Bull. Natl. Mus. Nat. Sci., Ser. B, 37(1), pp. 23–30, February 22, 2011

Genetic Structure and Diversity of the Eelgrass Zostera marina L.
(Zosteraceae) in Sagami Bay, Japan
Norio Tanaka1,* and Boon Keng Lim2
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki, 305–0005 Japan
2
Kajima Technical Research Institute, Kajima Corporation,
Isshiki 2400, Hayama-cho, Miura-gun, Kanagawa, 240–0111 Japan* E-mail: ntanaka@kahaku.go.jp(Received 16 November 2010; accepted 22 December 2010)

Abstract Over the past few decades, seagrass beds are declining worldwide. The eelgrass species
Zostera marina L. is the main component of seagrass beds and has been declining in some areas ofJapan, including Sagami Bay. This study examined the genetic structure and diversity in populations of this eelgrass in the Sagami Bay to establish guidelines for its restoration. Genotypes of 260
individuals from nine eelgrass beds were determined using five microsatellite markers. Genetic differentiation was observed among all eelgrass beds. A neighbor-joining tree based on FST values
among eelgrass beds revealed that they were grouped in two areas: the Miura Peninsula and the Izu
Peninsula. Significant positive correlation between genetic and geographical distances was detected among all beds as well as among beds of the Miura Peninsula. We suggest that restoring beds bytransplanting seeds and/or plants of eelgrass to other sites should be avoided; however, if eelgrassbeds have disappeared or severely declined, neighboring beds, except those of the Tenjin Island
and Nabeta, should be used as donors. The beds of the Tenjin Island and Nabeta are not suitable
donors since they lack genetic diversity.
Kew words : eelgrass, gene flow, genetic diversity, microsatellite marker, restoration, Sagami Bay,seagrass, Zostera marina.

Introduction
The eelgrass Zostera marina L. is an angiosperm that inhabits soft-bottom marine habitats ranging from the intertidal zone to depths ofapproximately 10 m in temperate latitudes (denHartog, 1970). It is widely distributed in thenorthern Pacific and northern Atlantic Oceans,and occurs from Kyusyu to Hokkaido in Japan.
Because of their high primary productivity andprovision of habitats for various associatedfauna, eelgrass beds are regarded as one of themost important components of coastal ecosystems (Kikuchi and Pérès, 1980; Jernakoff et al.,
1996; Duarte and Chiscano, 1999; Hemminga
and Duarte, 2000; Williams and Heck, 2001).
However, they are becoming more scarce world-

wide due to direct and indirect effects of humanactivity (Fortes, 1988; Shorts and Wyllie-Echeverria, 1996; Duarte, 2002; Moore and Jarvis,
2008; Rueda et al., 2009; Waycott et al., 2009;
Martin et al., 2010). Seagrass beds have been restored at many sites worldwide (Zimmerman etal., 1995; van Katwijk, 2000; Orth et al., 2006);
however, some problems associated with artificialtransplantation have become apparent. One concern is that transplantation of eelgrass seeds
and/or plants from remote areas may violate thegenetic composition of regional populations. In
addition, eelgrass beds with high genetic diversity are ideally suited as donors for restoration by
reintroduction. It has been demonstrated that levels of genetic diversity in species determines the
success of reintroduction (Martins and Jain,

24

Norio Tanaka and Boon Keng Lim

1979; Calero et al., 1998). This is also true for
eelgrass, which demonstrates better survivorshipand reproductive success with higher genetic diversity (Williams, 2001; Hughes and Stachowicz,
2004; Ehlers et al., 2008). Therefore an assessment of the genetic diversity and gene flow ofwild eelgrass populations is required before projects for restoration are initiated.
The Sagami Bay is located between the Miuraand Izu Peninsulas. All eelgrass beds are confirmed to be present only in the eastern part ofthe Bay (Tanaka et al., 2006). Kudo (1999) reported that eelgrass beds in the Odawa Bay have
reduced within the last two decades due to thedecline in water transparency, with the largest
remaining in the Sagami Bay. A possibility thateelgrass beds will continue to decrease exists.
In this study, we performed population genetic
analysis of eelgrass beds in the Sagami Bay using
microsatellite marker, codominant molecularmarkers that are suitable for detecting genetic
structure and diversity within a population andamong populations (Reusch, 2001; Olsen et al.,2004). Based on this data, we discussed thegenetic structure of eelgrass in the Sagami Bay

