Bull. Natl. Mus. Nat. Sci., Ser. B, 38(1), pp. 29–36, February 22, 2012

Genetic Diversity and Gene Flow of East Asian Seagrass,
Zostera caespitosa Miki (Zosteraceae), in Northeast Japan
Norio Tanaka1,*, Masahiro Nakaoka2, Yuji Omori3 and Keiko Aioi4
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
2
Hokkaido University, Akkeshi Marine Station, Field Science Center for Northern Biosphere,
Hokkaido University, Aikappu, Akkeshi, Hokkaido 088–1113, Japan
3
Yokosuka City Museum, Fukadadai 95, Yokosuka, Kanagawa 238–0016, Japan
4
Wetlands International Japan, Nihonbashi-Ningyocho 3–7–3, Chuo-ku, Tokyo 103–0013, Japan* E-mail: ntanaka@kahaku.go.jp(Received 15 November 2011; accepted 28 December 2011)

Abstract The genetic diversity and gene ﬂow of Zostera caespitosa Miki (Zosteraceae) in northeast Japan were evaluated. Twelve microsatellite markers designed for Z. marina were surveyed
for their applicability to Z. caespitosa. For four of these loci, 167 individuals from nine sites were
genotyped. Although a neighbor-joining tree based on FST values among Z. caespitosa sites
grouped them into four geographical areas: Otsuchi Bay, Yamada Bay, Mutsu Bay, and Notoro
Lake, analysis of molecular variance (AMOVA) did not reveal signiﬁcant genetic differentiation
among the four areas. No signiﬁcant correlation between genetic and geographical distances was
detected within either Otsuchi Bay or Yamada Bay. It was concluded that strong gene ﬂow occurs
in Z. caespitosa, at least in nearly closed environments such as bays.
Kew words : gene ﬂow, genetic diversity, microsatellite marker, seagrass, Zostera caespitosa.

Introduction
The genus Zostera (Zosteraceae), consisting of
12 species, is representative of seagrasses
adapted to brackish to salt water (den Hartog,
1970; Tanaka et al., 2003). The genus is distributed from the intertidal to subtidal zone, mainly
in subarctic to subtropical zones of both hemispheres. Most species show local distributions,
except for eelgrass Z. marina, which is widespread in the temperate to subarctic coastal areasof the northern hemisphere.
Because Z. marina beds are considered one of
the most important components of coastal ecosystems (Kikuchi and Pérès, 1980; Jernakoff etal., 1996; Duarte and Chiscano, 1999; Hemminga
and Duarte, 2000; Williams and Heck, 2001), thegenetic structure and diversity of Z. marina havebeen well studied in various regions by several

approaches. The development of microsatellite
markers has improved the understanding of thegenetic structure (Reusch, 2001; Olsen et al.,2004). Intensive research in both Europe andNorth America has revealed that the genetic
clonal structure of a single bed and genetic similarity among local populations vary greatly fromsite to site (Reusch et al., 1999a). The study
found that the size of Z. marina beds and their
genetic clonal structure were not always related.
Genetic similarity among Z. marina populations
decreases monotonically with geographic distance in some regions, such as the western Baltic
and the Gulf of California, whereas isolation by
distance (IBD) was not obvious for populations
in southern Europe and the Paciﬁc coast of Baja
California (Olsen et al., 2004; Muñiz-Salazar etal., 2005).
While studies of Z. marina are abundant, those30

Norio Tanaka et al.

of other Zostera species are scarce. The genetic
structure of Z. noltii, a European species, wascharacterized with microsatellite markers (Coyer
et al., 2004), while the genetic structures of Z.
muelleri in New Zealand (Jones et al., 2008) andZ. caespitosa in Japan (Tanaka et al., 2002) werecharacterized with random ampliﬁed polymorphic DNA (RAPD) markers.
Four Zostera species are endemic to the ocean
surrounding the Japanese archipelago. Of these
species, Z. caespitosa Miki is distributed innorthern Japan, Korea, and northeast China(Miki, 1933; Tanaka et al., 2009). The rhizomes
sub-erect and do not creep; their growth habit iscaespitose, which is rare character in seagrasses.
Because it is probable that individuals cannot
propagate vegetatively such as other Zostera spe-cies, comparison of the genetic structure of Z.
caespitosa with that of other Zostera species will
have important implications for the population
genetics of seagrasses.
In this study, we surveyed the applicability of
microsatellite markers described for Z. marina(Reusch et al., 1999b; Reusch, 2000) to Z. caespitosa. Using these markers, we performed population genetic analysis of Z. caespitosa in northeast
Japan.
Materials and Methods
Three Zostera caespitosa sites in the Otsuchi
Bay and three in the Yamada Bay, two in the
Mutsu Bay and one in the Notoro Lake were
sampled between 1999 and 2002 (Fig. 1 andFig. 1 Collection sites of Zostera caespitosa in this study. Closed circles indicate the positions of Z. caespitosa sites.
Related information of each site is described in Table 1.

