Rev Fish Biol Fisheries (2009) 19:265–293 DOI 10.1007/s11160-009-9107-4

RESEARCHPAPER

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 Ó Springer Science+Business Media B.V. 2009

Abstract Fish species identification is traditionally in molecular biology in the past 10 years has based on external morphological features. Yet, in rendered possible the study of DNA from virtually many cases fishes and especially their diverse any substrates, offering new perspectives for the developmental stages are difficult to identify by development of various applications, which will morphological characters. DNA-based identification likely continue to increase in the future. methods offer an analytically powerful addition or even an alternative. This work intends to provide an Keywords Mitochondrial DNA updated and extensive overview on the PCR-methods DNA barcoding Taxonomy PCRÁ Á Á Á for fish species identification. Among the ten main Food products Forensic Á 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 Introduction quantification of DNA and simultaneous detection of numerous species, respectively. Almost 500 species Fish species identification is traditionally based on have been targeted in the past decade, among which external morphological features, including body the most studied belong to gadoids, scombroids, and shape, pattern of colors, scale size and count, number salmonids. The mitochondrial cytochrome b gene and relative position of fins, number and type of fin was by far the most targeted DNA markers. The most rays, or various relative measurements of body parts common applications belonged to the forensic, tax- (Strauss and Bond 1990). Gill rakers are sometimes onomic, and ecological fields. At last, some key counted to differentiate very similar species (e.g., Iff problems, such as the degradation of DNA, the At 2002). Otoliths, are also occasionally used, reliability of sequences, and the use of scientific especially when none of the former features are names, likely to be encountered during the develop- available, e.g., to identify fossils or stomach contents ment of molecular identification methods are (Pierce and Boyle 1991; Granadeiro and Silva 2000). described. In conclusion, the tremendous advances Currently, some websites provide broad information on fish morphology (www.fishbase.org), or more specifically on otoliths (www.pescabase.org). Yet, in F. Teletchea (&) some cases morphological features are of limited UR AFPA, Nancy Universite´, MAN 34 Rue Sainte Catherine, 54000 Nancy, France value for identification and differentiation purposes, e-mail: [email protected] even with whole specimens, because they can show

123 266 Rev Fish Biol Fisheries (2009) 19:265–293 either considerable intraspecific variations or small systems are also normally raised against undenatured differences between species. For instance it is very proteins and thus useful for fresh samples only; even difficult to differentiate the eight Barbus species of though some antibodies were developed against the Iberian Peninsula based on morphological fea- thermostable proteins (Ansfield et al. 2000). This tures only (Callejas and Ochando 2001). Besides, results in that attention has now turned to DNA as a once most morphological features have been removed source of information. during either digestion (e.g., in stomach contents) or As an alternative to protein analysis, DNA-based various processing (e.g., canning, filleting), the identification methods have been developed, chiefly identification becomes difficult or even impossible. in the past decade (Teletchea et al. 2005). DNA Furthermore, the identification of early life stages presents several advantages over protein analysis. (egg and larvae) is even more complicated than adult First, even though DNA might be altered by various identification (Strauss and Bond 1990). Taken all processing (canning, heating), it is more resistant and together, these difficulties explained why researchers thermostable than proteins are and it is still possible have attempted to develop new methods for identi- to PCR (polymerase chain reaction) amplify small fying fish species without relying on morphological DNA fragments (with sufficient information to allow features. identification). Second, DNA could potentially be Traditional and official methods used in species retrieved from any substrate because it is present in identification, including fish, are based chiefly on the almost all cells of an organism. In addition, because separation and characterization of specific proteins of the degeneracy of the genetic code and the using electrophoretic techniques, such as isoelectric presence of many non-coding regions, DNA provides focusing: IEF (Rehbein 1990) and capillary electro- much more information than proteins do. This results phoresis: CE (Kvasnicˇka 2005), high performance in that the tremendous advances in molecular biol- liquid chromatography: HPLC (Hubalkova et al. ogy, driven by the clinical arena, have now rendered 2007), or immunoassay systems, such as Enzyme- possible the identification of any species in virtually Linked ImmunoSorbent Assay: ELISA (Asensio et al. any kind of organic substrate, such as muscle, fin, or 2008) (for a complete review see e.g., Mackie et al. blood (Lockley and Bardsley 2000; Teletchea et al. 1999; Civera 2003; Moretti et al. 2003; Hubalkova 2005). et al. 2007). Nowadays, different companies com- Focusing on the past decade (1997–2007), the mercialize ELISA diagnostic kits for various main goals of the present review are to provide: (a) a applications, such as the authentication of species in brief description and comparison of the main methods milk and cheese (Asensio et al. 2008). However, for applied to fish species identification, (b) an assess- fish species identification, no immunological test kit ment of the species and DNA markers targeted, (c) has yet been commercialized, probably because of the few examples of the possible applications, and potentially large number of species that might be (d) a summary of the key problems that may be involved (Mackie et al. 1999). Although most of encountered. these methods are of considerable value in certain instances, they are not suitable for routine sample analysis because proteins lose their biological activity Description and comparison of methods after animal death, and their presence and character- istic depend on the cell types (Asensio 2007). Most of the 153 studies reviewed here (Appendix) Besides, because most proteins are heat labile, they have clearly focused on three among the ten methods become irreversibly denatured when the flesh is applied to fish species identification in the past cooked (e.g., in food products) and thus can no longer decade, i.e., PCR-RFLP, PCR-sequencing and PCR- be examined by techniques suitable for their native specific primers (Table 1). On the opposite, five states. It is, however, possible to solubilise some methods were barely used, and some seems even heat-denatured proteins in denaturing solvents such abandoned today, namely PCR-SSCP, PCR-RAPD, as 2% sodium dodecylsulphate or 8 M urea to obtain PCR-DGGE, PCR-ALFP, and cloning and sequenc- species-specific profiles on electrophoresis (Etienne ing. More recently, two new methods have emerged et al. 2000). Antibodies used in immunoassay 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 analyzed. Lin and Hwang (2007) used this technique more used, while only one study has applied micro- to identify eight species of scombroids. Two sets of array technology to fish species identification in the primers were designed to amplify 126 and 146 bp of past decade (Babola et al. 2004). A brief description the mitochondrial cytochrome b gene, and five and comparison of all these methods are provided restriction enzymes were determined to analyze the below. short length fragments. The method was successfully applied to the authentication of species in 18 Descriptions of the methods commercial canned tuna.

PCR-RFLP PCR-sequencing

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

123 268 Rev Fish Biol Fisheries (2009) 19:265–293 discrimination between the four species. Balitzki- PCR-SSCP Korte et al. (2005) used a new sequencing method- ology, called pyrosequencing, which is well suited for PCR-SSCP (Single Strand Conformation Polymor- the sequencing of short stretches located next to the phism) is a technique based on the relationship sequencing primer. This technique is based on the between the electrophoretic mobility of a single- detection of pyrophosphate (PPi) that is released stranded DNA and its folded conformations, which in from dNTPs during the DNA synthesis. Based on a turn reflects the nucleotide sequence. The amplified cascade of enzymatic reactions, visible light is product is denatured to a single-stranded form generated and detected. The amount of light is (ssDNA) and electrophoresed on a non-denaturing directly related to the number of incorporated polyacrilamide gel (PAGE). The differential migra- nucleotides (Ronaghi 2001). Balitzki-Korte et al. tion of the ssDNA (in theory two bands of ssDNA (2005) demonstrated that the detection by pyrose- have to be expected, one per strand) originates in the quencing of only 20 nucleotides following the difference in DNA sequence between samples. PCR- sequencing primer, within a 149-bp fragment of the SSCP is fast and easy to perform. Besides, because mitochondrial 12S rDNA gene, was sufficient to even single base changes in a sequence are likely to identify the biological origin of samples by align- result in different conformations, highly close-species ment with a reference sequence database. may be accurately discriminated, even using very short fragments. Yet, this technique displays three PCR-specific primers major disadvantages: (a) the necessity to run refer- ences and samples side by side on the same gel Detailed sequence information has become available (because SSCP profiles are highly dependent on the for many species and consequently phylogenetically conditions of native PAGE), (b) intra-species varia- information single base polymorphisms may be iden- tion may result in different conformations leading to tified that enable species-specific primers to be wrong identification, and (c) sometimes more than designed. Under suitable stringent reaction conditions, two bands are visible, differing in intensity. The such primers generate a fragment, visualized by reason for this phenomenon may be that the ssDNA agarose gel electrophoresis, only in the presence of exists in several states of conformation depending on DNA from a given species. This procedure is applica- the electrophoretic conditions. Weder et al. (2001) ble only when some previous knowledge of the studied the interest of a SSCP method, originally material analyzed is available and identification is developed to identify tuna and bonito species, for made on a small set of putative species. Besides, discriminating other fish and animal species. Two to appropriate controls should be included to preclude the four strong ssDNA bands were obtained for several possibility of false positive or negative results being fish species (e.g., blue ling, carp), while other fish obtained (the lack of amplified fragment on the gel may species resulted in either weak (cod, spined dogfish) be due to technical problems rather than due to the or no ssDNA bands (halibut, herring). They demon- absence of the target DNA). Multiplex PCR is a variant strated that increasing the stringency of PCR of PCR-specific primers, which enables simultaneous conditions caused a more pronounced difference amplification of many targets of interest by using more between strong and weak ssDNA bands. Besides, than one pair of primers in one reaction tube. Michelini they also found that inter-laboratory reproducibility et al. (2007) developed a one-step triplex PCR-based (using different chemicals, such as PCR kits, gel assay to discriminate between three tuna species, rehydratation buffer) of the method was good. yellowfin tuna Thunnus albacares, bigeye tuna Thun- nus obesus, and skipjack tuna Katsuwonus pelamis. Real-time PCR The species of origin of the DNA was indicated by the distinctive size of the PCR product, i.e., 246 bp for Real-time PCR (also known as quantitative PCR, yellowfin tuna, 262 bp for bigeye tuna and 113 bp for Real-time quantitative PCR, or qPCR) is a method of skipjack tuna. Multipex PCR has the potential to simultaneous DNA amplification and quantification. produce considerable savings of time and effort within In real-time PCR a fluorescent reporter molecule is the laboratory without comprising test utility. included in the assay mix and this enables the

123 Rev Fish Biol Fisheries (2009) 19:265–293 269 products of the PCR reaction to be measured after simultaneously. Besides, because of its simplicity and each cycle once a threshold has been passed. This speed in execution, the RAPD procedure is very reporter molecule is an oligonucleotide that has a effective. However, RAPD analysis presents some fluorescent dye attached to the 50 end and a fluores- major disadvantages: (a) it may not be practical to cence quencher attached to the 30 end and is designed identify the species of origin in products containing to anneal to a position between the two primers. mixtures of species, and (b) it is not adequate for During amplification, the 50-30 exonuclease activity analysis of severally degraded material, because it is of the Taq polymerase digests the probe and releases highly susceptible to slight changes in target-DNA the reporter molecule that then fluoresces. The quality and quantity. Lakra et al. (2007) developed a amount of fluorescence produced is proportional to PCR-RAPD method to detect interspecific genetic the amount of amplicon produced during PCR. DNA variability and genetic relatedness among five Indian quantification is based on the threshold cycle (Ct), scianids. Among the 50 arbitrary primers tested, eight which is the cycle at which the fluorescence is were selected on the basis of reproducibility and detected at a predetermined value above the back- resolution of banding patterns in all five species. The ground. In practice, a Ct value is determined for both eight primers amplified a total of 85 loci in the size a normalizing target sequence that is non-discrimi- range from 40 to 4,697 bp. The number of stable and natory (acts as an internal standard) as well as for the clear RAPD bands generated per primers varied specific target. To improve precision, both assays between 9 and 12. should be performed simultaneously within the same tube by using different fluorescent reporter mole- PCR-DGGE cules. The accuracy of quantification obtained with PCR is largely dependent on the reference material PCR-DGGE (Denaturing Gradient Gel Electrophore- used to construct the standard curve (for a complete sis) is a method based on the separation of PCR description of qPCR techniques available see Lockley amplicons of the same size but different sequences. and Bardsley 2000). Casper et al. (2007) experimen- This is because these fragments can be separated in a tally compared the utility of real-time PCR for denaturing gradient gel based on their differential identifying consumption of captive Arctocephalus denaturation (melting) profiles. In DGGE gel, double- seals fed mixed prey diets (one squid and two fish strand DNA fragments are subjected to an increasing taxa) with the occurrence of hard part remains of prey denaturing environment as they encounter increasing in scats. They found that although all test prey had concentrations of the denaturing agents (urea or robust hard parts, detecting consumption during the formamide) and partially melt in discrete regions studied period was 1.4 and 5.8 times more likely called ‘‘melting domains’’. The melting temperature using genetic analysis than morphological analysis of (Tm) of these domains is sequence-specific. Once the scats. However, neither DNA nor hard part methods Tm of the lowest melting domain is reached, that part accurately reflected the relative importance of test of the fragment becomes partially melted, creating prey to diet using the frequency of occurrence index. branched ‘‘breaking’’ molecules. This behavior reduces the DNA mobility in the acrylamide gel. PCR-RAPD Therefore, DNA fragments of the same size but different base pair compositions will show a different PCR-RAPD (Random Amplified Polymorphic DNA) response to the denaturing gradient. The different consists in the amplification, by PCR, of random sequences of the DNA fragments will have melting segments of genomic DNA using a single short domains with different Tm values that will run primer of arbitrary sequence, thus one can expect to different distances in the DGGE (for a detailed scan the genome more randomly than using conven- description see Roelfsema and Peters 2005). This tional techniques. The two main advantages of using method has been chiefly used in several fields of RAPD are (a) it does not required previous knowl- microbial ecology (Ercolini 2004); yet, rarely for fish edge of DNA sequences and (b) it targets many species identification. Comi et al. (2005) provided a sequences in the DNA of the sample, producing rare example of a comparison between three different DNA patterns that allow comparison of many loci PCR methods (RFLP, SSCP and DGGE) used to

