Luke Bassett
Biology 303
10/26/14
“Population structure of three shark species and implications for conservation”
The clade Selachimorpha includes over 470 species of sharks that are distributed worldwide. Despite vast differences in morphology and behavior, many of these species share the
threat of extinction due to anthropogenic pressure. Humans affect shark populations through
habitat loss due to development and pollution, climate change, and the overfishing of their prey
species. However, the most significant threat to sharks is their direct fishing and fining (Freund,
2014). Overfishing had pushed many species towards the verge of extinction and threatens to
drive other species down a similar path. Sharks are essential predators in ecosystems worldwide
and exert top down control on many marine organisms. This predation results in an overall
benefit to prey populations as the more competitive members live on to reproduce. Shark
predation also prevents the over consumption of lower trophic level organisms (SharkSavers.org,
2014). For example, by consuming vast quantities of zooplankton, whale sharks ensure the
phytoplankton is not overly grazed upon. Due to these traits, sharks are recognized as keystone
species as well as ecosystem engineers. The importance of sharks has encouraged conservation
efforts worldwide as well as increased shark fisheries management.
In order to better understand species’ susceptibility to threat and extinction, the use of
genetic techniques has been implemented in order to study their gene flow, population structure,
and mating structure. Many species are extremely transient, so observation based behavior
studies often prove ineffective. Instead, the collection of genetic samples and the examination of
mtDNA and microsatellite DNA allows researchers to infer life history dynamics and prescribe
methods for conservation and management. Microsatellite DNA and mtDNA from neonates
(newly born sharks) and juveniles can even be used to recreate adult genotypes as well as infer
population dynamics of these adults.
Perhaps one of the widest spread and commercially important species is the blacktip
shark. Due to slow growth, low fecundity, and limited recruitment rates, black tip shark
populations are at risk of collapse due to overfishing. Although highly mobile, female blacktip
sharks often travel to nursery locations where they give birth to juveniles who will remain there
until water temperatures drop below 20oC (Keeney et al. 2005). The mobile nature of blacktip
sharks has led to the assumption that populations likely lack genetic structure. However, a
previous analysis of mitochondrial DNA by Keeney et al. (2003) suggests haplotype frequency
differences between neonates from nurseries on the Gulf Coast of Florida and the Atlantic coast
of South Carolina. The cause of these findings was interpreted as female philopatry to nursery
sites. Philopatry is the behavior of returning to the same home, mating, or nursing site. Keeney et
al. (2005) investigated this assumption by using mtDNA control region sequences and eight
nuclear microsatellite loci to further examine the genetic structure of black tip shark nurseries in
the northwestern Atlantic Ocean, Gulf of Mexico, and Caribbean Sea. Variation or heterogeneity
in these markers would indicate overall philopatry while homogeneity in the microsatellite loci
and heterogeneity in the mtDNA would indicate only female philopatry.
Neonate and young of year (healed umbilical scar) juvenile samples were collected from
one South Carolina nursery, three Florida Gulf Coast nurseries, one Texas Gulf Coast Nursery,
and one nursery off the coast of Quintna Roo, Mexico. Keeney et al. (2005) also collected sharks
from the Atlantic coast of Georgia and fish markets on the Caribbean Sea coast of Belize. Live
sharks were captured using gillnets and rods. The tip of the dorsal fin was then be removed and
used to isolate genomic DNA from the tissue. The mtDNA control regions and the microsateillte
loci were then amplified via PCR and sequenced. The control sequences were then read and
aligned using the CLUSTALW program while Arlequin 2.0 was used to determine the number of
unique haplotypes, haplotype frequency, the number of polymorphic sites, transitions and
transversions, nucleotide composition, haplotype diversity, and nucleotide sequence diversity.
An analysis of molecular variance was also conducted. GENEPOP was used to determine the
number of genes per locus, expected and observed heterozygotes, deviations from Hardy
Weinberg expectations for each locus in which nursery, and tests of linkage disequilibrium.
Wright’s f statistics were calculated using Amova (Keeney et al. 2005).
Figure 1. Neighbor joining
trees from genetic distance for
mtDNA control region
sequences and eight
microsatellite loci. (Keeney et.
al., 2005)
Table 1. Pairwise regional
AMOVA estimations for
control region sequences and
microsatellites. (Keeney et al.,
2005)
In comparisons between regions, genetic structure was detected among the nurseries with
mtDNA and microsatellites. The significant amount of mtDNA structure indicated that females
use their natal nurseries for parturition. Homogeneity between the years 2000 and 2001 supports
the assertion that genetic structure reflects geographical differences between the nurseries. This
along with observations in past studies provides further evidence for philopatry in females.
