Príncipe island hawksbills: Genetic isolation of an eastern Atlantic stock
C. Monzón-Argüello a, b,⁎ , N.S. Loureiro c, d, C. Delgado e, A. Marco b, J.M. Lopes d,
M.G. Gomes d, F.A. Abreu-Grobois f
a
Department of Biological Sciences, Institute of Environmental Sustainability, College of Science, Swansea University, Swansea SA2 8PP, UK
Estación Biológica de Doñana (CSIC), Américo Vespucio, s/n, 41092 Sevilla, Spain
Faculdade de Ciências e Tecnologia — DCTMA — Ed. 8, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
d
Programa SADA — Sustainable Conservation of the Hawksbill Population at Príncipe Island, Sao Tomé and Príncipe
e
CIIMAR — Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal
f
Laboratorio de Genética, Unidad Académica Mazatlán, Apdo. Postal 811, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Mazatlán, Sinaloa 82000,
Mexico
b
c
a b s t r a c t
Keywords: Connectivity
Eretmochelys imbricata
Mitochondrial DNA
Mixed stock analysis
Population structure
Western Africa
The hawksbill turtle is a critically endangered species that has been extensively exploited for centuries.
Príncipe Island off western Africa harbours one of the species' major nesting populations in the eastern
Atlantic, as well as hosting year-round foraging aggregations of juveniles, subadults and adult males. To gain
insight into the population's genetic structure and relationships with regional stocks, we analysed
mitochondrial DNA (mtDNA) sequences of nesting females (N = 9), foraging adult females (N = 11), adult
males (N = 32), subadults (N = 15) and juveniles (N = 80). The nesting population was found to be fixed for a
single haplotype (EATL), which had been previously reported in both western and eastern Atlantic hawksbill
foraging sites but had no known rookery source prior to this study. Thus it is now possible to confirm the
westward transoceanic movement by hawksbills originating from Príncipe Island. Our analyses demonstrated
that the Príncipe Island nesting colony is genetically distinct from breeding populations in the western
Atlantic and is phylogenetically linked with Indo-Pacific hawksbill clades, suggesting that Príncipe Island was
most likely colonised by migrants from the Indian Ocean via the Cape of Good Hope in southern Africa. Mixed
stock analyses revealed that the eastern Atlantic appears to be the primary foraging area for Príncipe
hawksbills (75%) while most of the foraging juveniles in Príncipe waters originate from the Príncipe rookery
(84%). Furthermore, the presence of Caribbean haplotypes at low frequencies (b 5%) suggests that eastward
transatlantic movements by juveniles to distant foraging and developmental habitats also take place.
Depleted hawksbill populations in the eastern Atlantic combined with the low genetic variability and high
genetic distinctiveness found in the Príncipe nesting and foraging aggregations with respect to the western
Atlantic, underscore the high degree of isolation and vulnerability of this eastern Atlantic stock. These
characteristics are highly relevant for the development of effective conservation programmes and highlight
the urgent need to consolidate international cooperation across regional boundaries.
.
1. Introduction
The hawksbill turtle (Eretmochelys imbricata) is a circumglobal
species that is considered critically endangered (Mortimer and
Donnelly, 2008 — IUCN 2010), although it is not as well studied as
other species such as the loggerhead turtle (Caretta caretta) and the
green turtle (Chelonia mydas). This study addressed a significant gap
in our understanding of the species by being the first genetic study to
focus on a rookery in the Eastern Atlantic.
⁎ Corresponding author at: Department of Biological Sciences, Institute of Environmental Sustainability, College of Science, Swansea University, Swansea SA2 8PP, UK.
Fax: +34 954 621 125.
E-mail address: catalinama@iccm.rcanaria.es (C. Monzón-Argüello).
Breeding sea turtles exhibit natal homing and this leads to strong
population structure between nesting colonies at a global scale,
although proximal rookeries may be interconnected (Bass et al., 1996;
Bowen and Karl, 2007; Broderick et al., 1994). While female sea turtles
exhibit strong philopatry and nest site fidelity, male-mediated gene
flow may connect discrete rookeries, thereby decreasing levels of
genetic differentiation at nuclear loci (Bass et al., 1996; Bowen et al.,
2005, 2007; Carreras et al., 2007; Karl et al., 1992). Maternally
inherited molecular markers (e.g., mitochondrial DNA — mtDNA
sequences) have been used extensively for identifying the nesting
origin of migratory animals (reviewed in Bowen and Karl, 2007 and
Lee, 2008), using mixed stock analysis (MSA; Pella and Masuda,
2001). More recently, a ‘many-to-many’ variation of MSA has been
developed that simultaneously estimates the origins and destinations
of individuals in a meta-population with several source populations
and many mixed stocks, with rookery size as a constraint in the
analysis (Bolker et al., 2007).
Similar to other marine turtles, after hatching on tropical nesting
beaches, hawksbill hatchlings are thought to undergo a pelagic
dispersal phase drifting within ocean currents prior to recruitment
into neritic areas, where they adjust to a benthic environment.
Although the age for this ontogenetic shift is not known, the smaller
sizes of the earliest benthic-staged hawksbills suggest that it occurs
earlier than in other sea turtle species (Bolten, 2003). Recruitment
into foraging sites may occur as a multistep process — distribution and
location of early staged juveniles being influenced by external forces
such as oceanic currents, while older juveniles, gaining increased
autonomy due to their greater swimming capacity, may be homing to
feeding habitats in the vicinity of their natal rookeries (Bass, 1999;
Bowen et al., 1996; Meylan, 1999; Vélez-Zuazo et al., 2008).