123 270 Rev Fish Biol Fisheries (2009) 19:265–293 differentiate eight species commonly used in the taxonomic characterization was performed either at production of cod-fish. They found that while RFLP the species or at the population level depending on and SSCP were not able to identify all the species the number of available individuals. 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) PCR-cloning and sequencing migrated in different positions in the gel, illustrating the discriminatory power of DGGE (similar results PCR-cloning and sequencing involves three main described by Deagle et al. 2005). steps. First, the target fragment is PCR amplified. Then, the PCR amplification products are cloned PCR-AFLP using various kits, such as the TOPO TA cloningÒ kit (Invitrogen). According to this protocol, PCR prod- PCR-AFLP (Amplified Fragment Length Polymor- ucts are combined with a TOPOÒ cloning vector, and phism) is a technique based on the selective PCR then transformed into competent Escherichia coli. amplification of restriction fragments from a total The bacteria are plated and positive transformants digest of genomic DNA (Vos et al. 1995). AFLP uses recognized using blue/white color selection (for a full restriction enzymes to cut genomic DNA, followed description see http://www.invitrogen.com/site/us/en/ by ligation of adaptors to the sticky ends of the home.html). At last, several clones per amplification restriction fragments. A subset of the restriction product are sequenced. Clones could also be analyzed fragments are then amplified using primers comple- by cheaper techniques than sequencing (especially mentary to the adaptor and part of the restriction site when the number of clones is very high), such as fragments. After final amplification, selectively DGGE (Deagle et al. 2005). The major advantage of amplified fragments are separated by gel electropho- this technique is that it allows detecting whether resis and visualized either through autoradiography or several different target DNA sequences, potentially fluorescence technologies. AFLP combines the belonging to different species, are present within the advantages of RAPD and RFLP, and thus displays studied sample. Yet, compared to the previous higher reproducibility, resolution, and sensitivity at molecular methods, PCR-cloning and sequencing is the whole genome level compared to these two much more expensive and time-consuming, and techniques. Besides, AFLP generates hundreds of explained why it has rarely been used for fish species informative genetic markers that increase the identification. Jarman et al. (2004) evaluated the probability of detection of species-specific and pop- interest of this technique for studying the feces of ulation-specific polymorphisms. At last, this various marine predators, among which a sample of a technique does not require any upfront knowledge fin whale (Balaenoptera physalus). They analyzed of the species dealing with. This technique has been the diversity of two clone libraries containing 46 used chiefly in population genetics to determine and 25 clones, which were obtained with primers slight differences within populations, but barely for designed to amplify any eukaryotic or Bilateria DNA, fish species identification. Maldini et al. (2006) respectively. In both cases, sequences from krill were assessed the potential use of the AFLP technology the only unequivocal prey item identified, even to determine fish and seafood species (molluscs and though the clone library generated with Bilateria- crustaceans) in processed commercial products and specific PCR primers was more useful (i.e., only 4 out domestics stocks. The ten primer combinations of the 46 clones analyzed corresponded to prey items produced fragments in the size range 70–600 bp. with the eukaryotic primers). Therefore, they advised The total number of fragments varied between 14 and to use group-specific primers rather than more gen- 90, and major differences were observed in different eral or ‘‘universal’’ primers (see section ‘‘DNA systematic groups. The comparison of informative markers used for species identification’’), because it markers between unknown frozen and fresh products allows a considerable reduction in the diversity of and references samples enabled the accurate identi- PCR products to those derived from a target group fication of 32 different species, 20 freshwater and of species, which can greatly simplify downstream marine fish, four crustaceans and eight molluscs. The analyses.

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Microarray he may choose between PCR-RFLP or PCR-specific primers (cheapest and fast results). Whereas, if one Microarrays (also known as DNA chips) are glass wants to develop a more general approach, targeting microscope slides on which oligonucleotide probes many species simultaneously, it may be more inter- are spotted that are complementary to the DNA target esting to develop his own DNA chip. Besides, sequences to be analyzed. DNA target, which is depending on the samples analyzed, the quality of usually fluorophore-labeled during PCR amplifica- the DNA varies greatly (see section ‘‘DNA markers tion, hybridises with the oligonucleotide probe on the used for species identification’’), thus some methods microarray and can be detected after washing steps by are not useful with highly degraded DNA, particu- its label. DNA microarrays enable the examination of larly PCR-RAPD or PCR-AFLP. Consequently, complex mixtures of PCR products and, potentially, depending on the objective of the study, kind of the identification of hundreds or even thousands of samples analyzed and available funds, one could species simultaneously. Even though applying DNA choose between the different methods based on their microarrays for gene expression has already reached respective advantages and disadvantages. In the last the routine level of high throughout systems, they years, advances in PCR-methods have led to rapid have been only recently applied for the identification development of different commercial kits for fish of species and only once for fish species during the species identification. PCR kits contain all the past decade. Indeed, Babola et al. (2004) were the first necessary reagents, controls and accessories for rapid to provide a microarray targeting 32 vertebrate testing (for more information see Asensio 2007; species, including 12 mammals, 5 birds and 15 fish. Rasmussen and Morrissey 2008). More recently, two new studies (Kochzius et al. 2008; Teletchea et al. 2008) further explored the potential of microarray for fish species identification. Teletchea Species and DNA markers targeted et al. (2008) developed a microarray-based method using cytochrome b-derived probes to identify 71 Species targeted commercial and/or endangered vertebrate species in both food and forensic samples (9 birds, 34 mammals, Overall, 478 species were targeted in the different 26 ray-finned fishes and 2 sharks). They demonstrated studies reviewed here. Yet, among these 478 species, that this microarray-based method was relatively fast, 291 were targeted only once and 141 between two completed within 2 days, easy to use, and that results and four times. On the opposite, 11 species were were straightforward (high ratio between signal/ targeted more than 10 times, among which the most background). It was also cost-effective, particularly used were Salmo salar (18 times), Oncorhynchus when compared with PCR-cloning and sequencing, mykiss (16), Thunnus albacares (16) and Gadus and provided more accurate results. This method was morhua (15). Hence, it emerged clearly that most successfully validated on both simple samples and studies have focused on few taxonomic groups, mixtures (containing up to five species) as well as on corresponding either to the most economically fresh and degraded substrates (hair, bone, blood, important (e.g., gadoids, scombroids, salmonids, muscle and foodstuffs). flatfishes), or among the most endangered ones (sturgeons; Table 3). Besides, much more studies Comparisons focused on marine rather than freshwater fishes. It is also obvious that the majority of studies targeted A variety a DNA based methods are potentially species inhabiting Europe and North America, while available for use in fish species identification. These only few focused on tropical species, such as red vary in their range of applications, complexity and snappers Lutjanus spp. (Zhang et al. 2004). The last costs (a comparison of the ten methods reviewed here category, called ‘‘miscellaneous’’, includes either are presented in Table 2). All these factors are liable freshwater fishes such as perch Perca spp. (Strange to influence the uptake of such tests by laboratory. and Stepien 2007) or marine fishes such as hairtail For instance, if one wants to routinely analyze fish Trichiurus spp. (Chakraborty et al. 2007). In numerous samples of the same four or five species, 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) 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 19:265–293 (2009) Fisheries Biol Fish Rev 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 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 characteristics that make its study easier and more that the number of species for which an identification straightforward. Lastly, mtDNA generally evolves method is developed will tremendously increase much faster than nuclear DNA and thus enables even in the coming years, particularly with the current closely related species to be differentiated and development of the DNA barcoding initiative (see identified. Besides, a suitable DNA marker for section ‘‘DNA barcoding’’). identification at the species level should be suffi- ciently variable between species (particularly the DNA markers used for species identification closest ones) and display either low or no-intra- specific variations across the geographic area. Ideally, Among the nine main DNA markers targeted, it this marker should be widely studied for a large appeared obvious that most studied have focused on number of species to enable comparison of the mitochondrial genes, and particularly on the cyto- nucleotide sequence from an unknown sample with chrome b gene (Table 4). Three main reasons explain reference sequences in a database. The gene encoding why most studies focused on mitochondrial DNA the cytochrome b satisfied most of these criteria and genome (mtDNA) rather than nuclear DNA. First, is by far the most studied gene for phylogeny. It was because there are several copies of mtDNA inside a certainly used in more than half of the phylogenetic cell (*1,000 9 the copies of nuclear DNA) it is studies published in the past decade (more than 140 more likely to amplify a fragment within this genome 000 sequences available in GenBank at the time of rather than within the nuclear genome (particularly this writing). In addition, this gene displayed both interesting when DNA is degraded, see section (a) conserved regions allowing the determination of ‘‘Study of degraded DNA’’). Besides, this small primers amplifying numerous species such as the circular genome (*16 kb in most vertebrate species) ‘‘universal’’ primers published by Kocher et al. displays maternal inheritance in most animal species, (1989), and regions with a high level of variability, is haploid, and does not undergo recombination— 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 Besides, most of these fish species are available the ‘‘miscellaneous’’ category, were NADH2 (Dalm- commercially after the removal of some external asso et al. 2006) and ND4 genes (McDowell and features as fresh (eviscerated, beheaded, skinned, Graves 2002) among others (see also Rasmussen and filleted, etc.) and processed products (marinated, Morrissey 2008). salted, smoked, caned, frozen, etc.), which makes the identification of species a difficult task. About 80% of fish and fishery products on the German Possible applications market are pretreated: smoked, canned, salted, or pickled fillets, or at least filleted (Horstkotte and It appeared obvious that most studies have focused on Rehbein 2003). Market globalization, large numbers three main fields of applications: forensic science, of both species exploited and processed fish products taxonomy and ecology (Table 5). Among these three, explained why the substitution of a less valuable forensic science and particularly food authentication species for a valuable one (representing a commercial is the most important. Others, such as the identifica- fraud), may be a common phenomenon difficult to tion of early life stages, are more recent and will detect. For instance, a study on food fish in the United certainly continue to develop further in the future. A States revealed that three-quarters of fish sold as the description and few examples of the different fields of threatened ‘‘red snapper’’ Lutjanus campechanus application are provided below. were mislabeled and belonged to other species (Marko et al. 2004). Likewise, Pepe et al. (2007) Forensic applications found that 84.2% (16/19) of surimi-based fish prod- ucts sold as Theragra chalcogramma were actually Food products prepared with species different from the one declared (more examples are provided in Rasmussen and The international fish market, estimate by the FAO at Morrissey 2008). The development of analytical around 60 billion/year, may imply more than 20,000 methods for fish species identification may help to species of fish (Rasmussen and Morrissey 2008). For detect and avoid wilful, as well as unintentional instance, about 420 species of fish are sold in substitution of different fish species and thus enforce the German market alone (Kochzius et al. 2008). 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- Identification of hazardous species in order to elim- cial circuit only if the commercial name, method of inate them from retail trade is one of the most production (farmed or wild) and capture area are important tasks of veterinary inspection of food clearly labeled (for more details see Asensio and products. Current laws ban the sale of both poisonous Montero 2008). Therefore, numerous methods have fish belonging to the families Tetraodontidae, Moli- been developed to authenticate various fish species in a dae, Diodontidae and Canthigasteridae and fishery wide range of food products, including soup and dried products containing biotoxins, such as ciguatoxins or fins (Hoelzel 2001), surimi (Weder et al. 2001; Pepe muscle paralyzing toxins (Civera 2003). Indeed, even et al. 2007), fish roe (Klossa-Kilia et al. 2002), spicy though these toxins are heat-labile, thus cooked food roe (Aranishi et al. 2005), fish tails (Sanjuan et al. should be safe, 100–200 people per year are seriously 2002), canned sardine and sardine-type products intoxicated, with a lethal outcome for half of them (Je´roˆme et al. 2003), and canned tuna (Rehbein et al. (Civera 2003). Hsieh and Hwang (2004) developed a 1999). The development of these molecular methods PCR-RFLP assay to identify 16 puffer fishes (Lago- helps not only to protect both consumers and producers cephalus spp., Takifugu spp.) from Taiwanese from frauds, but may also help to protect fish species seawaters. This new assay, complete within 24 h, from over-exploitation or illegal trafficking (Teletchea could be valuable to help wildlife officers to identify et al. 2005). Indeed, most exploited fish species these dangerous fish. Other species may also contain are currently endangered, among which sturgeons; some toxic substances, such as histamine in scom- because their eggs (caviar) are one of the most broid fishes. Tsai et al. (2007) reported two incidents exclusive and expensive fishery products (Wolf et al. of food-borne poisonings, causing illness in 59 and 43 1999). Based on survey of 95 lots of commercially victims due to the ingestion of billfish meats. available caviar in the New York City area, Birstein Analyses of histamine showed that the suspected et al. (1998) found that 23% of the designations made billfish samples contained more than 150 mg/100 g by caviar suppliers were mislabeled with respect to of histamine, which is higher than the hazard action species identification. Replacement of commercial level of 50 mg/100 g. Based on the results of a PCR- species with endangered and threatened species indi- RFLP of a fragment of the cytochrome b gene, they cates possible illegal harvest and poaching. were able to identify the two species implicated in

123 276 Rev Fish Biol Fisheries (2009) 19:265–293 the food poisonings, namely Makaira nigricans and holotype) and E. spinosus sexed turned out to be Xiphias gladius. males and females, respectively. The use of molec- ular genetic makers alongside traditional taxonomic methods can also help clarify relationships as they GMO detection ran reveal for example, the existence of cryptic species (taxa that cannot be distinguished morpho- Consumer concerns regarding genetically modified logically but are genetically distinct). While studying organisms GMOs have created a demand for labeling the genetic variability of Schindleria, believed to foods derived from transgenic products (Miraglia include one of the smallest and youngest reproducing et al. 2004). In anticipation of voluntary or mandatory vertebrates, Kon et al. (2007) found as many as 21 labeling, tests are being developed to identify GM genetically distinguishable species inhabiting the food. Current methodologies for the analysis of studied geographical areas. This discovery of many genetically modified organisms are focused on either cryptic species in Schindleria suggests that the use of one of two targets, the transgenic DNA inserted- or DNA sequences is necessary for species identification the novel protein(s) expressed- in a genetically of such morphologically conserved taxa. Conse- modified product. Masri et al. (2002) developed a quently they proposed that DNA-based designation PCR-specific primers protocol for the identification is necessary for such taxa in order to compile the full of genetically modified coho salmon (Oncorhynchus ‘‘species lists’’; yet there is presently no consensus for kisutch) carrying a growth hormone transgene. The the inclusion of DNA sequencing data in the formal primers were specific for the amplification of a gene descriptions of new species. construct present in transgenic salmon; no amplifica- Fish species identification is also traditionally tion from the wild-type fish. The optimized PCR based on morphology, using keys. Yet, in some method identified all samples tested (61 samples and cases conventional keys could be misleading or 17 controls) with 100% accuracy. Besides, the assay provide ambiguous diagnoses especially for individ- was sensitive enough to detect the transgenic in as uals with intermediate values in traits that are little as 1 ng of total DNA. The PCR test could be supposed to be discriminative. In certain taxa, it is performed in a few hours, using tissues from all parts indeed not unusual to encounter situations in which of the fish including blood, bones, scales, slime and individuals possess combination of character states other internal organs; thus it was possible to perform thought to be typical of different species, such as in the detection test without damaging or reducing the the palearctic coregonids (Politov et al. 2000). market value of the target fish. Besides, morphological characters are sometimes of limited value for identification and differentiation Taxonomic applications purposes because they show a considerable intraspe- cific variations and differences among species are Species discrimination and identification small. All these difficulties could result in misi- dentification. For example, adult rockfishes Sebastes Fish species taxonomy is traditionally based on mystinus, S. melanops, and S. ciliatus are so similar morphological and anatomical traits. Yet reliance that they are often misidentified on the field on morphology can sometimes present difficulties in (Gharrett et al. 2001). Reid and Wilson (2006) cases where species may be very similar morpholog- found a disagreement between 20% of samples ically or apparent differences are misleading. provided by various government agencies and spe- Byrkjedal et al. (2007) demonstrated that Eumicro- cies-specific digest patterns, indicating a need for tremus spinosus and E. eggvinii, currently considered greater care during field identifications of Moxos- as two valid species, are clearly separated by a toma species. In addition, the difficulty to identify considerable number of morphological characters; yet species can led to incomplete survey of the true they in fact constitute a single, sexually dimorphic fauna present in a given area (Tinti et al. 2003; species. Indeed, identical DNA sequences were found Schander and Willassen 2005) or to misunderstand in all E. eggvinii and E. spinosus samples studied. ecological interactions, such as marine bioinvasions Besides, all specimens of E. eggvinii (including the (Bucciarelli et al. 2002).

