Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

Mitochondrial DNA supports the low genetic diversity of Tursiops truncatus

(Artiodactyla: Delphinidae) in Bocas del Toro, Panama and exhibits

new Caribbean haplotypes

María Alejandra Duarte-Fajardo1, 2; https://orcid.org/0000-0002-6494-6941

Dalia C. Barragán-Barrera*1, 3, 4; https://orcid.org/0000-0003-4023-9908

Camilo A. Correa-Cárdenas1, 5; https://orcid.org/0000-0001-5009-6213

Betzi Pérez-Ortega6, 7, 8; https://orcid.org/0000-0001-5414-6329

Nohelia Farías-Curtidor9; https://orcid.org/0000-0002-2617-8988

Susana Caballero1; https://orcid.org/0000-0002-9285-3873

1. Laboratorio de Ecología Molecular de Vertebrados Acuáticos-LEMVA, Departamento de Ciencias Biológicas,

Universidad de los Andes, Bogotá, Colombia; aduarte108@gmail.com, daliac.barraganbarrera@gmail.com

(*Correspondence), camilocc510@gmail.com, sj.caballero26@uniandes.edu.co

2. Fundación Malpelo y otros Ecosistemas Marinos, Bogotá, Colombia; aduarte108@gmail.com

3. Instituto Javeriano del Agua, Pontificia Universidad Javeriana, Bogotá, Colombia; daliac.barraganbarrera@gmail.com

4. R&E Ocean Community Conservation Foundation, Oakville, Canada; daliac.barraganbarrera@gmail.com

5. Grupo de Investigación en Enfermedades Tropicales del Ejército (GINETEJ), Laboratorio de Referencia e Investigación,

Dirección de Sanidad, Ejército Nacional de Colombia, Bogotá, Colombia; camilocc510@gmail.com

6. Fundación Panacetacea Panamá, Ciudad de Panamá, Panamá; betziperez@yahoo.com

7. Biology Department and Redpath Museum – McGill University, Montreal, Canada; betziperez@yahoo.com

8. Instituto Smithsonian de Investigaciones Tropicales. Ciudad de Panamá, Panamá; betziperez@yahoo.com

9. Fundación Macuáticos Colombia, Medellín, Colombia; nohefa@gmail.com

Received 30-VII-2022. Corrected 13-II-2023. Accepted 09-VI-2023.

ABSTRACT

Introduction: The common bottlenose dolphin (Tursiops truncatus) is one of the most studied cetaceans world-

wide; however, information about the genetic structure of wild populations is scarce in some regions like Central

America and the Caribbean. There are two known genetic forms identified in the Caribbean based on mitochon-

drial DNA Control Region (mtDNA-CR) data: the ‘inshore (or coastal) form’ and the ‘Worldwide distributed

form’. In general, the inshore form refers to coastal and highly philopatric populations that show low genetic

diversity. Worldwide distributed form refers to highly mobile populations with coastal and oceanic individuals

that do not show philopatry and usually display high genetic diversity.

Objective: To determine the preliminary genetic status of common bottlenose dolphins in La Guajira, Colombian

Caribbean, using a hypervariable portion of mtDNA-CR. The obtained haplotypes were compared with samples

collected in Panama (likely ‘inshore form’) and with haplotypes previously found in other areas of the Caribbean.

Methods: In 2016, a total of 26 skin samples were obtained by remote biopsy system (PAXARMS) in two loca-

tions, La Guajira (Colombia, N=7) and Bocas del Toro (Panama, N=19). DNA was extracted, samples sexed,

and a segment of mtDNA-CR (~550-750 bp) was amplified by PCR. The successfully amplified DNA sequences

were manually reviewed and cleaned, and subsequently compared with 44 haplotypes previously reported for

the Caribbean.

Results: The mtDNA-CR sequences from Bocas del Toro shared the same unique inshore haplotype previously

reported for this population, while the samples from La Guajira represented six novel haplotypes, five belong-

ing to the Worldwide distributed form and one to the ‘inshore form.’ Population structure analysis revealed two

https://doi.org/10.15517/rev.biol.trop..v71iS4.57291

SUPPLEMENT • SMALL CETACEANS

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

phylogroups for the Caribbean (FST=0.1353, ΦST=0.3330) with high haplotype diversity: Panama(Bocas del Toro)-

Bahamas-Cuba-Mexico (h=0.8489, π=4.2536 %) and Colombia-Costa Rica-Honduras-Puerto Rico (h=0.8837,

π=4.2423 %).

