MARINE ECOLOGY PROGRESS SERIESMar Ecol Prog Ser

Vol. 424: 273–283, 2011
doi: 10.3354/meps08989

Published March 1

Population structure of the loggerhead turtle
Caretta caretta, a large marine carnivore thatexhibits alternative foraging behaviors
Kunihiro K. Watanabe1, 9, Hideo Hatase1,*, Masato Kinoshita2, Kazuyoshi Omuta3,
Takeharu Bando4, Naoki Kamezaki5, Katsufumi Sato6, Yoshimasa Matsuzawa5,
Kiyoshi Goto7, Yoshito Nakashima8, Hiroshi Takeshita8, Jun Aoyama1,
Katsumi Tsukamoto1
1

Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan
2
Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan
3
Yakushima Sea Turtle Research Group, 489-8 Nagata, Yakushima, Kagoshima 891-4201, Japan
4
Institute of Cetacean Research, 4-5 Toyomi, Chuo, Tokyo 104-0055, Japan5
Sea Turtle Association of Japan, 5-17-18-302 Nagao-motomachi, Hirakata, Osaka 573-0163, Japan
6
International Coastal Research Center, Atmosphere and Ocean Research Institute, The University of Tokyo, Otsuchi,
Iwate 028-1102, Japan
7
Minabe Sea Turtle Research Group, 278 Higashiyoshida, Minabe, Wakayama 645-0001, Japan
8
Miyazaki Wildlife Animal Research Group, 9779-8 Shimanouchi, Miyazaki 880-0121, Japan

9

Present address: National Institute for Land and Infrastructure Management, 1 Asahi, Tsukuba, Ibaraki 305-0804, Japan

ABSTRACT: Knowledge of detailed population genetic structure is crucial to conserve and manage
endangered species effectively. Size-related variation in feeding-habitat use (neritic vs. oceanic) by
adult loggerhead turtles Caretta caretta has been reported within several populations, and sympatricpopulation subdivision was suspected. In the present study, genetic differences between the 2
feeding-habitat groups within 2 Japanese nesting sites were assessed, using 5 microsatellite loci andmitochondrial (mt) DNA sequences. There were no genotypic or haplotype differences between thefeeding-habitat groups, which were defined by egg-yolk stable isotope ratios and body size, at both
nesting sites, suggesting that both neritic and oceanic individuals belong to the same genetic population. Differences in feeding-habitat use are unlikely to be a limiting factor for gene flow between
feeding-habitat groups and were thought to be the result of phenotypic plasticity rather than population subdivision. Gene flow among 5 nesting sites was assessed by pooling these feeding-habitatgroups at each nesting site. Significant genetic structure by female natal homing was observed at the
mtDNA level. However, no significant structure was found at the microsatellite DNA level, suggesting male-mediated gene flow caused by migration through courtship areas. Although nesting
beaches are connected by male-mediated gene flow, which might have evolved as a mechanism to
avoid genetic fragmentation by natal homing, extirpated beaches would not be easily recolonizedfrom other nesting populations due to female philopatry. Therefore, conservation of individual nesting beaches is still needed to maintain the overall genetic diversity of Japanese loggerheads.
KEY WORDS: Alternative life histories · Microsatellite DNA · Mitochondrial DNA · Phenotypic
plasticity · Reptile · Caretta caretta
Resale or republication not permitted without written consent of the publisher

INTRODUCTION
Information on population genetic structure isfundamental for recognizing management units ofendangered species (Moritz 1994). Because population
genetic structure depends on both the resolution andthe inheritance modes of genetic markers, it should beanalyzed through the combined use of multiple mark-*Corresponding author. Email: hatase@aori.u-tokyo.ac.jp© Inter-Research 2011 · www.int-res.com

274

Mar Ecol Prog Ser 424: 273–283, 2011ers. Sequences of maternally inherited mitochondrial(mt) DNA and microsatellites of biparentally inherited
nuclear DNA are the markers commonly used in recent
wildlife population genetics (Frankham et al. 2002). In
principle, due to their faster rate of evolution, the resolution of microsatellites is greater than that of mtDNA
sequences. Nevertheless, in some cases, populationdifferentiation inferred from mtDNA sequences isstronger than that from microsatellites (e.g. FitzSimmons et al. 1997b, Bowen et al. 2005, Carreras et al.
2007, Chen et al. 2008, Lukoschek et al. 2008, Okello et
al. 2008, Caparroz et al. 2009, Hefti-Gautschi et al.
2009, Portnoy et al. 2010), and this has been attributed
to sex differences in natal philopatry and dispersal.
Individuals sampled at a location are usually treatedas a single population in wildlife population genetics(Frankham et al. 2002). However, if they show intrapopulation polymorphisms in life history, sympatricpopulation subdivision should be assessed before examining overall population structure. Intra-population
polymorphisms in life history have been observed inseveral types of animals such as insects (Harrison1980), fishes (Tsukamoto et al. 1998), amphibians (Wilbur & Collins 1973) and birds (Lundberg 1988). For
example, some salmonid fishes have mature individuals of 2 types — sea/lake run and river resident types(Thorpe 1986, Olsson et al. 2006) — and the river resident type is smaller than the sea/lake run type.
Whether there are genetic partitions between alternative phenotypes varies among species (e.g. Berthold
1991, Voss 1995, Jones et al. 1997, Braendle et al. 2005,
Hoelzel et al. 2007). The occurrence and maintenanceof alternative life histories have become important
issues in evolutionary ecology (Gross 1996).
Sea turtles may be a good subject for addressing the
aforementioned issues related to both conservation/
population genetics and evolutionary ecology. In sea
turtles, intra-population polymorphisms in life history
were first reported as variation in feeding-habitat use
by adult female loggerhead turtles Caretta caretta
nesting in Japan (Hatase et al. 2002d, 2007). Loggerhead turtles are generally carnivorous throughout
their lives (Dodd 1988). Using stable isotope analysis
and satellite telemetry, these studies revealed that,during the non-nesting season, large females foraged
mainly on benthic items in neritic waters where water
depths are < 200 m, while small females, like the juveniles, foraged mainly on planktonic items in oceanic
waters where depths are > 200 m. Because female sea
turtles grow little after reaching sexual maturity(Hatase et al. 2004), this phenomenon does not meanthat adult females shift habitats with aging. Similar
foraging dichotomies have increasingly been reported
in loggerheads nesting at Cape Verde (Hawkes et al.
2006) and Florida, USA (Reich et al. 2010), and ingreen turtles Chelonia mydas nesting at Ogasawara
Islands, Japan (Hatase et al. 2006), as well as at the
Galápagos Islands, Ecuador (Seminoff et al. 2008).
Male loggerheads were also thought to show this typeof foraging dichotomy (Hatase et al. 2002c). Thesefindings suggest that intra-population polymorphisms
in life history might be common in sea turtles (McClellan & Read 2007, Caut et al. 2008, Mansfield et al.
2009, McClellan et al. 2010, Zbinden et al. 2011).
Alternative life histories sometimes have a genetic
basis coupled with assortative mating (e.g. Maekawa
et al. 1994, Bearhop et al. 2005, Ólafsdóttir et al. 2006).
Assortative mating may result from morphologicalmate choice and/or temporal reproductive isolation. In
the case of adult loggerheads, because oceanic andneritic foragers are different in body size, they might
use this character for mate choice. The timing of arrival
to courtship areas, which are generally near nesting
beaches (Limpus 1993), might also differ between the
2 feeding-habitat groups, because distances to thecourtship areas are different between feeding habitats(Hatase et al. 2002d, 2007). These phenotypic differences might contribute to assortative mating between
oceanic and neritic foragers, thus making population
subdivision possible.
Although loggerhead turtles are circumglobally distributed in tropical and temperate waters (Dodd 1988),
Japan is the only country that hosts nesting beaches forthis species within the North Pacific. A substantial
decline in the number of nests has been reported
among Japanese populations during the last half of the20th century (Sato et al. 1997, Hatase et al. 2002c,
Kamezaki et al. 2003), and actions for conservation areurgently needed. In the present study, we firstassessed the relationship between feeding-habitat use,which was reflected in egg-yolk stable isotope ratios
and body sizes, and genetic features within Japaneseloggerhead turtle nesting beaches, using both mtDNA
sequences and 5 microsatellite loci. Second, overall
population structure among nesting beaches was
examined with the same markers. Based on the resultsof these analyses, we discuss sex-biased gene flow andconservation of Japanese loggerheads.