and suggested guidelines for its restoration.
Materials and Methods
Between 2003 and 2004, seven eelgrass beds
on the west side of the Miura Peninsula and twoon the east side of the Izu Peninsula were sampled (Fig. 1 and Table 1). From each bed, 27 to
35 shoots of eelgrass were randomly collected.
To avoid collection of the same genet, only one
shoot was collected within a diameter of 3 m. In
the Tenjin Island and Nabeta beds, shoots werecollected at 1 m intervals because the beds
were small and their distribution was fragmented.
Collected shoots were rinsed with fresh water toremove 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 eelgrass leaf
tissue using the CTAB (hexadecyltrimethyl ammonium bromide) method of Doyle and Doyle(1987). Crude extracts of total DNA were purified with the Plasmid Mini Kit (QIAGEN,
Hilden, Germany) and preserved at 20°C.

Fig. 1. Collection sites of eelgrass (Zostera marina) in this study. Closed circles indicate the positions of eelgrass beds. Related information of each site is described in Table 1.

TNS-9524163, 9524169
TNS-9524170-9524173
E138°5827
E138°5643
* All voucher specimens are deposited in National Museum of Nature and Science, Japan (TNS).

N34°4024
N34°3953
Sotoura Bay, Shimoda, Shizuoka Pref.
Nabeta Bay, Shimoda, Shizuoka Pref.
30
29

800
100

TNS-9524126-9524131
TNS-9524132-9524137
TNS-9524138-9524142
TNS-9524180-9524183
TNS-9524143-9524148
TNS-9524149-9524155
TNS-9524156-9524162
E139°3322
E139°3412
E139°3439
E139°3618
E139°3715
E139°3736
E139°3955
N35°1746
N35°1618
N35°1513
N35°1317
N35°1234
N35°3944
N35°0843
500
1600
2300
200
40000
3400
8700
Kotsubo, Zushi, Kanagawa Pref.
Shinnase harbor, Hayama, Kanagawa Pref.
Chojagasaki, Hayama, Kanagawa Pref.
Tenjin-island, Yokosuka, Kanagawa Pref.
Odawa Bay, Yokosuka, Kanagawa Pref.
Koajiro Bay, Miura, Kanagawa Pref.
Ena Bay, Miura, Kanagawa Pref.
30
29
29
35
27
29
28

Miura Peninsula
KotsuboShinnase
Chojagasaki
Tenjin Island
Odawa Bay
KoajiroEna BayIzu Peninsula
Sotoura
NabetaLongitude
LatitudeArea (m2)Locality
Number ofindividuals
Collection
sites

Table 1.

Sample collection data and habitat characteristics for eelgrass used in this study. The positions of collection sites are indicated in Fig. 1

Voucher*

Genetic Diversity of Zostera marina in Sagami Bay

25

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.,
1999: Zosmar CT-12, Zosmar CT-19, Zosmar
CT-35, Zosmar CT-20, and Zosmar CT-17H;
Reusch, 2000). Based on the degree of polymorphism and stability of amplification, five loci
(Zosmar CT-3, Zosmar GA-1, Zosmar GA-3,
Zosmar GA-4, and Zosmar CT-19) were selected
for the following analyses.
PCR was carried out in a volume of 6.0 m lcontaining 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 3100Avant genetic analyzer (Applied
Biosystems). 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 eelgrass bed wasmeasured using clonal diversity, which was expressed as a function of the number of rametsand genets sampled (Olsen et al., 2004), thenumber of alleles, allelic richness (Pettitt et al.,1998), and average heterozygosity (Nei, 1987).
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 by hand.
Genetic differentiation and distances were
quantified by calculating the proportion of genetic variance (FST) (Weir and Cockerham, 1984)
using Arlequin 2.001 (Schneider et al., 2001) and26

Norio Tanaka and Boon Keng Lim

GENEPOP on the Web 3.4. A neighbor-joining(Saitou and Nei, 1987) tree based on FST values
was constructed using PAUP* 4.0 (Swofford,2001). To calculate variance components between groups, among beds, 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 geographical distance in kilometers between
pairs of populations. Geographical distances between populations were manually measured froma chart as either direct distance (when there wasno land) or coastal distance (when land was located in a direct line connecting the two points ofcollection). The Mantel test was performed using
the ISOLDE program of GENEPOP on the Web(Rousset, 1997).
Table 2.