Nonetheless, a significant portion of juveniles at foraging grounds
may still originate in distant rookeries (e.g., Mona Island, Puerto Rico;
Vélez-Zuazo et al., 2008). In hawksbill turtles, long distance
migrations are rare, yet a few transoceanic migrations have been
recorded in the Atlantic (Bellini et al., 2000; Bowen et al., 2007;
Grossman et al., 2007; Marcovaldi and Filippini, 1991; MonzónArgüello et al., 2010a), however with incomplete genetic coverage of
regional rookeries, no points of origin have been ascertained. For
example, in a recent mtDNA survey of a hawksbill foraging
aggregation in the eastern Atlantic (Cape Verde Islands; MonzónArgüello et al., 2010a) the vast majority of the sampled juveniles
(86%) had haplotypes of unknown origin, highlighting a critical need
for additional research in order to adequately understand the species'
dispersal and migratory patterns.
In the eastern Atlantic, hawksbills breed along the west coast of
central Africa, although fewer than 100 females are thought to nest
annually (an equivalent of about 350 nests/year; Mortimer and
Donnelly, 2008). The largest nesting populations are thought to
occur on Bioko Island and on the islands of São Tomé and Príncipe
(Abreu-Grobois and LeRoux, 2008; Fretey, 2001; Mortimer and
Donnelly, 2008). Bioko Island (Equatorial Guinea) has had less than
10 nesting females/season since 1997 (equivalent to about 35 nests/
season; Tomás et al., 2010). Although monitoring is opportunistic and
no reliable census exists for São Tomé and Príncipe, Príncipe Island is
estimated to have less than 50 nesting females/season (equivalent to
about 175 nests/season; Loureiro pers. comm.).
Príncipe Island is a small (114 km 2) island in the Gulf of Guinea, off
Central Africa (Fig. 1). Its location at 01°05′ N, 07°30′ E provides a
tropical moist climate and luxuriant vegetation. Hawksbill nesting
occurs from November to February and the main nesting beaches,
located along the northern, eastern and southern coasts of the island,
are: Ponta Marmita, Sundy, Mocotó, Ribeira Izé, Bom-Bom, Santa Rita,
Banana, Macaco, Boi, Grande, Grande do Infante, Sêca and Rio Sao
Tomé. Additionally, there is an important year-round foraging
aggregation composed mainly of juveniles; subadults and adult
males are also present although in lower numbers. Adult females
join the foraging aggregations but only during the breeding season
(November to February). Similar to other hawksbill populations
worldwide, Príncipe hawksbills have been overharvested for meat,
eggs and carapace scutes for decades if not centuries. In 2009, Príncipe
Island Autonomic Parliament decreed full protection for sea turtles,
and these laws are slowly being implemented (Decreto Legislativo
Regional no. 3/ALRAP, de 8 de Julho de 2009). Within this
Fig. 1. Map of sample locations used or referred to in this study involving hawksbill nesting populations (circles) and foraging aggregations (stars). Corresponding numbers and
location names are shown in Table 1. The circles are approximately proportional to the corresponding nesting population size, except for Venezuela (7) and Brazil (11). Rookery size
information was extracted from Mortimer and Donnelly (2008). The discontinuous square indicates the Príncipe study sites. The dotted lines represent the major genetic
discontinuities among Atlantic nesting (black) and foraging (red) aggregations as indicated by BARRIER (Manni et al., 2004).
conservation framework, Programa SADA is contributing to the
preservation of the sea turtle genetic heritage of the island, involving
local communities in a collaborative effort to identify, reduce or
eliminate the various threats that hawksbills face in this region.
In this paper, we analysed mtDNA sequences of Príncipe Island
hawksbills sampled from nesting (breeding females) and foraging
aggregations (female and male adults, subadults and juveniles) with
the following objectives: (1) undertake the first genetic characterization of an eastern Atlantic hawksbill nesting population; (2) test the
existence of male philopatry by measuring genetic differences
between sexes among the adults in the foraging aggregations; and
(3) evaluate the level of connectivity between the Príncipe foraging
aggregation and other Atlantic basin habitats by a) measuring the
geographic scope of eastern Atlantic juvenile dispersal through
haplotype profile comparisons between Príncipe and Cape Verde
juveniles and those from western Atlantic habitats, and b) evaluating
the Príncipe habitat as a sink given the low number of nesters
reported for the eastern Atlantic (Abreu-Grobois and LeRoux, 2008;
Fretey, 2001; Mortimer and Donnelly, 2008) and the occasional
transoceanic movements described for this species (Bellini et al.,
2000; Bowen et al., 2007; Grossman et al., 2007; Marcovaldi and
Filippini, 1991). Results from these studies provide important insights
into the genetic distinctiveness and geographic distributions of
Príncipe hawksbills, as well as uncovering aspects of the biology of
this poorly studied regional population, and ultimately informing
future management and conservation strategies.
feeding area. We classified bigger juveniles as “large” and were
considered to have the capacity to swim independently of oceanic
currents.
We injected a passive integrated transponder (PIT) tag (Avid®)
into the right front flipper of all sampled turtles and additionally we
tagged turtles with CCLmin N 45 cm in both front flippers using Inconel
style 681 tags provided by the Cooperative Marine Turtle Tagging
Program, Archie Carr Center for Sea Turtle Research.
