DNAマーカーに基づく西日本キチヌの集団構造解析

誌名誌名 水産増殖 = The aquiculture

ISSNISSN 03714217

著者著者

Ahmad Syazni, K.笘野, 哲史上野, 香菜子大原, 健一海野, 徹也

巻/号巻/号 63巻1号

掲載ページ掲載ページ p. 17-27

発行年月発行年月 2015年3月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Aquacult. Sci. 63 (1)， 17 -27 (2015)

Genetic structure of yellowfin black seabream

Acαnthotαgrus latus in western Japan based on

microsatellite and mtDNA marker analyses

Kamarudin AHMAD SYAZNl1，2， Satoshi TOMANOl， Kanako UENOl， Kenichi OHARA3 and Tetsuya UMIN01， *

Abstract: Yellow宣nblack seabream Acanthopagrus latus is an important宣shspecies found in出ecoastal waters along the Pacific coast of Japan. The genetic variability of A. 1，αtus was estimated by analyzing seven populations in western Japan， using highly variable microsatellite loci (n=312) and the sequence variability in the mitochondrial DNA (mtDNA) (n=42).官lemicrosatellite loci revealed a high level of genetic variability， wi出 themean number of alleles per locus ranging from

22 to 47 and the mean observed hetero勾gosityranging from 0.840 to 0.904 across populations.

τbe sequence variability in the control region (289 bp) of 42 individuals yielded 27 haplotypes.

官leglobal宣xationindex (F':訂)of -0.00024 (P> 0.05) and 0.0160 (P> 0.05) for microsatellites and

mtDNA， respectively， suggested that there was no significant differentiation among the seven putative populations. High gene flow caused by random dispersal of pelagic eggs and larvae likely

explains the occurrence of a single stock of A. 1αtus in western J apan. Information about the genetic population structure of A. latus can aid in designing proper management strategies for this species in the near future.

Key words: Acanthopagrus latus; Genetic structure; Homogeneity; Microsatellite loci

Genetic differentiation in marine fishes is

largely a function of their dispersal capabilities

のN"aples198η. For example， planktonic eggs and larvae can be passively dispersed over

great distances， leading to dynamic and seem-

ingly weak genetic differentiation (Waples 1998;

Cowen et al. 2000). Likewise， some marine

fishes migrate long distances and exhibit

non-homing behavior， thereby increasing the

probability of high gene flow among areas.τbis

can lead to the absence of or weak stock struc-

加reexcept at very large geographic scales.

The absence of strong barriers to dispersal in a marine environment and generally large

Received 1 May 2014; Accepted 4 November 2014.

population sizes tend to reduce the level of

genetic differentiation (Gyllensten 1985; Ward

et al. 1994).

Unfortunate私自elevel of genetic differen-tiation in many marine fishes remains unclear.

In particular， there is a critical need for genetic

markers that can distinguish between a we叫E

population structure and the absence of struc-

ture. Mitochondrial DNA (mtDNA) sequences

are basic DNA markers， which have been widely used during the past three decades

because of血ey訂 etechnically easy to use and

able to detect population history， species delin-

eation， and identification (Galtier et al. 2009).

1 Graduate School ofBiosphere Science， Hiroshima University， Higashi-Hiroshima， Hiroshima 739-8528， Japan. 2 Faculty of Agriculture， Biotechnology and Food Science， University Sultan Zainal Abidin， Kampus Tembila， 22200 Besut， Terengganu， Malaysia. 3G出 PrefecturalResearch Institute for Freshwater Fish and Aquatic Environments， Gero， Gi釦509-2592，Japan. * Corresponding author: Tel/Fax， (+81) 82-424-7944; Email， umino@hiroshima-u.ac必σ:Umino).

K. A. Sy招 ni，S. Tomano， K. Ueno， K. Ohara and T. Umino

countries such as China and Taiwan， there is

stil1 limited knowledge of由egenetic charac-

teristics of this species in J apan. Hence， our objective was to assess the ecological and bio-

logical framework of the wild stock s廿uctureof

A. latus across its geographical range in west-ern J apan. The results of this study wil1 assist in the conservation and management of an import-

ant and widely distributed species in J apan in the fu加re.

Materials and Methods

Sampling A total of 312 wild A. latus were sampled from

seven locations (Fig. 1) for microsatellite anal-

ysis. We selected mtDNA sequences from 42

individuals for analysisぐrable1). A pectoral

or caudal fin clip was taken仕omeach individ-

ual and stored in 99% ethanol. Total DNA was

-・・H4'

-・・l32・

Fig. 1. Sampling sites of Acanthotagrus latus in western ]apan.

