Fish Sci (2014) 80:11931204

DOI 10.1007/s12562-014-0789-8

ORIGINAL ARTICLE

Biology

Genetic population structure of the Pacific bluefin tuna Thunnus

orientalis and the yellowfin tuna Thunnus albacares in the North
Pacific Ocean
Shohei Nomura Toru Kobayashi Yasuo Agawa
Daniel Margulies Vernon Scholey
Yoshifumi Sawada Naoki Yagishita

Received: 20 March 2014 / Accepted: 11 July 2014 / Published online: 24 August 2014
Japanese Society of Fisheries Science 2014

Abstract The genetic population structure of the Pacific

bluefin tuna (PBF) Thunnus orientalis and the yellowfin
tuna (YFT) T. albacares in the North Pacific Ocean was
investigated. The polymorphism of microsatellite (SSR)
loci and sequences of mitochondrial DNA control region
(mtCR) were analyzed for 71 samples of PBF from Japan
and Mexico and 45 samples of YFT from Japan and Panama. In the SSR analyses, both single-locus (-0.010 to
0.008 in PBF and -0.023 to 0.020 in YFT) and global
multilocus (0.003 in PBF and -0.002 in YFT) FST values
among the geographic populations were low and not significant in these species. In the mtCR analyses, neither the
neighbor-joining tree nor the minimum spanning network
showed genetic differentiation among the geographic
populations in each species. The pairwise FST values
among the geographic populations of them (-0.005 in PBF
and -0.020 to -0.014 in YFT) were low and not significant. Our SSR and mtCR data suggested that genetic differentiations were not evident among the eastern and
western populations in the North Pacific Ocean either in
PBF or in YFT. Mismatch distributions, demographic
parameters, and neutrality tests suggested that sudden
population expansion of PBF and YFT in the North Pacific

S. Nomura  T. Kobayashi  N. Yagishita (&)

Laboratory for Aquatic Biology, Graduate School of Agriculture,
Kinki University, Nakamachi, Nara 631-8505, Japan
e-mail: yagi@nara.kindai.ac.jp
Y. Agawa  Y. Sawada
Fisheries Laboratories, Kinki University, Kushimoto,
Wakayama 649-3633, Japan
D. Margulies  V. Scholey
Inter-American Tropical Tuna Comission, La Jolla,
CA 92037-1508, USA

Ocean occurred 628,000731,000 and 450,000525,000

years ago, respectively.
Keywords Thunnus orientalis  Thunnus albacares 
Genetic population structure  Demographic history 
Mitochondrial DNA  Microsatellites

Introduction
The Pacific bluefin tuna (PBF) Thunnus orientalis is a
highly migratory species that mainly inhabits temperate
and sub-tropical regions of the North Pacific Ocean [1].
The species has higher commercial value than the other
species of Thunnus, and is mainly processed for traditional
Japanese foods, such as sushi and sashimi. In recent years,
the species has become increasingly commercially important world-wide and demands on the species are growing
[2]. The annual catch of PBF in the Pacific Ocean peaked at
40,383 t in 1956 and the lowest recorded to date was 8,653
t in 1990. After 1990, catches of the species in the Pacific
Ocean fluctuated between 11,000 t and 29,000 t with an
overall tendency to decrease because of overexploitation in
recent years [3, 4]. Estimated spawning stock biomass
(SSB) of PBF in the Pacific Ocean in 2010 was reported to
be at or near historically low levels since estimates of SSB
were started in 1950 [3, 4]. The yellowfin tuna (YFT) T.
albacares is globally distributed in the worlds tropical and
sub-tropical oceans [5], and commercially important
world-wide and the most harvested species within the
genus Thunnus [6]. The annual catch of the species in the
eastern Pacific Ocean increased from about 100,000 t in
1960 to over 400,000 t in the beginning of the 2000s, and
the SSB is reported to be holding at an intermediate level.
However, the recruitments of YFT in the eastern Pacific

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Ocean were estimated to be below average in recent years

