Conservation Genetics (2005) 6:715–728
Springer 2005

DOI 10.1007/s10592-005-9031-7

Mitochondrial and nuclear DNA analyses reveal fine scale geographic structure
in bottlenose dolphins (Tursiops truncatus) in the Gulf of Mexico

Anna B. Sellas1,3, Randall S. Wells2 & Patricia E. Rosel3,*

1
Ocean Sciences Department, University of California, Santa Cruz, CA, 95064, USA; 2Chicago Zoological
Society, c/o Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL, 34236, USA; 3National
Marine Fisheries Service, 646 Cajundome Blvd, Lafayette, LA, 70506, USA (*Corresponding author: Phone:
+1-337-291-2123; Fax: +1-337-291-2106; E-mail: Patricia.Rosel@noaa.gov)
Received 30 September 2004; accepted 10 February 2005

Key words: management, microsatellites, mitochondrial DNA, population structure, Tursiops truncatus

Abstract
There is a need for biological information to support current stock designations of bottlenose dolphins
(Tursiops truncatus) in the Gulf of Mexico. The existence of many inshore, resident ‘‘communities’’
raises questions as to the relationship these dolphins may hold with dolphins inhabiting neighboring
inshore and coastal areas. In this study, population subdivision was examined among four resident,
inshore bottlenose dolphin stocks (Sarasota Bay, FL, Tampa Bay, FL, Charlotte Harbor, FL and
Matagorda Bay, TX) and one coastal stock (1–12 km offshore) in the Gulf of Mexico. Evidence of
significant population structure among all areas was found on the basis of both mitochondrial DNA
(mtDNA) control region sequence data and nine nuclear microsatellite loci. Estimates of relatedness
showed no population contained a significantly high number of related individuals, while separate
AMOVAs for males and females indicated that both sexes exhibit a significant level of site philopatry.
Results presented here provide the first genetic evidence of population subdivision between the coastal
Gulf of Mexico and adjacent inshore areas along the central west coast of Florida. Such strong genetic
subdivision is surprising given the short geographical distance between many of these areas and the lack
of obvious geographic barriers to prevent gene flow. These findings support the current, separate
identification of stocks for bottlenose dolphins inhabiting the eastern coastal and inshore areas of the
Gulf of Mexico.

Introduction ences have been documented between coastal and

For cetacean species, Hoelzel (1998) suggested an
important mechanism for the formation of intra-
specific genetic differentiation within a geographic
region to be behavior specialization which, in turn,
may be the precursor to resource specialization
(Skúlason and Smith 1995). One example of
intraspecific differentiation within a geographic
region in cetacean species is found in bottlenose
dolphins (Tursiops truncatus) where strong differ-
offshore forms in both the Northwest Atlantic and
Gulf of Mexico on the basis of hematology, mor-
phology, stomach contents, parasite loads
and genetics (Hersh and Duffield 1990; Ross
and Cockcroft 1990; Banos and Odell 1990; Mead
and Potter 1995; Hoelzel et al. 1998; Kingston and
Rosel 2003). While the level of differentiation be-
tween coastal and offshore Tursiops is well docu-
mented, and may eventually lead to separate
taxonomic species designations, the distinctions
716
among inshore, and between inshore and coastal
areas, are less well studied. Here we define inshore
areas as bays, sounds or estuarine waters, bor-
dered by mainland on one side and barrier islands
on the other, and coastal waters as nearshore or
neritic waters from shore to 12 km seaward of
shore. From long-term photo-identification and
telemetry studies, it appears many inshore areas of
both the Gulf of Mexico and the Northwest
Atlantic harbor resident bottlenose dolphins that
generally remain in a discrete geographic area over
time (Irvine and Wells 1972; Shane et al. 1986;
Wells 1986; Würsig and Lynn 1996; Zolman 2002;
Wells 2003). From a management perspective, the
preservation of each of these resident stocks is
important since repopulation of any area would
theoretically require a recolonization event.
Therefore, in the Gulf of Mexico, each of 33
inshore areas is currently managed as a separate
stock (Waring et al. 2002). Along the eastern coast
of the Gulf of Mexico, running adjacent to many
inshore areas, a separate coastal stock has been
defined as ranging from shore to 9.3 km seaward
of the 18.3 m isobath (Waring et al. 2002). Two
additional bottlenose dolphin stocks range farther
offshore in the Gulf of Mexico (Waring et al. 2002)
but will not be considered in this paper. Due to the
many logistical obstacles involved in studying wild
cetaceans, biological data to support the discrete-
ness of most of these bottlenose dolphin stocks is
lacking.
Bottlenose dolphins inhabiting inshore waters
along the central west coast of Florida have been
the subjects of research since 1970 (Irvine and
Wells 1972; Wells et al. 1980; Scott et al. 1990;
Wells 1991; 2003). While these research efforts
have focused on the resident dolphins in Sara-
sota Bay, FL, considerable research has also
been conducted in the two adjacent inshore
areas, Tampa Bay, FL, to the north, and
Charlotte Harbor, FL (including Pine Island
Sound), to the south (Wells et al. 1996a; b;
Shane et al. 1986). In addition to these inshore
areas, photo-identification data have also been
collected in the neighboring coastal area of the
Gulf of Mexico from Tampa Bay down to
Lemon Bay, FL (Fazioli 1999) and, in the wes-
tern Gulf of Mexico, in Matagorda Bay, TX
(Würsig and Lynn 1996). These studies have all
documented long-term year-round use of each of
these areas by core groups of resident dolphins.
Nevertheless, for the inshore Florida localities,
interactions of dolphins between neighboring
areas are not uncommon. Wells (1986) estimated
17% of group sightings of resident Sarasota Bay
dolphins also included at least one non-resident.
In the adjacent coastal Gulf of Mexico waters,
Fazioli (1999) found 14% of group sightings of
coastal Gulf dolphins also involved at least one
resident Sarasota Bay dolphin. Similarly, mixed
groups containing Sarasota Bay and Tampa Bay
dolphins, and mixed groups containing Tampa
Bay and coastal Gulf dolphins, are also com-
monly observed (Weigle 1990; Wells 1991). These
mixed groups suggest a certain amount of
genetic exchange could occur among dolphins
inhabiting different inshore areas and/or between
dolphins in inshore and coastal areas.
Surprisingly few studies have investigated the
degree of genetic exchange that occurs between
these well documented inshore dolphin stocks.
From the few studies available, it has been
suggested that, while resident, inshore bottlenose
dolphins often remain in their local area over
time, gene flow between adjacent, inshore areas
remains high enough to maintain homogeneity
(Dowling and Brown 1993; Duffield and Wells
2002). Using whole mtDNA restriction fragment
length (RFLP) analysis and six endonucleases,
Dowling and Brown (1993) did not detect sig-
nificant subdivision among samples from the
Gulf of Mexico, although samples sizes were
small in some regions. Duffield and Wells (1991,
2002) found allele frequency differences at three
allozyme loci between Sarasota Bay and both
Tampa Bay and Charlotte Harbor, but did not
detect differences between the latter two bays. A
similar pattern in haplotype frequency differences
was seen using a HinfI digest of whole mtDNA.
(Duffield and Wells 1991, 2002). Consequently,
questions remain as to the level of genetic dif-
ferentiation that exists between coastal and
inshore areas and, on a finer scale, among in-
shore areas. Indeed the extreme mobility of these
dolphins, coupled with a lack of obvious geo-
graphic barriers and field data documenting
long-range movements and mixing of dolphins
from the inshore and coastal areas, would
imply that there is little reproductive isolation
between them. While this is a logical conclusion,
mixing of animals does not necessarily mean
that interbreeding is occurring. This study uses