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In conclusion, application of molecular tools can simple, generalized forms of most larvae provide provide valuable information for species identifica- relatively few consistent taxonomically important tion and complement the traditional taxonomic data. characters with which to characterize and describe morphological relationships among species. There Identification of body parts are two basic approaches to the study of fish eggs and larvae. The most common relies on field collections Many commercially important shark species are and works back from known adults, using diagnostics difficult to identify whole, and this task is more characters common to smaller individuals of a size daunting if individuals are processed (head, entrails, series. The other relies on eggs and larvae reared and fins removed); unfortunately at-sea processing is from known parents and works forward (Methven widespread in the industry (Greig et al. 2005). and McGowan 1998). Whenever fishes can be Although current US legislation prohibits the practice hatched and reared from eggs, the second method is of ‘‘finning’’ (where fins are retained and discarded at preferred because the specimens being described are sea), the landings of fins is allowed where carcasses known with certainty. and fins are off-loaded at the same time in no more Despite considerable effort, larval identification than 1:20 (fin-to-carcass) weight ratio. However, remains one of the major factors limiting the many serious problems can arise in matching off-loaded important questions that can be addressed by field- fins to processed carcasses, e.g., it might be tempting based ichthyoplankton studies, including the identi- to increase the fin-to-carcass ratio with spoiled meat fication of spawning locations and seasons of or ‘‘finning’’ target species out of season (and migrating species, the quantification of population subsequently attributing the fins to fish that are levels or biomass of fished species. For instance, only allowed to be caught during the season). Within this 15 species, among the 65 rockfish species Sebastes context, Greig et al. (2005) developed a PCR-FINS found along the California coast, can be separated at method to identify 35 shark species (Carcharhinus the larval stage by physical characters such as body spp., Sphyrna spp., Mustelus spp. among others) from shape, pigmentation patterns, and head spine devel- the Atlantic fishery. They found that the sequence of opment (Li et al. 2006). While increased effort in the 12S–16S region of the mtDNA contained ample developing morphological identification systems will information for discriminating between the shark likely be successful for many taxonomic groups of species studied, and thus planned to increase their fishes, it appears that the underlying limits have been database to the seventy-three species inhabiting the reached for many others (Richardson et al. 2007). United States territorial waters of the Atlantic Ocean, Therefore, the application of molecular-based iden- Gulf of Mexico and Caribbean Sea. Such accurate tification methods in ichthyoplankton surveys could and reliable species identification methods are para- significantly increase their accuracy, particularly for mount for law enforcement and sound shark species species producing eggs and larvae that cannot be management (most endangered). separated using classical methods. Based on genetic identification, Fox et al. (2005) found that the Ecological applications majority of eggs in the Irish Sea, wrongly believed to be from cod, were actually from whiting Merlan- Early life history stages determination gius merlangus, leading to an overestimation of cod stocks. The study of morphological variation among fish species has conventionally been based on compari- Trophic relationships sons of adults. This is true in part because young stages are usually difficult to collect and identify. Determining trophic relationships within an ecosys- However, another important reason for emphasizing tem is a key part of many ecological studies; adults is that many fishes have distinctive larvae that however, obtaining reliable data on diet composition are transitory forms, possessing very different body for most species is fraught with difficulties. On one shapes and numerous, sometimes bizarre, temporary hand, it is not always possible to track trophic organs (Strauss and Bond 1990). In addition, the very interactions between predators and prey by direct

123 278 Rev Fish Biol Fisheries (2009) 19:265–293 observation, particularly when observing small or salmonids are not common prey for seals in these elusive animals with cryptic food-wed ecology. On Scottish estuaries. the other hand, gut and/or fecal analysis can some- times allow prey remains to be identified visually but Other applications is only possible when a component of the diet is resistant to digestion, such as otoliths or skeletal DNA barcoding parts, which can be identified to genus and species level by experienced researchers (Pierce and Boyle Hebert et al. (2003) proposed standardizing the 1991; Smith et al. 2005; Casper et al. 2007). Besides, various approaches used in species identification in some cases there are no solid remains, and when through the establishment of a DNA barcoding there are it can lead to bias in interpretation of prey system, similar in practice to a supermarket barcode, choice. Consequently, numerous invasive and non- for all living organisms, based on a single sequence: a invasive methods have been developed to character- 648-bp portion of the mitochondrial gene cytochrome ize predator-prey interactions, among which c oxidase I (COI). Initial reactions to this DNA molecular methods (reviewed in Sheppard and Har- barcoding proposition have ranged from enthusiasm, wood 2005; King et al. 2008). Smith et al. (2005) especially from ecologists (Janzen 2004), to criti- developed a DNA sequencing method to identify cisms, chiefly concerning the identification of closely partially digested prey items taken from the gut related species using a single gene (Lipscomb et al. content of seven species of large pelagic fishes, such 2003; Mallet and Willmott 2003; Moritz and Cicero as marlins Makaira spp. or tunas Thunnus spp. All are 2004). In the meantime, Tautz et al. (2003) proposed large opportunistic predators feeding on pelagic to give DNA a central and mandatory role in fishes, squids and crustaceans. They found that the taxonomy for both identifying and defining species prey items taken from the 14 samples analyzed were (the DNA taxonomy concept), which is aimed at derived from six pelagic fish species from two replacing the traditional, time-consuming, chiefly families (Scombridae and Carangidae). Deagle et al. morphology-based system. This second proposition (2005) conducted a captive feeding trial to test has led to condemnation, particularly concerning the whether prey DNA could be reliably detected in scat concept of species (Mallet and Willmott 2003; samples from Steller sea lions (Eumetopias jubatus). Seberg et al. 2003; Will and Rubinoff 2004) and Two sea lions were fed a diet of fish (five species) has revived the old controversy in systematics about and squid (one species), and DNA was extracted from the relative importance of DNA technology versus the soft component of collected scats. Most of the morphological traits (Blaxter 2003, 2004; Wheeler DNA came from the predator, but prey DNA could be et al. 2004; DeSalle et al. 2005; Will et al. 2005). amplified using prey-specific primers, with very good Despite this initial lively debate, still ongoing today results. Thus, they concluded that even though (e.g., Paterlini 2007; Schlick-Steiner et al. 2007), a several aspects of the methodology need further new international program ‘‘Barcode of Life Initia- development before a complete picture of diet can be tive’’ has eventually been created for studies and constructed, molecular scatology to study diet has the molecular cataloging of species diversity of all potential to provide new insight into the diet of animals and plants of the Earth (http://www. vertebrate species. Similarly, Matejusova´ et al. barcoding.si.edu/). The major goals of this program (2008) applied quantitative PCR to examine the are to provide molecular identification of organisms presence of salmonids, Atlantic salmon (Salmo salar) using standardized DNA region (DNA barcode) and and sea trout (Salmo trutta), in the diet of grey seals to create a special database that would be more (Halichoerus grypus) and harbor seals (Phoca vitu- taxonomically accurate and would have more rigor- lina) in the Moray Firth, UK, during the summer of ous rules for entry of the data compared with the 2003 and 2005. The qPCR assay approach was shown existing databases, such as GenBank (for more details to be highly efficient and consistent in detection of see Hanner and Gregory 2007). In the past 5 years, salmonids from seal scats, and to be more sensitive several studies have evaluated the potential of DNA than conventional hard-parts analysis. Nevertheless, barcoding for identifying fish species, among which their results confirmed previous indicating that Australia’fishes (Ward et al. 2005; Pegg et al. 2006)

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(for a more complete review about DNA barcoding than 300 bp), they studied more than 20 salmon bone progresses see Waugh 2007). At the time of this samples (dated 7,000–2,000 BP) from the site of writing, more than 36000 COI barcode records are Namu of the central coast of British Columbia. Four deposited in the Barcode of Life Database (Ratnas- species, coho (Oncorhynchus kisutch), sockeye ingham and Hebert 2007) with representation from (O. nerka), pink (O. gorbuscha) and chum (O. keta) more than 6,500 species of actinopterygians. salmon, were identified from the remains, which was Another project that has been focused on sequence consistent with other lines of evidence. These results information for specific genes is the FishTrace demonstrated that the DNA can be well preserved in Consortium (http://www.fishtrace.org), which com- remains from the Pacific Northwest Coast of North prises 53 members from several European institutions America, and thus provide new data to interpret (Sevilla et al. 2007). The FishTrace database provides faunal remains at Namu. They concluded that these detailed information on a number of fish species workable protocols could be used to identify more common to Europe, along with barcoding data for the vertebra samples from more sites to study fishery gene mitochondrial cytochrome b and nuclear rho- activity in the region. 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 Key problems DNA barcodes longer in length will allow for increased efficiency of identification labels (Sevilla Study of degraded DNA et al. 2007). Besides, the combination of two genes that exhibit different genomic positions and rates of Depending on the kind of samples analyzed, the evolution was reported to be valuable for the effi- quantity and quality of DNA vary greatly. In ciency of DNA barcoding. Other DNA databases, unprocessed food products (such as fillets) or fresh among which the largest is Genbank (http:// samples (egg, larvae, body parts), DNA is generally www.ncbi.nlm.nih.gov) can also be accessed online unaltered and present in high quantity, thus large (reviewed in Rasmussen and Morrissey 2008). fragments of DNA (*1–2 kb) can potentially be amplified (in some cases the variability of the entire Ancient DNA analyses genome can be studied using PCR-RAPD, PCR- AFLP methods). In the opposite, in most samples Ancient DNA research, defined broadly as the encountered in both fishery market (canned products, retrieval of DNA sequences from museum specimens, surimi) and ecology (scats, gut contents), DNA is archaeological finds, fossil remains, and other degraded and present in small quantities that consid- unusual sources of DNA, was born about 20 years, erably reduce the number of DNA fragments with when DNA sequences were separately described suitable size for molecular analysis (Lockley and from the quagga (a type of zebra) and an ancient Bardsley 2000; Teletchea et al. 2005; King et al. Egyptian individual (Pa¨a¨bo et al. 2004). The retrieval 2008). Such damages to DNA are due to heat and analysis of such degraded DNA should apply key exposure, high pressure, low pH, irradiation, drying, criteria (appropriate laboratory facilities and controls, nucleases that cause enzymatic degradation, depuri- independent replication and cloning of amplification nation and hydrolysis, among others. Consequently, products) to ascertain to the greatest extent possible identification methods from degraded substrates that results represent authentic ancient DNA should be based on the analysis of very short sequences (Gilbert et al. 2005; Willerslev and Cooper mitochondrial fragments, preferably between 100 2005). Despite these significant constraints, numer- and 200 bp (explanations are given in section ous studies have been published in the past decade, ‘‘DNA markers used for species identification’’). particularly concerning mammals (Pa¨a¨bo et al. 2004; For instance, Quinteiro et al. (1998) found that DNA Millar et al. 2008). Yang et al. (2004) provided a rare was degraded during the canning process of fish example of applying ancient DNA analysis for muscle: DNA fragment sizes that ranged from \100 species identification of archaeological fish bone. up to 200 bp were obtained from canned tuna muscle, Targeting short mitochondrial DNA fragments (less whereas DNA sizes for frozen tuna muscle ranged