[7]. The annual catch of YFT in the western and central
Pacific Ocean increased from about 100,000 t in 1970 to
650,000 t in 2008 and recent recruitment is estimated to be
much lower than the long-term average [8, 9].
These trends have led to increased concern for the
proper management of PBF and YFT, and aquaculture of
these species, particularly PBF, may help relieve fishing
pressure. The whole life cycle of PBF under aquaculture
conditions was completed in 2002 [10]; however, present
tuna aquaculture practices world-wide depend on wildcaught fish for seed supply. In 2010 Kinki University
joined with the Aquatic Resources Authority of Panama
(ARAP) and the Inter-American Tropical Tuna Commission (IATTC) in a research project on the comparative
spawning ecology, reproductive biology, and early life
history of PBF and YFT. This project aims to gather more
information involved in managing natural populations in
order to assist in long-term sustainability for PBF and
YFT, and to establish the foundation for the aquaculture
of YFT.
Studies of the genetic population structures of PBF
and YFT will enhance the broodstock management for
aquaculture seed production and sustainable management
of these species. The PBF is known mainly to spawn
offshore from southern Japan to Taiwan from spring to
summer, and the young PBF show clockwise migration
patterns closely related to the ocean structure in and
around the Kuroshio-Oyashio Interfrontal Zone [3, 11,
12]. The young PBF show individual variation in
migration patterns, and some of the young PBF are
known to migrate from Japan to the western coast of
North America and then return to Japan to spawn after
staying in the eastern Pacific Ocean for some years [3,
11, 12]. However, to date we are unaware of any studies
about genetic population structure of PBF. The YFT is
believed to spawn throughout the year with peaks
occurring during the summer in the core area of distribution. Although lack of evidence for long-range east
west or northsouth migrations of adults was suggested
in the Pacific Ocean [5, 13], there has been no detailed
information about the long-range migration of YFT.
However, genetic analyses of the population structure in
the Pacific Ocean were carried out using numerous different approaches in YFT. Sharp [14] revealed differentiation of glucose phosphate isomerase polymorphism
between the western and eastern Pacific populations.
This conclusion was supported by Ward et al. [15, 16]
who suggested the eastern and western-central Pacific
samples were significantly different from each other in
one allozyme locus. However, three studies of YFT
using a restriction fragment length polymorphism (RFLP)
of mitochondrial (mt) DNA showed no evidence of

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Fish Sci (2014) 80:11931204

significant spatial differentiation in the Pacific Ocean

[1517]. More recently, there are two microsatellite
(simple sequence repeat: SSR) studies that estimated
genetic variations of YFT in the Pacific Ocean. Although
Appleyard et al. [18] concluded that the samples from
the Philippines and Fiji were significantly different from
the other areas in the West Pacific Ocean in one SSR
locus, they noted it was unclear whether the low, but
significant differentiation reflected noise in the data set,
or real population differentiation. Daz-Jaimes and UribeAlcocer [19] revealed significant spatial differentiation
between the samples from north- and south-equatorial
locations in the eastern Pacific Ocean. However, none of
these studies produced genetic information on YFT in
Northeast Asia (including Japan).
In this study, we investigated genetic population
structure of PBF and YFT in the North Pacific Ocean,
including samples collected near Japan, based on both
polymorphism of SSR loci and sequences of mtDNA
control region (CR), and evaluated the demographic history of the species. These species are congeners; however,
the annual catches of the two species in the Pacific Ocean
are quite different from each other as described above.
We also estimated the effective population sizes (Ne) of
PBF and YFT in the North Pacific Ocean after population
expansion during the Pleistocene Epoch, that might be
capable of explaining the difference in catches between
the two species.

Materials and methods

Sampling and DNA extraction
Samples of PBF were collected around Japan (n = 36) and
Mexico (n = 35) (Fig. 1; Table 1). Samples from Mie
(n = 33), Japan captured by commercial vessels were
bought at fish market. Samples from Hokkaido (n = 1),
Aomori (n = 1), and Iwate (n = 1), Japan captured by
commercial vessels, were provided by the Osaka Municipal
Wholesale Market Honjo, Osaka, Japan. Samples from
Mexico were kept for cultivation after capture from the
Pacific coast and provided by the Osaka Municipal
Wholesale Market Honjo, Osaka, Japan and the Tsukiji
Market, Tokyo, Japan. Samples of YFT were collected
from Japan (n = 25) and Panama (n = 20) (Fig. 1;
Table 1). Samples from Japan were collected from the
Pacific coast of Okinawa Island by line fishing. Samples
from Panama were captured on the Pacific coast off Pedasi,
Los Santos Province, and provided by recreational fishermen. A piece of white muscle was dissected from each fish
and stored either frozen at -20 C or in 99.5 % alcohol
until DNA extraction.

Fish Sci (2014) 80:11931204

1195

Fig. 1 Map of sampling areas

for Thunnus orientalis
( , Japan; , Mexico) and
T. albacares ( , Japan;
, Panama)

Table 1 Number of samples, body weight (mean), and sampling date

for Thunnus orientalis and T. albacares used in this study
Species

Location

Body weight
(kg)

Sampling date

T. orientalis

Japan

36

23112 (72)

Nov. 2012

Mie

33

733 (14)

Nov. 2007

Hokkaido

84

Nov. 2012

Aomori

112

Nov. 2012

Iwate

23

Nov. 2012

Mexico

35

1874 (36)

Oct. 2012Feb.
2013

25

13 (2)

March 2011July
2011

20

324 (15)

April 2011Nov.
2012

T. albacares

Japan
Okinawa
Panama
Los Santos

Total genomic DNA was isolated using either a standard

phenolchloroform extraction or the DNeasy Blood &
Tissue Kit (QIAGEN, Hilden, Germany).
Microsatellite analyses
We performed a polymerase chain reaction (PCR) to
amplify each of the following eight SSR loci for both PBF
and YFT: Ttho-1, Ttho-4, Ttho-6, and Ttho-7 [20]; and
cmrTa-113, cmrTa-125, cmrTa-144, and cmrTa-161 [18].
In addition, four SSR loci, Tth5, Tth8, Tth21, and Tth34
[21] were amplified and analyzed for PBF. The PCRs were
carried out using a PCR Thermal Cycler Dice Gradient
(TaKaRa Bio, Shiga, Japan), each with 10 ll of reaction
solution containing 10 ng template DNA, 0.5 lM of each
primer, 0.2 mM dNTP mixture, 1 9 PCR buffer [10 mM
TrisHCl (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2], and

