molecule is
included in the assay mix and this enables the
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products of the PCR reaction to be measured after
each cycle once a threshold has been passed. This
reporter molecule is an oligonucleotide that has a
fluorescent dye attached to the 50 end and a fluores-
cence quencher attached to the 30 end and is designed
to anneal to a position between the two primers.
During amplification, the 50 -30 exonuclease activity
of the Taq polymerase digests the probe and releases
the reporter molecule that then fluoresces. The
amount of fluorescence produced is proportional to
the amount of amplicon produced during PCR. DNA
quantification is based on the threshold cycle (Ct),
which is the cycle at which the fluorescence is
detected at a predetermined value above the back-
ground. In practice, a Ct value is determined for both
a normalizing target sequence that is non-discrimi-
natory (acts as an internal standard) as well as for the
specific target. To improve precision, both assays
should be performed simultaneously within the same
tube by using different fluorescent reporter mole-
cules. The accuracy of quantification obtained with
PCR is largely dependent on the reference material
used to construct the standard curve (for a complete
description of qPCR techniques available see Lockley
and Bardsley 2000). Casper et al. (2007) experimen-
tally compared the utility of real-time PCR for
identifying consumption of captive Arctocephalus
seals fed mixed prey diets (one squid and two fish
taxa) with the occurrence of hard part remains of prey
in scats. They found that although all test prey had
robust hard parts, detecting consumption during the
studied period was 1.4 and 5.8 times more likely
using genetic analysis than morphological analysis of
scats. However, neither DNA nor hard part methods
accurately reflected the relative importance of test
prey to diet using the frequency of occurrence index.
PCR-RAPD
PCR-RAPD (Random Amplified Polymorphic DNA)
consists in the amplification, by PCR, of random
segments of genomic DNA using a single short
primer of arbitrary sequence, thus one can expect to
scan the genome more randomly than using conven-
tional techniques. The two main advantages of using
RAPD are (a) it does not required previous knowl-
edge of DNA sequences and (b) it targets many
sequences in the DNA of the sample, producing
DNA patterns that allow comparison of many loci
269
simultaneously. Besides, because of its simplicity and
speed in execution, the RAPD procedure is very
effective. However, RAPD analysis presents some
major disadvantages: (a) it may not be practical to
identify the species of origin in products containing
mixtures of species, and (b) it is not adequate for
analysis of severally degraded material, because it is
highly susceptible to slight changes in target-DNA
quality and quantity. Lakra et al. (2007) developed a
PCR-RAPD method to detect interspecific genetic
variability and genetic relatedness among five Indian
scianids. Among the 50 arbitrary primers tested, eight
were selected on the basis of reproducibility and
resolution of banding patterns in all five species. The
eight primers amplified a total of 85 loci in the size
range from 40 to 4,697 bp. The number of stable and
clear RAPD bands generated per primers varied
between 9 and 12.
PCR-DGGE
PCR-DGGE (Denaturing Gradient Gel Electrophore-
sis) is a method based on the separation of PCR
amplicons of the same size but different sequences.
This is because these fragments can be separated in a
denaturing gradient gel based on their differential
denaturation (melting) profiles. In DGGE gel, double-
strand DNA fragments are subjected to an increasing
denaturing environment as they encounter increasing
concentrations of the denaturing agents (urea or
formamide) and partially melt in discrete regions
called ‘‘melting domains’’. The melting temperature
(Tm) of these domains is sequence-specific. Once the
Tm of the lowest melting domain is reached, that part
of the fragment becomes partially melted, creating
branched ‘‘breaking’’ molecules. This behavior
reduces the DNA mobility in the acrylamide gel.
Therefore, DNA fragments of the same size but
different base pair compositions will show a different
response to the denaturing gradient. The different
sequences of the DNA fragments will have melting
domains with different Tm values that will run
different distances in the DGGE (for a detailed
description see Roelfsema and Peters 2005). This
method has been chiefly used in several fields of
microbial ecology (Ercolini 2004); yet, rarely for fish
species identification. Comi et al. (2005) provided a
rare example of a comparison between three different
PCR methods (RFLP, SSCP and DGGE) used to
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differentiate eight species commonly used in the
production of cod-fish. They found that while RFLP
and SSCP were not able to identify all the species
tested, DGGE achieved better results. They also
found that, in some cases, samples (differing by only
one base) belonging to the same species (e.g., cod)
migrated in different positions in the gel, illustrating
the discriminatory power of DGGE (similar results
described by Deagle et al. 2005).
PCR-AFLP
PCR-AFLP (Amplified Fragment Length Polymor-
phism) is a technique based on the selective PCR
amplification of restriction fragments from a total
digest of genomic DNA (Vos et al. 1995). AFLP uses
restriction enzymes to cut genomic DNA, followed
by ligation of adaptors to the sticky ends of the
restriction fragments. A subset of the restriction
fragments are then amplified using primers comple-
mentary to the adaptor and part of the restriction site
fragments. After final amplification, selectively
amplified fragments are separated by gel electropho-
resis and visualized either through autoradiography or
fluorescence technologies. AFLP combines the
advantages of RAPD and RFLP, and thus displays
higher reproducibility, resolution, and sensitivity at
the whole genome level compared to these two
techniques. Besides, AFLP generates hundreds of
informative genetic markers that increase the
probability of detection of species-specific and pop-
ulation-specific polymorphisms. At last, this
technique does not require any upfront knowledge
of the species dealing with. This technique has been
used chiefly in population genetics to determine
slight differences within populations, but barely for
fish species identification. Maldini et al. (2006)
assessed the potential use of the AFLP technology
to determine fish and seafood species (molluscs and
crustaceans) in processed commercial products and
domestics stocks. The ten primer combinations
produced fragments in the size range 70–600 bp.
The total number of fragments varied between 14 and
90, and major differences were observed in different
systematic groups. The comparison of informative
markers between unknown frozen and fresh products
and references samples enabled the accurate identi-
fication of 32 different species, 20 freshwater and
marine fish, four crustaceans and eight molluscs. The
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taxonomic characterization was performed either at
the species or at the population level depending on
the number of available individuals.