MATERIALS AND METHODS
Sample collection and DNA extraction. From May toAugust 1994 to 2004, a total of 284 nesting females of theloggerhead turtle Caretta caretta were sampled from 5
nesting sites in southern Japan (Fig. 1, Table 1): Minabe(33° 46’ N, 135° 18’ E, 1994 and 1995; n = 115), Kamouda(33° 15’ N, 134° 45’ E, 2002 to 2004; n = 10), Miyazaki(31° 47’ to 32° 08’ N, 131° 28’ to 131° 32’ E, 1995 and 1999;
n = 46), Yakushima (30° 24’ N, 130° 26’ E, 1995 and 1999;

Watanabe et al.: Population structure of Japanese loggerheads

275

Muscle tissue samples were preserved in a high concentration urea-containing buffer (TNES-Urea: 6 or
8 M urea; 10 mM Tris-HCl, pH 7.5; 125 mM NaCl;
10 mM EDTA; 1% SDS; Asahida et al. 1996) or 99%
ethanol at room temperature, while blood samples
were preserved in a TNES buffer (100 mM Tris-HCl,
pH 8.0; 10 mM NaCl; 100 mM EDTA; 1% SDS; Bass etal. 1996) at room temperature. Total DNA was extracted from the samples using a standard phenol/
chloroform (1:1) treatment or DNeasy Tissue Kit (QIAGEN) and then suspended in TE buffer (10 mM TrisHCl, pH 8.0; 1 mM EDTA).
PCR amplification and genotype/haplotype determination. Five microsatellite markers — Cc7, Cc117,
Cc141, Cm84, and Ei8 (FitzSimmons et al. 1995, 1999,
FitzSimmons 1998) — were used for genotyping. Forward primers were labeled with fluorochrome. Polymerase chain reaction (PCR) amplification was carriedout in the Model 9700 thermal cycler (Applied Biosystems) with a 15 µl reaction volume containing 100 ng oftemplate DNA, 1 × PCR buffer (TaKaRa), 0.5 µM ofeach primer, 0.2 mM of each dNTP, and 0.4 U ExTaq
DNA polymerase (TaKaRa). The PCR amplification
condition consisted of initial denaturation at 94°C for
5 min, followed by 30 cycles consisting of 30 s at 94°C,
1 min at 55 or 59°C (optimal annealing temperature foreach primer pair), 30 s at 72°C, and a final extraction
for 7 min at 72°C. Allele sizes were resolved
on an ABI 3130xl genetic analyzer (Applied
45°N Biosystems). Individual genotypes were assigned using GeneMapper software Ver. 3.5(Applied Biosystems).
Details of mtDNA analysis are describedin Hatase et al. (2002b). A portion (350 base40°
pairs [bp]) of the mtDNA control region was
analyzed by direct sequencing of 111 samples collected in the first year at 4 nestingsites (Minabe, Miyazaki, Yakushima, and
Japan
Korea
Fukiagehama), and 7 polymorphic sites
35°
were detected as described by Bowen et al.
(1995). These polymorphisms resolved 3
Minabehaplotypes (A, B, and C: Bowen et al. 1995),
KamoudaChina
Fukiagehama
which differed by 4 or 5 nucleotide substituMiyazakitions. Haplotype A is the most dominant in30°
East
Yakushima
the South Pacific and is designated CCP1
China
there (Boyle et al. 2009). PCR restriction
Sea
Pacific Ocean
fragment length polymorphism (RFLP) ana200
lysis was alternatively used for 148 samples
25°
collected in the second year at the 4 nestingsites to assign the 3 haplotypes. Because the
500 kmTaiwansequenced length for checking polymorphism by Bowen et al. (1995) and Hatase et
120°E
125°
130°
135°
140°
al. (2002b) was short (350 bp), a longer fragment of 650 bp was sequenced for a prelimFig. 1. Caretta caretta. Locations of the 5 nesting sites sampled forloggerhead turtles in southern Japan. Contour line: 200 m depth
inary set of 20 individuals from Kamouda
n = 91), and Fukiagehama (31° 30’ N, 130° 18’ E, 1997 and1999; n = 22). Nesting females were identified by tagging,and their straight carapace lengths were measured withcalipers during nightly patrols (Kamezaki et al. 1997, Satoet al. 1997). Female loggerheads lay several clutches of
eggs (ca. 110 eggs clutch–1) on specific sandy beaches
every ca. 2 wk during a nesting season and remigrate
there with high fidelity from distant feeding grounds
every few years (Dodd 1988). In fact, there are no published records on the exchange of tagged females among
the above 5 sampled sites, despite the large number(> 2000) of tagged females (Kamezaki et al. 1997). Small
pieces of muscle tissue, which were secondarily recovered while punching front flippers of nesting females for
attachment of plastic tags, were collected for both
microsatellite and mtDNA analyses. In addition, blood
samples of 10 hatchlings derived from 10 individual nesting females (1 hatchling female–1) were used to determine mtDNA haplotypes of loggerheads nesting at
Kamouda; these were not used in the microsatellite
analysis. These hatchlings were collected when they
emerged from their nests, which had been marked during the nesting season and enclosed by nets just before
the emergence (ca. 60 d after the oviposition). The eggs
from 48 individual loggerhead turtles (1 egg female–1)
were collected at Yakushima for stable isotope analysis(Hatase et al. 2002d).