0.5 U Ex Taq DNA polymerase (TaKaRa Bio, Shiga,

Japan). The reaction conditions were as follows: an initial
incubation at 94 C for 5 min; 40 cycles of denaturing at
94 C for 30 s, annealing at an appropriate temperature for
each primer pair (5268 C) for 30 s, and extension at
72 C for 2 min; and a final extension at 72 C for 7 min.
Fragments were sized on an automated DNA sequencer
ABI Prism 3130 xl (Applied Biosystems, CA, USA) using
GeneScan-600 LIZ size standard (Applied Biosystems,
CA, USA), and scored using Peak Scanner ver. 1.0
(Applied Biosystems, CA, USA).
Micro-Checker ver. 2.2.1 [22] was used for identifying
possible genotyping errors (i.e. stuttering, large allele
dropout, and null-alleles) within the microsatellite data set
by performing 1,000 randomizations. The observed (HO)
and expected (HE) heterozygosities and deviations of
genotypic distributions from HardyWeinberg equilibrium
(exact test [23]) were calculated using Arlequin ver. 3.5
[24]. A sequential Bonferroni correction [25] was used to
adjust the significance level of the HardyWeinberg equilibrium. The extent of population differentiation was
quantified using FST [26] calculated using Arlequin ver.
3.5. The statistical significance of the FST was determined
by 10,000 permutations.
Mitochondrial DNA analyses
We performed a PCR to amplify approximately 450 base
pairs (bp) of the mtCR, using the following primers:
L15998 (50 -TAC CCC AAA CTC CCA AAG CTA-30 ) and
CSBDH (50 -TGA ATT AGG AAC CAG ATG CCA G-30 )
[27]. The PCRs were carried out using a PCR Thermal
Cycler Dice Gradient or Program Temp Control System
PC-818 (ASTEC, Fukuoka, Japan), each with 10 ll of
reaction solution containing 10 ng template DNA, 0.5 lM
of each primer, 0.2 mM dNTP mixture, 1 9 PCR buffer

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1196

[10 mM TrisHCl (pH 8.3), 50 mM KCl, and 1.5 mM

MgCl2], and 0.5 U Ex Taq DNA polymerase. The reaction
conditions were as follows: an initial incubation at 94 C
for 5 min; 35 cycles of denaturing at 94 C for 30 s,
annealing at 54 C for 30 s, and extension at 72 C for
2 min; and a final extension at 72 C for 7 min. The PCR
products purified by illustra Exostar PCR and Sequencing
Reaction Clean-Up kit (GE Healthcare Bio-Sciences,
Uppsala, Sweden), were sequenced on an ABI Prism 3130
xl, using the BigDye Terminator Cycle Sequencing Kit ver.
3.1 (Applied Biosystems, CA, USA) and the primers used
in the PCR. All sequences have been deposited in DDBJ/
EMBL/Genebank under accession numbers AB906703
AB906818.
In YFT, the published DNA sequences of 20 individuals
near the Philippines (acc. nos. AF301181AF301200) were
added in the following analyses. The DNA sequences of
PBF and YFT were aligned separately using ClustalW ver.
2.1 [28]. The statistical values such as haplotype diversity
(h [29]) and nucleotide diversity (p [29]) were calculated
for each geographic population in each species using Arlequin ver. 3.5. A neighbor-joining (NJ [30]) tree was
constructed from evolutionary distances calculated based
on Kimuras two-parameter model [31], using PHYLIP
ver. 3.65 [32]. The robustness of statistical support for the
branches was determined by 1,000 bootstrap replicates
[33]. The DNA sequences of T. maccoyii (acc. no.
AB906819) and T. obesus (AB906820) were determined
using the same methods described above to root the NJ
trees; the PBF tree was rooted with them and one individual of YFT (AB906774), and the YFT tree was rooted
with them and one individual of PBF (AB906706). A
minimum spanning network (MSN) based on the minimum
sequence differences between haplotypes was constructed
using TCS ver. 1.21 [34]. The pairwise FST values [26]
among the geographic populations were calculated by Arlequin ver. 3.5. The statistical significance of the FST was
determined by 10,000 permutations. Analysis of mismatch
distributions was performed independently for each geographic population and all the samples in each species with
Arlequin ver. 3.5 to infer the recent demographic history of
the samples and to test the fit for the sudden expansion
model [35]. The fit between the observed and expected
distributions was tested using the Harpendings raggedness
index (Hri [36]) and the sum of squared deviations (SSD
[37]). The demographic parameters of mutational time
scale (s), expected pairwise differences before (h0) and
after (h1) a rapid change in population size [37, 38] were
obtained using a parametric bootstrap approach with
10,000 replications. Tajimas D [39, 40] and Fus FS [41]
tests of selective neutrality were conducted, using Arlequin
ver. 3.5 with 10,000 simulated samples.