PCR-cloning and sequencing
PCR-cloning and sequencing involves three main
steps. First, the target fragment is PCR amplified.
Then, the PCR amplification products are cloned
using various kits, such as the TOPO TA cloning" kit
(Invitrogen). According to this protocol, PCR prod-
ucts are combined with a TOPO" cloning vector, and
then transformed into competent Escherichia coli.
The bacteria are plated and positive transformants
recognized using blue/white color selection (for a full
description see http://www.invitrogen.com/site/us/en/
home.html). At last, several clones per amplification
product are sequenced. Clones could also be analyzed
by cheaper techniques than sequencing (especially
when the number of clones is very high), such as
DGGE (Deagle et al. 2005). The major advantage of
this technique is that it allows detecting whether
several different target DNA sequences, potentially
belonging to different species, are present within the
studied sample. Yet, compared to the previous
molecular methods, PCR-cloning and sequencing is
much more expensive and time-consuming, and
explained why it has rarely been used for fish species
identification. Jarman et al. (2004) evaluated the
interest of this technique for studying the feces of
various marine predators, among which a sample of a
fin whale (Balaenoptera physalus). They analyzed
the diversity of two clone libraries containing 46
and 25 clones, which were obtained with primers
designed to amplify any eukaryotic or Bilateria DNA,
respectively. In both cases, sequences from krill were
the only unequivocal prey item identified, even
though the clone library generated with Bilateria-
specific PCR primers was more useful (i.e., only 4 out
of the 46 clones analyzed corresponded to prey items
with the eukaryotic primers). Therefore, they advised
to use group-specific primers rather than more gen-
eral or ‘‘universal’’ primers (see section ‘‘DNA
markers used for species identification’’), because it
allows a considerable reduction in the diversity of
PCR products to those derived from a target group
of species, which can greatly simplify downstream
analyses.
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Microarray
Microarrays (also known as DNA chips) are glass
microscope slides on which oligonucleotide probes
are spotted that are complementary to the DNA target
sequences to be analyzed. DNA target, which is
usually fluorophore-labeled during PCR amplifica-
tion, hybridises with the oligonucleotide probe on the
microarray and can be detected after washing steps by
its label. DNA microarrays enable the examination of
complex mixtures of PCR products and, potentially,
the identification of hundreds or even thousands of
species simultaneously. Even though applying DNA
microarrays for gene expression has already reached
the routine level of high throughout systems, they
have been only recently applied for the identification
of species and only once for fish species during the
past decade. Indeed, Babola et al. (2004) were the first
to provide a microarray targeting 32 vertebrate
species, including 12 mammals, 5 birds and 15 fish.
More recently, two new studies (Kochzius et al. 2008;
Teletchea et al. 2008) further explored the potential of
microarray for fish species identification. Teletchea
et al. (2008) developed a microarray-based method
using cytochrome b-derived probes to identify 71
commercial and/or endangered vertebrate species in
both food and forensic samples (9 birds, 34 mammals,
26 ray-finned fishes and 2 sharks). They demonstrated
that this microarray-based method was relatively fast,
completed within 2 days, easy to use, and that results
were straightforward (high ratio between signal/
background). It was also cost-effective, particularly
when compared with PCR-cloning and sequencing,
and provided more accurate results. This method was
successfully validated on both simple samples and
mixtures (containing up to five species) as well as on
fresh and degraded substrates (hair, bone, blood,
muscle and foodstuffs).
Comparisons
A variety a DNA based methods are potentially
available for use in fish species identification. These
vary in their range of applications, complexity and
costs (a comparison of the ten methods reviewed here
are presented in Table 2). All these factors are liable
to influence the uptake of such tests by laboratory.
For instance, if one wants to routinely analyze
numerous samples of the same four or five species,
271
he may choose between PCR-RFLP or PCR-specific
primers (cheapest and fast results). Whereas, if one
wants to develop a more general approach, targeting
many species simultaneously, it may be more inter-
esting to develop his own DNA chip. Besides,
depending on the samples analyzed, the quality of
the DNA varies greatly (see section ‘‘DNA markers
used for species identification’’), thus some methods
are not useful with highly degraded DNA, particu-
larly PCR-RAPD or PCR-AFLP. Consequently,
depending on the objective of the study, kind of
samples analyzed and available funds, one could
choose between the different methods based on their
respective advantages and disadvantages. In the last
years, advances in PCR-methods have led to rapid
development of different commercial kits for fish
species identification. PCR kits contain all the
necessary reagents, controls and accessories for rapid
testing (for more information see Asensio 2007;
Rasmussen and Morrissey 2008).