genetic methods to measure, indirectly, the level
of gene flow among four well documented in-
shore stocks (Tampa Bay, FL, Sarasota Bay,
FL, Charlotte Harbor, FL, and Matagorda Bay,
TX) and one putative coastal stock of bottlenose
dolphins in the Gulf of Mexico. Mitochondrial
DNA control region sequences and nuclear mi-
crosatellites were used to test the null hypothesis
that resident bottlenose dolphins of these inshore
areas and coastal bottlenose dolphins in the Gulf
of Mexico represent one homogeneous popula-
tion. Moreover, if significant genetic differences
do exist between these proximal areas, what
mechanism(s), such as behavior and/or resource
specialization, might explain this differentiation?
Methods
Sample collection
Skin samples from 223 dolphins were collected
between 1996 and 2002 (Figure 1). Skin samples
were collected using a biopsy dart method in
Figure 1. Map showing locations where samples were collected. Sampling areas are depicted by either solid black circles (inshore –
Tampa Bay, Sarasota Bay, Charlotte Harbor, Matagorda Bay) or a hashed box (coastal – Gulf of Mexico).
717
Tampa Bay, Charlotte Harbor, and the coastal
Gulf of Mexico. In Sarasota Bay and Matagorda
Bay, skin samples were collected during live cap-
ture-release projects using a surgical biopsy pro-
cedure. The coastal Gulf study area extended
approximately 12 km offshore from just outside of
Tampa Bay (27.62 N–82.91 W) to the south end
of Lemon Bay (26.84 N–82.49 W), a distance of
approximately 94 km (Figure 1). Genomic DNA
was extracted from the samples using standard
proteinase K digestion and phenol/chloroform
extraction protocols, as outlined in Rosel and
Block (1996). Samples collected via biopsy darting
were sexed using ZFX and SRY specific primers as
described in Rosel (2003). In Sarasota Bay, where
extensive genealogy data were available for many
dolphins, when samples were available from
mothers and their calves, only the sample from the
mother was included in the analyses.
MtDNA sequencing
A 450 bp fragment, including the proline tRNA
and the 5¢ end of the mitochondrial DNA control
718
region, was amplified via PCR using primers
L15824 (5¢- CCTCACTCCTCCCTAAGACT-3¢)
and H16265 (5¢-GCCCGGTGCGAGAAGAGG-
3¢) (Rosel et al. 1999b). Amplification reactions
contained 50 ng DNA, 20 mM Tris–HCl, pH 8.0,
50 mM KCl, 1.5 mM MgC12, 0.3 lM of each
primer, 150 lM dNTPs and 1.25 units of Taq
DNA polymerase. The thermal cycler profile
consisted of initial denaturation at 94 C for 30 s,
followed by 30 cycles of 94 C for 1 min, 52 C for
1 min and 72 C for 1 min, with a final extension
at 72 C for 10 min. PCR products were purified
by excision from a 0.8% SeaPlaque GTG low
melting point agarose gel (FMC, Rockland, ME)
and the agarose bands digested overnight at 40 C
with 5–10 units of agarase (Sigma, St. Louis, MO).
Cycle sequencing reactions were carried out using
the original forward and reverse primers with 3–
6 ll of the agarased product following general
protocols supplied from the manufacturer for ABI
Prism Big Dye Terminator Ready-reaction mix
(Applied Biosystems, Foster City, CA). Cycle
sequencing products were purified either by etha-
nol precipitation or using Centri-Sep spin columns
(Princeton Separations, Adelphia, NJ) and then
sequenced using an ABI 310 Genetic Analyzer.
Sequences were edited using the program Sequence
Navigator 1.0.1 (Applied Biosystems, Foster City,
CA). Consensus sequences were aligned by eye
using the program SeqPup 0.6 (Gilbert 1995) and
used for mtDNA analyses.
MtDNA sequence analysis
Nucleotide (p) and haplotypic (d) diversities (Nei
1987) were estimated for each stock using
Arlequin 2.0 (Schneider et al. 2000). To measure
the degree of population subdivision, an analysis
of molecular variance (AMOVA, Excoffier et al.
1992) was run using Arlequin 2.0. The AMOVA
calculates a measure of population subdivision
based on haplotype frequency data alone, FST
(Wright 1965), and a measure, FST, based on
both haplotype frequency and genetic distance
data. The Tamura–Nei model (Tamura and Nei
1993) was used to estimate the evolutionary dis-
tances between sequences (as recommended for
control region sequence data; Kumar et al. 1993)
with a gamma correction (a = 0.17) estimated by
maximum likelihood using PAUP 4.0b (Swofford
1998). To examine possible differences in gene
flow among the sexes, FST estimates were also
obtained for males and females separately. Sta-
tistical significance of FST and FST estimates was
estimated using a permutation simulation with
10,000 permutations of haplotypes among popu-
lations and the sequential Bonferroni test was
used to correct probability values for multiple
comparisons (Rice 1989). The extent of geo-
graphic heterogeneity in haplotype frequency
distributions was further assessed with Monte
Carlo simulations of v2 values as described in
Roff and Bentzen (1989) with 10,000 random-
izations of the original data matrix using the
program REAP (McElroy et al. 1992). This pro-
cess of performing repeated randomizations of
the original data matrix is intended to reduce the
effect of large numbers of small cell counts on the
validity of the v2 procedure (Roff and Bentzen
1989). Unlike Wright’s F-statistics, which are
parameters, v2 is a true test of statistical signifi-
cance and thus the use of this statistic has been
purported to be more powerful at detecting
haplotype frequency differences than sequence
based statistics (Hudson et al. 1992). To illustrate
the relationship among haplotypes, a minimum
spanning network was constructed using the
program Network 2.0 (Bandelt et al. 1999).
Microsatellite genotyping
All samples were genotyped at nine dinucleotide
microsatellite loci: Ttr04, Ttr11, Ttr19, Ttr34,
Ttr48, Ttr63 (Rosel et al. 2005); MK8 (Krützen
et al. 2001); EV14, EV37 (Valsecchi and Amos
1996). Amplification reactions contained 25–
50 ng DNA, 20 mM Tris–HCl, pH 8.0, 50 mM
KC1, 1.5 mM MgC12, 0.3 lM of each primer,
150 lM dNTPs and 0.75 units of Taq DNA
polymerase. The thermal cycler profile consisted
of initial denaturation at 94 C for 30 s, followed
by 30 cycles of 94 C for 20 s, 55–62 C for 20 s
and 72 C for 40 s, with a final extension at
72 C for 10 min. All reactions included both
positive and negative controls. Following ampli-
fication, samples were mixed with an internal size
standard and run on either an ABI 310 or ABI
3100 Genetic Analyzer. Genescan Analysis 3.1
and GeneMapper 3.5 software (Applied Biosys-
tems, Foster City, CA) were used for sizing of
allele fragments.

