Plant Species Biology (2009) 24, 202–208

doi: 10.1111/j.1442-1984.2009.00255.x

Reproduction dynamics of eelgrass (Zostera marina L.)
populations in Ise–Mikawa Bay, central Honshu, Japan

psbi_255

202..208

TSUKASA YOSHIDA,*† HIDEAKI OKAMOTO,† YOSHITO TANAKA,* TAKASHI HIBINO,* TERUAKI SUZUKI‡ and
TERUHIRO TAKABE§*Graduate School of Environmental and Human Sciences and §Research Institute, Meijo University, 1-501 Siogamaguchi,
Tenpaku-ku, Nagoya 468-8502, Japan, †Chateau Marine Survey Company, 1-4-2, Katamachi, Miyakojima, Osaka 534-0025,
Japan and ‡Marine Resources Research Center, Aichi Fisheries Research Institute, 97 Wakamiya, Miya-cho, Gamagori 443-0021,
Japan

Abstract
Suitable methods for the conservation and reconstruction of eelgrass populations areneeded because of decreasing eelgrass populations. For this, it is important to understandthe reproduction mechanism of eelgrass populations at a molecular level as well as at a
macro level. In the present study, we analyzed the dynamic changes in eelgrass populations in Ise–Mikawa Bay by monitoring their distribution from 1993 to 2005 and by
genetic analysis using microsatellite polymorphism. The distribution of eelgrass populations in the eastern part of Ise Bay showed a periodic shift with a cycle of 4 years, thatis, eelgrass populations appeared on the bar trough, gradually moved to the inshore sideof the habitat every year, and disappeared in the fourth year. Microsatellite analysis
revealed that the eelgrass populations in Ise–Mikawa Bay have high genetic diversity.
The eelgrass populations in Ise Bay and those in Mikawa Bay showed genetic differentiation and genetic connectivity. The importance of upstream parent populations, seedtransportation among local populations and genetic diversity were discussed for thesustainability of eelgrass populations.
Keywords: conservation, eelgrass, genetic diversity, Ise–Mikawa Bay, microsatellite, Zostera
marina.
Received 15 April 2009; accepted 6 August 2009

Introduction
Eelgrass (Zostera marina L.) forms dense populations onshallow sandy and muddy bottoms in inner bay areas,and plays an important role from an ecological and ﬁsheries standpoint (Heck et al. 1995). Eelgrass populations
are decreasing signiﬁcantly throughout the world as aresult of human activities such as reclamation. Once eelgrass populations have been decreased by human activities, it is generally very difﬁcult to restore the populations
by plantation (Suzuki 2006). Eelgrass is a plant that has
two breeding systems, vegetative propagation and sexual
reproduction (Phillips & Menez 1988). It is unclear inmany cases which reproduction mode works in eachhabitat. Therefore, plantation of foreign eelgrass without
consideration of the genetic diversity could easily lead toCorrespondence: Teruhiro Takabe
Email: takabe@ccmfs.meijo-u.ac.jp

long-term disturbance of the ecosystem (Williams 2001;
Hughes & Stachowicz 2004).
Ise–Mikawa Bay is a semi-enclosed bay located on
Central Honshu Island, Japan (Fig. 1). The surface area of
Ise Bay is 1738 km2. Large rivers, such as the Kiso River,ﬂow into the head of the bay. Low salinity water with lightdensity tends to ﬂow in the surface from the bay head tothe bay mouth. The surface area of Mikawa Bay is 604 km2.
It is connected to Ise Bay by the Nakayama and Morozaki
channels, which are 9 km wide. Ise Bay water and oceanic
water ﬂow into Mikawa Bay from Morozaki channel andﬂow out through Nakayama channel (Suzuki et al. 2002).
Until 1950, eelgrass populations covered most of theshallow water (until DL [datum level] -5 m) in Ise–
Mikawa Bay. However, the distribution of the eelgrass
populations has decreased signiﬁcantly as a result ofdevelopment in the coastal area and water pollution. At
present, the main eelgrass population in Aichi prefecture
is restricted to the east coast area in Ise Bay and to the© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology

R E P R O D U C T I O N D Y N A M I C S O F E E L G R A S S P O P U L AT I O N S

203

Seagrass bed

Tarumi

100m

Multiple bar trough part Seagrass bed
Fig. 2 An aerial photograph of Kosugaya and Tarumi. The redlines show the positions at which the degree of vegetation coverof eelgrass was examined. The red circles show the positions at
which the sampling area was set up for microsatellite analysis.