2.2. Laboratory procedures
We isolated genomic DNA using a DNeasy Tissue Kit (QIAGEN)
following the manufacturer's protocols. We used the polymerase
chain reaction (PCR) to amplify a fragment of approximately 800 base
pairs (bp) from the 5′ end of the mtDNA control region with primers
LCM15382 (5′-GCTTAACCCTAAAGCATTGG-3′) and
H950 (5′GTCTCGGATTTAGGGGGTTTG-3′) (Abreu-Grobois et al., 2006). We
typically performed PCR reactions in 20 μl volumes under the
following conditions: 94 °C for 5 min; followed by 30 cycles at 94 °C
for 1 min, 55 °C for 1 min, 72 °C for 1 min; with a final extension at
72 °C for 5 min. We conducted cycle sequencing reactions with Big
Dye fluorescent dye-terminator v. 3.1 (Applied Biosystems) and
analysed fragments on an automated sequencer (Applied Biosystems
Inc. model 3100). We sequenced new haplotypes in both forward and
reverse directions to ensure accuracy. We aligned chromatograms
using Bioedit Sequence Alignment Editor v.7.0.9 (http://www.mbio.
ncsu.edu/BioEdit/bioedit.html; Hall, 1999).
2. Material and methods
2.3. Statistical analyses
2.1. Sample and data collection
We collected a total of nine blood samples from different nesting
females (curved carapace lengths of 75–84 cm CCLmin; Bolten, 1999)
during the 2009/2010 nesting season (November to February) from
the five main nesting beaches: Praia Grande, Praia Ribeira Izé, Praia
Sêca, Ponta Marmita and Praia Bom-Bom. At sea, local spear-divers
captured alive individuals handly and took them ashore for tagging,
sampling and data recording. We collected a total of 138 blood
samples from individuals in marine habitats at different times from
May 2009 to May 2010. We stored samples in a 96% ethanol solution
at 4 °C. Based on their carapace size and secondary sex characters, we
classified the 138 individuals from the in-water captures as follows:
32 males (23% of the total; CCLmin 66.5–87 cm), 15 subadults (11%;
CCLmin 58–74.5 cm), and 80 neritic juveniles (58%; CCLmin 18–
57.5 cm) (Fig. 2). We also sampled 11 mature females (8%; CCLmin
76–87 cm) during the nesting season. We further classified juveniles
depending on their size. We classified juveniles below 35 cm CCLmin
as “small”, using the criteria of Vélez-Zuazo et al., 2008, to represent
juveniles with little swimming capacity to move independently of
oceanic currents and those that have recently recruited to the neritic
To assure broad comparisons of our results with previous studies,
we trimmed our control region sequence alignments to the 384 bp
reading frame employed by Bass et al. (1996). Haplotype designations
followed those of previous publications (Bass et al., 1996; Bowen et
al., 1996, 2007; Díaz-Fernández et al., 1999; Monzón-Argüello et al.,
2010a). In all genetic analyses, we treated a deletion in haplotype D at
position 59 and a 10-bp insertion at position 354 in haplotype Mx1a as
single substitutions following Bowen et al. (2007). We used
FindModel (http://www.hiv.lanl.gov) to determine that the best
model of nucleotide substitution for our data was the Tamura–Nei
model with no gamma correction. We identified unique haplotypes
from the sequences with DnaSP v. 5 (http://www.ub.es/dnasp/) and
used the sequence information to construct a haplotype network to
describe evolutionary relationships. We constructed a median-joining
network linking 30 Atlantic haplotypes using the programme
Network and default parameters (http://www.fluxus-technology.
com/). To simplify, we discarded the divergent Mx1a, with a 10-bp
insertion, along with haplotypes R, S, T and U that have only been
observed in E. imbricata × C. caretta hybrids (Bass et al., 1996; Bowen
et al., 2007; Lara-Ruiz et al., 2006). Furthermore, we conducted a
Fig. 2. Minimum curved carapace length (CCLmin) distribution and life cycle classification for hawksbills in this study (N = 145; 99% of all samples).
neighbour-joining tree (Saitou and Nei, 1987) of hawksbill haplotypes
and bootstrap analysis (10,000 replicates) with the software MEGA v.
4 (Tamura et al., 2007). For this analysis, we considered 31 Atlantic
haplotypes, discarding sequences from hybrids, and also 24 IndoPacific haplotypes (Okayama et al., 1999). We estimated sequence
divergence (d) between haplotypes using the Tamura–Nei model of
nucleotide substitution, which was designed for control region
sequences (Tamura and Nei, 1993). We linearised the gene tree
(Takezaki et al., 1995) in MEGA v.4, and converted sequence
divergence estimates to absolute time using a rate of 0.018 substitutions/site per million years (mean value of 0.012–0.024, as
estimated from the control region of green turtles; Encalada et al.,
1996). We calculated haplotype frequencies, Nei's (1987) haplotype
diversity (h) and nucleotide diversity (π) values of mtDNA sequences
using Arlequin v. 3.11 (Excoffier et al., 2005). We quantified genetic
differences between nesting and foraging Príncipe hawksbills with
respect to other breeding and feeding aggregations using the exact
test of population differentiation (Raymond and Rousset, 1995) and
Phi statistics (φST) (Excoffier et al., 1992), with 10,000 permutations
and the Tamura–Nei model of nucleotide substitutions with no
gamma correction. We carried out all computations using Arlequin v.
3.11 (Excoffier et al., 2005). We performed an analysis of molecular
variance (AMOVA; Arlequin v. 3.11) with φST distances and 10,000
permutations (Excoffier et al., 2005) to determine how the genetic
variation was partitioned among the nesting and foraging populations. We used BARRIER v. 2.2 (Manni et al., 2004), which is a
programme using Monmonier's maximum difference algorithm, to
identify the location of major genetic barriers between nesting
populations and foraging aggregations in the Atlantic Ocean.