Species and DNA markers targeted
Species targeted
Overall, 478 species were targeted in the different
studies reviewed here. Yet, among these 478 species,
291 were targeted only once and 141 between two
and four times. On the opposite, 11 species were
targeted more than 10 times, among which the most
used were Salmo salar (18 times), Oncorhynchus
mykiss (16), Thunnus albacares (16) and Gadus
morhua (15). Hence, it emerged clearly that most
studies have focused on few taxonomic groups,
corresponding either to the most economically
important (e.g., gadoids, scombroids, salmonids,
flatfishes), or among the most endangered ones
(sturgeons; Table 3). Besides, much more studies
focused on marine rather than freshwater fishes. It is
also obvious that the majority of studies targeted
species inhabiting Europe and North America, while
only few focused on tropical species, such as red
snappers Lutjanus spp. (Zhang et al. 2004). The last
category, called ‘‘miscellaneous’’, includes either
freshwater fishes such as perch Perca spp. (Strange
and Stepien 2007) or marine fishes such as hairtail
fish Trichiurus spp. (Chakraborty et al. 2007). In
conclusion, taking into account the ca. 30,000 species
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Table 2 Comparison of the ten methods applied to fish species identification in the past decade (1997–2007)

123
Techniques DNA Admixturesb Prior Quantitatived Intraspecific Reproductibilityf Developing Costh Speed Main Main
qualitya knowledgec variationse timeg analysisi advantagej disadvantagek

PCR-RLFP ? Yes Yes No Yes Yes ? ? ? Fast Incomplete

digestion
PCR- ? No Yes/No No No Yes ? ?? ?? Quantity of Cost
sequencing information
PCR-specific ? Yes Yes No Yes Yes ? ? ? Fast Low taxonomic
primers range
PCR-SSCP ? No Yes No Yes Yes/No ? ? ? Fast Reproducibility
Real-time PCR ?? No Yes Yes No Yes ?? ?? ? Quantitative Cost
PCR-RAPD ??? No Yes No No Yes/No ? ? ? Fast Quality of
genomic DNA
PCR-DGGE ?? No Yes No Yes Yes ? ? ? Fast Discriminatory
power
PCR-AFLP ??? No Yes No No Yes/No ? ?? ?? Sensitivity Quality of
genomic DNA
Cloning and ? Yes No ? No ? ?? ??? ??? Admixtures Cost
Sequecing
Microarray ? Yes No ? No Yes ????? ??? ??? Admixtures Developing time
‘‘?’’ indicates that according to current knowledge it is difficult to decisively conclude
a
DNA quality required
b
Admixtures: possibility to evaluate the presence of several DNA target
c
Prior knowledge: necessity to have an idea of the species or group of species targeted to apply the method
d
Quantitative: is the method allow quantifying the DNA target
e
Intraspecific variations: are the results influenced by intra-species variations
f
Reproducibility: given the same experimental conditions, will the method gives similar result
g
Developing time: time required to conceive, produce and experimentally validate the method
h
Cost: cost of the analysis (not including the developing time)
i
Speed analysis: time required from the extraction of DNA to result
j
Main advantage compared to the other available methods
k
Main disadvantage compared to the other available methods
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Rev Fish Biol Fisheries (2009) 19:265–293 273

Table 3 List of the main groups targeted in the past decade (1997–2007)
Environment Groups Number References

Freshwater Salmonids 27 [12, 13, 15, 22–24, 36, 45, 47, 57–59, 79, 82, 93, 95, 100, 105, 106, 109, 110,
120, 144–146, 149, 153]
Anguillids 13 [2, 13, 50, 58, 66, 70, 87, 93, 110, 112, 127, 143, 146]
Sturgeons 8 [14, 59, 82, 91, 110, 133, 147, 148]
Cyprinids 5 [19, 20, 42, 79, 144]
Miscellaneous 7 [10, 11, 82, 98, 121, 132, 144]
Marine Gadoids 29 [1, 5–8, 13, 21, 32, 39, 43, 45, 46, 48, 49, 55, 58, 82, 93, 94, 102–104, 108,
110, 119, 135, 141, 144, 146]
Scombroids 23 [3, 4, 13, 16, 33, 37, 40, 41, 59, 68, 69, 86, 89, 90, 93, 97, 99, 107, 111, 115,
116, 134, 136]
Flatfishes 14 [9, 26–28, 38, 48, 51, 58, 93, 103, 122, 130, 144, 146]
Clupeoids 10 [15, 45, 58, 73, 74, 101, 110, 124, 125, 144]
Sharks 10 [31, 34, 53, 54, 56, 93, 128, 131, 137, 144]
Billfishes 7 [60–62, 92, 96, 115, 139]
Rockfishes 6 [52, 84, 85, 117, 118, 144]
Puffers 5 [35, 63–65, 79]
Miscellaneous 32 [10, 17, 18, 22, 25, 29, 30, 36, 44, 45, 67, 72, 75–81, 83, 88, 93, 101, 113,
114, 123, 138, 140, 146, 150–152]
Number, number of studies focusing on a particular group (the number is superior to the 153 studies reviewed here because studies
can target more than one group)
References, numbers correspond to studies listed in the Appendix

already described (Froese and Pauly 2008), it is likely
that the number of species for which an identification
method is developed will tremendously increase
in the coming years, particularly with the current
development of the DNA barcoding initiative (see
section ‘‘DNA barcoding’’).
DNA markers used for species identification
Among the nine main DNA markers targeted, it
appeared obvious that most studied have focused on
mitochondrial genes, and particularly on the cyto-
chrome b gene (Table 4). Three main reasons explain
why most studies focused on mitochondrial DNA
genome (mtDNA) rather than nuclear DNA. First,
because there are several copies of mtDNA inside a
cell (*1,000 9 the copies of nuclear DNA) it is
more likely to amplify a fragment within this genome
rather than within the nuclear genome (particularly
interesting when DNA is degraded, see section
‘‘Study of degraded DNA’’). Besides, this small
circular genome (*16 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. Besides, a suitable DNA marker for
identification at the species level should be suffi-
ciently variable between species (particularly the
closest ones) and display either low or no-intra-
specific variations across the geographic area. Ideally,
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
the cytochrome b satisfied most of these criteria and
is by far the most studied gene for phylogeny. It was
certainly used in more than half of the phylogenetic
studies published in the past decade (more than 140
000 sequences available in GenBank at the time of
this writing). In addition, this gene displayed both
(a) conserved regions allowing the determination of
primers amplifying numerous species such as the
‘‘universal’’ primers published by Kocher et al.
(1989), and regions with a high level of variability,
which allows to discriminate even closely related

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Table 4 List of the main DNA targeted in the past decade (1997–2007)
DNA markers Number References

Cytochrome b 77 [1, 5, 6, 8, 13, 14, 16, 18, 21, 28, 31–37, 39, 47, 48, 54, 56–66, 68, 73–76, 78, 82, 86–89,
91, 96, 97, 99, 100, 102, 107, 109–112, 115–118, 120, 123–126, 129, 130, 132–134, 136,
139, 140, 144, 146–149, 153]
16S rRNA 31 [2, 15, 24, 25, 29, 30, 44–46, 50–53, 70, 71, 75, 77, 79–81, 84, 90, 94, 100, 103, 106,
119, 127, 137, 138, 143]
12S rRNA 16 [5, 7, 10, 12, 26, 38, 46, 52, 53, 68, 69, 79, 84, 87, 150, 151]
5S rRNA 10 [3, 4, 11, 22, 27, 40, 51, 67, 77, 78]
D-LOOP 10 [9, 17, 96, 101, 108, 114, 122, 133, 141, 149]
ATPase 6 [41, 49, 68, 96, 113, 135]
COI 6 [5, 18, 43, 71, 101, 142]
ND3/ND4 4 [23, 52, 84, 85]
ATPase 8 3 [49, 113, 135]
Miscellaneous 19 [18, 34, 40, 54–56, 69, 72, 92, 95, 96, 98, 104–106, 109, 126, 128, 145]
Number, number of studies focusing on a particular DNA marker (the number is superior to the 153 studies reviewed here because
studies can target more than one DNA marker)
References, numbers correspond to studies listed in the Appendix

species. The other DNA markers targeted, grouped in
the ‘‘miscellaneous’’ category, were NADH2 (Dalm-
asso et al. 2006) and ND4 genes (McDowell and
Graves 2002) among others (see also Rasmussen and
Morrissey 2008).