Conclusions: These findings support the results previously reported for common bottlenose dolphins in Bocas

del Toro-Panama and reinforce the need to protect this vulnerable ‘inshore’ population by treating it as a unique

population management unit. Mitochondrial DNA analysis of samples collected from La Guajira dolphins

provide the first insight into the genetic diversity of common bottlenose dolphins in this region, indicating the

presence of both inshore and Worldwide distributed genetic forms. The potential connectivity of this last form

among La Guajira-Colombia, Costa Rica, and Honduras in Central America highlights the need for more genetic

and ecological studies to determine the appropriate management units for this species in Central America and

the Caribbean.

Key words: bottlenose dolphin; cetaceans; mtDNA; genetic diversity; La Guajira, Colombia, Central America.

RESUMEN

ADN mitocondrial soporta la baja diversidad genética de Tursiops truncatus (Artiodactyla: Delphinidae)

en Bocas del Toro, Panamá y detecta nuevos haplotipos en el Caribe

Introducción: El delfín nariz de botella común (Tursiops truncatus) es uno de los cetáceos más estudiados a

nivel mundial. Sin embargo, la información sobre la estructura genética de sus poblaciones silvestres es escasa en

algunas regiones, como Centroamérica y el Caribe. Con base en datos de la Región de Control del ADN mito-

condrial (ADNmt-CR), dos formas genéticas han sido identificadas en el Caribe: la ‘forma inshore (o costera)’

y la ‘forma mundialmente distribuida’. En general, la forma costera se refiere a poblaciones costeras y altamente

filopátricas que muestran baja diversidad genética. La forma mundialmente distribuida se refiere a poblaciones

altamente móviles con individuos costeros y oceánicos que no muestran filopatría y generalmente muestran alta

diversidad genética.

Objetivo: Para determinar el estado genético preliminar de los delfines nariz de botella comunes en La Guajira,

Caribe colombiano, se realizó un análisis genético utilizando una porción hipervariable de ADNmt-CR. Los

haplotipos obtenidos se compararon con muestras recolectadas en Panamá (probablemente la forma costera) y

con haplotipos encontrados previamente en otras áreas del Caribe.

Métodos: En 2016 se obtuvo un total de 26 muestras de piel colectadas con el sistema de biopsia remota

(PAXARMS) en dos localidades, La Guajira (Colombia, N=7) y Bocas del Toro (Panamá, N=19). Se extrajo el

ADN, se sexaron las muestras, y un segmento de ADNmt-CR (~550-750 pb) se amplificó mediante PCR. Las

secuencias de ADN amplificadas con éxito se revisaron y limpiaron manualmente; posteriormente, se compara-

ron con 44 haplotipos reportados previamente en el Caribe.

Resultados: Las secuencias de ADNmt-CR de Bocas del Toro compartieron el mismo haplotipo costero único

reportado previamente para esta población, mientras que las muestras de La Guajira representaron seis haplo-

tipos nuevos, cinco pertenecientes a la forma mundialmente distribuida y uno a la forma costera. El análisis de

la estructura de la población reveló dos filogrupos para el Caribe (FST=0.1353, ΦST=0.3330) con alta diversidad

haplotípica: Panamá(Bocas del Toro)-Bahamas-Cuba-México (h=0.8489, π=4.2536 %) y Colombia-Costa Rica-

Honduras-Puerto Rico (h=0.8837, π=4.2423 %).

Conclusiones: Estos hallazgos respaldan los resultados previamente reportados para los delfines nariz de botella

comunes en Bocas del Toro-Panamá y refuerzan la necesidad de proteger a esta vulnerable población costera

tratándola como una unidad de manejo poblacional única. Para La Guajira, estos resultados de ADNmt resultan

en el primer esfuerzo por determinar la diversidad y estructura genética del delfín nariz de botella común en esta

región, los cuales sugieren que ambas formas genéticas están presentes en el área, siendo la forma mundialmente

distribuida la predominante. La conectividad potencial de esta última forma entre La Guajira-Colombia, Costa

Rica y Honduras en Centroamérica destaca la necesidad de realizar más estudios genéticos y ecológicos para

determinar las unidades de manejo apropiadas para esta especie en Centroamérica y el Caribe.

Palabras clave: delfín nariz de botella; cetáceos; ADNmt; diversidad genética; La Guajira, Colombia,

Centroamérica.

Nomenclature: SMT1: Supplementary material Table 1; SMF1: Supplementary material Figure 1.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

INTRODUCTION

The common bottlenose dolphin, Tursiops

truncatus (Montagu, 1821) is a cosmopolitan

species inhabiting a variety of nearshore and

oceanic environments in tropical and temper-

ate waters. It is currently listed as Least Concern

by the International Union for the Conserva-

tion of Nature (IUCN) (Wells et al., 2019).