Materials and methods

Measurement of the distribution of theeelgrass populations

Fig. 1 Map of the sites studied in the Ise–Mikawa Bay. The manmade island (shown by the open circle) is constructed to ofﬁng
3 km in 2000, and the area of the man-made island is 577 ha.

northern coast area in Mikawa Bay. Tarumi and Kosugaya
are typical foreshores of the Ise Bay east coast (Fig. 1).
Eelgrass populations at Tarumi are 1000 m wide. Kosugaya beach consists of a multiple bar-trough part and an
inshore ﬂat part. Eelgrass populations form a belt-shaped
distribution in the hollow of the multiple bar-trough partand an island-like colony in the inshore ﬂat part (Yoshida
et al. 2006). Isshiki in the northern coast area of Mikawa
Bay is located near the mouth of the Yahagi River. Eelgrass
populations at this beach are similar to those in Kosugaya.
Miya is a semi-enclosed sea area that is interrupted by abreakwater offshore. Eelgrass populations with a width of
300 m are observed in this area.
In the present study, the reproductive features of eelgrass populations were examined by monitoring changes
in the eelgrass populations in the east coast areas of Ise
Bay and by genetic analysis. Based on the data, we discuss
the genetic diversity of multiple eelgrass populations in
Ise–Mikawa Bay in relation to changes in the eelgrass
populations.
Plant Species Biology 24, 202–208

The degree of vegetation cover of eelgrass (proportion of
the area covered by eelgrass in a 50 cm ¥ 50 cm quadrat
was quantiﬁed into ﬁve classes) was estimated by scuba
diving from 1993 to May 2005 along a line at the center of
each population of eelgrass in Tarumi (34°51′59″N,
136°51′05″E) and Kosugaya (34°50′03″N, 136°51′48″E),
located on the east coast of Ise Bay (Fig. 1). The length ofthe lines from the position of DL ⫾ 0 in an offshore direction was 600 m at Kosugaya and 1200 m at Tarumi (Fig. 2).
The position of the sites was determined using a Deferential Global Positioning System ([DGPS]; Furuno Electric,
Nishinomiya, Japan).

Genetic analysis
Genetic polymorphism of microsatellite markers was analyzed in eelgrass populations of Isshiki (34°46′18″N,
137°01′37″E) and Miya (34°48′15″N, 137°15′24″E) in
Mikawa Bay, and in Tarumi and Kosugaya in Ise Bay(Fig. 1).
Leaf blades of eelgrass were harvested during May andJune 2005. The sampling area of 10 m ¥ 10 m (100 m2) was
set up in the center of the eelgrass populations in Tarumi,
Kosugaya, Isshiki and Miya. The sampling area wasdivided into meshes of 2 m with a rope and spindle. Leaf
blades of eelgrass were sampled at the center of each© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology

Results

Spatial and temporal dynamics of theeelgrass populations
As Tarumi and Kosugaya are major sites where large
meadows of eelgrass have remained, their eelgrass populations were examined from 1993 to 2005 (Fig. 1). The
degree of vegetation cover of eelgrass (proportion of thearea covered by eelgrass in a 50 cm ¥ 50 cm quadrat wasquantiﬁed into ﬁve classes) was estimated by scuba diving
along a line at the center of each population of eelgrass asshown by the red line in Fig. 2.
Figure 3 shows the changes in the distribution of theeelgrass populations on the survey line in Tarumi andVegetation
coveragerank