Furthermore， microsatellite markers， which

typical1y exhibit higher levels of polymorphism， may be more robust at detecting a weak popu-

lation structure in areas with high gene flow

(ぬmtsenet al. 2003; Umino et al. 2009). The

use of both microsatellite and mtDNA markers

is likely to be more informative when evaluat-

ing the population structure of yel10wfin black

seabream Acanthopagrus latus. A. latus is dis凶butedacross a wide geo・

graphic area from J apan to Australia and throughout the Western 1ndo-Pacificαia et

al. 2008). 1n J apan， A. latus is a popular food fish that inhabits shal10w and coastal waters，

often entering river mou白sand estuaries.

Understanding the pa仕ernof genetic diversity

and defining the unit at which a species is man-

aged is one of the critical first steps in fishery

management and conservation (FAO 1993). A

number of studies have shown that the abun-

dance of A. latus has decreased in several parts of their range， including in China (Xia et

al. 2008) and Australia (Shaw 2000; Hesp et al.

2004)， owing to overfishing. Although A. latus occupies a wide geographic range along the

western Pacific coast in J apan， the population is somewhat fragmented and is restricted pri-

marily to larger river mouths. Adults appear

to e対1ibita preference for brackish water and

are rarely found offshore. 1n addition， A. 1αtus larvae and juveniles are句rpical1yfound in es加-

aries (1wamoto et al. 2009). Together， these

observations suggest that their life cycle is com・

pleted entirely within close proximity to a river

mouth or estuary at the internal basin.

Despite several reports on the genetic vari-

ability and structure of A. latus in several

18

..l40・

-・・ ~36'

Table 1. Population location， collection timing， sample size， andω凶 lengthand weight of Acanthot旬 ruslatus screened for microsatellite and mitochondrial DNA markers

Number of samples Microsatellit廷 mtDNA

48

24

48

48

48

48

48

Body weight (g)

Total length (mm)

Population codes

201-1050

6ι438

Not measured 31-227

167-1650

Not measured Not measured

243-423

157-329

> 250

123-237

213-445

Not measured 150ー350

HIR MIY

AIC

KOC

SHI TOKU MIE

popOPOPOPO氏UPO

Sampling period

Hiroshima October 2011 Miyazaki August 2012 Aichi September 2012 Kochi September 2012 Shizuoka October 2012 Tokushima November 2012 Mie December 2012 mtDNA: mitochondrial DNA

Population

Genetic s仕uctureof yellowfin black seabream 19

extracted for microsatellite analysis using an

alkaline lysis method following the KOD F玄

N eo protocolσoyobo， ]apan). Total genomic

DNA was extracted for mtDNA analysis using a

standard phenol-chloroform extraction and eth-

anol precipitation (Sambrook et al. 1989).

Micγosα:telliteα%α:lysis

Ten microsatellite loci σF 6a， YF 6b， YF

9b， YF 11， YF 15， YF 16， YF 18， YF 19， YF 22，

and YF 2ηwere amplified using primer pairs，

which had been isolated previously in the same

species (Ahmad-Syazni et al. 2012).百lefor-

ward and reverse primers were labeled at the

5' end with FAM， HEX， or NED.τbe final reac-

tion volume for PCR amplifications was 5μl

and consisted of 0.7μ1 ddH20， 1X KOD FX

Neo Buffe乙0.04mM dNTPs， 0.2μM each for-

ward and reverse primer， 0.02 units/μ1 KOD

polymerase (Toyobo)， and 50 ng template DNA.

τbe PCR was performed in a Mastercycler

Gradient 96 well (Eppendorf， USA).官lether-

mal profile consisted of an initial denaturing

step at 940

C for 4 min， followed by 30 cycles of

940

C for 1 min， the locus四 specificannealing tem-

perature (Ahmad-Syazni et al. 2012) for 1 min，

and 720

C for 1 min. A final extension was car同

ried out at 720

C for 10 min.

