Bull. Natl. Mus. Nat. Sci., Ser. B, 40(2), pp. 95–100, May 22, 2014

Seed Germination Response to Storage Conditions of Eriocaulon
heleocharioides (Eriocaulaceae), an Extinct Species in the Wild
Norio Tanaka1,*, Mina Goto2, Kohei Suzuki2, Toshinari Godo3,
Juntaro Kato4 and Takashi Kamijo5
1

Depatment of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
2
Graduate School of Life and Environmental Sciences, University of Tsukuba,
Tennodai 1–1–1, Tsukuba, Ibaraki 305–8572, Japan
3
Botanic Gardens of Toyama,
Kamikutsuwada 42, Fuchu-machi, Toyama 939–2713, Japan
4
Department of Science Education, Aichi University of Education,
Hirosawa 1, Igaya, Kariya, Aichi 448–8542, Japan
5
Faculty of Life and Environmental Sciences, University of Tsukuba,
Tennodai 1–1–1, Tsukuba, Ibaraki 305–8572, Japan* E-mail: ntanaka@kahaku.go.jp(Received 12 February 2014; accepted 26 March 2014)

Abstract Seed germination experiments with Eriocaulon heleocharioides Satake (Eriocaulaceae) were performed to investigate the effect of air exposure on seeds during the storage period.
Although the seeds germinated both above and under water, the response and durability of germination differed from each other. Moreover, the seeds that were dried for 1 month and sown inwater showed the highest germination rate (84.4%) among all combinations of conditions. This
result shows that treatment by drying prior to sowing promotes the germination of this species.
Key words : aquatic plant, Eriocaulon heleocharioides, ex situ conservation, extinct in the wild,
re-introduction, seed germination.

Introduction
Eriocaulon heleocharioides Satake (Eriocaulaceae) is an annual aquatic herb endemic to Japanand an extinct species in the wild (Ministry of theEnvironment, 2012). The species was collected
for the ﬁrst time in Koshigaya, a city in central
Japan, in 1938 and described in the following year
(Satake, 1939). Thereafter, it was never found at
this or other sites, and hence, was consideredextinct (Satake, 1982). However, the species was
rediscovered, collected as herbarium specimens
and deposited in the Ibaraki Prefectural Museum,at Sanuma Lake, Shimotsuma, Ibaraki Prefecture,
central Japan in 1975. The lake is a large water
storage reservoir for paddy ﬁelds. Further, in 1994,

this species also became extinct in Sanuma Lake.
This species disappeared from its last habitat after
a change in water management in 1994 and has
been preserved only ex situ such as in botanicalgardens.
Ex situ conservation is essential for extinct
species in the wild. In a subsequent stage, reintroduction of extinct species to the wild is necessary, because an organism is a component of theecosystem and can be maintained as a wild species
with genetic diversity only in the wild (Maunder,
1992; Primack, 1995; Lawrence and Kaye, 2011).
It is necessary for a stable ex situ conservation
to reveal seed storage and germination behavior,especially for annual plants, such as E. heleocharioides. Although seeds can generally beNorio Tanaka et al.

