Vol. 30: 83–93, 2016
doi: 10.3354/esr00724

ENDANGERED SPECIES RESEARCH
Endang Species Res

Published April 14

OPEN
ACCESS

Fine-scale genetic population structure ofloggerhead turtles in the Northwest PacificY. Matsuzawa, N. Kamezaki, T. Ishihara, K. Omuta, H. Takeshita, K. Goto, T. Arata,
H. Honda, K. Kameda, Y. Kashima, M. Kayo, I. Kawazu, J. Kodama, Y. Kumazawa,
K. Kuroyanagi, K. Mizobuchi, K. Mizuno, K. Oki, K. K. Watanabe, A. Yamamoto,Y. Yamashita, T. Yamato, T. Hamabata, A. Ishizaki, P. H. Dutton*
For list of author affiliations, see Appendix

ABSTRACT: Effective conservation of globally distributed marine species relies on identification ofdemographically independent populations to ensure that management actions are directed at theappropriate scale. This identification is particularly challenging for species with complex life histories when local breeding populations have not been adequately sampled. We used mtDNA to analyze the population structure of loggerhead turtles from a total of 555 samples collected from 12
nesting sites in Japan in the Northwest Pacific, including previously unsampled rookeries in the
Ryukyu Archipelago, for a comprehensive coverage of the nesting distribution. We identified a totalof 9 haplotypes based on 820 bp of the mtDNA control region, including 5 variants of a single previously described 380 bp haplotype. We discovered that 1 haplotype (CcP1.1) previously rare in the
North Pacific is common in the Ryukyu Archipelago. Based on analysis of haplotype frequencies,
we found significant differentiation among regionally grouped nesting populations (analysis of molecular variance p < 0.0001, df = 8; pairwise FST ranging from 0.033 to 0.145). Our results provide evidence to support the recognition of 3 management units (MU) within the NW Pacific Regional
Management Unit (RMU). These include (1) the Ryukyu MU that includes Okinawa, Okinoerabu
and Amami, (2) Yakushima Island MU and (3) a Mainland MU that includes Bousou, Enshu-nada,
Shikoku, Kii and Eastern Kyushu. These new data from Japan will provide important baseline datafor global genetic stock assessments and contribute to our understanding of the population
structure, ecology and life history of this migratory marine species in the northern Pacific.
KEY WORDS: Mitochondrial DNA · Management units · Population differentiation · Caretta caretta

INTRODUCTION
Identifying the geographic range of demographically independent populations is important for conservation and management of endangered species,
and yet this task can often be challenging given thelack of any unifying set of criteria to define population boundaries, particularly for globally distributed
marine species with complex life histories (Waples &Gaggiotti 2006, Taylor et al. 2010). Sea turtles, which
exhibit natal philopatry, tend to have reproductively*Corresponding author: peter.dutton@noaa.gov

isolated nesting populations (or rookeries) characterized by differences in composition of matrilineal
genetic lineages that are maintained by recruitmentof nesting females to their natal beaches (Bowen &Karl 2007, Jensen et al. 2013). Maternally inherited
mitochondrial (mt) DNA markers are useful forstudying the stock structure of sea turtles. Management units (MUs) are defined as rookeries (or groupsof rookeries) with significant differences in mtDNA
haplotype frequencies (Moritz 1994), and these populations are considered to be demographically iso© The authors 2016. Open Access under Creative Commons by
Attribution Licence. Use, distribution and reproduction are unrestricted. Authors and original publication must be credited.
Publisher: Inter-Research · www.int-res.com

84

Endang Species Res 30: 83–93, 2016

lated with respect to female natal recruitment. Sincemale-mediated nuclear gene flow can occur between
MUs defined by mtDNA, it is preferable to use multiple genetic markers to obtain a holistic characterization of population structure (Bowen et al. 2005, Palsbøll et al. 2007). However, in the absence of nuclear
data, mtDNA analysis is adequate for defining rookeries as independent nesting populations because
female dispersal and recruitment is what shapes
rookeries demographically, regardless of the level ofmale-mediated gene flow. Rookery mtDNA haplotype frequencies are also important for providing
baseline data to inform mixed stock analyses, which
are used to estimate nesting population origins of foraging turtles (Bolker et al. 2007, Jensen et al. 2013).
For loggerhead turtles, at the global level, 9 regionally significant nesting population aggregations
have been recognized as Regional Management
Units (RMUs) based on genetic, demographic, geographic and oceanographic considerations: (1) Northwest
Atlantic Ocean, (2) Southwest Atlantic Ocean, (3)Northeast Atlantic Ocean, (4) Mediterranean Sea, (5)Southwest Indian Ocean, (6) Northwest Indian Ocean,(7) Southeast Indian Ocean, (8) North Pacific Oceanand (9) South Pacific Ocean, with a tenth putative
RMU proposed for the Northeast Indian Ocean forwhich genetic and biological data are lacking (Wallace et al. 2010). A recent study based on a 380 and~800 base pair (bp) fragment of the mitochondrial
control region has demonstrated genetic partitioning
within the Atlantic, Mediterranean and Southwest Indian Ocean RMUs (Shamblin et al. 2014). While the
mtDNA control region has been a useful marker for
detecting stock structure, previous studies haveshown that common and widespread haplotypes
make it harder to detect fine-scale structure for many
marine turtle species (Dutton et al. 1999, Dethmers etal. 2006, Jensen et al. 2013). Longer sequences might
have additional variation to help resolve those common haplotypes and provide increased resolution to
stock structure (Shamblin et al. 2012a, 2014, LeRouxet al. 2012, Dutton et al. 2013, Jensen et al. 2013).
Genetic stock assessments for loggerheads in the
North Pacific have been based on published data forJapan from Hatase at al. (2002a). Recently, the development of new primers has allowed production of
additional mtDNA sequence data that have uncovered additional genetic variation among Pacific loggerheads (LeRoux et al. 2008). However, the Japanese nesting populations have not been analyzed withthese new tools, so current stock assignments of loggerheads encountered as bycatch in fisheries andestimates of stock composition of foraging aggrega-tions of loggerheads are based on the limited data ofBowen et al. (1995), Hatase et al. (2002a) and
Nobetsu et al. (2004). Furthermore, Hatase et al.
(2002a) suggested that there was sub-structuring
among nesting populations in Japan, which implies
that risk factors could impact rookeries in Japan differently, depending on the status of each stock. The
evidence presented by Hatase et al. (2002a) for substructuring was weak, due to small sample sizes andlow genetic variation in the 384 bp sequence datathat were used (Dutton 2007). More recently Watanabe et al. (2011) reanalyzed the data from Hatase etal. (2002a) incorporating 384 bp sequence data from
2 additional nesting sites and found additional evidence of sub-structuring among 5 nesting sites. However, there is extensive loggerhead nesting throughout the Ryukyu Archipelago (Nansei-Shoto) in thesouthern region of Japan (Kamezaki et al. 2003,
Kamezaki 2012) that has not been included in previous genetic surveys. This region represents thesouthernmost portion of the nesting distribution ofloggerheads in the Northwest Pacific.
In this study, we use mtDNA markers to identify
the stock structure of loggerheads nesting in the
Northwest Pacific. We build on previous work by (1)reanalyzing available samples using ~800 bp sequences, (2) substantially increasing the sample sizefor previously analyzed rookeries and (3) including
samples from previously unsampled rookeries in the
Ryukyu Archipelago for a comprehensive coverageof the nesting distribution. Finally, we consider implications of our findings for conservation of this threatened species.

