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\begin{document}

\title{Odor of the achlorophyllous plant's seeds induce the seed-dispersing
ants}



\author[1]{Mikihisa Yamada}%
\author[2]{Masaru Hojo}%
\author[1]{Akio Imamura}%
\affil[1]{Hokkaido University of Education - Asahikawa Campus}%
\affil[2]{Kwansei Gakuin University School of Science and Technology Graduate School of Science and Technology}%


\vspace{-1em}



  
  \date{June 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Seed dispersal by ants is one of the important means of migration for
adherent plants. Although many myrmecochorous plants have seed with
elaiosome which is nutritional reward for ants, some seeds without
elaiosomes are also dispersed by ant species. We tested seed dispersal
by ants using the achlorophyllous and myco-heterotrophic herbaceous
plant Monotropastrum humile, whose seeds do not have elaiosome, and
require a fungal host for germination and ultimately survival. We
performed a bioassay using seeds of M. humile and the ant, Nylanderia
flavipes, to demonstrate ant-mediated seed dispersal. We also analyzed
the volatile odors emitted from M. humile seeds, and conducted bioassays
using dummy seeds coated with seed volatiles. Although elaiosomes were
absent from the M. humile seeds, the ants carried them to their nest.
They also carried the dummy seeds coated with the seed volatile mixture
to the nest, and left some dummy seeds inside the nest and discarded the
rest of the dummy seeds outside the nest with a bias towards locations
with moisture conditions conducive to germination. We concluded that
seeds of the myco-heterotrophic, herbaceous species were dispersed by
the ants, and that seed odors were sufficient to induce directed
dispersal even without elaiosomes. The flesh-fruit producing genus
Monotropastrum have probably evolved from the related anemochorous genus
Monotropa, which produces capsule fruit. This transformation from
anemochory to myrmecochory, presents a novel evolutionary pathway
towards ant-mediated seed dispersal in an achlorophyllous plant.%
\end{abstract}%



\sloppy


\textbf{Odor of~the achlorophyllous plant's seeds induce the
seed-dispersing ants}

Author information

Mikihisa YAMADA\textsuperscript{1}, Masaru K. HOJO\textsuperscript{2},
Akio IMAMURA\textsuperscript{1}

1. Hokkaido University of Education, Asahikawa Campus,

Hokumon-cho 9, Asahikawa, Hokkaido 070-8621, Japan

2. Department of Bioscience, School of Science and Technology, Kwansei
Gakuin University, Sanda, Hyogo 669-1337, Japan.

correspondence: Akio Imamura

E-mail: ginryou715@yahoo.co.jp

ORCiD ID 0000-0002-5208-1936

\textbf{Declarations}

Acknowledgments

We thank Takafumi Mizuno and Toshiharu Akino for their help with GC-MS
analyses. This work was supported by JSPS KAKENHI Grant no. 18H02512. We
would like to thank Editage (www.editage.com) for English language
editing.

Author contributions

A.I. conceived the study, conducted analyses, and wrote the manuscript.
K.Y. conducted experiments. M.K. Hojo performed a gas chromatograph
analysis and check the statistical analyses.

Funding

This work was supported by JSPS KAKENHI Grant no. 18H02512.

Conflicts of interest

There are no conflicts of interest to declare.

Data availability statement

All relevant data are within the paper and its Supporting Information
files.

\textbf{Abstract}

Seed dispersal by ants is one of the important means of migration for
adherent plants. Although many myrmecochorous plants have seed with
elaiosome which is nutritional reward for ants, some seeds without
elaiosomes are also dispersed by ant species. We tested seed dispersal
by ants using the achlorophyllous and myco-heterotrophic herbaceous
plant \emph{Monotropastrum humile} , whose seeds do not have elaiosome,
and require a fungal host for germination and ultimately survival. We
performed a bioassay using seeds of \emph{M. humile} and the
ant,\emph{Nylanderia flavipes,} to demonstrate ant-mediated seed
dispersal. We also analyzed the volatile odors emitted from \emph{M.
humile} seeds, and conducted bioassays using dummy seeds coated with
seed volatiles. Although elaiosomes were absent from the \emph{M.
humile} seeds, the ants carried them to their nest. They also carried
the dummy seeds coated with the seed volatile mixture to the nest, and
left some dummy seeds inside the nest and discarded the rest of the
dummy seeds outside the nest with a bias towards locations with moisture
conditions conducive to germination. We concluded that seeds of the
myco-heterotrophic, herbaceous species were dispersed by the ants, and
that seed odors were sufficient to induce directed dispersal even
without elaiosomes. The flesh-fruit producing genus\emph{Monotropastrum}
have probably evolved from the related anemochorous genus
\emph{Monotropa} , which produces capsule fruit. This transformation
from anemochory to myrmecochory, presents a novel evolutionary pathway
towards ant-mediated seed dispersal in an achlorophyllous plant.