276

Mar Ecol Prog Ser 424: 273–283, 2011

Table 1. Caretta caretta. Sample sizes for nesting sites and foraging-habitat groups of Japanese loggerhead turtles. Sample sizesof mtDNA analysis for Minabe (MN), Miyzaki, Yakushima (YK), and Fukiagehama are from Hatase et al. (2002b). Females from
Minabe were divided into 4 size groups by straight carapace length (mm) — MN-1: < 800; MN-2: 800 to 850; MN-3: 850 to 900;
MN-4: ≥900. Females from Yakushima were divided into 2 groups based on stable isotope ratios in egg yolks — YK-Oc: oceanic
forager; YK-Nr: neritic forager
Locus

mtDNA
Cc7
Cc117
Cc141
Cm84
Ei8

MinabeKamouda

102
105
111
110
109
112

20
10
8
9
9
10

Nesting site
Miyazaki Yakushima Fukiagehama
46
40
42
42
44
43

89
86
83
81
73
77

using the primer sets AKACB1 (Hatase et al. 2002b)
and TCR6 (Norman et al. 1994). Nineteen samples with
Haplotype B exhibited exactly the same 309 to 650 bp
sequence and could not be divided into any more
haplotypes. Although 1 female had a sequence corresponding to Haplotype C, only 2 sites were discovered
as new polymorphic sites outside the region sequenced
by Bowen et al. (1995) and Hatase et al. (2002b). Newly
determined 309 to 650 bp sequences of Haplotypes B
and C were deposited in the DNA Data Bank of Japan(DDBJ; Accession Numbers AB290590, AB290591,
AB548628, AB548629, and AB548631 to AB548646).
Classification into foraging-habitat groups. Stablecarbon and nitrogen isotope ratios (δ13C and δ15N) in
egg yolks from 48 females that nested at Yakushima
were measured (Hatase et al. 2002d); the females witha δ13C of less than –18 ‰ and a δ15N of less than 12 ‰were estimated to inhabit oceanic waters (the oceanic
Pacific, seafloor depth > 200 m), whereas the otherfemales were estimated to inhabit neritic waters (suchas the continental shelf in the East China Sea andcoastal waters of Japan, seafloor depth < 200 m). Basedon these findings, 8 females (mean ± SD straight carapace length: 826 ± 45 mm) were classified into an
oceanic foraging group, while the other 40 females(854 ± 43 mm) were classified into a neritic foraging
group (Table 1).
At Minabe, because stable isotope data were notavailable, straight carapace lengths of nesting females
were alternatively used for classification into foraginghabitat groups. A total of 112 females were classified
into 4 groups (< 800 mm, n = 20; 800 to 850 mm, n = 51;
850 to 900 mm, n = 29; ≥900 mm, n = 12; Table 1),
because there had been significant differences in both
egg-yolk δ13C and δ15N among these categories at bothMinabe and Yakushima (Hatase et al. 2002d). In thisclassification, small and large size groups roughly corresponded to oceanic and neritic foraging groups,
respectively, owing to high variations in both δ13C andδ15N within each size class.

22
21
19
21
18
21

MN-1
20
18
19
19
19
20

Foraging-habitat group
MN-2 MN-3 MN-4 YK-Oc
45
48
48
50
50
50

23
26
28
27
26
27

11
11
12
11
11
12

8
8
8
8
7
6

YK-Nr
40
40
40
40
37
39

Data analysis. Genetic diversities at microsatellite
loci were expressed as the number of alleles and observed/expected heterozygosities (HO/HE); the heterozygosities were calculated in the program GENEPOP
Ver. 3.4 (Raymond & Rousset 1995). Departure from
Hardy-Weinberg equilibrium at each locus within each
population and the linkage disequilibrium between lociwithin each population were tested by a Markov chain
method (10 000 dememorizations, 100 batches, and5000 or 10 000 iterations) with the above program. The
sequential Bonferroni method (Rice 1989) was applied
to correct for multiple simultaneous comparisons in theabove 2 tests. If deviations from Hardy-Weinberg equilibrium were found, the existence of null alleles andgenotyping errors was assessed (1000 randomizations)using MICRO-CHECKER Ver. 2.2.3 (Van Oosterhout etal. 2004). Genetic diversities at the mtDNA level wereexpressed as the number of haplotypes and the haplotype (h) and nucletotide diversities (π). The h and π
values were calculated using the method of Nei (1987),
taking into account the genetic distances between
haplotypes estimated by the Jukes-Cantor method(Jukes & Cantor 1969), in the program ARLEQUIN Ver.
2.000 (Schneider et al. 2000). Population size for eachnesting site, which was represented as the number ofnests per season, was cited from literature to explore
the relationship between it and genetic diversities.
Genetic differentiations between foraging-habitatgroups were tested using genotypic frequencies, because some loci might exhibit deviations from HardyWeinberg equilibrium. Microsatellite genotypes were
permutated among samples (10 000 randomizations)using a genotypic goodness-of-fit test (Goudet et al.
1996) that was implemented in FSTAT Ver. 2.9.3.2(Goudet et al. 2002). Differences in mtDNA haplotype
frequencies were examined using an exact test
(500 000 of steps in Markov chain and 1000 dememorization steps) that was implemented in ARLEQUIN
Ver. 2.000 (Schneider et al. 2000). For both mtDNA andmicrosatellite loci, an unbiased estimator of Wright’s

277

Watanabe et al.: Population structure of Japanese loggerheads(Table 3). Of 55 population/ locus combinations, 53
were in Hardy-Weinberg equilibrium after adjusting
the significance (α) level for the number of pairwise
comparisons of populations and loci (n = 55; α = 0.01;
Table 2). Although combinations of Cc7 and Cc141
within Yakushima exhibited heterozygote deficiency,
the existence of null alleles and genotyping errors was
not suggested. Linkage disequilibrium was notobserved between any loci in any population after
sequential Bonferroni correction (n = 102, test resultsof 8 pairwise comparisons were lacking due to small
sample sizes; α = 0.01). Therefore, all 5 loci in each
population were used for the following analyses.
Three mtDNA haplotypes (A, B, and C) were observed among a total of 279 samples from 5 nestingsites (Table 3), where Haplotype B was the most common and accounted for 89% of the total. The h and π
values varied among nesting sites, from 0.058 and
0.00083, respectively, at Minabe to 0.415 and 0.00598
at Miyazaki (Table 3). Overall h and π values were0.193 and 0.00277, respectively. Because there were no
significant differences in mtDNA haplotype frequencies between sampling years within each nesting site
(Hatase et al. 2002b), samples from different years
were pooled at each nesting site.

FST (θ) (Weir & Cockerham 1984) was calculated between foraging-habitat groups or among nesting sites
using 10 000 steps of permutation tests in ARLEQUIN
Ver. 2.000 (Schneider et al. 2000). For microsatellite
loci, an unbiased estimator of Slatkin’s RST (ρ) (Slatkin1995) was also calculated between foraging-habitatgroups or among nesting sites using 1000 steps of permutation tests with RST Calc (Goodman 1997). Whenthe numbers of alleles were compared among nestingsites, rarefaction analysis was done to investigate theeffect of undersampling using the Rarefaction Calculator (www2.biology.ualberta.ca/jbrzusto/rarefact.php;
accessed 15 September 2010).