Results
Clonal diversity varied from 0.37 (Tenjin Island) to 1.00 (Odawa Bay). The mean number ofalleles per locus and bed ranged from 2.0 (Nabeta) to 6.4 (Odawa Bay; Table 2). Allelic richness
ranged from 1.29 to 1.69. Private alleles wereobserved in beds from Kotsubo, Chojagasaki,
Odawa Bay, Ena Bay, and Sotoura. Average observed heterozygosity for all beds was between
0.379 and 0.690 and average expected heterozygosity was between 0.467 and 0.690 and waslowest at 0.364 (Nabeta) (Table 2). All beds, except the Tenjin Island beds (P0.0089) were in
Hardy-Weinberg equilibrium (HWE). Although
FST value varied from 0.033 to 0.468, significant
genetic differentiation was detected among all
beds (P0.001; Table 3). Analysis of the genetic
relationship among the nine beds using the

Genetic diversity and clonality in eelgrass of the Sagami Bay

Site

n

G

C

A

Ar

Pa

HO

HE

HWE

KotsuboShinnase
Chojagasaki
Tenjin Island
Odawa Bay
KoajiroEna Bay
Sotoura
Nabetaaverage

30
29
29
35
27
29
28
30
29
29.6

23
25
25
13
27
25
25
28
16
23.0

0.77
0.86
0.86
0.37
1.00
0.86
0.89
0.93
0.55
0.8

5.6
4.8
6.0
3.6
6.4
3.0
5.2
6.2
2.0
4.8

1.64
1.61
1.69
1.58
1.64
1.52
1.47
1.59
1.29
1.6

1
0
2
0
1
0
2
1
0
0.8

0.653
0.634
0.648
0.669
0.607
0.552
0.464
0.627
0.379
0.581

0.639
0.611
0.690
0.585
0.635
0.518
0.467
0.594
0.364
0.567

ns
ns
ns
P0.0089
ns
ns
ns
ns
ns

n, 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; HWE, deviation from HardyWeinberg equilibrium (nsnot significant).
Table 3. Pairwise FST -values (Weir and Cockerham, 1984) among nine eelgrass (Zostera marina) beds in the
Sagami Bay

Shinnase
Chojagasaki
Tenjin Island
Odawa Bay
KoajiroEna Bay
Sotoura
NabetaKotsuboShinnase Chojagasaki

0.033
0.057
0.119
0.078
0.085
0.209
0.101
0.319

0.107
0.092
0.100
0.125
0.253
0.131
0.359

0.114
0.043
0.082
0.201
0.112
0.294

TenjinIsland

Odawa
Bay

0.083
0.188
0.328
0.145
0.379

0.132
0.244
0.066
0.223

Genetic differentiation was significantly detected among all beds (P0.05).

Koajiro

0.186
0.193
0.430

Ena
Bay

0.266
0.468

Sotoura0.167

Genetic Diversity of Zostera marina in Sagami Bay

neighbor-joining tree revealed that the eelgrassbeds were grouped into two clusters, seven in thewaters of the Miura Peninsula and two in thewaters of the Izu Peninsula (Fig. 2). AMOVA revealed significant genetic variation at two spatial
levels (among eelgrass beds within each geographical group and among individuals withineach bed), excluding the Miura Peninsula and the
Izu Peninsula among groups (P0.026; Table 4).
Significant positive correlation between genetic
and geographical distances was detected among
all beds as well as among beds of the Miura
Peninsula (Mantel test, P0.01; Fig. 3).

Fig. 2. Neighbor-joining (NJ) tree based on FST
values (Weir and Cockerham, 1984) among
eelgrass beds in the Sagami Bay.

