Mar Biol (2012) 159:489498

DOI 10.1007/s00227-011-1828-y

ORIGINAL PAPER

Population genetics of the nurse shark (Ginglymostoma cirratum)

in the western Atlantic
Stephen A. Karl Andrey L. F. Castro
Ricardo C. Garla

Received: 29 June 2011 / Accepted: 19 October 2011 / Published online: 5 November 2011
Springer-Verlag 2011

Abstract The nurse shark, Ginglymostoma cirratum,

inhabits shallow, tropical, and subtropical waters in the
Atlantic and the eastern Pacific. Unlike many other species
of sharks, nurse sharks are remarkably sedentary. We
assayed the mitochondrial control region and eight microsatellite loci from individuals collected primarily in the
western Atlantic to estimate the degree of population
subdivision. Two individuals from the eastern Atlantic and
one from the Pacific coast of Panama also were genotyped.
Overall, the mtDNA haplotype (h = 48 5%) and
nucleotide (p = 0.08 0.06%) diversities were low. The
microsatellite data mirror the mitochondrial results with the

Communicated by M. I. Taylor.

average number of alleles (NA = 9) and observed heterozygosity (HO = 0.58) both low. The low levels of diversity
seen in both the mtDNA and the microsatellite may be due
to historical sea level fluctuations and concomitant loss of
shallow water habitat. Eight of the 10 pair-wise western
Atlantic FST estimates for mtDNA indicated significant
genetic subdivision. Pair-wise FST values for the microsatellite loci indicated a similar pattern as the mtDNA. The
western Atlantic population of nurse sharks is genetically
subdivided with the strongest separation seen between the
offshore islands and mainland Brazil, likely due to deep
water acting as a barrier to dispersal. The eastern and
western Atlantic populations were closely related. The
eastern Pacific individual is quite different from Atlantic
individuals and may be a cryptic, sister species.

Electronic supplementary material The online version of this

article (doi:10.1007/s00227-011-1828-y) contains supplementary
material, which is available to authorized users.

Introduction
S. A. Karl  A. L. F. Castro
Department of Biology, University of South Florida, SCA110,
4202 E. Fowler Ave., Tampa, FL 33620, USA
Present Address:
S. A. Karl (&)
Hawaii Institute of Marine Biology, University of Hawaii,
PO Box 1346, Manoa, Kaneohe, HI 96744, USA
e-mail: skarl@hawaii.edu
Present Address:
A. L. F. Castro
Departamento de Zootecnia, Universidade Federal de Sao Joao
del Rei, Av. Visconde do Rio Preto, S/N, Sao Joao Del Rei,
MG 36301-360, Brazil
R. C. Garla
Departamento de Botanica, Ecologia e Zoologia, Centro de
Biociencias, Universidade Federal do Rio Grande do Norte,
Campus Universitario, Lagoa Nova, Natal, RN 59072, Brazil

Understanding natural metapopulation structure can lend

insight into the evolution, behavior, and management of a
species. Both teleosts and elasmobranchs often occur in
shallow, coastal tropical, and subtropical waters (\100 m)
and are benthic and benthopelagic with relatively limited
dispersal abilities outside of the egg or larval stages.
Elasmobranchs, in particular, often are thought to have
reduced dispersal ability limited to actively swimming
juveniles and adults (Musick et al. 2004). Tagging and
acoustic monitoring studies of sharks, however, have
shown that many of the larger coastal species are able to
engage in long-distance movements (Kohler and Turner
2001; Pardini et al. 2001; Castro et al. 2007; Meyer et al.
2009). In these species, dispersal has been considered the
primary mechanism explaining their wide distributions and

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is thought to maintaining genetic connectivity among