123 280 Rev Fish Biol Fisheries (2009) 19:265–293 from \100 bp up to 20,000 bp. Carrera et al. (2000) DNA could induce artifactual results, e.g., chimeric found that DNA degradation produced by the cold molecules produced by jumping PCR (Pa¨a¨bo et al. smoking process did not prevent amplification of 1990). In conclusion, the retrieval and analysis of DNA fragments close to 1 kb in size. On the contrary, DNA in highly degraded samples require to apply nucleic acids were heavily degraded during the several criteria to ensure as much as possible the sterilization process. Yet, the precise effects of reliability of the results. various processing practices on the integrity of DNA in seafood are still largely unknown (Bossier Reliability of sequences in databases 1999). Because DNA is generally altered and present in low quantity in degraded samples and PCR such a Among the ten PCR methods reviewed here sensitive method, sometimes a single exogenous (Table 1), eight are developed from sequences, DNA molecule could be preferentially amplified usually taken from international database, such as instead of the degraded one (reviewed in Cooper Genbank. Yet, sequences deposited in these databases and Wayne 1998). Thus, degraded samples should be could sometimes be false (Forster 2003; Nilsson et al. manipulated (before PCR) in a dedicated laboratory 2006). These false sequences might be due to to avoid as much as possible contaminations and sequencing errors, misidentification of the target appropriate negative controls (extraction and PCR species or other laboratory problems. Indeed, PCR blanks) should be processed on every run to guaran- reactions are easily contaminated by carryover DNA tee the reliability of the results (Teletchea et al. from other organisms of by other DNAs in laboratory. 2005). Furthermore, the lack of amplification in Mislabeling of tubes can also lead to incorrect degraded samples (i.e., a negative readout) could be assignment of sequence data to species. While explained by the inhibition of the PCR amplification developing a PCR-RFLP method for the authentica- rather than by insufficient amount or absence of the tion of 11 carcharhiniform sharks, Heist and Gold target DNA. Such inhibition is due to the presence of (1999) detected numerous differences between the various products that are co-purified with the target restriction patterns predicted from the sequence of DNA (reviewed in Wilson 1997). Therefore extrac- Martin and Palumbi (1993) for tiger shark (Gale- tion methods should allow removals of inhibitors; yet ocerdo cuvier) collected near Hawaii and their tiger no general extraction method has proved useful with shark collected from the Gulf of Maxico (Atlantic) all the different matrices encountered. Therefore, and Hawaii and Australia (Pacific). They sequenced each new substrate requires the development of new the entire cytochrome b gene in tiger shark from extraction and amplification methods or the adapta- Florida and, in comparison with the ‘‘tiger shark’’ tion of existing ones (Woolfe and Primrose 2004; sequence reported by Martin and Palumbi (1993), Teletchea et al. 2005). Additional work should now found one nucleotide difference in the first (50-most) focus on extraction yield to improve the quality and 540 bp of the gene and 64 nucleotide differences in quantity of DNA retrieved, particularly from highly the remaining 606 bp of the gene. They hypothesized degraded samples (e.g., Chapela et al. 2007). At last, that the ‘‘tiger shark’’ sequence of Martin and DNA retrived in highly degraded samples are also Palumbi (1993) was a mixture that included sequence known to display in some cases chemical modifica- data from another species. Another source of false tions: modified DNA molecules might display breaks sequence when targeting mitochondrial genome to or artifactual mutations (reviewed in Poinar 2002; develop identification methods are the nuclear mito- Bower et al. 2005). Consequently, absence of DNA chondrial pseudogenes (generally referred to as degradation from highly degraded samples should be numts). Numts are nuclear inserted copies of mito- checked before using methods based on few variable chondrial origin exhibiting a high degree of similarity sites. These DNA modifications can indeed produce with mtDNA sequences, which might be amplified misidentification of species because the DNA inadvertently by the PCR in addition to or even sequence obtained is slightly different from the instead of the authentic target mtDNA, thus leading reference one (Teletchea et al. 2005). Similarly, to erroneous results. Within vertebrates, the majority non-invase studies have also shown that PCR of numts have been reported in mammals and birds, amplifcation starting from tiny amounts of altered but it is likely a consequence of the disproportionate

123 Rev Fish Biol Fisheries (2009) 19:265–293 281 attention these taxa receive relative to other groups identification. Similarly, while developing a micro- (Benesh et al. 2006). In fish species, numts have been array-based method for identifying various found in Fugu rubripes, Tetraodon nigroviridis, and vertebrates, Teletchea et al. (2008) found two diver- Danio rerio (Antunes and Ramos 2005), or in gent groups (diverging by 8.93%) within the john Gadiculus argenteus and Melanogrammus aeglefinus dory (Zeus faber), one cluster of two individuals from (Teletchea et al. 2006) among others. Bensasson et al. the Atlantic Ocean and the Mediterranean Sea, and (2001) described methods for detecting them (e.g. one cluster of three individuals from Japan, which checking for unique changes and odd substitution might correspond to two different and valid species. patterns, such as indels and/or stop codons). How- On the opposite, two valid species based on morpho- ever, current practices do not preclude inadvertent logical characters could be genetically similar. While analysis of numts and much caution should be studying the partial cytochrome b (401 bp) and exercised when using mitochondrial sequences for cytochrome oxidase I (495 bp) genes variability identification purposes. within the cod family, Carr et al. (1999) obtained In conclusion, Harris (2003) proposed several identical sequences between the Greenland cod solutions to check the quality of the published Gadus ogac and the Pacific cod Gadus macroceph- sequences and the ‘‘simplest’’ is to re-sequence them. alus. The authors concluded that these two species Another solution would be to use as many reference are in fact the same and should be synonymized. sequences as possible resulting from different studies Furthermore, hybridization between closely related (when available) and never base a method on only species (introgression) can occur if these species one reference sequence (Teletchea et al. 2005). The share overlapping habitats or sometimes through problem of false sequences will certainly be reduced human intervention (e.g. during captive breeding). with the current development of specific projects Hence, it has been found that several fish species dedicated to the identification of species, such as the show interspecific mitochondrial introgression: charr Consortium for the barcode of life, as it requires new (Doiron et al. 2002; Redenbach and Taylor 2003) or standards for the publication of sequences (Hanner sturgeon (Ludwig et al. 2003). These introgressions and Gregory 2007). could sometimes disturb molecular identification of closely related species (Moritz and Cicero 2004). The species problem For all these reasons, caution should be taken with the use of scientific names. One should indicate The literature about species concepts might be more clearly the species he studies, for example give the extensive than that about any other subject in name of the author of authority who identified the evolutionary biology and therefore it is obviously species (e.g., Gadus morhua Linnaeus, 1758), and out of the scope of the present review to discuss the also should provide geographical distribution and key validity of all these concepts (see e.g., Sites and biological information. Furthermore, researchers Marshall 2003). Rather, I illustrate here some prob- should be reminded that scientific names could refer lems usually encountered in fish taxonomy that could to different contents according to the progress of result in erroneous molecular identification methods science (Seberg et al. 2003) and that discrepancies if not take into account. The same scientific names could exit between taxonomical conclusions obtained could refer to highly divergent molecular groups. from morphological and molecular characters. This While studying the mitochondrial sequence variation implies that when new molecular-based clusters are (927 bp fragment of the ATPase and COX3 genes) found, only expert taxonomists can satisfactorily within and between tuna species (Thunnus spp.), resolve the relationship between them and species Takeyama et al. (2001) found two divergent groups (Schindel and Miller 2005). In conclusion, whatever (diverging by *4.5%) within the northern bluefin the species, a thorough analysis should be made each tuna, Thunnus thynnus (i.e. T. t. thunnus, living in time a new group is to be studied (ideally with the Atlantic ocean and T. t. orientalis, in the taxonomists of each group) and several samples from Pacific ocean). Thus, they showed that a re-evalua- the full distribution range should be taken into tion of previous RFLP methods was required to consideration to validate the method (Ruedas et al. avoid inconsistent profiles and erroneous tuna 2000; Comesana et al. 2003). Voucher specimens

123 282 Rev Fish Biol Fisheries (2009) 19:265–293 should ideally be preserved, and submitters of affinis (De Haan) using mitochondrial DNA sequences should conform to the ICZN (International analysis. J Exp Mar Biol Ecol 217:153–163. Commission on Zoological Nomenclature; http:// doi:10.1016/S0022-0981(97)00039-7. www.iczn.org/) rules of nomenclature (Ruedas et al. 10. Asensio L, Gonzalez I, Fernandez A, et al. 2000). (2001) PCR-SSCP: A simple method for the authentification of grouper (Epinephelus gu- Acknowledgements I thank two anonymous reviewers that aza), werck fish (Polyprion americanus), and helped to improve the manuscript. nile perch (Lates niloticus) fillets. J Agric Food Chem 49:1720–1723. doi:10.1021/jf001185w. 11. Asensio L, Gonza´lez I, Rodrı´guez M et al. (2004) PCR-ELISA for the semiquantitative Appendix: List of the studies reviewed detection of Nile perch (Lates niloticus) in sterilized fish muscle mixtures. J Agric Food 1. Akasaki T, Yanagimoto T, Yamakami K, et al. Chem 52:4419–4422. doi:10.1021/jf0350010. (2006) Species identification and PCR-RFLP 12. Babola O, Desvarenne S, Lacroix B, et al. analysis of cytochrome b gene in cod fish (2004) L’identification des espe`ces animales (Order Gadiformes) products. J Food Sci dans l’alimentation humaine et animale: un 71:190–195. exemple d’application de la technologie des 2. Aoyama J, Ishikawa S, Otake T, et al. (2001) puces a` ADN. Bull Soc Fr Micr 19:30–36. Molecular approach to species identification of 13. Balitzki-Korte B, Anslinger K, Bartsch C, et al. eggs with respect to determination of the (2005) Species identification by means of spawning site of the Japanese eel Anguilla pyrosequencing the mitochondrial 12S rRNA japonica. Fish Sci 67:761–763. doi:10.1046/ gene. Int J Legal Med 119:291–294. doi:10.1007/ j.1444-2906.2001.00288.x. s00414-005-0537-9. 3. Aranishi F (2005) PCR-RFLP analysis of 14. Birstein VJ, Doukakis P, Sorkin B, et al. (1998) nuclear nontranscribed spacer for mackerel Population aggregation analysis of three caviar- species identification. J Agric Food Chem 53: producing species of sturgeons and implications 508–511. doi:10.1021/jf0484881. for the species identification of black caviar. 4. Aranishi F (2005) Rapid PCR-RFLP method for Conserv Biol 12:766–775. doi:10.1046/j.1523- discrimination of imported and domestic mack- 1739.1998.97081.x. erel. Mar Biotechnol 7:571–575. doi:10.1007/ 15. Bottero MT, Civera T, Nucera D, et al. (2003) s10126-004-4102-1. Design of universal primers for the detection of 5. Aranishi F (2006) Single fish egg DNA extrac- animal tissues in feedstuff. Vet Res Commun tion for PCR amplification. Cons Gen 7:153– 27:667–669. doi:10.1023/B:VERC.0000014243. 156. doi:10.1007/s10592-005-5387-y. 75976.cd. 6. Aranishi F, Okimoto T, Izumi S (2005) Iden- 16. Bottero MT, Dalmasso A, Cappelletti M, et al. tification of gadoid fishes (Pisces, Gadidae) by (2007) Differentiation of five tuna species PCR-RFLP analysis. J Appl Genet 46:69–73. by a multiplex primer-extension assay. J Bio- 7. Aranishi F, Okimoto T, Ohkubo M (2006) A technol 129:575–580. doi:10.1016/j.jbiotec. short-cut DNA extraction from cod caviar. J Sci 2007.01.032. Food Agric 86:425–428. doi:10.1002/jsfa.2370. 17. Bucciarelli G, Golani D, Bernardi G (2002) 8. Aranishi F, Okimoto T, Ohkubo M, et al. Genetic cryptic species as biological invaders: (2005) Molecular identification of commercial the case of a Lessepsian fish migrant, the spicy pollack roe products by PCR-RFLP hardyhead silverside Atherinomorus lacunosus. analysis. J Food Sci 70:235–238. J Exp Mar Biol Ecol 273:143–149. doi:10.1016/ 9. Asahida T, Yamashita Y, Kobayashi T (1997) S0022-0981(02)00138-7. Identification of consumed stone flounder, 18. Byrkjedal I, Rees DJ, Willassen E (2007) Kareius bicoloratus (Basilewsky), from the Lumping lumpsuckers: molecular and morpho- stomach contents of sand shrimp, Crangon logical insights into the taxonomic status of

123 Rev Fish Biol Fisheries (2009) 19:265–293 283

Eumicrotremus spinosus (Fabricius, 1776) and 27. Ce´spedes A, Garcı´a T, Carrera E, et al. (1999) Eumicrotremus eggvinii Koefoed, 1956 (Teleo- Identification of Sole (Solea solea) and Green- stei: Cyclopteridae). J Fish Biol 71:111–131. land Halibut (Reinhardtius hippoglossoides) doi:10.1111/j.1095-8649.2007.01550.x. by PCR Amplification of the 5S rDNA Gene. 19. Callejas C, Ochando MD (1998) Identification J Agric Food Chem 49:1046–1050. doi:10.1021/ of Spanish barbel species using the RAPD jf9810970. technique. J Fish Biol 53:208–215. doi:10.1111/ 28. Ce´spedes A, Garcı´a T, Carrera E, et al. (1998) j.1095-8649.1998.tb00121.x. Identification of flatfish species using polymer- 20. Callejas C, Ochando MD (2001) Molecular ase chain reaction (PCR) amplification and identification (RAPD) of the eight species of the restriction analysis of the cytochrome b gene. genus Barbus (Cyprinidae) in the Iberian Pen- J Food Sci 63:206–209. insula. J Fish Biol 59:1589–1599. doi:10.1111/ 29. Chakraborty A, Aranishi F, Iwatsuki Y (2005) j.1095-8649.2001.tb00223.x. Molecular identification of hairtail species 21. Calo-Mata P, Sotelo CG, Pe´rez-Martı´n RI, et al. (Pisces: Trichiuridae) based on PCR-RFLP (2003) Identification of gadoid fish species analysis of the mitochondrial 16S rRNA gene. using DNA-based techniques. Eur Food Res J Appl Genet 46:381–385. Technol 217:259–264. doi:10.1007/s00217-003- 30. Chakraborty A, Aranishi F, Iwatsuki Y (2007) 0735-y. Polymerase chain reaction-restriction fragment 22. Carrera E, Garcı´a T, Ce´spedes A et al. (2000) length polymorphism analysis for species Differentiation of smoked Salmo salar, On- identification of hairtail fish fillets from super- corhynchus mykiss and Brama raii using the markets in Japan. Fish Sci 73:197–201. nuclear marker 5S rDNA. Int J Food Sci doi:10.1111/j.1444-2906.2007.01319.x. Technol 35:401–406. doi:10.1046/j.1365-2621. 31. Chan RWK, Dixon PI, Pepperell JG, et al. 2000.00404.x. (2003) Application of DNA-based techniques 23. Carrera E, Garcı´a T, Ce´spedes A et al. (1999) for the identification of whaler sharks (Carcha- PCR-RFLP of the mitochondrial cytochrome rhinus spp.) caught in protective beach meshing oxidase gene: a simple method for the discrim- and by recreational fisheries off the coast of ination between Atlantic salmon (Salmo salar) New South Wales. Fish Bull (Wash D C) and rainbow trout (Oncorhynchus mykiss). J Sci 101:910–914. Food Agric 79:1654–1658. doi:10.1002/(SICI) 32. ChapelaMJ,SanchezA,SuarezMI,etal.(2007)A 1097-0010(199909)79:12\1654::AID-JSFA414[ rapid methodology for screening hake species 3.0.CO;2-S. (Merlucciusspp.)bysingle-strandedconformation 24. Carrera E, Garcı´a T, Ce´spedes A et al. (1999) polymorphism analysis. J Agric Food Chem Salmon and trout analysis by PCR-RFLP for 55:6903–6909.doi:10.1021/jf070132c. identity authentication. J Food Sci 64:410–413. 33. Chapela MJ, Sotelo CG, Pe´rez-Martı´n RI, et al. doi:10.1111/j.1365-2621.1999.tb15053.x. (2007) Comparison of DNA extraction methods 25. Casper RM, Jarman SN, Deagle BE, et al. from muscle of canned tuna for species identifi- (2007) Detecting prey from DNA in predator cation. Food Contr 18:1211–1215. doi:10.1016/ scats: a comparison with morphological analy- j.foodcont.2006.07.016. sis, using Arctocephalus seals fed a known diet. 34. Chapman DD, Abercrombie DL, Douady CJ, J Exp Mar Biol Ecol 347:144–154. doi:10.1016/ et al. (2003) A streamlined, bi-organelle, multi- j.jembe.2007.04.002. plex PCR approach to species identification: 26. Ce´spedes A, Garcı´a T, Carrera E, et al. (2000) Application to global conservation and trade Genetic differentiation between sole (Solea so- monitoring of the great white shark, Carcharodon lea) and Greenland halibut (Reinhardtius carcharias. Cons Gen 4:415–425. doi:10.1023/ hippoglossoides) by PCR-RFLP analysis of a A:1024771215616. 12S rRNA gene fragment. J Sci Food Agric 35. Cheng C-A, Hsieh Y-W, Noguchi T, et al. 80:29–32. doi:10.1002/(SICI)1097-0010(20000101) (2001) Effect of processing on sequence of 80:1\29::AID-JSFA470[3.0.CO;2-4. cytochrome b gene and its restriction site in the

