Danielle Dyson

Dr. Elizabeth Lacey

MARS 2200

6 March 2019

Analysis of DNA barcoding methods in sharks

Introduction

Across the world, shark species wild stocks are in rapid decline with over 100 million
sharks being caught every year. The shark fin market has been very lucrative since its boom in
the 1970s, leading to an increase in catches as well as illegal, unreported, and unregulated (IUU)
shark fishing. Within Asian culture, shark fin is considered a high demand luxury; it acts as a
status symbol, showing wealth and prosperity for individuals. As the middle-class wealth
increases, shark fin is becoming easier for consumers to buy leading to increased demand (Ho
Shea and Wai Lun To 2017).

Increases in fishing pressure are stopping shark species from repopulating. Their late
maturation rate, long gestation period, and slow growth do not allow the species to repopulate
quickly. Moreover, a large number the shark species being caught are either listed as endangered
or threatened. Sharks are important to all marine ecosystems; they act as keystone species
meaning that they are vital to ecosystems and their removal would drastically change the
dynamic of their ecosystems. As more sharks are entering the fin trade, populations are
threatened like never before and are in drastic need of conservation practices (Sebastian et al.
2008).

In 1998, the United Nations Food and Agriculture Organization (FAO) called for any
nations within the shark fin trade to develop and implement their own national plans that would
ensure shark management and conservation. In compliance with this, the United States’ National
Marine Fisheries branch developed a National Plan of Action for the Conservation of Sharks
(Shivji et al. 2001). Both of these plans call for a reliable information about the data on shark
landings, catch data, and the assessment of population biomass.

However, we do not have an accurate assessment of shark population biomass nor do we

know which species are being targeted and sold. Little is known about the sharks that are seized
from catches due to data gaps from misidentification, cultural barriers, and incomplete databases.
Fishermen are entrusted to identify fins morphologically, meaning that simply by looking at a
fin, a fisherman is supposed to be able to recognize its species. However, with shark fins that are
so morphologically similar, this opens the door for misidentification (Sebastian et al. 2008).
 In addition, cultural barriers across the world may lead to fishermen in one area calling a
species by one name, while fishermen across the globe call it something else. Local species
names cause fins from the same species to be logged as two entirely different sharks when in fact
they are part of the same population. Consequently, this leads to several shortcomings within
worldwide databases as fins are not being logged correctly (Sebastian et al 2008).

In order to attempt to solve this issue, various versions of DNA barcoding have arisen.
DNA barcoding starts by obtaining a sample and completing a Polymerase Chain Reaction
(PCR) assay on it. In essence, the DNA within the sample is isolated and copied. The DNA is
then sequenced using primers. There are several methods to which primers are the most accurate
when it comes to sequencing. Universal primers, species specific primers, and COI gene
detection are all different methods that can be used to identify what species of shark a fin has
come from.

Two universal primers have been generated to identify shark species based on their
similarity to the pattern. Concurrently, a method was produced to use species specific primers
that would genetically match the sequenced DNA to a species logged in a database. In addition,
yet another method focuses on the isolation of the COI gene within shark DNA to identify shark
species. This paper will serve as a comparative analysis between these three variations of this
method of identification to assess which method provides the best results.

It is clear that shark populations are in dire need of help as the shark fin trade continues to
grow. If we do not act now to stop the slaughter of endangered sharks, many populations will go
extinct very soon (Ward et al. 2008). It is through the use of these methods that we hope to fix
data gaps caused by current methods of identification, and get reliable data on shark catches and
population biomass so that correct conservation methods can be put into place.

Main Analysis

Current Methods

The current data used to identify shark fins is greatly lacking. The methods being used
are not adequate, especially with new, more accurate technological advances. One major way
that fins are currently identified is morphologically. Fishermen usually just look at the landed
fins and are supposed to be able to correctly identify the species from which it came. However,
many shark fins share common morphology, especially when the body is removed (Figure 1).
Figure 1 represents a field test that was given to fishermen to have them identify. Out of the three
species present, fishermen were able to correctly identify 2 species. However, the third provided
issues for them in which it was correctly identified 50% of the time and incorrectly identified the
other 50% of the time (Sebastian et al. 2008).

