Food and forensic molecular identification: update and challenges

Review

TRENDS in Biotechnology

Vol.23 No.7 July 2005

Food and forensic molecular identification: update and challenges Fabrice Teletchea, Celia Maudet and Catherine Ha¨nni Centre de Ge´ne´tique Mole´culaire et Cellulaire, CNRS UMR5534, UCB-Lyon I, 16, Rue Raphael Dubois, 69622 Villeurbanne Cedex, France

The need for accurate and reliable methods for animal species identification has steadily increased during past decades, particularly with the recent food scares and the overall crisis of biodiversity primarily resulting from the huge ongoing illegal traffic of endangered species. A relatively new biotechnological field, known as species molecular identification, based on the amplification and analysis of DNA, offers promising solutions. Indeed, despite the fact that retrieval and analysis of DNA in processed products is a real challenge, numerous technically consistent methods are now available and allow the detection of animal species in almost any organic substrate. However, this field is currently facing a turning point and should rely more on knowledge primarily from three fundamental fields – paleogenetics, molecular evolution and systematics.

Introduction Recent food scares (e.g. BSE, avian flu, foot-and-mouth disease, etc.), malpractices of some food producers, religious reasons, food allergies and GMOS have tremendously reinforced public awareness regarding the composition of food products. However, because labels do not provide sufficient guarantee about the true contents of a product, it is necessary to identify and/or authenticate the components of processed food, thus protecting both consumers and producers from illegal substitutions [1]. In addition, trade of endangered species has contributed to severe depletion of biodiversity. Approximately 10–20% of all vertebrates and plant species are at risk of extinction over the next few decades (IUCN; http://www. redlist.org and CITES http://www.cites.org). Wildlife and their products represent the third greatest illegal traffic after drugs and arms [2] and one of the most serious threats to the survival of animal populations is poaching. Each year, millions of endangered animals are illegally killed or captured for private zoo collections, hunt trophies, ornamental objects (e.g. elephant ivory [3]), human consumption (e.g. sea turtles and their eggs [4]) or traditional medicine (e.g. tiger [5,6], rhinoceros [7]). Food authentication and protection of biodiversity both require reliable and accurate methods for determining, without ambiguity, the animal species in a wide array of degraded and processed substrates (Table 1). The Corresponding author: Ha¨nni, C. ([email protected]). Available online 31 May 2005

development of these methods should protect both consumers and producers from frauds, and protect animal species from over-exploitation or illegal trafficking. Molecular authentication or molecular traceability, which is based on the PCR amplification of DNA, has been developed in the past ten years and offers promising solutions for these issues. Furthermore, this field will probably experience tremendous development because: (i) Most DNA methods developed have proved useful on almost all organic substrates and will certainly become the new legal standards for identification. (ii) More regulations have introduced safety standards into the chain of food production (e.g. European Regulations such as the 2000/104/EC, which establishes that fish products can enter the commercial circuit only if the commercial name, method of production and capture area are clearly labelled or the 2002/1774/EC, which bans the intra-species recycling of animal by-products). (iii) During the past decade, molecular identification tests have only been developed for a few species but it is likely that this number will steadily increase, particularly among fish (Box 1). Therefore, it is now crucial to reassess the different molecular methods available for animal species identification, particularly in light of three fundamentals fields: paleogenetics, molecular evolution and systematics. We are convinced that these three fields could improve the potential of analysing DNA in degraded substrates, help to choose the most appropriate molecular markers and highlight some common problems encountered in systematics that could result in erroneous identification. Here we concentrate mainly on species identification and not in the recognition of distinct populations of the same species because the latter question requires different concepts at some point. DNA from food and forensic samples Fresh food products or forensic samples without processing are suitable for many types of molecular or protein analyses (traditional biochemical approaches based on proteins used either electrophoretic, chromatographic or immunological techniques; reviewed in [8]). Unfortunately, because most foodstuffs and forensic samples are processed, DNA is usually altered. However, several research fields had already worked with such DNA: ancient DNA studies or paleogenetics (studying DNA

www.sciencedirect.com 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.05.006

360

Review

TRENDS in Biotechnology

Vol.23 No.7 July 2005

Table 1. Examples of forensic or food substrates for molecular analysis Samples analyzed Food products Canned products

Species targeted

Extraction methods

Fragment targeted

Fragment size (bp)

Refs

Tuna

Cyt b

171

[58]

Canned products

Sardine

Chloroform, methanol, water Chelexa, phenol, chloroform, isoamyl alcohol Guanidium thiocyanate (GUSCN) Dneasy tissue kitb

152

[59]

145–313

[60]

Meat-and-bone meal in compound feeds Blood or meat meal, pet food, baby food ‘Mortara’ salami Goat cheese Foie gras

Beef, sheep, pig, chicken Ruminant, avian, fish and pig Goose Beef Goose and mule duck

Caviar

Sturgeon species

Genomic prep kitc Dneasy tissue kitb Phenol, chloroform, isoamyl alcohol Not indicated

Whale

Cyt b Lys

tRNA

, ATPase

12s rRNA, tRNAval, 16S rDNA Cyt b D-loop 5S rDNA

104–290

[61]

350 413 250–1000

[62] [63] [56]

Cyt b, 16S, 12S

Not indicated

[55]

Not indicated

D-loop

155–378

[64]

Human

Chelexa, QIAmpb

128–250

[65]

Chinese alligator Tiger

Phenol/chloroform Chelex1, phenol, chloroform

Microsatelittes, Amelogenin Cyt b Cyt b

180 165

[66] [6]

African elephant

QIAamp kitb

70–251

[3]

Faeces

Tiger

510

[67]