MATERIALS AND METHODS
Sample collection
A total of 555 samples were collected from 12 locations in Japan representing the main loggerhead turtle nesting sites (Fig. 1). Samples consisted of skinplugs from female loggerheads collected from tag
punches obtained while applying flipper tags to
identify nesting turtles, or dead hatchlings or embryos salvaged from nests that had emerged. Carewas taken to avoid duplicate sampling of nests laidby the same female by only sampling unknown nests
that were laid within a 10 d window. Tissue samples
were preserved in a concentrated urea buffer (TNESUrea: 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.

Matsuzawa et al.: Population structure of North Pacific loggerheads

85

Fig. 1. Nesting and sampling sites of loggerhead turtles Caretta caretta in the Northwest Pacific. Insets show details for (A)Eastern Kyushu and Yakushima and (B) the Ryukyu Archipelago. Black dots show nesting sites (>10 nests) and the relative
rookery size (number nests per year) based on 2010 data compiled by the Sea Turtle Association of Japan. Roman numerals
and dashed circles indicate the areas in this study, I: Bousou (Chiba, n = 10), II: Enshu-nada (Shizuoka, n = 38), III: Kii (Ise Bay,n = 11), IV: Kii (Minabe, n = 101), V: Shikoku (Shikoku, n = 48), VI: Eastern Kyushu (Miyazaki, n = 61), VII: Eastern Kyushu(Shibushi Bay, n = 25), VIII: Yakushima (Yakushima, n = 108), IX: Ryukyu Archipelago (Amami, n = 53), X: Ryukyu Archipelago (Okinoerabu, n = 24), XI: Ryukyu Archipelago (Okinawa Islands, n = 70), XII: Ryukyu Archipelago (Yaeyama, n = 6)

Laboratory analysis
Samples were processed and sequenced by a genetic service laboratory, Leave a Nest Co., in Tokyo,
Japan, and Akita Prefectural University Biotechnology Center, in Akita, Japan, and Laboratory of Systematic Zoology, Kyoto University, Kyoto, Japan.
Primers LCM-15382 and H950g (Abreu-Grobois etal. 2006) were used to generate approximately
820 bp of mtDNA sequence.

Statistical analysis
We assigned haplotypes by comparing aligned
sequences against a local reference library of~800 bp haplotype sequences using Geneious Pro
6.0.2 (Drummond et al. 2011) as well as searching thedatabase on GenBank (www.ncbi.nlm.nih.gov). We
standardized nomenclature of haplotypes based onthese ~800 bp alignments, assigning the CcP prefix
to numerically sequential names based on the original 384 bp alignments (Bowen et al. 1995, LeRoux etal. 2008) with a sequential numeric suffix to indicate
a variant resulting from polymorphism in the additional 386 bp region flanking the old shorter sequence (e.g. CcP2.1, CcP2.2 etc.). Unique sequenceswere then aligned with the ClustalW algorithm implemented in Geneious Pro 6.0.2 (Drummond et al.
2011). The alignment of each mtDNA segment waschecked and edited by eye separately. Haplotype (h)and nucleotide (π) diversity were calculated for eachrookery using Arlequin v3.5.1.2 (Excoffier & Lischer
2010). Haplotype diversity was estimated based on
Nei (1987), and nucleotide diversity was calculated
assuming the model of Tamura & Nei (1993). We con-Endang Species Res 30: 83–93, 201686

structed a minimum spanning network (Bandelt et al.
1999) using PopArt v1.7 (http://popart.otago.ac.nz) toillustrate the relationships between haplotypes.
Haplotype diversity (h) analysis, pairwise FST comparisons, pairwise exact tests of population differentiation and genetic distance-based analysis of molecular variance (AMOVA) were conducted using thesoftware Arlequin v3.5.1.2 (Excoffier & Lischer 2010).
Significance values for AMOVA were obtained from
10 000 permutations. Structure was examined using
frequency-based AMOVA, frequency-based pairwise FST comparisons and exact tests of populationdifferentiation with p-values < 0.05 considered significant. Exact tests of population differentiation
were conducted with 100 000 permutations and
10 000 dememorization steps (Raymond & Rousset
1995). We performed a Mantel test (Mantel 1967) toevaluate the relationship between geographic andgenetic (FST) distances in order to test for isolation by
distance. Geographic distances (km) were transformed using natural logs, while frequency-based
FST values were standardized using FST /(1 − FST) andrun in Arlequin using 10 000 permutations.