RESULTS
Genetic diversities at microsatellite loci and mtDNA
For Caretta caretta, the number of alleles per locus
ranged from 10 (locus Cc7) to 15 (loci Cc117 and Ei8),
with an average of 13.4 (Table 2). HO values ranged
from 0.500 (Cc7 in Kamouda, the 800 to 850 mm sizegroup of Minabe [MN-2], the oceanic group of Yakushima [YK-Oc], and Cc117 in YK-Oc) to 1.000 (Cc141
in the ≥900 mm size group of Minabe [MN-4]),
whereas HE values ranged from 0.511 (Ei8 in Kamouda) to 0.908 (Cc141 in Kamouda). Overall means of HO
and HE were 0.718 and 0.738, respectively. While HO at
Cc117 and Cm84 within Yakushima, the largest nesting population in Japan (Table 3), were 0.651 and
0.740, respectively, those within Kamouda were 0.750
and 0.889, despite its extremely small population size

Genetic differences between foraging-habitatgroups within nesting sites
At Yakushima, among the observed alleles, 1 or 2 specific alleles were predominant at each microsatellite locus (Fig. S1 in the supplement at www.intres.com/articles/suppl/m424p273_supp.pdf). Genotypic

Table 2. Caretta caretta. Genetic diversities at 5 microsatellite loci among nesting sites and foraging-habitat groups for Japaneseloggerhead turtles. Group abbreviations as defined in Table 1. K: number of alleles; HO: observed heterozygosity; HE: expected
heterozygosity
Site or group
K

Cc7
HO

HE

K

Cc117
HO

HE

K

Cc141
HO

HE

K

Cm84
HO

HE

K

Ei8
HO

HE

MinabeKamoudaMiyazaki
Yakushima
Fukiagehama

6
4
6
9
5

0.590
0.500
0.625
0.593a0.667

0.664 120.642 6
0.623 100.653 130.678 8

0.748
0.750
0.786
0.651
0.526

0.727
0.617
0.794
0.713
0.761

12
8
9
12
10

0.773
0.778
0.857
0.815a
0.762

0.825
0.908
0.778
0.857
0.807

12
7
9
11
10

0.789
0.889
0.727
0.740
0.889

0.765
0.817
0.753
0.753
0.813

13
5
11
12
7

0.732
0.600
0.674
0.649
0.714

0.772
0.511
0.628
0.700
0.679

MN-1
MN-2
MN-3
MN-4
YK-Oc
YK-Nr

5
6
6
5
4
7

0.667
0.500
0.731
0.636
0.500
0.600

0.694 7
0.666 100.689 70.662 70.575 70.616 9

0.842
0.694
0.821
0.833
0.500
0.700

0.802
0.725
0.721
0.736
0.692
0.674

8
10
11
8
8
11

0.789
0.720
0.778
1.000
0.875
0.800

0.848
0.815
0.847
0.853
0.842
0.869

8
10
11
5
5
11

0.789
0.760
0.808
0.818
0.571
0.748

0.785
0.761
0.770
0.736
0.758
0.793

9
13
8
7
5
11

0.750
0.740
0.704
0.667
0.667
0.667

0.701
0.813
0.734
0.768
0.667
0.724

Overall
Mean

10
0.601

0.651

0.714

0.724

0.813

0.841

0.775

0.773

0.688

0.700

a

15

13

14

15

Significant departure from Hardy-Weinberg equilibrium, based on exact tests after sequential Bonferroni correction (p < 0.01)

278

Mar Ecol Prog Ser 424: 273–283, 2011

Table 3. Caretta caretta. Distribution of mtDNA haplotypes, genetic diversities,
and population sizes among 5 nesting sites for Japanese loggerhead turtles. All
data, except for Kamouda, on mtDNA distributions and diversities are from
Hatase et al. (2002b). The numbers of nests per season were recorded during
sampling periods, i.e. in the 1990s for Minabe (Hatase et al. 2002a), Miyazaki(Kamezaki et al. 2003), Yakushima (Kamezaki et al. 2003), and Fukiagehama(Kagoshima University Sea Turtle Research Group 2002), and in the early 2000s
for Kamouda (Watanabe 2006)

Genetic differences among nestingsites

Global FST at mtDNA among 5 nesting sites was 0.094 (p < 0.001). Pairwise
FST between 2 nesting sites ranged
from –0.024 to 0.268, with 5 of 10 combinations being significantly different
Nesting
Haplotype
Total Haplotype Nucleotide Nests perfrom 0 (p < 0.05) (Table 4).
site
A
B
C
diversity (h ) diversity (π) season
At all microsatellite loci, distributions of allelic frequencies were almost
Minabe
0
99
3
102
0.058
0.00083
29–349the same among nesting sites (Fig. S3
Kamouda
0
19
1
20
0.100
0.00144
13–23
in the supplement at www.int-res.com/
Miyazaki
0
33 13
46
0.415
0.00598 200–900
Yakushima
1
77 11
89
0.239
0.00339 600–1600
articles/suppl/m424p273_supp.pdf). AlFukiagehama 0
21
1
22
0.091
0.00131
18–125though some alleles were not observed
Total
1 249 29279
0.193
0.00277
in Kamouda and Fukiagehama, thosemissing alleles were probably due tosmaller sample sizes rather than the existence of
frequencies were not significantly different between
beach-specific alleles, because those alleles had low
the 2 foraging-habitat groups at each microsatellite
frequencies in Minabe, Miyazaki, and Yakushima.
locus (Cc7, p = 0.945; Cc117, p = 0.732; Cc141, p =Rarefaction analysis confirmed this interpretation,
0.392; Cm84, p = 0.920; Ei8, p = 0.488; Fig. S1). FST and
because the numbers of alleles in subsamples from
RST over loci between foraging-habitat groups were
Minabe, Miyazaki, and Yakushima were quite similar–0.013 and –0.022, respectively. These FST and RST values were not significantly different from 0 (p = 0.877
to those in the samples from Kamouda and Fukiageand 0.847, respectively). Two mtDNA haplotypes (Bhama at all loci (Table 5). Global FST among the 5 nestand C) were observed in the analyzed samples (Fig.
ing sites did not exhibit significant departure from
S1). Haplotype frequencies were also not significantly
0 (FST = 0.003, p = 0.112). Pairwise FST between 2 nestdifferent between the 2 foraging-habitat groups (p =ing sites ranged from –0.013 to 0.011, with only 2 of
1.000), and FST was not significantly different from 0
10 combinations (Minabe–Fukiagehama and Yaku(FST = –0.081, p = 1.000).
shima–Fukiagehama) being significantly different
At Minabe, the allelic distributions in microsatellite
from 0 (p < 0.05) (Table 4). Pairwise RST between
loci were almost the same as at Yakushima (Fig. S2 in
2 nesting sites ranged from –0.020 to 0.009, with all
10 combinations not being significantly different fromthe supplement at www.int-res.com/articles/suppl/
m424p273_supp.pdf). There were no significant differ0 (p > 0.05) (Table 4). The original data on allelic andences in genotype frequencies among 4 size groups at
haplotype frequencies presented in Figs. S1 to S3 areeach microsatellite locus (Cc7, p = 0.953; Cc117, p =available as Table S1 in the supplement at www.int0.473; Cc141, p = 0.685; Cm84, p = 0.486; Ei8, p = 0.601;
res.com/articles/suppl/m424p273_supp.pdf.
Fig. S2). FST and RST over loci among
Table 4. Caretta caretta. Genetic partitions among 5 nesting sites for Japanesethe 4 size groups were –0.005 andloggerhead turtles. Above the diagonal are FST based on mtDNA sequence com–0.005, respectively. These FST and RST
parisons from Hatase et al. (2002b), with additional data from Kamouda; below
values were not significantly different
the diagonal are partitions based on 5 microsatellite loci (upper value: FST; lower
from 0 (p = 0.887 and 0.514, respecvalue: RST). Significant values (p < 0.05) based on permutation tests are indicated by asterisks
tively). Two mtDNA haplotypes (B andC) were observed in the analyzed samples (Fig. S2). Haplotype frequencies
Minabe Kamouda Miyazaki Yakushima Fukiagehama
were not significantly different among
Minabe–0.0240.268*
0.056*–0.024the 4 size groups (p = 1.000), and FST
Kamouda–0.0070.117*–0.001–0.050among the groups was not significantly–0.005different from 0 (FST = –0.025, p =Miyazaki
0.003–0.0090.061*
0.128*
0.874). Because there was no genetic
0.004–0.019differentiation
betweenforagingYakushima–0.006–0.013–0.0000.005
habitat groups at the 2 nesting sites,
0.000
0.006
0.000
samples at each nesting site were
Fukiagehama 0.009*
0.001
0.003
0.011*
pooled and used in the following inter0.006–0.020–0.0100.009
population comparisons.