Muscle, blood, eggs, skin Soup, dried fin, cartilage pills

Sea turtles Shark

Guanidium thiocyanate (GUSCN) Phenol-chloroform; Dneasyb Phenol-chloroform; Dneasyb

12S, cyt b, microsatellites Cyt b Cyt b Cyt b

875–876 155–188

[4] [68]

Forensic products Dried, salted and unfrozen strips of meat Formalin fixed paraffin embded tissues Skin, tanned hide, scales Pills and plasters made with tiger’s bone Elephant tusk (Ivory)

a

BioRad (http://www.bio-rad.com). Applied Biosystems (http://www.appliedbiosystems.com). Amersham Biosciences (http://www1.amershambiosciences.com).

b c

from fossil bones or ancient organic remains), human forensics (studying DNA from hairs, saliva, blood etc. from crime scenes), non-invasive ecological studies (studying DNA from animal faeces or hairs found in the field) and more recently, food authentication. Taken together, these fields have demonstrated that, (i) despite being altered, DNA is more resistant and thermostable than proteins are and it is still possible to PCR amplify small DNA fragments (with sufficient information to allow identification) and (ii) DNA could potentially be retrieved from any substrate because it is present in almost all cells of an organism. In addition, molecular evolution and phylogenetics have shown that, because of the degeneracy of the

genetic code and the presence of many non-coding regions, DNA provides much more information than proteins do. However, in processed products, DNA is altered and displays several particular features that must be taken into account. Substrates, DNA quality and contaminations Short DNA fragments. First, during production processes, food products might be subject to thermal treatments (cooking, pasteurization, etc.), high pressure, pH modifications, irradiation, drying and so on. For example, many food products are heated up to 1008C for 10–60 min and are exposed to a pH!4. Consequently, molecular

Box 1. Review of species identified To evaluate the species for which at least one method is currently available, we attempted to collate comprehensively, but not exhaustively, all studies published in the past decade. It seemed that almost all of these studies (w100 reviewed) had focused on a few species belonging to three main groups of vertebrates: mammals (36%), actinopterygian fishes (34%) and birds (20%). † Within mammals, more than one-third (41%) of the studies only dealt with one or all of the same four livestock species (cattle, pig, sheep and goat). The remaining mammal species studied are usually endangered ones, such as seven species of animals for bushmeat [69], rhinoceroses [7] or tigers [5]. † Within birds, two-thirds (60%) only dealt with chicken and/or turkey, few dealt with other birds such as goose and duck [70] or ostrich [71]. † Within fishes, the number of species studied is much higher than in the two previous groups. This was expected, because the number of exploited species is far higher than either in mammals or birds. Indeed www.sciencedirect.com

O1200 species are fished (http://www.fishbase.org/search.cfm) and w220 are farmed (both numbers include shellfish) [72]. Nevertheless, as with the two previous groups, we observed a strong bias towards several species, such as tuna (20%), salmon (16%) and sturgeon (6%). By contrast, the most valuable fish have hardly been studied, such as sardines [73,59] and cod [74], which are the first (22 472 563 metric tons in 2002) and second (8 392 479 metric tons in 2002) most important groups ‘harvested’ worldwide (http://www.fao.org), respectively. † Finally, we found several studies (10%) that focused on other groups, such as clam species [75] or sea turtles [4]. Consequently, despite the relatively high number of studies published, the number of species for which a method of detection is currently available is still small, probably less than 100 animal species overall. Thus it seems likely that in the future the number of species studied will increase significantly, particularly within the fish.

Review

TRENDS in Biotechnology

Box 2. Example of simultaneous detection of mixed species in foodstuff DNA was first extracted from a pet food can labelled ‘meat flavour’ (Figure I, [76]) and was amplified using trans-vertebrates (enabling the amplification of all vertebrate species) or ‘universal primers’ designed by Kocher et al. [26]. The amplified DNA (a 380 bp portion of the Cytochrome b gene) was then cloned using a commercial cloning kit (TOPO TA cloning, Invitrogen; http://www.invitrogen. com/) to separate each molecule of DNA, and 30 clones were sequenced using a direct sequencing method. Sequences obtained were compared to those in the sequence bank GenBank (http://www. ncbi.nlm.nih.gov/Genbank/index.html). A mix of five species sequences was observed: seven clones of beef (Bos taurus), four clones of pig (Sus scrofa), five clones of duck (Cairina moschata), seven clones of chicken (Gallus gallus), and seven clones of sea trout (Salmo trutta). Today, this approach is the only one (with DNA chips) that enables broad identification of animal species used in a food product, even if, to our knowledge, it has rarely been used.

Canned pet food DNA isolation PCR amplification using trans-vertebrates primers Cloning and sequencing of 30 clones

TRENDS in Biotechnology

Figure I.

identification methods from such highly degraded substrates should be based on the analysis of very short DNA fragments, preferably between 100–200 base pairs (e.g. researchers were not able to amplify fragments longer that 200 bp from canned tunas [9] or in processed animal meals [10,11]). Low amounts of DNA. DNA is not only degraded but is also present in small quantities that considerably reduce the number of DNA fragments with suitable size for molecular analysis. Thus, paleogenetics and non-invasive molecular studies have shown that increasing the number of PCR cycles, up to 45 or 55 cycles, is often required to get enough amplified DNA for subsequent analysis. However, DNA from other components, fraudulently or accidentally included, and from minority constituents, could be present in very small quantities. Species detection methods should therefore be very specific to provide reliable results. Contaminations. Because PCR is such a sensitive method, sometimes a single exogenous DNA molecule could be preferentially amplified instead of the degraded one (reviewed in [12]). Thus, food and forensic samples should be manipulated (before PCR) in a dedicated www.sciencedirect.com