We computed a Bayesian mixed stock analysis (MSA) using the
many-to-many approach (Bolker et al., 2007) where rookery abundances (number of annual nesting females) were used as our prior,
assuming that the contribution of each rookery is proportional to its
relative size. We took rookery sizes from Mortimer and Donnelly (2008)
(Table 1). For the Príncipe rookery we assumed that there are 50 nesting
females/season (Loureiro pers. comm.). We used the available mtDNA
haplotype profiles for eleven rookeries: Cuba, Puerto Rico, US Virgin
Islands, Antigua, Barbados Leeward and Windward, Venezuela, Costa
Rica, Belize, Mexico and Príncipe (Bass et al., 1996; Bowen et al., 2007;
Browne et al., 2009; Díaz-Fernández et al., 1999; Vélez-Zuazo et al.,
2008; present study) (Table 1). For rookeries with more than one
survey, we combined the datasets. We excluded the Brazilian rookery
since we did not detect any hybrid or haplotypes exclusive to Brazil
(Lara-Ruiz et al., 2006). We excluded from the analysis haplotypes
found at foraging grounds but not at rookeries (orphan haplotypes), and
all haplotypes found only at a single rookery were lumped together.
Using the rookery-centric option in the many-to-many MSA, we first
estimated the extent that individuals from Príncipe disperse to each of
the ten previously studied hawksbill foraging areas in the Atlantic
(Bowen et al., 2007; Díaz-Fernández et al., 1999; Monzón-Argüello et al.,
2010a; Vélez-Zuazo et al., 2008). Second, we computed a foraging
ground-centric analysis to determine the contribution of regional stocks
to the Príncipe feeding aggregation. After using the Markov chain Monte
Carlo (MCMC) method to obtain the posterior distribution of the
parameters of interest, we used values lower than 1.2 from the Gelman–
Rubin diagnostic test to confirm chain convergence in the posterior
distribution (Gelman and Rubin, 1992).
3. Results
great importance is the fact that prior to this study, no origin was
known for the EATL haplotype, despite being detected in a large
proportion of juveniles feeding off the Cape Verde Islands (68% of
sampled individuals; Monzón-Argüello et al., 2010a), as well as being
present in a sample of four turtles from a market in a neighbouring
island of São Tomé (Bowen et al., 2007). This haplotype has also been
reported in the western Atlantic but only from a single juvenile
feeding off the U.S. Virgin Islands and representing barely 0.13% of all
sampled juveniles in the western Atlantic; Table 1. Interestingly, the
EATL haplotype was also reported in foraging samples from the
Seychelles in the Indian Ocean (haplotype 24 in Okayama et al., 1999).
Surprisingly, haplotype A, which was found at very low frequencies in
the present study, occurs widely and at high frequency in many of the
western Atlantic rookeries (e.g., Cuba, Antigua, Venezuela and
Barbados) and foraging aggregations (e.g., Bahamas, Cuba, Puerto
Rico and U.S. Virgin Islands) (Table 1).
All sampled females had the EATL haplotype, both from the nesting
(N = 9) and the in-water (N = 11) surveys. This haplotype was also
fixed in all male (N = 32) and subadult (N = 15) samples. Of the 80
juveniles, the majority had haplotype EATL (92%), with haplotypes A
and Ei-A87 at very low frequencies (N = 3, 4% each).
A median-joining network revealed that haplotypes EATL and EiA87 form part of a distinct haplotype clade that is highly divergent from
both Caribbean and western Atlantic sequences, but closely related to
other haplotypes detected in the eastern Atlantic (EiA-82, EiA-49)
(Fig. 3). The phylogenetic tree generated by the neighbour-joining
method revealed two main clusters. The first cluster consisted of three
subclades: the first and the second with haplotypes previously
reported only from foraging habitats in the Indo-Pacific region (with
the exception of Pac which was found in the Caribbean) (Okayama et
al., 1999) although it is evidently evolutionarily linked with the IndoPacific. The last of these subclades contained sequences from the
Indian Ocean (Seychelles) and from the eastern Atlantic (Cape Verde
and Príncipe Island). The second cluster included haplotypes from the
western Atlantic (Fig. 4).
Since all in-water mature females were captured during the
nesting season (November–February), both nesting (N = 9) and inwater mature females (N = 11) fell within a similar size range (74–
84 cm and 76–87 cm CCLmin, respectively) and haplotype EATL was
fixed in both sample sets, we considered the entire group of animals
to be part of a single Príncipe Island breeding female population. Thus,
we pooled the data from the two sets of female samples for further
analyses (N = 20) (Table 1). All adult males from the foraging
aggregation (N = 32) were also fixed for the same haplotype (exact
P = 1.00; p = 0.99; in all comparisons) and they appeared to belong to
the same genetic stock.
As with some Caribbean hawksbill rookeries, such as those nesting
in Venezuela or on the leeward Barbados beaches, the Príncipe nesting
population presented no haplotype (h) or nucleotide (π) variation as
a consequence of having a single haplotype (Table 1). All population
pairwise comparisons between Príncipe and all studied Atlantic
nesting populations revealed statistically significant differences
(exact P b 0.01; φST N 0.89, p b 0.01) (Table 2). The AMOVA based on
φST values attributed 84% of the total genetic variation to the
differences between eastern (Príncipe) and western Atlantic breeding
populations, 10% to differences among rookeries, and only 6% to
differences within rookeries. Furthermore, the BARRIER programme
assigned the major genetic barrier, computed on the φST distance
matrix, between the eastern Atlantic (Príncipe Island) and all nesting
populations in the western Atlantic (Fig. 1).