Possible applications
It appeared obvious that most studies have focused on
three main fields of applications: forensic science,
taxonomy and ecology (Table 5). Among these three,
forensic science and particularly food authentication
is the most important. Others, such as the identifica-
tion of early life stages, are more recent and will
certainly continue to develop further in the future. A
description and few examples of the different fields of
application are provided below.
Forensic applications
Food products
The international fish market, estimate by the FAO at
around 60 billion/year, may imply more than 20,000
species of fish (Rasmussen and Morrissey 2008). For
instance, about 420 species of fish are sold in
the German market alone (Kochzius et al. 2008).
Besides, most of these fish species are available
commercially after the removal of some external
features as fresh (eviscerated, beheaded, skinned,
filleted, etc.) and processed products (marinated,
salted, smoked, caned, frozen, etc.), which makes
the identification of species a difficult task. About
80% of fish and fishery products on the German
market are pretreated: smoked, canned, salted, or
pickled fillets, or at least filleted (Horstkotte and
Rehbein 2003). Market globalization, large numbers
of both species exploited and processed fish products
explained why the substitution of a less valuable
species for a valuable one (representing a commercial
fraud), may be a common phenomenon difficult to
detect. For instance, a study on food fish in the United
States revealed that three-quarters of fish sold as the
threatened ‘‘red snapper’’ Lutjanus campechanus
were mislabeled and belonged to other species
(Marko et al. 2004). Likewise, Pepe et al. (2007)
found that 84.2% (16/19) of surimi-based fish prod-
ucts sold as Theragra chalcogramma were actually
prepared with species different from the one declared
(more examples are provided in Rasmussen and
Morrissey 2008). The development of analytical
methods for fish species identification may help to
detect and avoid wilful, as well as unintentional
substitution of different fish species and thus enforce
labeling regulations (Lockley and Bardsley 2000;

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Table 5 List of the main applications found in the past decade (1997–2007)
Types of applications Number References

Forensic Food products 84 [1, 3, 4, 6–8, 10–16, 21–24, 26–30, 32, 33, 36–41, 43, 46–48, 55–63, 66,
68–70, 73, 74, 78–80, 86, 89–91, 93, 94, 97, 99, 102, 104, 107–112,
120, 124, 125, 127, 130, 132, 135, 136, 138, 144, 146–148, 150, 151, 153]
Food poisoning 4 [35, 64, 65, 139]
Genetically modified 3 [95, 98, 114]
organisms
Taxonomic Species discrimination 21 [17–20, 42, 44, 50–52, 75, 81, 83, 87, 105, 113, 121, 131, 134, 137, 140, 145]
and identification
Body parts 9 [31, 34, 53, 54, 82, 96, 123, 128, 133]
Ecological Early life history stages 19 [2, 5, 49, 67, 76, 77, 84, 85, 88, 92, 101, 103, 115–118, 141, 143, 152]
Trophic relationships 9 [9, 25, 45, 72, 100, 106, 119, 122, 129]
Others DNA barcoding 3 [71, 126, 142]
Ancient DNA analysis 1 [149]
Number, number of studies focusing on a particular application
References, numbers correspond to studies listed in the Appendix

Teletchea et al. 2005; Asensio 2007; Mafra et al. Food poisoning

2008). For instance, the European Union Reg.104/
2000 establishes that fish products enter the commer-
cial circuit only if the commercial name, method of
production (farmed or wild) and capture area are
clearly labeled (for more details see Asensio and
Montero 2008). Therefore, numerous methods have
been developed to authenticate various fish species in a
wide range of food products, including soup and dried
fins (Hoelzel 2001), surimi (Weder et al. 2001; Pepe
et al. 2007), fish roe (Klossa-Kilia et al. 2002), spicy
roe (Aranishi et al. 2005), fish tails (Sanjuan et al.
2002), canned sardine and sardine-type products
(Jérôme et al. 2003), and canned tuna (Rehbein et al.
1999). The development of these molecular methods
helps not only to protect both consumers and producers
from frauds, but may also help to protect fish species
from over-exploitation or illegal trafficking (Teletchea
et al. 2005). Indeed, most exploited fish species
are currently endangered, among which sturgeons;
because their eggs (caviar) are one of the most
exclusive and expensive fishery products (Wolf et al.
1999). Based on survey of 95 lots of commercially
available caviar in the New York City area, Birstein
et al. (1998) found that 23% of the designations made
by caviar suppliers were mislabeled with respect to
species identification. Replacement of commercial
species with endangered and threatened species indi-
cates possible illegal harvest and poaching.
Identification of hazardous species in order to elim-
inate them from retail trade is one of the most
important tasks of veterinary inspection of food
products. Current laws ban the sale of both poisonous
fish belonging to the families Tetraodontidae, Moli-
dae, Diodontidae and Canthigasteridae and fishery
products containing biotoxins, such as ciguatoxins or
muscle paralyzing toxins (Civera 2003). Indeed, even
though these toxins are heat-labile, thus cooked food
should be safe, 100–200 people per year are seriously
intoxicated, with a lethal outcome for half of them
(Civera 2003). Hsieh and Hwang (2004) developed a
PCR-RFLP assay to identify 16 puffer fishes (Lago-
cephalus spp., Takifugu spp.) from Taiwanese
seawaters. This new assay, complete within 24 h,
could be valuable to help wildlife officers to identify
these dangerous fish. Other species may also contain
some toxic substances, such as histamine in scom-
broid fishes. Tsai et al. (2007) reported two incidents
of food-borne poisonings, causing illness in 59 and 43
victims due to the ingestion of billfish meats.