Watanabe et al.: Population structure of Japanese loggerheads

Table 5. Caretta caretta. Results of rarefaction analysis on
number of alleles at 3 larger nesting sites (Minabe, Miyazaki,
and Yakushima). K: number of alleles; KKamouda andKFukiagehama: number of alleles (mean ± SD) in subsamples thesize of which is the same as at Kamouda and Fukiagehama,respectively
Locus

Nesting siteK

KKamouda

KFukiagehama

Cc7

MinabeKamoudaMiyazaki
Yakushima
Fukiagehama
MinabeKamoudaMiyazaki
Yakushima
Fukiagehama
MinabeKamoudaMiyazaki
Yakushima
Fukiagehama
MinabeKamoudaMiyazaki
Yakushima
Fukiagehama
MinabeKamoudaMiyazaki
Yakushima
Fukiagehama

6
4
6
9
5
12
6
10
13
8
12
8
9
12
10
12
7
9
11
10
13
5
11
12
7

4.4 ± 0.8

5.2 ± 0.7

4.4 ± 0.8
4.7 ± 1.0

5.3 ± 0.7
6.0 ± 1.1

5.8 ± 1.1

7.8 ± 1.1

6.6 ± 1.1
6.1 ± 1.3

8.9 ± 0.8
9.0 ± 1.3

7.3 ± 1.2

9.4 ± 1.0

6.9 ± 1.0
7.8 ± 1.2

8.7 ± 0.5
10.2 ± 1.0

6.2 ± 1.2

8.0 ± 1.2

6.1 ± 1.0
6.2 ± 1.1

7.5 ± 0.9
7.7 ± 1.1

7.2 ± 1.3

9.5 ± 1.2

6.1 ± 1.2
7.0 ± 1.3

8.4 ± 1.1
9.5 ± 1.1

Cc117

Cc141

Cm84

Ei8

DISCUSSION
The present study did not provide evidence of sympatric population subdivision in a large marine reptilethat exhibits alternative foraging behaviors. This result
may be reasonable when considering that sea turtles
are solitary and aggregate only during the breeding
season. In social marine mammals, such as killer
whales Orcinus orca, sympatric foraging specialists(ecotypes) are genetically distinct (Hoelzel et al. 2007).
Foraging skills are learned and transmitted amongindividuals within respective ecotype societies. In
contrast, solitary sea turtles develop foraging skills bythemselves. Therefore, the extent of sympatric genetic
structure may be dependent on the degree of sociality
among large marine animals. As observed in other terrestrial and marine animals (e.g. Okello et al. 2008,
Portnoy et al. 2010), gene flow among nesting sites
examined by genetic markers with different resolutions and modes of inheritance showed contrasting
patterns: greater gene flow at microsatellite DNA thanat mtDNA. Because we obtained similar results withother sea turtle population studies using a comparable
number of microsatellie loci (5 in the present study vs.

279

4 in FitzSimmons et al. 1997b, 5 in Bowen et al. 2005,
and 7 in Carreras et al. 2007), it is unlikely that thefindings in the present study would change dramatically with the use of more markers. In fact, althoughfine-scale population structure was assessed for loggerhead turtles nesting at the Cape Verde Islands with11 microsatellite loci, no significant differentiation was
found among the islands (Monzón-Argüello et al.
2010). We discuss these results in light of the evolutionof sea turtle behaviors and the conservation of Japanese loggerheads Caretta caretta.

Genetic similarity between foraging-habitat groups
within nesting sites
The lack of significant differences in genotype
and haplotype frequencies between foraging-habitatgroups within nesting sites suggests that large neritic
and small oceanic individuals nesting at the same
beach belong to the same genetic population. Sizerelated differences in feeding-habitat use would not bea limiting factor for gene flow between foraginghabitat groups. This genetic homogeneity is probably
attributed to gene flow at mating habitats. Male loggerheads are also thought to show size-related differences in their use of feeding habitats (Hatase et al.
2002c). Individuals of the 2 groups would share a common mating habitat spatio-temporally. Mating generally occurs near nesting beaches prior to the nestingseason (Limpus 1993), and mate choice based on foraging habitats would not occur there. Eastern Australiangreen turtles did not select mates by body size (Limpus
1993), and the same may be true for Japanese loggerheads. The absence of differentiation in mtDNA haplotype frequencies between foraging-habitat groups was
also reported in female loggerheads nesting in Florida,
USA (Reich et al. 2010).
Individual variation in growth rates at oceanic developmental stages was hypothesized as a key factor for
determining whether Japanese loggerhead turtles stayin oceanic waters or recruit to neritic waters (Hatase etal. 2004, 2006, 2010). This model was analogous to themodel proposed for salmonids, in which growth rate
determines whether they stay in a river or go down tothe ocean/lake during immature periods (Thorpe1986). Life-history polymorphisms within some salmonid populations appeared to result from phenotypic
plasticity expressed by individuals with the same
genotypes (Jones et al. 1997, Olsson et al. 2006). The
lack of genetic differences between feeding-habitatgroups revealed in our study similarly suggests thatvariation in feeding-habitat use by adult loggerheads
results from phenotypic plasticity. The fact that incubation environment greatly affects loggerhead hatchling

280

Mar Ecol Prog Ser 424: 273–283, 2011

phenotypes, such as body sizes and activity levels(Carthy et al. 2003), might support this conclusion.