In geno匂rping，the PCR products were

diluted 10 times with ddH20. Exactly 1μl

of the dilute PCR product was mixed with

8.8μ1 HiDi Formamide (Applied Biosystems，

USA) and 0.2μ1 of the size standard 400HD

ROX (Applied Biosystems， USA). The mix-

加rewas白endenatured at 950

C for 3 min and

quickly chilled on ice. The宣nalmixtures were

sequenced using an ABI 313白d Sequencer

(Applied Biosystems， USA)， and the allele size

was estimated using Peak Scanner So立ware

v1.0 (Applied Biosystems， USA).

The genetic diversity at each locus and for

each population of A. latus was estimated based

on the number of alleles (NA) using FSTAT

v2.9.3 (Goudet 1995). Observed and expected

heterozygosity (Ho and HE) were calculated

using Microsate11ite Toolkit add-ins in Microsoft Excel 2007 (Park 2001). Allelic richness (AR)

was adapted to compare the smallest sample

(n=24) using the rarefaction method in F訂AT

v2.9.3 (Goudet 1995).百四 programMicro-

Checker version 2.2.3 (van Oosterhout et al.

2004) was used to test for null alleles， large

allele dropout， and scoring errors due to stut-

ter peaks. Genotype frequencies at each locus

throughout the population were tested for con-

formity to Hardy-Weinberg equilibrium (ill町E)

using a Markov chain method in Arlequin v3.0

(Excoffier et al. 2005). Bonferroni adjustments

of P-values were applied whenever multiple

tests were performed (悶ce1989). Linkage dis-

equi1ibrium (LD) was estimated wi血 Genepopversion 4.1 (Raymond and Rousset 1995;

Rousset 2008).百leinbreeding coefficient (Frs)

(Weir and Cockerham 1984) was calculated

using FSTATv2.9.3 (Goudet 1995). Wright's fix-

ation index (F.訂)(Weir and Cockerham 1984)

was used to analyze population differen世ation

because it is more reliable when fewer than

20 loci are considered (Gaggiotti et al. 1999).

Global analysis of molecular variance (AMOVA)

was used to estimate F sr and pairwise F sr values for all possible population combinations

and their significance levels using Arlequin 3.0

(Excoffier et al. 2005).

Mitochondrial DNAαηα句sis

The 289-bp mtDNA control region was

amplified by PCR using the primers 16086F

(5可寸AGTATGGTGACAATGCA下3')and 16621R

(5'-GACACCA1寸AACITAτ'GCAA-3') (Liu et al.

2004). The final reaction volume for PCR ampli-fications was 10.05μ1 and consisted of 6.8μl

ddH20， 1.0μ1 10X ExTaq Buffer σAKARA， ]apan)， 0.2 mM dNTPs， 0.2 mM forward and

reverse primers， 0.125 units/μ1 ExTaq poly-

meraseσAKARA， ] apan) ， and 50 ng template

DNA. The PCR thermal profile consisted of an initial denaturing step for 4 min at 94

0

C;

30 cycles of 1 min at 940

C， 1 min at 520

C， and

1 min at 720

C; and a final extension for 10 min

at 720

C. The reactions were carried out in a

Mastercycler Gradient 96 well (Eppendorf，

Germany). The amplified products were puri-

fied using an ExoSAP-IT kit (Amersham

Bioscience， N]， USA). All sequencing reactions

were performed according to the manufacturer' s

20 KASy田 ni，S. Tomano， K Ueno， K Ohara and T. Umino

Table 2. Genetic variability ofAcanthotagrus latus among seven locations in ]apan

Iρcus Iρcation

YF6a YF6b YF9b YF11 YF15 YF16 YF18 YF19 YF22 YF27 Mean

Hiroshima NA 34 23 30 15 20 26 29 19 17 11 AR 26.925 19.278 23.582 12.885 17.291 20.293 21.243 15.457 15.674 8.883 Ho 1.000 0.938 0.875* 0.917 0.771 * 0.813* 0.854 0.833 0.896 0.688 0.858 HE 0.972 0.951 0.963 0.902 0.934 0.944 0.940 0.907 0.933 0.758 0.920 Frs ー0.029* 0.014 ー0.016 0.092 0.040 0.094 0.030 P(HWE) 0.940 0.768 0.044 0.684 0.010 0.021 0.229 0.566 0.126 0.507

Miyazaki NA 25 17 22 12 16 21 19 14 18 14 AR 25.000 17.000 22.000 12.000 16.000 21.000 19.000 14.000 18.000 14.000 Ho 0.917 0.958 0.958 1.000 0.917 0.833 0.958 0.917 0.875 0.708 0.904 HE 0.957 0.943 0.955 0.906 0.921 0.933 0.957 0.874 0.932 0.817 0.920 Frs 0.043 -0.016 -0.106 ー0.002 0.062 0.136 0.018 P但WE) 0.636 0.862 0.919 0.096 0.767 0.440 0.639 0.706 0.574 0.197