Microsatellite differentiation was present, but less significant than mtDNA (Table 1) (Keeney et
al., 2005). Homogeneity was present throughout the Gulf and northwestern Atlantic, but
differentiation was found between the Yucatan and Belize sites despite their close proximity
(Table 1). Philopatry is probably more prevalent among females while males may be more prone
to stray between sites, mating with females from different nurseries. The lack of microsatellite
structure among the Gulf and Atlantic areas may be due to a recent separation of populations
associated with rises in seas level in the last 10,000-15,000 years. This sea level rise could have
increased the distances between populations and caused the geographical genetic structure
present in the mtDNA. These findings suggest that black tip populations are largely based on
geography with straying males conserving the gene flow between nurseries (Keeney et al. 2005).
However, movement is limited primarily to coastlines which restricts gene flow resulting in
genetic stock structure between populations in the northwestern Atlantic and Gulf, northwestern
Atlantic and Belize, Gulf and Belize, and within the Gulf. Figure 1 illustrates the genetic
distances between the locations sampled with the two Belize locations are (Belize City and
Dangriga) clearly the most unique when compared to the other locations.
Keeney’s et al. (2005) findings highlight the need for conservation and stock
management practices at the local level. Philopatry and high rates of neonate mortality indicate
that nurseries are critical for recruitment. Protection efforts focused on the conservation of
nurseries would help to improve population health and maintain regional gene flow. For the
population to subsist at a persist at a healthy number, legislation and regulation must focus on
limiting the direct fishing of nurseries and commercial development that threatens to damage
them.
A similar study was conducted by DiBattista et al. (2008) to assess the population and
mating structure of lemon sharks. Lemon sharks are another abundant population of sharks that
is found throughout the coasts of the Atlantic Ocean as well as the tropical west coast of the
Americas. Female lemon sharks, like black tip sharks, make use of nursery habits where they
give birth to young who remain in the shallow sheltered areas until a seasonal shift occurs. A
past study of a population in Bimini, Bahamas had revealed that female lemon sharks also
exhibit philopatry to their natal nursery site. The female lemon sharks of the Bimini population
also practiced polyandry, the act of mating with multiple males which is exhibited more
frequently in older and larger females (Feldman et al. 2002). To improve the overall
understanding of lemon shark mating systems, a second lemon shark study was conducted in a
nursery in Marquesas Key, Florida. Marquesas Key is a very similar habitat to the Bimini
habitat, but shark life histories vary significantly between the two regions. Despite occasional
inbreeding between the two populations, sharks in Marquesas Key grow faster and larger while
fast growing sharks are selected against in Bimini. In order to better understand the consistency
between the two mating systems, 11 microsatellite markers and recently developed
reconstruction methods were used by DiBattista et al (2008) to evaluate polyandry and breeding
site fidelity.
Sampling took place on the Marquesas Key nursery which houses seventy five to one
hundred juveniles per year. Gill nets and fishing gear were used to catch juveniles after pupping
by adults through 1998-2000 and 2002-2006 for periods of seven to thirty days. Juveniles were
early newly born neonates or one year old juveniles which were used to provide data for previous
years. DNA was then extracted from fin samples using a salting out protocol and genotyped
using a combination of six dinucleotide microsatellite primer pairs and five new tetra-nucleotide
microsatellite loci (DiBattista et al., 2008). Genotypes were then generated for 408 sharks for at
least ten of the eleven loci. The program Colony 1.2 was then used to group individuals into full
and half sibling families and reconstruct the genotypes of parents for the sibling groups. The
degree of polyandry was also estimated based on the inferred family structure calculated by
Colony (DiBattista et al., 2008).
Figure 2. Degree of polyandrous
lemon shark litters per year from the
family structure determined by
colony. (DiBattista et al. (2008))
Figure 3 Proportion of sharks
assigned to the same sibling
group using Kinship 1.3.
(DiBattista et al. (2008))
The Colony program identified a mean litter size of 4.29 sharks with 46 unique mothers
and 163 different fathers, indicating clear polyandry in the populations. These findings are
demonstrated in Figure 4 which consistently shows a larger proportion of neonates related
maternally rather than paternally (DiBattista et al. 2008). Twenty of the mother genotypes were
fully reconstructed by the program while eight father genotypes were fully reconstructed. The
large number of half siblings in the same year also indicated that multiple male paternity of
litters occurred, likely due to sperm storage by females. In fact, 81% of litters had multiple
fathers with 1.95 sires per litter. As indicated in Figure 3, the proportion of polyandrous litters
per year was highly variable, possibly due to measurement error (DiBattista et al. 2008). The
years sampled with the greatest confidence (1999, 2000, and 2003) all had high levels of
polyandry hinting actual polyandry may be higher than what the results indicate for the other
years. This polyandrous behavior likely occurs out of convenience for females. It’s easier to
simply mate with multiple males rather than deal with harassment from rebuffed aggressive
males (Portnoy et al. 2007). Other patterns were also discovered in the population. Females were
found to have fidelity to the nursery site and typically produced litters there in a two year cycle.