Microsatellite analyses
Allele frequencies, observed heterozygosity (Ho),
expected heterozygosity (He), Hardy–Weinberg
exact tests (Guo and Thompson 1992) and tests for
heterozygote deficiency were carried out using
GENEPOP 3.3 (Raymond and Rousset 1995).
Estimates of allelic richness per population, a
measure of the number of alleles per locus inde-
pendent of sample size (Petit et al. 1998), were
calculated using the program FSTAT (Goudet
2002). To test whether the microsatellite loci were
independently inherited, tests for linkage disequi-
librium were carried out using Arlequin 2.0
(Schneider et al. 2000). For tests of Hardy–Wein-
berg equilibrium (HWE) and linkage disequilib-
rium, the sequential Bonferroni correction was
applied to correct probability values for multiple
comparisons (Rice 1989). For each area where
samples were collected via biopsy darting, indi-
vidual genotypes were compared against each
other to identify possible duplicate samples. The
probability of identity calculation from Paetkau
et al. (1995) was then used to obtain the proba-
bility that two different individuals in our dataset
could share the same genotype across all loci.
The inbreeding coefficient, FIS (Weir and
Cockerham 1984), was estimated for each popu-
lation using FSTAT where significance values were
obtained by permuting alleles 1000 times within
populations (Goudet 2002). As a further means of
testing whether samples collected from each of the
populations contained a significantly high number
of related individuals, Queller and Goodnight’s
index of relatedness was used to obtain average
and pairwise relatedness values for individuals of
each population (R; Queller and Goodnight 1989).
The distribution of pairwise R values per popula-
tion was then compared to a distribution gener-
ated from the random simulation of 1000
unrelated pairs using each population’s allele fre-
quencies. The simulations were carried out using
KINSHIP 1.2 (Goodnight and Queller 1999) and
the distributions were statistically compared using
a Komogorov–Smirnov two-sample test.
The common estimator of population subdivi-
sion, FST (Wright 1965), which assumes the infinite
allele model (IAM, Crow and Kimura 1970), and a
statistic more specific to microsatellite data, RST
(Slatkin 1995), which assumes a stepwise mutation
model (SMM, Kimura and Ohta 1978), were used
719
to estimate population subdivision using the
microsatellite data. Based on the differences in
these models, FST takes into account allele fre-
quencies, whereas RST takes into account both
allele frequencies and genetic distance. To obtain
an FST estimate, an AMOVA was run using the
program Arlequin 2.0 (Schneider et al. 2000). As
with the mtDNA data, FST estimates were also
obtained for males and females separately to
examine possible differences in gene flow among
the sexes. An unbiased version of RST, RhoST, in
which allele sizes are transformed to standard
variances, was calculated using the program RST
CALC (Goodman 1997). For probability values of
both FST and RhoST estimates, the sequential
Bonferroni test was used to correct for multiple
comparisons (Rice 1989).
We used the program MIGRATE 2.0.3 to
estimate asymmetric rates of migration between
the populations using the microsatellite data set to
the Brownian mutation model (Beerli and Fel-
senstein 2001). MIGRATE uses a coalescent-
based maximum likelihood approach to estimate
migration assuming constant effective population
size while allowing for unequal subpopulation si-
zes (Beerli and Felsenstein 2001). An initial run
used FST based estimates of Nm and h and 10 short
chains with 10,000 sampled genealogies and three
long chains with 100,000 sampled genealogies
(with a burn-in of 10,000). Resulting Nm and h
values were then inputted as initial parameters for
a second run using 20 short chains with 20,000
sampled genealogies and five long chains with
200,000 sampled genealogies.
Results
Mitochondrial DNA
After all consensus sequences were aligned and
tRNA sequence trimmed, a final 359 bp fragment
of the mtDNA control region was used for
analyses. A total of 15 polymorphic sites defined
11 unique haplotypes among the 223 individuals.
One site contained an insertion/deletion while the
remaining 14 polymorphic sites were transition
mutations. The most common haplotype was
shared among the Sarasota Bay, Tampa Bay,
Charlotte Harbor and coastal Gulf populations,
but this haplotype was not found in the Matag-
720