Aichi NA 32 19 30 17 16 25 23 20 20 12 AR 24.115 16.253 22.957 13.921 14.665 19.608 18.136 15.887 16.732 9.355 Ho 0.958 0.896 0.938 0.917 0.938 0.750** 0.938 0.917 0.917 0.771 0.894 HE 0.958 0.923 0.960 0.900 0.923 0.943 0.922 0.892 0.937 0.749 0.911 Frs 0.000 0.030 -0.018 ー0.017 0.022 -0.030 -0.001 P但WE) 0.833 0.351 0.251 0.693 0.625 0.000 0.969 0.483 0.293 0.576

Kochi NA 30 20 26 14 21 30 26 20 20 13 AR 24.040 16.608 21.416 12.419 16.783 21.857 19.271 16.491 16.498 10.008 Ho 0.917 0.917 0.813* 0.896 0.896 0.792* 0.938 0.917 0.833 0.688* 0.860 HE 0.964 0.927 0.953 0.896 0.921 0.943 0.937 0.926 0.931 0.718 0.912 Frs 0.049 0.011 0.001 0.000 0.106* 0.042 0.035* P但WE) 0.126 0.089 0.011 0.864 0.618 0.046 0.225 0.502 0.340 0.043

Shizuoka NA 30 22 29 14 20 31 22 17 18 13 AR 23.766 17.376 22.897 12.794 16.117 22.759 17.475 13.986 15.735 10.190 Ho 0.938 0.938 0.896 0.938 0.958 0.875 0.958 0.896 0.854 * 0.792 0.904 HE 0.961 0.933 0.959 0.905 0.922 0.947 0.927 0.904 0.928 0.783 0.917 Frs 0.025 ー0.005 ー0.037 -0.034 0.080 -0.011 0.004 P(HWE) 0.190 0.077 0.059 0.442 0.445 0.146 0.875 0.145 0.045 0.478

Tokushima NA 35 21 25 17 24 27 24 17 21 16 AR 25.482 17.033 22.028 13.999 18.770 22.358 18.933 14.476 17.189 11.701 Ho 0.917 0.875 0.792** 0.917 0.938 0.813* 0.896 0.833 0.938 0.813 0.873 HE 0.961 0.928 0.962 0.895 0.934 0.955 0.942 0.905 0.933 0.786 0.920 Frs 0.047 0.058 ー0.025 0.050 ー0.005 ー0.034 0.017 P(HWE) 0.187 0.109 0.001 0.453 0.400 0.008 0.594 0.051 0.628 0.309

Mie NA 38 23 27 14 18 24 24 15 21 18 AR 26.915 19.213 22.487 11.977 15.162 19.339 19.185 12.709 16.991 12.569 Ho 0.896 0.917 0.938 0.938 0.729* 0.750** 0.917 0.729* 0.833 0.750 0.840 HE 0.966 0.940 0.961 0.895 0.918 0.947 0.933 0.879 0.928 0.797 0.916 Frs 0.073* 0.025 -0.048 0.017 0.103* 0.060 0.039*

P(HVI屯) 0.184 0.392 0.584 0.466 0.005 0.004 0.067 0.006 0.384 0.702

Mean NA 47 27 34 22 25 36 35 30 26 25 AR 25.195 17.737 22.995 12.829 16.401 21.332 19.122 14.945 16.543 10.674

キ， significant at P= 0.05; * *， significant at P= 0.005 (after Bonferroni correction); NA> number of alleles; AR' allelic richness; Ho， observed heterozygosity; HE' expected heterozygosity; FIS' inbreeding coefficient; P但WE)，件-valuefor Hardy-Weinberg equilibrium.

Genetic s仕uc卸reof yellowfin black seabream 21

instructions using a BigDye Terminator v3.1

Cycle Sequencing Kit (Applied Biosystems，

USA). Sequences were aligned using ClustalW

(http://clus回lw.ddbj.nig.ac.jpj). Nucleotide

sequences were registered in GenBank under

accession numbers AB91677 4-AB916800. Another

111 additional mtDNA sequences used in this

study were published in Xia et al. (2008) (GenBank

accession numbers EF506765-EF506875). The

relationship among mitochondrial haplotypes

within Japan and between Japan and China were

depicted by a median joining network calcu-

lated using NE1WORK (Bandelt et al. 1999).