RESULTS
We identified a total of 9 haplotypes based on the820 bp sequences, including 5 variants of the previously described 380 bp haplotype ‘B’ (CcP2) and 2

variants of Haplotype ‘C’ (CcP3) (Bowen et al. 1995)(Table 1; GenBank ID AB830477−482, AB831106−
107 and AB842487−88, respectively). Both CcP1 and
CcP3 were relatively distinct from each other and the
CcP2 haplogroup (Fig. 2). In the absence of significant differentiation based on initial pairwise tests(not shown), data from adjacent nesting sites of Ise
Bay and Minabe were combined to represent a single
population for Kii, and data from Miyazaki and Shibushi were combined to represent Eastern Kyushu.
Subsequent stock structure analysis was performed
using these combined data for turtles nesting in a
total of 9 different areas (Table 1). Data from
Yaeyama were not included in further analysis due tothe small sample size and the uncertainty overwhether they are representative of the relative nesting abundance for that distant island.
There was a latitudinal shift in haplotype frequencies, with CcP1.1 relatively common at the southern
nesting sites and rare or absent at the northern nesting sites (Table 1). Haplotype diversity ranged from
0.44 to 0.78, and nucleotide diversity averaged 0.003(Table 2). Results of the Mantel tests revealed a significant positive relationship between geographicaland genetic distances (r = 0.261; p < 0.05). The
AMOVA indicated significant structuring within ourstudy area (p < 0.0001, df = 8). The pairwise tests
indicated the following groupings based on consistent lack of differentiation: (1) Bousou + Enshu-nada+ Shikoku + Eastern Kyushu; (2) Kii; (3) Yakushima

Table 1. Frequencies of mtDNA haplotypes for loggerhead turtles Caretta caretta sampled at 12 Northwest Pacific nestingsites based on 820 bp. Equivalent haplotypes (A, B and C) published for shorter 384 bp sequences (Bowen et al. 1995, Hatase
et al. 2002a) are shown. Totals for regions are shown in bold. Roman numerals correspond to nesting sites indicated in Fig. 1.
Ryukyu total does not include numbers from Yaeyama
Location

BousouChiba (I)
Enshu-nadaShizuoka (II)Kii
Ise Bay (III)Minabe (IV)Shikoku (V)Eastern Kyushu
Miyazaki (VI)
Shibushi Bay (VII)Yakushima (VIII)
Ryukyu Archipelago
Amami (IX)
Okinoerabu (X)Okinawa Island & Zamami (XI)Yaeyama (XII)(A)
CcP1.1
–
–
–
–
–
–
–
–
–
–
–
4
34
6
6
22
2

CcP2 (B)
CcP2.1 CcP2.2 CcP2.3 CcP2.4 CcP2.5
4
4
20
20
78
4
74
28
55
37
18
78
62
23
11
28
2

2
2
5
5
10
1
9
7
5
5
–
1
–
–
–
–
–

1
1
3
3
16
3
13
13
11
9
2
3
30
17
4
9
–

–
–
–
–
1
–
1
–
–
–
–
–
–
–
–
–
–

–
–
–
–
–
–
–
–
–
–
–
–
1
1
–
–
–

CcP3 (C)CcP3.1 CcP3.2
3
3
9
9
5
3
2
7
15
10
5
22
20
6
3
11
2

–
–
1
1
2
–
2
–
–
–
–
–
–
–
–
–
–

Total

10
10
38
38
112
11
101
55
86
61
25
10
147
53
24
70
6

Matsuzawa et al.: Population structure of North Pacific loggerheads

87

and (4) Okinawa + Okinoerabu +Amami (Table 3). Although Okinawa
and Amami were significantly differentiated (p < 0.05), neither Amami nor
Okinawa were distinct from Okinoerabu, a geographic intermediary between the other 2 rookeries.

DISCUSSION

Fig. 2. Most parsimonious minimum spanning network of the 820 bp Japaneseloggerhead haplotypes. Number of mutations between haplotypes is illustrated by dashes in connecting lines. The size of the circles represents haplotype frequency in the overall sample set, and proportion of each haplotype isindicated for each of the 3 Japanese loggerhead management units (MUs)identified in our study
Table 2. Genetic diversity parameters for the 11 loggerhead rookeries in
the Northwest Pacific based on 820 bp mtDNA control region. The tableshows sample size (n), number of haplotypes (H), nucleotide (π) and haplotype(h) diversity and their standard deviation (SD). Results for the 7 regions(see Fig. 1) are shown in bold. Yaeyama is not included due to the small
sample size
Location

n

H

π

SD

h

SD

BousouEnshu-nadaKii
Ise Bay
MinabeShikoku
Eastern Kyushu
Miyazaki
Shibushi Bay
Yakushima
Ryukyu Archipelago
Amami
Okinoerabu
Okinawa Island &Zamami

10
38
112
11
101
55
86
61
25
108
147
53
24
70

4
5
6
4
6
4
4
4
3
5
5
5
4
4

0.00486
0.00404
0.00166
0.004450.00130
0.00271
0.00292
0.002930.00295
0.003150.001480.003280.004100.004390.00299
0.002350.001140.002740.00095
0.001680.001770.001780.001840.001880.001070.001960.002420.002490.77780.66000.4886
0.78180.4422
0.66460.5472
0.58630.45330.43870.71330.69590.71380.71010.09070.04990.05210.07490.05650.04680.05240.05910.10220.04950.01950.0376
0.06130.0266The southern Japanese rookeries in
the Ryukyu Archipelago make up a
separate MU that is demographicallydistinct from the Yakushima Islandand mainland nesting populations.
The finding that CcP1.1 is common in
the Ryukyu Archipelago rookeries is asignificant discovery because this
haplotype was previously assumed tocharacterize the South Pacific loggerhead populations nesting in Australia
and New Caledonia (Bowen et al.
1995, P. H. Dutton et al. unpubl. data).
In Japan, CcP1 was previously reported only at Yakushima, at relatively
low frequency (Hatase et al. 2002a,
Watanabe et al. 2011). The presenceof CcP1.1 at Yakushima is one reason
this site is also clearly differentiatedfrom the northern rookeries; however,the boundaries between the southern
Archipelago, Yakushima and themainland MUs are slightly tenuous.
Based on FST results, we identified
3 MUs within the NW Pacific RMU,
representing a mainland Japan regional MU, the Yakushima Island
population and the Ryukyu Archipelago in the southern part of the nesting
range. While results indicated that Kii
was differentiated from the otherrookeries to the south and from Bousou and Enshu-nada in the north, this
distinction was tenuous and should beinterpreted with caution. The similarity in haplotype frequencies and discrepancy between the FST and exact
test results of pairwise comparisons
between Kii and the other northernand central rookeries lend uncertainty
to designation of Kii as a separate MU.
Tagging studies show movement ofEndang Species Res 30: 83–93, 2016

