Genetic Identification of Pelagic Shark Body Parts for ... Event/Resources... · 1036 Conservation...

1036

Conservation Biology, Pages 1036–1047Volume 16, No. 4, August 2002

Genetic Identification of Pelagic Shark Body Parts for Conservation and Trade Monitoring

MAHMOOD SHIVJI,* SHELLEY CLARKE,† MELISSA PANK,* LISA NATANSON,‡ NANCY KOHLER,‡ AND MICHAEL STANHOPE§

*Guy Harvey Research Institute, Oceanographic Center, Nova Southeastern University, 8000 North Ocean Drive, Dania Beach, FL 33004, U.S.A., email [email protected]†T.H. Huxley School, Imperial College, London SW7 2BP, United Kingdom‡National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 28 Tarzwell Drive, Narragansett, RI 02882, U.S.A.§Evolutionary Bioinformatics, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, UP1345, Collegeville, PA 19426–0989, U.S.A.

Abstract:

The conservation and management of sharks on a species-specific basis is a pressing need becauseof the escalating demand for shark fins and the recognition that individual shark species respond differentlyto exploitation. Difficulties with the identification of many commonly fished sharks and their body parts hasresulted in a global dearth of catch and trade information, making reliable assessment of exploitation effectsand conservation needs for individual species nearly impossible. We developed and tested a highly stream-lined molecular genetic approach based on species-specific, polymerase-chain-reaction primers in an eight-primer multiplex format to discriminate simultaneously between body parts from six shark species commonin worldwide pelagic fisheries. The species-specific primers are based on DNA sequence differences among spe-cies in the nuclear ribosomal internal transcribed spacer 2 locus. The primers and multiplex format accu-rately and sensitively distinguished samples from each of three lamnid (

Isurus oxyrinchus

,

Isurus paucus

, and

Lamna nasus

) and three carcharhinid (

Prionace glauca

,

Carcharhinus obscurus

, and

Carcharhinus falciformis

)species from all but one other shark species encountered in the North Atlantic fishery. Furthermore, the threelamnid primers were robust enough in their discriminatory power to be useful for species diagnosis on a glo-bal scale. Preliminary testing of dried fins from Asian and Mediterranean commercial markets suggests thatour genetic approach will be useful for determining the species of origin of detached fins, thus allowing themonitoring of trade in shark fins for conservation assessment. Our approach will also facilitate detection ofproducts from protected and other at-risk shark species and may prove useful as a model for development ofthe high-throughput, genetic, species-diagnosis methods typically required in conservation and managementcontexts.

Identificación Genética de las Partes del Cuerpo de un Tiburón Pelágico para la Conservación y Monitoreo de suComercialización

Resumen:

La conservación y manejo de tiburones fundamentado a nivel de especie es una necesidad imper-ativa debido a la creciente demanda de aletas de tiburón y el reconocimiento de que las especies individualesde tiburones responden de manera distinta a la explotación. Las dificultades para la identificación de mu-chos tiburones capturados comúnmente, así como de partes de su cuerpo, han resultado en una escasez glo-bal de información sobre capturas y comercialización, haciendo casi imposible el poder realizar evalua-ciones de los efectos de la explotación y de las necesidades de conservación. Desarrollamos y evaluamos unmétodo altamente estilizado de genética molecular basado en detonadores de la reacción en cadena de lapolimerasa, especie-específicos, en un formato múltiple de ocho detonadores para discriminar simultánea-mente entre las partes del cuerpo de seis especies de tiburones provenientes de pesquerías pelágicas mundi-

Paper submitted April 19, 2001; revised manuscript accepted September 5, 2001.

Conservation BiologyVolume 16, No. 4, August 2002

Shivji et al. Genetic Identification of Pelagic Sharks

1037

ales comunes. Los detonadores especie-específicos están basados en diferencias en las secuencias de ADN en-tre especies del locus espaciador 2 nuclear, ribosomal, transcrito. Los detonadores y el formato múltipledistinguen muestras con precisión y sensitividad de cada uno de los tres lámnidos (

Isurus oxyrinchus

,

Isuruspaucus

y

Lamna nasus

) y tres especies de carcarínidos (

Prionace glauca

,


y

Carcharhinusfalciformis

) especies todas encontradas en las pesquerías de Norteamérica, excepto una. Mas aún, los detona-dores de los tres lamnidos fueron lo suficientemente robustos en su poder discriminante como para ser usa-dos para el diagnóstico de especies a escala mundial. Las pruebas preliminares de aletas secas de los merca-dos comerciales de Asia y el Mediterráneo sugieren que nuestro método genético puede ser útil paradeterminar la especie de origen de las aletas separadas, permitiendo así usar el monitoreo de las aletas de ti-burón para evaluaciones de conservación. Nuestro método también podría facilitar la detección de produc-tos provenientes de especies protegidas o en riesgo y podría resultar útil como un modelo para el desarrollode métodos genéticos de alto rendimiento para el diagnóstico de especies, métodos típicamente requeridos en

los contextos de conservación y manejo.

Introduction

Conservation and effective management of the world’sexploited shark populations have become issues of con-siderable concern on an international scale as a result ofgreatly expanded commercial fishing efforts over thepast two decades (Bonfil 1994; Weber & Fordham 1997;Food and Agriculture Organization [FAO] 1998, 2000;National Marine Fisheries Service [NMFS] 2001). The sta-tus of many of the world’s shark populations is poorlyknown, hampering the development and implementa-tion of appropriate conservation measures aimed at sus-taining populations over the long term. This lack ofknowledge stems from several political and biologicalfactors, including traditionally low levels of managementinterest and research funding, the general public’s mis-conception of sharks as man-eaters and therefore not de-serving of conservation, and logistical difficulties associ-ated with studying these animals. The population statusof pelagic sharks—defined here as species most com-monly, but not exclusively, encountered in offshore andoceanic habitats—in particular is extremely poorlyknown. This is likely due, in addition to the factors iden-tified above, to the offshore habitat of these species,their typically highly mobile nature resulting in the rou-tine crossing of political boundaries, and what appear tobe complex life-history and migratory characteristics.

The recent international attention being directed atshark conservation and management stems from the re-alization that sharks, with life-history characteristicsmore similar to those of mammals (e.g., slow growth,late reproductive maturity, relatively few young) than ofteleost fishes, are unlikely to respond well to the ex-panded fishing pressure they are experiencing ( FAO1998, 2000). The increased exploitation of sharks overthe past decade is reflected in the escalating quantitiesof shark fins exported to Asian markets to feed the grow-ing demand for shark-fin soup, a delicacy served whenentertaining or celebrating with guests. The muchhigher price garnered by fins than by the flesh of most

shark species has resulted in an escalation by several na-tions in the practice of “finning,” whereby only the finsfrom a shark are kept for market and the rest of the ani-mal is discarded at sea (Rose 1996; Camhi 1999). Pelagicsharks, which are caught in directed fisheries and as in-cidental catch in offshore long-line fisheries targetingtuna and billfishes, are now commonly exploited fortheir fins worldwide ( Rose 1996; Weber & Fordham1997).