Distance(m)mesh. Twenty-ﬁve samples per each population (i.e. a
total of 100 samples) were used for the analysis.
The samples were stored at -80°C until use. DNA wasextracted from 0.1 to 0.2 g fresh weight of leaf tissue using
the hexadecyltrimethylammonium bromide (CTAB)
method (Stacey & Isaac 1994). The DNA samples were
genotyped by six microsatellite loci (Reusch et al. 1999;
Reusch 2000a,b). The thermocycling proﬁle of the polymerase chain reaction (PCR) consisted of a 10 min denaturation at 95°C, followed by 30 cycles of 1 min at 95°C,
1 min at 57°C and 1 min at 72°C, and then an additional
7 min at 72°C. Twenty nanograms of DNA was used as atemplate for the PCR. The PCR products were diluted to
10% with 12 mL HiDi formamide (+1/40 vol. of Gene Scan
-500 Rox size standard) and subjected to electrophoresis(Applied Biosystems). A capillary sequencer, 310 Genetic
Analyzer (Applied Biosystems, California, USA), wasused. The size (bp) of the microsatellite markers was calculated using an electropherogram with 310 Gene Scan
2.1 using the Gene Scan -500 Rox size standard.
Average heterozygosities (Ho and He: Nei 1987) and theinbreeding coefﬁcient (FIS: Wright 1951, 1965) were used
to evaluate the genetic diversity of each group. A c2 test
was used to test goodness of ﬁt for deviation of genotypic
frequencies from Hardy–Weinberg expectation. The
genetic differentiation between the populations wasdetermined from the coefﬁcient of pairwise genetic differentiation (FST: Nei 1987). A signiﬁcance test of the FST value
for each dataset was done by 105-fold permutation.
Genetic connectivity was examined using the genetic
distance (D: Nei 1987) between populations using a phylogenetic tree made by the average distance method(UPGMA). He, Ho, FIS, FST and D were calculated by microsatellite analyser (Dieringer & Schlotterer 2003). The
data of genetic polymorphism at Kosugaya (34°50′03″N,
136°51′43″E: Kosugaya2004) in 2004 (Yoshida et al. 2006)
were also used. The genetic assignment of populations as
the origin of individuals was done using GeneClass2 (Piryet al. 2004).

Distance(m)204 T. Y O S H I D A E T A L .

Vegetation cover degree of eelgrass
5 75%<
4 50%~75%
3 25%~50%
2 5~25%
<1.5%
0

Green
Lime
Bright green
Light green
Light blue
White

Fig. 3 Change in the distribution of Zostera marina populations
on the survey lines in Tarumi and Kosugaya, on the east coast
area of Ise Bay, from 1993 to 2005. The changes in the center ofgravity of the eelgrass population are shown with the arrows.

Kosugaya. In Tarumi, class 3 or more of vegetation cover
was observed in an area 300 m and 500 m from the shoreline in 1993. Interestingly, the distribution of eelgrasschanged to an area of 200 m in 1994, 100–610 m in 1996,
80–450 m in 1997, 80–340 m in 1998, 140–850 m and1000 m in 2000, 80–580 m and 780–810 m in 2001 and 2002,
70–370 m and 760–910 m in 2003, 160–510 m and 630–
1040 m in 2004, and 0–460 m and 790–920 m in 2005. It
was remarkable that the vegetation cover degree was very
low, class 2 or less, over the whole survey line in 1995 and1999. Similar changes in vegetation cover were also
observed at Kosugaya. In Tarumi, the degree of vegetation
cover of eelgrass populations on the survey line became
uniformly high in 2000. Since 2000 a high degree of vegetation cover in Tarumi has been maintained.
We estimated the center of the eelgrass population fromthe distribution and vegetation cover degree. Arrows in
Fig. 3 show a change in the center of the eelgrass population. It is apparent that periodic change in the distributionof eelgrass populations was observed in Tarumi and© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology

Plant Species Biology 24, 202–208

R E P R O D U C T I O N D Y N A M I C S O F E E L G R A S S P O P U L AT I O N S
Table 1 Genetic diversity of Zostera marina populations in Ise–
Mikawa Bay calculated with allele data at six microsatellite loci

Location
Tarumi
KosugayaIsshiki
Miya
Kosugaya2004

Mean no.
alleles

Ho

He

FIS

11.3
11.0
11.8
10.5
12.0

0.840
0.853
0.773
0.693
0.842

0.836
0.848
0.794
0.696
0.807

-0.005-0.0070.026
0.004-0.043

All FIS values did not show a signiﬁcant difference to expectation zero (Hardy–Weinberg equilibrium, P > 0.05). FIS, inbreeding
coefﬁcient; He, expected heterozygosity; Ho, observed
heterozygosity.