123 284 Rev Fish Biol Fisheries (2009) 19:265–293

meat of puffer Takifugu rubripes. J Food Drug 45. Deagle B, Tollit D, Jarman S, et al. (2005) Anal 9:232–237. Molecular scatology as a tool to study diet: 36. Cocolin I, D’Agaro E, Manzano M, et al. (2000) Analysis of prey DNA in scats from captive Rapid PCR-RFLP method for the identification Steller sea lions. Mol Ecol 14:1831–1842. of marine fish fillets (Seabass, Seabream, doi:10.1111/j.1365-294X.2005.02531.x. Umbrine, and Dentex). J Food Sci 65:1315– 46. Di Finizio A, Guerriero G, Russo GL, et al. 1317. doi:10.1111/j.1365-2621.2000.tb10604.x. (2007) Identification of gadoid species (Pisces, 37. Colombo F, Mangiagalli G, Renon P (2005) Gadidae) by sequencing and PCR-RFLP anal- Identification of tuna species by computer- ysis of mitochondrial 12S and 16S rRNA gene assisted and cluster analysis of PCR-SSCP fragments. Eur Food Res Technol 225:337–344. electrophoretic patterns. Food Contr 16:51–53. doi:10.1007/s00217-006-0420-z. doi:10.1016/j.foodcont.2003.11.006. 47. Dooley J, Sage H, Brown H, et al. (2005) 38. Comesana AS, Abella P, Sanjuan A (2003) Improved fish species identification by use of Molecular identification of five commercial lab-on-a-chip technology. Food Contr 16:601– flatfish species by PCR-RFLP analysis of a 12 607. doi:10.1016/j.foodcont.2004.06.022. rRNA gene fragment. J Sci Food Agric 83:752– 48. Dooley J, Sage H, Clarke M-A, et al. (2005) 759. doi:10.1002/jsfa.1368. Fish species identification using PCR-RFLP 39. Comi G, Iacumin L, Rantsiou K, et al. (2005) analysis and lab-on-a-chip capillary electropho- Molecular methods for the differentiation of resis: application to detect white fish species in species used in the production of cod-fish can food products and an interlaboratory study. J detect commercial frauds. Food Contr 16:37– Agric Food Chem 53:3348–3357. doi:10.1021/ 42. doi:10.1016/j.foodcont.2003.11.003. jf047917s. 40. Dalmasso A, Civera T, Bottero MT (2006) 49. Fox CJ, Taylor MI, Pereyra R, et al. (2005) Biomolecular approaches for the identification TaqMan DNA technology confirms likely over- of tuna species. Vet Res Commun 30:179–181. estimation of cod (Gadus morhua L.) egg doi:10.1007/s11259-006-0035-7. abundance in the Irish Sea: implications for 41. Dalmasso A, Fontanella E, Piatti P, et al. (2007) the assessment of the cod stock and mapping of Identification of four tuna species by means of spawning areas using egg-based methods. Mol real-time PCR and melting curve analysis. Vet Ecol 14:879–884. doi:10.1111/j.1365-294X.2005. Res Commun 31:355–357. doi:10.1007/s11259- 02439.x. 007-0036-1. 50. Gagnaire PA, Tsukamoto K, Aoyama J, et al. 42. Das P, Prasad H, Meher PK, et al. (2005) (2007) RFLP and semi-multiplex PCR-based Evaluation of genetic relationship among six identification of four eel species from the south- Labeo species using randomly amplified poly- western Indian Ocean region. J Fish Biol 71:279– morphic DNA (RAPD). Aquacult Res 36:564– 287. doi:10.1111/j.1095-8649.2007.01605.x. 569. doi:10.1111/j.1365-2109.2005.01254.x. 51. Garcia-Vazquez E, Izquierdo JI, Perez J, et al. 43. Dawnay N, Ogden R, McEwing R, et al. (2007) (2006) Genetic variation at ribosomal genes Validation of the barcoding gene COI for use in supports the existence of two different Euro- forensic genetic species identification. Forensic pean subspecies in the megrim Lepidorhombus Sci Int 173:1–6. doi:10.1016/j.forsciint.2006. whiffiagonis. J Sea Res 56:59–64. doi:10.1016/ 09.013. j.seares.2006.04.001. 44. de los Angeles Barriga-Sosa I, Pe´rez-Ramı´rez M, 52. Gharrett AJ, Gray AK, Heifetz J (2001) Iden- Soto-Aguirre F, et al. (2005) Inter-specific vari- tification of rockfish (Sebastes spp.) by ation of the mitochondrial r16S gene among restriction site analysis of the mitochondrial silversides, ‘‘Peces Blancos’’ (Atherinopsidae: ND-3/ND-4 and 12S/16S rRNA gene regions. Menidiinae) and its utilization for species identi- Fish Bull (Wash D C) 99:49–62. fication. Aquaculture 250:637–651. doi:10.1016/ 53. Greig TW, Moore MK, Woodley CM (2005) j.aquaculture.2005.05.003. Mitochondrial gene sequences useful for

123 Rev Fish Biol Fisheries (2009) 19:265–293 285

species identification of western North Atlantic an adulterated dried mullet roe implicated Ocean sharks. Fish Bull (Wash D C) 103: in food poisoning. J Food Sci 68:142–146. 516–523. doi:10.1111/j.1365-2621.2003.tb14130.x. 54. Heist EJ, Gold JR (1999) Genetic identification 65. Hsieh YW, Shiu YC, Cheng CA, et al. (2002) of sharks in the U.S. Atlantic large coastal shark Identification of toxin and fish species in fishery. Fish Bull (Wash D C) 97:53–61. cooked fish liver implicated in food poisoning. 55. Hird HJ, Hold GL, Chisholm J, et al. (2005) J Food Sci 67:948–952. doi:10.1111/j.1365- Development of a method for the quantification 2621.2002.tb09433.x. of haddock (Melanogrammus aeglefinus) in 66. Hwang D-F, Jen H–C, Hsieh Y-W, et al. (2004) commercial products using real-time PCR. Eur Applying DNA techniques to the identification Food Res Technol 220:663–637. of the species of dressed toasted eel products. J 56. Hoelzel AR (2001) Shark fishing in fin soup. Cons Agric Food Chem 52:5972–5977. doi:10.1021/ Gen 2:69–72. doi:10.1023/A:1011590517389. jf034525x. 57. Hold GL, Russell VJ, Pryde SE, et al. (2001) 67. Imsiridou A, Minos G, Katsares V, et al. (2007) Validation of a PCR-RFLP based method for Genetic identification and phylogenetic infer- the identification of salmon species in food ences in different Mugilidae species using 5S products. Eur Food Res Technol 212:385–389. rDNA markers. Aquacult Res 38:1370–1379. doi:10.1007/s002170000237. doi:10.1111/j.1365-2109.2007.01808.x. 58. Hold GL, Russell VJ, Pryde SE, et al. (2001) 68. Infante C, Catanese G, Ponce M, et al. (2004) Development of a DNA-Based method aimed at Novel method for the authentication of frigate identifying the fish species present in food tunas (Auxis thazard and Auxis rochei) in products. J Agric Food Chem 49:1175–1179. commercial canned products. J Agric Food doi:10.1021/jf001149x. Chem 52:7435–7443. doi:10.1021/jf0492868. 59. Horstkotte B, Rehbein H (2003) Fish species 69. Infante C, Crespo A, Zuasti E, et al. (2006) PCR- identification by means of restriction fragment based methodology for the authentication of the length polymorphism and high-performance atlantic mackerel Scomber scombrus in com- liquid chromatography. J Food Sci 68:2658– mercial canned products. Food Res Int 39:1023– 2666. doi:10.1111/j.1365-2621.2003.tb05785.x. 1028. doi:10.1016/j.foodres.2006.02.006. 60. Hsieh HS, Chai T, Cheng CA, et al. (2004) 70. Itoi S, Nakaya M, Kaneko G, et al. (2005) Application of DNA technique for identifying Rapid identification of eels Anguilla japonica the species of different processed products of and Anguilla anguilla by polymerase chain swordfish meat. J Food Sci 69:FCT1–FCT6. reaction with single nucleotide polymorphism- doi:10.1111/j.1365-2621.2004.tb17847.x. based specific probes. Fish Sci 71:1356–1364. 61. Hsieh HS, Chai T, Hwang DF (2005) Rapid doi:10.1111/j.1444-2906.2005.01102.x. PCR-RFLP method for the identification of 5 71. Ivanova N, Zemlak T, Hanner R, et al. (2007) billfish species. J Food Sci 70:246–249. Universal primer cocktails for fish DNA bar- 62. Hsieh HS, Chai T, Hwang DF (2007) Using the coding. Mol Ecol Notes 7:544–548. doi: PCR-RFLP method to identify the species of 10.1111/j.1471-8286.2007.01748.x. different processed products to billfish meats. 72. Jarman S, Deagle B, Gales N (2004) Group- Food Contr 18:369–374. doi:10.1016/j.foodcont. specific polymerase chain reaction for DNA- 2005.11.002. based analysis of species diversity and identity 63. Hsieh HS, Hwang DF (2004) Molecular phylo- in dietary samples. Mol Ecol 13:1313–1322. genetic relationships of puffer fish inferred from doi:10.1111/j.1365-294X.2004.02109.x. partial sequences of cytochrome b gene and 73. Je´roˆme M, Lemaire C, Bautista JM, et al. restriction fragment length polymorphism anal- (2003) Molecular phylogeny and species iden- ysis. J Agric Food Chem 52:4159–4165. doi: tification of sardines. J Agric Food Chem 10.1021/jf035462l. 51:43–50. doi:10.1021/jf020713w. 64. Hsieh YW, Hwang PA, Pan HH, et al. (2003) 74. Je´roˆme M, Lemaire C, Verrez-Bagnis V, et al. Identification of tetradotoxin and fish species in (2003) Direct sequencing method for species

123 286 Rev Fish Biol Fisheries (2009) 19:265–293

identification of canned sardine and sardine- mitochondrial DNA restriction fragment analy- type products. J Agric Food Chem 51:7326– sis. Fish Bull (Wash D C) 104:182–196. 7332. doi:10.1021/jf034652t. 85. Li Z, Nishimoto MM, Love MS, et al. (2006) 75. Karaiskou N, Apostolidis AP, Triantafyllidis A, Comparing the identification of southern Cali- et al. (2003) Genetic identification and phylog- fornia juvenile rockfish (genus Sebastes spp.) eny of three species of the genus Trachurus by restriction site analysis of the mitochondrial based on mitochondrial DNA analysis. Mar ND3/ND4 region and by morphological char- Biotechnol 5:493–504. doi:10.1007/s10126-002- acteristics. Fish Bull (Wash D C) 104:376–382. 0099-5. 86. Lin W–F, Hwang D-F (2007) Application of 76. Karaiskou N, Triantafyllidis A, Alvarez P, et al. PCR-RFLP analysis on species identification (2007) Horse mackerel egg identification using of canned tuna. Food Contr 18:1050–1057. DNA methodology. Mar Ecol (Berl) 28:429– doi:10.1016/j.foodcont.2006.07.001. 434. doi:10.1111/j.1439-0485.2007.00190.x. 87. Lin Y-S, Poh Y-P, Lin S-M, et al. (2002) 77. Karaiskou N, Triantafyllidis A, Margaroni M, Molecular techniques to identify freshwater et al. (2005) A double DNA approach for eels: RFLP analyses of PCR-amplified DNA identifying Macrorhamphosus scolopax (Pisces, fragments and allele-specific PCR from mito- Centriscidae). ICES J Mar Sci 62:1683–1690. chondrial DNA. Zool Stud 41:421–430. doi:10.1016/j.icesjms.2005.05.011. 88. Lindstrom D (1999) Molecular species identifi- 78. Karaiskou N, Triantafyllidis A, Triantaphyllidis cation of newly hatched Hawaiian amphidr- C (2003) Discrimination of three Trachurus omous gobioid larvae. Mar Biotechnol 1:167– species using both mitochondrial-and nuclear- 174. doi:10.1007/PL00011764. based DNA approaches. J Agric Food Chem 89. Lockley A, Bardsley R (2000) Novel Method 51:4935–4940. doi:10.1021/jf034063n. for the Discrimination of Tuna (Thunnus 79. Kitano T, Umetsu K, Tian W, et al. (2007) Two thynnus) and Bonito (Sarda sarda) DNA. J universal primer sets for species identification Agric Food Chem 48:4463–4468. doi:10.1021/ among vertebrates. Int J Legal Med 121:423– jf000387p. 427. doi:10.1007/s00414-006-0113-y. 90. Lopez I, Pardo M (2005) Application of relative 80. Klossa-Kilia E, Papasotiropoulos V, Kilias G, quantification TaqMan Real-Time polymerase et al. (2002) Authentication of Messolongi chain reaction technology for the identification (Greece) fish roe using PCR-RFLP analysis of and quantification of Thunnus alalunga and 16s rRNA mtDNA segment. Food Contr 13:169– Thunnus albacares. J Agric Food Chem 53: 172. doi:10.1016/S0956-7135(01)00097-4. 4554–4560. doi:10.1021/jf0500841. 81. Kon T, Yoshino T, Mukai T, et al. (2007) DNA 91. Ludwig A, Debus L, Jenneckens I (2002) A sequences identify numerous cryptic species of molecular approach to Controlol the interna- vertebrate: a lesson from the gobioid fish tional trade in black caviar. Inter Rev Hydrobiol Schindleria. Mol Phylogenet Evol 44:53–62. 87:661–674. doi:10.1002/1522-2632(200211)87: doi:10.1016/j.ympev.2006.12.007. 5/6\661::AID-IROH661[3.0.CO;2-S. 82. Kyle CJ, Wilson CC (2007) Mitochondrial 92. Luthy SA, Cowen RK, Serafy JE, et al. (2005) DNA identification of game harvested freshwa- Toward identification of larval sailfish (Istio- ter fish species. Forensic Sci Int 166:68–76. phorus platypterus), white marlin (Tetrapterus doi:10.1016/j.forsciint.2006.03.025. albidus), and blue marlin (Makaira nigricans) 83. Lakra WS, Goswami M, Mohindra V, et al. in the western North Atlantic Ocean. Fish Bull (2007) Molecular identification of five indian (Wash D C) 103:588–600. sciaenids (Pisces: Perciformes, Sciaenidae) 93. Maldini M, Marzano F, Fortes G, et al. (2006) using RAPD markes. Hydrobiologia 585:359– Fish and seafood traceability based on AFLP 363. doi:10.1007/s10750-006-0480-x. markers: elaboration of a species database. Aqua- 84. Li Z, Gray AK, Love MS, et al. (2006) A key to culture 261:487–494. doi:10.1016/j.aquaculture. selected rockfishes (Sebastes spp.) based on 2006.07.010.