geographically separated groups. Regional and global
surveys of genetic variation in coastal sharks, however,
have shown that the genetic exchange among populations
can be lower than predicted based simply on presumed
dispersal ability. Studies of large coastal or migratory
species have found evidence of significant genetic structure
among Atlantic shark populations (e.g., scalloped hammerhead, Sphyrna lewini, Duncan et al., 2006; lemon
shark, Negaprion brevirostris, Feldheim et al., 2001;
sharptooth lemon shark, N. acutidens, Schultz et al., 2008;
bull shark, Carcharhinus leucas, Karl et al., 2011; blacktip
shark, Carcharhinus limbatus, Keeney et al., 2005). Many
shark species have complex, multi-phasic life histories
where mating, pupping, and maturation may occur in
separate, discrete locations (Pratt and Carrier 2001; Hueter
et al. 2005; Heupel et al. 2007). The presence of these
developmental habitats combined with natal site philopatry
can result in strong population subdivision in spite of high
intrinsic dispersal potential. Unfortunately, very little is
generally known about the life history attributes of many
shark species or to what extent developmental habitats
exist (Heupel et al. 2007).
One species of elasmobranch with a wide but disjunct
distribution is the nurse shark, Ginglymostoma cirratum
Bonnaterre, 1788. Nurse sharks inhabit shallow, warm
waters along both the western (Rhode Island to Southern
Brazil) and the eastern (Senegal to Gabon) coasts of the
Atlantic Ocean (Pillans 2003) as well as in the eastern
Pacific (Mexico to Peru; Pillans 2003). Nurse sharks have
been reported as the most abundant shark species in coastal
shallow waters (Castro 2000; Hazin et al. 2000; Castro and
Rosa 2005; Heithaus et al. 2007). Unlike other species of
sharks, however, nurse sharks are remarkably sedentary
and tagging studies have shown that individuals have
highly restricted movement patterns (Kato and Carvallo
1967; Pratt and Carrier 2001). Kohler and Turner (2001)
reported the longest distance ever travelled by a nurse

Fig. 1 Sample sizes (in

parentheses) and geographic
locations of nurse sharks
collected from Sao Tome,
Africa (AFR); Fernando de
Noronha, Brazil (FNR); Atol
das Rocas, Brazil (ADR); Natal,
Brazil (NTL); Bimini, Bahamas
(BMI); Florida, United States
(FLD); Glovers Reef, Belize
(BLZ) and Punta Culebra,
Panama (PNM). The Brazilian
Islands (BRI) are considered as
one location in the analyses (see
text for details)

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Mar Biol (2012) 159:489498

shark with an animal tagged in the Dry Tortugas, in

extreme southern Florida, being recaptured 7.8 years later
off Palm Bay in east-central Florida; a minimum 541 km
journey. Because of their wide geographic distribution but
low vagility, sedentary life style, and association with
shallow waters, questions remain about the level of genetic
connectivity among regional populations of nurse sharks.
As with lemon sharks (Negaprion brevirostris, Schultz
et al., 2008), it seems likely that nurse shark populations
connected by continuous coastline (i.e., north- and southwestern Atlantic Ocean) would be more genetically
homogenous than populations isolated by large expanses of
deep water (i.e., eastern and western Atlantic Ocean or
offshore islands). Even so, as with other shark species,
nurse sharks may demonstrate fidelity to developmental
habitats, which could greatly disrupt gene flow even among
geographically proximate locations.
Here, we present a survey of the genetic variation in
nurse shark populations sampled primarily from the western Atlantic (two individuals from the eastern Atlantic and
one from the eastern Pacific are also discussed) using both
mitochondrial control regions (CR) and nuclear microsatellite markers. The goal was to evaluate the genetic
diversity and population structure of this sedentary, coastal
species.

Materials and methods

Sampling and laboratory procedures
Sampling
We collected a total of 153 individuals in the western
Atlantic (Fig. 1). Tissue samples were obtained along the
northeastern coast of Brazil from Atol das Rocas Biological Reserve (ADR, n = 9), Fernando de Noronha National
Park (FNR, n = 34), and Natal City, Rio Grande do Norte