3.1. Haplotype profiles
3.2. Distribution of juveniles from Príncipe
We detected only three haplotypes during our surveys: two
previously described, EATL and A, and a new one, Ei-A87 (GenBank
accession n. GU138123). Haplotype Ei-A87 was closely related to
EATL, differing only in a single transition at position 53 (Fig. 3). Of
The many-to-many rookery-centric MSA suggested that turtles
hatched at Príncipe distribute to areas located mainly in the eastern
Atlantic. On the basis of results from studied foraging grounds,
Location
N
A
B
C
Nesting areas
1. Cuba (W)
2. P. Rico (W)
3. USVI (W)
4. Antigua (W)
5. Barbados L. (W)
6. Barbados W. (W)
7. Venezuela (W)
8. Costa Rica (W)
9. Belize (W)
10. Mexico (W)
11. Brazil (W)
12. Príncipe (E)
Total
70
128
50
15
54
30
7
57
14
69
5(18*)
20
514
62
2
5
9
54
3
7
0
0
0
5
0
147
0
0
1
4
0
0
0
0
0
0
0
0
5
0
0
0
2
0
0
0
0
0
0
0
0
2
Foraging grounds
13. Texas (W)
14. Bahamas (W)
15. Cuba A (W)
16. Cuba B (W)
17. Cuba D (W)
18. Dominican R. (W)
19. P. Rico pooled (W)
20. USVI (W)
21. Cape Verde (E)
22. Príncipe (E)
TOTAL
42
78
43
111
56
90
256
68
28
80
852
0
28
28
46
18
42
88
28
2
3
283
0
1
0
0
1
0
4
2
1
0
9
0
0
0
0
0
0
0
0
0
0
0
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
BI1
α
β
γ
Cu3
Cu4
Cum
DR1
DR2
Mx1a
EATL
A49
A82
A87
NF
h
π
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
1
1
0
0
0
0
0
0
0
0
400–833
0.213
0.0038
71
1
0
0
2
1
2
2
38
7
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
250–417
0.605
0.0048
43
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
86–278
0.255
0.0048
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100–125
0.590
0.0089
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
483
0
0
27
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.186
0.0035
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
32–53
0
0
33
6
0
0
0
0
4
0
0
0
0
0
0
0
0
0
0
10
0
0
4
0
0
0
0
0
0
0
0
0
10
0.624
0.0099
11
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8–56
0.396
0.0064
0
0
0
0
0
0
0
0
0
0
3
64
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
0
0
534–891
0.139
0.0011
0
0
0
0
0
0
0
0
0
0
0
0
9
2
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
350–585
0(0.693*)
0(0.0454*)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
0
0
0
50
0
0
186
8
1
1
2
1
6
2
38
7
3
66
9
2
1
1
2
10
0
5
5
1
0
0
0
1
20
0
0
0
3
0
0
0
0
0
0
0
0
0
0
38
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0.180
0.0003
20
0
0
0
0
0
0
0
2
0
1
21
0
0
0
0
0
1
0
0
3
0
0
1
0
0
0
0
0
0
0.740
0.0091
6
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
2
0
1
2
0
0
1
0
0
0
0
0
0
0.559
0.0080
34
0
0
0
0
0
1
0
10
0
0
11
0
0
0
0
0
1
0
5
2
0
0
1
0
0
0
0
0
0
0.720
0.0100
13
1
0
0
0
0
2
0
3
0
0
8
0
0
0
0
0
2
0
5
0
2
1
0
0
0
0
0
0
0
0.821
0.0101
30
2
0
0
1
0
1
0
0
0
0
6
0
0
0
0
0
6
0
0
0
0
0
1
1
0
0
0
0
0
0.669
0.0099
110
0
0
0
0
0
5
0
11
0
1
25
0
0
0
0
0
8
1
1
2
0
0
0
0
0
0
0
0
0
0.687
0.0095
17
0
0
0
0
0
0
0
6
0
0
9
0
0
0
0
0
3
0
1
0
0
1
0
1
0
1
0
0
0
0.757
0.0119
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
19
4
1
0
0.529
0.0168
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
74
0
0
3
0.143
0.0045
234
3
0
0
1
0
9
0
32
0
2
121
0
0
0
0
0
23
1
13
9
2
2
4
2
1
94
4
1
3
C. Monzón-Argüello et al. / Journal of Experimental Marine Biology and Ecology 407 (2011) 345–354
Table 1
Haplotype frequencies in nesting and foraging populations used in the many-to-many mixed stock analysis (except Brazil) with sample sizes (N) and current population sizes based on mean nesting females/year (NF). Note that foraging
ground haplotype data from Príncipe only includes results from juveniles. Population sizes were taken from Mortimer and Donnelly (2008). * indicates the total number of samples, haplotype (h) and nucleotide (π) diversities, including
hawksbill-loggerhead hybrid haplotypes (R, S, T and U). Haplotypes D and E have been eliminated since they have been identified as sequencing errors. Abbreviations: P. Rico, Puerto Rico; USVI, U.S. Virgin Islands; Barbados L., Barbados
Leeward; Barbados W., Barbados windward; Dominican R., Dominican Republic. (W) and (E) indicate populations situated in the western or eastern Atlantic, respectively. References: Bass, 1999; Bass et al., 1996; Bowen et al., 2007; Browne
et al., 2009; Díaz-Fernández et al., 1999; Monzón-Argüello et al., 2010a,b; Vélez-Zuazo et al., 2008).
349
350
C. Monzón-Argüello et al. / Journal of Experimental Marine Biology and Ecology 407 (2011) 345–354
Fig. 3. Median-joining network of 384 bp Atlantic hawksbill haplotypes. Grey circles
represent haplotypes found at Príncipe. Hypothetical intermediates or unobserved
haplotypes are shown in black circles. Haplotypes R, S, T, U and Mx1a were not included
(see text for details).
approximately 40% of the hawksbills from Príncipe utilise the Príncipe
foraging ground, while 35% migrate to the nearby Cape Verde feeding
area (Fig. 5A).