In addition to incorrectly identifying shark species, this test revealed another issue with
current identification methods. Fishermen across the world come from different cultural
backgrounds. Because of these cultural differences, a language barrier appears when naming
shark species. For example, while fishermen within the United States would call a Galeocerdo
cuvier a tiger shark, fishermen in Chilean waters would call it a ‘Gatuzo’ (Sebastian et al. 2008).
In addition, misspelling of common and scientific names could lead to further misinterpretation
of species.

Moreover, in Hong Kong, the leading shark fin capitol of the world, fishermen are
expected to label shark fins through codes. Each code stands for a related product that comes
across their boat. The most common shark codes are listed in Table 1 (Ho Shea and Wai Lun To
2017). Each code differs only slightly and can thus be misinterpreted easily. In addition, the final
code in table 1 stands for shark fins not specified elsewhere. This code is dangerous because if
fishermen cannot identify a fin, they can simply place it under this code and the data is not
logged or recorded. Consequently, that sample is lost indefinitely, and the shark it came from is
not included in the catch.

Similarly, in other places across the world scientists rely on fishermen log books.
Fishermen have bound journals in which they record and tally all the sharks that they bring
aboard their ships (Sebastian et al. 2008). However, there are several problems associated with
this method of identification. First, it relies on the fact that the book will not be lost by the
fishermen. Then, it also relies on the log to not be destroyed by the harsh elements of the sea.
Finally, it relies on the fishermen’s competency to spell correctly and write eligibly enough for a
scientist to decipher the information.
 All of these factors come into play when logging each shark species. It is unreliable to
depend on these methods of identification because of the falsified information they provide as
well as the favorable but unrealistic conditions they require. The inconsistent use of species
names and mislabeling directly affect and skew the data, thus making it impossible to get a
reliable assessment of the biomass of shark populations. New data forms are needed to properly
get an assessment of shark population biomass and fill this data gap so that proper conservation
methods may be put into place.

Barcoding Methods

Within the DNA barcoding method approach, there are several different ways to achieve
the end result of the species name. Different variations of the same common method have arisen
and each of them have positives and negatives associated with them. Three of these approaches
include: the use of universal primers, the use of species-specific primers, and isolation of the
COI gene.

All of these methods begin by the same process, a polymerase chain reaction (PCR)
assay. First, a sample is taken from the seized fin. That sample is then heated to open the tissues
and spun within a centrifuge to isolate the DNA. The DNA then undergoes the PCR assay in
which the DNA segment is essentially copied (Holmes et al. 2008). The process then differs
depending on which method is used.

Universal Primers

Under the universal primer method, after the basic PCR assay is conducted, two universal
primers, FishF1 and FishR1, are then added to the DNA to amplify regions of the mitochondrial
genome. If no regions were amplified, then the FishF2 primer may be used to further isolate the
DNA bands of closely related species (Table 2). The primers bind to an inverse DNA strand and
allow for copying of DNA to begin. The bands of DNA then form on an agarose gel and are
sequenced in both directions. The sequenced DNA is then entered into a bootstrapped tree
database that houses a list of the DNA sequences of sharks and their relation to one another
(Holmes et al. 2008).
 To begin with, out of the percentage of fins gathered 8.5% could not be sequenced or
identified due to DNA that had degraded (Holmes et al. 2008). Of the DNA that could be
sequenced, this method yielded results that 61% (118 out of 193 samples) of the time gave a
99%-100% identification record. However, for 40% of the time (75 out of 193 samples) yielded
a 76% identification record value or lower. Broken down even further, 42 out of the 193 samples
had a 76% identification record, 6 out of the 193 samples had a 60% identification record, and 27
out of the 193 samples had an identification record of 49% (Holmes et al. 2008).

In essence, these values mean that correct identification with 99% accuracy could only be
guaranteed 61% of the time when using universal primers. The rest of the time, species could
only be identified at most with a 76% accuracy rate. This poses a problem for this method as
several shark species are very similar genetically. If on a small scale, this method can only
produce a very accurate identification 61% of the time, it may be hard to implement on a world-
wide scale for the fact that it may also lead to misidentification (Holmes et al. 2008).