Vol.23 No.7 July 2005

361

laboratory [physically isolated from those where unaltered DNA (from fresh samples) and amplified DNA are handled] and appropriate negative controls (extraction and PCR blanks) should be processed on every run. PCR inhibitors. Many food or forensic constituent products could be co-purified with the target DNA and are known to be inhibitors of DNA amplification. These include organic and phenolic compounds, polysaccharides (or products of the Maillard reaction), glycogen, fats, milk proteins, collagen, iron, cobalt or fulvic acids (reviewed in [13] and [14]). Other more widespread inhibitors include bacterial cells, non-target DNA and exogenous contaminants [13]. Therefore, the absence of amplification in food or forensic samples (i.e. a negative readout) could be explained by the inhibition of the PCR amplification rather than by an insufficient amount or the absence of the target DNA. Thus, to detect a species in a food product, DNA isolation methods should allow removal of inhibitors (e.g. [15]). Despite the numerous protocols already described (examples are given in Table 1) to date, no general extraction method has proved useful with all the different matrices encountered [16]. Therefore, each new substrate requires the development of new extraction and amplification methods or the adaptation of existing ones. Nevertheless, several ancient DNA studies have shown that the use of the PTB (N-phenecylthiazolium bromide) allows cleaveage of cross-links between proteins and DNA (Maillard reaction) and thus enhances the subsequent PCR reaction by permitting the movement of the Taq polymerase on the DNA molecule [17,18]. Moreover, it is also possible to neutralize some of these inhibitors by the addition of bovine serum albumin (BSA) or spermidine to the PCR reagents [19,20]. Chemical modifications and PCR artefacts These are probably the least known features of DNA in food and forensic applications but paleogenetic studies showed that environment (acidity, UV light, moisture, etc.) could induce chemical modifications of the DNA molecule. In this case, modified DNA molecules might display breaks or artifactual mutations (reviewed in [21]). Consequently, absence of DNA degradation from a food product, particularly for extensively processed ones, should be checked before using methods based on few variable sites. These DNA modifications can indeed produce misidentification of species because the DNA sequence obtained is slightly different from the reference one. Although these chemical degradations have never been studied in food products, Ram et al. [22] found inexplicable variations in DNA extracted from canned tuna; this variability could be because of chemical modifications of the DNA molecule studied. Paleogenetic and non-invasive studies have also shown that PCR amplification starting from tiny amounts of altered DNA could induce artifactual results (e.g. allelic drop-out in microsatellite analysis [23] or chimeric molecules produced by jumping PCR [24]). Mix of individuals and species DNA extracted from a food product is a mix of DNA of many origins: bacterial, vegetable, animal and fungi.

362

Review

TRENDS in Biotechnology

Furthermore, several individuals of the same species (sometimes from different lineages) could be used in a food product. However, unfortunately almost none of the available methods allow the simultaneous identification of a wide array of species (or individuals) mixed in food products. This simultaneous detection of several species is certainly one of the greatest challenges in the field, but still remains unresolved. Two methods have the potential to offer convincing and reliable solutions – cloning and sequencing (Box 2) or DNA chips. DNA chips (also known as DNA macroarray or DNA microarray) allow the examination of complex mixtures of PCR products and, potentially, the identification of hundreds or thousands of species simultaneously. Such methods have already been used in numerous fields such as in ecology (e.g. for the simultaneous detection of five marine fish pathogens [25]) but not extensively for species identification in food and forensic samples (F. Teletchea et al., unpublished data). Despite all of these technical constraints and difficulties, it has been shown that DNA could be retrieved and analysed with sufficient size and information to allow the correct identification of species from a wide array of substrates by using different extraction protocols (Table 1, Box 3). DNA markers for species identification DNA retrieved from food and forensic products displays several specific features that complicate its detection. These characteristics underline why, for species identification in these kinds of products, the mitochondrial DNA genome (mtDNA) is preferentially targeted compared to nuclear DNA. Indeed, because there are several copies of mtDNA inside a cell (w1000!the copies of nuclear DNA) it is more likely to amplify a fragment within this genome rather than within the nuclear genome. Besides, this small circular genome (w16 kb in most vertebrate species) displays maternal inheritance in most animal species, is haploid, and does not undergo recombination – characteristics that make its study easier and more straightforward. Lastly, mtDNA generally evolves much faster than nuclear DNA and thus enables even closely related species to be differentiated and identified.

Vol.23 No.7 July 2005

More precisely, a suitable DNA marker for identification at the species level should be sufficiently variable between species (particularly between the closest ones) and display either low or no intra-specific variations across the geographic distribution area. In addition, this marker should be widely studied for a large number of species to enable comparison of the nucleotide sequence from an unknown sample with reference sequences in a database. The gene encoding cytochrome b satisfied most of these criteria and is by far the most studied gene for phylogeny. It is certainly used in more than half of the phylogenetic studies published in the past decade (more than 50 000 sequences available in GenBank in 2004). This gene also displayed both (i) conserved regions allowing the determination of primers such as ‘universal’ primers published by Kocher et al. [26], which could be used to amplify a wide range of vertebrate species (see Box 2), and (ii) regions with a high level of variability, which allows the evolutionary studies of even closely related species. Nevertheless, in the cases of identification of breeds, geographic origins, or individual assignments, markers should be different and those showing an important intra-specific variability will be very useful (e.g. D-loop [27]; microsatellites [28,29]; or coding region [30]). Thus, in some cases, a strong haplotypic structure within a species can allow allocation of an individual to a particular geographic population. The large number of reconstructed phylogeography using mtDNA genes clearly illustrated that infraspecific information can be used to improve identification and potentially to identify geographic origins of new invasive species. Thus, as expected, among approximately one hundred molecular identification studies (collated from the literature comprehensively but not exhaustively in the past decade) almost half of them targeted the cytochrome b (44%), then the 12S (11%), 16S (8%) and D-loop (8%). Lastly, about fifteen other markers have occasionally been used, mostly located within mtDNA [e.g. cytochrome c oxidase II (COX2)]. Besides, despite the technical problem of analyzing DNA in these highly degraded samples (as mentioned above), few studies targeted nuclear markers such as microsatelittes (Table 1).