Based on these findings, females can be classified as exhibiting breeding site fidelity (DiBattista
et al. 2008). However, it is still unclear whether males exhibit breeding site philopatry or if
neonates exhibit philopatry to their birth site. Males often only contribute to one litter per year
hinting that they either are far more abundant in number or mate at multiple nurseries.
Overall, results were found to be fairly consistent with the better sampled Bimini site
which showed polyandry in 89% of litters. The differences in life history are more likely related
to environmental factors rather than mating structures. The faster growth rates in Florida may
simply occur due to decreased pressure in juvenile life stages and Bimini lemon sharks may
exhibit compensatory growth outside the nursery. Alternatively, lemons sharks in Marquesas
Key may face more predatory pressure outside their nursery so they focus more on early
development. In both nurseries, however, it has been difficult to determine the level of philopatry
exhibited among the sharks (DiBattista et al. 2008). Variation is too low among the sharks to use
the same mtDNA methods used with the black tip sharks. Individual-based pedigree analysis has
been used in other shark species as a determinate of philopatry and could be applied to lemon
sharks as well.
Based on these results, local management programs should be applied to lemon shark
nurseries rather than wide scale oceanic conservation programs. Like blacktip sharks, nurseries
seem to be crucial to the survival of the species and are conserved across multiple populations.
Unfortunately, a degree of uncertainty still remain concerning philopatry in neonates and
additional studies are recommended.
Both blacktip and lemon sharks show some degree of population localization to nursery
based populations. However, this behavior is certainly not conserved among all sharks. Whale
sharks, unlike blacktip and lemon sharks, are not coastal species, but instead oceanic, existing in
massive basin scale populations. Unfortunately, they do share the threat of overfishing with the
other two species. In 2002, it was estimated that 20%-50% of whale sharks would be lost over
three generations if the current overfishing trends continued (Norman 2000). This threat has
prompted an increase in the necessity to understand their population structure. Whale sharks are
found in the Indian, Pacific, and Atlantic oceans and genetic evidence suggests connectivity in
the Indian and Pacific population. There is still uncertainty regarding the possibility of mixing
between Atlantic and Indo-Pacific sharks. It has been theorized that one of the two populations
could rebound from input from the other population, but past studies showed significant genetic
differentiation between the two groups (Castro et al. 2007 & Schmidt et al. 2009).
Vignaud et al. (2014) proposed that a more comprehensive study of whale sharks must be
undertaken to more accurately understand their population structure. DNA was collected from
634 whale sharks from a variety of Indo-pacific locations and Isla Holbox in the Caribbean.
PCR was performed on the fragments and programs were used to determine Allelic richness,
Amova values, Fst values, and rate of mutation. A DACP analysis and a neutrality analysis were
also performed on the mtDNA.
Table 2.
Indices of
genetic
diversity for
whale sharks
for
microsatellites
and mtDNA.
(Vignaud et al.
(2014))
Table 3.
Pairwise Fst
values for
microsatellites
and mtDNA.
Values range
from 0-1 with 0
implying no
probability of
inbreeding and
1 implying
complete
inbreeding of
the
populations.
(Vignaud et al.
(2014))
Figure 4. DACP
scatterplot of
microsatellite DNA.
Ellipses are present at
the mean for each
location. (Vignaud et
al. 2014)
It was found that high diversity was present among the mtDNA samples taken at the
locations excluding Isla Holbox. Allelic richness mirrored the trend of mtDNA as well. Richness
ranged from 4.37 in the Gulf of California to 4.82 in Djibouti, but only had a value of 3.95 in Isla
Holbox. Isla Holbox also had the lowest mean number of alleles over loci (5.71), the lowest
expected heterozygosity (0.6), and the lowest haplotype diversity (0.752) as shown in Table 2.
Microsatellite and mtDNA Fst values of population differentiation (Table 3) revealed that the
lowest probability of inbreeding occurred between Isla Holbox and the other sampled locations.
The DACP microsatellite analysis (Figure 4) also shows that while none of the samples were
tightly grouped, Isla Holbox were by far the most unique.(Vignaud et al. 2014).