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Fish Sci (2014) 80:11931204

Results
Microsatellite analyses
The summary of statistics for PBF and YFT are shown in
Tables 2 and 3, respectively; the FST values for PBF and
YFT are shown in Tables 4 and 5, respectively.
In PBF, the number of alleles per SSR locus within
samples varied from one at locus cmrTa-144 in both Japan
and Mexico, to 18 at locus Ttho-6 in Japan (Table 2). No
polymorphism was revealed at locus cmrTa-144, and it was
not included in further analyses. Mean number of alleles
across loci were 9.82 in Japan and 9.27 in Mexico. The HO
varied from 0.400 at locus Ttho-1 in Mexico to 0.914 at
locus cmrTa-125 in Mexico, and HE varied from 0.347 at
locus Ttho-1 in Mexico to 0.914 at locus Ttho-6 in Japan.
Mean HO and HE across loci were estimated to be 0.661
and 0.751 in Japan, and 0.639 and 0.731 in Mexico,
respectively. Significant deviation from HardyWeinberg
expectations due to homozygote excess was observed at
five loci (cmrTa-113, cmrTa-161, Ttho-4, Ttho-6, and Ttho7). As these loci were deduced to be affected by nullalleles, they were also excluded from further analyses. The
single-locus FST values (-0.0100.008) and global multilocus FST value (0.003) between Japan and Mexico were
low and not significant (Table 4).
In YFT, the number of alleles per SSR locus within
samples varied from two at locus cmrTa-144 in Japan and
Panama to 18 at locus cmrTa-161 in Japan (Table 3). Mean
number of alleles across loci were 8.62 in Japan and 7.62 in
Panama. The HO varied from 0.080 at locus cmrTa-144 in
Japan to 0.920 at locus cmrTa-161 in Japan, and HE varied
from 0.216 at locus cmrTa-144 in Japan to 0.926 at locus
cmrTa-161 in Japan. Mean HO and HE across loci were
estimated to be 0.525 and 0.622 in Japan, and 0.581 and
0.623 in Panama, respectively. Significant deviation from
HardyWeinberg expectations due to homozygote excess
was observed at locus Ttho-7. As the locus was deduced to
be affected by null-alleles, it was not included in further
analyses. The single-locus FST values (-0.0230.020) and
global multilocus FST value (-0.002) between Japan and
Panama were low and not significant (Table 5).
Mitochondrial DNA analyses
Number of variable sites (S), number of haplotypes (H),
haplotype diversity (h), and nucleotide diversity (p) for
PBF and YFT are shown in Table 6. The FST values for
PBF and YFT are shown in Tables 7 and 8, respectively.
Demographic parameters with the results of neutrality tests
for PBF and YFT are shown in Tables 9 and 10,
respectively.

0.236

115131

0.806

0.853

0.261

35

14

109135

0.514

0.892

0.000

as

HO

HE

HW
Mexico

as

HO

HE

HW

0.816

168

35

168

36

cmrTa-144

0.003

0.872

0.771

184206

10

1.000

0.347

0.400

181v191

35

0.911

35

0.506

0.000

0.556

181195

36

Ttho-1

0.885

0.639

184214

11

36

cmrTa-161

0.002

0.756

0.657

126150

10

35

0.521

0.739

0.694

126150

12

36

Ttho-4

The loci, cmrTa-144 was excluded from the estimation

Significant after sequential Bonferroni correction (initial a = 0.05/11 = 0.0045)

0.914

149167

35

0.169

0.761

0.694

149167

36

36

cmrTa-113

cmrTa-125

Locus

Japan

Sample

0.000

0.822

0.457

123171

16

35

0.000

0.914

0.583

119181

18

36

Ttho-6

0.022

0.895

0.828

193223

14

35

0.000

0.885

0.583

193225

14

36

Ttho-7

1.000

0.525

0.514

125133

35

0.801

0.570

0.543

121137

35

Tth5

0.011

0.772

0.571

296340

35

0.347

0.801

0.743

296348

10

35

Tth8

0.726

0.492

0.543

123127

35

0.572

0.531

0.628

123131

35

Tth21

0.241

0.855

0.857

97161

14

35

0.648

0.818

0.800

97153

13

35

Tth34

0.731

0.639

9.27a

0.751

0.661

9.82a

Average across loci

Table 2 Summary of statistics for 12 SSR loci in Thunnus orientalis: number of alleles (a), allele size range in base pairs (as), observed heterozygosity (HO), expected heterozygosity (HE), and
probability values of HardyWeinberg equilibrium (HW)

Fish Sci (2014) 80:11931204

1197

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Fish Sci (2014) 80:11931204

Table 3 Summary of statistics for eight SSR loci in Thunnus albacares: number of alleles (a), allele size range in base pairs (as), observed
heterozygosity (HO), expected heterozygosity (HE), and probability values of HardyWeinberg equilibrium (HW)
Sample