Box 3. Examples of food or forensic molecular identification Three molecular identification methods today represent O90% of all studies published: PCR-RFLP, Species-specific PCR and PCR-FINS. PCR-RFLP and species-specific PCR successes could be explained by the short development time necessary to design molecular markers, low cost and straightforward results. PCR-RFLP (restriction fragment length polymorphism). For example, Hold et al. [77] differentiated 36 fish species by digesting a 464 bp cytochrome b fragment with six different restriction enzymes. They showed that this method was still useful with processed products (1008C during 15 min) and also when several species were mixed. Species-specific PCR. Wand and Fang [5] analyzed tiger DNA in meat, faeces and dried skin and therefore showed that this protected animal was still illegally traded. Moreover, using a multiplex of species-specific primers Dalmasso et al. [61] proposed to identify the presence of the most used vertebrate groups in feedstuff products (ruminant, poultry, fish and pork). PCR-FINS (forensically informative nucleotide sequencing) is the www.sciencedirect.com

most direct means for obtaining information from PCR products, (i.e. by sequencing). Compared to the two previous methods, this method presents two main advantages: (i) it is not too sensitive to intraspecific variations; and (ii) by using cloning (Box 2) it is possible to detect several species in the same product. Je´roˆme et al. [59] used direct sequencing of a 103-bp diagnostic sequence to differentiate 14 sardine species used in food preparation. This method was successfully applied to 45 out of 47 commercial canned sardine and sardinetype products tested. New methods such as real time PCR (see [78] for a detailed presentation) and DNA chips have not been developed significantly for species identification to date. This low interest is particularly because of longer developing times and costs compared with the previous methods. However, in our laboratory, we have developed a DNA chip that allows the simultaneous detection of numerous vertebrate species in processed food and forensic samples (F. Teletchea et al., unpublished data).

Review

TRENDS in Biotechnology

Caution with mtDNA: the Numts The vast majority of identification methods developed in the past decade were based on mitochondrial sequences. However, we would like to emphasize that mitochondrial sequences could be obtained from a nuclear copy of the mtDNA rather than from endogenous mitochondrial DNA. Indeed, translocated pieces of mitochondrial DNA (known as Numts) are sometimes integrated into the nuclear genome. Thus, during analysis, these Numts might be amplified inadvertently by the PCR in addition to or even instead of the authentic target mtDNA and therefore led to erroneous results [31]. For example, while studying the snapping shrimp genus Alpheus, Williams and Knowlton [32] found multiple copies of the COX1 gene in at least ten species. They observed that within a single animal, differences between the real mtDNA and pseudogene sequences ranged from 0.2–18.8%. Thalmann et al. [31] found similar results within gorillas. Bensassson et al. [33] provided a detailed census of Numts in eukaryotic groups (they found Numts in 82 different species) and described methods for detecting them (e.g. checking for unique changes and odd substitution patterns). However, current practices do not preclude inadvertent analysis of Numts and much caution should be exercised when using mitochondrial sequences for identification purposes – explicit measures need to be taken to authenticate mtDNA sequences [31]. Species concepts The literature about species concepts might be more extensive than that about any other subject in evolutionary biology and therefore it is obviously out of the scope of the present review to discuss the validity of all these concepts (see [34]). Rather, we illustrate the three main problems usually encountered in systematics that could result in erroneous molecular identification methods if current practices do not take them into account. Complexes of cryptic species, closely related species and introgression The same scientific names could refer to highly divergent molecular groups. While studying the mitochondrial sequence variation (927 pb fragment of the ATPase and COX3 genes) within and between tuna species (Thunnus spp.), Takeyama et al., [35] found two divergent groups (diverging by w4,5%) within the northern bluefin tuna, Thunnus thynnus, (i.e. T. t. thunnus, living in the Atlantic ocean and T. t. orientalis, in the Pacific ocean). Thus, they showed that a re-evaluation of previous restriction methods was required to avoid inconsistent profiles and erroneous tuna identification. This result has considerable consequences as Thunnus thynnus is one of the most highly prized fish worldwide and Atlantic populations are on the red list. Similarly, Ludt et al. [36] found two distinct groups of red deer diverging by 5–6% (complete cytochrome b gene) and concluded that the Cervus elaphus species is clearly subdivided giving two valid species Cervus canadensis (occurring in Asia and North America) and Cervus elaphus (inhabiting Europe). Hird et al. [37] also found that the sequences of the partial cytochrome b www.sciencedirect.com