\textbf{KEYWORDS}

directed seed dispersal, elaiosome, myco-heterotrophic plant,
myrmecochory, seed volatiles

\textbf{1 \textbar{} INTRODUCTION}

Various seed and fruit traits such as size and color tend to be
correlated, forming what are known as ``dispersal syndromes'' that may
have arisen to attract particular dispersers (Brodie, 2017; Valenta and
Nevo, 2020) or may facilitate transport from their parent plants and
move by wind or currents (Nilsson et al., 1991; Bullock \& Claeke, 2000;
Ohnishi et al., 2008; Nathan et al., 2011). Among seed dispersal by
animals, dispersal depending on ants is specifically called
myrmecochory. Myrmecochory is widespread among angiosperms and
ecologically important; over 11,000 species of myrmecochorous plants in
77 families and 334 genera, participate in myrmecochory across various
ecosystems (Lengyel et al., 2010). One of the evolutionary significance
of myrmecochory is considered as directed seed dispersal. The advantages
of the directed ant-dispersal hypothesis are that ant nests maintain
moist conditions and the seeds carried to the nest can escape
desiccation and have a higher seedling survival (Levey \& Byrne, 1993),
and the ants bury the seeds at depths where humidity and temperature are
suitable for germination (Gibson, 1993).

Seeds of many myrmecochorous plants possess appendages, such as
elaiosomes containing various fatty acids and proteins. These are likely
to be nutritional rewards for ants who disperse the seeds (Brew et al.,
1989; Lanza et al., 1992). If the seeds are carried to an ant nest, the
elaiosomes will likely be consumed inside the nest before the seeds are
discarded (Culver \& Beattie, 1978). Some granivorous ants, such as
species of genera \emph{Messor,Pheidole,} and \emph{Tetramorium} , lose
or abandon their seeds during transportation and thus also disperse
seeds without elaiosomes (Retana et al., 2004; Kobayashi, 2009). Various
studies have shown that nutritional contents, such as lipids, amino
acids, and proteins, in the elaiosome are important for inducing the
seed dispersal. In contrast, how seeds without elaiosomes enable
efficient dispersal by ants is less investigated, although some chemical
signaling might be involved (Youngsteadt et al., 2008). It is also
unclear whether the seeds are discarded on suitable sites in a directed
manner in species without elaiosomes. In this study, we investigated
seed dispersal without specialized nutritional rewards to ants. We also
tested the possibility of directed seed dispersal by ants by examining
whether abandonment of dummy seeds is directionally biased towards
suitable location using an achlorophyllous plant whose seeds do not have
elaiosomes.

We used the plant \emph{Monotropastrum humile} (D. Don) H. Hara
(Ericaceae) and the ant \emph{Nylanderia flavipes} Smith (Formicidae) to
test the hypothesis. \emph{M. humile} is an achlorophyllous and
myco-heterotrophic plant that inhabits forest floors in the temperate
regions of Asia (Ohashi et al. 2016). The plant is about 10 cm height
and does not perform photosynthesis and parasitizes the fungi
of\emph{Russula} and \emph{Lactarius} species of Russulaceae (Bidartondo
and Bruns, 2001). \emph{M. humile} is a full myco-heterotrophic species
(Merckx, 2013). Seeds are smaller than 0.5 mm in length (Uehara \&
Sugiura, 2017) and require their host fungi for germination, thus, seed
destination is a determinant of survival. Their host, the mycorrhizal
fungi of \emph{Russula} species, inhabit the shallow areas of the forest
floor (Rachel, 2004; Courty et al., 2008), and Imamura and Kurogi (2003)
reported that \emph{M. humile} roots inhabit the forest floor to a depth
of 5--10 cm. For seed dispersal, \emph{M. humile} uses the insects
wandering on the ground when their berries fall to the ground during the
fruiting season. Cockroaches (Uehara \& Sugiura, 2017) and camel
crickets (Suetsugu, 2017) have been reported as their seed dispersers.
According to these studies, the \emph{M. humile} berries fall to the
ground during the fruiting season, the wandering insects are presumed to
consume pulpy flesh around the seeds and consequently contribute to the
dispersal of seeds involved in their foraging behavior.

Although \emph{M. humile} seeds have no elaiosomes and seem to be
non-myrmecocohorous plants (Suetsugu, 2017; Uehara \& Sugiura, 2017),
Suetsugu (2017) and Uehara and Sugiura (2017) reported that several ant
species, including \emph{N} . \emph{flavipes} , frequently visited
the\emph{M. humile} fruits. In addition, we have observed, by using a
fixed-point camera, that \emph{N. flavipes} ants were attracted to the
fruits and seeds of \emph{M. humile} and that a part of the seeds,
although not many, were certainly carried by them\emph{.} Since the
seeds are coated with a phlegmatic organic layer, it is possible
that\emph{M. humile} attract the ants with the fruit- and seed odors and
that their seed are carried by the ants. The seeds might be abandoned
after the outer components are consumed. Consequently, the seed could be
dispersed to the suitable, host-inhabited sites on the forest floor.