3.3. Príncipe Island foraging ground
We found no genetic differentiation between juveniles and mature
females (nesting and in-water; exact P = 1.00; p = 0.99; in both
cases), or between juveniles and males (exact P = 0.47; p = 0.30).
However, when the haplotype composition of small and large
juveniles were tested separately they revealed statistically significant
differences between them (exact P = 0.02; p = 0.02), and between
mature animals (both sexes grouped) and small juveniles (exact
P b 0.01; p b 0.01). These genetic differences are evidently due to the
Fig. 4. Phylogenetic tree based on the neighbour-joining method for 31 haplotypes
reported in the Atlantic (see Table 1 for references) and for 24 haplotypes in the IndoPacific (Okayama et al., 1999). Bootstrap values for the main nodes are shown. For
sample locations see Table 1 (Atlantic) and Okayama et al. (1999) (Indo-Pacific). In red
are all the haplotypes found in Príncipe nesting (N) or foraging habitats (FG). Symbols:
*haplotypes found in both western Atlantic nesting habitats and eastern Atlantic
foraging grounds (Cape Verde or Príncipe); **haplotypes found in both eastern Atlantic
nesting habitats (Príncipe only) and western Atlantic foraging grounds; ***haplotypes
reported only from foraging habitats of both Indo-Pacific and eastern Atlantic; ****one
haplotype found only in a foraging ground in the western Atlantic but belonging to an
eastern Atlantic clade. Grey boxes show all haplotypes with known origins (rookeries).
Sequences for IndoPac24 and EATL are identical at the 384 bp reading frame. CV = Cape
Verde (Monzón-Argüello et al., 2010a,b), P = Príncipe (this study). The scale below the
tree represents sequence divergence estimates.
C. Monzón-Argüello et al. / Journal of Experimental Marine Biology and Ecology 407 (2011) 345–354
351
Table 2
Pairwise comparisons of Atlantic hawksbill nesting populations. φST values are shown above the diagonal and FST values below the diagonal. All significantly different comparisons
(p b 0.05) are shown in bold. Significant pairwise comparisons based on the exact test of population differentiation are labelled with a (*). Abbreviations: P. Rico, Puerto Rico; USVI,
U.S. Virgin Islands; Barb. L. and Barb. W., Barbados leeward and windward; Venez., Venezuela; and C. Rica, Costa Rica. Genetic data for all rookeries except Príncipe was taken from
publications indicated in Table 1.
Cuba
P. Rico
USVI
Antigua
Barb. L.
Barb. W.
Venez.
C. Rica
Belize
Mexico
Brazil
Príncipe
Cuba
P. Rico
USVI
Antigua
Barb. L.
Barb. W.
Venez.
C. Rica
Belize
Mexico
Brazil
Príncipe
–
0.549
0.743
0.218
0.058
0.773
− 0.032
0.591
0.740
0.824
− 0.064
0.842
0.733*
–
0.150
0.394
0.614
0.157
0.526
0.093
0.088
0.580
0.515
0.566
0.734*
0.039*
–
0.612
0.863
− 0.022
0.769
0.121
0.010
0.810
0.761
0.826
0.224*
0.549*
0.500*
–
0.491
0.637
0.164
0.388
0.505
0.749
0.121
0.739
0.078
0.819*
0.866*
0.577*
–
0.925
0.000
0.682
0.912
0.922
0.000
1.000
0.762*
0.038*
− 0.023
0.531*
0.925*
–
0.838
0.129
0.023
0.845
0.829
0.889
− 0.016
0.767*
0.773*
0.239
0.000
0.838*
–
0.535
0.744
0.878
0.000
1.000
0.516*
0.124*
0.076*
0.216*
0.651*
0.076*
0.498*
–
0.036
0.633
0.520
0.593
0.716*
0.037*
− 0.024
0.389*
0.916*
− 0.027*
0.752*
0.016
–
0.804
0.719
0.834
0.835*
0.389*
0.453*
0.761*
0.963*
0.554*
0.940*
0.318*
0.535*
–
0.874
0.895
− 0.049
0.762*
0.765*
0.196
0.000
0.829*
0.000
0.481*
0.728*
0.939*
–
1.000
0.950*
0.937*
0.949*
0.938*
1.000*
0.968*
1.000*
0.890*
0.960*
0.987*
1.000*
–
presence of haplotype A (N = 3) that exists exclusively in the group of
juveniles smaller than 35 cm (CCLmin). The juvenile aggregation
recruiting into Príncipe Island waters exhibited one of the lowest h
and π values of all studied hawksbill foraging grounds in the Atlantic.
The situation is similar to that reported for hawksbills stranding along
the Texas coast, for which the main contributing rookery appeared to
be Mexico (84% from our many-to-many MSA; Bowen et al., 2007).
All pairwise comparisons between the Príncipe foraging aggregation and previously studied feeding areas, except Cape Verde,
revealed highly significant differences (exact test of differentiation
P b 0.01; φST N 0.87, p b 0.01). Differentiation with respect to the nearby
Cape Verde Islands foraging ground was lower although still
significant (exact test of differentiation P b 0.001; φST = 0.068,
p = 0.02). The AMOVA based on φST values showed a strong genetic
structure dividing foraging grounds located in the western and
eastern Atlantic, with 84% of the genetic variation accounted for by
differences between the two groups, with 15% of the total variation
attributed to the differences within foraging grounds. The Monmonier's algorithm computed in BARRIER, based on the φST distance
matrix, showed that the first genetic barrier isolates Príncipe and Cape
Fig. 5. Many-to-many mixed stock analysis of Príncipe Island. A) Estimated contributions by different nesting populations to the Príncipe foraging aggregation. Values and their
confidence intervals were generated using a foraging ground-centric approach and prior probabilities weighted by rookery sizes. B) Estimates of the contribution by the Príncipe
nesting population to different Atlantic feeding areas. Values and their confidence intervals were generated using a rookery-centric approach and prior probabilities weighted by
rookery sizes.