Arlequin 3.0 (Excoffier et al. 2005) was used to

estimate the haplotype diversity and nucleotide

diversity. Arlequin 3.0 (Excoffier et al. 2005)

was also used to calculate the global AMOVA

to estimate F sr and pairwise F sr values for all

possible population combinations and their sig-

nificance levels. Phylogenetic pa仕ernswere

analyzed based on unweighted pair-group

method wi血 arithmeticaverage (UPGMA) ，

and Tamura-Nei distance was calculated with

MEGA version 2.1 (Kumar et al. 2001).

Resul胎

Of the 10 microsatellite loci analyzed in the

312 individuals among seven populations， all

loci exhibited a high level of polymorphism

σ'able 2). The degree of variability differed

considerably among出e10 loci with the NA across all populations ranging from 22 to 47.

For instance， locus YF 6a exhibited a wide

range in the NA among populations (25-38).

Additionallぁ theAR value of A. latus at each

locus across the populations differed markedly

from 8.883 at YF 27 to 26.925 at YF 6a， both

values from the Hiroshima (HIR) population.

官官 levelof genetic diversity of A. latus in west-

ern Japan was high， based on the mean Ho and

HE values across populations (range: 0.840-

0.904 and 0.911-0.920， respectively).

A signi宜cant deviation from HWE was

observed in 13 of 70 single loci (Pく0.05).

Howeve乙afterBonferroni correction (P < 0.005)，

only three loci signi自cantlydeviated企omHWE

among the populations: Aichi (AIC: YF 16)，

TokushimaσOKU: YF 9b)， and Mie (MIE: YF

16). LD was not evident among all possible pair-

wise locus comparisons (P>0.05). The results

from the Micro-Checker analysis suggested

that some loci from HIRσF 16， YF 15)， AIC

σF 16)， Kochi (KOC; YF 9b， YF 16)， TOKU

σF 9b， YF 16)， and MIEσF 15， YF 16， YF 19)

may be affected by null alleles after Bonferroni

correction. Therefore， four loci， YF 9b， YF 15，

YF 16， and YF 19， were discarded for FIS and

九 analysis.τbetotal FIS for sIx populations was positive and was significantly greater than

zero in two populations (KOC， MIE) based on

permutation testing (Pく0.05)(Table 2).

We calculated a global F sr of -0.00024

(P> 0.05) based on genotyping 312 individuals

among seven populations using sIx microsat-

ellite loci. Besides出at，the pairwise F sr at the

sIx microsatellite loci ranged from 0.0025 (AIC

versus KOC population) to -0.0028 [Miyazaki

(M町)versus TOKU population]ぐfable3).官le

pairwise F sr was not significantly different for all combinations of populations (P> 0.05).

The 289-bp con仕01region in the mtDNA

analysis among 42 individuals from the western

J apanese population marked 42 polymorphic

Table 3. Distance method， pairwise fixation index (Fsr) ofAcanthotagrus la伽 inwesternJapan using mtDNA (above diag-onal) and microsatel1ite DNA (below diagonal)

Population Hiroshima Miyazaki Aichi Kochi Shizuoka Tokushima Mie

Hiroshima 0.1608 0.2000 -0.0464 -0.0710 0.0162 -0.0605 Miyazaki ー0.0008 0.0845 0.1094 0.0328 0.0109 0.1840 Aichi 0.0013 0.0002 0.0769 0.0693 -0.0161 0.1236 Kochi -0.0016 0.0007 0.0025 ー0.1116 ー0.0920 ー0.1336Shizuoka ー0.0026 0.0016 ー0.0003 -0.0004 -0.0871 -0.0804 Tokushima -0.0011 ー0.0028 -0.0013 0.0016 0.0011 -0.0323 Mie -0.0012 0.0014 0.0015 0.0011 -0.0008 0.0004

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22

Table 5. Haplotype frequencies and nucleotide diversities (μ) (and standard deviation) of出emtDNA ∞ntrol region in Acant，加ttagruslatus from seven populations (n = 42)