88

Table 3. Pairwise comparisons based on 820 base pair (bp) control region haplotype frequencies for 9 loggerhead turtle nesting populations in Japan. Nesting population locations are shown in Fig. 1. FST values are above the diagonal with significant
values (without correction for multiple tests): *p < 0.05; **p < 0.01. Pairwise exact test p values are below the diagonal.
BOU: Bousou; ENS: Enshu-nada; KII: Kii; SHI: Shikoku; KYU: Eastern Kyushu; YAK: Yakushima; AMA: Amami; OBU:
Okinoerabu; OWA: Okinawa Islands
BOU
BOU
ENS
KII
SHI
KYU
YAK
AMA
OBU
OWA

0.85430
0.04676
0.40258
0.164140.01300
0.02435
0.076110.00504

ENS

KII

SHI

KYU

YAK

AMA

OBU

OWA

0.00000.0989*0.0476*0.01100.00620.0334*0.02610.00270.01070.01500.10170.0402*0.0403**0.0853**0.01150.03670.0572*
0.0912**0.01250.0631**0.1389**0.02490.0416
0.0928**0.03240.0579*0.1061**0.00080.05150.0757**0.1450**0.0781**0.1039**0.1419**0.0392*0.00000.011150.153340.22023
0.002190.000070.004030.00000

0.07197
0.034240.00000
0.00000
0.00000
0.00000

0.133150.00000
0.002540.00208
0.00000

some individual female loggerheads between nesting sites at Kyushu, Shikoku, Kii and Enshunada,
whereas nesting at other sites is extremely rare forfemales tagged at Yakushima (Watanabe 2006). Thispattern is consistent with our genetic findings. In
their study based on the shorter 384 bp sequences,
Watanabe et al. (2011) also found significant differentiation between Yakushima and Miyazaki (Eastern
Kyushu). We were able to split CcP2 and also CcP3
into several new haplotypes based on the longer
sequences, providing finer resolution and further
reinforcing the significant differentiation between
Yakushima and the other rookeries. Haplotype diversities were substantially lower based on shorter
sequences (average of 0.039) reported by Watanabe
et al. (2011).
Despite the increased resolution of longer sequences, there could be fine-scale demographic substructuring that our mtDNA markers were not able
to detect. Dutton et al. (2013) demonstrated that
mtDNA did not have sufficient power to detect the
weak population structure (FST < 0.005) that characterizes proximate nesting populations in the case ofleatherbacks, when compared with an array of informative nuclear (microsatellite) markers. A limited
study (that did not include the Ryukyu Islands orsome of the northern Mainland sites) using 5 microsatellite markers failed to detect significant substructuring within Japan (Watanabe et al. 2011).
However, a more comprehensive study with larger
numbers of independent loci would help determine
whether there is any weak demographic structuring.
Additionally, data from tagging studies may provide
information in the future on nesting dispersal patterns of females that could be used to better define
connectivity (over shorter time scales than genetics)
between different nesting sites within these MUs.

0.006500.000120.00032
0.00000

0.00000
0.00033
0.00000

0.41868
0.00867

0.88371

Although our study represents a notable expansionof the geographic scope compared to that of previous
studies (Hatase et al. 2002a, Watanabe et al. 2011),
such that all the major nesting sites were adequately
covered, there were still gaps in the nesting range,
resulting from our inability to comprehensively sample areas of low-level sporadic nesting. This limitation may have created artificial ‘breaks’ in the nesting distribution, which is actually almost continuousalong the central and northern coasts of Japan(Fig. 1). This may explain the lack of clear structureevident among the central and northern rookeries,
and further sampling of some of the under-sampledintermediate sites will be needed to accurately characterize the level of genetic connectivity amongrookeries in this region.
Our findings regarding the nesting distribution in
Japan are similar to the NW Atlantic, where loggerhead nesting is almost continuous along the US coast,and it is difficult to identify MUs with clear boundaries based on genetic data. Mitochondrial DNA
studies show a gradual shift, or ‘cline’, in haplotype
frequencies with extensive haplotype sharing, suchthat rookeries at the northern and southern portion of
the US nesting distribution are significantly differentiated, but intermediate nesting sites are not (Encalada et al. 1998, Shamblin et al. 2011, 2014). Recent
advances using expanded analysis of the mitogenome may provide tools for further resolving finescale structure among the Japanese rookeries(Shamblin et al. 2012b).
The general demographic structuring indicated inour study is consistent with geographic and physicalfeatures that likely shaped the patterns of reproductive isolation and natal homing. The Ryukyu nestingsites are geographically isolated from the other 2
MUs. There are likely factors other than geographic

Matsuzawa et al.: Population structure of North Pacific loggerheads

isolation that contribute to the genetic isolation
between other MUs. Yakushima Island, while closer
to some of the Mainland MU nesting sites, is separated by a channel strongly influenced by the Kuroshio Current, which may act as an oceanographic
barrier (Fig. 3). The high mountain topography of theisland (~2000 m height) and distinct wide sandbeaches along with proximity to the Kuroshio Current may also facilitate natal homing by reproductive
females. The visual cues of the mountains andbeaches are thought to play a role in supplementing
the proven magnetic and olfactory mechanisms that
adult sea turtles use for long-range homing to natal
nesting beaches (Carr 1984, Lohmann et al. 1999).
It should also be noted that male-mediated geneflow will provide a higher level of genetic connectivity between rookeries than is evident from female
mtDNA lineage segregation (Watanabe et al. 2011)
and should be considered in the broader conservation genetics context and for a more holistic picture of
the genetic connectivity within the NW Pacific loggerhead RMU. However, for the purposes of defining
nesting population MUs, our mtDNA results are ofprimary interest.
The 3 haplogroups illustrated in our study (Fig. 2),
correspond to the 3 haplogroups described by Hatase
et al. (2002a) with shorter 384 bp sequences. Using a
Bayesian molecular clock framework, Shamblin et al.
(2014) estimated the deepest bifurcation among