In addition, in order for this method to work, it must have a very selective environment.
First the DNA must be intact enough to be sequenced using the primers. This means that old or
dried fins could pose several problems when their identification is needed as the DNA within
them is not as intact. Thus, it may prove impossible to identify with 99% accuracy the species
from which an older or dried fin has come.

Furthermore, there must be low levels of intraspecific variation but acceptable levels of
interspecific variation of the DNA. This means that the DNA sample must have low levels of
variation found within a single species group while also having enough variation from other
groups. In essence, the DNA must not be similar between two species such as two species of
hammerhead shark, but also have enough variation from another group such as a tiger shark. All
of these qualifications may be hard to replicate every time, especially with the hybridization of
shark species causing less distinct variation between species (Holmes et al. 2008). This would
prove detrimental to a method that already only has a 61% accuracy of identification as it would
lead to lower identification accuracy. Thus, the conditions needed for this method greatly cause
several drawbacks that may prove to make this method inapplicable on a large-scale system.
 Conversely, there are a few positives associated with this method as well. It would create
a universal standard under which all research could be conducted. In other words, the use of
these two universal primers for all shark identification would mean that across the world
scientists would rely on the same identification method and thus equalize the reliability of the
results. If all scientists used these two primers on DNA samples, they should all receive the same
results and had to have conducted the sequencing under the same specific conditions. This in
turn, would allow for a standard species identification to be made unlike using fishermen to
identify species (Holmes et al. 2008).

Species-Specific Primers

With the use of species-specific primers, after the initial polymerase chain reaction assay,
there is no need for sequencing. Instead, the primers are added to the newly formed Polymerase
Chain Reaction DNA in a single-reaction-tube to test for six common species of sharks. The six
species of sharks included in this database are: porbeagle, shortfin Mako, longfin Mako, blue
shark, dusky shark, and silky shark. All of these species are common in North Atlantic waters
and among the fin market worldwide (Shivji et al. 2002).

Each primer was made from derivations based on nucleotide sequence differences in the
ITS2 gene. The species-specific primer would match up with the DNA of the compatible species.
Once this happened, the fin would be identified and able to be logged into a database to monitor
its population and biomass. Species specific primers used are recorded in Table 3 by representing
the genetic makeup or presence of adenine, thymine, guanine, and cytosine (Shivji 2002).

The primers demonstrated complete species specificity when tested against the target and
non-target species. Figure 2 shows the results of the assay. Within group A, Longfin Mako (Lm)
and Dusky Shark (Dk) were tested against non-target species. In group B, Porbeagle (Pb) and
Shortfin Mako (Sm) were tested against other non-target species. Finally, group C tested Blue
Shark (Bl) and Silky Shark (Sk) against other non-target species. All groups only tested positive
for their specific species and showed no DNA bands for any non-target species. Thus, the
primers were able to identify the correct species without any traces of other similar DNA from
different species. This proves the accuracy of species-specific primers in their targeting abilities
(Shivji et al. 2002).
 In addition, this method offers a cheaper way of preforming the DNA analysis by cutting
out the sequencing process and database use. Scientists do not have to pay to use a national
database or the expensive sequencing step. Moreover, the gene that was isolated, the ITS2, is
incredibly resistant and does not degrade easily. Instead of DNA being too degraded to sequence,
this method would allow the primers to stick to this gene that is highly conserved and could lead
to better results in older fins (Shivji 2002). Thus, the lifespan of the DNA genes within the
species would be prolonged and could be sued to identify older or dried fins.

Although these primers are very effective, there are still some negative aspects associated
with this method. The main problem associated with this method is the lack of species-specific
primers. As of right now, there are only six species-specific primers. Furthermore, all of these
species are not found worldwide (Shivji et al. 2002). For this highly effective testing method to
become commonplace, more species would have to be added. With hundreds of shark species,
there definitely needs to be more than the six albeit common species in the Atlantic.

However, once those primers are added, the process of testing for which primer is the
correct species could potentially increase and make the process longer. Perhaps, the addition of
more species would make the primers too genetically similar and could lead to increased errors
in reading. If it is applied to species that are only contain a one to five difference in base pairs,
then the door for error is significantly opened (Shivji et al. 2002). This problem would thus
downgrade the reliability of this method; however, this problem cannot be proven at this time
and more research would be needed to conclude if it truly would be a large problem.