Mar Biol (2012) 159:489498

(NTL, n = 15). We also obtained tissue samples from

Bimini, Bahamas (BMN, n = 23), southern Florida,
mainly Big Pine Island, USA (FLD, n = 37), and from the
Caribbean coast of Belize (BLZ, n = 35). Two additional
individuals were sampled from the African coast off Sao
Tome and Principe, western Africa (AFR), as well as one
individual from the Pacific coast of Panama (PNM). All
samples were preserved in 95% ethanol or salt-saturated
dimethyl sulphoxide (DMSO) solution and stored at room
temperature. Total cell DNA was extracted using a phenol
chloroformisoamyl alcohol protocol (Sambrook et al.
1989) or using the QIAGEN DNeasy kit, according to the
manufacturers instructions (QIAGEN, Inc., Valencia, CA,
USA).
Mitochondrial control region
By searching for conserved regions in aligned mtDNA
sequences available in GenBank, we developed two sets of
primers to amplify the CR of the nurse shark. The forward
primer, PR1 (50 -CATATTAAACCCGAATGATAYTT-30 ),
primes in the cytochrome b gene (cyt b) approximately 230
nucleotides (nt) from the cyt btRNAThrtRNAProCR
junction. The reverse primer, PR2 (50 -GCTGAGTAAA
GCTTAATATG-30 ), primes in the CR approximately 350 nt
from the tRNAProCR junction. This primer pair amplifies
part of cyt b, all of tRNAPro and tRNAThr, as well as part of
the CR. A second forward primer, PR3 (50 -TTGG
CTCCCAAAGCCAAGATTCTACC-30 ), primes in the
tRNAPro, and a second reverse primer, PR4 (50 -CCGACT
AAGCAGGAATGTGC-30 ), primes in the CR approximately 795 nt from the tRNAProCR junction. With both
primer pairs, we amplified the entire 50 end of the CR, which
is though to be the most variable part of that gene (Kocher
et al. 1989; Lee et al. 1995).
PCRs were performed in 50 lL volumes consisting of
19 IMMOLASE buffer (Bioline Inc., Boston, MA, USA),
2 units of IMMOLASE DNA polymerase, 0.4 mM dNTPs,
4 mM MgCl2, 0.3 lM of each primer, 0.08 lg lL-1 bovine
serum albumen, and 0.751.00 lL of total cell DNA. PCR
cycling conditions consisted of 95C for 7 m, 40 cycles of
95C for 45 s, 58C for 60 s, and 72C for 90 s and one
cycle of 72C for 7 m. Amplicons were purified with
QIAquick kit (QIAGEN, Inc., Valencia, CA, USA) following the manufacturers instruction. Sequencing reactions were performed in both directions and resolved on an
ABI 3730XL Genetic analyzer (Applied Biosystems, Inc.,
Foster City, CA, USA).
Microsatellite loci
A total of eight polymorphic microsatellite loci were
amplified using primers developed for the nurse shark by

491

Heist et al. (2003). The forward primers for loci Gc-04,

Gc-07, Gc-10, Gc-11, Gc-18, Gc-25, Gc-35, and Gc-37
were fluorescently labeled (Applied Biosystems, Inc.,
Foster City, CA, USA). Amplification reactions were
12.5 lL consisting of 19 IMMOLASE buffer (Bioline
USA Inc., Boston, MA, USA), *2 to 10 units of Taq or
IMMOLASE DNA polymerase, 0.2 mM dNTPs, 23 mM
MgCl2, 0.10.3 lM of each primer, 0.4 lg lL-1 bovine
serum albumen, and 12 lL of total cell DNA. Cycling
conditions were as in Heist et al. (2003). PCR products
were resolved with an ABI 3100 automated sequencer and
visualized using GeneMarker software version 1.7 (Soft
Genetics LLC, State College, PA, USA).
Mitochondrial cytochrome c oxidase 1
To frame the potentially deeper divergence among the
western and eastern Atlantic and the eastern Pacific
samples (see Results for details) with respect to other
species in the order, we sequenced a region of the mitochondrial cytochrome c oxidase 1 gene (COI), which is
commonly used to rapidly and accurately identify species
(i.e., DNA bar coding; Hebert et al. 2003). We sequenced
63 western Atlantic (FNR = 12, ADR = 8, NTL = 12,
BMN = 10, FLD = 11, and BLZ = 10), one eastern
Atlantic, and one eastern Pacific nurse shark. We used the
FishF2-t1 and FishR2-t1 primers developed by Ward et al.
(2005) in 50 lL amplification reactions consisting of
19 IMMOLASE buffer (Bioline Inc., Boston, MA, USA),
2 units of IMMOLASE DNA polymerase, 0.4 mM dNTPs,
4 mM MgCl2, 0.3 lM of each primer, 0.1 lg lL-1 bovine
serum albumen, and 0.751 lL of total cell DNA. PCR
cycling conditions consisted of 95C for 7 m, 30 cycles of
94C for 30 s, 50C for 90 s, and 72C for 45 s and one
cycle of 72C for 3 m. Amplicons were purified with a
QIAquick kit (QIAGEN, Inc., Valencia, CA, USA) following the manufacturers instruction. Sequences were
performed in both directions and resolved on an ABI
3730XL Genetic analyzer (Applied Biosystems, Inc., Foster City, CA, USA).
Data analysis
mtDNA control region
Sequences were aligned with Sequencher 4.1 (Gene Codes,
Ann Arbor, MI, USA), and alignments were checked by
eye and edited when necessary. Numbers of haplotypes,
polymorphic sites, transitions, and transversions and haplotype frequencies and nucleotide composition were estimated using ARLEQUIN 3.5.1.2 (Excoffier and Lischer
2010). Due to geographic proximity and genetic similarity