In response to concerns about the sustainable healthof shark populations on a global scale, the United Na-tions Food and Agriculture Organization (FAO) devel-oped an International Plan of Action for the Conserva-tion and Management of Sharks that calls for all membernations participating in shark fisheries to develop andimplement their own national plans of action aimed atensuring the conservation and management of sharkstocks (FAO 1998). To comply with the FAO plan direc-tive, the United States has recently developed its ownNational Plan of Action for the Conservation and Man-agement of Sharks (National Marine Fisheries [NMFS]2001).

A major and recurring recommendation in both theFAO and U.S. plans is the collection of data on sharkcatch, landings, and the assessment of shark stocks on aspecies-specific basis ( FAO 1998, 2000; NMFS 2001).This recommendation has its roots in (1) the historicalabsence of reliable data on shark catch and trade on aspecies-specific basis, making robust stock assessmentsand identification of overfished and potentially threat-ened populations and species nearly impossible in mostcases and (2) the recognition that individual species dif-fer in their life-history characteristics and therefore intheir susceptibility to exploitation (Bonfil 1994; Smith etal. 1998; Castro et al. 1999).

A major obstacle to obtaining data on shark catch andtrade and implementating conservation and manage-ment plans on a species-specific basis, however, is thedifficulty of accurately identifying many commonly ex-ploited species. Shark species within the economically

1038

Genetic Identification of Pelagic Sharks Shivji et al.


important families Carcharhinidae and Lamnidae (here-after referred to as carcharhinids and lamnids, respec-tively), for example, can differ only slightly in their mor-phology and are often confused with each other (Castro1993; Bonfil 1994; Castro et al. 1999). The species-iden-tification problem is exacerbated by the practice wide-spread in commercial fisheries of removing the head,tail, and most fins from the landed sharks while at sea tominimize storage needs and prevent spoiling. This re-moval of the primary species-distinguishing characteris-tics leaves shark carcasses ( known as “logs”) that areoften challenging for fishery management and enforce-ment personnel to identify accurately.

Species-specific conservation and management diffi-culties have been further compounded by the rapidlyexpanding trade in shark fins. Accurate identification ofspecies based on morphological characteristics of de-tached shark fins is difficult in most cases (a possible ex-ception is blue shark [

Prionace glauca

] fins [S.C., per-sonal observations]) ( Vannuccini 1999), and onlypreliminary morphological keys exist (Nakano & Kita-mura 1998; Anonymous 1999). These species-identifica-tion difficulties have been a major factor contributing tothe worldwide scarcity of species-specific records onshark catch and trade (Bonfil 1994; Castro et al. 1999).There is a pressing need to solve this problem so thateffective conservation and management of sharks canproceed on a species-specific basis (FAO 1998; NMFS2001).

The DNA–based methods for species identification, al-though widely and routinely employed in biomedicaland agricultural practices (Clapp 1993; Hendolin et al.2000; Zarlenga et al. 2000), have not been implementedwidely in fish management and conservation. This ispartly due to the perception that DNA methods, al-though accurate, are slow and expensive and thereforeunlikely to be suitable in monitoring and enforcementcontexts where large numbers of samples need to bescreened relatively quickly. Routine adoption of DNAidentification methods for fish conservation and man-agement therefore awaits the availability of simpler,quicker and more easily implemented approaches.

For sharks, the genetic identification methods devel-oped thus far have been based on conventional polymer-ase-chain reaction (PCR) amplification of mitochondrialor nuclear loci, followed by subsequent restriction-endo-nuclease digestion of the amplicons to yield species-diag-nostic restriction-fragment-length-polymorphism patterns( Martin 1993; Shivji et al. 1996; Heist & Gold 1999).These methods, although effective for species identifica-tion, require multiple steps (PCR followed by restriction-endonuclease digestion with multiple enzymes) and aretherefore relatively slow, labor intensive, and expensive.

We present an alternative, accurate, sensitive, quick,and easy-to-implement genetic approach for identifica-tion of pelagic shark species. The approach is highly

streamlined and therefore likely to be amenable to rou-tine use in conservation and management contexts. Spe-cies diagnosis is achieved by PCR only, without the needfor further manipulation of the amplicons by restriction-endonuclease digestion, sequencing, or DNA hybridiza-tion steps. Our approach relies on the use of multiplespecies-specific PCR primers that can be used in a sin-gle-reaction-tube, multiplex setting to simultaneouslydiscriminate between six shark species (three lamnids:porbeagle (

Lamna nasus

), shortfin mako (

Isurus ox-yrinchus

), and longfin mako (

Isurus paucus

); and threecarcharhinids: blue (

Prionace glauca

), dusky (

Carcha-rhinus obscurus

) and silky (


)commonly encountered in North Atlantic and world-wide pelagic fisheries. These six species are also fre-quently utilized in the global fin market (Shivji & Clarke,unpublished data).

The U.S. National Marine Fisheries Service (NMFS) hasrecently prohibited landings of dusky and longfin makosharks from the U.S. Atlantic fishery (NMFS 1999). Thedusky shark has also been categorized as vulnerable toextinction in North America by the American FisheriesSociety (Musick et al. 2000) and, depending on the pop-ulation, is in the vulnerable or lower risk–near threat-ened category of the World Conservation Union’s (IUCN )2000 Red List of Threatened Animals (www.redlist.org).All six shark species are widely distributed globally(Compagno 1984). For five of the six species (porbeaglesamples outside the northwest Atlantic were unavailablefor testing), our PCR primers may be suitable for identifi-cation of animals from globally distributed populations.

Methods

Sources of Shark Tissue Samples

We collected tissue samples (reference samples) usedfor DNA sequencing and species-specific PCR primer de-sign during fishery-independent surveys of shark popula-tion abundance and tagging conducted by the NMFS offthe U.S. Atlantic coast. Shark tissue samples (test sam-ples) that we used for testing the diagnostic utility of ourspecies-specific primers were collected from the Atlan-tic, Mediterranean, and Pacific by us or other shark ex-perts who identified the whole animals. Table 1 containsthe shark reference and test species analyzed and theirgeographic origins and sample sizes. Dried shark finswere obtained by us from the Hong Kong fin market andfrom the FAO as part of a separate study.

The reference samples we used for DNA sequencingconsisted of white muscle. Test samples were fin and mus-cle tissues. All tissues, with the exception of dried fins,were kept in SED preservative (saturated NaCl, EDTA,DMSO; Seutin & White 1991) or 95% ethanol at room tem-



1039

perature for short-term storage or at 4

�

C for long-termstorage. Dried fins were stored at room temperature.