This broadly distributed species is one of the

most studied cetaceans worldwide (Reynolds

et al., 2000). However, genetic information is

still scarce for populations in some regions,

particularly in coastal areas of Central America

and the Caribbean, where few studies have been

conducted. The lack of information regarding

population structure may result in poor man-

agement practices, since coastal populations

show a high degree of genetic differentiation

and face more anthropogenic threats due to

the proximity to human populations (e.g., Bar-

ragán-Barrera et al., 2017; Caballero et al., 2012;

Fruet et al., 2014; Natoli et al., 2004; Parsons et

al., 2006; Tezanos-Pinto et al., 2009). Therefore,

targeted studies like this one, address critical

data gaps for geographic regions of interest and

are key for ensuring effective conservation and

management programs.

Common bottlenose dolphins are highly

adaptable to different environmental condi-

tions, where habitat selection and resource spe-

cialisation likely shape patterns of movement,

gene flow, and population structure (Hoelzel

et al., 1998; Wiszniewski et al., 2010). For

example, ‘inshore’ and ‘offshore’ ecotypes have

been described in the Gulf of Mexico and the

Northwest Atlantic based on a variety of fac-

tors such as genetics, haemoglobin profile, and

morphometry (Duffield et al., 1983; Hersh &

Duffield, 1990; Hoelzel et al., 1998; Natoli et al.,

2004; Sellas et al., 2005). However, Tezanos-

Pinto et al. (2009) provided a worldwide phy-

logeographic perspective highlighting that the

‘offshore’ of the Northwest Atlantic is geneti-

cally interconnected to several mitochondrial

DNA (mtDNA) haplotypes distributed world-

wide regardless of the habitat where samples

were collected (coastal or pelagic) or ocean

basin. Therefore, although the ‘inshore form’

is present in the Wider Caribbean, only the

populations located in Florida-western North

Atlantic, the Bahamas (Caballero et al., 2012;

Natoli et al., 2004; Tezanos-Pinto et al., 2009),

and Bocas del Toro Archipelago, Panama (Bar-

ragán-Barrera et al., 2017) have been success-

fully classified as entire ‘inshore form’.

The inshore populations found in the west-

ern North Atlantic, which are highly struc-

tured among bays (e.g., Sellas et al., 2005),

have recently been proposed as a new species

based on an integrative approach comparing

mitochondrial and nuclear DNA with mor-

phometrics (Costa et al., 2022). These findings

indicate that inshore bottlenose dolphins are

likely under-characterized and deserve addi-

tional attention. Given this, it is imperative

to conduct studies on dolphin populations in

the Caribbean and Central America regions.

This research should take into consideration

that the inshore form is primarily found in the

Wider Caribbean, specifically in Bocas del Toro

(Barragán-Barrera et al., 2017; Caballero et al.,

2012; Tezanos-Pinto et al., 2009).

The ‘inshore’ population found in Bocas

del Toro exhibits a strong population struc-

ture and low genetic diversity (all individuals

possess a single mitochondrial DNA Control

Region (mtDNA-CR) haplotype) compared to

other populations in the Caribbean (Barragán-

Barrera et al., 2017). The population is small,

shows high site fidelity, and restricted coastal

feeding habits; all characteristics usually attrib-

uted to the inshore ecotype (Barragán-Barre-

ra, Luna-Acosta et al., 2019; May-Collado et

al., 2015, May-Collado et al., 2017). However,

recent observations in the field suggest that

oceanic individuals may be entering within the

north-west region, which may result in poten-

tial genetic flow (B. Pérez, personal communi-

cation, 2022). Additional analyses are needed

to better understand the genetic isolation and

the ‘inshore form’ genetic connectivity between

the Bocas del Toro dolphins and other dolphin

populations in the Caribbean.

Little is known about the population found

in La Guajira (Colombian Caribbean). To date,

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

most of the studies have focused on habitat use

and occurrence (Combatt & González, 2007;

Palacios et al., 2013). In the absence of data on

genetic diversity and population structure, it

is unknown if bottlenose dolphins in La Gua-

jira belong to the inshore form or ‘Worldwide

distributed form’, or if both forms coexist. This

information gap prevents adequate manage-

ment plans for this population, despite bottle-

nose dolphins in Colombia are facing threats

related to bycatch, interactions with fishing

gear, direct capture, vessel traffic/transit, pol-

lution, and pathogens (Avila & Giraldo, 2022).

Here, we evaluate the genetic diversity and

population structure of the common bottle-

nose dolphin in Bocas del Toro and La Guajira

by comparing the mtDNA-CR marker to 44

haplotypes previously published from various

locations in the Caribbean Sea (Barragán-Bar-

rera et al., 2017; Caballero et al., 2012). Results

from this work will contribute to the under-

standing of the genetic structure of bottlenose

dolphins in the Central American Caribbean

and represent a critical first step to local con-

servation efforts.