Gene flow among nesting sites
Global FST and pairwise RST at microsatellite loci did
not show significant departure from 0, and genetic
differences were not detected in most pairwise FST comparisons. These results were in contrast to those of the
mtDNA analysis, which found significant global FST andsignificant pairwise FST in half of all 10 combinations
among 5 nesting sites. The level of gene flow at microsatellite loci may be greater than that for mtDNA
among Japanese populations. Because FST values depend on heterozygosities within populations, contrasting
FST values between DNA markers may also be attributed
to differences in the heterozygosities of the markers(Hellberg 2009). In fact, intra-population HE values at
5 microsatellite loci (0.511 to 0.908) were higher thanintra-population h values at the mtDNA level (0.058 to
0.415). Furthermore, differences in effective population
size between nuclear DNA and mtDNA (i.e. 4-foldsmaller at the mtDNA level) may be responsible for thecontrasting FST values (Birky et al. 1983). Similar results
were reported in loggerhead turtles inhabiting the western North Atlantic (Bowen et al. 2005) and the Mediterranean (Carreras et al. 2007), and male-mediatedgene flow was considered as a reasonable explanation.
FitzSimmons et al. (1997b) reported similar results forAustralian green turtles and discussed the mechanismsof male-mediated gene flow. Male-biased dispersal
was unlikely to be the cause, because male turtles had
the same level of fidelity to courtship areas near natalbeaches as did female turtles (Limpus 1993, FitzSimmons et al. 1997a). Mating outside the courtship areas
was thought to be the cause of gene flow between nesting sites. However, mating in feeding habitats, usuallyshared by several nesting populations, would not occur,
because the receptive period of breeding females
may be < 2 wk (Comuzzie & Owens 1990). Thus, only
migration corridors were hypothesized to be the places
where interbreeding between females and males fromdifferent nesting sites occurs.
Such a situation may also apply to loggerhead turtles
nesting in Japan. The East China Sea is a major feeding habitat for Japanese loggerheads (Kamezaki et al.
1997), and coastal areas around Yakushima Island may
be an important migration pathway for individuals thatnest on beaches facing the Pacific Ocean (Hatase et al.
2002d). Females from Minabe, Kamouda, and Miyazaki could mate with males from Yakushima, andmales from these 3 locations could mate with females
from Yakushima in the course of migration to courtshipareas near natal beaches.

Implications for conservation
The heavy decline in nesting female loggerheads at
Minabe in recent years was thought to result from adecline in oceanic small females possibly due topelagic fisheries (Hatase et al. 2002a). Our results suggest that the decline in oceanic females could also
affect neritic females, because they belong to the same
genetic population. Efforts to remove the threats tooceanic females are also needed to conserve neritic
females. Conversely, protecting neritic females frombycatch in coastal fisheries will also contribute to thesubsistence of oceanic females.
The present study suggests that male-mediated geneflow is a general feature in sea turtles rather than specific for regional populations (FitzSimmons et al.
1997b, Bowen et al. 2005, Carreras et al. 2007). Natalhoming of female turtles limits gene flow between
nesting sites (Allard et al. 1994). Such limitation shouldbe critical for Japanese loggerhead populations, because there were only 15 beaches where >10 clutches
were laid annually in the 1990s (Kamezaki et al. 2003).
In small populations of threatened species, inbreeding
depression is considered to be the most immediate
genetic threat (Frankham et al. 2002). Franklin (1980)
suggested that effective population size should not be< 50 in the short term. Loss of genetic diversity caused
by genetic isolation of small populations was suspected
for Japanese populations. In fact, only 3 mtDNA haplotypes were detected among 279 individuals, and overall h and π values for Japanese loggerheads (0.193 and0.00277, respectively) were lower than those for theirsoutheastern USA counterpart (0.320 and 0.01437),
which had 5 haplotypes among 197 individuals (Encalada et al. 1998). While genetic diversities at the
mtDNA level were low, genetic diversities in microsatellite loci for nesting females at Kamouda (a small
nesting beach, where only ca. 20 clutches were laidannually) were similar to those at Yakushima, thelargest nesting population in Japan (Table 3). This
indicates that male-mediated gene flow plays animportant role in preventing genetic fragmentation
between nesting sites. Male-mediated gene flow might
have evolved as a general feature in sea turtles to compensate for the risk of genetic fragmentation caused bynatal homing. Varied mtDNA diversities among nesting sites with significant global FST, in contrast to similar microsatellite diversities among nesting sites with
non-significant global FST, imply that the collapse ofeven a small nesting beach like Kamouda would affectoverall genetic diversity of Japanese loggerhead turtles, and that extirpated beaches would not be easily
recolonized from other nesting populations due tofemale philopatry. The decline of the Miyazaki population, the second largest loggerhead rookery in Japan

Watanabe et al.: Population structure of Japanese loggerheads(Table 3), would obviously cause a dramatic decreasein the overall genetic diversity of Japanese loggerheads, because it had the highest h and π values
among the 5 nesting sites and significant pairwise FSTat the mtDNA level was found between it and the4 other nesting sites. The uniqueness of Miyazaki implies that this population might have been a source
during the postglacial range expansion of this species
near the Japanese Archipelago. Conservation efforts
are still needed for individual nesting populations inorder to maintain the overall genetic diversity ofJapanese loggerheads.

Acknowledgements. We thank T. Kamata, M. Okamoto,R. Iguchi, J. Nii, and F. Sakai, for field assistance and logistical support at Kamouda beach. We also thank H. Tanaka, S.
Minamikawa, F. Fukuhara, the volunteers of the Kyoto University Sea Turtle Research Team, members of Yakushima
Sea Turtle Research Group, members of Miyazaki Wild Animal Research Group, K. Nozaki, K. Tanaka, and members of
Kagoshima University Sea Turtle Research Group for help
with the collection and supply of samples. The Sea Turtle
Association of Japan supplied tags, tagging pliers, andcalipers. M. J. Miller helped to improve the manuscript.
Y. Minegishi provided technical advice on our DNA analyses.
Three anonymous referees provided constructive comments.
This work was partly supported by grants from the Research
Fellowships of the Japan Society for the Promotion of Science(JSPS) for Young Scientists (K.K.W., H.H., K.S., and Y.M.),
from the JSPS 21st Century COE Program (Biodiversity andEcosystem Restoration) at The University of Tokyo to K.K.W.
and H.H., and from the Nippon Foundation to H.H. All experiments described here complied with current Japanese laws.

LITERATURE CITED➤ Allard MW, Miyamoto MM, Bjorndal KA, Bolten AB, Bowen

BW (1994) Support for natal homing in green turtles frommitochondrial DNA sequences. Copeia 1994:34–41
Asahida T, Kobayashi T, Saitoh K, Nakayama I (1996) Tissuepreservation and total DNA extraction from fish stored atambient temperature using buffers containing high concentration of urea. Fish Sci 62:727–730➤ Bass AL, Good DA, Bjorndal KA, Richardson JI, Hillis ZM,
Horrocks JA, Bowen BW (1996) Testing models of female
reproductive migratory behavior and population structurein the Caribbean hawksbill turtle, Eretmochelys imbricata, with mtDNA sequences. Mol Ecol 5:321–328➤ Bearhop S, Fiedler W, Furness RW, Votier SC and others(2005) Assortative mating as a mechanism for rapid evolution of a migratory divide. Science 310:502–504➤ Berthold P (1991) Genetic control of migratory behaviour inbirds. Trends Ecol Evol 6:254–257
Birky
CW Jr, Maruyama T, Fuerst P (1983) An approach to➤population and evolutionary genetic theory for genes inmitochondria and chloroplasts, and some results. Genetics
103:513–527➤ Bowen BW, Abreu-Grobois FA, Balazs GH, Kamezaki N,
Limpus CJ, Ferl RJ (1995) Trans-Pacific migrations of theloggerhead turtle (Caretta caretta) demonstrated withmitochondrial DNA markers. Proc Natl Acad Sci USA 92:
3731–3734