DNA Extraction, PCR Amplification, and DNA Sequencing

Genomic DNA was extracted from 25 mg of tissue withthe QIAmp Tissue Kit (QIAGEN Inc., Valencia, California)and stored at

�

20

�

C until use. A PCR fragment (hereafterreferred to as a positive control amplicon) of approxi-mately 1350 bp from the lamnid sharks ( longfin mako,shortfin mako, porbeagle) and 1470 bp from the car-charhinid sharks (blue, dusky, silky) containing the en-tire nuclear ribosomal DNA internal transcribed spacer(ITS2) region plus portions of the flanking 5.8S (approx-imately 160 bp) and 28S (approximately 60 bp) riboso-mal RNA genes were amplified from the six shark spe-cies by standard PCR, employing the shark universalprimers FISH5.8SF (forward primer 5

�

-TTAGCGGTGGATCACTCGGCTCGT-3

�

) and FISH28SR (reverse primer5

�

-TCCTCCGCTTAGTAATATGCTTAAATTCAGC-3

�

).Following PCR amplification, both strands of the posi-tive control amplicon were sequenced according tostandard protocols on an automated sequencer, with in-ternal sequencing primers designed as necessary. Refer-ence sequences for this locus for the six species weredeposited in GenBank under the following accessionnumbers: porbeagle, AF515444; shortfin mako, AF515442;longfin mako, AF515443; blue, AF515441; silky, AF513986;and dusky, AY033819.

Design of Species-Specific Primers and the MultiplexPCR Assay

We aligned the ITS2 sequences obtained from referenceshark samples with the alignment program ClustalX(Thompson et al. 1997) and manually adjusted the align-

Table 1. Shark reference and test species tested using the six species-specific primers in triplex and octaplex-PCR formats,and their geographic origins.

SpeciesGeographicorigin (

n

)

*

Reference samplesIsurus paucus

(longfin mako) NW Atlantic (1)

Isurus oxyrinchus

(shortfin mako) NW Atlantic (1)

Lamna nasus

(porbeagle) NW Atlantic (1)


(dusky) NW Atlantic (1)


(silky) NW Atlantic (1)

Prionace glauca

(blue) NW Atlantic (1)

Test samples

longfin mako NW Atlantic (5)longfin mako W Pacific (3)shortfin mako NW Atlantic (8)shortfin mako NE Pacific (3)shortfin mako W Pacific (9)porbeagle NW Atlantic (17)dusky NW Atlantic (12)dusky SW Pacific (3)dusky W Pacific (6)silky NW Atlantic (13)silky W Pacific (8)blue NW Atlantic (8)blue Mediterranean (24)blue NE Pacific (6)blue W Pacific (5)blue SW Pacific (3)

Alopias vulpinus

(thresher) NW Atlantic (6)

Alopias superciliosus

(bigeye thresher) NE Pacific (1)W Pacific (6)

Alopias pelagicus

(pelagic thresher) NE Pacific (2)W Pacific (2)

Carcharodon carcharias

(white) NW Atlantic (4)NE Pacific (2)Indian Ocean (4)

Lamna ditropis

(salmon) NE Pacific (17)

Carcharias taurus

(sandtiger) NW Atlantic (3)

Carcharhinus altimus

(bignose) NW Atlantic (5)

Carcharhinus longimanus

(oceanic whitetip) NW Atlantic (1)

NE Pacific (2)W Pacific (3)

Carcharhinus signatus

(night) NW Atlantic (2)

Carcharhinus plumbeus

(sandbar) NW Atlantic (5)Central Pacific (2)

Carcharhinus limbatus

(blacktip) NW Atlantic (8)

Carcharhinus leucas

(bull) NW Atlantic (6)

Carcharhinus brevipinna

(spinner) NW Atlantic (5)W Pacific (3)

Carcharhinus isodon

(finetooth) NW Atlantic (5)

Carcharhinus acronotus

(blacknose) NW Atlantic (6)

Carcharhinus perezi

(caribbean reef) NW Atlantic (3)SW Atlantic (3)

Carcharhinus amboinensis

( java) SW Pacific (2)

Carcharhinus tilstoni

(australian blacktip) SW Pacific (2)

Carcharhinus sorrah

(spot-tail) SW Pacific (2)

Carcharhinus amblyrhynchos

(grey reef) Central Pacific (2)

Negaprion brevirostris

(lemon) NW Atlantic (2)SW Atlantic (2)

Continued

Table 1. Continued

SpeciesGeographicorigin (

n

)

*

Negaprion acutidens

(sicklefin lemon) SW Pacific (2)

Galeocerdo cuvier

(tiger) NW Atlantic (3)W Pacific (3)

Triaenodon obesus

(whitetip reef ) Central Pacific (2)

Rhizoprionodon terranovae

(atlantic sharpnose) NW Atlantic (6)

Sphyrna mokkaran

(great hammerhead) NW Atlantic (3)

SW Pacific (1)

Sphyrna lewini

(scalloped hammerhead) NW Atlantic (3)

W Pacific (3)

Sphyrna zygaena

(smooth hammerhead) NW Atlantic (2)

NE Pacific (3)W Pacific (3)

*

Number of animals tested from each geographic location.

1040



ment using the sequence-editing program GeneDoc(Nicholas & Nicholas 1997).

Five to seven PCR primers putatively specific for eachof the six pelagic species were designed based on nucle-otide-sequence differences in the ITS2. Each primer wasfirst tested individually on geographically widespreadsamples (from both Atlantic and Pacific Oceans whenpossible; Table 1) of the species for which the primerwas designed (hereafter referred to as the target spe-cies). To test each primer, we used a combination ofthree primers simultaneously—one putatively species-specific primer plus the shark forward and reverse uni-versal primers—in multiplex (triplex) PCR reactions( Fig. 1). Our a priori measure of primer success in spe-cies identification was the expectation that a three-primer, multiplex combination containing, for example,the primer for shortfin mako would produce two ampli-cons when used to amplify the target-species (i.e., short-fin mako) genomic DNA: (1) a 1350-bp positive-controlamplicon generated from the two shark universal prim-ers and (2) a smaller 771-bp amplicon diagnostic forshortfin mako generated from the forward primer spe-cific to the shortfin mako and the shark universal re-verse primer (Fig. 1). In contrast, this combination ofprimers, when tested against genomic DNA from anyother (nontarget) shark species would produce only thepositive-control amplicon because of failure of theprimer specific to shortfin mako to anneal to DNAs fromthese other species. Analogous test results would be ex-pected for the other five shark species when a three-primer combination is used that includes a different spe-cies-specific primer.