Analyses of histamine showed that the suspected
billfish samples contained more than 150 mg/100 g
of histamine, which is higher than the hazard action
level of 50 mg/100 g. Based on the results of a PCR-
RFLP of a fragment of the cytochrome b gene, they
were able to identify the two species implicated in

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the food poisonings, namely Makaira nigricans and
Xiphias gladius.
GMO detection
Consumer concerns regarding genetically modified
organisms GMOs have created a demand for labeling
foods derived from transgenic products (Miraglia
et al. 2004). In anticipation of voluntary or mandatory
labeling, tests are being developed to identify GM
food. Current methodologies for the analysis of
genetically modified organisms are focused on either
one of two targets, the transgenic DNA inserted- or
the novel protein(s) expressed- in a genetically
modified product. Masri et al. (2002) developed a
PCR-specific primers protocol for the identification
of genetically modified coho salmon (Oncorhynchus
kisutch) carrying a growth hormone transgene. The
primers were specific for the amplification of a gene
construct present in transgenic salmon; no amplifica-
tion from the wild-type fish. The optimized PCR
method identified all samples tested (61 samples and
17 controls) with 100% accuracy. Besides, the assay
was sensitive enough to detect the transgenic in as
little as 1 ng of total DNA. The PCR test could be
performed in a few hours, using tissues from all parts
of the fish including blood, bones, scales, slime and
other internal organs; thus it was possible to perform
the detection test without damaging or reducing the
market value of the target fish.
Taxonomic applications
Species discrimination and identification
Fish species taxonomy is traditionally based on
morphological and anatomical traits. Yet reliance
on morphology can sometimes present difficulties in
cases where species may be very similar morpholog-
ically or apparent differences are misleading.
Byrkjedal et al. (2007) demonstrated that Eumicro-
tremus spinosus and E. eggvinii, currently considered
as two valid species, are clearly separated by a
considerable number of morphological characters; yet
they in fact constitute a single, sexually dimorphic
species. Indeed, identical DNA sequences were found
in all E. eggvinii and E. spinosus samples studied.
Besides, all specimens of E. eggvinii (including the
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holotype) and E. spinosus sexed turned out to be
males and females, respectively. The use of molec-
ular genetic makers alongside traditional taxonomic
methods can also help clarify relationships as they
ran reveal for example, the existence of cryptic
species (taxa that cannot be distinguished morpho-
logically but are genetically distinct). While studying
the genetic variability of Schindleria, believed to
include one of the smallest and youngest reproducing
vertebrates, Kon et al. (2007) found as many as 21
genetically distinguishable species inhabiting the
studied geographical areas. This discovery of many
cryptic species in Schindleria suggests that the use of
DNA sequences is necessary for species identification
of such morphologically conserved taxa. Conse-
quently they proposed that DNA-based designation
is necessary for such taxa in order to compile the full
‘‘species lists’’; yet there is presently no consensus for
the inclusion of DNA sequencing data in the formal
descriptions of new species.
Fish species identification is also traditionally
based on morphology, using keys. Yet, in some
cases conventional keys could be misleading or
provide ambiguous diagnoses especially for individ-
uals with intermediate values in traits that are
supposed to be discriminative. In certain taxa, it is
indeed not unusual to encounter situations in which
individuals possess combination of character states
thought to be typical of different species, such as in
the palearctic coregonids (Politov et al. 2000).
Besides, morphological characters are sometimes of
limited value for identification and differentiation
purposes because they show a considerable intraspe-
cific variations and differences among species are
small. All these difficulties could result in misi-
dentification. For example, adult rockfishes Sebastes
mystinus, S. melanops, and S. ciliatus are so similar
that they are often misidentified on the field
(Gharrett et al. 2001). Reid and Wilson (2006)
found a disagreement between 20% of samples
provided by various government agencies and spe-
cies-specific digest patterns, indicating a need for
greater care during field identifications of Moxos-
toma species. In addition, the difficulty to identify
species can led to incomplete survey of the true
fauna present in a given area (Tinti et al. 2003;
Schander and Willassen 2005) or to misunderstand
ecological interactions, such as marine bioinvasions
(Bucciarelli et al. 2002).
Rev Fish Biol Fisheries (2009) 19:265–293
In conclusion, application of molecular tools can
provide valuable information for species identifica-
tion and complement the traditional taxonomic data.
Identification of body parts
Many commercially important shark species are
difficult to identify whole, and this task is more
daunting if individuals are processed (head, entrails,
and fins removed); unfortunately at-sea processing is
widespread in the industry (Greig et al. 2005).
Although current US legislation prohibits the practice
of ‘‘finning’’ (where fins are retained and discarded at
sea), the landings of fins is allowed where carcasses
and fins are off-loaded at the same time in no more
than 1:20 (fin-to-carcass) weight ratio. However,
serious problems can arise in matching off-loaded
fins to processed carcasses, e.g., it might be tempting
to increase the fin-to-carcass ratio with spoiled meat
or ‘‘finning’’ target species out of season (and
subsequently attributing the fins to fish that are
allowed to be caught during the season). Within this
context, Greig et al. (2005) developed a PCR-FINS
method to identify 35 shark species (Carcharhinus
spp., Sphyrna spp., Mustelus spp. among others) from
the Atlantic fishery. They found that the sequence of
the 12S–16S region of the mtDNA contained ample
information for discriminating between the shark
species studied, and thus planned to increase their
database to the seventy-three species inhabiting the
United States territorial waters of the Atlantic Ocean,
Gulf of Mexico and Caribbean Sea. Such accurate
and reliable species identification methods are para-
mount for law enforcement and sound shark species
management (most endangered).
Ecological applications
Early life history stages determination
The study of morphological variation among fish
species has conventionally been based on compari-
sons of adults. This is true in part because young
stages are usually difficult to collect and identify.