Locus
cmrTa-113

cmrTa-125

cmrTa-144

cmrTa-161

Ttho-1

Ttho-4

Ttho-6

Ttho-7

Average
across loci

Japan
n

25

25

25

25

25

25

25

25

a
as

13
105139

4
155165

2
170172

18
182224

5
182194

7
129143

4
109121

16
191239

8.62

HO

0.680

0.400

0.080

0.920

0.520

0.760

0.200

0.640

0.525

HE

0.849

0.520

0.216

0.926

0.684

0.655

0.257

0.869

0.622

HW

0.010

0.308

0.020

0.308

0.167

0.270

0.249

0.000

Panama
n

20

20

20

20

20

20

20

20

11

14

14

as

105135

155165

170172

184224

182194

129143

109123

119225

HO

0.800

0.650

0.150

0.700

0.650

0.600

0.250

0.850

0.581

HE

0.912

0.546

0.224

0.882

0.603

0.682

0.233

0.903

0.623

HW

0.102

0.619

0.246

0.067

0.075

0.435

1.000

0.527

7.62

Significant after sequential Bonferroni correction (initial a = 0.05/8 = 0.0062)

Table 4 The single-locus and

global multilocus FST values
between Japan and Mexico for
Thunnus orientalis based on
microsatellite data

Locus

FST

P value

cmrTa-125

0.006

0.196

Ttho-1

0.008

0.149

Tth5

-0.008

0.611

Tth8

0.002

0.060

Tth21

-0.010

Tth34
Multilocus

Table 6 Number of variable sites (S), number of haplotypes (H),

haplotype diversity (h), and nucleotide diversity (p) for Thunnus
orientalis and T. albacares from the North Pacific Ocean estimated
based on sequences of mitochondrial control region
Species

Population

T. orientalis

All the
samples

71

90

62

0.996 0.003

0.037

0.637

-0.004

0.675

Japan

36

76

31

0.990 0.009

0.050

0.003

0.242

Mexico
All the
samples

35
65

67
89

31
65

0.993 0.009
1.000 0.003

0.049
0.026

P value

Japan

25

53

25

1.000 0.011

0.029

T. albacares
Table 5 The single-locus and
global multilocus FST values
loci between Japan and Panama
for Thunnus albacares based on
microsatellite data

Locus

FST

46

20

1.000 0.016

0.034

0.007

0.318

The
Philippines

20

cmrTa-113
cmrTa-125

-0.021

0.976

Panama

20

67

20

1.000 0.016

0.045

cmrTa-144

-0.023

1.000

cmrTa-161

0.020

0.067

Ttho-1

-0.010

0.637

Ttho-4

-0.019

0.703

Ttho-6

-0.018

0.932

Multilocus

-0.002

0.718

Table 7 Pairwise FST (above diagonal) and significance value

(below diagonal) between Japan and Mexico for Thunnus orientalis
estimated from sequences of mitochondrial control region
Population

Japan

Japan
Mexico

In PBF, the alignment consisting of 411 bp fragments

was obtained from the 71 individuals (Table 6). In total, 90
variable sites (S = 76 in Japan and 67 in Mexico; 75 transitions and 11 transversions), and five gaps ranging from
one to 11 were observed. A total of 62 haplotypes were
defined (H = 31 in Japan and 31 in Mexico). Haplotype
diversity (h) was high in each geographic population
(0.990 0.009 in Japan and 0.993 0.009 in Mexico).

123

Mexico
-0.005

0.577

Nucleotide diversity (p) was 0.050 in Japan and 0.049 in

Mexico. Neither the NJ tree (Fig. 2a) nor the MSN (Fig. 3a)
showed genetic differentiation among the geographic populations. The pairwise FST value between Japan and Mexico
was low and not significant [FST = -0.005 (P = 0.577);
Table 7]. The mismatch distributions for all the samples of
Japan and Mexico, only Japan, and only Mexico (Fig. 4ac)

Fish Sci (2014) 80:11931204

1199

revealed rough bimodal curves. The SSD and Hri values in

each of them were low, and the values except for SSD in
Japan indicated that the observed distributions were not
significantly different from the distribution predicted by the
sudden expansion model [SSD = 0.004 (P [ 0.05) and
Hri = 0.002 (P [ 0.05) for all the samples; SSD = 0.036
(P = 0.003) and Hri = 0.005 (P [ 0.05) for Japan;
SSD = 0.004 (P [ 0.05) and Hri = 0.006 (P [ 0.05) for
Mexico; Table 9]. Estimated effective female population
size after expansion was suggested to be much higher than
before expansion in all the samples, Japan, and Mexico
(h0 = 0.000 and h1 = 54.766 in all the samples;
h0 = 0.000 and h1 = 46.953 in Japan; h0 = 3.574 and
Table 8 Pairwise FST (above diagonal) and significance values
(below diagonal) among Japan, Panama, and the Philippines for
Thunnus albacares estimated from sequences of mitochondrial control region
Population

