J. CETACEAN RES. MANAGE. 7(1):65–70, 2005

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A preliminary note on population structure in eastern South
Pacific common bottlenose dolphins, Tursiops truncatus
G. PAOLO SANINO*+ , KOEN VAN WAEREBEEK#*, MARIE-FRANÇOISE VAN BRESSEM# AND LUIS A. PASTENE++
Contact e-mail: research@leviathanchile.org
ABSTRACTPrevious studies of eastern South Pacific common bottlenose dolphins, Tursiops truncatus, defined offshore and inshore ecotypes in Peru
based on cranial and tooth morphology, documented the presence of a single resident inshore community (‘pod-R’) in central-north Chile,and confirmed the presence of offshore bottlenose dolphins off Chile. Here, mtDNA control region (331bp) was examined to evaluate
genetic relationships between four geographic areas: inshore pod-R (n=8), Chilean offshore population (n=8), Peruvian inshore (n=3) and
offshore (n=12) ecotypes. This is the first genetic analysis of T. truncatus in this ocean basin. Phylogenetic analysis grouped the three
Peruvian specimens morphologically identified as inshore ecotype in an independent cluster, supported by 100% bootstrap value. The net
genetic distance between Peruvian inshore and Peruvian offshore ecotypes was estimated at 2.9%, and even higher when compared with
Chilean bottlenose dolphins. Morphological and mtDNA evidence combined argues for considering inshore and offshore ecotypes as
evolutionary significant units, to be managed accordingly. Despite its inshore behavioural ecology, pod-R presented a high divergence fromthe Peruvian inshore ecotype and a relatively closer affinity with the Chilean offshore stock (3.41% and 0.87% net interpopulational
distance, respectively). However, homogeneity tests showed significant genetic differences of pod-R with all other groups, including
Chilean offshore. This, combined with a low nucleotide diversity (0.0069) and behavioural observations, suggest that pod-R may be
reproductively isolated and active protection measures are recommended. Only one haplotype from a total of 21 was shared by Peruvian
and Chilean offshore animals. Their net genetic distance was estimated at 0.024 and no significant differences were found in haplotype
frequencies, suggesting a single, wide-ranging ‘Peru-Chile offshore stock’.
KEYWORDS: GENETICS; STOCK IDENTITY; COMMON BOTTLENOSE DOLPHIN; PACIFIC OCEAN; SOUTH AMERICA;TAXONOMY; CONSERVATION; MANAGEMENTINTRODUCTION
While the taxonomic status of the Tursiops genus(bottlenose dolphins) is still under discussion, most authors
including ourselves presently recognise two species, one ofcosmopolitan distribution, the common bottlenose dolphin,T. truncatus, Montagu, 1821 and the Indo-Pacific bottlenose
dolphin, T. aduncus, Ehrenberg, 1833 (Ross, 1977; Curry,
1997; LeDuc, 1997; Hale et al., 2000; Krützen et al., 2001;
Shirakihara et al., 2003 and Wang et al., 1999). However the
existence of other species or subspecies cannot be ruled
out.
Using general comparative morphology, cranial, diet andparasite load differences, as well as mtDNA analysis, twodistinct common bottlenose dolphin ecotypes, offshore andinshore (syn. coastal), have been described for the eastern
North Pacific (USA), western North Pacific (China andJapan), western North Atlantic (USA), eastern South Pacific(Peru), western South and Indo-Pacific (Australia andSolomon Islands), eastern South Atlantic (Namibia, South
Africa), and the east coast of South Africa (Ross, 1977;
1984; Walker, 1981; Duffield et al., 1983; Ross andCockcroft, 1990; Van Waerebeek et al., 1990; Findlay et al.,
1992; Mead and Potter, 1995; Hoelzel et al., 1998 and Wanget al., 1999).
In the Southeast Pacific Ocean, common bottlenose
dolphins are known to occur from the Galápagos
archipelago, continental Ecuador, the entire coast of Peru,
northern and central Chile south to at least Quenu Island, off