352
C. Monzón-Argüello et al. / Journal of Experimental Marine Biology and Ecology 407 (2011) 345–354
Verde foraging grounds (eastern Atlantic) from all other studied
feeding areas (western Atlantic) (Fig. 1). These results confirmed
those obtained with the many-to-many rookery-centric mixed stock
analysis, where juveniles from eastern Atlantic rookeries distributed
mainly to eastern Atlantic feeding grounds.
Finally, the many-to-many foraging ground-centric MSA indicated
that the vast majority of the juveniles at the studied site come from
the Príncipe nesting population (mean value = 84%), with a small
proportion originating from Caribbean rookeries (Fig. 5B), although
all non-Príncipe sources included values of 0 within their confidence
intervals. On the basis of the identity of non-Príncipe haplotypes,
Caribbean source candidates would need to be restricted to those with
haplotype A at high frequencies. Notably, two of the largest rookeries
in the Caribbean (Barbados and Cuba) are thus characterised.
Nonetheless, the presence of haplotype Ei-A87 in three individuals,
which had to be removed from the MSA due to lack of information of
its source of origin, strongly suggests additional recruitment from
regional sources aside from Príncipe (Table 1).
4. Discussion
This study represents the first mtDNA characterization for a
hawksbill nesting population in the eastern Atlantic. Our results have
finally identified the natal origin of haplotype EATL, previously
identified in foraging or market samples as an orphan haplotype.
Interestingly, haplotype EATL fits within the Indo-Pacific phylogenetic
clade (Fig. 4) and has also been reported in a foraging ground off the
Seychelles (Indian Ocean; haplotype 24 in Okayama et al., 1999). This
result suggests additional transoceanic connectivity or a historical longdistance migration that might be clarified as genetic surveys are
extended in the Indo-Pacific. This would not be the first time that
transoceanic migrations have been described for a tropical species. For
example, Bourjea et al. (2007) found Atlantic haplotypes in Indo-Pacific
green turtle nesting populations, indicating recent matrilineal gene flow
from the Atlantic Ocean into the Indian Ocean via the Cape of Good
Hope. Similarly, satellite tracking studies have shown that leatherback
turtles (Dermochelys coriacea) move between the two ocean basins
around southern Africa (Luschi et al., 2006). In our study, the neighbourjoining tree results suggested gene flow from the Indian to the Atlantic
Ocean, since haplotypes from the eastern Atlantic were grouped into the
Pacific phylogroup. Several examples of marine dispersal connecting
Indian Ocean and eastern Atlantic species may be found in the literature,
such as for bigeye tuna (Chow et al., 2000; Durand et al., 2005) and
hammerhead sharks (Duncan et al., 2006). The movement of individuals
from the Indian Ocean to the Atlantic is certainly assisted by the flow of
the warm water of the Agulhas Current. Thus, we hypothesise that
hawksbills from the Indian Ocean colonised Príncipe Island by migrating
around southern Africa.
The low genetic variability detected in the Príncipe nesting
population might be due to the relatively low sample size, but more
likely it may be the result of an isolated founder event compounded by
strong natal homing. An alternative and non-exclusive explanation
may be a recent bottleneck brought about by population collapses
caused by high exploitation by local and regional fishing operations.
All population pairwise comparisons revealed that Príncipe is a
significantly independent unit, statistically different from all studied
Atlantic nesting populations (Table 2). The genetic distinctiveness of
this population together with its low genetic variability substantiates
the need to prioritise conservation and management plans for the
species in the eastern Atlantic.
The lack of differentiation between mature females and males at
Príncipe Island (exact test of differentiation P = 1.00; p = 0.99; in both
cases), suggests that males are as philopatric as the breeding females.
Direct long-term tracking has recently shown that for some species,
males travel directly back to the same nesting area across different
years (Hays et al., 2010a). Further, finding haplotype profiles in large
juveniles that are identical to those of the adults suggests a strong
natal homing behaviour in juveniles as well. However, we cannot rule
out the presence of regional rookeries with identical haplotype
profiles in combination with migration to Príncipe.
The presence of haplotype A (so far only reported in Caribbean
rookeries), which was found at low frequency at the Príncipe foraging
ground (4%) and only in the small juveniles (b 35 cm CCL),
corroborates the finding that eastward transatlantic movements
during early developmental stages do occur in hawksbills, though
only occasionally (Bellini et al., 2000; Bowen et al., 2007; Grossman et
al., 2007; Marcovaldi and Filippini, 1991; Monzón-Argüello et al.,
2010a). These results are consistent with the passive drift hypothesis
of early stages that have been shown in other species (Hays et al.,
2010b; Hays and Marsh, 1997; Monzón-Argüello et al., 2010b). As no
Caribbean haplotypes were found among adult individuals, our results
are consistent with a high degree of genetic isolation of the Príncipe
population, punctuated by very low levels of transoceanic migrations
(b 5%) from western Atlantic sources. These movements could remain
during early developmental phases prior to returning to their natal
sites at some point without interbreeding with East Atlantic hawksbill
stocks. Nonetheless, juveniles hatched at Príncipe Island appear to
distribute themselves regionally but only within eastern Atlantic
habitats (75% of the juveniles from Príncipe migrated to nearby
Príncipe and Cape Verde feeding areas). These conclusions are also
supported by the high degree of genetic differentiation found
between eastern and western Atlantic feeding aggregations (84% of
the variation in AMOVA; φST = 0.852, p b 0.001) and the Monmonier's
algorithm, where the major genetic barrier divides eastern from all
western Atlantic foraging areas. The genetic isolation of eastern
Atlantic hawksbill rookeries in combination with very low levels of
eastward transoceanic dispersal of early-staged animals is reminiscent of the migratory behaviour of other species such as the
loggerhead or the green turtle. Western Atlantic loggerhead populations are known to undertake transoceanic migrations during their
first years of life (Bjorndal et al., 2000; Bolten et al., 1993, 1998; Carr,
1986; Monzón-Argüello et al., 2009). Juvenile loggerheads are found
in various eastern Atlantic foraging habitats, with a very high
percentage (N 80%) of individuals originating in western Atlantic
rookeries (Bolten et al., 1998; Monzón-Argüello et al., 2009).