Fig. 3. Kuroshio Current (large arrow) and its branch current(small arrow). These currents may influence nesting site
fidelity within management units (MUs)

89

Pacific loggerhead lineages (CcP1 vs. CcP2) to be
1.4 million years before present (YBP) (95% highest
posterior density interval: 0.3 to 2.12). Our haplotype
network illustrates that the additional diversity
detected using longer sequences in our study represents relatively recent accumulation of new mutations within the 3 main mtDNA lineages, primarily
CcP2. The sequence divergence between CcP2.1 and
CcP2.2 (0.002318) provides a reasonable basis to estimate coalescence for this haplogroup to 105 000 to
136 000 YBP using a molecular clock proposed for amarine turtle mtDNA control region of approximately 1.7 to 2.2% per million years (Encalada et al.
1996, Dutton et al. 1999). This suggests that the
recent evolutionary history of the NW Pacific loggerhead RMU has been shaped by processes occurringat minimum within the last 150 000 yr. A more comprehensive analysis with additional data for theSouth Pacific nesting populations is needed to provide more detailed insights into the evolution, phylogeography and demographic history of loggerheads.
Of note here is that the presence of older lineages
among all 3 MUs indicate the importance of these
Japanese nesting populations for maintaining genetic diversity among loggerheads both in the Pacificand globally.

Conservation implications
Our results have implications with regard to theappropriate scale of units needed for the effetive conservation of this population in that they provide evidence to support the recognition of at least 3 MUs
within the NW Pacific RMU. These are: (1) Ryukyu
MU that includes Okinawa, Okinoerabu and Amami,(2) Yakushima Island MU and (3) a Mainland MU
that includes Bousou, Enshu-nada, Shikoku andEastern Kyushu. Due to the uncertainty in the resultsfor Kii with respect to its distinction from the othercentral/northern rookeries, recognition of Kii as aseparate MU is not warranted. However, because it isdifferentiated from Yakushima and the southern MU,
Kii should be provisionally included in the Mainland
MU. These MU designations recognize that on a
local scale, these groups of rookeries are sufficiently
isolated demographically to be treated as independent populations for conservation. Conservation actions on nesting beaches designed to increase hatchling production are expected to result in increased
recruitment to the local nesting population as females mature and return to nest at their natal beaches (Dutton et al. 2005).

90

Endang Species Res 30: 83–93, 2016

The age to maturity of female loggerheads in the
North Pacific is ~40 yr on average (Ishihara 2011).
Until the 1970s, before a ban on harvesting, 80 to
90% of eggs were poached or legally collected inYakushima and Miyazaki (Kanno 1976, Uchida 1976,
Takeshita 2006). After > 4 decades of loggerheadconservation, there has been a dramatic increase innesting, particularly at Yakushima (Omuta 2011,
Kamezaki 2012) that is consistent with natal homing
expectations for this MU. This increase on Yakushima over the last decade contrasts with the nesting
trends observed at a majority of the rookeries within
the Mainland MU, which have remained depleted or,at best, slightly increased (Kamezaki 2012, BCJ 2014;see also Yakushima Umigame-kan 2011). However,within the Mainland MU, there is a general lack ofconsistency between trends at local rookeries. The
nesting trend at Miyazaki (Eastern Kyushu), whichshows a marked increase, differs from that in theother Mainland regions, including Shikoku, Kii and
Enshunada, suggesting that the demographic relationships between these local rookeries may be more
complex than is apparent from the genetic results
and emphasizing the need to further examine finescale structure.
Despite the lack of a detectable genetic signal todifferentiate rookeries within the Mainland MU, inpractice, nesting sites are managed as separate populations based on geopolitical and legislative factors.
Loggerhead nesting populations in Japan are regulated and managed at local scales ranging fromgovernment prefectures, municipalities as well as
National Parks and Monuments (Matsuzawa &Kamezaki 2012) that are generally smaller than the
MU identified in our study. Along with geography,
demography and physical characteristics, these divisions provide a basis for subdividing the Mainland
MU into ‘recovery units’, similar to the approach
used to manage loggerhead nesting populations in
the Southeast USA. In the absence of clear MU
boundaries (based solely on genetic differentiation)
between adjacent rookeries along the Atlantic coastof the USA, a combination of geographic distributionof nesting densities, geographic separation and geopolitical boundaries was used to designate 5 recovery
units for conservation and management purposes(NMFS and USFWS 2008). Although there are no
obvious geographic breaks in the distribution of nesting habitat in the Mainland Japan MU, generally features of beaches and adjacent landscape differ
among regions. River plains and long stretch ofbeaches with fine sand are common in Miyazaki and
Enshunada, where females nest sparsely. In contrast,

