Bull. Natl. Mus. Nat. Sci., Ser. B, 46(4), pp. 177–183, November 20, 2020

Eﬀect of Herbivore Predation and Water-Wave Disturbance on theSurvival of Wild Extinct Aquatic Plant Eriocaulon heleocharioides(Eriocaulaceae) in the Reintroduction Site
Norio Tanaka1,*, Kyoka Tsuda2, Yuju Horiuchi3, Shoh Nagata4,
Takashi Kamijo3, and Jun Nishihiro5
1

Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan2
Department of Environmental Science, Faculty of science, Toho University,
2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan
3
Graduate School of Life and Environmental Sciences, University of Tsukuba,
1–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan
4
Speciﬁed Nonproﬁt Corporation AQUA CAMP, 670–126 Shimohiro-oka,
Tsukuba, Ibaraki 305–0042, Japan
5
National Institute for environmental studies, Japan, 16–2 Onogawa,
Tsukuba, Ibaraki 305–8506, Japan*E-mail: ntanaka@kahaku.go.jp(Received 11 August 2020; accepted 23 September 2020)

Abstract The eﬀects of two factors, an alien herbivore and water-wave disturbance, on the
decline of a wild extinct annual amphibious herb, Eriocaulon heleocharioides (Eriocaulaceae),
was examined in a reintroduction site where the species existed. The numbers of surviving individuals after sowing seeds in three plot types were measured every two weeks. Surviving individuals
were recorded only in plots sheltered by a net from all sides (Close). There were no surviving individuals to the reproductive stage in sheltered plots with opened corners (Semi-Close) and the plots
without sheltering (Open). The eﬀect of sheltering and vanishing water-wave structure (VWS)
revealed that plot closing prevents aquatic organisms and mitigates the water waves. However,Semi-Close allowed aquatic organisms enter and water-wave disturbance as well. Thus, the overall
survival is mainly promoted by the prevention of aquatic organisms to enter. The water-wave mitigation could additionally reduce the decline of the plants. To prevent herbivores is most essential
for the design and management of the reintroduction site. The mitigation of water-wave disturbance could further help to reduce the decline of this plant.
Key words : Aquatic plant, Eriocaulon heleocharioides, extinct in the wild, herbivore, reintroduction.

Introduction
Eriocaulon heleocharioides Satake (Eriocaulaceae) is an annual amphibious herb that is
endemic to Japan and extinct in the wild (Ministryof the Environment, 2019). Since 2008, a research
project for ex situ conservation and reintroduc© 2020 National Museum of Nature and Sciencetion of the species to its last known habitat in the
Sanuma Lake, Ibaraki Prefecture, has been
carried out by the Tsukuba Botanical Garden,
National Museum of Nature and Science, Japan(Tanaka et al., 2014, 2015, 2018, 2020). Sowing
seeds of the species in the reintroduction site produced hundreds to thousands of surviving individuals in the ﬁrst few years of the project. Then,the number of individuals gradually decreased

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despite the continued sowing each year (Tanaka
et al., 2018).
Various factors – methodological, environmental and biological – are thought to cause the difference between success and failure (Guerrant
and Kaye, 2007; Menges, 2008; Godefroid et al.,2011). In the case of Eriocaulon heleocharioides,
predation by alien aquatic organisms such as bullfrog tadpoles (Rana catesbeiana) and red swamp
crayﬁsh (Procambarus clarkii), is presumed as theleading harmful factors (Tanaka et al., 2018). A
net sheltering of the sowing site at the experimental area, located in the last habitat of Sanuma
Lake, improved the survival of E. heleocharioides seedlings (Tanaka et al., 2018, 2020). The
net sheltering could have protected the plantsfrom herbivore predation or water-wave disturbance. Tanaka et al. (2018) reported a lower
impact of water wave disturbance on the survival
than that of herbivores. However, it was an estimate based only on observation and there was noevidence to support it.
These previous researches revealed that E.
heleocharioides cannot survive to the reproductive stage without protection from the present
natural environment of Sanuma Lake. The
construction design needs the identiﬁcation offactors inhibiting plant survival and their degrees