METHODS

Study area: The Bocas del Toro Archi-

pelago is located on the western Caribbean

coast of Panama (Fig. 1), and has great marine

biodiversity associated with coral reefs, sea-

grasses, and mangroves (Coates et al., 2005;

Guzmán et al., 2005). La Guajira, in the west-

ern coast of Colombia, is the northernmost

area of South America (Fig. 1). It is one of

the most biodiverse regions in Colombia; it

holds several coastal and marine ecosystems

such as mangroves, coral reefs, coastal wetland,

sandy beaches, and seagrasses (Corporación

Autónoma Regional de La Guajira & Instituto

de Investigaciones Marinas y Costeras, 2012).

Sample collection: Tissue samples were

collected from wild common bottlenose dol-

phins using remote biopsy darts fired from a

Fig. 1. Sample locations in Bocas del Toro Archipelago, Panama (orange dots) and La Guajira, Colombia (purple triangles)

in the Caribbean Sea. Red dot indicates the location of Cabo de la Vela in La Guajira.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

modified rifle (PAXARMS) (Krützen et al.,

2002). This is a standardised methodology for

collecting small skin biopsies from small ceta-

ceans (Tezanos-Pinto & Baker, 2012). Samples

were collected in February, March, and Octo-

ber of 2016 in Bocas del Toro, and in May

and June of 2016 in La Guajira. GPS data and

photographs of the dolphin biopsied were also

collected. The latter allowed individual identifi-

cation avoiding re-sampling (Fruet et al., 2014;

Krützen et al., 2002). Samples were preserved

in 70 % ethanol and stored at −20 °C (Amos &

Hoelzel, 1991) for further laboratory analysis.

DNA extraction, PCR, sequencing,

and sexing: DNA was extracted from skin

samples using the DNeasy kit (QIAGEN).

Fragments of approximately 535 - 684 bp

of mtDNA-CR were amplified through the

polymerase chain reaction (PCR), using the

primers Dlp8G (5’-CCATCGWAGATGTCT-

TATTTAAGRARTTCTA-3’) or Dlp5G (5’-

GGAGTACTATGTCCTGTAACCA-3’) and

Dlp1.5 (5’-TCACCCAAAGCTGRARTTC-

TA-3’) (Baker et al., 1998). Amplification fol-

lowed this protocol: an initial pre-denaturation

step of 94 ºC as denaturation for 2 min, with

34 cycles of 2–4 repeat times of 30 s at 94 ºC,

followed by 45 s at 55 ºC as annealing and

an extension at 72 ºC, with a final elongation

after the last cycle of 10 min at 72 ºC (Baker

et al., 1998). PCR products were visualized

on agarose gel before sequencing. DNA was

cleaned and sequenced in both directions with

the Sanger sequencing method (Sanger & Coul-

son, 1975) on an ABI 3500 DNA automated

sequencer using the Big DyeTM Terminator

v.3.1 Cycle Sequencing kit. In order to identify

the sex of the samples collected, we used male-

specific SRY gene and ZFY/ZFX genes of males

and females (Gilson et al., 1998). Fragments

were amplified by PCR using the protocol pro-

posed by Gilson et al. (1998) and visualised on

agarose gel to determine sex.

Data analysis: All mitochondrial sequenc-

es were checked, cleaned manually and aligned

using the software Geneious v. 2022.1.1

(Drummond et al., 2009). To confirm that the

samples collected were from common bottle-

nose dolphins and not from other delphinid

species of the delphinid complex Delphinus-

Stenella-Tursiops, we used BLAST in NCBI

(https://blast.ncbi.nlm.nih.gov/Blast.cgi) to

compare our sequences to publicly available

reference sequences and confirm species of

origin. Unique haplotypes were assigned using

the R script RemoveRedundantTaxa in RStudio

(v. 2022.02.3+492). Haplotypes obtained were

compared to 44 previously published sequences

on NCBI GenBank from the Bahamas, Colom-

bia, Costa Rica, Cuba, Honduras, Jamaica, Mex-

ico, Puerto Rico, and Virgins Islands (Accession

numbers: JN596281-JN596321) (Caballero et

al., 2012), Costa Rica (Accession numbers:

KY817220-KY817221), and Panama (Bocas del

Toro) (Accession number: KX833116) (Bar-

ragán-Barrera et al., 2017). The complete align-

ment was 386 bp.