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(i.e., nonsignificant FST values; data not shown), ADR and

FNH were pooled and treated as a single sample, hereafter
called Brazilian islands (BRI). Genetic diversity within
samples was estimated as the number of mitochondrial
haplotypes, haplotype diversity (h), and nucleotide diversity (p) estimated with Neis corrected average genetic
divergence (Nei 1987) incorporating Tamura and Neis
(1993) model of sequence evolution with ARLEQUIN. The
overall genetic diversity was calculated for each of the
western Atlantic samples, all western Atlantic samples
combined, and overall including the African and Pacific
samples. Evolutionary divergence between haplotypes was
estimated using the maximum-likelihood (ML) method as
implemented in PAUP 4.0b10 (Swofford 2000). The hierarchical likelihood-ratio test of MODELTEST (version
3.06; Posada and Crandall 1998) was used to determine the
most likely model of sequence evolution for the entire
mtDNA sequence (1,166 bp). A minimum spanning haplotype network was constructed using TCS 1.2.1 (Clement
et al. 2000).
Microsatellites
For microsatellite analysis, individuals were binned into
the same five geographic regions as in the CR analysis.
Numbers of alleles per locus, expected and observed heterozygosities, deviations from HardyWeinberg expectations for each locus within each sample, and tests of
linkage disequilibrium between all pairs of loci within and
over all regions were calculated with GENEPOP (version
3.3; Raymond and Rousset 1995). Significances of deviations from HardyWeinberg expectations were determined
using the Markov chain exact probability test of Guo and
Thompson (1992), and significances of linkage disequilibrium values were determined with Fishers exact test as
implemented in GENEPOP. The program BOTTLENECK
(Piry et al. 1999) was used to look for signs of recent
changes in population size. We used the stepwise mutation
(SMM) and two-phase models (TPM) of evolution but not
the infinite alleles model because the first two are most
appropriate when evaluating microsatellite data (diRienzo
et al. 1994). We performed 1,000 iterations and tested
significance with the Wilcoxon signed rank test (Wilcoxon
1945), and for the TP model, we used 95% SMM with a
variance of 5.
For both mtDNA haplotypes and microsatellite genotypes, population subdivision was estimated using an
analysis of molecular variance (AMOVA; Excoffier et al.
1992), and pair-wise population FST (Cockerham and Weir
1993) as implemented in ARLEQUIN. Significance of FST
was determined via nonparametric permutation (Excoffier
et al. 1992) with 10,000 permutations. For mtDNA only,
population differentiation also was tested using a Raymond

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Mar Biol (2012) 159:489498

and Rousset (1995) exact test of sample differentiation

based on haplotype frequencies.
mtDNA COI region
The degree of COI sequence divergence was calculated in
MEGA 4.0 (Tamura et al. 2007) using a Kimura
2-parameter distance model (K2P; Kimura 1980). We
chose this model because it allows us to compare our
results to Ward et al. (2008) who evaluate the divergence
of COI sequences among 210 chondrichthyan species. To
illustrate the range of similarities and divergences found in
the Orectolobiformes order, COI sequences from other
species available in the Barcode of Life Database (Ratnasingham and Hebert 2007) also were included in the
analysis. These included the tawny nurse shark, Nebrius
ferrugineus (GenBank accession numbers BWA3013 and
BWA2717), whale shark, Rhincodon typus (BWA3307),
zebra shark, Stegostoma fasciatum (BWA2566 and
BWA2567), brownbanded bamboo shark, Chiloscyllium
punctatum (BW-2144 and BW-2146), Indonesian bamboo
shark, C. hasselti (BW2127 and BW2130), slender bamboo
shark, C. indicum (BW2122 and BW2123), white spotted
bamboo shark, C. plagiosum (BW2137 and BW2138),
spotted wobbegong shark, Orectolobus maculates (BW2
068 and BW2070), and western wobbegong shark,
O. hutchinsi (BWA2620 and BWA2621). A neighborjoining (NJ) tree based on the K2P distances was created
using MEGA 4.0 and data bootstrapped 1,000 times to
indicate the support for nodes.