Vol.23 No.7 July 2005

363

gene (447 bp) differed by 3% between the English and USA breeds of turkey (Meleagris gallopavo). Two valid species could be genetically closely related. While studying the complete cytochrome b gene variability within the bear family, Talbot and Shields [38] found that brown bears (Ursus arctos) inhabiting the islands of southeastern Alaska seemed to be more closely related to polar bears (Ursus maritimus) than to any other brown bear populations. Similar conclusions were obtained between the Greenland cod Gadus ogac and the Pacific cod Gadus macrocephalus, in which sequences of partial cytochrome b gene (401 bp) and COX1 (495 bp) were identical [39]. The authors concluded that these two species are in fact the same and should be synonymized. Hybridization between closely related species (introgression) can occur if these species share overlapping habitats or sometimes through human intervention (e.g. during captive breeding). Within the group of the Bovini (comprising cattle-like species) several hybrids have been described (e.g. [40–42]). Similarly, several fish species show interspecific mitochondrial introgression (charr [43,44]; sturgeon [45]). These introgressions could sometimes disturb molecular identification of closely related species [46]. For all these reasons, more caution should be taken in this area. First with the use of scientific names, indeed most authors were quite elusive about the species they studied, for example neither the name of the author of authority who identified the species (e.g. Gadus morhua Linnaeus, 1758) nor geographical distribution and biological information were indicated; and sometimes only common names were given (e.g. [47,48]). Second, researchers should be reminded that scientific names could refer to different contents according to the progress of science [49] and that discrepancies could exits between taxonomical conclusions obtained from morphological and molecular characters even in well known groups, as shown here. Therefore identification methods cannot only rely on one or several sequences per species as was the case in the methods reviewed here. All the problems highlighted here are particularly critical within fish, where there are numerous examples of such unclear species boundaries and species complexes [50]. In addition, because this group is likely to become the most studied one in the next decades, it is likely that these kinds of situations will also become more and more common in the molecular identification field (for a detailed review of species already analyzed see Box 1). Recommendations Whatever the species, a thorough analysis should be made each time a new group is to be studied (ideally with taxonomists of each group) and several samples from the full distribution range should be taken into consideration to validate the method [51,52]. Voucher specimens should ideally be preserved, and submitters of sequences should conform to the ICZN (International Commission on Zoological Nomenclature; http://www.iczn.org/) rules of nomenclature [51]. Finally, because sequences from international banks are usually taken as reference, we would like to emphasize

364

Review

TRENDS in Biotechnology

Box 4. What is DNA taxonomy, and why is it so controversial? What is DNA barcoding? Herbert et al. [79] recently proposed a DNA-based barcoding system for all animal species, similar in practice to a supermarket barcode. According to the proponents of this system, the basic procedures of DNA barcoding would be straightforward [80]. A tissue sample is taken from a collected individual and DNA is extracted from this. This DNA serves as the reference sample from which one or several gene regions are, as a first approximation, an identification tag for the species from which the respective individual was derived. This sequence is then made available via appropriate references, against which sequences from sampled individuals can be compared (http:// www.barcodinglife.com/). Put simply, given the long history of use of molecular markers for molecular identification of species Moritz and Cicero [46] considered that there is nothing fundamentally new in this DNA barcoding concept, except increased scale and proposed standardization.

Why is it so controversial? However, the main problem with the DNA barcoding initiative is that the proponents of it propose not only to use DNA for identifying species (the ‘DNA barcode’, which seems to be generally accepted) but also for defining species, and therefore they want to give DNA a central and mandatory role in taxonomy (the so-called ‘DNA taxonomy’). Indeed they think that this is the only way to overcome the current impediment of taxonomy and particularly they consider that (i) it is impossible to describe biological diversity with traditional approaches [81], (ii) the current system depends heavily on specialists, whose knowledge is frequently lost when they retire [80], and (iii) species identification based on morphological characters has several significant limitations [79]. Such remarks have already received outright condemnation, chiefly from taxonomists, on both the theoretical (e.g. [49,82,83]) and practical level (e.g. [46,84]). Among the numerous criticisms, the main one is that DNA sequences, as morphological characters, are only data and could not serve to define a species on their own. In fact, what defines a species is an intractable debate that cannot be resolved satisfactorily using part of a single gene. The circumscription of a species will therefore always remain an opinion based on all data available [49,84]. Finally, molecular identification, using mtDNA, can be misled by Numts, introgression (as we have illustrated here) but also by incomplete lineage sorting, retention of polymorphism or absence of polymorphism (see [85] for a more complete review). For all these reasons, most taxonomists consider that relegating taxonomy, rich in theory and knowledge, to a high-tech service industry would decidedly be a setback for science. They argued that, instead of discarding more than 250 years of knowledge, new generations of taxonomists must be trained in initiatives such as the Partnerships for Enhancing Expertise in Taxonomy, PEET program ([86]; http://web.nhm.ku.edu/peet/).

that they could contain false sequences. For example, Forster [53] found that half of all published studies of human mtDNA sequences contain mistakes, not to mention Numts (as described above). Therefore, Harris [54] proposed several solutions to check the quality of the published sequences and the ‘simplest’ is to re-sequence them. Another solution would be to use as many reference sequences as possible resulting from different studies (when available) and never base a method on only one reference sequence. Consequently, even though there is a great need for molecular identification in numerous fields, protection of the biodiversity, food traceability and also ecology [46], much more caution should be taken in developing molecular identification methods and more www.sciencedirect.com

Vol.23 No.7 July 2005

generally in implementing the recent call for a ‘DNA taxonomy’ (Box 4). Conclusion Despite these technical and conceptual challenges, molecular species identification in food products and forensic samples is likely to increase exponentially. Indeed, numerous reliable methods now exist for identifying species in almost all kinds of substrates (Table 1 and Box 3) and some approaches have already produced significant and interesting results, for example in gourmet food (such as species identification in caviar [55] or in foie gras [56]) and on forensic samples made from endangered species (e.g. tiger [5] or rhinoceros [7]). Molecular identification has already proven useful in court [4,57] and some methods are currently used in industry [1,76]. Nevertheless, to become more accurate and reliable, this biotechnological field will have to take into account the technical and conceptual knowledge of its nearest fundamental fields. Acknowledgements We thank our team for constructive discussions, Brian B. Rudkin and Vincent Laudet for critical reading of the article, and CNRS UCB-Lyon1, Re´gion Rhoˆne-Alpes and Ministe` re de l’Education Nationale, de l’Enseignement Supe´rieur et de la Recherche for financial support.