Table 1. Distribution of mtDNA haplotypes among sampling locations

Mitochondrial DNA haplotype

Population 1 2 3 4 5 6 7 8 9 10 11

Gulf of Mexico 1 3 18 5 15 8 6 0 0 0 0
Sarasota Bay 0 0 18 0 0 3 15 0 0 0 0
Tampa Bay 0 1 34 0 0 6 3 1 1 0 0
Charlotte Harbor 0 0 30 0 0 7 13 1 0 0 0
Matagorda Bay 0 0 0 0 0 1 10 0 0 16 7

Table 2. Total number of females (F) and males (M) sampled per population as well as the total individuals sampled (N) MtDNA
genetic diversity estimates +/) standard error.

Location F M N Haplotype diversity Nucleotide diversity

Gulf of Mexico 24 32 56 0.796±0.028 0.009±0.005

Sarasota Bay 20 16 36 0.586±0.042 0.011±0.006
Tampa Bay 17 29 46 0.441±0.085 0.007±0.004
Charlotte Harbor 9 42 51 0.581±0.055 0.010±0.006
Matagorda Bay 15 19 34 0.668±0.046 0.003±0.002

orda Bay population (Table 1). Unique haplotypes
were found in the Tampa Bay, Matagorda Bay,
and coastal Gulf populations, but not in Sarasota
Bay or Charlotte Harbor. Haplotype diversity
estimates ranged from 0.441 in Tampa Bay to
0.796 in the coastal Gulf of Mexico. Nucleotide
diversity estimates ranged from 0.003 in Matag-
orda Bay to 0.011 in Sarasota Bay (Table 2).
Relationships among the eleven unique haplotypes
and their relative frequency are shown as a mini-
mum spanning network in Figure 2. Sequences
were deposited into GenBank under Accession
numbers AY997307 to AY997311.
Results from the AMOVA analyses using the
mtDNA sequence data indicated a significant
amount of genetic variation among all areas using
both frequency information alone (FST = 0.186,
P < 0.001), and haplotype frequency and genetic
distance information combined (FST = 0.323,
P < 0.001). After sequential Bonferroni correc-
tion, FST values showed significant differentiation
for all pairwise population comparisons except for
the comparisons of Sarasota Bay to Charlotte
Harbor and Tampa Bay to Charlotte Harbor
(Table 3). Although fewer pairwise comparisons
were found to be significant when both haplotype
frequency and genetic distance data were com-
bined (FST), the overall partitioning of the genetic
variation among areas was greater using this esti-
mate. Based on v2 randomizations, the observed