COI Barcoding

After conducting the Polymerase Chain Reaction, instead of using a primer to bind to
isolate the DNA, a specific gene is isolated. The mitochondrial cytochrome c oxidase 1 gene
(cox1) is sequenced for to make the DNA copies within the reaction. The sequenced DNA is
then entered into the Barcode of Life Database (BOLD) to identify the species. This database is
different from others that may be used in that it houses DNA sequences for thousands of shark
species, and can therefore better match the sequence exactly to a species. It is a large and rapidly
growing resource that can be used to house all of the COI DNA barcoding sequences needed
(Ward et al. 2008).
 The results of this method are 99% accurate. Within a test on 210 species of sharks, all
sharks were identified and matched 99% to a known species sequence within the Barcode of Life
Database (BOLD). This method proved to be the most accurate while also being the most
accessible across the nation. The use of a universal database allows for a standard to be presented
worldwide that way every nation has access to the same records and standards. This in turn can
then be used to log all of the species caught and finned and get a correct population biomass
estimation (Ward et al. 2008).

However, there are a few drawbacks related to COI barcoding. The method itself is very
pricy and may be unaffordable to developing nations that largely partake within the shark fin
trade. Furthermore, the database as of now is managed by the public. Therefore, it could contain
unvalidated results or records that do not reach species level identification (Ward et al. 2008).
This raises the question that if this database is going to be used worldwide for all shark species
identification, should it be managed solely by scientists.

In turn, this then begs the question of which scientists should have access to input
information. In essence, which nations should be able to input data into the BOLD database.
This could potentially pose an issue if a nation wanted to cross check the results of an analysis or
if the nation in charge would be able to falsify statistics for their own benefits. Consequently,
although this method was incredibly accurate in identifying species, several issues regarding the
BOLD database would have to be ironed out before this method was able to be implemented on a
worldwide scale.

Conservation Data

In order for a method to be used readily for conservation data, it needs to come from a
reliable source. By using any DNA barcoding method, the resulting data is more reliable and
comes directly from a scientific point of view. Thus, it can be used to set a standard for
conservation (Ho Shea and Wai Lun To 2017).

The data gained through these barcoding methods would be able to provide scientists
with a more accurate measurement of the population biomass of several shark species. Without
having to rely on inconsistent data, scientists can truly get a measurement of how threatened
shark species are across the world. Within one test, the results supplied data on the species
composition and proportion of several fishing fleets. These fisheries contained no data on any of
this information despite the Chilean prominence within the shark fin trade (Sebastain et al.
2008). Consequently, the information gathered from the barcoding was able to fill a data gap
within the global database quickly and accurately. This data can then in turn be used to apply the
correct conservation methods and legal action required to save these sharks.

In addition, the tests run within these projects all concluded that a large portion of the fins
identified were taken from threatened or endangered species. At least a quarter of all fins
identified in the tests were determined to be of an endangered or threatened species (Ward et al.
2008). These results are inconsistent with the current submitted data for these species across the
world. By gaining access to these more valid results, there is a better handle for scientists on the
exact number of endangered sharks still being used in the shark fin trade (Ho Shea and Wai Lun
To 2017). Consequently, by collecting this data scientists can then make an educated, well
informed decisions on the status of several shark species. Perhaps they could move certain
species from threatened to endangered or vice versa to accurately represent their involvement in
the shark fin trade. Consequently, the use of this data would allow scientists to ban several more
species from the shark fin trade due to unhealthy population biomass.

Moreover, this data provides a location for the illegal fishing. If boat catches were to be
sampled using this technology, then the illegal fishing could be traced to a point of origin. There
would be no guessing about what boat or dock the illegal shark fins came from or where along
the supply chain they were passed on. This, in turn, would allow for more direct consequences
for shark fin suppliers (Sebastian et al. 2008). If they were to be caught with illegal fins aboard,
then they could be fined or have legal action taken against them right away. This would open the
door for stricter regulations on the shark fin trade and allow for more direct and enforced
consequences for catching and selling illegal shark fins.