96

preserved under low temperature and dry conditions (Roos and Davidson, 1992), information
about seed storage and germination of this species
and genus is insufﬁcient. Thompson et al. (1997)
showed that E. aquaticum formed a short-term
persistent soil seed bank, with seeds surviving inthe soil for at least 1 year but no more than 5
years. Miyamoto (1995, 1997) found that seedstorage in water in the dark at 4°C is suitable forE. heleocharioides and that after 35 days of thistreatment, germination is promoted on dampened
ﬁlter paper in the light at 25°C. In contrast, weobserved that air exposure for several weeks may
improve the seed germination rate. In papaya(Carica papaya, Caricaceae), drying treatment
prior to sowing has also been reported to be promoted germination (Hore and Sen, 1993; Sippel
and Claassens, 1993). In some plant species, seedgermination after exposure to cycles of hydrationand dehydration has been reported to be delayed(Allen et al., 1993; Downs and Cavers, 2000;
Ren and Tao, 2003) or reduced (Vincent andCavers, 1978; Downs and Cavers, 2000).
This study aims to determine the effect of air
exposure during the storage period on seed germination of E. heleocharioides.
Materials and Methods
The seeds for this study were collected in
November 2009, 2010 and 2011 from multiple
individuals of E. heleocharioides Satake (Eriocaulaceae) cultivated in the nursery of the Tsukuba
Botanical Garden, National Museum of Natureand Science (TBG accession no. 137574). The
plants were originally collected from Sanuma
Lake in 1994 and have since been continuously
subcultured every year by seed collection frompropagated plants and seedings.
The experiment was performed from August2012 to January 2013 and consisted of three different treatment methods after collection ofseeds: Treatment 1, preservation in water; Treatment 2, air exposure; Treatment 3, preservationin water after air exposure. The germinated seeds
were counted and removed daily until seven con-secutive days without seed germination. The
detailed methods are as follows, and combinations of experimental conditions are listed inTable 1. Two iterations of ﬁfty seeds were used
for each combination.
Treatment 1: Before the experiments, the seeds
collected in November 2009, 2010 and 2011were preserved in water in the dark at 4°C. Using
these seeds, two sowing environments were prepared, and they were kept in the light at 25°C:“wet,” placed on dampened ﬁlter paper in a 9-cm
Petri dish, and “water”, placed in water. Dormancy
breaking was not applied in Treatment 1, because
the storage period after collection functioned as
dormancy breaking.
Treatment 2: The seeds exposed to air at room
temperature since their collection in November
2011 were used for the germination test. The
seeds were divided into two groups: with andwithout dormancy breaking (in water in the dark
at 4°C for 35 days) and then sown in either the“wet” or “water” environment, as described forTreatment 1.
Treatment 3: The seeds collected in November
2009, 2010 and 2011 were preserved in water inthe dark at 4°C. They were then exposed to air for
1–3 months before dormancy breaking and sownin either of the two environments, as describedfor Treatment 1. The air exposure was applied foreach combination of temperature (4°C or 35°C)
and illumination (light or dark). For air exposure,the moisture content of seeds was adjusted to 12%
in a desiccator.
For Treatment 3, a classiﬁcation tree model(CTM) (Breiman et al., 1984) was used to detect
the important conditions for germination rate
among the multiple combinations of experimental conditions. The germination rate was used as
the response variable. Duration, temperature andillumination in seed drying, dormancy breaking,and sowing environment were used as predictor
variables. The most appropriate tree size in the
CTM was obtained by cross-validation. Package“mvpart” of R 2.14.2 (R Development Core Team,
2012) was used for the analysis.
To conﬁrm the viability of non-germinatedSeed Germination of Eriocaulon heleocharioides
Table 1.

97

Germination rate of Eriocaulon heleocharioides seeds in each treatment conditions
Conditions of drying seeds

Duration(month)Temperature(°C)

Treatment 1: preservation in water—0—0Illumination(light or dark)

Dormancy
breaking(with or without)

Sowingenvironment(water or wet)

Germination
rate
(%)

—
—

with
with

water
wet

37.0
44.0

with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
with
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without
without

water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet
water
wet

72.0
50.0
85.3
58.0
96.7
82.0
96.7
86.7
54.7
35.3
80.7
49.3
33.3
22.0
68.7
57.3
64.0
42.7
85.3
75.3
0.7
0.0
48.0
44.7
67.0
18.0
82.0
30.0
100.0
43.0
69.0
51.0
47.0
10.0
52.0
33.0
19.0
12.0
58.0
33.0
45.0
19.0
71.0
8.0
0.0
0.0
14.0
3.0

Treatment 3: preservation in water after air exposure
1
4
light
1
4
light
1
4
dark
1
4
dark
1
25
light
1
25
light
1
25
dark
1
25
dark
2
4
light
2
4
light
2
4
dark
2
4
dark
2
25
light
2
25
light
2
25
dark
2
25
dark
3
4
light
3
4
light
3
4
dark
3
4
dark
3
25
light
3
25
light
3
25
dark
3
25
dark
1
4
light
1
4
light
1
4
dark
1
4
dark
1
25
light
1
25
light
1
25
dark
1
25
dark
2
4
light
2
4
light
2
4
dark
2
4
dark
2
25
light
2
25
light
2
25
dark
2
25
dark
3
4
light
3
4
light
3
4
dark
3
4
dark
3
25
light
3
25
light
3
25
dark
3
25
dark

Note: Data for Treatment 2 and for 2009 and 2010 seeds were not shown, because none of their seeds germinatedas described in Results.

Norio Tanaka et al.