of impact. This study aims to examine the degreeof impact of two factors, e.g., aquatic herbivore
predation and water-wave disturbance, on thesurvival decline of E. heleocharioides in its reintroduction site.
Materials and Methods
Study sites and data measurements
This experiment was conducted within a semiprotected area constructed in Yanagi Wando,
Sanuma Lake, Shimotsuma City, Ibaraki Prefecture, Japan. The area was made of timber andwaterproof sheets to hold soil and water. It consists of three plots in eight beds in two sites(Fig. 1). Site A was used for the experiment in2017 (only Bed-1 and -2; Tanaka et al., 2018) and2018 (Bed-1, -2, -3 and -4; Tanaka et al., 2020).
Site B was newly constructed in March, 2019. All
beds, except Bed-1 and Bed-5 that were isolated
from the water of the lake, were used for thisstudy. Bed-2 and -6, Bed-3 and -7, and Bed-4 and-8 have the same ground levels – 28, 42 and 57 cm
below the maximum water levels of the lake,
respectively. Each site has nine plots including 25
sub-plots of 0.048 m2 (0.3×0.16 m). All eighteen
plots were categorized into three types of treatments, e.g. Open, Semi-Close and Close. The

Fig. 1. Schematic layout of the semi-protected area used for this study. MWD means maximum water depth on
the soil surface at each bed.

Eﬀect of herbivore predation and water-wave disturbance on Eriocaulon heleocharioides

Close treatment (Plots-1, -6, -7, -10, -15 and -16)
was fenced all around by nets with 2-mm mesh
to prevent herbivores from entering them, whilein the Semi-Close treatment, the two diagonalcorners were left open (Plots-2, -4, -9, -11, -13
and -18). No fencing was used in the Open treatment (Plots-3, -5, -8, -12, -14 and -17). In eachbed, the three treatment types area were randomly arranged.
In March 2018, the soil of Bed-3 and Bed-4 ofsite A (constructed in March 2017) has been
replaced with new soil from a location 10 m oﬀshore. That of Bed-2 had been partly left
unchanged, partly replaced with new soil, andpartly plowed to replace the surface soil with
subsoil. In 2019 (for this study), the surface soil
up to 10 cm depth at all beds in site A was
replaced with new soil from 10 m oﬀshore. Site
B was constructed in March 2019 at about 3 m
next to the site A. At the same time, the soils of
Bed-6, Bed-7, and Bed-8 in the Site B was newlytransported from a location 10 meters oﬀshore.
Eleven vanishing water-wave structures (VWS)
were constructed to surround site B to mitigate
the water-wave disturbance. The VWS is 182 cm
wide and 115 cm high and consisted with 13
timbers 7.5 cm high, 182 cm wide and 9 mm
thick, boarded with a gap of 5 mm. The mouth of
the Yanagi Wando holding the study site directs
to west and the winds often blow from west or
north west and produces water-waves to thestudy site, especially in Spring and Autumn (byauthorsʼ observation).
On March 29, 2019, 151 seeds of Eriocaulon
heleocharioides that were collected in the fall of
2018 from individuals raised in the semi-protectedarea (Bed-1 in Fig. 1) of Sanuma Lake, weresown in each sub-plot (Tanaka et al., 2018,2020). The numbers of individuals were counted
in nine sub-plots, randomly selected from 25
sub-plots per plot, every two weeks from April 15
to October 21, 2019. Surviving individuals until
October 21 were considered as ﬁnal surviving
individuals. Most of them ﬂower by October andproduce seeds by November.

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Statistical Analysis
To quantify the eﬀect of Net-sheltering and
VWS on the number of surviving individuals,
analysis was performed using a Poisson generalized linear mixed model (GLMM) across thesurveyed days. We designated the number ofsurviving individuals as the response variable,
net-sheltering (three category: Open, Semi-Close,
and Close), VWS (two category: set and no set)and Bed for experiment (Upper, Middle andLower) as the predictor and each plot as random
eﬀects term using lme4 package (version 1.1-19,
Bates et al., 2015). These analyses were performed using R software (version 3.5.3, R Development Core Team, 2019).
Results
Final surviving individuals of Eriocaulon
heleocharioides were found only in Plot-1 (2.0
individuals/sub-plot), Plot-6 (13.7), Plot-10 (3.6)
and Plot-15 (1.6), which are all of the Close

Fig. 2. Mean number of the ﬁnal surviving individuals for sub-plots in each plot. Bars indicate
SD. Each plot includes the combination of threeelements: VWS (vanishing water-wave structure) is set or not, the water depth is Upper(Bed-2 and -6), Middle (Bed-3 and -7) or
Lower (Bed-4 and -8), and the sheltering type isOpen, Semi-Close or Close.