Haplotype ID HIR MIY AIC KOC Total

Ju

ρ-u cu a

hu e

u

eo VA e

LU m

u

n

n

o

--53

CAM e

c

c

a

bh

n

a

B

n

e

G

2mw一間

3

2一世

111600一ω

OAO-一d，‘、一n一a

一弘

、，r一AU

円

anU一U

3

2一式

1111---pooo--

nvQV7u

〆e

・、--FL

Et

一n

一0

8η一姐

-11--F----6位旧一個

ハ札口以一

b

〆t

、一AU一e

-

V

A

一e一ιEL

l-

88一i-

-11---------6ω旧一時

。uηU十一

e

dE

、-VA

一e

一w

、，，-AU

Qυ

ワ臼一ゆ

4141-cf

-11-----------6AMO一日

nvnu一Qd

(一叩

、‘，ノ一『，t

6

0一アl

lh'141i-tJ

-Il-------

同

-----POOO一目

0.ω一蜘臥

一ち

ω

、J一臼円山戸

197b

EIl-m--m--

同------目

6mm一一山川

nvnv一n

e

r--、一

n-m

一

日

u

F三

一

i:

-

6

3

7

F

一郎均

一

ct

一心

f

一9unu

一k叩

012345678901234567

一Mf

1ムワ白

qaAせFDnb7RUQU1it--ょ1ょti--1A1ム1i1ょっ臼ワ白ワ臼ワ白ワ白ワ臼

9uワ

山

一

以

陀

QQQQQQQQQQQQQQQQQQQQQQQQQQQ}一副仕

ssssssssssssssssssssssssssSIE--

非

非

井

非

井

井

非

非

非

井

非

#

井

非

非

#

井

非

#

非

非

井

#

井

井

井

弁

n

，rS一Gω

GenBank accession numbers

EF506769/AB916774 EF506783/AB916775 AB916776 EF506815/ AB916777 EF506838/ AB916778 EF506840/ AB916779 AB916780 AB916781 EF506830/ AB916782 EF506833/ AB916783 AB916784 AB916785 AB916786 AB916787 AB916788 AB916789 AB916790 AB916791 AB916792 AB91679③ AB916794 AB916795 AB916796 AB916797 AB916798 AB916799 AB916800

643222221111111111111111111必

MIE

1

1

1

TOKU

1

1

SHI

2

1

1

1

1

1

1

-121

1 1

2

2

1 1

Genetic s仕uctureof yel10wfin black seabream 23

Fig. 2. Haplotype median-joining network of Acanthopagrus latus from Japan and China. Black circles and black area within a circle indicate the J apan population， while open circles and white area within a circle indicate the China population. Circle sizes represent the haplotype frequencド

sites， and transversions only occurred at 90

bpぐrable4).百lemtD NA variation among 42

individuals revealed 27 haplotypes， with eight

haplotypes being shared田nong23 individu-

alsぐrable5). Nucleotide diversity ranged from

0.016 (M町)to 0.032 (MIE) ， while haplotype

diversities were recorded as 1.00 for all popula-

tions.

Medianてjoiningnetworks of 27 haplotypes

reveal a high degree of complexity. There are

two separated haplotype network groups found

町nongwestern J apan individuals， where one

group shared a haplotype with northern China

and another group shared a haplotype with

sou血ernChina σig.2).官lem司orityof genetic

variance occurred within populations， while

the variance among western J apan populations

(品tIOVA;F.訂=0.0160， P= 0.31) was virtually insignifi.cant.τ'he pairwise F';IT using mtDNA sequences revealed no signifi.cant difference for

all population combinationsぐrable3).

Of the 27 haplotypes in the western J apan

population， 20 haplotypes were unique to the

Fig. 3. Haplo勿pephylogenetic仕eeby the unweighted pair-group method wi血 arithmeticmean and Tamura-Nei distance method.τne Japan population is represented by open rectangles and sequence numbers SQl to SQ27 (accession numbers AB916774-AB916800)， while the China population is represented by closed triangles and sequence numbers SQ765ωSQ875 (accession numbers

EF50676ιEF506875).

J apanese population， while seven haplotypes

were shared wi白 previoushaplotypes deposited

in GenBank from a Chinese population (Xia et

al. 2008). Haplotype EF506769 from the Chinese

population of A. l，αtus was found in 14.3% of indi-

viduals in western Japanσable 5). InterestinglぁUPGMA (Fig. 3) using the Tamura-Nei distance

revealed由atthe phylogenetic pa仕ernconsisted

of a mixture of haplotypes from both J apan and

China (GenBank accession numbers EF506765-

EF506875).