However, another important reason for emphasizing
adults is that many fishes have distinctive larvae that
are transitory forms, possessing very different body
shapes and numerous, sometimes bizarre, temporary
organs (Strauss and Bond 1990). In addition, the very
277
simple, generalized forms of most larvae provide
relatively few consistent taxonomically important
characters with which to characterize and describe
morphological relationships among species. There
are two basic approaches to the study of fish eggs and
larvae. The most common relies on field collections
and works back from known adults, using diagnostics
characters common to smaller individuals of a size
series. The other relies on eggs and larvae reared
from known parents and works forward (Methven
and McGowan 1998). Whenever fishes can be
hatched and reared from eggs, the second method is
preferred because the specimens being described are
known with certainty.
Despite considerable effort, larval identification
remains one of the major factors limiting the many
important questions that can be addressed by field-
based ichthyoplankton studies, including the identi-
fication of spawning locations and seasons of
migrating species, the quantification of population
levels or biomass of fished species. For instance, only
15 species, among the 65 rockfish species Sebastes
found along the California coast, can be separated at
the larval stage by physical characters such as body
shape, pigmentation patterns, and head spine devel-
opment (Li et al. 2006). While increased effort in
developing morphological identification systems will
likely be successful for many taxonomic groups of
fishes, it appears that the underlying limits have been
reached for many others (Richardson et al. 2007).
Therefore, the application of molecular-based iden-
tification methods in ichthyoplankton surveys could
significantly increase their accuracy, particularly for
species producing eggs and larvae that cannot be
separated using classical methods. Based on genetic
identification, Fox et al. (2005) found that the
majority of eggs in the Irish Sea, wrongly believed
to be from cod, were actually from whiting Merlan-
gius merlangus, leading to an overestimation of cod
stocks.
Trophic relationships
Determining trophic relationships within an ecosys-
tem is a key part of many ecological studies;
however, obtaining reliable data on diet composition
for most species is fraught with difficulties. On one
hand, it is not always possible to track trophic
interactions between predators and prey by direct
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observation, particularly when observing small or
elusive animals with cryptic food-wed ecology. On
the other hand, gut and/or fecal analysis can some-
times allow prey remains to be identified visually but
is only possible when a component of the diet is
resistant to digestion, such as otoliths or skeletal
parts, which can be identified to genus and species
level by experienced researchers (Pierce and Boyle
1991; Smith et al. 2005; Casper et al. 2007). Besides,
in some cases there are no solid remains, and when
there are it can lead to bias in interpretation of prey
choice. Consequently, numerous invasive and non-
invasive methods have been developed to character-
ize predator-prey interactions, among which
molecular methods (reviewed in Sheppard and Har-
wood 2005; King et al. 2008). Smith et al. (2005)
developed a DNA sequencing method to identify
partially digested prey items taken from the gut
content of seven species of large pelagic fishes, such
as marlins Makaira spp. or tunas Thunnus spp. All are
large opportunistic predators feeding on pelagic
fishes, squids and crustaceans. They found that the
prey items taken from the 14 samples analyzed were
derived from six pelagic fish species from two
families (Scombridae and Carangidae). Deagle et al.
(2005) conducted a captive feeding trial to test
whether prey DNA could be reliably detected in scat
samples from Steller sea lions (Eumetopias jubatus).
Two sea lions were fed a diet of fish (five species)
and squid (one species), and DNA was extracted from
the soft component of collected scats. Most of the
DNA came from the predator, but prey DNA could be
amplified using prey-specific primers, with very good
results. Thus, they concluded that even though
several aspects of the methodology need further
development before a complete picture of diet can be
constructed, molecular scatology to study diet has the
potential to provide new insight into the diet of
vertebrate species. Similarly, Matejusová et al.
(2008) applied quantitative PCR to examine the
presence of salmonids, Atlantic salmon (Salmo salar)
and sea trout (Salmo trutta), in the diet of grey seals
(Halichoerus grypus) and harbor seals (Phoca vitu-
lina) in the Moray Firth, UK, during the summer of
2003 and 2005. The qPCR assay approach was shown
to be highly efficient and consistent in detection of
salmonids from seal scats, and to be more sensitive
than conventional hard-parts analysis. Nevertheless,
their results confirmed previous indicating that
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salmonids are not common prey for seals in these
Scottish estuaries.
Other applications
DNA barcoding
Hebert et al. (2003) proposed standardizing the
various approaches used in species identification
through the establishment of a DNA barcoding
system, similar in practice to a supermarket barcode,
for all living organisms, based on a single sequence: a
648-bp portion of the mitochondrial gene cytochrome
c oxidase I (COI). Initial reactions to this DNA
barcoding proposition have ranged from enthusiasm,
especially from ecologists (Janzen 2004), to criti-
cisms, chiefly concerning the identification of closely
related species using a single gene (Lipscomb et al.
2003; Mallet and Willmott 2003; Moritz and Cicero
2004). In the meantime, Tautz et al. (2003) proposed
to give DNA a central and mandatory role in
taxonomy for both identifying and defining species
(the DNA taxonomy concept), which is aimed at
replacing the traditional, time-consuming, chiefly
morphology-based system. This second proposition
has led to condemnation, particularly concerning the
concept of species (Mallet and Willmott 2003;
Seberg et al. 2003; Will and Rubinoff 2004) and
has revived the old controversy in systematics about
the relative importance of DNA technology versus
morphological traits (Blaxter 2003, 2004; Wheeler
et al. 2004; DeSalle et al. 2005; Will et al. 2005).