205

Table 2 Matrix of pairwise genetic differentiation (FST) among
populations of Zostera marina in Ise–Mikawa Bay
Location

Tarumi Kosugaya Isshiki Miya Kosugaya2004

Tarumi
KosugayaIsshiki
Miya
Kosugaya2004—
0.000†
0.045
0.086
0.004*

—
0.040
0.072
0.000*—
0.026—
0.037 0.053—† FST did not show a signiﬁcant difference to expectation zero
(P > 0.01).

Kosugaya. The center of the eelgrass populations shifted
approximately 100–200 m every year. The degree of vegetation cover of the eelgrass populations decreased withthe shift in the center of the populations over 3 years. The
closest distance from the shoreline was 400–500 m in
Tarumi and 200–300 m in Kosugaya. Once again, eelgrass
populations showed regeneration with movement of thecenter of the populations in the direction of the shore fromthe next year.
Fig. 4 Neighbor-joining tree based on Nei’s genetic distance (D).

Genetic diversity
Table 1 shows the parameters of genetic diversity. The
average number of alleles that was detected in these six
loci was 10.5–12.0 for the ﬁve populations. The average
heterozygosity (Ho) shows that the genetic diversity was
higher than 0.800 for the populations in Tarumi, Kosugaya
and Kosugaya2004, 0.773 for the population in Isshiki and
0.693 for the population in Miya. The inbreeding coefﬁcient (FIS) did not show a signiﬁcant difference from an
expectation of zero (Hardy–Weinberg equilibrium) in all
populations.

Gene ﬂow
Table 2 shows the coefﬁcient of pairwise genetic differentiation (FST). Tarumi–Kosugaya, Tarumi–Kosugaya2004,
Kosugaya–Kosugaya2004 and Isshiki–Miya did not show
genetic differentiation. The FST of Tamrumi–Isshiki,
Tamrumi–Miya, Kosugaya–Isshiki and Kosugaya–Miya,
which corresponds to the FST between the site of Ise Bay
and the site of Mikawa Bay, showed a value larger thanzero (P < 0.01). Figure 4 shows Nei’s genetic distance (D)between the eelgrass populations. The eelgrass populations of Ise–Mikawa Bay could be divided into twogroups, Tarumi and Kosugaya, and Isshiki and Miya.
Table 3 shows the numbers of individuals assigned toeach population as the most probable origin. Among thePlant Species Biology 24, 202–208

Table 3 Numbers of individuals of eelgrass assigned to each
population of Tarumi, Kosugaya, Isshiki and Miya as the origin

Location

Tarumi

Tarumi
KosugayaIsshiki
Miya

12
13
6
0

Location assigned as the origin
KosugayaIsshiki
7
12
0
1

5
0
13
7

Miya
1
0
6
17

individuals sampled at Tarumi, seven individuals wereassigned to Kosugaya. In addition, 13 individuals
sampled at Kosugaya were assigned to Tarumi. Six individuals at Isshiki were assigned to Miya and seven individuals at Miya were assigned to Isshiki. Five individuals
at Tarumi were assigned to Isshiki and six individuals at
Isshiki were assigned to Tarumi, but individuals at Kosugaya were assigned to neither Isshiki nor Miya.