281➤ Bowen BW, Bass AL, Soares L, Toonen RJ (2005) Conserva-

tion implications of complex population structure: lessons
from the loggerhead turtle (Caretta caretta). Mol Ecol 14:
2389–2402➤ Boyle MC, FitzSimmons NN, Limpus CJ, Kelez S, VelezZuazo X, Waycott M (2009) Evidence for transoceanic
migrations by loggerhead sea turtles in the southern
Pacific Ocean. Proc Biol Sci 276:1993–1999➤ Braendle C, Friebe I, Caillaud MC, Stern DL (2005) Genetic
variation for an aphid wing polyphenism is geneticallylinked to a naturally occurring wing polymorphism. Proc
Biol Sci 272:657–664➤ Caparroz R, Miyaki CY, Baker AJ (2009) Contrasting phylogeographic patterns in mitochondrial DNA and microsatellites: evidence of female philopatry and male-biased
gene flow among regional populations of the blue-andyellow macaw (Psittaciformes: Ara ararauna) in Brazil.
Auk 126:359–370
Carreras C, Pascual M, Cardona L, Aguilar A and others(2007) The genetic structure of the loggerhead sea turtle(Caretta caretta) in the Mediterranean as revealed by
nuclear and mitochondrial DNA and its conservation
implications. Conserv Genet 8:761–775
Carthy RR, Foley AM, Matsuzawa Y (2003) Incubation environment of loggerhead turtle nests: effects on hatching
success and hatchling characteristics. In: Bolten AB, Witherington BE (eds) Loggerhead sea turtles. Smithsonian
Books, Washington, DC, p 144–153
Caut S, Guirlet E, Angulo E, Das K, Girondot M (2008) Isotope
analysis reveals foraging area dichotomy for Atlantic
leatherback turtles PLoS ONE 3:e1845➤ Chen SF, Jones G, Rossiter SJ (2008) Sex-biased gene flow
and colonization in the Formosan lesser horseshoe bat:inference from nuclear and mitochondrial markers. J Zool
(Lond) 274:207–215
Comuzzie DKC, Owens DW (1990) A quantitative analysis ofcourtship behavior in captive green sea turtles (Chelonia
mydas). Herpetologica 46:195–202
Dodd CK Jr (1988) Synopsis of the biological data on the loggerhead sea turtle Caretta caretta (Linnaeus, 1758). Biol
Rep US Fish Wildl Serv 88:1–110➤ Encalada SE, Bjorndal KA, Bolten AB, Zurita JC and others(1998) Population structure of loggerhead turtle (Caretta
caretta) nesting colonies in the Atlantic and Mediterranean as inferred from mitochondrial DNA control region
sequences. Mar Biol 130:567–575➤ FitzSimmons NN (1998) Single paternity of clutches andsperm storage in the promiscuous green turtle (Chelonia
mydas). Mol Ecol 7:575–584➤ FitzSimmons NN, Moritz C, Moore SS (1995) Conservationand dynamics of microsatellite loci over 300 million yearsof marine turtle evolution. Mol Biol Evol 12:432–440➤ FitzSimmons NN, Limpus CJ, Norman JA, Goldizen AR,
Miller JD, Mortitz C (1997a) Philopatry of male marine turtles inferred from mitochondrial DNA markers. Proc Natl
Acad Sci USA 94:8912–8917
FitzSimmons NN, Moritz C, Limpus CJ, Pope L, Prince R(1997b) Geographical structure of mitochondrial andnuclear gene polymorphisms in Australian green turtle
populations and male-biased gene flow. Genetics 14:
1843–1854
FitzSimmons NN, Moritz C, Bowen BW (1999) Population
identification. In: Eckert KL, Bjorndal KA, Abreu-Grobois
FA, Donnelly M (eds) Research and management techniques for the conservation of sea turtles. Marine Turtle
Specialist Group Publication No. 4, IUCN/SSC, Gland,p 72–79