Calbuco, X Region (41°45’S, 73°10’W)1 and the offshore
Chilean archipelagos of San Ambrosio and San Félix, Salas
and Gómez Islands and the Juan Fernández Islands (e.g.
Lévèque, 1963; Aguayo, 1975; Donovan, 1984; Guerra etal., 1987; González et al., 1989; Van Waerebeek et al., 1990;
Félix, 1994; Félix and Samaniego, 1994; Findlay et al.,
1998; Sanino and Yáñez, 2001).
Van Waerebeek et al. (1990) found distinct cranialdifferences between offshore and inshore ecotypes ofbottlenose dolphins in Peru, as well as clear differences indiets and helminth parasite loads, suggesting reproductively
isolated populations. Santillán (2003), studying a larger
sample, confirmed cranial differences. Recent management
concerns, including direct takes (Sanino and Yáñez, 2000;
2001) led us to biopsy animals from an inshore dwelling
community, named pod-R, at Choros Island (29°15’S,71°26’W) in central-north Chile. These were originally
thought to form part of a wider coastal population ofbottlenose dolphins.
Video-identification studies revealed high site-fidelity of
the 28 pod-R members, as well as morphological andbehavioural differences in observed pods of offshore
bottlenose dolphins (Sanino and Yáñez, 2001). The issue
was raised as to whether pod-R bottlenose dolphins are
1 Live-stranding of two adult bottlenose dolphins, trapped by fastreceding tide, on 2 August 2004 (CMMR files, positive identificationfrom photographic evidence published in El Llanquihue No. 36.642, 4August 2004; Sociedad Periodística Araucanía, Puerto Montt, Chile).

* Centre for Marine Mammals Research 2LEVIATHAN, Chile.
+ National Museum of Natural History 2MNHN, Casilla 787, Santiago, Chile.
# Peruvian Centre for Cetacean Research (CEPEC), Museo de Delfines, Pucusana, Peru.
++ The Institute of Cetacean Research, 4-5 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan.

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largely reproductively isolated or are part of, and mix with,other communities to form a wide-ranging Chilean coastal
population.
In this study2 mtDNA control region sequences were used
to examine genetic diversity and phylogenetic relationships
among common bottlenose dolphins from different
localities in the southeast Pacific Ocean, with particularemphasis given to pod-R dolphins in central-north Chile and
the Peruvian inshore ecotype.
MATERIALS AND METHODS
Samples and localities
Sampling localities and sample sizes for the bottlenose
dolphins are as follows (Fig. 1): inshore Choros Island(Chile) (CL-I, n=8), offshore Chile (CL-O, n=8), Peruvian
inshore (PE-I, n=3) and samples from Peru that included
individuals from both confirmed offshore ecotype andindeterminate but like-offshore specimens for which noskulls were collected (PE-O, n=12). Morphologicalcharacteristics used to distinguish ecotypes in Peru include
tooth diameter, the morphology of pterygoids, palatine
bones, antorbital process and the separation of occipital
condyles (Van Waerebeek et al., 1990). Accompanying
fisheries data were also taken into account, especially if thelanding process was monitored by one of the authors. All
tissue samples from Peru (PE-I and PE-O) were taken fromeither freshly landed specimens captured in a variety offisheries, or from body remains found on beaches nearfishing towns. Most were stored in dimethylsulphoxide(DMSO) solution with the remainder being stored in 70%
ethanol.
CL-I skin samples were collected using a ‘Golden Bear’
long-bow with modified darts, as described in IWC (1991),
mounted with a 6mm (diameter) tip. The samples were
soaked in 70% ethanol for three weeks, after which thehypodermis was eliminated and the epidermis/dermis wastransferred to a DMSO saturated saline solution.
Examination of photographs and videotapes for individual
identification precluded the possibility of more than one
sample coming from the same animal. CL-O samples werecollected with a Barnett crossbow and tethered bolts (IWC,1991) from the bow of the R/V Shonan Maru 2 during thethird blue whale cruise of the IWC/SOWER programme(Findlay et al., 1998). Sampling error was considerednegligible as biopsies of bowriding specimens were taken
near-randomly over a wide area (8° of latitude).
Extraction of DNA
Total cell DNA was extracted from samples of skin or othertissue. DNA extractions followed phenol/chloroform/
isoamyl/alcohol protocols as described by Sambrook et al.
(1989). Extracted DNA was resuspended in 500ml 0.1M
Tris-HCl (pH 8.0), 0.05 mM EDTA.
Amplification of mtDNA control region
The first 500 nucleotides at the 5’ end of the mitochondrial
control region were amplified by the polymerase chain
reaction. The oligonucleotides employed in the PCR
amplification were MT4 (Arnason et al., 1993%) and P2R(5’-GAA GAG GGA TCC CTG CCA AGC GG-3’).
Reactions were carried out in 50 mL volumes containing 100
mM KCl, 20 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT;
0.5% Tween R20, 0.5% Nonidet RP-40, 200 mM dNTPs, 2.5
2 An earlier version of this paper (SC/55/SM22) was presented to the