Kii and Shikoku are characterized by many small
pocket beaches with coarse sand along rocky reefs,
where females come ashore to nest densely on somebeaches (such as Minabe) but not on others. These
geomorphic characteristics may separate Kii from
Enshunada and other regions.
The Kagoshima prefecture spans 3 different MUsof Mainland, Yakushima and Ryukyu. Although sea
turtle conservation in the water in these 3 geographic
areas is controlled separately by different regional
fisheries management units, conservation on thebeach is covered across-the-board by the Kagoshima
Prefectural Sea Turtle Conservation Act. These 3
MUs in this prefecture could be assessed and managed separately to avoid neglecting differences innesting trends and area-specific threats. Conversely,
our proposed MUs provide a framework for neighboring local governments and conservation teams
within a MU to share information and cooperate for
efficient conservation of the appropriate demographic units based on our genetic results.
Decade-long monitoring has revealed that nesting
trends differ among regions (Kamezaki 2012, BCJ
2014; see also Yakushima Umigame-kan 2011). Such
differences may reflect differences in MU-specificthreats and mortalities not only for eggs and hatchlings on the beach but also for turtles in the water.
Contrary to threats on the beach (e.g. Matsuzawa et
al. 2002, Kudo et al. 2003), little is known about
threats in the water (but see Hatase et al. 2002b,
Omuta 2011, Ishihara et al. 2014). Threats resulting
in higher mortalities should be identified andaddressed to ensure recovery of loggerhead stocks in
the North Pacific. This study now provides a baseline
to reassess MU-specific threats at sea by enabling
more informative mixed stock analysis (see, forexample, LaCasella et al. 2013).
Acknowledgements. Tissue samples were collected following guidelines authorized under the Act on the Protection ofFisheries Resources, Natural Parks Act, Cultural Assets
Preservation Act and Kagoshima Sea Turtle Protection Ordinance, with the great cooperation of Kuroshima Research
Station and Keiichi Ebi in Yaeyama Islands; Umima-ru(Shinya Inoue and Asuka Takamatsu), Shigeo Kobayashi,
Rumi Payne, Hisashi Inoue, Churagame (Sea Turtle
Research Group of University of Ryukyus), Okinawa
Churashima Foundation and Kunio Komesu in Okinawa
Islands; Yoron-to Co., Ltd., Yoron Town, Okinoerabu Sea
Turtle Network, Doren Campsite and Amami Marine Life
Research Association in Amami Islands; Yakushima
Umigame-kan, Ryoko Masuyama and the Turtle Crew inOsumi Islands; Miyazaki Wild Life Research Group in mainland Kyushu; Shigeru Noichi, Himeji City Aquarium, Hiroki
Tanaka and Hiwasa Chelonian Museum in Shikoku; Akashi
City, Minabe Sea Turtle Research Group, Sea Turtle Associ-Matsuzawa et al.: Population structure of North Pacific loggerheads

ation of Japan, Kameppuri (Sea Turtle Research Group of
Mie University), Kenji Kuroyanagi, Minamichita Beachland
Aquarium, Omaezaki City, Kingdom of Kamehameha, Ichinomiya Sea Turtle Research Group, Fukashi Moriya, KatoriMoriya, Akemi Watabe, and Aqua World Ibaraki Prefectural
Oarai Aquarium in Honshu. This study was funded by Western Pacific Regional Fishery Management Council, Keidanren Nature Conservation Fund, Sea Turtle Association ofJapan, and NOAA-Southwest Fisheries Science Center. Erin
LaCasella provided assistance with data review, andMichael Jensen and Robin LeRoux provided helpful input toearlier drafts of this manuscript.

LITERATURE CITED
Abreu-Grobois FA, Horrocks J, Formia A, LeRoux R andothers (2006) New mtDNA dloop primers which work fora variety of marine turtle species may increase the resolution capacity of mixed stock analyses. In: Frick M,
Panagopoulou A, Rees AF, Williams K (compilers) Book
of Abstracts. 26th Annu Symp Sea Turtle Biol Conserv.
International Sea Turtle Society, Athens, p 179➤ 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➤ Bandelt H, Forster P, Röhl A (1999) Median-joining networks for inferring intraspecific phylogenies. Mol Biol
Evol 16:37–48
BCJ (Biodiversity Center of Japan) (2014) Overview of sea
turtle survey (2004-2012) in the Monitoring-Site 1000
project. Ministry of the Environment, Fujiyoshida City,
p 55 (in Japanese with English Abstract)➤ Bolker BM, Okayama T, Bjorndal KA, Bolten AB (2007)
Incorporating multiple mixed stocks in mixed stock
analysis: ‘many-to-many’ analyses. Mol Ecol 16:
685−695➤ Bowen BW, Karl SA (2007) Population genetics and phylogeography of sea turtles. Mol Ecol 16:4886−4907➤ Bowen BW, Abreu-Grobois FA, Balazs GH, Kamezaki N,
Limpus CJ, Ferl RJ (1995) Trans-Pacific migrations of theloggerhead turtles (Caretta caretta) demonstrated withmitochondrial DNA markers. Proc Natl Acad Sci USA
92:3731−3734➤ Bowen BW, Bass AL, Soares LS, Toonen RJ (2005) Conservation implications of complex population structure: lessons from the loggerhead turtle (Caretta caretta). Mol
Ecol 14:2389−2402
Carr A (1984) The sea turtle: so excellent a fishe. Universityof Texas Press, Austin, TX➤ Dethmers KEM, Broderick D, Moritz C and others (2006)
The genetic structure of Australasian green turtles (Chelonia mydas): exploring the geographical scale of genetic
exchange. Mol Ecol 15:3931−3946
Drummond AJ, Ashton B, Buxton S and others (2011)
Geneious v.5.4. www.geneious.com/
Dutton P (2007) Genetic stock composition of loggerheads(Caretta caretta) encountered in the Hawaii-based longline fishery using mtDNA analysis. In: North Pacific Loggerhead Sea Turtle Expert Workshop, 19−20 December
2007. A report of the Western Pacific Regional Fishery
Management Council pursuant to National Oceanic andAtmospheric Administration Award NOAA Grant FNA05NMF4411092, Honolulu, HI, p 17−19