Similarly, an important proportion of juvenile green turtles feeding
off the Cape Verde Islands (38%) come from Suriname, in the western
Atlantic (Monzón-Argüello et al., 2010b). Furthermore, the clustering
analysis of green turtle feeding grounds failed to produce any
consistent results, and the AMOVA showed that the variation was
equally derived from differences within (49%) and among feeding
grounds (51%) (φST = 0.507, p b 0.001). Consequently, though our
results confirmed the existence of eastward transoceanic movements
by Atlantic hawksbills, this transport is much less frequent than in
other sea turtle species (Bolker et al., 2007; Bolten et al., 1998;
Carreras et al., 2006; Casale et al., 2002; Monzón-Argüello et al., 2009;
Monzón-Argüello et al., 2010a). We cannot rule out that the lower
levels of reported transoceanic movements in early staged hawksbills
may be due to much smaller population sizes when compared to the
other two species (the Atlantic-wide population size for hawksbills is
estimated to be less than 3050 breeding females per year) (Mortimer
and Donnelly, 2008), which is ~ 10 times lower than that for
loggerheads at 27,000 individuals (Ehrhart et al., 2003; Marcovaldi
and Chaloupka, 2007) and green turtles at 38,000 (Seminoff, 2004). A
further possibility is that hawksbills are more restricted to tropical
environments and hence less able to undertake long-distance
movements that require trajectories through high latitude habitats.
Additional studies are needed to refine our knowledge of the
genetic makeup of remaining hawksbill rookeries in the eastern
Atlantic. The fact that orphan haplotypes were discovered in the
Príncipe foraging habitat is an indication that further sampling of this
and other populations is required.
C. Monzón-Argüello et al. / Journal of Experimental Marine Biology and Ecology 407 (2011) 345–354
4.1. Conservation implications and future research
Although hawksbills are critically endangered worldwide (Mortimer
and Donnelly, 2008 — IUCN 2010), important knowledge gaps remain in
the conservation biology of this species. While Caribbean hawksbill
turtles are relatively well-studied for population genetics and connectivity (Bowen and Karl, 2007), this study represents the first mtDNA
description of a nesting population in the eastern Atlantic. Our finding
that hawksbills from Príncipe show a high degree of genetic isolation
compared to other Atlantic nesting populations, yet retain an
evolutionary link with southwestern Indian Ocean turtles, is intriguing.
More extensive rookery-based information is needed from western
Atlantic habitats to determine if the haplotype clade observed in the
Príncipe and Cape Verde aggregations is widely distributed and retains a
link with the Indo-Pacific or if instead, it represents an independent
phylogenetic branch associated exclusively with the eastern Atlantic.
The existence of orphan haplotypes in the Príncipe foraging
aggregation (Table 1) underscores the necessity of additional
sampling of the Príncipe rookery (to rule out a sampling artefact),
but also highlights the need to expand the genetic analysis to other
nesting populations along the western coast of Africa to include
unsampled areas. This may reveal the true extent to which animals
from regional rookeries interact in developmental habitats and the
relevance of threats to the survival of the Príncipe rookery.
Nevertheless, the genetic distinctiveness and the low genetic
variability found in the Príncipe population are evidence that there is
a need to conserve the rookery as a priority not only for the
population, but also for the species in the eastern Atlantic basin.
Furthermore, the genetic evidence signifying the migratory and
dispersal links between Príncipe and other habitats such as Cape
Verde, illustrates that international collaboration will be essential to
ensure comprehensive protection of all life stages to minimise
hawksbill mortality in the region and ensure the long-term protection
of this unique eastern Atlantic stock.
Acknowledgements
This paper is an output of the Programa SADA — Sustainable
Conservation of the Hawksbill Population at Príncipe Island (www.
tartarugasstomeprincipe.org), supported by the Oceanário de Lisboa,
and by a Marine Turtle Conservation Act — U.S. Fish & Wildlife Service
grant. We thank the President of the Autonomic Government of Príncipe
Island for the strong support to the sea turtle protection efforts in the
region, N. Pereira, from the Oceanário de Lisboa, for continual help and
support. We are grateful to D. Power and R. Castilho, both from the
University of Algarve, for valuable suggestions during the initial
campaigns, N. Viegas and T. Pires, from LeArt Castelo S.A., D. Matos,
from Príncipe Fisheries Service, D. Ramos, from Príncipe Island Natural
Park, volunteers A. Almeida, P. Patrício, R. Ferreira, and all the other
people that contributed to field work and sampling. We also thank the
Instituto Canario de Ciencias Marinas, Estación Biológica de Doñana, and
Fundación BBVA for help with laboratory equipment and Centre of
Marine and Environmental Research (CIMAR) for help with field
sampling equipment. Collection, transport and processing of samples
were conducted under the cooperative agreement between the
University of Algarve (Portugal) and the Autonomic Government of
Príncipe Island. We are also indebted to G. Hays, an anonymous
reviewer, K. Stewart and P. Dutton for improvements to the original
manuscript. [SS]
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