Discussion

Reproduction modes of the eelgrass populations
As shown in Fig. 3, we found that eelgrass populations in
Tarumi and Kosugaya, in the east coast area of Ise Bay,© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology

206 T. Y O S H I D A E T A L .
showed dynamic regeneration over a period of 4 years
with an accompanying shift in the population center tothe shore. We call this phenomenon a ‘walking meadow’.
The observed shift in the eelgrass population center toshore was approximately 100 m per year (Fig. 3). As the
expansion of rhizomes by vegetative propagation is signiﬁcantly smaller than 100 m per year (Kawabata et al.
1993), the transport of seeds by rip currents is a key factor
in the shift of the eelgrass population center to the shore.
When eelgrass seeds are dispersed, they fall onto the sea
bottom near the parents by gravity because they are negatively buoyant (Orth et al. 1994). The rip current would
carry the eelgrass seeds a short distance and would cause
mixing of seeds within eelgrass populations. The rip
current in the bay during June and July, which is theperiod of seed dispersal in Tarumi and Kosugaya, is in the
direction from offshore to inshore by a south-west wind(Japan Fisheries Resource Conservation Association(JFRCA) 1997). As a result, seeds are transported by therip current to shore. Eelgrass seeds of various origins are
physically mixed during the transport process by the rip
current.
As shown in Fig. 2, a multiple bar trough in Kosugaya isformed by wind waves from the north-west in winter(JFRCA 1997). Seeds transported by the rip current would
be trapped more easily in this trough. This transport ofeelgrass seeds to shore is repeated every year. A phenomenon similar to that of Kosugaya was also observed in
Tarumi before a large man-made island (Fig. 1) was built
in 2000.
Table 1 shows that the genetic diversity of the eelgrass
populations of Tarumi and Kosugaya was high comparedwith the diversity on the Atlantic and Californian coasts(Reusch et al. 2000; Hammerli & Reusch 2003; MunizSalazar et al. 2005). Hence, it is believed that the highgenetic diversity of eelgrass populations in these areas isachieved by sexual reproduction as well as the physicalmixing and transport of seeds.
In Tarumi, the degree of vegetation cover of the eelgrass
populations on the survey line became high in 2000,
approximately 800 m from the shore (Fig. 3). A highdegree of vegetation cover has been maintained since
2000. This could be attributed to a weaker rip current insummer caused by the large man-made island (577 ha;
Fig. 1), which was built 3 km offshore in 2000 (JFRCA
1997). The weaker rip current in summer will decrease thefrequency of transport of eelgrass seeds to shore, increase
the probability of seeds settling in the area, and decrease
the loss of seedlings by wind waves in winter. All thesefactors contribute to an increase in the growth of eelgrass
populations in the short term, but reduce the genetic
diversity achieved by the physical mixing of the seeds inthe long term. Moreover, when the large man-madeisland blocks the tidal residual current from the inner bay

areas to the bay mouth, and the watercourse is changed,
the seed supply from Tarumi, the largest eelgrass population in Ise Bay, to Kosugaya and Mikawa Bay in thedownstream would decrease. This would be one reason
for the decline in eelgrass populations in Ise–Mikawa Bay.
At present, it is unclear why eelgrass populations in
Tarumi and Kosugaya showed dynamic regeneration overa period of 4 years. We conjecture as follows. In summer,
the seeds are transported to the trough near the shore by
the rip current from offshore to inshore, and the center ofthe eelgrass populations in Tarumi and Kosugaya moves
toward the shore. In the third year, the eelgrass populations reach the marginal areas of the tidal ﬂat. The shift in
the population center stops here because the marginalareas of the tidal ﬂat are not suitable for eelgrass growth
because they dry up and the water temperature is high(Kawasaki 1988). Over this period of time, the offshoreeelgrass populations, whose vegetation cover degree islow because of the inevitable transport of seeds to shore,
recover to the original levels based primarily on vegetative propagation. Because it is presumed that 4 years are
required to supply a sufﬁcient amount of seeds (Izumi
et al. 2002), the overall degree of vegetation cover
increases every fourth year. It is thought that this ‘walking
meadow’ phenomenon with a cycle of 4 years is attributed to the physical characteristics of the waters (including the rip current to shore in summer and a multiple bar
trough formed in winter) as well as to the characteristicsof the regeneration process of eelgrass, which grows byboth vegetative propagation and sexual reproduction.