Japan

Japan
The Philippines

0.898

Panama

0.950

The Philippines

Panama

-0.014

-0.016
-0.020

0.975

Table 9 Demographic parameters and the results of neutrality tests

for Thunnus orientalis
All the samples

Japan

Mexico

Demographic parameters
SSD (P)

0.004 (0.384)

0.036 (0.003)

0.004 (0.439)

Hri (P)

0.002 (0.991)

0.005 (0.942)

0.006 (0.682)

h0

0.000

0.000

3.574

h1

54.766

46.953

100.762

14.721

10.477

11.359

Neutrality tests
Tajimas D
-0.789

-0.671

-0.463

-11.529*

-13.755*

Fus Fs
*

-24.210*

Significant values at P \ 0.01

h1 = 100.762 in Mexico). Tajimas D values were negative

but not significant for all the samples, Japan, and Mexico
(Tajimas D = -0.789 for all the samples, -0.671 for Japan,
and -0.463 for Mexico). Fus Fs values were negative and
significant for each of them (Fus Fs = -24.210 for all the
samples, -11.529 for Japan, and -13.755 for Mexico).
In YFT, the alignment consisting of 397 bp fragments
was obtained from 65 individuals (Table 6). In total, 89
variable sites (S = 53 in Japan, 46 in the Philippines, and
67 in Panama; 80 transitions and 10 transversions), and
three gaps ranging from one to two were observed. A total
of 65 haplotypes were defined (H = 25 in Japan, 20 in the
Philippines, and 20 in Panama). Haplotype diversity
(h) was high in each geographic population (1.000 0.011
in Japan, 1.000 0.016 in the Philippines, and
1.000 0.016 in Panama). Nucleotide diversity (p) was
0.029 in Japan, 0.034 in the Philippines, and 0.045 in
Panama. The NJ tree (Fig. 2b) showed two major clades
[exceptionally, an individual from Panama (acc. no.
AB906815) deviated from the two clades]; however,
bootstrap values supporting the clades were low (\50 %).
Neither the NJ tree nor the MSN (Fig. 3b) showed genetic
differentiation among the geographic populations. The
pairwise FST values among the geographic populations
were low and not significant [FST = -0.014 (P = 0.898)
between Japan and the Philippines, -0.016 (0.950)
between Japan and Panama, and -0.020 (0.975) between
the Philippines and Panama; Table 8]. The mismatch distributions for all the samples (Fig. 4d) and only Panama
(Fig. 4g) showed indistinct bimodal curves with frequency
of the first (left-hand) mode much larger, and the distributions for only Japan (Fig. 4e) and only the Philippines
(Fig. 4f) showed unimodal (although rough in the latter)
curves. The SSD and Hri values in them were low, and the
values except for SSD in all the samples indicated that the
observed distributions were not significantly different from
the distribution predicted by the sudden expansion model
[SSD = 0.004 (P = 0.042) and Hri = 0.008 (P [ 0.05) for
all the samples; SSD = 0.008 (P [ 0.05) and Hri = 0.014

Table 10 Demographic parameters and the results of neutrality tests for Thunnus albacares
All the samples

Japan

The Philippines

Panama

Demographic parameters
SSD (P)

0.004 (0.042)

0.008 (0.162)

0.009 (0.216)

0.010 (0.203)

Hri (P)

0.008 (0.120)

0.014 (0.395)

0.018 (0.313)

0.020 (0.219)

h0

0.000

0.000

0.000

0.002

h1

99,999

99,999

99,999

99,999

10.201

9.188

10.635

11.547

Tajimas D

-1.478*

-1.407

-0.765

-1.340

Fus Fs

-24.559*

-19.760*

-12.097*

-10.701*

Neutrality tests

Significant values at P \ 0.01

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Fish Sci (2014) 80:11931204

Fig. 2 Neighbor-joining tree

constructed from sequences of
mitochondrial control region of
(a) Thunnus orientalis ( ,
Japan; , Mexico) and (b) T.
albacares ( , Japan; , the
Philippines; , Panama). The
evolutionary distances were
calculated based on Kimuras
two-parameter model. Numbers
at nodes indicate the bootstrap
values (only values [50 %
shown). Asterisk indicates the
individual of T. albacares from
Panama deviating from the
major two clades

(P [ 0.05) for Japan; SSD = 0.009 (P [ 0.05) and

Hri = 0.018 (P [ 0.05) for the Philippines; SSD = 0.010
(P [ 0.05) and Hri = 0.020 (P [ 0.05) for Panama;
Table 10]. Estimated effective female population size after
expansion was suggested to be greatly higher than before
expansion in all the samples, Japan, the Philippines, and
Panama (h0 = 0.000 and h1 = 99,999 in all the samples;
h0 = 0.000 and h1 = 99,999 in Japan; h0 = 0.000 and
h1 = 99,999 in the Philippines; h0 = 0.002 and
h1 = 99,999 in Panama). Tajimas D values were negative
and significant for all the samples, while negative, but not
significant for Japan, the Philippines, and Panama (Tajimas
D = -1.478 for all the samples, -1.407 for Japan, -0.765
for the Philippines, and -1.340 for Panama). Fus Fs values
were negative and significant for each of them (Fus
Fs = -24.559 for all the samples, -19.760 for Japan,
-12.097 for the Philippines, and -10.701 for Panama).