Results
We sequenced a 1,166 nt fragment of mtDNA containing
the 30 end of the cyt b gene, both tRNAPro and tRNAThr and
the 50 end of the control region from a total of 146 nurse
sharks. On average, this fragment was of 13.1% guanine,
33.9% adenine, 31.3% thymine, and 21.6% cytosine. There
were 34 polymorphic sites consisting of 32 substitutions
(25 transitions and seven transversions) and two indels,
resolving 12 haplotypes (Table 1; Fig. 2; Table S1; GenBank Accession numbers JF950288-JF950299). The vast
majority of the polymorphism was found between the
Pacific (haplotype H12) and Atlantic haplotypes with 19
substitutions (14 transitions and five transversions) and two
indels. The overall haplotype (h) and nucleotide diversities
(p) for the mtDNA of the western Atlantic populations
were 43 5% (standard deviation) and 0.04 0.04%,
respectively (Table 2). Considering all samples (i.e.,
including the eastern Atlantic and Pacific), h and p were
slightly larger (Table 2). H1 was the most prevalent
haplotype, found in all western Atlantic locations and in

Mar Biol (2012) 159:489498

493

68% of the individuals, but not in AFR or PNM (Table 1).

Only two other haplotypes were shared among populations
(H2 and H4; Table 1).
The minimum spanning haplotype network shows no
evidence of population partitioning of haplotypes in the

Table 2 Location, number of individuals (N), number of haplotypes

(n), haplotype (h) and nucleotide (p) diversity estimates, and standard
deviations observed in the control region of the nurse shark
Location

BRI

37

0.43 0.06

0.0004 0.0004

NTL

15

0.56 0.09

0.0005 0.0005

Table 1 Geographic distribution of mtDNA haplotypes found in

nurse sharks. Sample locations are as in Fig. 1

BMN
FLD

21
37

6
4

0.43 0.13
0.16 0.08

0.0004 0.0004
0.0001 0.0002

Haplotype

BLZ

Geographic location
BRI

NTL

BMN

FLD

BLZ

AFR

PNM

Total

H1

26

16

34

20

105

H2

13

19

H3
H4

11

11
2

H5

H6

H7

H8

H9

H10

H11

H12

Total

37

15

21

37

33

146

33

0.49 0.04

0.0004 0.0004

Western Atlantic

143

10

0.43 0.05

0.0004 0.0004

Total

146

12

0.48 0.05

0.0008 0.0006

Note that overall values are presented for the western Atlantic and for
the entire sample set (i.e., including samples from East Pacific and
East Atlantic). Locations abbreviations are as in Fig. 1

western Atlantic sharks (Fig. 2). The AMOVA indicated a

moderate and significant genetic structure in haplotype
frequencies among the western Atlantic populations for the
mtDNA (P B 0.05), with a large amount of the genetic
variability found within populations (78.2%). Eight of the
10 pair-wise FST comparisons for mtDNA show statistically significant population differentiation (P B 0.05;
Table 3). The exact test of population differentiation based
on haplotype frequencies (Raymond and Rousset, 1995)
revealed the same pattern of genetic structure (data not
shown).
The hierarchical likelihood-ratio test of MODELTEST
(Posada and Crandall 1998) indicated the HasegawaKishino-Yano 85 distance (HKY85; Hasegawa et al. 1985)
as the most likely model of sequence evolution, and this
model was used to estimate divergence among mtDNA
haplotypes. The magnitude of the divergence among haplotypes reflected a pattern similar to the TCS network
(Table S2; Fig. 2). There was approximately a *12-fold
higher divergence between the Atlantic and eastern Pacific
haplotypes (1.90%; Table S2) than there was among any of
the western Atlantic haplotypes (0.16%), and divergence
between the western and eastern Atlantic haplotypes was
intermediate (0.42%).
Table 3 Estimate of pair-wise genetic distance (FST) among nurse
shark samples for control region sequences (above diagonal) and
microsatellite loci (below)