References 1 Pascal, G. and Mahe, S. (2001) Identity, traceability, acceptability and substantial equivalence of food. Cell Mol. Biol. 47, 1329–1342 2 Manel, S. et al. (2002) Detecting wildlife poaching: identifiying the origin of individuals with Bayesian assignment test and multilocus genotypes. Conserv. Biol. 16, 650–659 3 Comstock, K. et al. (2003) Amplifying nuclear and mitochondrial DNA from African elephant ivory: a tool for monitoring the ivory trade. Conserv. Biol. 17, 1840–1843 4 Moore, M. et al. (2003) Use of restriction fragment length polymorphisms to identify sea turtle eggs and cooked meats to species. Conserv. Genet. 4, 95–103 5 Wan, Q.H. and Fang, S.G. (2003) Application of species-specific polymerase chain reaction in the forensic identification of tiger species. Forensic Sci. Int. 131, 75–78 6 Wetton, J. et al. (2004) An extremely sensitive species-specific ARMs PCR test for the presence of tiger bone DNA. Forensic Sci. Int. 140, 139–145 7 Hseih, H.M. et al. (2003) Species identification of rhinoceros horns using the Cytochrome b gene. Forensic Sci. Int. 136, 1–11 8 Civera, T. (2003) Species identification and safety of fish products. Vet. Res. Commun. 27 (Suppl. 1), 481–489 9 Quinteiro, J. et al. (1998) Use of mtDNA direct polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism methodologies in species identification of canned tuna. J. Agric. Food Chem. 46, 1662–1669 10 Frezza, D. et al. (2003) A competitive polymerase chain reaction-based approach for the identification and semiquantification of mitochondrial DNA in differently heat-treated bovine meat and bone meal. J. Food Prot. 66, 103–109 11 Prado, M. et al. (2004) Application of a polymerase chain (PCR) method as a complementary tool to microscopic analysis for the detection of bones and other animal tissues in home-made animal meals. J. Sci. Food Agric. 84, 505–512 12 Cooper, A. and Wayne, R. (1998) New uses for old DNA. Curr. Opin. Biotechnol. 9, 49–53 13 Wilson, I.G. (1997) Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63, 3741–3751 14 Scholz, M. et al. (1998) A polymerase chain reaction inhibitor of ancient hard and soft tissue DNA extract is determined as human collagen type I. Anal. Biochem. 259, 283–286

Review

TRENDS in Biotechnology

15 Ha¨nni, C. et al. (1995) Isopropanol precipitation removes PCR inhibitors from ancient bone extract. Nucleic Acids Res. 23, 881–882 16 Woolfe, M. and Primrose, S. (2004) Food forensics: using DNA technology to combat misdescription and fraud. Trends Biotechnol. 22, 222–226 17 Hofreiter, M. et al. (2001) Ancient DNA. Nat. Rev. Genet. 2, 353–359 18 Poinar, H.N. et al. (1998) Molecular coproscopy: dung and diet of the extinct ground sloth Nothrotheriops shastensis. Science 281, 402–406 19 Pa¨a¨bo, S. et al. (1988) Mitochondrial DNA sequences from a 7000-year old brain. Nucleic Acids Res. 16, 9775–9787 20 Orlando, L. et al. (2002) Ancient DNA and the population genetics of cave bears (Ursus spelaeus) through space and time. Mol. Biol. Evol. 19, 1920–1933 21 Poinar, H.N. (2002) The genetic secrets some fossils hold. Acc. Chem. Res. 35, 676–684 22 Ram, J.L. et al. (1996) Authentication of canned tuna and bonito by sequence and restriction site analysis of polymerase chain reaction products of mitochondrial DNA. J. Agric. Food Chem. 44, 2460–2467 23 Goossens, B. et al. (1998) Plucked hair samples as a source of DNA: reliability of dinucleotide microsatellite genotyping. Mol. Ecol. 7, 1237–1241 24 Pa¨a¨bo, S. et al. (1990) DNA damage promotes jumping between templates during enzymatic amplification. J. Biol. Chem. 265, 4718–4721 25 Gonzalez, S.F. et al. (2004) Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray. J. Clin. Microbiol. 42, 1414–1419 26 Kocher, T.D. et al. (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. U. S. A. 86, 6196–6200 27 Gongora, J. et al. (2004) Phylogenetic relationships of Australian and New Zealand feral pigs assessed by mitochondrial control region sequence and nuclear GPIP genotype. Mol. Phylogenet. Evol. 33, 339–348 28 Nielsen, E.E. et al. (2001) Fisheries. Population of origin of Atlantic cod. Nature 413, 272 29 Cunningham, E.P. and Meghen, C.M. (2001) Biological identification systems: genetic markers. Rev. Sci. Tech. 20, 491–499 30 Maudet, C. and Taberlet, P. (2002) Holstein’s milk detection in cheeses inferred from melanocortin receptor 1 (MC1R) gene polymorphism. J. Dairy Sci. 85, 707–715 31 Thalmann, O. et al. (2004) Unreliable mtDNA data due to nuclear insertions: a cautionary tale from analysis of humans and other great apes. Mol. Ecol. 13, 321–335 32 Williams, S.T. and Knowlton, N. (2001) Mitochondrial pseudogenes are pervasive and often insidious in the snapping shrimp genus Alpheus. Mol. Biol. Evol. 18, 1484–1493 33 Bensasson, D. et al. (2001) Mitochondrial pseudogenes: evolution’s misplaced witnesses. Trends Ecol. Evol. 16, 314–321 34 Sites, J.W. and Marshall, J.C. (2003) Delimiting species: a renaissance issue in systematic biology. Trends Ecol. Evol. 18, 462–470 35 Takeyama, H. et al. (2001) Mitochondrial DNA sequence variation within and between tuna Thunnus species and its application to species identification. J. Fish Biol. 58, 1646–1657 36 Ludt, C.J. et al. (2004) Mitochondrial DNA phylogeography of red deer (Cervus elaphus). Mol. Phylogenet. Evol. 31, 1064–1083 37 Hird, H. et al. (2003) Rapid detection of chicken and turkey in heated meat products using the polymerase chain reaction followed by amplicon visualisatoin vista green. Meat Sci. 65, 1117–1123 38 Talbot, S.L. and Shields, G.F. (1996) Phylogeography of brown bears (Ursus arctos) of Alaska and paraphyly within the Ursidae. Mol. Phylogenet. Evol. 5, 477–494 39 Carr, S.M. et al. (1999) Molecular systematics of gadid fishes: implications for the biogeographic origins of pacific species. Can. J. Zool. 77, 19–26 40 Nijman, I.J. et al. (2003) Hybridization of banteng (Bos javanicus) and zebu (Bos indicus) revealed by mitochondrial DNA, satellite DNA, AFLP and microsatellites. Heredity 90, 10–16 41 Kikkawa, Y. et al. (2003) Phylogenies using mtDNA and SRY provide evidence for male-mediated introgression in Asian domestic cattle. Anim. Genet. 34, 96–101 42 Verkaar, E.L. et al. (2003) Paternally inherited markers in bovine hybrid populations. Heredity 91, 565–569 www.sciencedirect.com