Discussion

The NA per locus across all populations in

this study (22 to 47 loci) was higher than that

reported in other species of Sparidae (Perez-

Enriquez and Taniguchi 1999; Perez-Enriquez

et al. 2001; Jeong et al. 2003， 2007; Blanco

Gonzalez and Umino 2009). In red seabream，

Pagrus mαrjor， microsatellite surveys revealed

between four and 41 alleles at three loci among

eight populations off J apan and出eSouthwest

24 KASy招 ni，S. Tomano， K Ueno， K Ohara and T. Umino

Paci:fic (perez.主nriquezand Taniguchi 1999)，

whi1e the N A per locus ranged from 15 to 32 alleles at three loci in a western Japanese pop-ulation (perez-Enriquez et al. 2001). Moreover，

the NA in western Japan and South Korean pop-

ulations of black sea bream A. schlegelii ranged between six and 21 at four loci (J eong et al.

2003). Simi1arly， seven to 18 alleles were仕acedat seven loci for parental assignment using 51

broodstock of A. schlegelii (J eong et al. 2007). According to Blanco Gonzalez and Umino

(2009)， the NA per locus ranged from 7 to 24 at six loci in A. schlegelii由atwere released into Hiroshima Bay. Furthermore， the range of HE for two commonly studied Sparidae in J apan， P. major and A. schlegelii (perez-Enriquez and Taniguchi 1999; Jeong et al. 2003) was 0.69-0.92

and 0.77-0.83， respectively， which was consis-

tent with the range of values in our study (HE

range: 0.911-0.920). DeWoody and Avise (2000)

noted that the mean H-r. for microsatellite mark-E ers in 12 0仕lermarine species was 0.77. Taken

toge血er，these observations suggest that the

microsatellite markers for A. latus in this study had a sufficient power of resolution.

百lreeof 70 (4.3%) single loci departed from

HWE， suggesting that there was only a slight

heterozygote deficiency. Simi1ar deviations from HWE at microsatellite loci have been reported

in marine fishes such as P. major (Perez-Enriquez and Taniguchi 1999)， A. schlegelii (T eong et al. 2003)， black rockfish Sebastes inermis (An et al. 2011)， Paci:fic cod Gadus macrocePhalus (Kim et al. 2010)， and gi1thead seabream Sparusαurata (De lnnocentiis et al.

200の.Furthermore， the Frs among A. latus in several populations in western J apan revealed

evidence for the occurrence of weak， but

signi:ficant， inbreeding. This phenomenon also

indicates the excess of homozygosity in some

populations.τ'he global F'5I' value suggests由at

there is no signi:ficant population differentiation

in western J apan， meaning the genetic struc-

ture of A. latus can be characterized as a single panmictic group.

The lack of signi:ficant genetic differentiation

between the wi1d populations of A. 1，αtus in our study could be due to high rates of gene宜ow

during their pelagic phase. This is consistent

with the life cycle of A. latus， which includes a relatively long pelagic larval and early juveni1e stage that would allow mixing (Chang et al.

2002). Furthermore， this species spawns eggs

that float企eely，a廿aitthat facilitates larg令 scale

larval dispersal， resulting in high levels of gene

flow among population， particularly those由at

are geographically close. J ean et al. (2000) con戸

cluded that A. latus individuals surrounding Taiwan were represented by a single population

wi血 alow degree of geographic divergence，

and they suggested that there was considerable

gene flow due to the surrounding currents.τ'he

northeasterly winter monsoon in September

drives the Paci:fic Ocean surface waters south-

ward and is responsible for the dispersal of A.

latus larvae and juveni1es. Moreover， both the Kuroshio and Kuroshio countercurrents in血e

coastal Paci:fic Ocean also possibly facilitate the

dispersion of pelagic eggs and larvae of fish

such as A. latus. Jeong et al. (2003) concluded that the lack of genetic divergence between A.

schlegelii populations in western J apan was due to the random dispersal of pelagic eggs and

larvae or the extensive migrations of adult fish.

Taken together， these observations suggest

that there is a high level of gene flow between

A.lαtus wild populations in western J apan. Some members of the Sparidae family undergo

extensive migrations in a limited area during

their life cycle (T ean et al. 2000; J eong et al.