Despite this initial lively debate, still ongoing today
(e.g., Paterlini 2007; Schlick-Steiner et al. 2007), a
new international program ‘‘Barcode of Life Initia-
tive’’ has eventually been created for studies and
molecular cataloging of species diversity of all
animals and plants of the Earth (http://www.
barcoding.si.edu/). The major goals of this program
are to provide molecular identification of organisms
using standardized DNA region (DNA barcode) and
to create a special database that would be more
taxonomically accurate and would have more rigor-
ous rules for entry of the data compared with the
existing databases, such as GenBank (for more details
see Hanner and Gregory 2007). In the past 5 years,
several studies have evaluated the potential of DNA
barcoding for identifying fish species, among which
Australia’fishes (Ward et al. 2005; Pegg et al. 2006)
Rev Fish Biol Fisheries (2009) 19:265–293
(for a more complete review about DNA barcoding
progresses see Waugh 2007). At the time of this
writing, more than 36000 COI barcode records are
deposited in the Barcode of Life Database (Ratnas-
ingham and Hebert 2007) with representation from
more than 6,500 species of actinopterygians.
Another project that has been focused on sequence
information for specific genes is the FishTrace
Consortium (http://www.fishtrace.org), which com-
prises 53 members from several European institutions
(Sevilla et al. 2007). The FishTrace database provides
detailed information on a number of fish species
common to Europe, along with barcoding data for the
gene mitochondrial cytochrome b and nuclear rho-
dopsin. The barcoding information used by FishTrace
includes a longer DNA sequence than that used in
COI studies, and is has been argued that the use of
DNA barcodes longer in length will allow for
increased efficiency of identification labels (Sevilla
et al. 2007). Besides, the combination of two genes
that exhibit different genomic positions and rates of
evolution was reported to be valuable for the effi-
ciency of DNA barcoding. Other DNA databases,
among which the largest is Genbank (http://
www.ncbi.nlm.nih.gov) can also be accessed online
(reviewed in Rasmussen and Morrissey 2008).
Ancient DNA analyses
Ancient DNA research, defined broadly as the
retrieval of DNA sequences from museum specimens,
archaeological finds, fossil remains, and other
unusual sources of DNA, was born about 20 years,
when DNA sequences were separately described
from the quagga (a type of zebra) and an ancient
Egyptian individual (Pääbo et al. 2004). The retrieval
and analysis of such degraded DNA should apply key
criteria (appropriate laboratory facilities and controls,
independent replication and cloning of amplification
products) to ascertain to the greatest extent possible
that results represent authentic ancient DNA
sequences (Gilbert et al. 2005; Willerslev and Cooper
2005). Despite these significant constraints, numer-
ous studies have been published in the past decade,
particularly concerning mammals (Pääbo et al. 2004;
Millar et al. 2008). Yang et al. (2004) provided a rare
example of applying ancient DNA analysis for
species identification of archaeological fish bone.
Targeting short mitochondrial DNA fragments (less
279
than 300 bp), they studied more than 20 salmon bone
samples (dated 7,000–2,000 BP) from the site of
Namu of the central coast of British Columbia. Four
species, coho (Oncorhynchus kisutch), sockeye
(O. nerka), pink (O. gorbuscha) and chum (O. keta)
salmon, were identified from the remains, which was
consistent with other lines of evidence. These results
demonstrated that the DNA can be well preserved in
remains from the Pacific Northwest Coast of North
America, and thus provide new data to interpret
faunal remains at Namu. They concluded that these
workable protocols could be used to identify more
vertebra samples from more sites to study fishery
activity in the region.
Key problems
Study of degraded DNA
Depending on the kind of samples analyzed, the
quantity and quality of DNA vary greatly. In
unprocessed food products (such as fillets) or fresh
samples (egg, larvae, body parts), DNA is generally
unaltered and present in high quantity, thus large
fragments of DNA (*1–2 kb) can potentially be
amplified (in some cases the variability of the entire
genome can be studied using PCR-RAPD, PCR-
AFLP methods). In the opposite, in most samples
encountered in both fishery market (canned products,
surimi) and ecology (scats, gut contents), DNA is
degraded and present in small quantities that consid-
erably reduce the number of DNA fragments with
suitable size for molecular analysis (Lockley and
Bardsley 2000; Teletchea et al. 2005; King et al.
2008). Such damages to DNA are due to heat
exposure, high pressure, low pH, irradiation, drying,
nucleases that cause enzymatic degradation, depuri-
nation and hydrolysis, among others. Consequently,
identification methods from degraded substrates
should be based on the analysis of very short
mitochondrial fragments, preferably between 100
and 200 bp (explanations are given in section
‘‘DNA markers used for species identification’’).
For instance, Quinteiro et al. (1998) found that DNA
was degraded during the canning process of fish
muscle: DNA fragment sizes that ranged from \100
up to 200 bp were obtained from canned tuna muscle,
whereas DNA sizes for frozen tuna muscle ranged
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from \100 bp up to 20,000 bp. Carrera et al. (2000)
found that DNA degradation produced by the cold
smoking process did not prevent amplification of
DNA fragments close to 1 kb in size. On the contrary,
nucleic acids were heavily degraded during the
sterilization process. Yet, the precise effects of
various processing practices on the integrity of
DNA in seafood are still largely unknown (Bossier
1999). Because DNA is generally altered and present
in low quantity in degraded samples and PCR such a
sensitive method, sometimes a single exogenous
DNA molecule could be preferentially amplified
instead of the degraded one (reviewed in Cooper
and Wayne 1998). Thus, degraded samples should be
manipulated (before PCR) in a dedicated laboratory
to avoid as much as possible contaminations and
appropriate negative controls (extraction and PCR
blanks) should be processed on every run to guaran-
tee the reliability of the results (Teletchea et al.
2005). Furthermore, the lack of amplification in
degraded samples (i.e., a negative readout) could be
explained by the inhibition of the PCR amplification
rather than by insufficient amount or absence of the
target DNA. Such inhibition is due to the presence of
various products that are co-purified with the target
DNA (reviewed in Wilson 1997). Therefore extrac-
tion methods should allow removals of inhibitors; yet
no general extraction method has proved useful with
all the different matrices encountered. Therefore,
each new substrate requires the development of new
extraction and amplification methods or the adapta-
tion of existing ones (Woolfe and Primrose 2004;
Teletchea et al. 2005). Additional work should now
focus on extraction yield to improve the quality and
quantity of DNA retrieved, particularly from highly
degraded samples (e.g., Chapela et al. 2007). At last,
DNA retrived in highly degraded samples are also
known to display in some cases chemical modifica-
tions: modified DNA molecules might display breaks
or artifactual mutations (reviewed in Poinar 2002;
Bower et al. 2005). Consequently, absence of DNA
degradation from highly degraded samples 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 (Teletchea et al. 2005). Similarly,
non-invase studies have also shown that PCR
amplifcation starting from tiny amounts of altered
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Rev Fish Biol Fisheries (2009) 19:265–293
DNA could induce artifactual results, e.g., chimeric
molecules produced by jumping PCR (Pääbo et al.