BRI

Fig. 2 Statistical parsimony network of haplotypes for nurse shark

mitochondrial DNA sequences. All haplotypes are separated by one
mutation, and solid black circles represent hypothetical haplotypes
not sampled in the study. The size of the circles is proportional to the
haplotype frequency except for H1, which corresponds to 68% of the
individuals

BRI

NTL

0.275

BMN

FLD

0.157
a

BLZ

0.218

0.334

NTL

0.012

0.109

0.266

-0.034

BMN
FLD

0.020
0.016

0.030
0.011

-0.003

0.003

0.185
0.318

BLZ

0.009

0.005

0.006

-0.004

Italicized values are significant at P B 0.05 and were not Bonferroni

corrected. Location abbreviations are as in Fig. 1
a

P = 0.06

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Mar Biol (2012) 159:489498

Fig. 3 Neighbor-joining tree of

cytochrome oxidase I sequences
for orectolobiform sharks using
Kimura 2-parameter distances.
Scale bar is substitutions per
site. The numbers at the nodes
are percent bootstrap support
and the numbers in parentheses
correspond to the Barcode of
Life database accession
numbers

Genotypes of 152 nurse sharks were determined at eight

microsatellite loci. Number of individuals analyzed (N),
number of alleles (NA), observed (HO) and expected (HE,)
heterozygosity, and deviation from the HardyWeinberg
expectation (FIS) for each locus within each location are
provided as supplemental material (Table S3). The total
number of alleles and observed heterozygosities per locus
ranged from three to 20 (NA = 9.00) and 0.200.92
(HO = 0.58), respectively. Two out of the 40 locus-bysample tests of HardyWeinberg equilibrium indicated
significant heterozygote deficits (P B 0.05). With sequential Bonferroni adjustment (a = 0.007), only locus Gc-07
in one sample (NTL) was significant (P = 0.0062; Table
S3). Globally, there was no significant linkage disequilibrium among the western Atlantic samples, which supports
the independent assortment of alleles at different loci (data
not shown). The pair-wise population FST estimates for the
microsatellite data supported much of the basic genetic
structure observed with the mtDNA with the notable
exception that the locations in the northwest Atlantic (i.e.,
FLD, BMN, and BLZ) are not significantly differentiated
(Table 3).
For the COI gene, no differences were found among the
western Atlantic nurse shark sequences. A single substitution (transition) was observed between the western and
eastern Atlantic haplotypes. The Pacific nurse shark haplotype had 10 substitutions (nine transitions and one

123

transversion) when compared to the western Atlantic haplotypes. The K2P distance estimates indicate an average of
1.6% divergence between the Pacific and the western
Atlantic haplotypes. This is much smaller than the divergence found among the four species in the Genus Chiloscyllium (7.59.8%), but similar to the divergence between
the two species of Orectolobus (1.8%, Fig. 3). Interestingly, the Orectolobus spp. are thought to have formed *2
million years ago (Corrigan and Beheregaray 2009), which
is fairly close to the estimated age of the closing of the
Isthmus of Panama (*3.5 million years ago; Coates et al.
1992) that would have divided the Pacific and western
Atlantic nurse shark populations.

Discussion
Genetic diversity
Nurse sharks are reported to be the most abundant shark
species in shallow, coastal waters (Castro 2000; Hazin
et al. 2000; Castro and Rosa 2005; Heithaus et al. 2007).
Precise estimates of population sizes are mostly lacking,
but in the Atol das Rocas Marine Reserve, Brazil, there are
at least 400 individuals living in one *6 km2 area (Castro
and Rosa 2005). Even so, nurse sharks exhibit the fourth
lowest overall mtDNA haplotype (h = 43%) and lowest