To identify which of the two genetic forms

of common bottlenose dolphins the samples

analysed belonged to, a Maximum Likelihood

phylogenetic tree was constructed with the evo-

lutionary model of Generalised-Time-Revers-

ible γ + Invariant (general reversible time

substitution model GTR γ + I) substitution and

1 000 bootstrap replicates using RAxML-HPC

BlackBox tool in CIPRES Science Gateway V.

3.3 (Miller et al., 2010). As outgroup we defined

a sample from an Atlantic spotted dolphin,

Stenella frontalis (Cuvier, 1829) collected in La

Guajira in 2016, since bootstrap value indicates

100 support segregating T. truncatus from S.

frontalis, and this species has been used before

as outgroup for mtDNA analysis on bottlenose

dolphins (e.g., Wang et al., 1999). Also, a hap-

lotype network was constructed using the TCS

method in PopART software (Clement et al.,

2000) to visualise to what population the hap-

lotypes used in this study belonged and their

frequency. Finally, we defined the number of

phylogroups for the Caribbean by calculating

FCT using Arlequin v. 3.5 (Excoffier & Lischer,

2010). We used the same software to assess

estimates of genetic structure (FST and ΦST),

nucleotide (π), and haplotype diversity (h).

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

Here, samples from Jamaica and the US Virgin

Islands were excluded due to the low number of

samples (N = 1).

RESULTS

A total of 26 tissue samples from common

bottlenose dolphins were collected; seven in La

Guajira, Colombia, and 19 in Bocas del Toro,

Panama. All samples from Panama were suc-

cessfully amplified, sequenced and included

in genetic structure and diversity analyses.

Among these samples, only a single haplotype

was found. Six out of the seven samples from La

Guajira were successfully amplified, each repre-

senting a unique haplotype. Overall, ten males

and nine females were identified in Bocas del

Toro samples, and four males and three females

were identified in La Guajira samples.

When the sequences obtained from this

study were compared with 44 previously Carib-

bean haplotypes (Barragán-Barrera et al., 2017;

Caballero et al., 2012; SMT1), all samples from

La Guajira resulted in new haplotypes not

previously reported for the Caribbean, while

the only haplotype found in the samples from

Bocas del Toro was identified as the same

unique inshore haplotype reported previously

(Barragán-Barrera et al., 2017). New T. trun-

catus haplotypes were submitted to GenBank

(https://www.ncbi.nlm.nih.gov/genbank/) as

accession numbers OR090913-OR090918 and

the new S. frontalis (outgroup) haplotype was

submitted to GenBank as accession number

OR090919.

Further comparisons conducted through

phylogenetic analyses, showed samples from

Bocas del Toro nested with the previously

reported haplotype BOC (Accession number:

KX833116), which had been reported by Bar-

ragán-Barrera et al. (2017) as ‘inshore.’ Five

out of six samples from La Guajira nested with

the Worldwide distributed form, and only one

sample nested with the inshore form, group-

ing with the inshore Q haplotype from Cuba

(Fig. 2). The haplotype network (Fig. 3) shows

the relationships among haplotypes, frequency,

and locations.

In the genetic structure analysis using the

new sequences found here with the previous

ones reported in the Caribbean, we evaluated

from two to five phylogroups. Based on the

most probable phylogroups, we defined two

(FCT = 0.2655, P = 0.0352). One phylogroup

formed by Bocas del Toro (Panama)-Bahamas-

Cuba-Mexico (mostly formed by inshore hap-

lotypes) and the other one by Colombia-Costa

Rica-Honduras-Puerto Rico (mostly formed

by Worldwide distributed form haplotypes).

We reported similar values of haplotype and

nucleotide diversity between both phylogroups

(Table 1), but values of FST and ΦST showed

strong population structure between them,

especially at the nucleotide level (FST = 0.1353,

ΦST = 0.3330, P < 0.0001, Table 1). We found

relatively high haplotype and nucleotide diver-

sity in both phylogroups considered in this

analysis, with the highest haplotype diversity

found in the Colombia-Costa Rica-Honduras-

Puerto Rico phylogroup (Table 1).

Table 1

Estimates of mitochondrial differentiation among the two forms (‘inshore form’ and ‘Worldwide distributed form’) of

common bottlenose dolphin (Tursiops truncatus) located in the Caribbean Sea. FST value is shown above diagonal and ΦST

below diagonal. P-value is indicated under each value. Haplotype (h) and nucleotide diversity (π) are shown on the diagonal

for each phylogroup.