Vignaund et al. (2014), also investigated the presence of a recent population expansion as
well. Through a mismatch and neutrality analysis as well as a Bayesian plot of mtDNA, effective
population size was shown to be an order of magnitude higher than in the past. This expansion
likely corresponded to the rise of sea levels and phytoplankton productivity in the early Holocene
era. A trend of decreasing genetic diversity was also noticed in the Ningaloo population over the
course of the study. Causes for this could possibly be exploitation, changing of breeding
locations, non-random mating, poor sampling, or an increase in the average age of breeding
females.
The data collected in this project very strongly supports the theory that the Isla Holbox
location is separate from the Indo-pacific populations and little to no breeding occurs between
the two. This contradicts earlier theories that in the event of a population decline, mixing from
one basin could aid in replenishing the population of the other. Vingaud et al. (2014) asserts that
if only a few individuals dispersed between the basins every two to four years, far lower levels of
genetic structure would be observed. Unfortunately, whale sharks are at the greatest risk of
overfishing in the Indo-Pacific. If population numbers continue to decrease due to overfishing by
nations such as China, genetic diversity would be permanently affected and gene flow from the
Atlantic could not counteract the negative effects of genetic drift. These findings encourage
conservationists to view the two populations as separate entities that in which specific methods
must be undertaken to ensure their survival.
Shark overfishing represents one of the largest environmental challenges currently faced
by mankind. On average, 100 million sharks are killed globally each year primarily due to the fin
trade (Fairclough, 2013). This dramatically unsustainable removal of these apex predators has
already have dramatic effects on the ecosystem which will only become direr without regulation.
The use of genetic techniques to assess population dynamics has grown increasing prevalent with
study scales increasing over the years. In each of the three studies above, the conclusion was
reached that populations should be managed over smaller versus larger regions; whether it be on
the scale of nurseries rather than oceans for lemon and blacktips or on basin scales rather than
worldwide scales for whale sharks. Accurate population management would limit the risk of
deleterious genetic trends occurring due to genetic drift with population decrease. Whether the
issue of shark population health is approached from a resource management perspective or
conservation perspective, it can be universally agreed that the benefits associated with the
maintained health of these species easily outweighs the few cons. Hopefully, the use of genetic
methods to assess population and mating structure will continue to provide insight that will allow
more accurate management, resulting in healthier populations.
Works Cited
Journal Sources
Castro A.L.F. et al. (2007) Population Genetic structure of Earth’s largest fish, the whale shark (Rhincodon typus).
Molecular Ecology: 16:5183-5192
DiBattista J. D., Feldheim K.A., Thibert-Plante X., Gruber S. H., & Hendry A.P. (2008) A genetic assessment of
polyandry and breeding-site fidelity in lemon sharks. Molecular Ecology. 17: 3337-3351
Keeney D.B., Heuprel M.R., Hueter R.E., & Heist E. J. (2005) Microsatellite and mitochondrial DNA analyses of
the genetic structure of black tip shark (Carcharhinus limbatus) nurseries in the northwestern Atlantic, Gulf
of Mexico, and Caribbean Sea. Molecular Ecology. 14:1911-1923
Keeney D.B., Heuprel M.R., Hueter R.E., & Heist E. J. (2003) Genetic heterogeneity among blacktip shark,
Carcharhinus limbatus, continental nurseries along the US Atlantic and Gulf of Mexico. Marine Biology.
143:1039-1046
Portnoy D.S., Piercy A.N., Musick J.A., Burgess G.H., & Graves J.E. (2007) Genetic polyandry and sexual conflict
in the sandbar shark, Carcharinus plumbeus in the western North Atlantic and Gulf of Mexico. Molecular
Ecology. 16:187-197
Schmidt J.V. et al. (2009) Low genetic differentiation across three major ocean populations of the whale shark,
Rhincodon typus. Plos One. 4:e4988
Web Sources:
Fairclough C. (2013) “Shark Finning: Sharks Turned Prey” SI.edu. Smithsonian Institution. 14
<http://ocean.si.edu/ocean-news/shark-finning-sharks-turned-prey>
Freund J. (2014) “Sharks” WWF.org. WWF.
http://wwf.panda.org/about_our_earth/species/profiles/fish_marine/shark2/
Norman B. (2000) Rhincodon typus. IUCN 2007. 2007 IUCN Red List of Threatened Species. <Iucnredlist.org>
SharkSavers (2014) “Sharks’ Role in the Ocean” SharkSavers.org WILDAID.
<http://www.sharksavers.org/en/education/the-value-of-sharks/sharks-role-in-the-ocean/>