Amplifications were initially performed in a Master-cycler Gradient (Eppendorf Inc., Westbury, New York)thermal cycler with the temperature gradient option todetermine the most stringent annealing temperature(range used: 55

�

–68

�

C) that could be used for each pu-tatively species-specific primer to amplify its target spe-

cies. Total amplification-reaction volumes were 50

�

Land contained 1

�

L of the extracted genomic DNA, 12.5pmol of each primer, 1X PCR buffer (QIAGEN Inc.), 40

�

M dNTPs, and 1 unit of HotStar Taq DNA Polymerase(QIAGEN Inc.). The PCR thermal cycling profile weused was 94

�

C initial heating for 15 minutes to activatethe DNA polymerase, followed by 35 cycles of 94

�

C for1 minute, 58

�

–68

�

C (gradient) for 1 minute, 72

�

C for 2minutes, and a 5-minute final extension step at 72

�

C.Completed reactions were kept at 4

�

or

�

20

�

C untilchecked by gel electrophoresis on 1.2% agarose gels.Each primer that successfully amplified its target speciesat an annealing temperature of 65

�

C or higher was sub-sequently tested at 65

�

C (all other PCR conditions as de-scribed above) for its amplification performance againstvarious nontarget shark species typically or occasionallyencountered in the U.S. Atlantic pelagic fishery. Forthose nontarget species that are globally distributed,these primers were also tested on Pacific representativeswhere available (Table 1) to verify that potential popula-tion-level sequence polymorphisms in the ITS2 locuswould not reduce the diagnostic performance of theprimers. To further evaluate the discriminatory power ofthe three carcharhinid primers in worldwide pelagic-shark fisheries, they were also tested against closely re-lated, nontarget carcharhinid species endemic to theIndo-Pacific, when samples were available ( Table 1).These amplifications were performed in MastercyclerGradient (gradient option off) or MJ Research PTC-100(MJ Research, Inc., Waltham, Massachusetts) thermal cy-clers.

From these amplification tests, we selected the one“optimized” primer for each of the six pelagic speciesthat demonstrated the best performance in the triplexPCR assay according to the following criteria: (1) theprimer demonstrated the greatest species specificity inits amplification behavior at the high-stringency anneal-ing temperature of 65

�

C and (2) the primer produced a

Figure 1. (a) Schematic represen-tation of the shark nuclear 5.8S and 28S ribosomal RNA genes and ITS2 locus showing relative an-nealing sites and orientation of primers used in the triplex–PCR as-says. The shark universal primers (FISH5.8SF and FISH28SR) are shown as solid arrows. The short-fin mako shark primer (open ar-row) is an example of a species-spe-cific primer. (b) Spatial coverage of the two amplicons expected to be produced using this combina-tion of three primers when tested against shortfin mako DNA.



1041

species-specific amplicon that was diagnostic in size foreach of the six species.

To further streamline the species-identification assay,these optimized primers were evaluated for their diag-nostic performance in a more extensive multiplex (octa-plex) PCR reaction that used all eight primers simulta-neously (i.e., the two-shark, universal forward andreverse primers and all six species-specific primers) in asingle-tube amplification reaction. Shark test samples an-alyzed (target and nontarget species) were the same asabove (Table 1). The PCR conditions for the octaplex re-actions were as described above, with the followingmodifications: annealing temperature was 65

�

C for allspecies, and HotStar Taq DNA polymerase was used intwo amounts, 0.5 and 1 unit per reaction, in separate tri-als to assess the effect of the amount of DNA polymeraseon the performance of the eight-primer octaplex assay.

To test the primers and multiplex approach for theirutility in amplifying DNA from dried fins obtained fromthe Hong Kong market, we used the octaplex assay on12 fins designated by the fin traders as blue shark, 8 finsdesignated as “mako” shark (species not identified), and55 fins designated as silky shark. The 31 dried fin sam-ples obtained from the FAO were identified as part of ablind test by the octaplex assay, with the exception thatthe primer for longfin mako was replaced by a primerfor the thresher shark (

Alopias vulpinus

; primer detailsto be published separately) because we suspected thepresence of thresher shark fins in the samples based onthe Mediterranean location of the fin fishery.

Results

Testing each Species-Specific Primer in the Three-Primer Multiplex Assay

The ITS2 loci in the three lamnid and three carcharhinidsharks we sequenced ranged from 1123 to 1136 bp and1233 to 1269 bp in size, respectively. Sequence diver-gence (p-distance, expressed as absolute percent differ-

ence between sequences) between each pair of speciesranged from 11% to 15% among the lamnids, 9% to 13%among the carcharhinids, and 55% to 58% between thelamnids and carcharhinids. Annealing sites for each ofthe six optimized species-specific PCR primers were in-ternal to the forward and reverse shark universal prim-ers (Fig. 2).

In the three-primer triplex assays, each species-spe-cific primer demonstrated complete species specificity( porbeagle, shortfin mako, longfin mako, silky, blueprimers) or nearly complete species specificity (duskyprimer, see below) species specificity when testedagainst target and nontarget species ( Fig. 3). The se-quence of each species-specific primer is given in Table2. Each of the six species-specific primers amplified a di-agnostic-sized amplicon from its target species (Fig. 3).As expected, the coamplification of a positive controlamplicon occurred in all cases in longfin mako, shortfinmako, porbeagle, and dusky sharks. Co-amplification ofthe positive control amplicon from blue and silky sharkswas inconsistent, however, typically occurring in loweryields, when the blue and silky primers were included inthe PCR (see discussion section on the ramifications ofthis inconsistency). In the case of all nontarget species,only the positive control amplicon was amplified (Fig. 3;remaining gels not shown). The only exception to thecomplete species specificity of each primer was ob-served with the dusky primer, which amplified bothdusky and oceanic whitetip DNA.

Testing the Eight-Primer Octaplex Assay

Combining the six pelagic shark primers with the twoshark universal primers in an eight-primer, single-tubeamplification reaction at 65

�

C annealing temperatureconsistently yielded a species-diagnostic-sized ampliconfrom geographically widespread samples of each ofthe six pelagic species (Fig. 4). Each amplicon had a size(Table 2; size inferred from the ITS2 sequences of refer-ence animals) easily distinguishable from that of theother amplicons on a 1.2% agarose gel.

Figure 2. Schematic representa-tion of the shark nuclear 5.8S and 28S ribosomal RNA genes and ITS2 locus showing relative annealing sites and orientation of primers used in the octaplex–PCR assays. Shark universal primers (FISH5.8SF and FISH28SR) are shown as solid arrows. The six spe-cies-specific primers are shown as open arrows. Abbreviations: SF Mako, shortfin mako; LF Mako, longfin mako.