FST

ΦST

Bocas del Toro (Panama)-

Bahamas-Cuba-México

Colombia-Costa Rica-

Honduras-Puerto Rico

Bocas del Toro (Panama)-

Bahamas-Cuba-México

h = 0.8489 +/- 0.0179

π = 4.2536% +/- 0.021 0.1353*

Colombia-Costa Rica-

Honduras-Puerto Rico 0.3330* h = 0.8837 +/- 0.0337

π = 4.2423% +/- 0.0214

Significant values (p < 0.05) are indicated with an asterisk (*).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

DISCUSSION

This study provides new insights into the

population structure and genetic diversity of

common bottlenose dolphins from La Gua-

jira (Colombia) and Bocas del Toro (Panama)

in the western Caribbean using mtDNA-CR

data. The results confirm that a single unique

haplotype is present among the individuals

sampled in Bocas del Toro, and it is unique

to the Caribbean Sea (Barragán-Barrera et al.,

2017). This population has low mitochondrial

diversity and both sexes appear to be highly

philopatric (Barragán-Barrera et al., 2017). Pre-

vious photo-ID data supports high site fidelity

(e.g., May-Collado et al. 2012, 2015, 2019), and

genetic data shows a strong population struc-

ture based on nuclear data (nine microsatellite

loci), as well as that both sexes share the same

unique inshore haplotype not reported in other

Caribbean area (Barragán-Barrera et al., 2017).

This site fidelity may be explained by the prey

availability for dolphins within the Archipelago

(Barragán-Barrera, Luna-Acosta et al., 2019), as

well as for the shallow areas suitable as nursery

(May-Collado et al., 2019). Even though most

studies have demonstrated male-biased disper-

sal (Baker et al. 1993; Escorza-Trevino & Dizon,

2000; Möller et al. 2004; Rosel et al. 1999),

philopatry in both sexes has been documented

Fig. 2. Phylogenetic reconstruction by Maximum Likelihood of Control Region haplotypes from common bottlenose

dolphins (Tursiops truncatus) in the Caribbean Sea (N = 70, 386 bp). Phylogeny in a circular polar form shows bootstrap

support in branches with percentages > 50 %. Purple line groups the ‘inshore form’ and the green line groups the ‘Worldwide

distributed form’. Red letters represent the samples used for this study and the braces indicate the new haplotypes. Outgroup:

Atlantic spotted dolphin (Stenella frontalis).

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before in odontocetes. For example, in resi-

dent killer whales located in British Colum-

bia and Washington State (Bigg et al., 1987)

and pilot whales located in the Faeroe Islands

(Amos et al., 1993). In some bottlenose dol-

phin populations, males are more mobile than

females even in resident populations (Möller &

Beheregaray, 2004).

Conversely, the genetic pattern of the pop-

ulation of La Guajira is very different. Here, we

infer that dolphins are mainly of the World-

wide distributed form, in addition to a few

inshore dolphins. A previous study (Combatt

& González, 2007) suggested that dolphins in

La Guajira probably belonged to the inshore

form (based on observations and occurrence

data). However, in general, La Guajira Penin-

sula does not have closed and protected bays

as does the Bocas del Toro Archipelago. This

geography and oceanic foraging may promote

admixture among different forms. Nonetheless,

the area is influenced by upwelling conditions,

mainly in the low (south) and high (north)

Guajira, offering nutrients to dolphin prey

species (Barragán-Barrera, Luna-Acosta et al.,

2019; Gutiérrez Leones et al., 2015; Paramo

et al., 2011). Although this upwelling is vari-

able throughout the year and between years

(Andrade & Barton, 2005), it has been related

to presence of dolphins in La Guajira (Farías-

Curtidor et al., 2017). The area appears to be

important for transit, and consequently, a very

mobile common bottlenose dolphin population

may visit La Guajira when oceanographic con-

ditions are favourable. Nevertheless, the indi-

vidual nested with ‘inshore form’ haplotypes

could suggest the potential presence of inshore

populations in the high Guajira (where the

sample was collected), or admixture between

forms. The sampled inshore individual was

a male in a group of 15 individuals that were

feeding close to a fishing boat in the Cabo de la

Fig. 3. Haplotype network reconstruction for common bottlenose dolphins (Tursiops truncatus) in the Caribbean Sea under

parsimony criteria with the TCS algorithm (N = 50, 386 bp). The size of the circles indicates the frequency of each haplotype.

Black dots indicate the hypothetical ancestral haplotype, and the perpendicular lines between the haplotypes refer to the

number of nucleotide substitutions between them. New haplotypes are highlighted within a red square.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

Vela (12.113333º N & -72.294533º W) (Fig. 1).

In this area, mainly in the north, some enclosed

bays may offer adequate habitats for dolphins,

but sufficient monitoring data to confirm this

assumption does not exist.