Genetic Diversity of Zostera caespitosa in Northeast Japan
Table 1.

31

Zostera caespitosa sites used in this study. The positions of collection sites are indicated in Fig. 1

Collection sites
Otsuchi Bay
Akahama
Hakosaki
Murohama
Yamada Bay
Kumagasaki
Namiitasaki
OhshimaMutsu Bay
AsamushiNoheji
Notoro Lake
Notoro Lake

Locality

LatitudeLongitude

Akahama, Otsuchi, Iwate Pref.
Hakosaki, Kamaishi, Iwate Pref.
Murohama, Kamaishi, Iwate Pref.

N39°21′02″
N39°19′54″
N39°20′25″

E141°55′56″
E141°55′16″
E141°54′49″

Kumagasaki, Yamada, Iwate Pref.
Namiitasaki, Yamada, Iwate Pref.
Ohshima, Yamada, Iwate Pref.

N39°26′57″
N39°26′57″
N39°27′46″

E141°58′57″
E141°58′08″
E141°58′21″

Asamushi, Aomori, Aomori Pref.
Noheji, Aomori Pref.

N40°53′58″
N40°52′25″

E140°51′20″
E141°07′46″

Notoro Lake, Abashiri, Hokkaido Pref.

N44°00′59″

E144°07′31″

Table 1). From each site, 8 to 24 plants of Z.
caespitosa were randomly selected and theirleaves were collected. Collected shoots wererinsed with fresh water to remove epiphytic
algae, and pieces of leaves were cut and frozen at−80°C within 24 h of collection.
Genomic DNA was extracted from approximately 0.02–0.04 g dry weight of leaf tissue
using the CTAB (hexadecyltrimethyl ammonium
bromide) method of Doyle and Doyle (1987).
A preliminary test using polymerase chain
reaction (PCR) ampliﬁcation was conducted for
12 microsatellite loci (Zosmar CT-3, Zosmar
GA-1, Zosmar GA-2, Zosmar GA-3, Zosmar
GA-4, Zosmar GA5-5, and Zosmar GA-6;
Reusch et al., 1999b: Zosmar CT-12, Zosmar
CT-19, Zosmar CT-35, Zosmar CT-20, and Zosmar CT-17H; Reusch, 2000).
PCR was carried out in a volume of 6.0 μL
containing 3 ng of template DNA, 1.2 pmole ofeach primer, 0.2 mM of each dNTP, 1x PCR buffer, and 0.3 units of ExTaq polymerase (Takara
Bio Inc., Shiga, Japan). PCR was performed in a
GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The PCR program
employed consisted of a 5 min denaturing step at
95°C, followed by 30 cycles at the following
times and temperatures: 30 s at 95°C, 1 min at
55°C, and 1 min at 72°C.
Size separation of PCR products was carriedout using capillary electrophoresis on an ABIPRISM 3100 genetic analyzer (Applied Biosys-tems). Size sorting of banding patterns and genotyping was performed in a semi-automated way
using the software program GeneMapper version
3.5 (Applied Biosystems).
Genetic diversity of each site was measured
using clonal diversity, which was expressed as afunction of the number of ramets and genets
sampled (Olsen et al., 2004), the number ofalleles, allelic richness (Pettitt et al., 1998), average heterozygosity (Nei, 1987), and level ofinbreeding (FIS; Weir and Cockerham, 1984).
Deviation from the Hardy–Weinberg equilibrium(HWE) was tested using the Markov chain algorithm developed by Guo and Thompson (1992).
These calculations were performed using GENEPOP on the Web 3.4 (Raymond and Rousset,
1995) and FSTAT (Goudet, 1995) only for allelicrichness.
Genetic differentiation and distances were
quantiﬁed by calculating the proportion ofgenetic variance (FST) (Weir and Cockerham,
1984) using Arlequin 3.11 (Excofﬁer et al.,2005). A neighbor-joining (Saitou and Nei, 1987)
tree based on FST values was constructed using
PAUP* 4.0 (Swofford, 2001). To calculate variance components among bays and lake (Otsuchi
Bay, Yamada Bay, Mutsu Bay and Notoro Lake),among sites, and among individuals, an analysis
of molecular variance (AMOVA; Excofﬁer et al.,1992) was performed. Isolation by distance(IBD) was detected using the Mantel test
(Mantel, 1967) between two matrices: FST andNorio Tanaka et al.