1042



The amount of DNA polymerase used in the reactionhad a clear influence on the amplification yield of thepositive control amplicon. In the presence of target spe-cies, 0.5 units of enzyme per octaplex–PCR reaction pro-duced consistent and clearly recognizable amplificationof the species-diagnostic amplicon, but usually no ampli-fication of the positive control amplicon (gels not shown).Increasing the amount of DNA polymerase to one unit

per reaction produced both the species-diagnostic andpositive control amplicons from target species in thecase of longfin mako, shortfin mako, porbeagle, anddusky sharks. As with the triplex assay, however, coam-plification of the positive-control amplicon was inconsis-tent in the case of blue and silky sharks, usually occur-ring in low yields (Fig. 4). With nontarget species, onlythe positive-control amplicon was amplified in the octa-

Figure 3. Results of amplification reactions with the three-primer multiplex combination of two shark universal primers and one species-specific primer. (a) Testing specific primers for longfin mako and dusky sharks against their target species (lanes 1–3 and 9–11, respectively) and nontarget species (lanes 4–8 and 12–16, respectively). Geographic origin of the target animals: At, Atlantic; Pa, Pacific. Nontarget species: lane 4, porbeagle; 5, shortfin mako; 6, silky; 7, dusky; 8, blue; 12, porbeagle; 13, shortfin mako; 14, longfin mako; 15, silky; 16, blue. Arrows la-beled Lm and Dk show the longfin mako and dusky species-specific amplicons, respectively. Arrow labeled � indi-cates the positive control amplicon. Lanes labeled N contain the negative control reactions (no shark DNA). Lanes labeled M contain the molecular size standard (Gibco-Life Technologies 123 bp ladder standard). Sizes (bp) of in-dividual molecular size-standard bands spanning the species-specific and positive control amplicons are indicated to the right of the gel picture. (b) Testing of specific primers for porbeagle and shortfin mako sharks against their target and nontarget species. Nontarget species: lane 4, longfin mako; 5, shortfin mako; 6, silky; 7, dusky; 8, blue; 12, porbeagle; 13, longfin mako; 14, silky; 15, dusky; 16, blue. Arrows labeled Pb and Sm show the porbeagle and shortfin mako species-specific amplicons, respectively. All other annotation is as in figure A. (c) Testing specific primers for blue and silky sharks against their target and nontarget species. Nontarget species: lane 4, porbeagle; 5, shortfin mako; 6, longfin mako; 7, silky; 8, dusky; 12, porbeagle; 13, shortfin mako; 14, longfin mako; 15, dusky; 16, blue. Arrows labeled Bl and Sk show the blue and silky species-specific amplicons, respectively. All other anno-tation is as in figure A.



1043

plex assay, regardless of the amount of DNA polymeraseused.

Overall, for the sample sizes and geographic originstested here, the octaplex assay proved 100% accurateand sensitive in its ability to discriminate among samplesof known identity of the six pelagic shark species. Nofalse-positive amplifications occurred, with the excep-tion of the dusky primer also amplifying DNA from itscongener, the oceanic whitetip shark.

Testing Dried Shark Fins

The octaplex assay corroborated the Hong Kong trader’sdesignation of blue shark (Ya jian chi) fins in all cases (

n

�

12). Of the eight fins designated as “mako” (Qing lianchi) by the traders, seven fins were genetically identifiedas belonging to shortfin mako. Analysis of the one“mako” fin resulted in amplification of only the positive-control amplicon, indicating that the fin was not from ashortfin or longfin mako or any of the four remainingspecies tested with the octaplex assay. Of the 55 finsdesignated as silky shark (Wu yang chi) by fin traders,45 fins were genetically typed as silky, with the remain-ing 10 fins producing only the positive-control ampli-con. The 31 FAO shark-fin samples were identified with100% accuracy in the blind test ( D. Bartley, personalcommunication), with 24 samples turning out to be bluesharks and 7 thresher sharks.

Figure 4. Results of octaplex–PCR testing on pelagic-shark tar-get and nontarget species. Lanes 1–6 show octaplex–PCR amplifi-cation products from the six tar-get species: 1, longfin mako; 2, dusky; 3, porbeagle; 4, shortfin mako; 5, blue, 6, silky. Geographic origin: At, Atlantic; Pa, Pacific. The species-diagnostic amplicons are indicated by arrows. Lanes 7–13 show octaplex–PCR ampli-fication products from nontarget species: 7, night; 8, bignose; 9, sandbar; 10, tiger; 11, thresher; 12, pelagic thresher; 13, bigeye thresher. The � indicates the positive control amplicons. Lane labeled N contains the negative-control reaction (no shark DNA). Lanes labeled M contain the molecular size-standard (Gibco-Life Technologies 123 bp ladder standard). Sizes (bp) of individual, molecular size-stan-dard bands spanning the spe-cies-diagnostic and positive con-trol amplicons are indicated to the left of the gel picture.

Table 2. Species-specific primer sequences and size of the species-diagnostic amplicon produced.

Shark species Primer sequence Amplicon size (bp)

Longfin mako primer 5�-CCTCAACGACACCCAACGCGTTC-3� 418Dusky primer* 5�-GTGCCTTCCCACCTTTTGGCG-3� 480Porbeagle primer 5�-GTCGTCGGCGCCAGCCTTCTAAC-3� 554Shortfin mako primer 5�-AGGTGCCTGTAGTGCTGGTAGACACA-3� 771Blue primer 5�-AGAAGTGGAGCGACTGTCTTCGCC-3� 929Silky primer 5�-ACCGTGTGGGCCAGGGTC-3� 1085

*Dusky primer sequence reported previously by Pank et al. (2001).

1044 Genetic Identification of Pelagic Sharks Shivji et al.


Discussion

The predominant recent approaches to vertebrate speciesidentification for ecological, conservation, management,and forensic questions involve either PCR amplification ofa specific locus followed by restriction endonucleaseanalysis (the PCR–RFLP approach; e.g., Innes et al. 1998;Heist & Gold 1999; Lindstrom 1999; Asensio et al. 2000;Gharrett et al. 2001) or phylogenetic reconstructions todetermine relationships between DNA sequences fromunknown samples and known species (the phylogeneticapproach; e.g., Baker & Palumbi 1994; Malik et al. 1997;Palumbi & Cipriano 1998; Dizon et al. 2000). Both ap-proaches, although generally robust and effective for spe-cies identification, are relatively time consuming and ex-pensive, requiring downstream analysis of the amplifiedproducts after the PCR step (i.e., restriction endonucleaseanalysis and DNA sequencing, respectively).

We present an alternative, efficient approach to spe-cies identification which requires only PCR withoutadditional downstream manipulation of the amplifiedproducts. Given international concerns about the eco-logical health of exploited shark populations, we dem-onstrate the utility of this method for species identifica-tion of shark body parts, including dried fins.

The ITS2 locus was targeted for species discriminationbased on our observations (Pank et al. 2001; Shivji et al.unpublished data) that this locus is highly conservedwithin shark species, but sufficiently variable even be-tween congeners to provide species-diagnostic nucle-otide polymorphisms. Furthermore, the ITS2 is part of aclass of repetitive elements in eukaryotes (Lewin 2000),thus providing an abundant target for primer annealingand enhancing the efficiency of PCR amplification.

Multiplex Amplification Format

The initial three-primer triplex amplifications (i.e., onespecies-specific and two shark universal primers) werestructured to allow inclusion of an internal positive con-trol in each PCR reaction. The rationale behind this ap-proach is described in detail by Pank et al. (2001). To re-capitulate briefly, the internal positive control wasincluded to prevent the complete absence of any ampli-fication (due, for example, to inhibitory substances inthe starting DNA or to errors in setting up the reaction)from being interpreted as the absence of the target spe-cies (i.e., a false-negative result). Inclusion of an internalpositive control also reduced costs of the assay by cir-cumventing the need for separate positive-control reac-tions aimed at verifying the integrity of the PCR chemi-cal components and DNA quality.