La Guajira appears to be an important

transit area for common bottlenose dolphins in

the Wider Caribbean, where the presence of the

Worldwide distributed form individuals formed

by dolphins with both coastal and oceanic hab-

its, may allow for genetic flow among inshore

populations. In the Wider Caribbean, two dif-

ferentiated phylogroups were detected based

on the high FST and ΦST values: the phylogroup

formed by Bocas del Toro (Panama)-Bahamas-

Cuba-Mexico that is mostly represented by the

inshore form, and the one formed by Colom-

bia-Costa Rica-Honduras-Puerto Rico which

is mostly represented by Worldwide distributed

form individuals. Despite this differentiation,

similar haplotype and nucleotide values were

found between the two phylogroups, likely

because some countries such as Colombia (La

Guajira), Cuba, Mexico, and Puerto Rico shel-

ter both genetic forms (Caballero et al., 2012).

Furthermore, three haplotypes (CG3, CG4.1,

and CG4.2) from three samples obtained in

La Guajira seem to be intermediate haplotypes

between inshore and Worldwide distributed

forms (Fig. 2 and Fig. 3). Bootstrap support

values segregating both forms are high (Fig. 2),

but more extensive sampling for genetic and

genomic analyses are needed to elucidate the

origin of these intermediate haplotypes. Here,

we hypothesize these haplotypes could be either

conserved haplotypes or the result of the genet-

ic exchange between inshore and Worldwide

distributed forms. It is interesting to note that

the HH and LL haplotypes form Puerto Rico

also nest in this intermediate zone. A previ-

ous study using mitochondrial DNA suggested

possible ancestral connectivity between Puerto

Rico and the Mediterranean Sea (Tezanos-

Pinto et al., 2009); therefore, this may support

our second hypothesis.

The common bottlenose dolphin is likely

at risk in Colombia and worldwide (Avila et

al., 2018; Avila & Giraldo, 2022), particularly

coastal populations that are more vulnerable

to human activities (Avila et al., 2018). In the

Caribbean, a recent review documented at least

68 threats for common bottlenose dolphins:

in Aruba (2 threats), Bahamas (2), Bermu-

da (1), Colombia (2), Cuba (42), Dominican

Republic (5), Guyana (3), Haiti (2), Honduras

(1), Mexico (1), Puerto Rico (2), and Venezu-

ela (3) (Avila et al., 2018). These threats are

related to anthropogenic activities that include

fisheries (reported as interaction with fishing

activities in six cases), hunting (25), tourism

(23), scientific research (7), unreported direct

human activities (1), urban development (1),

and unidentified sources (5) (Avila et al., 2018).

However, common bottlenose dolphins in the

Caribbean could be exposed to more threats

in other countries that were not included in

the review. These threats include contamina-

tion by mercury exposure, reported in Bocas

del Toro, Panama (Barragán-Barrera, Luna-

Acosta et al., 2019), and in Colombia and Belize

for this species and other small delphinids

(Barragán-Barrera, Farías-Curtidor et al., 2019;

González-Velásquez et al., 2020), as well as

boat traffic, reported extensively in Bocas del

Toro-Panama (e.g., Kassamali-Fox et al., 2020;

May-Collado et al., 2012, 2017; May-Collado

& Quiñones-Lebrón, 2014; Pérez-Ortega et

al., 2021). The general lack of monitoring in

the Central American Caribbean may further

threaten inshore populations of common bot-

tlenose dolphins.

Considering the threats that bottlenose

dolphins face in the Wider Caribbean, identify-

ing genetically distinct dolphin populations is

critical to designing and implementing man-

agement plans. The case of common bottlenose

dolphins in Bocas del Toro exemplifies this

situation, where a small (seemingly isolated)

population that is vulnerable to contaminant

exposure and boat traffic from dolphin-watch-

ing activities (Barragán-Barrera, Luna-Acosta

et al., 2019; May-Collado et al., 2017), has

been proposed to be categorised as endan-

gered at the local level based on ecological and

genetic studies (Barragán-Barrera et al., 2017,

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

Barragán-Barrera, Luna-Acosta et al. 2019;

May-Collado et al., 2017).

The definition of population structure is

required for effective conservation planning.

Considering the threatened level for the com-

mon bottlenose dolphins in the Central Amer-

ican Caribbean, further genetic studies are

needed. The ‘inshore form,’ previously reported

in the Caribbean (Barragán-Barrera et al., 2017;

Caballero et al., 2012; Tezanos-Pinto et al.,

2009), needs to be studied in detail (particularly

in Central America) to detect the inshore form

level of isolation from the western North Atlan-

tic (Costa et al., 2022). The results of population

structure studies would provide critical infor-

mation for local management plans. Converse-

ly, in the case of Worldwide distributed form

dolphins, a regional plan at the Caribbean level

is urgent to maintain the genetic flow between

populations. At the national level, Colombia

and Panama must strengthen research and

develop effective management plans to accom-

plish better boat and fishing practices, and

reduce anthropogenic stressors for the inshore

populations. La Guajira deserves special atten-

tion as a potentially important transit area for

both common bottlenose dolphins’ forms in the

Caribbean Sea.