Figure 2. Minimum spanning network showing the relationships among 11 bottlenose dolphin haplotypes. Haplotype numbers
correspond to those listed in Table 1. The size of each circle approximates the relative frequency of the haplotype in the total sample.
Each hash mark represents one mutational event.
 721

Table 3. Estimates of population differentiation for all pairwise population comparisons for mtDNA and microsatellite data where
*P<0.05, **P<0.01, ***P<0.001 following sequential Bonferroni correction

Populations Mitochondrial DNA Microsatellites

FST FST v2 FST RST

SB–TB 0.137*** 0.185*** 15.94* 0.027*** 0.003

SB–CHB 0.008 0.005 3.25 0.032*** 0.008
TB–CHB 0.036 0.073 8.34 0.024*** 0.022**
GOM–SB 0.113*** 0.115** 27.06* 0.042*** 0.030**
GOM–TB 0.155*** 0.062 29.51* 0.030*** 0.056***
GOM–CHB 0.098** 0.051 30.48* 0.026*** 0.022**
MTB–SB 0.284*** 0.491*** 42.98* 0.043*** 0.048**
MTB–TB 0.441*** 0.753*** 67.05* 0.042*** 0.060***
MTB–CHB 0.325*** 0.553*** 57.80* 0.032*** 0.035**
MTB–GOM 0.236*** 0.642*** 70.27* 0.050*** 0.029**

SB: Sarasota Bay, TB: Tampa Bay, CHB: Charlotte Harbor, GOM: coastal Gulf of Mexico, MTB: Matagorda Bay.

distribution of haplotypes was significantly heter-
ogeneous for all pairwise population comparisons
except for the comparisons of Sarasota Bay to
Charlotte Harbor and Tampa Bay to Charlotte
Harbor (Table 3). Sample sizes per sex, as deter-
mined either in the field or using genetics, are
shown in Table 2. When analyzed separately, both
males and females showed significant differentia-
tion (FST = 0.153, P < 0.001 and FST = 0.240,
P < 0.001, respectively).
Microsatellite results
The number of alleles per microsatellite locus
ranged from 2 for Ttr19 in the Sarasota Bay
population to 20 for EV37 in the coastal Gulf
population. No locus showed significant evidence
of genotypic disequilibrium nor deviated signifi-
cantly from Hardy–Weinberg equilibrium (HWE)
in any population following sequential Bonferroni
correction. The average number of alleles per locus
was highest in the coastal Gulf population and
lowest in Sarasota Bay (Table 4). Out of a total of
27 unique alleles, eleven were found in the coastal
Gulf population, nine were found in Matagorda
Bay, five were found in Charlotte Harbor, and two
were found in Tampa Bay. For eight out of nine
loci, all populations shared the most common al-
lele except the Matagorda Bay population which
had a different most common allele at four of the
nine loci. Average observed heterozygosity values
ranged from 0.580 in Matagorda Bay to 0.700 in
the coastal Gulf of Mexico (Table 4). FIS estimates
and their corresponding P-values showed no
indication of significant inbreeding in any popu-
lation (Table 5).
From the probability of identity estimates, the
probability of two dolphins sharing the same
genotype across all nine loci was highest for Sara-
sota Bay (3.710)8) and lowest for the coastal Gulf
of Mexico (6.210)10, Table 4). In total, nine pairs
of biopsy samples were identified as having identi-
cal genotypes across all nine loci. Based on the low
probability of identity values for all populations,
one of each of these pairs was considered to be a
duplicate sample and removed from all analyses.
Average relatedness values per population ranged
from )0.005 to 0.001 (Table 5). Results from the
comparison of pairwise R value distributions per
population to distributions generated from the

Table 4. Estimates of the average number of alleles (A), allelic richness (RS), observed heterozygosity (Ho), expected heterozygosity
(He), and probability of identity (PID) per population

A Rs Ho He P(ID)

Gulf of Mexico 9.4 8.6 0.700 0.720 6.210)10

Sarasota Bay 6.4 6.4 0.627 0.636 3.710)8
Tampa Bay 8.1 7.6 0.597 0.627 3.510)8
Charlotte Harbor 8.4 7.7 0.601 0.633 1.210)8
Matagorda Bay 7.3 7.3 0.580 0.624 1.710)8
722

Table 5. FIS values and corresponding significance values estimated by FSTAT, average relatedness estimates (R) per population +/)
the standard error and significance values from Kolmogorov–Smirnov two-sample tests comparing the distribution of pairwise R
values per population to the distribution generated from a random simulation of 1000 pairs using the population allele frequencies

Location FIS P-value R ± SE P-value

Gulf of Mexico 0.022 0.17 )0.001 ± 0.005 0.06

Sarasota Bay 0.005 0.48 )0.005 ± 0.009 0.36
Tampa Bay 0.038 0.09 0.001 ± 0.007 0.37
Charlotte Harbor 0.039 0.06 )0.002 ± 0.005 0.24
Matagorda Bay 0.053 0.06 )0.004 ± 0.009 0.76

Table 6. MIGRATE asymmetric migration rates (4Nem)

Source population Receiving population

GOM SB TB CHB MTB

GOM – 25.2 9.7 22.7 20.8

SB 16.7 – 23.3 12 7.7
TB 7.5 22.4 – 11.3 10.1
CHB 11.2 17.5 17 – 13.7
MTB 14.1 8.5 11.4 14.7 –

SB: Sarasota Bay, TB: Tampa Bay, CHB: Charlotte Harbor, GOM: coastal Gulf of Mexico, MTB: Matagorda Bay.