Discussion
Genetic variation of microsatellite loci
and mitochondrial DNA control region
Mean HE of PBF was estimated to be 0.751 in Japan and
0.731 in Mexico, which were similar to the results of

123

Takagi et al. [20] who examined four SSR loci for PBF in
the Pacific Ocean and suggested mean HE to be 0.788.
Mean HE of YFT was estimated to be 0.622 in Japan and
0.623 in Panama, which were also similar to the mean HE
of three previous studies for YFT in the Pacific Ocean:
0.667 [20], 0.619 [18], and 0.576 [19]. In both PBF and
YFT, each geographic population in the Pacific Ocean was
characterized by high haplotype diversity (Table 6), corresponding to the results of previous studies for the
Atlantic bluefin tuna (ABF) T. thynnus in the Mediterranean Sea (h = 0.9820.996 [42] and 0.992 [43]) and the
North Atlantic Ocean (h = 0.988 [43]), and for YFT in the
Pacific Ocean (h = 0.999 [44] and 0.992 [45]).
Genetic differentiation among populations
In the SSR, both the single-locus and global multilocus FST
values among the geographic populations of PBF and YFT
were low and not significant (Tables 4, 5). In the mtCR
analyses, neither the NJ tree (Fig. 2) nor the MSN (Fig. 3)
showed genetic differentiation among the geographic
populations, and the pairwise FST values among the geographic populations were low and not significant (Tables 7,
8). Based on the SSR and mtCR analyses, it does not seem
that individuals from the eastern and western North Pacific
Ocean, including Japan, exhibit genetic differentiations in

Fish Sci (2014) 80:11931204

Fig. 3 Minimum spanning network among haplotypes of mitochondrial control region of (a) Thunnus orientalis ( , Japan; , Mexico)
, the Philippines;
, Panama).
and (b) T. albacares ( , Japan;
Node equals one nucleotide substitution. Numbers in the circles
indicate the number of individuals. Small open circles indicate
missing intermediate. Asterisk indicates the same individual of T.
albacares as shown in Fig. 2

either PBF or YFT. This may result from significant levels

of gene flow and/or physical mixing of individuals between
the eastern and western areas in the North Pacific Ocean for
both PBF and YFT. Our result for YFT agreed with Ward
et al. [15, 16], Scoles and Graves [17], and Appleyard et al.
[18] who revealed no genetic differentiation between
individual YFT from the eastern and western Pacific Ocean
based on mtDNA variation. However, Sharp [14] and Ward
et al. [15, 16] detected differentiation of an allozyme locus
between the western and eastern Pacific populations of
YFT. Considering the lack of SSR differentiation between
individuals from the western and eastern Pacific Ocean
revealed in this study, differential selection in the presence
of gene flow is implicated as the cause of the differentiation
of the allozyme locus as discussed by Ward et al. [16].
Demographic history
In PBF, both Tajimas D and Fus Fs were negative for all
the samples, Japan, and Mexico (Table 9). While Tajimas
D was not significant, Fus Fs, which is the most powerful

1201

test for detecting population growth [46], was significant

for each region. Although the SSD value for Japan was
significant, SSD values for all the samples and Mexico and
Hri value for each region was low and not significant
(Table 9). The significant and negative Fus Fs, low and
non-significant Hri values, and the large difference
between h0 and h1 (Table 9), suggested the sudden
expansion [37, 38, 47] of all the samples as well as for
Japan and Mexico. For estimating years from the population expansion in the North Pacific Ocean, we assumed an
average divergence rate of 4.95.7 % per million years as
this rate was suggested to be reasonable for the mtCR of
ABF and the swordfish Xiphias gladius [43]. Estimated
using the sequence length of 411 bp, the s-value of 14.721
(Table 9), and the divergence rate of 4.95.7 %, the population expansion of PBF (all the samples) likely occurred
between 731,000 and 628,000 years ago. Alvarado Bremer
et al. [43] suggested the population expansion of ABF in
the North Atlantic Ocean occurred between 450,000 and
390,000 years ago. The population expansion of PBF based
on our analysis was likely to occur much earlier before the
population expansion of ABF in the North Atlantic Ocean.
In the Pleistocene Epoch (from 2,580,000 to 10,000 years
ago), there were several cyclical climatic changes of
approximately 40,000100,000 years in the earth [48, 49].
The population expansion of PBF was most likely caused
by the climatic change in the Pleistocene Epoch.
In YFT, the mismatch distributions of all the samples
(Fig. 4d) and Panama (Fig. 4g) showed more numbers of
pairwise differences than Japan (Fig. 4e) and the Philippines (Fig. 4f), due to existence of the individual from
Panama deviating from the other individuals in the NJ tree
(Fig. 2b) and MSN (Fig. 3b). Ely et al. [44] revealed a
unimodal curve of the mismatch distribution based on the
sequences of mtCR of samples from the Atlantic and Indian
Oceans in addition to the Pacific Ocean. Wu et al. [45] also
revealed a unimodal curve of the mismatch distribution
based on the sequences of mtCR of samples from the
western Pacific and the western Indian Oceans. The first
(left hand) mode of all the samples (Fig. 4d) is at approximately 10 of the number of pairwise differences, almost
corresponding to the mismatch distributions estimated by
Ely et al. [44] and Wu et al. [45]. Tajimas D was negative
for all the samples, Japan, the Philippines, and Panama
(Table 10), but only significant for all the samples. However, Fus Fs for each region was negative and significant.
While the SSD value for all the samples was significant,
SSD values for Japan, the Philippines, and Panama and the
Hri value for each region was low and not significant. The
significant and negative Fus Fs, low and non-significant
Hri values, and the large difference between h0 and h1
(Table 10), suggested the sudden expansion [37, 38, 47] of
all the samples as well as for Japan, the Philippines, and