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Fig. 3. Temporal change of mean number of surviving individuals for sub-plots in each plot. Bars indicate SD.

treatments in Bed-2 and Bed-3 (Fig. 2). Regardless of the presence or absence of the VWS, no
individuals survived until the reproductive stage
in any of the Bed-4 and the Open and Semi-

Close treatments of Bed-2 and -3.
The temporal change of surviving individual
number is shown in Fig. 3. The number of individuals was remarkably changed during the early

Eﬀect of herbivore predation and water-wave disturbance on Eriocaulon heleocharioides

Fig. 4. Temporal change of the eﬀects of net-sheltering in the Close and Semi-Close treatments on
the number of surviving individuals. The coeﬃcient values were shown on the vertical axis.
Bars indicate SD. Asterisks mean the signiﬁcantlevels (***P＜0.001, **P＜0.01, *P＜0.05).
Data after June 14th was excluded from theanalysis because the number of surviving individuals became zero at most of plots.

Fig. 5. Temporal change of the eﬀects of VWS on
the number of surviving individuals. The coeﬃcient values were shown on the vertical axis.
Bars indicate SD. Asterisks mean the signiﬁcantlevels (***P＜0.001, **P＜0.01, *P＜0.05).
Data after June 14th was excluded from theanalysis because the number of surviving
individuals became zero at most of plots.

181

stage of their life cycle – from April to June.
GLMM analysis shows the positive eﬀect ofClose treatment throughout the period on theplant survival compared to the Open treatment,
especially after the middle of May (p＜0.01–
0.001, Fig. 4). In contrast, no eﬀect was detectedin the Semi-Closed treatment throughout theperiod.
GLMM analysis showed that the VWS had apositive eﬀect on plant survival in all plots from
April to May (Fig. 5). Comparing the types ofshelter treatment, deﬁnitive eﬀects of the VWS
are shown in Open from April to May, whileslight eﬀect only in April 15 in Semi-Close, andno eﬀects were detected in Close throughout theperiod (Fig. 6).
Discussion
The eﬀect of the three sheltering types
The number of the ﬁnal surviving individuals
that can produce seeds for the next generation
is an important measure for the suitability ofenvironmental conditions for the reintroductionof an annual species, Eriocaulon heleocharioides.
In this study, the ﬁnal surviving individuals wererecorded only in four plots. All of them were
found in the Close treatments (among all 18
plots) (Fig. 2). The higher survival in the Close
treatment compared to the Open treatment agrees
with the results of the 2017 and 2018 experiments (Tanaka et al., 2018, 2020).
On the other hand, there was no signiﬁcanteﬀect in the Semi-Close treatment with two open
corners (Fig. 4). Since the lateral side of thesemi-closed treatments was about 93% coveredby the net, they were set to allow most of theaquatic organisms to enter the plot, but also to
mitigate water waves. However, since thestrength of water waves was not recorded in eachplot, the actual eﬀect of water-wave mitigation
was not clearly shown in this experiment. Therefore, it may reﬂect not only the eﬀect of aquaticorganisms but also the eﬀect of being unable tomitigate water waves in the Semi-Close treatment structure. Here, the eﬀect of the VWS in182

Norio Tanaka et al.

the ﬁrst half of May (Fig. 6). In general, theseedling stage is the most vulnerable part of theplant life-cycle (Primack and Drayton, 1997),
because during that period the seedlings could
easily be aﬀected by environmental disturbancessuch as water waves. Many early seedlings of E.
heleocharioides were observed by the authors in
the Semi Protection area of the Sanuma Lake todetach from the soil and to ﬂoat on the water
surface. The VWS may be eﬀective in the otherseason as well. However, further discussion is
not possible at this time because no plant individuals survive after the early growth stage except
those in the closed treatment.