IWC Scientific Committee in 2003.

Fig. 1. Distributions of common bottlenose dolphins sampled in Peru
and Chile.

pM each oligonucleotide and one unit of Taq DNA
polymerase. After an initial denaturation step at 95° C for 5
minutes, a PCR amplification regime of 30 seconds at 94°C,
followed by 30 seconds at 50°C and 30 seconds at 72°C wasrepeated 30 times. The amplification was completed with afinal extension step of 10 minutes at 72°C. Subsequent cycle
sequencing reactions were performed with 100ng ofproducts generated in the above PCR amplifications using
the PrismTM dRhodamine Terminator Cycle Sequencing Kit(Applied Biosystems, Inc.). The oligonucleotides used toprime the cycle sequencing reaction were the same as
employed in the initial PCR amplification listed above. A
total of 25 cycles with 10 seconds at 96°C, 20 seconds at
56°C and four minutes at 60°C were performed. The
nucleotide sequence of each cycle sequencing reaction wasdetermined by electrophoresis through a 5% Long
RangerTM (FMC, Inc.) denaturing polyacrylamide matrix on
a DNA PrismTM 377 DNA Sequencer (Applied Biosystems,Inc.) under standard conditions. Both strands weresequenced in their entirety for all samples.
Sequence analysis
Sequencing was performed with 100ng PCR products using
the PRISMTM Ready Reaction Dye Deoxy Terminator Kit(Applied Biosystems) (ABI). Primers used for sequencing
were the same as indicated above. The reaction was
performed through 25 cycles of 96°C for 10sec, 56°C for
20sec and 60°C for 4min. The nucleotide sequence for each
amplification was determined by electrophoresis through a
5% Long RangerTM polyacrylamide matrix on an ABI DNA

J. CETACEAN RES. MANAGE. 7(1):65–70, 2005

67

PrismTM 377, following the manufacturer protocols. For
each sample both forward and reverse strands weresequenced. Sequences were aligned using the DNA
sequence comparison software ‘Sequence Navigator’developed by ABI.

very near to haplotype ‘14’ (with 100% bootstrap support).
Two other clusters with a branch supported by a highbootstrap value (70%) were identified, however theseclusters included individuals from different localities, but no
Peruvian inshore specimens.

Levels of polymorphism
Genetic distances among different haplotypes were
estimated using Kimura’s two parameters method based on
genetic distance among haplotypes (Kimura, 1980).
Nucleotide diversity (p) was estimated following equation
10.5 of Nei (1987). The net genetic distance between
populations (dA) was estimated by subtracting the averagelevel of variation within each population, following
equation 10.21 of Nei (1987).

Net inter-populational distances
Table 2 shows the net inter-populational distances between
areas. Individuals with haplotypes ‘4’ or ‘14’ were identified
as the Peruvian inshore type based on morphologicalfeatures (Van Waerebeek et al., 1990) and capture
circumstances. All pairwise comparisons involving thisecotype (PE-I, n=3) showed large genetic distances ranging
from 0.02900 to 0.03412. The other pairwise comparisons
resulted in genetic distances between 0.00024 and 0.00870.
The smallest genetic distance was found between CL-O and
PE-O.