123 Rev Fish Biol Fisheries (2009) 19:265–293 287

94. Maretto F, Reffo E, Dalvit C, et al. (2007) ICES J Mar Sci 62:908–914. doi:10.1016/ Finding 16S rRNA gene-based SNPs for the j.icesjms.2005.04.001. genetic traceability of commercial species 104. Perez M, Vieites J, Presa P (2005) ITS1- belonging to Gadiformes. Ital J Anim Sci rDNA-based methodology to identify world- 6:161–163. wide hake species of the genus Merluccius.J 95. Masri S, Rast H, Ripley T, et al. (2002) Agric Food Chem 53:5239–5247. doi:10.1021/ Detection of genetically modified coho salmon jf048012h. using polymerase chain reaction (PCR) ampli- 105. Politov D, Gordon N, Afanasiev K, et al. fication. J Agric Food Chem 50:3161–3164. (2000) Identification of palearctic coregonid doi:10.1021/jf011606p. fish species using mtDNA and allozyme 96. McDowell JR, Graves JE (2002) Nuclear and genetic markers. J Fish Biol 74:51–71. mitochondrial DNA markers for specific iden- 106. Purcell M, Mackey G, LaHood E (2004) tification of istiophorid and xiphiid billfishes. Molecular methods for the genetic identifica- Fish Bull (Wash D C) 100:537–544. tion of salmonid prey from Pacific harbor seal 97. Michelini E, Cevenini L, Mezzanotte L, et al. (Phoca vitulina richardsi) scat. Fish Bull (2007) One-step triplex-polymerase chain reac- (Wash D C) 102:213–220. tion assay fot the authentication of yellowfin 107. Quinteiro J, Sotelo CG, Rehbein H, et al. (1998) (Thunnus albacares), bigeye (Thunnus obesus), Use of mtDNA direct polymerase chain reac- and skipjack (Katsuwonus pelamis) tuna DNA tion (PCR) sequencing and PCR-restriction from fresh, frozen, and canned tuna samples. J fragment length polymorphism methodologies Agric Food Chem 55:7638–7647. doi:10.1021/ in species identification of canned tuna. J Agric jf070902k. Food Chem 46:1662–1669. doi:10.1021/jf970 98. Nam YK, Park JE, Kim KK, et al. (2003) A 552?. rapid and simple PCR-based method for the 108. Quinteiro J, Vidal R, Izquierdo M, et al. (2001) analysis of transgenic fish using a restricted Identification of hake species (Merluccius amount of fin tissue. Transgenic Res 12:523– genus) using sequencing and PCR-RFLP anal- 525. doi:10.1023/A:1024274508052. ysis of mitochondrial DNA Controlol region 99. Pardo M, Pe´rez-Villareal B (2004) Identification sequences. J Agric Food Chem 49:5108–5114. of commercial canned tuna species by restriction doi:10.1021/jf010421f. site analysis of mitochondrial DNA products 109. Rehbein H (2005) Identification of the fish obtained by nested primer PCR. Food Chem species of raw or cold-smoked salmon and 86:143–150. doi:10.1016/j.foodchem.2003.09. salmon caviar by single-strand conformation 024. polymorphism (SSCP) analysis. Eur Food Res 100. Parsons K, Piertney S, Middlemas S, et al. Technol 220:625–632. doi:10.1007/s00217- (2005) DNA-based identification of salmonid 004-1067-2. prey species in seal faeces. J Zool (Lond) 110. Rehbein H, Kress G, Schmidt T (1997) 266:275–281. doi:10.1017/S0952836905006 Application of PCR-SSCP to species identifi- 904. cation of fishery products. J Sci Food Agric 101. Pegg GH, Sinclair B, Briskey L, et al. (2006) 74:35–41. doi:10.1002/(SICI)1097-0010(199705) MtDNA barcode identification of fish larvae in 74:1\35::AID-JSFA765[3.0.CO;2-2. the southern Great Barrier Reef, Australia. Sci 111. Rehbein H, Mackie I, Pryde S, et al. (1999) Mar 70:7–12. Fish species identification in canned tuna by 102. Pepe T, Trotta M, Di Marco I, et al. (2007) PCR-SSCP: validation by a collaborative study Fish species identification in surimi-based and investigation of intra-species variability of products. J Agric Food Chem 55:3681–3685. the DNA-patterns. Food Chem 64:263–268. doi:10.1021/jf063321o. doi:10.1016/S0308-8146(98)00125-3. 103. Perez J, Alvarez P, Martinez J, et al. (2005) 112. Rehbein H, Sotelo C, Perez-Martin R, et al. Genetic identification of hake and megrim (2002) Differentiation of raw and processed eel eggs in formaldehyde-fixed plankton samples. by PCR-based techniques: restriction fragment

123 288 Rev Fish Biol Fisheries (2009) 19:265–293

length polymorphism analysis (RFLP) and 122. Saitoh K, Takagaki M, Yamashita Y (2003) single strand conformation polymorphism anal- Detection of Japanese flounder-specific DNA ysis (SSCP). Eur Food Res Technol 214:171– from gut contents of potential predators in the 177. doi:10.1007/s00217-001-0457-y. field. Fish Sci 69:473–477. doi:10.1046/ 113. Reid SM, Wilson CC (2006) PCR-RFLP based j.1444-2906.2003.00647.x. diagnostic tests for Moxostoma species in 123. Sanjuan A, Raposo-Guillan J, Comesana A Ontario. Conserv Genet 7:997–1000. doi:10.1007/ (2002) Genetic identification of Lophius s10592-006-9113-1. budegassa and L. piscatorius by PCR-RFLP 114. Renshaw M, Saillant E, Broughton R, et al. analysis of a mitochondrial tRNAGLU/Cyto- (2006) Application of hypervariable genetic chrome b segment. J Food Sci 67:2644–2648. markers to forensic identification of ‘wild’ from doi:10.1111/j.1365-2621.2002.tb08792.x. hatchery-raised red drum, Sciaenops ocellatus. 124. Santaclara F, Cabado A, Vieites J (2006) Forensic Sci Int 156:9–15. doi:10.1016/j.forsc Development of a method for genetic identi- iint.2005.05.038. fication of four species of anchovies: E. 115. Richardson D, Vanwye J, Exum A, et al. encrasicolus, E. anchoita, E. ringens, and E. (2007) High-throughput species identification: japonicus. Eur Food Res Technol 223:609– from DNA isolation to bioinformatics. Mol 614. doi:10.1007/s00217-005-0241-5. Ecol Notes 7:199–207. doi:10.1111/j.1471- 125. Sebastio P, Zanelli P, Neri T (2001) Identifi- 8286.2006.01620.x. cation of Anchovy (Engraulis encrasicholus 116. Robertson M, Ovenden J, Barker S (2007) L.) and gilt sardine (Sardinella aurita) by Identification of small juvenile scombrids from polymerase chain reaction, sequence of their northwest tropical Australia using mitochondrial mitochondrial cytochrome b gene, and restric- DNA cytochrome b sequences. Ichthyol Res tion analysis of polymerase chain reaction 54:246–252. doi:10.1007/s10228-007-0397-z. products in semipreserves. J Agric Food Chem 117. Rocha-Olivares A (1998) Multiplex haplo- 49:1194–1199. doi:10.1021/jf000875x. type-specific PCR: a new approach for species 126. Sevilla R, Diez A, Noren M, et al. (2007) identification of the early life stages of rockf- Primers and polymerase chain reaction condi- ishes of the species-rich genus Sebastes tions for DNA barcoding teleost fish based on Cuvier. J Exp Mar Biol Ecol 231:279–290. the mitochondrial cytochrome b and nuclear doi:10.1016/S0022-0981(98)00098-7. rhodopsin genes. Mol Ecol Notes 7:730–734. 118. Rocha-Olivares A, Moser H, Stannard J (2000) doi:10.1111/j.1471-8286.2007.01863.x. Molecular identification and description of 127. Sezaki K, Itoi S, Watabe S (2005) A simple pelagic young of the rockfishes Sebastes method to distinguish two commercially valu- constellatus and Sebastes ensifer. Fish Bull able eel species in Japan Anguilla japonica (Wash D C) 98:353–363. and A. anguilla using polymerase chain reac- 119. Rosel P, Kocher T (2002) DNA-based identi- tion strategy with a species-specific primers. fication of larval cod in stomach contents of Fish Sci 71:414–421. doi:10.1111/j.1444-2906. predatory fishes. J Exp Mar Biol Ecol 267:75– 2005.00979.x. 88. doi:10.1016/S0022-0981(01)00359-8. 128. Shivji M, Clarke S, Pank M, et al. (2002) 120. Russell V, Hold G, Pryde S, et al. (2000) Use Genetic identification of pelagic shark body of restriction fragment length polymorphism parts for conservation and trade monitoring. to distinguish between salmon species. J Conserv Biol 16:1036–1047. doi:10.1046/ Agric Food Chem 48:2184–2188. doi:10.1021/ j.1523-1739.2002.01188.x. jf991213e. 129. Smith P, MCveagh S, Allain V et al. (2005) 121. Ruzainah A, Siti Azizah M, Patimah I, et al. DNA identification of gut contents of large (2003) RAPD fingerprinting of the eel-loaches pelagic fishes. J Fish Biol 67:1178–1183. Pangio filinaris and Pangio piperata: prelimin- doi:10.1111/j.0022-1112.2005.00804.x. ary evaluation. Aquacult Res 34:959–965. 130. Sotelo C, Calo-Mata P, Chapela M, et al. (2001) doi:10.1046/j.1365-2109.2003.00951.x. Identification of flatfish (Pleuronectiforme)

123 Rev Fish Biol Fisheries (2009) 19:265–293 289

species using DNA-based techniques. J Agric Food Chem 53:2039–2045. doi:10.1021/ Food Chem 49:4562–4569. doi:10.1021/jf01 jf048542d. 0452a. 139. Tsai Y-H, Hsieh H, Chen H–C, et al. (2007) 131. Spies IB, Gaichas S, Stevenson D, et al. (2006) Histamine level and species identification of DNA-based identification of Alaska skates billfish meats implicated in two food-borne (Amblyraja, Bathyraja and Raja: Rajidae) poisonings. Food Chem 104:1366–1371. using cytochrome c oxydase subunit I (coI) doi:10.1016/j.foodchem.2007.01.052. variation. J Fish Biol 69:283–292. doi:10.1111 140. Tzeng CH, Chen CS, Chiu TS (2007) Analysis /j.1095-8649.2006.01286.x. of morphometry and mitochrodrial DNA 132. Strange R, Stepien C (2007) Yellow (Perca sequences from two Trichiurus species in flavescens) and Eurasian (P. fluviatilis) perch waters of the western North Pacific: taxonomic distinguished in fried fish samples by DNA assessment and population structure. J Fish analysis. Fish Bull (Wash D C) 105:292–295. Biol 70:165–176. doi:10.1111/j.1095-8649.2007. 133. Tagliavini J, Conterio F, Gandolfi G, et al. (1999) 01368.x. Mitochondrial DNA sequences of six sturgeons 141. Von Der Heyden S, Lipinski M, Matthee C species and phylogenetic relationships within (2007) Species-specific genetic markers for Acipenseridae. J Appl Ichthyology 15:17–22. identification of early-life history stages of doi:10.1111/j.1439-0426.1999.tb00198.x. Cape hakes, Merluccius capensis and Merluc- 134. Takeyama H, Chow S, Tsuzuki H, et al. (2001) cius paradoxus in the southern Benguela Mitochondrial DNA sequence variation within current. J Fish Biol 70:262–268. doi:10.1111/ and between tuna Thunnus species and its j.1095-8649.2007.01409.x. application to species identification. J Fish 142. Ward R, Zemlak T, Innes B, et al. (2005) DNA Biol 58:1646–1657. doi:10.1111/j.1095-8649. barcoding Australia’s fish species. Philos 2001.tb02319.x. Trans R Soc B-Biol Sci 360:1847-1857. 135. Taylor M, Fox C, Rico C (2002) Species- 143. Watanabe S, Minegishi Y, Yoshinaga T, et al. specific TaqMan probes for simultaneous (2004) A quick method for species identifica- identification of (Gadus morhua L.), had- tion of Japanese eel (Anguilla japonica) using dock (Melanogrammus aeglefinus L.) and real-time PCR: an onboard application for use whithing (Merlangius merlangus L.). Mol Ecol during sampling surveys. Mar Biotechnol Notes 2:599–601. doi:10.1046/j.1471-8286. 6:566–574. doi:10.1007/s10126-004-1000-5. 2002.00269.x. 144. Weder J, Rehbein H, Kaiser K–P (2001) On 136. Terol J, Mascarell R, Fernandez-Pedrosa V, the specificity of tuna-directed primers in et al. (2002) Statistical validation of the iden- PCR-SSCP analysis of fish and meat. Eur tification of tuna species: bootstrap analysis of Food Res Technol 213:139–144. doi:10.1007/ mitochondrial DNA sequences. J Agric Food s002170100339. Chem 50:963–969. doi:10.1021/jf011032o. 145. Whithler R, Candy J, Beacham T, et al. (2004) 137. Tinti F, Ungaro N, Pasolini P, et al. (2003) Forensic DNA analysis of Pacific salmonid Development of molecular and morphological samples for species and stock identification. markers to improve species-specific monitor- Environ Biol Fishes 69:275–285. doi:10.1023/ ing and systematics of Northeast Atlantic and B:EBFI.0000022901.26754.0b. Mediterranean skates (Rajiformes). J Exp Mar 146. Wolf C, Burgener M, Lu¨thy J (2000) PCR- Biol Ecol 288:149–165. doi:10.1016/S0022- RFLP Analysis of Mitochondrial DNA: Dif- 0981(03)00021-2. ferentiation of Fish Species. LWT-Food Sci. 138. Trotta M, Scho¨nhuth S, Pepe T, et al. (2005) Technol 33:144–150. Multiplex PCR method for use in real-time 147. Wolf C, Hu¨bner P, Lu¨thy J (1999) Differen- PCR for identification of fish fillets from tiation of sturgeon species by PCR-RFLP. Grouper (Epinephelus and Mycteroperca spe- Food Res Int 32:699–705. doi:10.1016/S0963- cies) and common substitute species. J Agric 9969(99)00150-7.