Metapopulation structure
As the pairwise FST of the Tarumi and Kosugaya populations is zero, there is no genetic differentiation among
these two populations. Therefore, it might be consideredthat the rate of gene ﬂow between these two populations
is high. However, it is plausible that Tarumi and Kosugaya were one large population until recent years so thatthey maintain high genetic diversity although their populations have been rapidly declined recently. Negligible
values of FST for Kosugaya2004 with Tarumi or with
Kosugaya (Table 2) also support the genetic connectivityof these two populations.
In addition to the short-distance transport of seeds,
eelgrass has a system of long-distance transport of seeds
by residual tidal current. As spadices containing seeds canﬂoat on the sea surface when they are detached from theshoots (Harwell & Orth 2002), seeds can be transported bydrift. The long-distance transport of seeds by the drift ofspadix of eelgrass can explain the cross-assignment oforigin for individuals between Tarumi and Kosugaya
populations and between Isshiki and Miya populations.
The existence of a residual tidal current in Ise–Mikawa Bay© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology

Plant Species Biology 24, 202–208

R E P R O D U C T I O N D Y N A M I C S O F E E L G R A S S P O P U L AT I O N S
supports the long-distance transport of seeds by drift ofthe spadix of eelgrass.

Important factors for the sustainability ofeelgrass populations
It has been revealed that the gene ﬂow between the eelgrass populations in Ise–Mikawa Bay can be attributed to
the long-distance transport of seeds by the residual tidal
current. The rapid reduction in the distribution area of
eelgrass populations since 1960 (Takeda 2005) falls overthe period in which large-scale reclamation projects inline with harbor development in the inner bay areas andlarge-scale levee protection works (including high tide
breakwater) were undertaken. Thus, it is likely that the
rapid decrease in eelgrass populations in Ise–Mikawa Bay
since 1960 can be attributed to an extreme reduction in the
supply of seeds to downstream, which would result in thedisappearance of eelgrass populations, which had been
widely distributed in the inner bay areas (i.e. upstream of
the residual tidal current). Even today, reduction and fragmentation in the distribution of eelgrass populations arein progress in Ise–Mikawa Bay because of reclamation andother factors. A reduction and fragmentation in the distribution inhibit the transport of seeds to the entire bay andhave a quite signiﬁcant negative impact on the genetic
diversity levels that are currently maintained by the eelgrass populations. It is feared that lower eelgrass seedsupply to downstream and declining genetic diversity
may accelerate a reduction in the eelgrass populations in
the entire bay in the future.
Conventionally, efforts to restore eelgrass populations,based primarily on vegetative propagation, have been
focused on small-scale eelgrass populations in foreshores
as a compensatory measure against reclamation andlarge-scale development. For this reason, there has been atendency to neglect the maintenance of a connected structure among local populations and genetic diversity. Targeting eelgrass populations in individual foreshores alone
will be ineffective. It is important to have a concept of
restoring the entire local populations in a ‘self-sustained’
manner for the whole bay. To preserve the eelgrass populations, it is essential to take proactive preservation measures, such as securing large-scale parent populations in
the upstream waters of the residual tidal current that supplies seeds, selecting settlement areas for the stably supplied seeds based on ﬂow analyses, and improving thehabitat environment. These efforts will enable eelgrass
populations in different foreshores of Ise–Mikawa Bay tosurvive as local populations in a self-sustained manner
while maintaining connectivity, and will also lead to thepreservation of the genetic diversity of eelgrass populations. It is noteworthy that numerical simulations that
Plant Species Biology 24, 202–208

207

have been carried out in developing nurseries for littleneck clams Ruditapes philippinarum take into consideration
the relationship between the supply of larvae and thewater ﬂow of the entire bay (Suzuki et al. 2002). Likewise,
the preservation of eelgrass populations will require
efforts from a macroscopic view that are similar to thosemade in preserving a tidal ﬂat.

Acknowledgments
This work was supported in part by the Salt Science
Research Foundation, Grants-in-Aid for ScientiﬁcResearch from the Ministry of Education, Science andCulture of Japan, and the High-Tech Research Center of
Meijo University.

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253–258.

© 2009 The Authors
Journal compilation © 2009 The Society for the Study of Species Biology

Plant Species Biology 24, 202–208