MtDNA genealogy
Phylogenetic reconstruction of haplotypes was made using
the neighbour-joining method (Saitou and Nei, 1987). To
evaluate the confidence limits of phylogenies, 1000
bootstrap simulations were conducted (Felsenstein, 1985).
The phylogenies were rooted using the homologous
sequence from a common dolphin (Delphinus sp.; GenBank
accession number: U02652).
Homogeneity test
Homogeneity tests were conducted using the sequence(Kst*) and haplotype (Hst) statistics proposed by Hudson etal. (1992). The degree of divergence was inferred as being
larger than zero, if an equal or more extreme value of theKst* or Hst was observed in less than 5% of 10,000 Monte
Carlo simulations.
RESULTS
Level of polymorphism
The first 331 nucleotides were determined in the mtDNA
control region for each of the 31 samples. A total of 32
polymorphic sites were detected (30 transitions, one
transversion and one deletion), which defined 21 unique
haplotypes (Table 1).
Nucleotide diversity for the whole sample population was
estimated to be 0.02193, and the nucleotide diversity within
a single sample ranged from 0.00201 in the Peruvian inshore(PE-I), to 0.02007 in the Chilean offshore group (CL-O)(Table 2).
Geographic distribution of haplotypes
The frequency of haplotypes in the bottlenose dolphin
samples is shown in Fig. 2. Apart from haplotype ‘9’, which
was shared by CL-O and PE-O, no shared haplotype
occurred among CL-I, CL-O, PE-I and PE-O. All
individuals in the PE-O group showed a different haplotype.
Six haplotypes were defined in eight CL-O individuals,
while only two haplotypes were defined in the eight CL-I
animals. Two individuals of the Peruvian inshore ecotype
shared the same haplotype (‘14’), although they were landedin ports 556km apart (Chimbote and San Andrés).
MtDNA haplotype genealogy
A neighbour-joining-based phylogenetic tree of the
haplotypes (Fig. 2) shows haplotypes ‘4’ and ‘14’ as highly
divergent from all others. Haplotype ‘14’ includes two
individuals from the Peruvian inshore ecotype. Haplotype‘4’ was represented by a single inshore individual (MFB465), as determined by cranial and capture data. This was
supported by sequencing results since MFB-465 clustered

Homogeneity tests
Pairwise comparisons between areas predominately resulted
in significant genetic differences. However, the comparison
between CL-O and PE-O, showed no significant difference
and the comparison between CL-I and PE-I was only nearto-significant for Hst. The latter is presumably due to thesmall PE-I sample size since the corresponding Kst* washighly significant (Table 3).
DISCUSSION
The three bottlenose dolphin specimens from Peru identified
as the Peruvian Inshore ecotype through the evaluation of
cranial characteristics, tooth diameters (Van Waerebeek etal., 1990) and fisheries data (e.g. Van Waerebeek et al.,
1997; 2002), were found to be phylogenetically distinct
from all other bottlenose dolphins studied in the easternSouth Pacific. Furthermore we found a high net interpopulational distance of 2.9% between Peruvian inshore andoffshore ecotypes, and even higher values when comparedwith another area (Chile). These results suggest that the
Peruvian inshore ecotype should be considered an
evolutionarily significant unit, and should be managed
separately from the offshore ecotype, coinciding withmorphological and ecological evidence (Van Waerebeek etal., 1990; Santillán, 2003).
Inshore Chilean bottlenose dolphins (pod-R) were highly
divergent from the Peruvian inshore ecotype based on
phylogenetic analysis and the net inter-population distance.
This is surprising since pod-R dolphins reside inshore withhigh site fidelity, which are characteristic of all inshore
bottlenose dolphins. In fact, pod-R appeared more closely
related to the Chilean offshore stock. However,homogeneity tests for pod-R in pairwise comparison with
the Chilean offshore and the Peruvian offshore groups
revealed significant genetic differences. Furthermore, theeight CL-I individuals showed only two haplotypes (Fig. 2)
and a concomitant low nucleotide diversity. Strictly
speaking, mtDNA divergence does not necessarily signify
reproductive isolation since mtDNA is maternally inherited.
However, these mtDNA analysis results are consistent withintensive field observations on pod-R, which suggest thatthe group may be reproductively isolated (Sanino andYáñez, 2001), and a study to ascertain whether this is thecase using nuclear DNA markers is planned. If reproductive
isolation is indeed confirmed, the long-term survival of thiscommunity of some 30 individuals looks uncertain (Sanino
and Yáñez, 2001). Pod-R might actually constitute the only
remnant pod of a population. To date there is no evidence for68