The typically low-yield (or occasionally visually unde-tectable) coamplification of the positive-control ampli-con when silky and blue sharks were the target speciesdeserves comment. We are not certain of the reason for

this occurrence but have also observed it with othershark species when the annealing site of the species-spe-cific forward primer is relatively close to the annealingsite of the shark universal 5.8SF primer (Fig. 2; Pank etal. 2001). We speculate that this phenomenon may re-sult from some form of primer competition, possibly forDNA polymerase binding, between the closely annealedforward primers. This putative “primer-proximity” com-petitive interaction is suggested by the observation thatthe positive control amplicon was amplified efficientlyfrom both silky and blue shark DNA in all instanceswhen their respective species-specific primers were ab-sent from the multiplex reaction (Fig. 3).

This low-yield or inconsistent amplification of the posi-tive control amplicon does not detract from the diagnos-tic utility of the multiplex assay for silky and blue sharks.As indicated above, the internal positive control is in-cluded in the assay only for the purpose of preventingfalse-negative results from being interpreted as the ab-sence of the target species. In fact, its coamplificationalong with the species-diagnostic amplicon is quite un-necessary for species identification because identifica-tion of the unknown sample can be reliably accomplishedsimply by scoring for the appearance of a species-diag-nostic amplicon by itself. Absence of any amplificationsignals a problem with the integrity of the reaction. Inthis context, the internal positive control achieved itsgoal remarkably well in all cases, amplifying robustly inthe absence of the target species (Figs. 3 & 4).

Combining the eight primers in an octaplex–PCR as-say further streamlines the identification process by re-quiring only a single PCR amplification to distinguishamong the six shark species simultaneously, instead ofindividual tests of the unknown samples with each spe-cies-specific primer in separate reactions. For this octa-plex assay to work reliably, the species-specific andshark universal primers had to be designed to functionunder stringent PCR conditions (65� C annealing tem-perature) to (1) prevent or reduce primer-primer anneal-ing when relatively large numbers of primers are mixedtogether and (2) maintain species-specific primer an-nealing to the genomic DNA. The latter is especially im-portant in cases where the sequence of the species-spe-cific primer differs from its orthologous sequence inclosely related, nontarget species by only one or two nu-cleotides (Pank et al. 2001).

A second requirement for the octaplex–PCR assay towork efficiently was that each of the six species-diagnos-tic amplicons needed to be sufficiently different in sizefrom one another and the positive control ampliconneeded to be easily identifiable on an agarose gel. To ac-commodate this requirement, we were careful to designprimers that annealed to the ITS2 locus sufficiently farapart to yield unambiguously scorable bands on visualinspection of the gel (Fig. 4), circumventing the needfor more sophisticated analysis of band size by special-


Shivji et al. Genetic Identification of Pelagic Sharks 1045

ized software. This design provides the advantage of in-creasing speed and simplicity of gel interpretation andtherefore rapidity of the assay.

Applications to Shark Conservation and Management

Our species-specific primers have been developed pri-marily for use in the conservation and management ofmultinational shark fisheries in the North Atlantic. Thesix shark species in this study form a reasonably cohe-sive group in that they are most commonly encounteredoffshore either as bycatch in the tuna and swordfish fish-eries (i.e., shortfin and longfin makos, blue, silky, dusky;Castro 1993; Bonfil 1994; Buencuerpo et al. 1998; Beer-kircher 2000) or in the pelagic, directed fishery (porbea-gle, shortfin mako; NMFS 2001). In an applications con-text, we envision use of this suite of eight primers as afirst attempt to determine the species identity of un-known samples obtained from North Atlantic pelagicfisheries, because there is a high probability that the un-known samples belong to one of these six species.

But we recognize that, depending on the region, otherspecies such as the oceanic whitetip, night, thresher,and bigeye thresher sharks can also be common in theAtlantic pelagic fishery (Castro 1993). Furthermore, be-cause there is overlap in some species’ distributions,several other mostly nearshore shark species are also oc-casionally encountered in offshore fisheries (Castro1993; Bonfil 1994; Beerkircher 2000). In recognition ofthis possibility, we made a considerable effort to de-velop primers for each of the six pelagic species whichare species specific (or nearly species-specific in thecase of the dusky primer) when tested against nontargetspecies that might also be encountered, frequently oroccasionally, in the North Atlantic fishery.

For two reasons, we are confident that the suite of sixprimers we have developed will provide a reliablemeans of accurately identifying longfin mako, shortfinmako, porbeagle, dusky, silky, and blue shark tissues ob-tained from the North Atlantic pelagic fishery. First, wehave verified the species specificity of each primeragainst 21 other shark species that might be encoun-tered in the North Atlantic pelagic fishery. Second, eventhough we have not tested these primers against all pos-sible shark species found in the North Atlantic, it is un-necessary to do so for the following reasons. As non-cod-ing regions, ribosomal ITS loci are typically highlyvariable at the DNA sequence level (Miller et al. 1996;Harris & Crandall 2000). In sharks, the ITS2 is highly di-vergent between taxa above the genus level (Shivji et al.this paper and unpublished data), making it extremelyunlikely that these three lamnid and three carcharhinidprimers will amplify DNA from more distantly relatedshark species, especially at the high PCR stringency con-ditions we used. Closely related nontarget species (allother lamnids and most carcharhinids, including conge-

ners) that occur in the North Atlantic (Compagno 1984)have been tested and, with the one exception (i.e., oce-anic white-tip), do not cross amplify with the six pe-lagic-shark primers. Other nontarget species, including afew carcharhinids not included in the set of speciestested, occupy different habitats and are unlikely to beencountered in the pelagic swordfish-tuna and directedshark fisheries (Buencuerpo et al. 1998; Beerkircher 2000).

The observation that the dusky primer, althoughnearly species-specific, also amplifies its congener, theoceanic whitetip, can lead to some ambiguity in the dis-crimination of samples from these two species becausethe latter is also commonly encountered in the pelagicfishery. This potential ambiguity is ameliorated, how-ever, by the fact that the dusky and oceanic whitetip car-casses are relatively easily distinguished from each otherby morphology, and the oceanic whitetip shark’s large,paddle-like fins with rounded white-tips are unmistak-able (Castro 1993).