Mar Biol (2012) 159:489498

nucleotide (p = 0. 04%) diversities of any shark species

assessed to date (Karl et al. 2011). The lowest mtDNA p
found in other sharks was in the sharptooth lemon shark,
Negaprion acutidens (h = 28%, p = 0.06%; Schultz et al.
2008). Although the population size estimates for this
species are unknown, it is undoubtedly quite small since
N. acutidens is listed by the International Union for the
Conservation of Nature (IUCN 2003) as extirpated from
India and Thailand, endangered in southeast Asia, and
vulnerable in the rest of its distribution (Pillans 2003).
Nurse shark diversity values were comparable to those seen
in the gray nurse shark (Carcharias taurus; h = 32%,
p = 0.08%; Ahonen et al. 2009;), which is listed as globally vulnerable by the IUCN. Even with a limited understanding of the actual population size for nurse sharks,
there is a clear discordance with this presumably abundant
species possessing genetic diversity levels similar to species of conservation concern due to exceptionally small
population sizes. The general lack of both nuclear and
mitochondrial DNA variation and the large population
sizes may indicate that nurse shark populations may have
recently expanded in size. The analysis with BOTTLENECK does indicate that two of the locations (BRI and
FLD) deviate from expected levels of heterozygosity with a
heterozygosity deficiency (P B 0.01) consistent with a
recent population expansion (Luikart and Cornuet 1998).
None of the other locations, however, indicated significant
changes in population sizes. It should be noted, however,
that the power of this test is reduced due to the modest
number of loci used (Cornuet and Luikart 1996; Luikart
and Cornuet 1998).
Population structure
A number of tagging studies have shown that the nurse
shark is a remarkably sedentary species (Kato and Carvallo
1967; Kohler and Turner 2001; Castro and Rosa 2005;
Heithaus et al. 2007). This and its shallow, warm coastal
water habitat suggest that dispersal through open, deep,
pelagic waters may be uncommon. Our data show moderate, significant genetic structure for the mtDNA among
western Atlantic populations (Table 3). With mtDNA,
there are three populations consisting of the offshore
Brazilian islands (BRI), the geographically proximate
populations in Florida and the Bahamas (FLD and BMN),
and populations connected by continuous coastline (NTL
and BLZ). A similar pattern is seen in the microsatellite
data; however, all three northwestern Atlantic samples
(BLZ, FLD, and BMN) constitute a singe group (Table 3).
Other shark species show remarkably different patterns for
nuclear versus mitochondrial markers likely due to female
philopatry (Feldheim et al. 2002; Pardini et al. 2001; Hueter et al. 2005; Keeney et al. 2005; Schultz et al. 2008;

495

Karl et al. 2011). Tagging studies have reported site fidelity

in nurse sharks of different ages (Kohler and Turner 2001;
Castro and Rosa 2005; Heithaus et al. 2007). Pratt and
Carrier (2001) also found sex-specific philopatry in adult
nurse sharks, where females return to mating sites in the
Dry Tortugas, FL, USA every other year. Our data indicate,
however, that males and females do not differ in the pattern
of population subdivision and do not support differential
philopatry in nurse sharks. Apparently, the relatively short,
but deep ocean expanse between mainland and the offshore
islands of Brazil is a significant barrier. As the shark
swims, the distance between Florida and the Bahamas is
about as far as the Brazilian Islands are offshore
(*300 km). Between Florida and the Bahamas, however,
the water is rarely deeper than *0.8 km and when it is, it is
only for short stretches (i.e., \100 km). To the contrary,
mainland Brazil and the offshore islands are separated by
water *4.0 km deep for nearly the entire distance. As has
been shown for other coastal sharks (e.g., sharptooth lemon
sharks; Schultz et al. 2008), water depth can be more of a
barrier to movement than distance.
Considering the average divergence calculated among
nurse shark populations, and if we assume that the transisthmian divergence was 3.5 million years ago, (Coates
et al. 2004) the nurse shark mtDNA CR is diverging at a
rate of about 0.54% per million years. This is very similar
to what was found for N. brevirostris (0.67%; Schultz
et al., 2008) and S. lewini (0.80%; Duncan et al., 2006). At
this rate, the western Atlantic haplotypes coalesce in the
late Pleistocene (*300 000 years ago). During this period,
sea levels fluctuated substantially and accounted for several
major marine extinctions (Page and Lydeard 1994; Briggs
1995; Floeter et al. 2008). During times of low sea level,
shallow water habitats were severely restricted and population sizes for the organisms dependent on them likely
shrank concomitantly. It is reasonable to assume that nurse
sharks suffered from population bottlenecks as a consequence of the sea level decrease in the Late Pleistocene. It
is important to point out, however, that very little is known
about historical population sizes of nurse sharks and that
there was no indications of a significant excess of heterozygosity in the BOTTLENECK analysis. The generally
low heterozygosity level and small number of loci, however, limit the power of our data to detect anything but very
large changes in population size (Cornuet and Luikart
1996; Luikart and Cornuet 1998).
Phylogeography
Musick et al. (2004) hypothesized that vicariant events
could be responsible for the disjunct nurse shark distribution on either side of the Atlantic. Using our slowest
mutation rate (i.e., the longest time), the divergence among