32

geographical distance in kilometers between
pairs of populations. Geographical distances
between populations were manually measured
from a chart as either direct distance (when there
was no land) or coastal distance (when land was
located in a direct line connecting the two pointsof collection). The Mantel test was performed
using the ISOLDE program of GENEPOP on the
Web (Rousset, 1997).
Results
Of the 12 microsatellite markers tested for
PCR ampliﬁcation, four markers (Zosmar CT-3,
Zosmar GA-1, Zosmar GA-2, and Zosmar GA-6)
showed stable ampliﬁcation and polymorphism.
These four loci were selected for subsequent
analyses.
Clonal diversity varied from 0.48 (NotoroLake) to 1.00 (Noheji). The mean number ofalleles per locus and site ranged from 8 (AkaTable 2.

hama) to 17 (Asamushi; Table 2). Allelic richness ranged from 1.77 (Akahama) to 2.46
(Ohshima). Private alleles were observed in sites
from Ohshima, Asamushi, and Noheji. Averageobserved heterozygosity for all sites was between
0.375 (Kumagasaki) and 0.600 (Noheji), andaverage expected heterozygosity (HE) was
between 0.343 (Kumagasaki) and 0.544 (Noheji;
Table 2). All sites were in Hardy–Weinberg equilibrium (HWE). Although FST value varied from0.002 to 0.258, signiﬁcant genetic differentiation
was detected among all sites, except between
Akahama and Murohama, Namiitasaki and
Ohshima, and Asamushi and Noheji (P＞0.05;
Table 3). Analysis of the genetic relationshipamong the nine sites using a neighbor-joining(NJ) tree revealed that the sites fell into fourgroups: three in Otsuchi Bay, three in Yamada
Bay, two in Mutsu Bay, and the Notoro Lake site
(Fig. 2). AMOVA revealed signiﬁcant genetic
variation at two spatial levels: among sites within

Genetic diversity and clonality of Zostera caespitosa in this study

Site

n

G

C

A

Ar

Pa

HO

HE

FIS

Akahama
Hakosaki
Murohama
Kumagasaki
Namiitasaki
Ohshima
AsamushiNoheji
Notoro Lake
Average

8
19
21
24
17
21
24
10
23
18.6

7
13
12
20
16
18
20
10
11
14.1

0.88
0.68
0.57
0.83
0.94
0.86
0.83
1.00
0.48
0.79

8
11
10
15
16
16
17
14
10
13.0

1.77
1.98
1.74
2.03
2.30
2.46
2.25
2.33
1.82
2.08

0
0
0
0
0
1
2
1
0
0.4

0.500
0.500
0.429
0.375
0.456
0.571
0.417
0.600
0.435
0.476

0.453
0.411
0.379
0.343
0.436
0.501
0.392
0.544
0.420
0.431

−0.113−0.225−0.136−0.096−0.048−0.144−0.064−0.11−0.037−0.108n, number of ramets; G, number of genotypes; C, clonal diversity; A, number of alleles; Ar, allelic richness; Pa,number of private alleles; HO, observed heterozygosity; HE, expected heterozygosity; FIS, inbreeding coefﬁcient.
Table 3.
Hakosaki
Murohama
Kumagasaki
Namiitasaki
Ohshima
AsamushiNoheji
NotoroLake

Pairwise Fst-values (Weir and Cockerham, 1984) among nine Zostera caespitosa sites in this study
Akahama

Hakosaki

Murohama

Kumagasaki

Namiitasaki

Ohshima

AsamushiNoheji

0.128
0.029*
0.066
0.112
0.064
0.059
0.077
0.153

0.109
0.223
0.173
0.120
0.149
0.139
0.258

0.183
0.222
0.138
0.084
0.108
0.216

0.021
0.038
0.051
0.065
0.059

0.020*
0.090
0.079
0.128

0.034
0.053
0.092

0.002*
0.042

0.078

* Genetic differentiation was not detected (P＞0.05).

Genetic Diversity of Zostera caespitosa in Northeast Japan

Fig. 2 Neighbor-joining (NJ) tree based on FST values(Weir and Cockerham, 1984) among Zostera caespitosa sites in this study.

Table 4. Analysis
ofmolecular
variance(AMOVA) for Zostera caespitosa sites
Source ofvariation
Among bays
and lake
Sites/bays
and lake
Individuals/
sites

d. f.