SANINO et al.: SOUTH PACIFIC COMMON BOTTLENOSE DOLPHINS

Antofagasta (Guerra et al., 1987), and unconfirmed reportsof dolphins in the surfzone off beaches around Iquiquerequire further investigation.
No significant differences were found between Peruvian
and Chilean offshore bottlenose dolphins, but only one
haplotype was shared between them (No. 9, Fig. 2). Thesedolphins often travel at great speeds with steady bearing,
performing high, energetic jumps (personal observations)and are thought to cover great distances with ease. Chilean
and Peruvian offshore bottlenose dolphins probably form asingle wide-ranging population, which we haveprovisionally named the ‘Peru-Chile offshore bottlenose
dolphin stock’. Affinities with other nominal bottlenose
dolphin species described from the eastern Pacific Ocean3
should be established, including insular animals foundaround archipelagos.
The mtDNA results presented here, together withmorphological data and parasite load differences (Van
Waerebeek et al., 1990) show that the Chilean and Peruvian
inshore stocks of bottlenose dolphins should each be
managed as distinct reproductive units.

ACKNOWLEDGEMENTS

the existence of a widely distributed inshore bottlenose
dolphin population in Chile south of Punta Coloso(23°43’S), near Antofagasta (see Aguayo, 1975; Sielfeld,
1980; 1983; Guerra et al., 1987; Van Waerebeek et al.,1990). North of Punta Coloso, several undetermined
ecotype specimens are curated at the University ofWe thank Joanna Alfaro-Shigueto, Ruth Bello, Aquiles
García-Godos and Robert Pitman for their contributions tosample collection. The International Whaling Commissionis acknowledged for access to SOWER samples; G.P.
Donovan and H. Kato have been particularly helpful. We
thank M. Goto for assistance with data analysis. Field
3 In particular Tursiops nuuanu Andrews, 1911 of the eastern tropical
Pacific and T. truncatus gillii Dall, 1873 of southern and Baja
California (see comparative discussion by Andrews, 1911).

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69

Fig. 2. Phylogenetic tree and frequencies of haplotypes (H) for common bottlenose dolphins from the four study areas (CL-I: Inshore Chile, CL-O:
offshore Chile, PE-I: inshore Peru, PE-O: offshore Peru). In circles are the bootstrap values over 50% 2in 1,000 Monte Carlo simulations.

research by KVW and MFB, and CEPEC collection
maintenance, was supported by the Leopold-III Fonds voor
Natuuronderzoek en Natuurbehoud, Gesellschaft zum
Schutz der Meeressäugetiere, New England Aquarium,
Chicago Zoological Society and AGCD (Belgian Agency
for Development Aid). P. Clapham, P. Deimer, R. Reeves
and J. Van Goethem were instrumental in recommending
this funding. Field research by GPS was supported by the
Chilean National Museum of Natural History MNHN and
the CMMR Leviathan. We also thank the Stadio Italiano,
Santiago, for having allowed us the use of their facilities forfour months to prepare the 130 volunteers and CMMR
Leviathan members who made the TURSIOPS98/99 andTurismo Seguro projects successful. Special thanks to the
local fishermen from Punta de Choros and Caleta Chañaral
de Aceituno. The comments of two anonymous reviewers
helped considerably in improving the manuscript.

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