Our primers also prove useful for identification ofdried shark fins. Trade in shark fins, although wide-spread, is poorly documented on a species-specific level(Vannuccini 1999). Consequently, reliable quantitativeassessment of the current level and impact of the shark-fin harvest on the status of individual pelagic shark spe-cies is impossible. Our dried-fin test results suggest thatit should be possible to determine the species identity ofthe fins using our methods and to begin to apply themto the problem of monitoring trade in these products.Our preliminary surveys of dried fins in the Hong Kongmarket suggest that, at least for blue, silky, and possiblyshortfin mako sharks, the fin traders we surveyed werereasonably accurate in their assessment of the fins’ spe-cies of origin. Interestingly, 9 of the 10 fins misidentifiedas “silky” came from a single fin trader. The trader andfin sample sizes we have surveyed thus far, however, aretoo small to permit firm conclusions about the large-scale accuracy of identifications made by traders. We arecurrently developing species-specific primers for addi-tional shark species that figure prominently in the globalshark trade (Vannuccini 1999), and we are investigatingthe concordance between fin trader-based (visual) andgenetics-based identifications.

Utility of the Six Pelagic Shark Primers for GlobalScale Surveys

In light of the global distribution of the six pelagic spe-cies, we asked whether these primers would also am-plify their target species from globally widespread areas,including the Atlantic and Indo-Pacific oceans. In thecase of the porbeagle, we were unable to obtain testsamples from outside the northwestern Atlantic andtherefore could not address this issue. For the other fivespecies, however, we had representatives from globallywidespread areas (Table 1), and our results indicate that

1046 Genetic Identification of Pelagic Sharks Shivji et al.


the primers will successfully amplify individuals of thetarget species from as far apart as the western Atlanticand western Pacific ocean basins (Figs. 3 & 4). These re-sults suggest that low intraspecific variation exists in theITS2 locus in globally distributed populations of at leastfive of the six species.

Due to difficulties in obtaining samples from all appro-priate nontarget shark species worldwide, we have beenunable to test every one of the six pelagic-shark primersfor its species specificity against every closely relatednontarget species in the Indo-Pacific. This raises the ques-tion of whether all six primers are as robustly species-specific on a global scale as they are for North Atlanticpopulations. We found the three lamnid-species primersrobustly species specific in tests against other closely re-lated nontarget taxa likely to be encountered in Indo-Pacific pelagic fisheries (i.e., other lamniform sharks:salmon shark, white shark, common thresher, bigeyethresher, and pelagic thresher). Furthermore, the highITS2 sequence divergence between taxa above the ge-nus level and the high stringency PCR conditions usedmakes it extremely unlikely that these lamnid primerswill amplify more distantly related species not alreadytested. Consequently, we suggest that the shortfin andlongfin mako primers will be reliable for identificationof these two species on a global scale. The porbeagleprimer may be useful on a global scale as well. The onlycaveat is that, because we have not tested this primer onporbeagle samples outside the northwestern Atlantic, thereis a small ( but unlikely) possibility that sufficient in-traspecific variation exists in the primer ITS2 annealingsite within this species on a global scale to reduce theprimer’s diagnostic utility outside the northwest Atlantic.

The global-scale utility of the three carcharhinid prim-ers is more uncertain because there are several carchar-hinid species in the Indo-Pacific against which we havenot tested these primers. Despite this uncertainty, fortwo reasons these primers may turn out to be of diag-nostic utility worldwide after all. First, our results showeach of these three carcharhinid primers is species-spe-cific even when tested against 21 other carcharhinidspecies (including 16 congeners from the genus Car-charhinus; Table 1), with only one cross-species amplifi-cation occurring (i.e., dusky primer also amplifying theoceanic whitetip). Although we cannot discount thepossibility of some cross-species amplification occurringon a global scale with the 14 remaining Indo-Pacific Car-charhinus species not tested in this study, the very lowincidence of cross-species amplification observed thusfar even with congeners suggests that these primers willlikely continue to demonstrate species specificity whentested against most, if not all, of the remaining Carcha-rhinus species. Second, in the practical application ofidentification of sharks caught in pelagic fisheries, mostof the carcharhinid species not yet tested are unlikely tobe encountered frequently in these fisheries because

they typically occupy nearshore habitats (Compagno1984). These two factors taken together will reduce thepotential for misidentification of sharks caught in world-wide pelagic fisheries when this suite of primers is used.

Use of the six species-specific primers either individu-ally or in various combinations of one to six primers in amultiplex–PCR format provides an efficient way toachieve accurate and rapid identification of pelagic-shark body parts, including dried fins. Furthermore, thestreamlined octaplex approach may prove useful as amodel for development of rapid species-diagnosis assaysfor a wide diversity of taxa that are hunted or requireconservation action.

Acknowledgments

We thank D. Abercrombie, D. Chapman, and J. Magnus-sen for laboratory assistance. For assistance with sharksample collection we thank D. Abercrombie, L. Beer-kircher, D. Chapman, C. Conrath, K. Duncan, B. Faulkner,K. Goldman, M. Grace, D. Grubbs, E. Heist, D. Holts, R.Martin, A. Pardini, M. Stroud, S. Van Sommeran, B. Weth-erbee, and the Pelagic Shark Research Foundation. Thisresearch was supported by the Wildlife Conservation So-ciety, the David and Lucile Packard Foundation, the HaiStiftung/Shark Foundation, the PADI Foundation Interna-tional, The Florida Sea Grant Program, and the U.S. Na-tional Marine Fisheries Service.

Literature Cited

Anonymous. 1999. Characterization of morphology of shark fin prod-ucts: a guide of the identification of shark fin caught by tuna long-line fishery. Fisheries Agency of Japan, Tokyo.

Asensio, L., I. Gonzalez, A. Fernandez, A. Cespedes, P. Hernandez, T.Garcia, and R. Martin. 2000. Identification of Nile perch (Lates no-liticus), grouper (Epinephelus guaza), and wreck fish (Polyprionamericanus) by polymerase chain reaction-restriction fragmentlength polymorphism of a 12S rRNA gene fragment. Journal ofFood Protection 63:1248–1252.

Baker, C. S., and S. R. Palumbi. 1994. Which whales are hunted? A mo-lecular genetic approach to monitoring whaling. Science 265:1538–1539.

Beerkircher, L. 2000. Elasmobranch bycatch observed on pelagic long-lines off the Southeastern U.S. coast, 1992–1997. Masters thesis.Nova Southeastern University Oceanographic Center, Dania Beach,Florida.

Bonfil, R. 1994. Overview of world elasmobranch fisheries. Fisheriestechnical paper 341. Food and Agriculture Organization, Rome.

Buencuerpo, V., R. Santiago, and J. Moron. 1998. Pelagic sharks associ-ated with the swordfish, Xiphias gladius, fishery in the easternNorth Atlantic Ocean and the Strait of Gibraltar. Fishery Bulletin96:667–685.

Camhi, M. 1999. Sharks on the line. II. An analysis of Pacific state sharkfisheries. Living Oceans Program report. National Audubon Soci-ety, Islip, New York.

Castro, J. I. 1993. A field guide to the sharks commonly caught in com-mercial fisheries of the southeastern United States. Technical mem-


Shivji et al. Genetic Identification of Pelagic Sharks 1047

orandum NMFS-SEFSC-338. National Oceanic and Atmospheric Ad-ministration, Miami.