Variancecomponent%Totalvariance

P-value

3

0.065

7.54

P＝0.002

5

0.039

4.47

＜0.001

325

0.759

87.99

＜0.001each bay and lake and among individuals withineach site. Some genetic variation between bays
and lake was detected (P＝0.002; Table 4). Signiﬁcant correlation between genetic and geographical distances was not detected among allsites (Mantel test, P＞0.05), whereas signiﬁcantcorrelation was detected among the six sites in
Otsuchi Bay and Yamada Bay (P＜0.01; Fig. 3).
Discussion
Genetic diversity (HE) of Z. marina is reported

33

to range from 0.15 to 0.69 in North America andEurope (Olsen et al., 2004), 0.491 to 0.563 (average＝0.539) in San Quintin Bay, Mexico(Muñiz-Salazar et al., 2006), 0.312 to 0.541 in
the Southern California Bight, Mexico (Coyer etal., 2008), 0.464 to 0.708 (average＝0.620) in
Tokyo Bay, Japan (Tanaka et al., 2011), and
0.364 to 0.690 (average＝0.567) in Sagami Bay,
Japan (Tanaka and Lim, 2011). Coyer et al.
(2004) showed the diversity of Z. noltii to lie
between 0.297 and 0.667. The genetic diversityof Z. caespitosa in this study (0.343 to 0.544) fellwithin these ranges (Table 2). However, the average value for Z. caespitosa is 0.431, which islower than that of Z. marina reported in the previous studies. Because the rhizomes of Z. caespitosa sub-erect and do not creep unlike other
Zostera species, we believe that this species
increases mainly by sexual propagation. Accordingly, we predicted a higher genetic diversity inthe experimental populations than in other Zostera species. However, the genetic diversity inthis study was low. Genetic variation of Z. caespitosa based on RAPD analysis was also characteristic of inbreeding plant species (Tanaka et al.,2002). Although we observed that Z. caespitosais protogynous similar to Z. marina (De Cock,1980), it may be geitonogamous, given that anindividual generates several ﬂowering shoots
simultaneously. However, because the clonal
diversity was lower than that of Z. marina inTokyo Bay (0.9) and Sagami Bay (0.8), and allsites in this study were in HWE, the low genetic
diversity could result from vegetative reproduction rather than from geitonogamy. Further studies are needed to analyze other sites and regions
using more loci.
Genetic differentiation was detected among
most sites, though not between Akahama andMurohama, Namiitasaki and Ohshima, and Asamushi and Noheji (Table 3). In a NJ tree basedon FST values, all sites were grouped into fourclusters, which were consistent with the geographical distribution (Fig. 2), although AMOVA
did not show signiﬁcant genetic differentiation
among bays and lake (Table 4). By RAPD analy-

Norio Tanaka et al.

34

Fig. 3. Isolation-by-distance (IBD) used Mantel tests for Zostera caespitosa sites in this study based on FST as genetic
distance. a) Six sites within the Otsuchi Bay and Yamada Bay: b) All Z. caespitosa sites. Pairwise genetic differentiation between populations were linearized and plotted against the geographic distance.

sis, in contrast, sites of Otsuchi Bay were notgrouped into a single cluster (Tanaka et al.,2002). Lougheed et al. (2000) asserted that microsatellites were superior to RAPD markers fordiscerning ﬁne-scale genetic differentiation
between subpopulations separated by tens ofkilometers. Their assertion favors acceptance ofthe genetic relationships among sites of Otsuchi
Bay and Yamada Bay observed in the present
study.
Seeds of Z. caespitosa are probably dispersedby means of detached generative shoots that drift
on the surface of water similar to those of Z.
marina. Often no IBD is observed when strong

gene ﬂow occurs among populations within aslightly closed region such as a gulf or a bay(Muñiz-Salazar et al., 2005, 2006; Tanaka et al.,2011). This could be due to nearly saturated geneﬂow in a closed environment. IBD was detectedamong the six populations of Otsuchi Bay and
Yamada Bay (Fig. 3b), but no IBD was detectedamong the sites within each bay (P＞0.05; result
not shown). There was also no genetic differentiation between the two sites of Mutsu Bay. We
conclude that strong gene ﬂow occurs in Z. caespitosa, at least in nearly closed environments
such as bays. IBD was also not detected among
all sites (Fig. 3a). Besides the present study sites,

Genetic Diversity of Zostera caespitosa in Northeast JapanZ. caespitosa sites occur in many other regionsof Japan (Tanaka et al., 2009). To evaluate largescale genetic structure such as that among bays
and regions, it will be necessary to sample manysites in other regions using not only microsatellite analysis but also haplotype analysis based on
DNA sequence.
The present situation of Z. caespitosa sites in
Otsuchi Bay and Yamada Bay is unknown after
the tsunami of March 11, 2011. The results of ourstudy may be useful for evaluating the inﬂuenceof the tsunami on the genetic structure of the species, owing to its disappearance or decline andsubsequent regeneration by growth and/or reintroduction.
Acknowledgments
The authors thank Koichi Morita, Jun Michimata, Seiichi Tamura, Naoko Kouchi, Masako
Watanabe for help with collecting plant materials, and Chikako Ishii for analysis of the data.
The authors are thankful for grants to N. T. (No.
12740476 and 21710248) and M. N. (21241055)
from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan, and to N. T.
from Fujiwara Natural History Fundation.
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