Vol.23 No.7 July 2005

365

43 Redenbach, Z. and Taylor, E.B. (2003) Evidence for bimodal hybrid zones between two species of charr (Pisces: Salvelinus) in northwestern North America. J. Evol. Biol. 16, 1135–1148 44 Doiron, S. et al. (2002) A comparative mitogenomic analysis of the potential adaptive value of Arctic charr mtDNA introgression in brook charr populations (Salvelinus fontinalis Mitchill). Mol. Biol. Evol. 19, 1902–1909 45 Ludwig, A. et al. (2003) Nonconcordant evolutionary history of maternal and paternal lineages in Adriatic sturgeon. Mol. Ecol. 12, 3253–3264 46 Moritz, C. and Cicero, C. (2004) DNA barcoding: promise and pitfalls. PLoS Biol. 2, 1529–1531 47 Sun, Y.L. and Lin, C.S. (2003) Establishment and application of a fluorescent polymerase chain-reaction fragment length polymorphism (PCR-RFLP) method for identifying porcine, caprine, and bovine meats. J. Agric. Food Chem. 51, 1771–1776 48 Saez, R. et al. (2004) PCR-based fingerprinting techniques for rapid detection of animal species in meat products. Meat Sci. 66, 659–665 49 Seberg, O. et al. (2003) Shortcuts in systematics? A commentary on DNA-based taxonomy. Trends Ecol. Evol. 18, 63–65 50 Sweijd, N.A. et al. (2000) Molecular genetics and the management and conservation of marine organisms. Hydrobiologia 420, 153–164 51 Ruedas, L.A. et al. (2000) The importance of being earnest: what, if anything, constitutes a “specimen examined? Mol. Phylogenet. Evol. 17, 129–132 52 Comesana, A.S. et al. (2003) Molecular identification of five commercial flatfish species by PCR-RFLP analysis of a 12 rRNA gene fragment. J. Sci. Food Agric. 83, 752–759 53 Forster, P. (2003) To err is human. Ann. Hum. Genet. 67, 2–4 54 Harris, D.J. (2003) Can you bank on GenBank? Trends Ecol. Evol. 18, 317–319 55 DeSalle, R. and Birstein, V. (1996) PCR identification of black caviar. Nature 381, 197–198 56 Rodriguez, M.A. et al. (2003) Identification of goose, mule duck, chicken, turkey, and swine in foie gras by species-specific polymerase chain reaction. J. Agric. Food Chem. 51, 1524–1529 57 Whitler, R.E. et al. (2004) Forensic DNA analysis of pacific salmonid samples for species and stock identification. Environmental biology of fishes 69, 275–285 58 Terol, J. et al. (2002) Statistical validation of the identification of tuna species: bootstrap analysis of mitochondrial DNA sequences. J. Agric. Food Chem. 50, 963–969 59 Jerome, M. et al. (2003) Direct sequencing method for species identification of canned sardine and sardine-type products. J. Agric. Food Chem. 51, 7326–7332 60 Krcmar, P. and Rencova, E. (2003) Identification of species-specific DNA in feedstuffs. J. Agric. Food Chem. 51, 7655–7658 61 Dalmasso, A. et al. (2004) A multiplex PCR assay for the identification of animal species in feedstuffs. Mol. Cell. Probes 18, 81–87 62 Colombo, C. et al. (2002) Identification of the goose (Anser anser) in italian “Mortara” salami by DNA sequencing and a polymerase chain reaction with an original primer pair. Meat Sci. 61, 291–294 63 Maudet, C. and Taberlet, P. (2001) Detection of cows ‘milk in goats’ cheeses inferred from mitochondrial DNA polymorphism. J. Dairy Res. 68, 229–235 64 Baker, C.S. et al. (2003) www.DNA-surveillance: applied molecular taxonomy for species conservation and discovery. Trends Ecol. Evol. 18, 271–272 65 Legrand, B. et al. (2002) DNA genotyping of unbuffered formalin fixed paraffin embedded tissues. Forensic Sci. Int. 125, 205–211 66 Yan, P. et al. (2004) Identification of chinese alligators (Alligator sinensis) meat by diagnostic PCR of the mitochondrial Cytochrome b gene. Biological conservation 67 Verma, S.K. et al. (2003) Was elusive carnivore a panther? DNA typing of faeces reveals the mystery. Forensic Sci. Int. 137, 16–20 68 Hoelzel, A.R. (2001) Shark fishing in fin soup. Cons Genet 2, 69–72 69 Kelly, J. et al. (2003) Forensic identification of non-human mammals involved in the bushmeat trade using restriction digests of PCR amplified mitochondrial DNA. Forensic Sci. Int. 136, 378 70 Calvo, J.H. et al. (2001) Random amplified polymorphic DNA fingerprints for identification of species in poultry pate. Poult. Sci. 80, 522–524