random simulation of 1000 unrelated pairs showed
that no population contained a significantly higher
number of related individuals than expected in a
randomly mating population (Table 5).
AMOVA results from the microsatellite
data indicate significant genetic structure among
all five populations (FST = 0.034, P < 0.0001;
RhoST = 0.032, P < 0.0001) and all pairwise pop-
ulation comparisons resulted in significant FST
values (Table 3). As with the mtDNA data, when
analyzed separately, both males and females
showed significant differentiation (FST = 0.032,
P < 0.001 and FST = 0.046, P < 0.001, respec-
tively). All pairwise population comparisons were
also significant using the RhoST estimator except for
the comparisons of Sarasota Bay to Tampa Bay and
Sarasota Bay to Charlotte Harbor (Table 3).
Asymmetrical migration rate estimates produced by
MIGRATE ranged from 7.5 to 25.2 (4Nem,
Table 6).
Discussion
Population structure
Results from this study reveal fine scale geographic
structure of bottlenose dolphins in the Gulf of
Mexico. Based on the mtDNA data, when haplo-
type frequency and genetic distance are consid-
ered, approximately 32% of the total variation
detected can be attributed to differences among
these populations (FST = 0.323). In context of
other mtDNA studies, this level of differentiation
is similar to that found among beluga whales of
five summer areas in Alaska and north–west
Canada (O’ Corry-Crowe et al. 1997), and greater
than that found among many other cetacean
populations (Dizon et al. 1994; Rosel et al. 1999a;
Escorza-Trevino and Dizon 2000). When just
haplotype frequency information was considered
for population structure analyses, the percentage
of variation attributed to differences among pop-
ulations was reduced but remained significant (FST
0.186, P < 0.001). The FST estimator is strongly
influenced by the presence of the geographically
isolated Matagorda Bay samples. These samples
are dominated by a haplotype not found in any of
the eastern populations and this haplotype differs
from the most common haplotype in the other
populations by a relatively high number of nucle-
otide substitutions (see Figure 2). The microsatel-
lite data also revealed significant differentiation
between Matagorda Bay and all Florida inshore
populations. These results suggest there has been
very little recent genetic exchange between the
Matagorda Bay and eastern Gulf populations.
Interestingly, from the minimum spanning net-