1990). In conclusion, the retrieval and analysis of
DNA in highly degraded samples require to apply
several criteria to ensure as much as possible the
reliability of the results.
Reliability of sequences in databases
Among the ten PCR methods reviewed here
(Table 1), eight are developed from sequences,
usually taken from international database, such as
Genbank. Yet, sequences deposited in these databases
could sometimes be false (Forster 2003; Nilsson et al.
2006). These false sequences might be due to
sequencing errors, misidentification of the target
species or other laboratory problems. Indeed, PCR
reactions are easily contaminated by carryover DNA
from other organisms of by other DNAs in laboratory.
Mislabeling of tubes can also lead to incorrect
assignment of sequence data to species. While
developing a PCR-RFLP method for the authentica-
tion of 11 carcharhiniform sharks, Heist and Gold
(1999) detected numerous differences between the
restriction patterns predicted from the sequence of
Martin and Palumbi (1993) for tiger shark (Gale-
ocerdo cuvier) collected near Hawaii and their tiger
shark collected from the Gulf of Maxico (Atlantic)
and Hawaii and Australia (Pacific). They sequenced
the entire cytochrome b gene in tiger shark from
Florida and, in comparison with the ‘‘tiger shark’’
sequence reported by Martin and Palumbi (1993),
found one nucleotide difference in the first (50 -most)
540 bp of the gene and 64 nucleotide differences in
the remaining 606 bp of the gene. They hypothesized
that the ‘‘tiger shark’’ sequence of Martin and
Palumbi (1993) was a mixture that included sequence
data from another species. Another source of false
sequence when targeting mitochondrial genome to
develop identification methods are the nuclear mito-
chondrial pseudogenes (generally referred to as
numts). Numts are nuclear inserted copies of mito-
chondrial origin exhibiting a high degree of similarity
with mtDNA sequences, which might be amplified
inadvertently by the PCR in addition to or even
instead of the authentic target mtDNA, thus leading
to erroneous results. Within vertebrates, the majority
of numts have been reported in mammals and birds,
but it is likely a consequence of the disproportionate
Rev Fish Biol Fisheries (2009) 19:265–293
attention these taxa receive relative to other groups
(Benesh et al. 2006). In fish species, numts have been
found in Fugu rubripes, Tetraodon nigroviridis, and
Danio rerio (Antunes and Ramos 2005), or in
Gadiculus argenteus and Melanogrammus aeglefinus
(Teletchea et al. 2006) among others. Bensasson et al.
(2001) described methods for detecting them (e.g.
checking for unique changes and odd substitution
patterns, such as indels and/or stop codons). How-
ever, current practices do not preclude inadvertent
analysis of numts and much caution should be
exercised when using mitochondrial sequences for
identification purposes.
In conclusion, Harris (2003) 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 (Teletchea et al. 2005). The
problem of false sequences will certainly be reduced
with the current development of specific projects
dedicated to the identification of species, such as the
Consortium for the barcode of life, as it requires new
standards for the publication of sequences (Hanner
and Gregory 2007).
The species problem
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 e.g., Sites and
Marshall 2003). Rather, I illustrate here some prob-
lems usually encountered in fish taxonomy that could
result in erroneous molecular identification methods
if not take into account. The same scientific names
could refer to highly divergent molecular groups.
While studying the mitochondrial sequence variation
(927 bp fragment of the ATPase and COX3 genes)
within and between tuna species (Thunnus spp.),
Takeyama et al. (2001) found two divergent groups
(diverging by *4.5%) 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-evalua-
tion of previous RFLP methods was required to
avoid inconsistent profiles and erroneous tuna
281
identification. Similarly, while developing a micro-
array-based method for identifying various
vertebrates, Teletchea et al. (2008) found two diver-
gent groups (diverging by 8.93%) within the john
dory (Zeus faber), one cluster of two individuals from
the Atlantic Ocean and the Mediterranean Sea, and
one cluster of three individuals from Japan, which
might correspond to two different and valid species.
On the opposite, two valid species based on morpho-
logical characters could be genetically similar. While
studying the partial cytochrome b (401 bp) and
cytochrome oxidase I (495 bp) genes variability
within the cod family, Carr et al. (1999) obtained
identical sequences between the Greenland cod
Gadus ogac and the Pacific cod Gadus macroceph-
alus. The authors concluded that these two species
are in fact the same and should be synonymized.
Furthermore, hybridization between closely related
species (introgression) can occur if these species
share overlapping habitats or sometimes through
human intervention (e.g. during captive breeding).
Hence, it has been found that several fish species
show interspecific mitochondrial introgression: charr
(Doiron et al. 2002; Redenbach and Taylor 2003) or
sturgeon (Ludwig et al. 2003). These introgressions
could sometimes disturb molecular identification of
closely related species (Moritz and Cicero 2004).
For all these reasons, caution should be taken with
the use of scientific names. One should indicate
clearly the species he studies, for example give the
name of the author of authority who identified the
species (e.g., Gadus morhua Linnaeus, 1758), and
also should provide geographical distribution and key
biological information. Furthermore, researchers
should be reminded that scientific names could refer
to different contents according to the progress of
science (Seberg et al. 2003) and that discrepancies
could exit between taxonomical conclusions obtained
from morphological and molecular characters. This
implies that when new molecular-based clusters are
found, only expert taxonomists can satisfactorily
resolve the relationship between them and species
(Schindel and Miller 2005). In conclusion, 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 (Ruedas et al.
2000; Comesana et al. 2003). Voucher specimens
123
282
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 (Ruedas et al.
2000).
Acknowledgements I thank two anonymous reviewers that
helped to improve the manuscript.
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