123

1202

Fish Sci (2014) 80:11931204

Fig. 4 Mismatch distributions

based on haplotypes of the
mitochondrial control region for
Thunnus orientalis (ac) and T.
albacares (dg). The solid and
dotted lines represent observed
distribution and simulated
distribution under the sudden
expansion model, respectively.
a All the samples of Japan and
Mexico. b Only Japan. c Only
Mexico. d All the samples of
Japan, Panama, and the
Philippines. e Only Japan.
f Only the Philippines. g Only
Panama

Panama. Estimated using the sequence length of 397 bp, the

s-value of 10.201 (Table 10), and the divergence rate of
4.95.7 % (as discussed above), the population expansion
of YFT in the Pacific Ocean (all the samples) likely
occurred between 525,000 and 450,000 years ago, nearly
corresponding to Ely et al.s [44] estimate which suggested
the global population expansion of YFT occurred
522,000 years ago. The population expansion of YFT was
also most likely related to climatic change 200,000 years
after the population expansion of PBF.
Effective population size
In PBF, the female Ne after expansion was estimated to be
2,700,000 using the sequence length of 411 bp, the h1value of 54.766 (Table 9), and the divergence rate of 4.9 %

123

per million years. On the other hand, our estimation of the

h1-value of YFT for the samples from the Pacific Ocean
was 99,999 (=?) and the specific value of Ne could not be
calculated. However, the h1-value of 99,999 (=?) suggested the female Ne of YFT to be considerably large.
Genetic diversity is known to increase with the Ne [50], and
the highest haplotype diversity of YFT (h = 1.000;
Table 6) also indicated the Ne of YFT to be extremely
large. Presumably, we calculated the female Ne of YFT in
the Pacific Ocean using the same parameter values as used
in Ely et al. [44] (the h1-value of 6,655, the sequence length
of 333, and the divergence rate of 4.9 %) and the female Ne
of YFT in the Pacific Ocean after expansion was estimated
to be 407,900,000. In addition, the female Ne of ABF after
expansion was estimated to be 5,400,000, calculated based
on the same parameter values as used in Alvarado Bremer

Fish Sci (2014) 80:11931204

et al. [43] (h1-value of 89.141 and sequence length of

338 bp). The SSB in the 1950s when dramatic changes of
abundance of these tuna species had not yet occurred, was
estimated to be 40,000130,000 t [3],300,000 t [51], and
3,300,000 t [7, 8] in PBF, ABF, and YFT in the Pacific
Ocean, respectively. As census population size can be
expected to be proportional to Ne in tuna species discussed
by Ely et al. [44], the SSB estimated from fisheries data
corresponded well with our estimation of the female Ne,
which was much larger in YFT in the Pacific Ocean than in
PBF and ABF, and several times larger in ABF than in
PBF. The maturity rates for PBF were reported to be 20,
50, and 100 % at the ages of 3, 4, and 5 years, respectively
[4]. On the other hand, the first maturing fish of YFT in the
western and central Pacific Ocean occurs at the age of 23
years and 100 % maturity occurs at the age of less than 4
years [52], and in the eastern Pacific Ocean the age at
maturity for YFT is approximately 2 years of age [53, 54].
The earlier maturity, wider spawning area, longer spawning period, and wider distribution of YFT (see Introduction) are likely related to the larger population size
estimated for this species.
Acknowledgments We thank Mr. Ryuichi Kojima at the Tsukiji
Market, Tokyo, Japan, Mr. Seiji Sakamoto at the Osaka Municipal
Wholesale Market Honjo, Osaka, Japan, Mr. Asanari Gima in Nanjocity, Okinawa Pref., Japan, Mr. Yousuke Matsumoto and Dr. Rui
Matsumoto at Okinawa Churaumi Aquarium, Japan, and Ms. Susana
Cusatti at IATTC, USA for their help in collecting the samples of the
Pacific bluefin tuna and the yellowfin tuna. This research was supported in part by a Grant-in-Aid for the SATREPS program in Japan.

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