Here, we provide further evidence that

dolphins in Bocas del Toro belonged to the

‘inshore form,’ and for the first-time provide

genetic evidence that the dolphins sampled in

La Guajira include both genetic forms, mainly

the Worldwide distributed form. The single

inshore haplotype in La Guajira may indicate

that the northern portion of the Colombian

Caribbean may be occupied by ‘inshore’ indi-

viduals. However, there is insufficient data to

confirm this assumption or establish the com-

mon bottlenose dolphin conservation status,

unlike in Bocas del Toro.

This study constitutes the first step to

decreasing the knowledge gaps related to the

genetic status of common bottlenose dolphins

in the western Caribbean and highlights the

urgent need of conducting more studies in

Colombia and in the Central American Carib-

bean. Confirming the real conservation status

of common bottlenose dolphins and the genetic

connectivity between populations is key to

determine at which scale management must

be implemented. Under this context, further

research is critical for conservation manage-

ment of inshore populations that could be

isolated and at high risk, but their status is cur-

rently unknown.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

See supplementary material

a14v71s4-MS1

ACKNOWLEDGMENTS

We thank Roger Ayala, Tomás Ayala, and

fishermen for their support on the small-scale

surveys out of La Guajira, as well as Demetrio

Georget for his support in Bocas del Toro. We

also thank the STRI Marine Biological Station

staff and Panacetacea team for their logistical

support We want to thank Ester Quintana-

Rizzo and Laura May-Collado for leading this

special issue as scientific editors. We thank

the anonymous reviewers whose comments

improved the final version of this manuscript.

Colombian samples were collected under

Resolution 1177 of 2014 - General Permit

for Specimen Collection of Wildlife Biodiver-

sity Non-Commercial Purposes of Scientific

Research. This permit was provided by the

National Authority for Environmental Licenses

(ANLA) to Universidad de Los Andes. Panama-

nian samples were collected under permission

from the Autoridad Nacional del Ambiente–

Panamá (ANAM, now Ministerio de Ambiente;

permits SC/A-11-12, SC/A-43-12, SC/A-17-14;

SE/A-101-16, SE/AO-6-16). This study was

supported by two Small Grants in Aid of

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71(S4): e57291, diciembre 2023 (Publicado Nov. 01, 2023)

Research from the Society for Marine Mam-

malogy (D. Barragán, 2011; 2014). The Sci-

ences Faculty of Universidad de Los Andes

provided three Research Grants to D. Barragán:

“Proyecto Semilla – 2013-2 Call for Funding

of Research Category: Master and Doctoral

students, project ‘Genetic structure and diver-

sity of bottlenose dolphins Tursiops truncatus

(Montagu, 1821) (Cetacea: Delphinidae) in La

Guajira, Colombian Caribbean’” from Univer-

sidad de Los Andes (D. Barragán, 2014); the

“Proyecto Semilla – 2015-1 Call for Funding

of Research Category: Master and Doctoral

students, project ‘Occurrence, distribution and

preliminary genetic status of delphinids in

La Guajira, Colombian Caribbean’” (D. Bar-

ragán, 2015); “Proyecto Semilla – 2017-1 Call

for Funding of Research Category: Candidates

Ph.D. students, project “Mercury concentra-

tions in bottlenose dolphins Tursiops truncatus

(Montagu, 1821) (Cetacea: Delphinidae) in

Bocas del Toro, Caribbean Coast of Panama”

(D. Barragán, 2016). The Rufford Foundation

provided three grants for this research: the

Booster Grant (D. Barragán, 2015), the Sec-

ond Booster Grant (D. Barragán, 2017), and

another Rufford Small Grant (N. Farías, 2016).

Colciencias/Minciencias also supported this

study through a Research Grant awarded by the

National Ph.D. Call 727 (D. Barragán, 2015),

as well as the National Secretary of Science

and Technology of Panama (SENACYT) and

Department of Biology, Redpath Museum Class

66 Award, Neotropical Environment Option

(NEO) and Biodiversity, Ecosystem Services

and Sustainability (BESS) program form McGill

University (B. Pérez, 2016). The Vicerrectoría

de Investigaciones from Pontificia Universidad

Javeriana is acknowledged by providing a Post-

doctoral Grant (Call 2021-2) to D. Barragán

(2022), who also thanks the Instituto Javeriano

del Agua for its support during this stay. The

funders had no role in the study design, data

collection, analysis, decision to publish, or

preparation of the manuscript.

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