work, it appears that two of the three most com-
mon haplotypes (6 and 7) among the Florida in-
shore populations are genetically distant from the
third (3), and appear to be more closely related to
the two haplotypes unique to Matagorda Bay (10
and 11) than the other haplotypes found in the
eastern Gulf. One explanation would be that
haplotypes 3, 6, and 7 were the most common
haplotypes of the source population from which
these inshore populations were founded (discussed
below). The fact that the less common haplotypes
in the coastal Gulf, Tampa Bay and Charlotte
Harbor populations appear to have radiated from
the most common haplotype in these populations,
haplotype 3, also supports this hypothesis.
Excluding Matagorda Bay, significant popula-
tion differentiation was also found solely among
the eastern Gulf sampling sites. However, the
mtDNA and microsatellite data differ in their
estimates for two inshore population pairwise
comparisons: Sarasota Bay to Charlotte Harbor
and Tampa Bay to Charlotte Harbor (Table 3).
Although one possible explanation for this could
be female-biased dispersal, higher overall FST
estimates for females alone (vs. males) and differ-
entiation detected with both mtDNA and nuclear
markers for all other pairwise comparisons argue
against this. Instead, two characteristics of the
data may explain the discrepancies between the
mtDNA and nuclear population structure esti-
mates. First, low haplotype variability (11 unique
haplotypes in 223 samples) compared to generally
high microsatellite diversity may translate to the
microsatellites being a more sensitive indicator of
population structure. The low level of variability
in the mtDNA sequences could be an artifact of
recent colonization of the inshore bays following a
founder event (discussed below). This effect of low
haplotypic diversity on the population structure
estimates helps to explain why previous studies of
population structure of bottlenose dolphins in this
area, using RFLP analysis of mtDNA markers
(Duffield and Wells 1991, 2002; Dowling and
Brown 1993), did not detect the differences
reported here using microsatellite loci. A second
factor that is likely contributing to these results is
the bias in sample size for males (n = 42) vs.
females (n = 9) in the Charlotte Harbor sample.
Not only is much power lost in the very small
sample size for females but, concurrently, if some
males are moving between populations, the effect
723
of sampling a male from a neighboring area and
erroneously including it in the Charlotte Harbor
sample would further reduce the accuracy of ge-
netic diversity estimates in the population, thereby
effecting estimates of genetic structure. In the ab-
sence of additional female samples from this
population, we cannot rule out the effect this bias
is having on the population comparisons involving
Charlotte Harbor. These discrepancies between
mtDNA and microsatellite FST estimates illustrate
the importance of utilizing more than one type of
marker when making inferences about the genetic
structure of populations.
The significant differentiation detected between
all eastern Gulf sampling sites using the micro-
satellite data is surprising given the relative
mobility of these dolphins, the close proximity of
these populations, the evidence for mixed groups
(Wells 1986; Fazioli 1999) and reported move-
ments of animals (both males and females) be-
tween these different areas (Duffield and Wells
1991). Wells (1986) and Fazioli (1999) have doc-
umented mixed groups containing both inshore
and coastal bottlenose dolphins in these areas.
Though Wells (1986) did note that, for Sarasota
Bay dolphins, mixing was more frequent outside of
the primary breeding season. While mixed groups
may provide significant opportunity for genetic
exchange to occur between the coastal Gulf and
Sarasota Bay populations, genetic results indicate
that, although the opportunity may be present,
little interbreeding is occurring. Similar results
have been found for other cetacean species where
significant population subdivision was detected
despite observational data documenting a high
occurrence of mixed groups (in beluga whales,
Brennin et al. 1997; in humpback whales, Valsec-
chi et al. 1997 and in Tursiops sp., Wang et al.
1999). One potential concern is that the population
structure detected here among the inshore popu-
lations reflects a high degree of relatedness among
sampled animals within each bay rather than true
population structure between bays. Genealogy
data are only available for Sarasota Bay dolphins
and, based on these data, we can say that no
known first-order relatives were included in that
dataset. Tests for significant FIS values did not
reveal evidence for inbreeding in any of the pop-
ulations sampled. Furthermore, it is unlikely that
these bays simply contain a single family group of
dolphins that has moved into the area for only a
724
generation or two. The observation of at least four
generations in Sarasota Bay is documented (Duf-
field and Wells 2002) and the abundance estimate
of 120 Sarasota Bay resident dolphins given by
Wells (2003) is too large to be comprised of a
single family group. Finally, in examining the
distributions of relatedness values for each popu-
lation, we found no evidence for significantly ele-
vated R values within populations, as no
population had a distribution that differed signif-
icantly from that of a randomly generated popu-
lation of unrelated individuals (Table 5). Taken
together, this evidence indicates that these are
populations subjected to evolutionary forces
rather than family groups occupying the inshore
habitats for only a generation or two.
Population history
Overall, the coastal Gulf population had the
highest genetic diversity as measured by haplo-
typic diversity, allelic richness, and microsatellite
heterozygosities. Although abundance estimates
are not available for all areas, it is generally
thought that the coastal Gulf population is larger
in size than the resident inshore populations.
Wells (2003) estimates 120 resident dolphins
currently inhabit Sarasota Bay, while abundance
for bottlenose dolphins in the eastern coastal
Gulf of Mexico stock (between 84 W longitude
and Key West, Florida) has been estimated at
9912 dolphins (Waring et al. 2002). The larger
population in the coastal Gulf would be able to
maintain a higher genetic diversity than the
smaller, insular inshore populations. It is also
possible that the inshore bottlenose dolphin
populations along the central west coast of
Florida represent founder populations originating
from the larger coastal population. This hypoth-
esis would help to explain the fact that the
majority of unique haplotypes and microsatellite
alleles were found in the coastal Gulf rather than
the inshore populations. That is, the inshore
populations would carry only a small proportion
of the genetic variation from the larger source
population, as certain alleles and haplotypes that
were present in the larger population would have
been lost during the founding events. The founder
hypothesis has been suggested by other studies
investigating population structure of bottlenose
dolphins in the Gulf of Mexico (Natoli et al.
2004) and western North Atlantic (Hoelzel et al.
1998).
The fact that no fixed differences were found
between the eastern inshore and coastal Gulf
populations suggests that either isolation was a
relatively recent event and/or a low level of gene
flow is still occurring among them. From geolog-
ical data, it is clear that the formation of the
inshore bays and estuaries along Florida’s west
coast, and therefore the founding of these areas by
bottlenose dolphins, were relatively recent events
(Davis 1997). This may explain the lack of fixed
differences between the eastern inshore and coastal
Gulf populations. Alternatively, low levels of
genetic exchange may also be ongoing. Indirect
estimates of migration from the genetic data are
concordant with those documented from obser-
vational data (Wells 1986) and relatively low, but
non-zero. Migration estimates presented here do
need to be taken with caution as certain assump-
tions of the underlying models, such as that of
constant effective population size and sampling of
all source populations, are potentially violated
with the data at hand. Nevertheless, private alleles
are seen in four of the five populations suggesting
that the low level of gene flow is not enough to
homogenize these populations.
Resource specialization and social structure
While founder events may have originally estab-
lished the inshore populations, what has main-
tained their isolation from the coastal Gulf? We
suggest differences in habitat and resource use are
mechanisms by which this differentiation has
evolved and/or is maintained. Although the in-
shore dolphins have easy access to the nearby
more open coastal Gulf waters, they spend the
majority of their time in the shallower, more
confined waters of the bays and estuaries. It may
be that these areas provide more protection from
predators (sharks) or that particular prey species
are more abundant in the inshore habitats (Wells
et al. 1980; Connor et al. 2000). A certain degree
of resource specialization with respect to prey has
been documented for the Sarasota Bay and coastal
Gulf populations. In a Barros and Wells (1998)
study, the distribution of Sarasota Bay feeding
events was correlated with prey habitat distribu-
tion and results indicated that feeding behaviors
were most often observed near seagrasses. As