2003). Hindell et al. (2008) documented the

movement of A. butcheri among major estua-rine rivers， with some individuals moving up to

30 kilometer per day (km/ day) (annual mean:

8.7 km/day). ConverselぁKerwathet al. (2007)

concluded白atChrysoblephus laticeps individuals were confined to a small home range of

between 1，000 and 3，000 m2• Simi1arly， Diplodus sargus tended to remain within a relatively small home range (Abecasis et al. 2013). Together，

based on the behavior of Sparidae fish， we sug-

gest that A. latus adults migrate extensively but only within a short distance around their home

range.τ'hus， gene flow is most likely to occur

because of random dispersal of pelagic eggs

and larvae.

Genetic s仕uc加reof yellowfin black seabream 25

According to Grant and Bowen (1998)，

species with high nuc1eotide and haplotype diversities can be categorized into one group，

suggesting that the specimens have been

impacted by secondary contact with the previ-

ously differentiated lineage or have a long evo・

lutionary history of a stable population. Thus，

results in this study showed that haplotypes

企omtwo groups wi白inthe J apanese popula-

tions， based on a median-joining network and

nuc1eotide and haplotype diversities， are not affected by the populations from which the indi-

viduals were sampled， possibly due to second-

訂 ycontact between populations. In spite of出e

presence of two groups based on a median-

joining network， the genetic s甘ucturerevealed a single population wi由inthe J apanese popula-

tions. In con廿ast，the genetic structure of A.

latus from the Chinese coast is characterized by two genetically differentiated geographic units，

a northern and southern population， based on

analysis of mtD NA markers αia et aL 2008).

Xia et aL (2008) also noted that the distance sep同

arating the two genetic groups was >1，000 km.

τbe J apanese population shared a higher pro-

portion of haplotypes with the northern China

population than the southern China population，

which is suggestive of gene flow between J apan

and China. Shared haplotypes between J apan

and China are likely due to historical， periodic，

and long-distance dispersal between A. latus populations. Fig.3 shows that the haplotype

mixed consisting of individuals from J apan

and China， with low bootstrap support (く50%).

Given this， we speculate that A. latus from both countries experienced a long evolutionary his-

tory in large stable populations.

In J apan， A. latus is a relatively uncommon Sparidae species relative to A. schlegelii and P. m句ior.Therefore， information from this study can be used to identify the genetic structure of

A. latus in J apan and provide insight into the unit at which the population can be managed

and conserved. Based on microsatellite and

mtDNA analysis， the conservation of A. 1αtus can be managed in a single management unit. In conc1usion， the microsatellite loci used

in this study revealed high levels of genetic

V訂iabilityofA. latus in western Japan. Moreover， the microsatellite and mtDNA markers revealed

no significant genetic structure in the A. latus samples in western J apan， despite samples

being collected from fish with different sea

basin origins.

Acknowledgmen白

We thank Mrs. Tomoe Hikosaka for her help

in sequencing with the ABI 3130xl Sequencer

at the Gene Science Division， Natural Science

Centre for Basic Research and Development， Hiroshima University. We also thank Mr.

Masahiro Maekawa (Mie) ， Mr. Makoto

Watanabe (Miyazaki)， Dr. Satoshi Tasumi (Sh包uoka)，世lelate Mr. Masayuki Takeyama

(Kochi) ， Dr. Yukio Uetaぐrokushima)，and Mr.

Mitsuo Kawane (Aichi) for assistance collecting

samples.

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73， 1979-1992.

DNAマーカーに基づく西日本キチヌの集団構造解析

Kamarudin AHMAD SYAZNI・宮野哲史・上野香菜子・大原健一・海野徹也

キチヌ Acanthopagruslatusは日本の太平洋沿岸に生息し，重要な漁業資源となっている。本研究

では高感度マイクロサテライト DNAマーカー 10座 (n=312)および mtDNA非コード領域の部分配

列 (n=42)を用いて，西日本7集団の遺伝的多様性と集団構造の推定を行った。その結果， MSマー

カーでは有効アリル数22~47，平均ヘテロ接合度の観測値0.840~0.904 とすべての集団で高い遺伝的

変異を示した。同様に mtDNAも高い多様性を示した。 7集団を単集団と仮定した AMOVA解析では，

遺伝的分化指数 (GlobalFST) はMSマーカーで一0.00024(P> 0.05)， mtDNAで0.016(P > 0.05) と

なり，集団間での有意な遺伝的分化は認められなかった。本種の河口周辺への依存性は高いが，発育

初期の浮遊期に起因する遺伝子流動によって集団間の分化が妨げられていると考えられた。