Consequently, the data gathered through these barcoding methods can be used for
conservation efforts. It provides accurate representations of biomass data and species
identification that can lead to scientists to make informed decisions about the fate of certain
species. In doing so, more laws can be put into place that would ban certain species from being
sold in the shark fin trade. Furthermore, stricter consequences for illegal shark finning could also
be put into place that would deter this from happening. Thus, the use of DNA barcoding would
provide reliable information that could be used to aid in the management and conservation of
shark species across the world.

Conclusion

Current methods have been failing for years and need to be revamped. Relying on
fishermen to identify and log the correct species of shark that they are catching leads to immense
data gaps within the system. DNA barcoding methods aim to fix this data gap by relying on
science to correctly identify the shark species rather than untrained fishermen. When all of the
DNA Barcoding methods are presented and analyzed, they all present both positive and negative
attributes. However, in some of them the positives far outweigh the negatives.

If you assess the universal primers, the conditions under which the primers may work are
hard to come by especially in sharks spawned through hybridization (Holmes et al. 2008). With
species-specific primers, it would take a long period of time to increase the known primers to
accommodate for more species within the populations (Shivji et al. 2002). Moreover, adding
more species to run samples against may cause several unforeseen factors that could decrease the
accuracy of this testing method. Finally, COI barcoding’s reliance upon the Barcode of Life
Database (BOLD) could cause several problems if implemented on a worldwide scale (Ward et
al. 2008).

However, when the options are weighed it is clear that COI barcoding is the best option
for species identification. It correctly identifies shark species to a 99% match and houses the
largest database to scan sequenced DNA into (Ward et al. 2008). In addition, the issue of price
will be addressed as the method becomes more mainstream. As it increases in popularity, the
price will drop to become more manageable for more developing countries to use. Moreover, if
nations agreed to share responsibility of the BOLD database and restrict access to highly
qualified scientists then the threats of misuse of it are eliminated. If all interested nations had one
lab that was granted access to the database, then inputting data would not be a problem across the
world. Those scientists would be able to input their own data and cross check the results of other
scientists to ensure accuracy of DNA sequencing.
 Furthermore, if this method is put into place, there can be strong evidence as to which
species of shark need urgent help to save their populations. With a 99% accuracy rate, the results
of these test will almost always yield the correct results, thus ensuring reliable data. As this data
improves from use of COI barcoding, new biomass charts can be drawn up and conservation
efforts can be put into place (Ward et al. 2008). With these new efforts perhaps, the species that
are endangered can have the chance to come back. However, this can only happen if our data
records improve greatly, as they would through the use of COI barcoding.
 Figure 1: Pictures of different shark species

Code as of 2006 Code as of 2012 Product Description

16042011 - Shark fins, prepared or
preserved, canned
16042091 - Shark fins, prepared or
preserved, not canned
- 305711 Shark fins, dried, with
cartilage
- 3057112 Shark fins, dried, without
cartilage
- 3057122 Shark fins, not dried,
without cartilage
- 03028100 Dogfish and other sharks,
fresh or chilled
 - 3038100 Dogfish and other sharks,
frozen
- 03057190 Shark fins not elsewhere
specified of included

Table 1: Common Shark Fin Codes

Primers DNA Sequence (5’-3’)

FishF1 (fwd) TCA ACC AAC CAC AAA GAC ATT

GGC AC
FishF2 (fwd) TCG ACT AAT CAT AAA GAT ATC GGC
AC
FishR1 (fwd) TAG ACT TCT GGG TGG CCA AAG
AAT CA

Table 2: Sequences of universal primers.

Shark Species Primer Sequence 5’- 3’

Longfin mako primer CCT CAA CGA CAC CCA ACG CGT TC

Dusky primer GTG CCT TCC CAC CTT TTG GCG

Porbeagle primer GTC GTC GGC GCC AGC CTT CTA AC

Shortfin mako primer AGG TGC CTG TAG TGC TGG TAG ACA CA

Blue primer AGA AGT GGA GCG ACT GTC TTC GCC

Silky primer ACC GTG TGG GCC AGG GTC

Table 3: Species-Specific Primers

Figure 2: Results of Species-Specific Assay
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