pinfish are often the dominant fish species in
Florida seagrass meadows (Stoner 1983), and a
primary prey species of Sarasota resident dolphins
(Barros and Wells 1998), one likely factor con-
tributing to the distribution of resident Sarasota
Bay dolphins is the distribution of the seagrass
meadows harboring their primary prey. In con-
trast, analysis of stomach contents of dolphins
stranded on Gulf coastal beaches indicates that,
from 1985 to 1996, 21% of the dolphins consumed
cephalopods, a prey item almost entirely absent in
the Sarasota Bay residents (Barros and Wells
1998). Similar differences have been found for
bottlenose dolphin populations on the east coast
of Florida where dolphins in the Indian River
Lagoon system are almost exclusively piscivorous,
whereas dolphins found in the adjacent coastal
waters have a mixed diet of fish and squid (Barros
1993). These differences, though not as extreme,
are not unlike differences found in the diets of
resident and transient killer whales (Matkin and
Saulitis 1994). Hoelzel and Dover (1991) suggested
that differing foraging strategies between sympat-
ric killer whale populations served as a behavioral
isolating mechanism by which genetic differences
evolved. As with killer whales, resource special-
ization with respect to prey is most likely a key
factor contributing to the separation of inshore
and coastal bottlenose dolphin populations.
Among the inshore areas, it is likely that the
complex social structure of these dolphins is con-
tributing to heterogeneity. Long-term social rela-
tionships documented in bottlenose dolphins
include the formation of male pairs and female
bands in Sarasota Bay (Wells 1991; Owen et al.
2002; Wells 2003) and first-order and second-order
male alliances in Shark Bay, Western Australia
(Connor et al. 1992). Still some bottlenose dol-
phins in these populations, both males and fe-
males, appear to remain more solitary, without
forming any strong social relationships (Wells
1991). Given these social affiliations, it is not sur-
prising that our genetic data support previous data
from field observations, that bottlenose dolphins
in these populations exhibit site philopatry (Wells
1991). Though male-biased dispersal is well doc-
umented in many other cetacean species (Baker
et al. 1993; Rosel et al. 1999a; Escorza-Trevino
and Dizon 2000), and recently in bottlenose dol-
phins (T. aduncus Möller et al. 2004), our results
indicate that both males and females exhibit some
725
degree of natal site philopatry, as indicated by
significant FST values for each sex based on both
mtDNA and nuclear microsatellite data. These
results are similar to those reported for bottlenose
dolphins in Shark Bay, Western Australia, where
males have been shown to disperse farther than
females yet still exhibit natal philopatry (Tursiops
sp. Krützen et al. 2004) and, more recently, to a
global study of bottlenose dolphins where signifi-
cant structure was detected using both mtDNA
and nuclear markers (Natoli et al. 2004).
Management implications
Current threats to bottlenose dolphins in the Gulf
of Mexico include entanglement in recreational
and commercial fishing gear, large scale die-offs
due to epizootic events and undetermined causes,
environmental contaminants and other pollu-
tants, and disturbance and collisions from
increasing boat traffic in coastal areas. The
anthropogenic threats mentioned are especially
relevant to the inshore dolphin populations on
the central west coast of Florida where human
population size, development, and boat traffic are
increasing at an alarming rate. Although bottle-
nose dolphins are not endangered, the long-term
research conducted on these populations allows a
unique opportunity to monitor not only the
health of these populations, but also the health of
their habitat and ecosystem over long periods of
time. The genetic data presented here indicate
that inshore waters do harbor semi-isolated
populations of bottlenose dolphins. As a result,
ecosystem health and monitoring of a single
location used as a proxy for other adjacent areas
may not be adequate. Furthermore, the relatively
low estimates of migration presented here, cou-
pled with a generation time of approximately
10 years when estimated as age at first repro-
duction (Wells 2003), means that, should one of
these populations be lost, recolonization to
genetically healthy population sizes could take on
the order of several hundred years. Such an event
would violate the mandate under the US Marine
Mammal Protection Act requiring that popula-
tions remain functioning elements of their eco-
systems. Thus, genetic results from this study
support the current identification of separate
management units (Waring et al. 2002) for dif-
ferent embayments in inshore areas of the Gulf of
726
Mexico. These results also indicate the occurrence
of a separate eastern coastal Gulf stock for dol-
phins inhabiting the nearshore waters along the
west coast of Florida. Whether the current putative
eastern coastal Gulf stock is comprised of more
than one population needs to be examined. In
addition, future studies will reveal whether similar
genetic differentiation exists among the other 29
defined stocks of bottlenose dolphins in the bays,
sounds and estuaries of the Gulf of Mexico, and
whether some of the larger bays may harbor more
than one genetically distinct stock or population.
Acknowledgments
We are grateful to the following people for their
assistance with sample collection: E. Zolman, J.
Thera, M. Alsina, T. Speakman, K. Fazioli, A.
Westgate, K. Hull, S. Hofmann and the entire staff
of the Sarasota Dolphin Research Program. This
work also benefited from discussions with and
assistance from L. Adams, S. Kingston, K. Tolley,
N. Barros, M. Krützen, L. Schwacke, A. Strand
and D. Queller. Funding and/or logistical support
for this research was provided by the Florida Fish
and Wildlife Conservation Commission, Chicago
Zoological Society, Dolphin Quest, Mote Marine
Laboratory, Sarasota Dolphin Research Program
and the U. S. National Marine Fisheries Service
(NMFS). Biopsy sampling was carried out under
NMFS Scientific Research Permits No. 522-1527
and No. 522-1569.
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