98

seeds after the germination test, both ﬂuoresceindiacetate (FDA) staining and a germination test
using gibberellic acid (GA) were performed. For
FDA staining, washed seeds were placed on 1%
SeaKem agarose in a 9-cm Petri dish and incubated at room temperature for 1 day. Further, the
FDA solution was poured in the Petri dish. The
dish was then exposed to 470 nm illuminationfrom a 470-nm EX Flash Light (Bio Tools Co.
Ltd.) and observed through a yellow ﬁlter. Moreover, for GA test, seeds were placed on a ﬁlter
paper dampened with 200 mg/l GA solution in a
9-cm Petri dish, and the dish was placed in thelight at 25°C.
Results
In the present study, none of the seeds collected
in 2009 and 2010 germinated under any of thetreatments. Therefore, only the results for 2011
seeds are presented in Table 1.
In Treatment 1, preservation in water, theseeds germinated in both “wet” and “water” environments (Table 1). The cumulative number ofgerminated seeds is shown in Fig. 1. In the seeds
placed in “water,” the ﬁrst germination wasobserved on day 3 after the start of the experimentand it continued until day 7 (ﬁnal germination

Fig. 1. Germination rate of Eriocaulon heleocharioides in Treatment 1. Cumulative percentages
for “wet” (▲) and “water”(●) environments areshown.

rate, 37.0%). In the “wet” treatment, germination
began on day 5 and continued until day 20 (ﬁnalgermination rate, 44.0%).
In Treatment 2, air exposure, no seeds germinated under any condition (data not shown in
Table 1).
In Treatment 3, preservation in water after air
exposure, germination was observed under mostexperimental conditions (Table 1). The mostappropriate tree is shown in Fig. 2. The highest
germination rate (84.4%) among all combinationsof conditions was obtained by drying for 1 monthand sowing in “water”. Moreover this rate ishighest also compared with Treatment 1. In eachnode, sowing with dormancy breaking and in“water” resulted in higher rates than that without
dormancy breaking and in “wet”, respectively.
FDA staining was invalid for E. heleocharioides
seeds, because the embryo is too small for the
staining to be visually recognizable. In addition,
the staining of an esterase-like enzyme, exuded
from living seeds was not observed on the agar
medium. The non-germinated seeds in the experiments did not germinate with GA treatment,
although 100% of the control seeds germinated.

Fig. 2. The classiﬁcation tree diagram for germination rate in Treatment 3. The number beneathnode represents the germination rate of the node.

Seed Germination of Eriocaulon heleocharioides

Discussion
In the present study, none of the seeds collected
in 2009 and 2010 germinated under any test condition, indicating that E. heleocharioides seeds
cannot be preserved for more than two years
under the storage conditions of this study. This
short-term survival behavior is consistent withthat of other Eriocaulon species (Thompson etal., 1997).
In this study, drying the seeds for 1 month prior
to sowing promoted their germination. However,in some plants, exposure to cycles of hydrationand dehydration is known to delay and/or reduceseed germination (Allen et al., 1993; Downs andCavers, 2000; Ren and Tao, 2003; Vincent andCavers, 1978). Promoting germination by dehydration of seeds, as in the present study, has also
been reported for papaya. In papaya, drying
treatment prior to sowing promoted germination(Hore and Sen, 1993; Sippel and Claassens,
1993). The only two natural habitats of E. heleocharioides, in which the species has ever beenrecorded, become marshy from late September toearly April due to lowering of water level. Therefore, most of the seeds are never submerged forseveral months after seed maturation. Moreover,some of the ﬂower stalks remain in an uprightposition for several weeks, supporting the seeds
that are exposed to air. This environmental condition is similar to that for dehydration prior tosowing. Therefore the seed germination behaviorof E. heleocharioides revealed in this study isbelieved to relate to these habitat environments.
The seeds in “water” germinated faster thanthose in “wet” and quickly reached the ﬁnal germination rate. In contrast, seed germination in“wet” was slow but sporadic. Kagaya et al. (2005)
reported that hydration and dehydration cycles ofseeds render the germination of Aster kantoensis(Asteraceae) sporadic. They considered thisbehavior to be associated with the plant's habitat,
where the seeds are exposed to several cycles ofhydration and dehydration. Moreover, in someaquatic plants, seed germination is markedly promoted by immersion in water (Monochoria kor-

99

sakowii Regel and Maack.; Godo, 1997) and bysowing in deoxygenated water (Zostera marina
L.; Moore et al., 1993). In the case of M. korsakowii, exposing the seeds above water strongly
reduces their germination. In contrast, germination of E. heleocharioides seeds seems to be controlled by some factors. This behavior may be
associated with a habitat in which the moisture
content of environment changes in a complex withthe difference of relative elevation, soil texture,
and weather.
Acknowledgments
The authors thank Shoh Nagata, Tomoko Obata,
Saori Ichikawa and Toru Nakamura for help with
conducting the research project; Kazuo Mochizuki
and Futoshi Miyamoto for providing seeds from
Sanuma Lake; Yoshiyuki Sugaya, Fumio Uchiyama and Shimotsuma city for improving thewater management. The authors are thankful for
grants to Tsukuba Botanical Garden from the
Ministry of Environment (The ex situ conservation model project).
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