Castro, J. I., C. M. Woodley, and R. L. Brudeck. 1999. A preliminaryevaluation of the status of shark species. Fisheries technical paper380. Food and Agriculture Organization, Rome.

Clapp, J. P. 1993. Species diagnostic protocols: methods in molecularbiology. Volume 50. Humana Press, Totowa, New Jersey.

Compagno, L. J. V. 1984. FAO species catalog: sharks of the world.Fisheries synopsis 125, Volume 4. Food and Agriculture Organiza-tion, Rome.

Dizon, A., C. S. Baker, F. Cipriano, G. Lento, P. Palsboll, and R. Reeves,editors. 2000. Molecular genetic identification of whales, dolphins,and porpoises: proceedings of a workshop on the forensic use ofmolecular techniques to identify wildlife products in the market-place. Technical memorandum NOAA-TM-NMFS-SWFSC-286. Na-tional Oceanic and Atmospheric Administration, La Jolla, California.

Food and Agriculture Organization (FAO). 1998. International plan ofaction for the conservation and management of sharks. DocumentFI:CSS/98/3. FAO, Rome.

Food and Agriculture Organization (FAO). 2000. Fisheries manage-ment. 1. Conservation and management of sharks. Technical guide-lines for responsible fisheries 4, supplement 1. FAO, Rome.

Gharrett, A. J., A. K. Gray, and J. Heifetz. 2001. Identification of rockfish(Sebastes spp.) by restriction site analysis of the mitochondrial ND-3/ND-4 and 12S/16S rRNA gene regions. Fishery Bulletin 99:49–62.

Harris, D. J., and K. A. Crandall. 2000. Intragenomic variation withinITS1 and ITS2 of freshwater crayfishes (Decapoda: Cambaridae):implications for phylogenetic and microsatellite studies. MolecularBiology & Evolution 17:284–291.

Heist, E. J., and J. R. Gold. 1999. Genetic identification of sharks in theU.S. Atlantic large coastal shark fishery. Fishery Bulletin 97:53–61.

Hendolin, P. H., L. Paulin, and J. Ylikoski. 2000. Clinically applicablemultiplex PCR for four middle ear pathogens. Journal of ClinicalMicrobiology 38:125–132.

Innes, B. H., P. M. Grewe, and R. D. Ward. 1998. PCR-based geneticidentification of marlin and other billfish. Marine & Freshwater Re-search 49:383–388.

Lewin, B. 2000. Genes VII. Oxford University Press, New York.Lindstrom, D. 1999. Molecular species identification of newly hatched

Hawaiian amphidromous gobioid larvae. Marine Biotechnology 1:167–174.

Malik, S., P. J. Wilson, R. J. Smith, D. M. Lavigne, and B. N. White.1997. Pinneped penises in trade: a molecular-genetic investigation.Conservation Biology 11:1365–1374.

Martin, A. P. 1993. Application of mitochondrial DNA sequence analy-sis to the problem of species identification of sharks. Pages 53–59in S. Branstetter, editor. Conservation biology of elasmobranchs.Technical report NMFS-115. National Oceanic and AtmosphericAdministration, Springfield, Virginia.

Miller, B. R., M. B. Crabtree, and H. M. Savage. 1996. Phylogeny offourteen Culex mosquito species, including the Culex pipienscomplex, inferred from the internal transcribed spacers of riboso-mal DNA. Insect Molecular Biology 5:93–107.

Musick, J. A., et al. 2000. Marine, estuarine, and diadromous fish stocksat risk of extinction in North America (exclusive of Pacific salmo-nids). Fisheries 25:6–30.

Nakano, H., and T. Kitamura. 1998. Identification of eleven sharkscaught by tuna long-line using morphological characters of theirfins. Information paper of the FAO Technical Working Group onthe conservation and management of sharks. Food and AgricultureOrganization, Rome.

National Marine Fisheries Service. 1999. Final fishery managementplan for Atlantic tuna, swordfish, and sharks. Highly Migratory Spe-cies Division, Silver Springs, Maryland.

National Marine Fisheries Service. 2001. Final United States nationalplan of action for the conservation and management of sharks.Highly Migratory Species Division, National Marine Fisheries Ser-vice, Silver Spring, Maryland.

Nicholas, K. B., and H. B. Nicholas Jr. 1997. GeneDoc: analysis and vi-sualization of genetic variation. Program available from GeneDochome page http://www.psc.edu/biomed/genedoc/.

Palumbi, S. R., and F. Cipriano. 1998. Species identification using ge-netic tools: the value of nuclear and mitochondrial gene sequencesin whale conservation. Journal of Heredity 89:459–464.

Pank, M., M. Stanhope, L. Natanson, N. Kohler, and M. Shivji. 2001.Rapid and simultaneous identification of body parts from the mor-phologically similar sharks Carcharhinus obscurus and Carcharhi-nus plumbeus (Carcharhinidae) using multiplex PCR. Marine Bio-technology 3:231–240.

Rose, D. 1996. An overview of world trade in sharks and other cartilag-inous fishes. TRAFFIC International, Cambridge, United Kingdom.

Seutin, G., and B. N. White. 1991. Preservation of avian blood and tissuesamples for DNA analysis. Canadian Journal of Zoology 69:82–90.

Shivji, M. S., C. Tagliaro, L. Natanson, N. Kohler, S. Rogers, and M.Stanhope. 1996. Utility of ribosomal DNA ITS2 for deriving sharkspecies–diagnostic identification markers. Pages 87–93 in E. M.Donaldson and D. D. MacKinlay, editors. Proceedings of the inter-national congress on the biology of fishes. American Fisheries Soci-ety, San Francisco, California.

Smith, S. E., D. W. Au, and C. Show. 1998. Intrinsic rebound potentialsof 26 species of Pacific sharks. Marine & Freshwater Research 49:663–678.

Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G.Higgins. 1997. The ClustalX windows interface: flexible strategiesfor multiple sequence alignment aided by quality analysis tools. Nu-cleic Acids Research 24:4876–4882.

Vannuccini, S. 1999. Shark utilization, marketing and trade. Fisheriestechnical paper 389. Food and Agriculture Organization, Rome.

Weber, M. L., and S. V. Fordham. 1997. Managing shark fisheries: op-portunities for international conservation. TRAFFIC Internationaland Center for Marine Conservation Report, Cambridge, UnitedKingdom.

Zarlenga, D. S., M. B. Chute, A. Martin, and C. M. Kapel. 2000. A multi-plex PCR for unequivocal differentiation of all encapsulated andnon-encapsulated genotypes of Trichinella. International Journalfor Parasitology 29:1859–1867.

Genetic Identification of Pelagic Shark Body Parts for ... Event/Resources... · 1036 Conservation...

Documents

Transcript of Genetic Identification of Pelagic Shark Body Parts for ... Event/Resources... · 1036 Conservation...