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haplotypes indicated that the western and eastern Atlantic

populations separated *780,000 years ago. Given that the
opening of the Atlantic Ocean occurred *1.0 million years
ago, this age would be inconsistent with the vicariant
hypothesis for the amphi-Atlantic distribution of nurse
sharks. Although broad expanses of deep water appear to
be a barrier to dispersal, clearly they are not an absolute
one over evolutionary time. The eastern and western
Atlantic and the off shore Brazilian island and mainland
Brazil are separated by deep open ocean; nonetheless,
nurse sharks inhabit all of these areas and thus must have
traversed the deep open ocean at some point. The maximum divergence observed between the Panamanian and
the western Atlantic CR haplotypes (d = 1.92%) was
smaller, although not dramatically so, than the transisthmian divergences found in other sharks (e.g., d = 2.8% in
S. lewini, Duncan et al. 2006; d = 2.4% in N. brevirostris,
Schultz et al. 2008). In COI sequences, there was, on
average, 1.6% divergence between haplotypes collected on
either side of the Isthmus of Panama. Ward et al. (2008),
analyzing 210 chondrichthyan species, found that the mean
intraspecific divergence in COI was 0.37%, similar to what
we saw among the western Atlantic haplotypes. The value
we see between eastern Pacific and western Atlantic nurse
shark COI sequences, however, is considerably larger.
Castro et al. (2003) have previously suggested a unique
specific status for Pacific Ocean nurse sharks based on a
series of morphological characters, such as body proportions, tooth morphology, and dental formula. Combined
with the genetic data, there is an indication that the eastern
Pacific nurse shark may be a species unique from G. cirratum. We caution, however, that we are basing this on a
single specimen and much larger sample sizes would be
necessary for a robust conclusion.
Overall, we found that the nurse shark is genetically
depauperate relative to many other shark species, which is
likely a consequence of historical population size fluctuations. Both mtDNA and microsatellite loci indicate population structuring with large expanses of open ocean a
significantly stronger barrier to dispersal than long distances of shallow water. Geographically localized groups
may constitute semi-isolated populations and may be sensitive to over-exploitation. This is an important issue for
the species in the southwestern Atlantic because of the
large and unregulated artisanal fishery along the Brazilian
coast. The amphi-Atlantic distribution appears to be due to
dispersal and not vicariance. The divergence seen between
the eastern Pacific and Atlantic is on par with congeners for
other shark species and the eastern Pacific nurse shark may
be a cryptic, sister species. Given the sedentary habits and
restricted movement patterns of nurse sharks, it is unlikely
that depleted populations will be replenished by continuous

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Mar Biol (2012) 159:489498

coastline and transoceanic dispersal. Therefore, immediate

action is needed to prevent local extinction and should
focus on the conservation of developmental habits in
coastal waters and protection against over fishing.
Acknowledgments We thank D. Chapman, K. Feldheim, S. Gruber,
E. Heist, L Rocha, R. Rosa, M. Shivji for providing samples and
A. Bass, B. Bowen, C. Curtis, K. Gorospe, C. Puchulutegui, C. Rocha,
L. Rocha, R. Toonen, and J. Zamzow for general assistance and
helpful comments on the manuscript. Nurse shark samples from Atol
das Rocas Marine Reserve and Fernando de Noronha National Park
were collected during the 19992001 international cooperative program between the University of Miami and Universidade Federal da
Paraba for the study of the natural history of the lemon shark in the
Atol das Rocas Marine Reserve and Fernando de Noronha National
Park and Conselho Nacional de Desenvolvimento Cientfico e
Tecnologico, CNPq. This study partially fulfilled the requirements for
ALFCs Ph.D. dissertation at University of South Florida and was
supported, in part, by Coordenacao de Aperfeicoamento de Pessoal de
Nvel Superior Fellowship BEX 1277-02-2 to ALFC. Funding for this
project also was provided by National Science Foundation grants
DEB 03-21924 and OCE 06-27299 to SAK. This is the University of
Hawaii, Manoa, School of Ocean and Earth Science and Technology
contribution No. 8503 and the Hawaii Institute of Marine Biology
contribution No. 1470.

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