91➤ Dutton PH, Bowen BW, Owens DW, Barragan AR, Davis SK(1999) Global phylogeography of the leatherback turtle(Dermochelys coriacea). J Zool (Lond) 248:397−409➤ Dutton DL, Dutton PH, Chaloupka M, Boulon R (2005)
Increase of a Caribbean leatherback turtle Dermochelys
coriacea nesting population linked to long-term nest protection. Biol Conserv 126:186−194➤ Dutton PH, Roden SE, Stewart KR, LaCasella EL, Tiwari M,
Formia A and others (2013) Population stock structure ofleatherback turtles (Dermochelys coriacea) in the
Atlantic revealed using mtDNA and microsatellite markers. Conserv Genet 14:625−636➤ Encalada S, Lahanas P, Bjorndal K, Bolten A, Miyamoto M,
Bowen B (1996) Phylogeography and population structure of the Atlantic and Mediterranean green turtle Chelonia mydas: a mitochondrial DNA control region sequence assessment. Mol Ecol 5:473–483➤ Encalada SE, Bjorndal KA, Bolten AB, Zurita JC, Schroeder
BA and others (1998) Population structure of loggerhead
turtle (Caretta caretta) nesting populations in the
Atlantic and Mediterranean as inferred from mitochondrial DNA control region sequences. Mar Biol 130:567−575➤ Excoffier L, Lischer HEL (2010) Arlequin suite v.3.5: a newseries of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10:
564−567➤ Hatase H, Kinoshita M, Bando T, Kamezaki N and others(2002a) Population structure of loggerhead turtles,
Caretta caretta, nesting in Japan: bottlenecks on the
Pacific population. Mar Biol 141:299−305➤ Hatase H, Takai N, Matsuzawa Y, Sakamoto W and others(2002b) 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
Ishihara T (2011) Life history in maturation process of loggerhead turtles (Caretta caretta) in North Pacific. PhD
dissertation, University of Tokyo (in Japanese)
Ishihara T, Kamezaki N, Matsuzawa Y, Ishizaki A (2014)
Assessing the status of Japanese coastal fisheries and sea
turtle bycatch. Wildl Human Soc 2:23−35 (in Japanesewith English Abstract)
Jensen MP, FitzSimmons NN, Dutton PH (2013) Molecular
genetics of sea turtles. In: Wyneken J, Lohmann KJ,
Musick JA (eds) The biology of sea turtles, Vol 3. CRC
Press, Boca Raton, FL, p 153−154
Kamezaki N (2012) Loggerhead turtles in Japan — ecology
and conservation. In: Kamezaki N (ed) Natural historyof sea turtles in Japan. Tokyo University Press, Tokyo,
p 281−301 (in Japanese)
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
Kanno T (1976) Sea turtles in Yakushima Island. Shizenhogo
174:24−26 (in Japanese)
Kudo H, Murakami A, Watanabe S (2003) Effects of sandhardness and human beach use on emergence success ofloggerhead sea turtles on Yakushima Island, Japan. Chelonian Conserv Biol 4:695−696➤ LaCasella EL, Epperly SP, Jensen MP, Stokes L, Dutton PH(2013) Genetic stock composition of loggerhead turtles
Caretta caretta bycaught in the pelagic waters of theNorth Atlantic. Endang Species Res 22:73−84

92Endang Species Res 30: 83–93, 2016

LeRoux RA, Pease VL, LaCasella EL, Frey A, Dutton PH(2008) Longer mtDNA sequences uncover additional
genetic variation among North Pacific loggerheads. In:
Rees AF, Frick M, Panagopoulou A, Williams K (compilers) Proc 27th Ann Symp Sea Turtle Biol Conserv. NOAA
Tech Memo NMFS-SEFSC-569, Silver Spring, MD,
p 129−130➤ LeRoux RA, Dutton PH, Abreu-Grobois AF and others (2012)
Re-examination of population structure and phylogeography of hawksbill turtles in the wider Caribbean using
longer mtDNA sequences. J Hered 103:806−820➤ Lohmann KJ, Hester JT, Lohmann CMF (1999) Longdistance navigation in sea turtles. Ethol Ecol Evol 11:1−23➤ Mantel N (1967) The detection of disease clustering anda generalized regression approach. Cancer Res 27:
209−220
Matsuzawa Y, Kamezaki N (2012) Conservation — conserving endangered species. In: Kamezaki N (ed) Natural
history of sea turtles in Japan. Tokyo University Press,
Tokyo, p 227−254 (in Japanese)➤ Matsuzawa Y, Sato K, Sakamoto W, Bjorndal KA (2002) Seasonal fluctuations in sand temperture: effects on the
incubation period and mortality of loggerhead sea turtle(Caretta caretta) pre-emergent hatchlings in Minabe,
Japan. Mar Biol 140:639−646➤ Moritz C (1994) Defining ‘evolutionarily significant units’ forconservation. Trends Ecol Evol 9:373−375
Nei M (1987) Molecular evolutionary genetics. Columbia
University Press, New York, NY
NMFS (National Marine Fisheries Service) and USFWS (USFish and Wildlife Service) (2008) Recovery plan for the
Northwest Atlantic population of the loggerhead sea turtle (Caretta caretta), 2nd revsn. National Marine Fisheries Service, Silver Spring, MD
Nobetsu T, Minami H, Matsunaga H, Kiyota M, Yokota K(2004) Nesting and post-nesting studies of loggerhead
turtles (Caretta caretta) at Omaezaki, Japan. In: Proc 5th
SEASTAR2000 Workshop Dec 13–15, 2004. Kyoto University, Kyoto, p 30−33
Omuta K (2011) Conservation project on Yakushima Island:
the biggest nesting site in Japan. In: Dutton PH, Squires
D, Ahmed M (eds) Conservation of Pacific sea turtles.
University of Hawaii Press, Honolulu, HI, p 108−119➤ Palsbøll PJ, Berube M, Allendorf FW (2007) Identification ofmanagement units using population genetic data. Trends
Ecol Evol 22:11−16➤ Raymond M, Rousset F (1995) An exact test for populationdifferentiation. Evolution 49:1280−1283➤ Shamblin BM, Dodd MG, Bagley DA, Ehrhart LM, Tucker
AD and others (2011) Genetic structure of the southeastern United States loggerhead turtle nesting aggregation:evidence of additional structure within the peninsular
Florida recovery unit. Mar Biol 158:571−587➤ Shamblin BM, Bolten AB, Bjorndal KA, Dutton PH, Nielsen
JT and others (2012a) Expanded mitochondrial control
region sequences increase resolution of stock structure
among North Atlantic loggerhead turtle rookeries. Mar
Ecol Prog Ser 469:145−160➤ Shamblin BM, Bjorndal KA, Bolten AB and others (2012b)
Mitogenomic sequences better resolve stock structure ofsouthern Greater Caribbean green turtle rookeries. Mol
Ecol 21:2330−2340➤ Shamblin BM, Bolten AB, Abreu-Grobois FA, Bjorndal KA,
Cardona L and others (2014) Geographic patterns ofgenetic variation in a broadly distributed marine vertebrate: new insights into loggerhead turtle stock structurefrom expanded mitochondrial DNA sequences. PLoS
ONE 9:e85956
Takeshita H (2006) The current status of loggerhead sea turtle rookeries in Miyazaki, Japan. In: Kinan I (ed) Proc
Western Pacific Sea Turtle Coop Research and Management Workshop. Vol II: North Pacific loggerhead sea
turtles. Western Pacific Regional Fishery Management
Council, Honolulu, HI, p 27−29➤ Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial
DNA in humans and chimpanzees. Mol Biol Evol 10:
512−526
Taylor BL, Martien K, Morin P (2010) Identifying units toconserve using genetic data. In: Boyd IL, Bowen WD,
Iverson SJ (eds) Marine mammal ecology and conservation — a handbook of techniques. Oxford University
Press, Oxford, p 306−344
Uchida I (1976) My impressions of Kanno’s article. Shizenhogo 174:26 (in Japanese)➤ Wallace BP, DiMatteo AD, Hurley BJ, Finkbeiner EM,
Bolten AB and others (2010) Regional management units
for marine turtles: a novel framework for prioritizing conservation and research across multiple scales. PLoS ONE
5:e15465➤ Waples RS, Gaggiotti O (2006) What is a population? An
empirical evaluation of some genetic methods for identifying the number of gene pools and their degree of connectivity. Mol Ecol 15:1419−1439
Watanabe KK (2006) Studies on gene flow and migratory
ecology in Japanese loggerhead turtles (Caretta caretta).
PhD dissertation, University of Tokyo, Tokyo (in Japanese)➤ Watanabe KK, Hatase H, Kinoshita M, Omuta K and others(2011) Population structure of the loggerhead turtle
Caretta caretta, a large marine carnivore that exhibits
alternative foraging behaviors. Mar Ecol Prog Ser
424:273−283
Yakushima Umigame-kan (2011) Report on sea turtle
research in Yakuhshima Island (1985-2009). Yakushima
Umigame-kan, Kagoshima, p 101 (in Japanese)