123 290 Rev Fish Biol Fisheries (2009) 19:265–293

148. Wuertz S, Belay M, Kirschbaum F (2007) On Asensio L, Montero A (2008) Analysis of fresh fish labelling in the risk of criminal contamination in caviar Spanish fish retail shops. Food Control 19:795–799. doi:10.1016/j.foodcont.2007.08.005 trade by intended contamination of caviar Asensio L, Gonza´lez I, Garcı´a T et al (2008) Determination of with PCR products. Aquaculture 269:130–134. food authenticity by enzyme-linked immunosorbent assay doi:10.1016/j.aquaculture.2007.05.027. (ELISA). Food Control 19:1–8. doi:10.1016/j.foodcont. 149. Yang D, Cannon A, Saunders S (2004) DNA 2007.02.010 Babola O, Desvarenne S, Lacroix B et al (2004) L’identifica- species identification of archaelogical salmon tion des espe`ces animales dans l’alimentation humaine et bone from the Pacific Northwest Coast of animale: un exemple d’application de la technologie des North America. J Archaeol Sci 31:619–631. puces a` ADN. Bull Soc Fr Micr 19:30–36 doi:10.1016/j.jas.2003.10.008. Balitzki-Korte B, Anslinger K, Bartsch C, Rolf (2005) Species identification by means of pyrosequencing the mitochon- 150. Zhang J, Huang H, Cai Z, et al. (2007) Species drial 12S rRNA gene. Int J Legal Med 119:291–294. identification in salted products of red snap- doi:10.1007/s00414-005-0537-9 pers by semi-nested PCR-RFLP based on the Bartlett S, Davidson W (1992) FINS (forensically informative mitochondria 12S rRNA gene sequence. Food nucleotide sequencing): a procedure for identifying the animal origin of biological specimens. Biotechniques Contr 18:1331–1336. doi:10.1016/j.foodcont. 3:408–411 2005.01.001. Benesh DP, Hasu T, Suomalainen L-R et al (2006) Reliability 151. Zhang J, Huang H, Cai Z, et al. (2006) Species of mitochondrial DNA in an acanthocephalan: the prob- identification in salted products of red snap- lem of pseudogenes. Int J Parasitol 36:247–254. doi: 10.1016/j.ijpara.2005.09.008 pers by semi-nested PCR-RFLP based on the Bensasson D, Zhang D, Hartl DL (2001) Mitochondrial pseu- mitochondrial 12S rRNA gene sequence. Food dogenes: evolution’s misplaced witnesses. Trends Ecol Contr 17:557–563. doi:10.1016/j.foodcont.2005. Evol 16:314–321. doi:10.1016/S0169-5347(01)02151-6 01.011. Birstein VJ, Doukakis P, Sorkin B et al (1998) Population aggregation analysis of three caviar-producing species of 152. Zhang J, Huang L, Huo H (2004) Larval sturgeons and implications for the species identification of identification of Lutjanus Bloch in Nansha black caviar. Conserv Biol 12:766–775. doi:10.1046/ coral reefs by AFLP molecular method. J Exp j.1523-1739.1998.97081.x Mar Biol Ecol 298:3–20. doi:10.1016/S0022- Blaxter M (2003) Molecular systematics: counting angels with DNA. Nature 421:122–124. doi:10.1038/421122a 0981(03)00341-1. Blaxter M (2004) The promise of a DNA taxonomy. Philos 153. Zhang J, Wang H, Cai Z (2007) The applica- Trans R Soc Lond B Biol Sci 359:669–679. doi:10.1098/ tion of DGGE and AFLP-derived SCAR rstb.2003.1447 for discrimination between Atlantic salmon Bossier P (1999) Authentication of seafood products by DNA patterns. J Food Sci 64:189–193. doi:10.1111/j.1365- (Salmo salar) and rainbow trout (Oncorhyn- 2621.1999.tb15862.x chus mykiss). Food Contr 18:672–676. doi: Bower MA, Spencer M, Matsumura S et al (2005) How many 10.1016/j.foodcont.2006.02.015. clones need to be sequenced from a single forensic or ancient DNA sample in order to determine a reliable consensus sequence? Nucleic Acids Res 33:2549–2556. doi:10.1093/nar/gki550 References Bucciarelli G, Golani D, Bernardi G (2002) Genetic cryptic species as biological invaders: the case of a Lessepsian Ansfield M, Reaney SD, Jackman R (2000) Production of a fish migrant, the hardyhead silverside Atherinomorus la- sensitive immunoassay for detection of ruminant and cunosus. J Exp Mar Biol Ecol 273:143–149. doi:10.1016/ porcine proteins, heated to [130°C at 2.7 bar, in com- S0022-0981(02)00138-7 pound animal feedstuffs. Food Agric Immunol 12:273– Byrkjedal I, Rees DJ, Willassen E (2007) Lumping lump- 284. doi:10.1080/09540100020008146 suckers: molecular and morphological insights into the Antunes A, Ramos MJ (2005) Discovery of a large number taxonomic status of Eumicrotremus spinosus (Fabricius, of previously unrecognized mitochondrial pseudogenes 1776) and Eumicrotremus eggvinii Koefoed, 1956 (Tele- in fish genomes. Genomics 86:708–717. doi:10.1016/ ostei: Cyclopteridae). J Fish Biol 71:111–131. doi: j.ygeno.2005.08.002 10.1111/j.1095-8649.2007.01550.x Aranishi F, Okimoto T, Ohkubo M (2005) Molecular identifi- Callejas C, Ochando MD (2001) Molecular identification cation of commercial spicy pollack roe products by PCR- (RAPD) of the eight species of the genus Barbus RFLP analysis. J Food Sci 70:235–238 (Cyprinidae) in the Iberian Peninsula. J Fish Biol Asensio L (2007) PCR-based methods for fish and fishery 59:1589–1599. doi:10.1111/j.1095-8649.2001.tb00223.x products authentication. Trends Food Sci Technol 18: Carr SM, Kivlichan DS, Pepin P et al (1999) Molecular 558–566. doi:10.1016/j.tifs.2007.04.016 systematics of gadid fishes: implications for the

123 Rev Fish Biol Fisheries (2009) 19:265–293 291

biogeographic origins of Pacific species. Can J Zool for the assessment of the cod stock and mapping of 77:19–26. doi:10.1139/cjz-77-1-19 spawning areas using egg-based methods. Mol Ecol Carrera E, Garcı´a T, Ce´spedes A et al (2000) Differentiation of 14:879–884. doi:10.1111/j.1365-294X.2005.02439.x smoked Salmo salar, Oncorhynchus mykiss and Brama raii Froese R, Pauly P (eds) (2008) FishBase. World Wide Web using the nuclear marker 5S rDNA. Int J Food Sci Technol electronic publication. www.fishbase.org, version (06/ 35:401–406. doi:10.1046/j.1365-2621.2000.00404.x 2008) Casper RM, Jarman SN, Deagle BE et al (2007) Detecting prey Gharrett AJ, Gray AK, Heifetz J (2001) Identification of from DNA in predator scats: a comparison with mor- rockfish (Sebastes spp.) by restriction site analysis of the phological analysis, using Arctocephalus seals fed a know mitochondrial ND-3/ND-4 and 12S/16S rRNA gene diet. J Exp Mar Biol Ecol 347:144–154. doi:10.1016/ regions. Fish Bull (Wash D C) 99:49–62 j.jembe.2007.04.002 Gilbert MT, Bandelt H-J, Hofreiter M et al (2005) Assessing Chakraborty A, Aranishi F, Iwatsuki Y (2007) Polymerase ancient DNA studies. Trends Ecol Evol 20:541–544. chain reaction-restriction fragment length polymorphism doi:10.1016/j.tree.2005.07.005 analysis for species identification of hairtail fish fillets Granadeiro JP, Silva MA (2000) The use of otoliths and ver- from supermarkets in Japan. Fish Sci 73:197–201. tebrae in the identification and size-estimation of fish in doi:10.1111/j.1444-2906.2007.01319.x predator-prey studies. Cybium 24:383–393 Chapela MJ, Sotelo CG, Pe´rez-Martı´n RI et al (2007) Com- Greig TW, Moore MK, Woodley CM (2005) Mitochondrial parison of DNA extraction methods from muscle of gene sequences useful for species identification of western canned tuna for species identification. Food Control North Atlantic Ocean sharks. Fish Bull (Wash D C) 18:1211–1215. doi:10.1016/j.foodcont.2006.07.016 103:516–523 Civera T (2003) Species identification and safety of fish Hanner RH, Gregory TR (2007) Genomic diversity research products. Vet Res Commun 27:481–489. doi:10.1023/ and the role of biorepositories. Cell Pres Tech 5:93–103. B:VERC.0000014205.87859.ab doi:10.1089/cpt.2007.9993 Comesana AS, Abella P, Sanjuan A (2003) Molecular identi- Harris DJ (2003) Can you bank on GenBank? Trends Ecol Evol fication of five commercial flatfish species by PCR-RFLP 18:317–319. doi:10.1016/S0169-5347(03)00150-2 analysis of a 12 rRNA gene fragment. J Sci Food Agric Hebert PDN, Cywinska A, Ball SL et al (2003) Biological 83:752–759. doi:10.1002/jsfa.1368 identifications through DNA barcodes. Proc R Soc Lond Comi G, Lacumi L, Rantsiou (2005) Molecular methods for the Ser B Biol Sci 270:313–321. doi:10.1098/rspb.2002.2218 differentiation of species used in production of cod-fish Heist EJ, Gold JR (1999) Genetic identification of sharks in the can detect commercial frauds. Food Control 16:37–42. U.S. Atlantic large coastal shark fishery. Fish Bull (Wash doi:10.1016/j.foodcont.2003.11.003 D C) 97:53–61 Cooper A, Wayne R (1998) New uses for old DNA. Curr Opin Hoelzel AR (2001) Shark fishing in fin soup. Conserv Genet Biotechnol 9:49–53. doi:10.1016/S0958-1669(98)80083-9 2:69–72. doi:10.1023/A:1011590517389 Dalmasso A, Civera T, Bottero MT (2006) Biomolecular Horstkotte B, Rehbein H (2003) Fish species identification approaches for the identification of tuna species. Vet Res by means of restriction fragment length polymor- Commun 30:179–181. doi:10.1007/s11259-006-0035-7 phism and high-performance liquid chromatography. J Deagle B, Tollit D, Jarman S et al (2005) Molecular scatology Food Sci 68:2658–2666. doi:10.1111/j.1365-2621.2003. as a tool to study diet: Analysis of prey DNA in scats from tb05785.x captive Steller sea lions. Mol Ecol 14:1831–1842. Hsieh HS, Hwang DF (2004) Molecular phylogenetic rela- doi:10.1111/j.1365-294X.2005.02531.x tionships of puffer fish inferred from partial sequences of DeSalle R, Egan MG, Siddall M (2005) The unholy trinity: cytochrome b gene and restriction fragment length poly- taxonomy, species delimitation and DNA barcoding. morphism analysis. J Agric Food Chem 52:4159–4165. Philos Trans R Soc B 360:1905–1916. doi:10.1098/ doi:10.1021/jf035462l rstb.2005.1722 Hubalkova Z, Kralik P, Tremlova B et al (2007) Methods of Doiron S, Bernatchez L, Blier P (2002) A comparative mi- gadoid fish species identification in food and their eco- togenomic analysis of the potential adaptive value of nomic impact in the Czech Republic: a review. Vet Med Arctic charr mtDNA introgression in brook charr popu- 52:273–292 lations (Salvelinus fontinalis Mitchill). Mol Biol Evol Iff At F (2002) Mullets of Korangi Creek, Karachi. Rec Zool 19:1902–1909 Surv Pak 14:11–18 Ercolini D (2004) PCR-DGGE fingerprinting: novel strategies Janzen DH (2004) Now is the time. Philos Trans R Soc Lond B for detection of microbes in food. J Microbiol Methods Biol Sci 359:731–732. doi:10.1098/rstb.2003.1444 56:297–314. doi:10.1016/j.mimet.2003.11.006 Jarman S, Deagle B, Gales N (2004) Group-specific polymer- Etienne M, Jerome M, Fleurence J et al (2000) Identification of ase chain reaction for DNA-based analysis of species fish species after cooking by SDS–PAGE and urea IEF: a diversity and identity in dietary samples. Mol Ecol collaborative study. J Agric Food Chem 48:2653–2658. 13:1313–1322. doi:10.1111/j.1365-294X.2004.02109.x doi:10.1021/jf990907k Je´roˆme M, Lemaire C, Verrez-Bagnis V et al (2003) Direct Forster P (2003) To err is human. Ann Hum Genet 67:2–4. sequencing method for species identification of canned doi:10.1046/j.1469-1809.2003.00002.x sardine and sardine-type products. J Agric Food Chem Fox CJ, Taylor MI, Pereyra R et al (2005) TaqMan DNA 51:7326–7332. doi:10.1021/jf034652t technology confirms likely overestimation of cod (Gadus King RA, Read DS, Traugott M et al (2008) Molecular analysis morhua L.) egg abundance in the Irish Sea: implications of predation: a review of best practice for DNA-based