282

Mar Ecol Prog Ser 424: 273–283, 2011

Frankham R, Ballou JD, Briscoe DA (2002) Introduction toconservation genetics. Cambridge University Press,
Cambridge
Franklin IR (1980) Evolutionary change in small populations.
In: Soulé ME, Wilcox BA (eds) Conservation biology: anevolutionary–ecological perspective. Sinauer Associates,
Sunderland, MA, p 135–149➤ Goodman SJ (1997) RST Calc: a collection of computer programs for calculating estimates of genetic differentiationfrom microsatellite data and determining their significance. Mol Ecol 6:881–885➤ Goudet J, Raymond M, Meeus T, Rousset F (1996) Testing
differentiation in diploid populations. Genetics 144:
1933–1940➤ Goudet J, Perrin N, Waser P (2002) Tests for sex-biased dispersal using bi-parentally inherited genetic markers. Mol
Ecol 11:1103–1114➤ Gross MR (1996) Alternative reproductive strategies and tactics: diversity within sexes. Trends Ecol Evol 11:92–98➤ Harrison RG (1980) Dispersal polymorphisms in insects. Annu
Rev Ecol Syst 11:95–118➤ Hatase H, Goto K, Sato K, Bando T, Matsuzawa Y, Sakamoto
W (2002a) Using annual body size fluctuations to explore
potential causes for the decline in a nesting population of
the loggerhead turtle Caretta caretta at Senri Beach,
Japan. Mar Ecol Prog Ser 245:299–304➤ Hatase H, Kinoshita M, Bando T, Kamezaki N and others(2002b) Population structure of loggerhead turtles,
Caretta caretta, nesting in Japan: bottlenecks on the
Pacific population. Mar Biol 141:299–305➤ Hatase H, Matsuzawa Y, Sakamoto W, Baba N, Miyawaki I(2002c) Pelagic habitat use of an adult Japanese maleloggerhead turtle Caretta caretta examined by the Argos
satellite system. Fish Sci 68:945–947➤ Hatase H, Takai N, Matsuzawa Y, Sakamoto W and others(2002d) Size-related differences in feeding habitat use of
adult female loggerhead turtles Caretta caretta around
Japan determined by stable isotope analyses and satellite
telemetry. Mar Ecol Prog Ser 233:273–281➤ Hatase H, Matsuzawa Y, Sato K, Bando T, Goto K (2004) Remigration and growth of loggerhead turtles (Caretta caretta)
nesting on Senri Beach in Minabe, Japan: life-history
polymorphism in a sea turtle population. Mar Biol 144:
807–811➤ Hatase H, Sato K, Yamaguchi M, Takahashi K, Tsukamoto K(2006) Individual variation in feeding habitat use by adultfemale green sea turtles (Chelonia mydas): Are they
obligately neritic herbivores? Oecologia 149:52–64➤ Hatase H, Omuta K, Tsukamoto K (2007) Bottom or midwater:alternative foraging behaviours in adult female loggerhead sea turtles. J Zool (Lond) 273:46–55➤ Hatase H, Omuta K, Tsukamoto K (2010) Oceanic residents,
neritic migrants: a possible mechanism underlying foraging dichotomy in adult female loggerhead turtles (Caretta
caretta). Mar Biol 157:1337–1342➤ Hawkes LA, Broderick AC, Coyne MS, Godfrey MH and others (2006) Phenotypically linked dichotomy in sea turtle
foraging requires multiple conservation approaches. Curr
Biol 16:990–995
Hefti-Gautschi B, Pfunder M, Jenni L, Keller V, Ellegren H(2009) Identification of conservation units in the European
Mergus merganser based on nuclear and mitochondrial
DNA markers. Conserv Genet 10:87–99
Hellberg ME (2009) Gene flow and isolation among populationsof marine animals. Annu Rev Ecol Evol Syst 40: 291–310➤ Hoelzel AR, Hey J, Dahlheim ME, Nicholson C, Burkanov V,
Black N (2007) Evolution of population structure in ahighly social top predator, the killer whale. Mol Biol Evol
24:1407–1415➤ Jones MW, Danzmann RG, Clay D (1997) Genetic relationships among populations of wild resident, and wild andhatchery anadromous brook charr. J Fish Biol 51:29–40
Jukes TH, Cantor CR (1969) Evolution of protein molecules.
In: Munro HN (ed) Mammalian protein metabolism. Academic Press, New York, NY, p 21–132
Kagoshima University Sea Turtle Research Group (2002)
Fukiagehama. In: Kamezaki N, Toji Y, Matsuzawa Y (eds)
Current status of Japanese loggerhead turtle nesting andbeach environment. Sea Turtle Association of Japan,
Osaka, p 54–55 (in Japanese)
Kamezaki N, Miyawaki I, Suganuma H, Omuta K and others(1997) Post-nesting migration of Japanese loggerhead turtle, Caretta caretta. Wildl Conserv Jpn 3:29–39 (in Japanese with English abstract)
Kamezaki N, Matsuzawa Y, Abe O, Asakawa H and others(2003) Loggerhead turtles nesting in Japan. In: Bolten AB,
Witherington BE (eds) Loggerhead sea turtles. Smithsonian Books, Washington, DC, p 210–217➤ Limpus CJ (1993) The green turtle, Chelonia mydas, inQueensland: breeding males in the southern Great Barrier
Reef. Wildl Res 20:513–523➤ Lukoschek V, Waycott M, Keogh JS (2008) Relative information content of polymorphic microsatellites and mitochondrial DNA for inferring dispersal and population genetic
structure in the olive sea snake, Aipysurus laevis. Mol Ecol
17:3062–3077➤ Lundberg P (1988) The evolution of partial migration in birds.
Trends Ecol Evol 3:172–175➤ Maekawa K, Nakano S, Yamamoto S (1994) Spawning behaviour and size-assortative mating of Japanese charr in an
artificial lake–inlet stream system. Environ Biol Fishes
39:109–117➤ Mansfield KL, Saba VS, Keinath JA, Musick JA (2009) Satellite tracking reveals a dichotomy in migration strategies
among juvenile loggerhead turtles in the northwest
Atlantic. Mar Biol 156:2555–2570➤ McClellan CM, Read AJ (2007) Complexity and variation inloggerhead sea turtle life history. Biol Lett 3:592–594➤ McClellan CM, Braun-McNeill J, Avens L, Wallace BP, Read
AJ (2010) Stable isotopes confirm a foraging dichotomy injuvenile loggerhead sea turtles. J Exp Mar Biol Ecol 387:
44–51
➤ Monzón-Argüello C, Rico C, Naro-Maciel E, Varo-Cruz N,
López P, Marco A, López-Jurado LF (2010) Population
structure and conservation implications for the loggerhead
sea turtle of the Cape Verde Islands. Conserv Genet 11:
1871–1884➤ Moritz C (1994) Applications of mitochondrial DNA analysis
in conservation: a critical review. Mol Ecol 3:401–411
Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New York, NY➤ Norman JA, Moritz C, Limpus CJ (1994) Mitochondrial DNA
control region polymorphisms: genetic markers for ecological studies of marine turtles. Mol Ecol 3:363–373➤ Okello JBA, Masembe C, Rasmussen HB, Wittemyer G andothers (2008) Population genetic structure of savannahelephants in Kenya: conservation and management implications. J Hered 99:443–452➤ Ólafsdóttir GÁ, Ritchie MG, Snorrason SS (2006) Positive
assortative mating between recently described sympatricmorphs of Icelandic sticklebacks. Biol Lett 2:250–252➤ Olsson IC, Greenberg LA, Bergman E, Wysujack K (2006)
Environmentally induced migration: the importance offood. Ecol Lett 9:645–651

Watanabe et al.: Population structure of Japanese loggerheads➤ Portnoy DS, McDowell JR, Heist EJ, Musick JA, Graves JE(2010) World phylogeography and male-mediated geneflow in the sandbar shark, Carcharhinus plumbeus. Mol
Ecol 19:1994–2010
Raymond M, Rousset F (1995) GENEPOP (Version 1.2): population genetics software for exact tests and ecumenicism.
J Hered 86:248–249
Reich KJ, Bjorndal KA, Frick MG, Witherington BE, Johnson
C, Bolten AB (2010) Polymodal foraging in adult female
loggerheads (Caretta caretta). Mar Biol 157:113–121➤ Rice WR (1989) Analyzing tables of statistical tests. Evolution
43:223–225
Sato K, Bando T, Matsuzawa Y, Tanaka H, Sakamoto W,
Minamikawa S, Goto K (1997) Decline of the loggerhead
turtle, Caretta caretta, nesting on Senri Beach in Minabe,
Wakayama, Japan. Chelonian Conserv Biol 2:600–603
Schneider S, Roessli D, Excoffier L (2000) ARLEQUIN
Ver.2.000: a software for population genetics data analysis.
Genetics and Biometry Laboratory, University of Geneva,
Geneva➤ Seminoff JA, Zárate P, Coyne M, Foley DG, Parker D, Lyon
BN, Dutton PH (2008) Post-nesting migrations of Galápagos green turtles Chelonia mydas in relation to oceanographic conditions: integrating satellite telemetry withremotely sensed ocean data. Endang Species Res 4:57–72
SlatkinM (1995) A measure of population subdivision based➤
Editorial responsibility: Inna Sokolova,
Charlotte, North Carolina, USA283

on microsatellite allele frequencies. Genetics 139:457–462
Thorpe JE (1986) Age at first maturity in Atlantic salmon,
Salmo salar : freshwater period influences and conflicts
with smolting. Publ Spec Can Sci Halieut Aquat 89:7–14➤ Tsukamoto K, Nakai I, Tesch WV (1998) Do all freshwater eels
migrate? Nature 396:635–636➤ Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P(2004) MICRO-CHECKER: software for identifying andcorrecting genotyping errors in microsatellite data. Mol
Ecol Notes 4:535–538
Voss SR (1995) Genetic control of paedomorphosis in theaxolotl, Ambystoma mexicanum: a test of the single-gene
hypothesis. J Hered 86:441–447
Watanabe KK (2006) Studies on gene flow and migratory
ecology in Japanese loggerhead turtles (Caretta caretta).
PhD dissertation, The University of Tokyo, Tokyo (inJapanese)➤ Weir BS, Cockerham CC (1984) Estimating F-statistics for the
analysis of population structure. Evolution 38:1358–1370➤ Wilbur HM, Collins JP (1973) Ecological aspects of amphibian
metamorphosis. Science 182:1305–1314➤ Zbinden JA, Bearhop S, Bradshaw P, Gill B, Margaritoulis D,
Newton J, Godley BJ (2011) Migratory dichotomy andassociated phenotypic variation in marine turtles revealedby satellite tracking and stable isotope analysis. Mar Ecol
Prog Ser 421:291–302
Submitted: August 10, 2010; Accepted: December 10, 2010
Proofs received from author(s): February 18, 2011