Matsuzawa et al.: Population structure of North Pacific loggerheads

93

Appendix. Complete list of author addresses
Y. Matsuzawa1, 2, N. Kamezaki2, 3, T. Ishihara1, 2, K. Omuta4, H. Takeshita5, K. Goto6, T. Arata7, H. Honda8, K. Kameda9,Y. Kashima10, M. Kayo1, I. Kawazu11, J. Kodama5, Y. Kumazawa12, K. Kuroyanagi13, K. Mizobuchi14, K. Mizuno15, K. Oki15,
K. K. Watanabe16, A. Yamamoto17, Y. Yamashita18, T. Yamato1, T. Hamabata19, A. Ishizaki20, P. H. Dutton21,*
1

Sea Turtle Association of Japan, 5-17-18 Nagaomotomati, Hirakata, Osaka 573-0163, Japan2
Suma Aqualife Park Kobe, 1-3-5 Wakamiya-cho, Suma, Kobe, Hyogo 654-0049, Japan
3
Okayama University of Science, 1-1 Ridai-cho, Okayama, Okayama 700-0005, Japan
4
Yakushima Umigame-Kan, 489-8 Nagata, Yakushima, Kumage-gun, Kagoshima 891-4201, Japan
5
Miyazaki Wildlife Research Group, 1-5-14 GakuenKihanadaikita, Miyazaki, Miyazaki 889-2152, Japan
6
Minabe Sea Turtle Research Group, 278 Higashiyoshida, Minabe, Wakayama 645-0001, Japan
7
Doren Camp Site, 2887-9 Ankyaba, Tatsugo, Oshima-gun, Kagoshima 894-0323
8
Churagami University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
9
Kuroshima Research Institute, Sea Turtle Association of Japan, 136 Kuroshima, Taketomi, Yaeyama,
Okinawa 907-1311, Japan10
193 Miyanomoto, Mugiura, Mugi, Kaifu-gun, Tokushima 775-0012, Japan
11
Okinawa Churashima Research Center, 888 Ishikawa, Motobu, Okinawa 905-0206, Japan
12
3302-1 Higashimorogi, Haruno, Kochi 781-0315, Japan
13
Minamichita Beachland Aquarium, 428-1 Okuda, Mihama, Chita, Aichi 470-3233, Japan
14
372-4 Fuwa, Shimanto, Kochi 787-0017, Japan
15
Amami Marine Life Research Association, 99-1 Hiramatsucho Naze, Amami, Oshima, Kagoshima 894-0045, Japan
16
National Institute for Land and Infastructure Management, 1 Asahi, Tsukuba, Ibaraki 305-0804, Japan
17
Kingdom of Kamehameha, 508-3 Hadu, Makinohara, Shizuoka 421-0523, Japan
18
Okinoerabujima Sea Turtle Network, 952 Furusato, Wadomari, Oshimagun, Kagoshima 891-9111, Japan
19
Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo, Kyoto 606-8502, Japan
20
Western Pacific Regional Fishery Management Council, 1164 Bishop St 1400, Honolulu, HI 96813, USA
21
Marine Mammal and Turtle Division, Southwest Fisheries Science Center, National Marine Fisheries Service,
National Oceanic and Atmospheric Administration, 8901 La Jolla Shores Drive, La Jolla, California 92037, USA

Editorial responsibility: Kartik Shanker,
Bangalore, India

Submitted: August 19, 2015; Accepted: January 23, 2016
Proofs received from author(s): March 26, 2016