27

Discussion
Genetic diversity of eelgrass beds
In previous studies of eelgrass, heterozygosity
ranged from 0.15 to 0.69 in North America andEurope (Olsen et al., 2004), 0.491 to 0.563 (average, 0.539) in the San Quintin Bay, Mexico(Muniz-Salazar et al., 2006), 0.312 to 0.541 in
the Southern California Bight, Mexico (Coyer etal., 2008), and 0.464 to 0.708 (average, 0.620) in
the Tokyo Bay (Tanaka et al., 2011). In comparison with these data, the genetic diversity of eelgrass in the Sagami Bay (heterozygosity:
0.364–0.690: Table 2) did not differ from that atother sites.
Procaccini et al. (2007) suggested the importance of considering both genetic (allelic) andgenotypic (clonal) diversity for a seagrassecosystem. The Odawa Bay had above average
allelic diversity (allelic richness and heterozygosity) and high clonal diversity despite the noticeable decline in the eelgrass beds in the 1980s and1990s (Kudo, 1999). Among the Miura Peninsula, above average allelic diversity and low clonal
diversity were detected in the Tenjin Island beds.
These results indicate that the Tenjin Island beds
may have been affected by the method by whichshoots were collected at intervals closer thanthose in other beds; however, the Tenjin Island
beds, consisting of several small patches, deviated from HWE. Several causes for deviation are
known (e.g., inbreeding, assortative mating, subdivision of a population, and selection) (Nei,
1987). Deviation in the Tenjin Island beds probably occurred because of subdivision of the population. A contrast was observed between the two
beds of the Izu Peninsula. The Nabeta beds
showed low genetic and clonal diversities compared to the other beds, while above average statistics were observed in the Sotoura beds, which
are located in the neighborhood of Nabeta. There
are few eelgrass beds within approximately 200
km west of Nabeta (Tanaka et al., 2009) and thestrong sea current, Kuroshio, flows south of the
Izu Peninsula from west to east. Although low
clonal diversity may be affected by the sampling28

Norio Tanaka and Boon Keng Lim
Table 4.

Analysis of molecular variance (AMOVA) for eelgrass (Zostera marina) beds in the Sagami Bay

Source of variationd.f.

Variance component%Total variance

P-value

Between groups
Eelgrass beds/groups
Individuals/eelgrass beds

1
7
266

0.197
0.238
1.440

10.76
12.97
78.52

0.026
<0.001<0.001

Fig. 3. Isolation-by-distance (IBD) used Mantel
tests for eelgrass populations in the Sagami
Bay based on FST as genetic distance. a) All
nine eelgrass beds: b) Seven beds within theMiura Peninsula (Kotsubo, Shinnase, Chojagasaki, Tenjin Island, Odawa Bay, Koajiro, Ena
Bay). Pairwise genetic differentiation between
populations were linearized and plotted against
the geographic distance.

method, low allelic diversity may be caused by
the small population size (area100 m2; Table 1)
and the geographical location. Consequently, theeelgrass beds, except for those of Tenjin Island
and Nabeta, fulfill the necessary conditions as
donor sites for restoration in terms of allelic andclonal diversities.

Genetic differentiation and gene flow among eelgrass beds
In this study, genetic differentiation was observed among all eelgrass beds (Table 3). The
eelgrass beds are grouped into two clusters: thosein the waters of the Miura Peninsula and those in
the water of the Izu Peninsula, although the genetic difference is not strong because AMOVA
analysis does not support a significant genetic
variation between the two geographical groups.
Isolation by distance (IBD) analysis showed significant correlation between genetic and geographic distances among all beds as well as
among beds of the Miura Peninsula. This result isin contrast to that obtained in the Tokyo Bay, located east and adjacent to the Sagami Bay. In the
inner part of the Tokyo Bay, correlation between
genetic and geographic distances was not detected among eelgrass beds (Tanaka et al., 2011). No
IBD was observed when strong gene flow
occurred among populations within a slightly
closed region such as a gulf or a bay (MunizSalazar et al., 2005, 2006). This could be due tothe nearly saturated gene flow that easily occurs
in a closed environment. The Sagami Bay is not
as closed as the Tokyo Bay. We believe that thesegeographical traits of the Sagami Bay caused thegenetic differences among the beds and thegenetic structure following IBD.
Implications for the restoration of eelgrass bedsin the Sagami Bay
In the Sagami Bay, all eelgrass beds are genetically different from each other; therefore, we believe that transplanting seeds and/or plants of eelgrass to other sites should be avoided. However,
if eelgrass beds have disappeared or severely declined, neighboring beds, except for those of Tenjin Island and Nabeta, should be used as donors.

Genetic Diversity of Zostera marina in Sagami Bay

The beds of Tenjin Island and Nabeta are notsuitable donors since they lack genetic diversity.
Acknowledgements
The authors thank Shimoda Floating Aquarium and fisheries cooperative associations of theMiura Peninsula for help with collecting plantmaterials, and Chikako Ishii, Shoh Nagata, Haruka Matsuyoshi for analysis of the data and drawing figures.
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