Fig. 6. Diﬀerence of the eﬀect of VWS on thenumber of surviving individuals in the three
types of net sheltering: Open, Semi-Close andClose. The coeﬃcient values were shown on thevertical axis. Bars indicate SD. Asterisks mean
the signiﬁcant levels (***P＜0.001, **P＜0.01,*P＜0.05). Data after June 14th was excluded
from the analysis because the number of surviving individuals became zero at most of plots.

each treatment (Fig. 5) seems to help this discussion. The clear eﬀect of VWS in the open treatment and the lacking eﬀect in the closed treatment indicate that the closed treatment mitigates
the water waves properly. On the other hand, theweak eﬀect observed only in the initial period in
the Semi-Close treatment indicates that it has awave mitigating eﬀect, too. However, it is
weaker than that of the closed treatment.
Judging from the above, the positive eﬀect on
the survival of individuals in Close treatment,
especially the clear eﬀect on the ﬁnal surviving
individual number, is mainly due to the prevention of aquatic organismsʼ entrance. Also, thewater-wave disturbance could additionallyinhibit the decline of plants. Water-wave mitigation was shown to improve the survival at leastin the early stage since the eﬀect of the VWS
was clearly detected (Fig. 5) and the diﬀerence
between the eﬀects of Semi-Close and that ofOpen treatments was small in the early stage – until

Water depth eﬀect
Regardless of the net-sheltering of the closed
treatment, there were no surviving individuals in
the lowest bed (Bed-4). Although the previous
experiment of 2018 showed that the individuals
could survive in the Lower bed (Tanaka et al.,2020), there were no surviving individuals at thesame depth in this study. Water depth is generally
known to have a strong eﬀect on the survival andgrowth of aquatic plants, particularly those thatare submerged (Havens, 2003; Bornette andPuijjalson, 2011). In the reintroduction site of E.
heleocharioides, the limitation of water depth onplant growth can be ampliﬁed by factors withannual ﬂuctuations, such as changes in watertransparency and water level. They can aﬀect theamount of transmitted light to the bottom andwater edge position where the physical disturbance is stronger. For reintroduction planningand management, it is necessary to consider thewater depth that allows for annual ﬂuctuations.
Design of reintroduction site
Herbivore predation is probably the main
factor that inhibits the survival of E. heleocharioides in the reintroduction site, while water-wave
disturbance is less harmful. In a review paper
considering the reintroduction cases of 249 plant
species worldwide (Godefroid et al., 2011), theauthors have reported that plant reintroduction inprotected areas signiﬁcantly increased the sur-

Eﬀect of herbivore predation and water-wave disturbance on Eriocaulon heleocharioides

vival rate compared to the reintroduction in
unprotected areas. They have also suggested thatthe management of the out-planting site througheither preparation for planting (e.g. fencing) or
post-planting management (e.g. ﬁre, reduced
competition) increased the probability of reintroduction success. Since it is diﬃcult to remove or
mitigate negative factors, such as herbivore predation and water-wave disturbance, throughout
the whole Sanuma Lake (having 0.55 km2 area),
the creation of a suitable environment at the reintroduction site can only be an applicable method
under the present conditions. So far, the reintroduction site design for this wild extinct plantneeds to prevent herbivores from accessing theplants, which is the most essential task. Then,water-wave disturbance mitigation could help toinhibit the decline of the seedlings.
Acknowledgments
The authors wish to thank Yoshiyuki Sugaya,
Fumio Uchiyama and Shimotsuma City for continuous cooperation on the reintroduction projectof Eriocaulon heleocharioides, and for theirhelpful experimental works, Chikako Ishii,
Keisuke Kutsuwada, Taichi Umino, Tatsuaki Ito,
Kazumi Ando, Mao Sugai, Akihisa Suzuki,
Tobimaru Tsutsui, Yuki Fukuda, and all participants in the trial lectures for conservation of E.
heleocharioides held at the Tsukuba Botanical
Garden in 2019. This study was ﬁnancially supported by JSPS KAKENHI Grant Number
17K07275 to NT and the integrated research
project “Integrated analysis of natural history
collections for conservation of highly endangered species” by the National Museum of
Nature and Science, Japan.
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