123 292 Rev Fish Biol Fisheries (2009) 19:265–293

approaches. Mol Ecol 17:947–963. doi:10.1111/j.1365- Matejusova´ I, Doig F, Middlemas SJ et al (2008) Using 294X.2007.03613.x quantitative real-time PCR to detect salmonid prey in Klossa-Kilia E, Papasotiropoulos V, Kilias G et al (2002) scats of grey Halichoerus grypus and harbour Phoca vi- Authentication of Messolongi (Greece) fish roe using PCR- tulina seals in Scotland—an experimental and field study. RFLP analysis of 16s RNA mtDNA segment. Food Control J Appl Ecol 45:632–640 13:169–172. doi:10.1016/S0956-7135(01)00097-4 McDowell JR, Graves JE (2002) Nuclear and mitochondrial Kocher TD, Thomas WK, Meyer A et al (1989) Dynamics of DNA markers for specific identification of istiophorid and mitochondrial DNA evolution in animals: amplification xiphiid billfishes. Fish Bull 100:537–544 and sequencing with conserved primers. Proc Natl Acad Methven DA, McGowan C (1998) Distinguishing small juve- Sci USA 86:6196–6200. doi:10.1073/pnas.86.16.6196 nile Atlantic cod (Gadus morhua) from Greenland cod Kochzius M, No¨lte M, Weber H et al (2008) DNA microarrays (Gadus ogac) by comparing meristic characters and dis- for identifying fishes. Mar Biotechnol 10:207–217. criminant function analysis of morphometric data. Can J doi:10.1007/s10126-007-9068-3 Zool 76:1054–1062 Kon T, Yoshino T, Mukai T et al (2007) DNA sequences Michelini E, Cevenini L, Mezzanotte L et al (2007) One-step identify numerous cryptic species of vertebrate: a lesson triplex-polymerase chain reaction assay fot the authenti- from the gobioid fish Schindleria. Mol Phylogenet Evol cation of yellowfin (Thunnus albacares), bigeye (Thunnus 44:53–62. doi:10.1016/j.ympev.2006.12.007 obesus), and skipjack (Katsuwonus pelamis) tuna DNA Kvasnicˇka F (2005) Capillary electrophoresis in food authen- from fresh, frozen, and canned tuna samples. J Agric Food ticity. J Sep Sci 28:813–825 Chem 55:7638–7647 Lakra WS, Goswami M, Mohindra V, Lal KK, Punia P (2007) Millar CD, Huynen L, Subramanian S et al (2008) New Molecular identification of five Indian scianids (pisces: developments in ancient genomics. Trends Ecol Evol perciformes, sciaenidae) using RAPD markers. Hydrobi- 23:386–393 ologia 583:359–363 Miraglia M, Berdal KG, Brera C (2004) Detection and trace- Li Z, Gray AK, Love MS et al (2006) A key to selected ability of genetically modified organisms in the food rockfishes (Sebastes spp.) based on mitochondrial DNA production chain. Food Chem Toxicol 42:1157–1180 restriction fragment analysis. Fish Bull 104:182–196 Moretti VM, Turchini GM, Bellagamba F et al (2003) Trace- Lin W-F, Hwang D-F (2007) Application of PCR-RFLP ability issues in fishery and aquaculture products. Vet Res analysis on species identification of canned tuna. Food Comm 27:497–505 Control 18:1050–1057 Moritz C, Cicero C (2004) DNA barcoding: promise and pit- Lipscomb D, Platnick N, Wheeler Q (2003) The intellectual falls. PLoS Biol 2:1529–1531 content of taxonomy: a comment on DNA taxonomy. Nilsson RH, Ryberg M, Kristiansson E (2006) Taxonomic Trends Ecol Evol 18:65–66 reliability of DNA sequences in public sequence dat- Lockley AK, Bardsley RG (2000) DNA-based methods for abases: a fungal perspective. PLoS ONE 1:e59 food authentication. Trends Food Sci Tech 11:67–77 Pa¨a¨bo S, Irwin DM, Wilson AC (1990) DNA damage promotes Ludwig A, Congiu L, Pitra C (2003) Nonconcordant evolu- jumping between templates during enzymatic amplifica- tionary history of maternal and paternal lineages in tion. J Biol Chem 265:4718–4721 Adriatic sturgeon. Mol Ecol 12:3253–3264 Pa¨a¨bo S, Poinar H, Serre D et al (2004) Genetic analyses from Mackie IM, Pryde SE, Gonzales-Sotelo C et al (1999) Chal- ancient DNA. Annu Rev Genet 38:645–679 lenges in the identification of species of canned fish. Paterlini M (2007) There shall be order. The legacy of Lin- Trends Food Sci Tech 10:9–14 naeus in the age of molecular biology. EMBO Rep 8:814– Mafra I, Ferreira I, Beatriz M et al (2008) Food authentication 816 by PCR-based methods. Eur Food Res Technol 227: Pegg GH, Sinclair B, Briskey L et al (2006) MtDNA barcode 649–665 identification of fish larvae in the southern Great Barrier Maldini M, Marzano FN, Fortes GG et al (2006) Fish and Reef, Australia. Sci Mar 70:7–12 seafood traceability based on AFLP markers: elaboration Pepe T, Trotta M, Di Marco I et al (2007) Fish species iden- of a species database. Aquaculture 261:487–494 tification in surimi-based products. J Agric Food Chem Mallet J, Willmott K (2003) Taxonomy: renaissance or Tower 55:3681–3685 of Babel? Trends Ecol Evol 18:57–59 Pierce GJ, Boyle PR (1991) A review of methods for diet Maretto F, Reffo E, Dalvit C et al (2007) Finding 16S rRNA analysis in piscivorous marine mammals. Oceanogr Mar gene-based SNPs for the genetic traceability of commer- Biol Ann Rev (Lond) 29:409–486 cial species belonging to Gadiformes. Ital J Anim Sci Poinar HN (2002) The genetic secrets some fossils hold. Acc 6:161–163 Chem Res 35:676–684 Marko PB, Lee SC, Rice AM (2004) Fisheries: mislabelling of Politov D, Gordon N, Afanasiev K et al (2000) Identification of a depleted reef fish. Nature 430:309–310 palearctic coregonid fish species using mtDNA and allo- Martin AP, Palumbi SR (1993) Protein evolution in different zyme genetic markers. J Fish Biol 74:51–71 cellular environments: cytochrome b in sharks and Quinteiro J, Sotelo CG, Rehbein H et al (1998) Use of mtDNA mammals. Mol Biol Evol 10:873–891 direct polymerase chain reaction (PCR) sequencing and Masri S, Rast H, Ripley T et al (2002) Detection of genetically PCR-restriction fragment length polymorphism method- modified coho salmon using polymerase chain reaction ologies in species identification of canned tuna. J Agric (PCR) amplification. J Agric Food Chem 50:3161–3164 Food Chem 46:1662–1669

123 Rev Fish Biol Fisheries (2009) 19:265–293 293

Rasmussen RS, Morrissey MT (2008) DNA-based methods for Takeyama H, Chow S, Tsuzuki H et al (2001) Mitochondrial the identification of commercial fish and seafood species. DNA sequence variation within and between tuna Thun- Compr Rev Food Sci Food Saf 7:280–295 nus species and its application to species identification. Ratnasingham S, Hebert PDN (2007) BOLD: the barcode of J Fish Biol 58:1646–1657 Life Data systems (www.barcodinglife.org). Mol Ecol Tautz D, Arctander P, Minelli A et al (2003) A plea for DNA Notes 7:355–364 taxonomy. Trends Ecol Evol 18:70–74 Redenbach Z, Taylor EB (2003) Evidence for bimodal hybrid Teletchea T, Maudet C, Ha¨nni C (2005) Food and forensic zones between two species of charr (Pisces: Salvelinus) in molecular identification: update and challenges. Trends northwestern North America. J Evol Biol 16:1135–1148 Biotech 23:359–366 Rehbein H (1990) Electrophoretic techniques for species Teletchea T, Laudet V, Ha¨nni C (2006) Phylogeny of the identification of fishery products. Z Lebensm Unters Gadidae (sensu Svetovidov, 1948) based on their mor- Forsch 191:1–10 phology and two mitochondrial genes. Mol Phyl Evol Rehbein H, Mackie I, Pryde S et al (1999) Fish species iden- 38:189–199 tification in canned tuna by PCR-SSCP: validation by a Teletchea F, Bernillon J, Duffraisse M et al (2008) Molecular collaborative study and investigation of intra-species identification of vertebrate species by oligonucleotide variability of the DNA-patterns. Food Chem 64:263–268 microarray in food and forensic samples. J Appl Ecol Reid SM, Wilson CC (2006) PCR-RFLP based diagnostic tests for 45:967–975 Moxostoma species in Ontario. Conserv Genet 7:997–1000 Tinti F, Ungaro N, Pasolini P et al (2003) Development of Richardson D, Vanwye J, Exum A et al (2007) High- molecular and morphological markers to improve species- throughput species identification: from DNA isolation to specific monitoring and systematics of Northeast Atlantic bioinformatics. Mol Ecol Notes 7:199–207 and Mediterranean skates (Rajiformes). J Exp Mar Biol Roelfsema JH, Peters DJ (2005) Denaturing gradient gel elec- Ecol 288:149–165 trophoresis (DGGE). In: Walker JM, Rapley R (eds) Medical Tsai Y-H, Hsieh H, Chen H-C et al (2007) Histamine level and biomethods handbook. Humana Press, New York, pp 79–86 species identification of billfish meats implicated in two Ronaghi M (2001) Pyrosequencing sheds light on DNA food-borne poisonings. Food Chem 104:1366–1371 sequencing. Genome Res 11:3–11 Vos P, Hogers R, Bleeker M et al (1995) AFLP: a new tech- Ruedas LA, Salazar-Bravo J, Dragoo JW (2000) The impor- nique for DNA fingerprinting. Nucleic Acids Res tance of being earnest: what, if anything, constitutes a 23:4407–4414 ‘‘specimen examined? Mol Phylogenet Evol 17:129–132 Ward RD, Zemlak TS, Innes BH et al (2005) DNA barcoding Sanjuan A, Raposo-Guillan J, Comesana A (2002) Genetic Australia’s fish species. Philos Trans R Soc Lond B Biol identification of Lophius budegassa and L. piscatorius by Sci 360:1847–1857 PCR-RFLP analysis of a mitochondrial tRNAGLU/Cyto- Waugh J (2007) DNA barcoding in animal species: progress, chrome b segment. J Food Sci 67:2644–2648 potential and pitfalls. BioEssays 29:188–197 Schander C, Willassen E (2005) What can biological barcoding Weder JKP, Rehbein H, Kaiszer KP (2001) On the specificity do for marine biology? Mar Biol Res 1:79–83 of tuna-directed primes in PCR-SSCP analysis of fish and Schindel DE, Miller SE (2005) DNA barcoding a useful tool meat. Eur Food Res Technol 213:139–144 for taxonomists. Nature 435:17 Wheeler QD, Raven PH, Wilson EO (2004) Taxonomy: Schlick-Steiner BC, Seifert B, Stauffer C et al (2007) Without impediment or expedient? Science 303:285 morphology, cryptic species stay in taxonomic crypsis Will KW, Rubinoff D (2004) Myth of the molecule: DNA following discovery. Trends Ecol Evol 22:391–392 barcodes for species cannot replace morphology for Seberg O, Humphries CJ, Knapp S et al (2003) Shortcuts in identification and classification. Cladistics 20:47–55 systematics? A commentary on DNA-based taxonomy. Will KW, Mishler BD, Wheeler QD (2005) The perils of DNA Trends Ecol Evol 18:63–65 barcoding and the need for integrative taxonomy. Syst Sevilla R, Diez A, Noren M et al (2007) Primers and poly- Biol 54:844–851 merase chain reaction conditions for DNA barcoding Willerslev E, Cooper A (2005) Ancient DNA. Proc R Soc Lond teleost fish based on the mitochondrial cytochrome b and Ser B Biol Sci 272:3–16 nuclear rhodopsin genes. Mol Ecol Notes 7:730–734 Wilson IG (1997) Inhibition and facilitation of nucleic acid Sheppard SK, Harwood JD (2005) Advances in molecular amplification. Appl Environ Microbiol 63:3741–3751 ecology: tracking trophic links through predator-prey Wolf C, Hu¨bner P, Lu¨thy J (1999) Differentiation of sturgeon food-webs. Funct Ecol 19:751–762 species by PCR-RFLP. Food Res Int 32:699–705 Sites JW, Marshall JC (2003) Delimiting species: a renaissance Woolfe M, Primrose S (2004) Food forensics: using DNA issue in systematic biology. Trends Ecol Evol 18:462–470 technology to combat misdescription and fraud. Trends Smith P, MCveagh S, Allain V et al (2005) DNA identification Biotechnol 22:222–226 of gut contents of large pelagic fishes. J Fish Biol Yang D, Cannon A, Saunders S (2004) DNA species identifi- 67:1178–1183 cation of archaeological salmon bone from the Pacific Strange R, Stepien C (2007) Yellow (Perca flavescens) and Northwest Coast of North America. J Archaeol Sci Eurasian (P. fluviatilis) perch distinguished in fried fish 31:619–631 samples by DNA analysis. Fish Bull 105:292–295 Zhang J, Huang L, Huo H (2004) Larval identification of Strauss RE, Bond CE (1990) Taxonomic methods: morphology. Lutjanus Bloch in Nansha coral reefs by AFLP molecular In: Schreck CB, Moyle PB (eds) Methods for fish biology. method. J Exp Mar Biol Ecol 298:3–20 American Fisheries Society, Maryland, pp 109–140

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