366

Review

TRENDS in Biotechnology

71 Abdulmawjood, A. and Bu¨lte, M. (2002) Identification of ostrich meat by restriction fragment length polymorphism (RFLP) analysis of Cytochrome b gene. J. Food Sci. 67, 1688–1691 72 Naylor, R.L. et al. (2000) Effect of aquaculture on world fish supplies. Nature 405, 1017–1024 73 Je´roˆme, M. et al. (2003) Molecular phylogeny and species identification. J. Agric. Food Chem. 51, 43–50 74 Comi, G. et al. (2004) Molecular methods for the differentiation of species used in production of cod-fish can detect commercial frauds. Food Contr. 16, 37–42 75 Fernandez, A. et al. (2002) Genetic differentiation between the clam species Rudipates decussatus (grooved carpet shell) and Venerupis pullustra (pullet carpet shell). J. Sci. Food Agric. 82, 881–885 76 Donne-Gousse´, C. et al. (2002) Method of determining the existence of animals or vegetals mixtures in organic substances. Patent FR2826022 77 Hold, G.L. et al. (2001) Development of a DNA-based method aimed at identifying the fish species present in food products. J. Agric. Food Chem. 49, 1175–1179

Vol.23 No.7 July 2005

78 Lockley, A.K. and Bardsley, R.G. (2000) DNA-based methods for food authentification. Trends Food Sci. Technol. 11, 67–77 79 Hebert, P.D. et al. (2003) Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B. Biol. Sci. 270, 313–321 80 Tautz, D. et al. (2003) A plea for DNA taxonomy. Trends Ecol. Evol. 18, 70–74 81 Blaxter, M. (2003) Molecular systematics: Counting angels with DNA. Nature 421, 122–124 82 Lipscomb, D. et al. (2003) The intellectual content of taxonomy: a comment on DNA taxonomy. Trends Ecol. Evol. 18, 65–66 83 Mallet, J. and Willmott, K. (2003) Taxonomy: renaissance or tower of babel? Trends Ecol. Evol. 18, 57–59 84 Will, K.W. and Rubinoff, D. (2004) Myth of the molecule: DNA barcodes for species cannot replace morphology for identification and classification. Cladistics 20, 47–55 85 Ballard, J.W.O. and Whitlock, M.C. (2004) The incomplete natural history of mitochondria. Mol. Ecol. 13, 729–744 86 Rodman, J.E. and Cody, J.H. (2003) The taxonomic Impediment overcome: NSF’s parterships for enhancing expertise in taxonomy (PEET) as a model. Syst. Biol. 52, 428–435

Elsevier celebrates two anniversaries with gift to university libraries in the developing world In 1580, the Elzevir family began their printing and bookselling business in the Netherlands, publishing works by scholars such as John Locke, Galileo Galilei and Hugo Grotius. On 4 March 1880, Jacobus George Robbers founded the modern Elsevier company intending, just like the original Elzevir family, to reproduce fine editions of literary classics for the edification of others who shared his passion, other ’Elzevirians’. Robbers co-opted the Elzevir family’s old printer’s mark, visually stamping the new Elsevier products with a classic old symbol of the symbiotic relationship between publisher and scholar. Elsevier has since become a leader in the dissemination of scientific, technical and medical (STM) information, building a reputation for excellence in publishing, new product innovation and commitment to its STM communities. In celebration of the House of Elzevir’s 425th anniversary and the 125th anniversary of the modern Elsevier company, Elsevier will donate books to 10 university libraries in the developing world. Entitled ‘A Book in Your Name’, each of the 6 700 Elsevier employees worldwide has been invited to select one of the chosen libraries to receive a book donated by Elsevier. The core gift collection contains the company’s most important and widely used STM publications including Gray’s Anatomy, Dorland’s Illustrated Medical Dictionary, Essential Medical Physiology, Cecil Essentials of Medicine, Mosby’s Medical, Nursing and Allied Health Dictionary, The Vaccine Book, Fundamentals of Neuroscience, and Myles Textbook for Midwives. The 10 beneficiary libraries are located in Africa, South America and Asia. They include the Library of the Sciences of the University of Sierra Leone; the library of the Muhimbili University College of Health Sciences of the University of Dar es Salaam, Tanzania; the library of the College of Medicine of the University of Malawi; and the libraries of the University of Zambia, Universite du Mali, Universidade Eduardo Mondlane, Mozambique; Makerere University, Uganda; Universidad San Francisco de Quito, Ecuador; Universidad Francisco Marroquin, Guatemala; and the National Centre for Scientific and Technological Information (NACESTI), Vietnam. Through ‘A Book in Your Name’, the 10 libraries will receive approximately 700 books at a retail value of approximately 1 million US dollars.

For more information, visit www.elsevier.com www.sciencedirect.com