To validate directed seed dispersal by ants, it is necessary to track
where the seeds are carried to and discarded. However, the seeds of
myrmecochorous plants are small and difficult to track in the field.
Thus, at first, we performed the bioassay using intact and odorless
seeds to verify whether the seed odors of \emph{M. humile} are key
signals for dispersal by ants. Afterwards, bioassays were performed
using small pieces of the paper coated with the odor component
of\emph{M. humile} seeds as dummy seeds. We tested whether the dummy
seeds were carried into the nests and were carried out of the nests by
the ants.

\textbf{2 \textbar{} MATERIALS AND METHODS}

\textbf{2.1 \textbar{} Plant and ant materials}

We used the ant species \emph{N. flavipes} , which is polyphagous and
collects plant seeds in the field (Tanaka \& Tokuda 2016; Hosoishi et
al., 2019). Ten \emph{N. flavipes} colonies were collected from June to
July 2019 from the sites at the Yuminariyama mountain and the Hokkaido
University of Education Asahikawa Campus in Hokkaido, Japan. All the
colonies collected were queenless, consisting of approximately 60
workers and numerous eggs, pupae, and larvae. The colonies of \emph{M.
humile} and \emph{N. flavipes} coexist sympatrically at Yuminariyama
mountain and we also collected \emph{M. humile} fruits at Yuminariyama
mountain in August 2019 and stored them in a screw tube at 4 .

The ant colonies were kept in a container that consisted of an
artificial nest and a foraging arena (Figure 1). The polypropylene box
(200 mm length \selectlanguage{ngerman}× 136 mm width × 68 mm height) was used as a foraging
arena. Fluon was applied to the upper 3 cm inside the feeding arena to
prevent ant escape. A vinyl tube (10 mm diam. × 120 mm length) was used
as an artificial nest and connected to the side wall of the container at
15 mm height. To prevent the artificial nest from drying out, an
absorbent piece of cotton was inserted at the wall-side of the nest and
moistened with distilled water. The absorbent cotton was replaced every
two weeks.

The feeding arena was divided into four equivalent sections to
investigate whether the ants discard seeds in areas with different
conditions. The four sections were respectively filled with dry glass
beads (1.5-2.5 mm diam.), moistened glass beads, dry cotton wool, and
moistened cotton wool.

Glass beads simulated an environment with inorganic substances like sand
or small pebbles in the forest floor, which were filled with about 10 g
of beads in each section. Cotton wool simulated an environment with
organic substances such as fallen and decomposing litter, which were
filled with about 0.25 g of cotton in each section. The entrance of the
nest was placed at the center of the arena so that the four sections
were equally aligned for the ants. The entrance of nest floats was
bridged with filter paper at the center of the feeding arena (Figure
1a). In the bioassay, the seeds or the dummy seeds were placed at the
center of the arena near where the bridge connected the different
sections (Figure 1b).

To moisten two of the sections, 5 mL of distilled water was added every
three days. All the containers were kept indoors at 25 \selectlanguage{english}. The ants were
fed with tuna and honey.

\textbf{2.2 \textbar{} Volatile collections and chemical analyses}

The volatiles emitted from \emph{M. humile} seeds were collected using
solid phase micro extraction (SPME) using a 66-\selectlanguage{greek}µ\selectlanguage{english}m PDMS/DVB fiber
(Supelco, Bellefonte, PA, USA). The seeds with pulp were placed in 4-mL
glass vials and sealed with aluminum foils. The SPME fiber was inserted
into the covered vial and volatiles were sampled for 1 h at room
temperature. Blank assays with empty vials were performed before each
volatile collection session. Three independent samples were collected
and analyzed.

Immediately after sampling, the SPME fiber was injected into the
split/splitless injector of a gas chromatograph (GC17A; Shimadzu, Japan)
equipped with a DB-WAX column (30 m length \selectlanguage{ngerman}× 0.25 mm inner diameter ×
0.25 \selectlanguage{greek}µ\selectlanguage{english}m film thickness; J \& W Scientific Inc., Folsom, CA, USA) and
mass spectrometry detectors (QP5000; Shimadzu, Japan) with electron
impact ionization (70 eV). The oven temperature was maintained at 40 \selectlanguage{ngerman}°C
for 5 mins, programmed to increase to 220\selectlanguage{english} degC at a rate of 10 degC
min\textsuperscript{-1}, and held isothermal for 10\selectlanguage{english} min. The SPME fiber
was desorbed in splitless mode for 1\selectlanguage{english} min, while the injector and
interface temperatures were 220\selectlanguage{english} degC. Helium was used as carrier gas, and
the column head pressure was 100\selectlanguage{english} kPa. Volatile compounds were
tentatively identified by the matches with the NIS mass spectral
database, and identifications were confirmed by matching mass spectra
and Kovat's retention index with those of commercially available
authentic standards.

\textbf{2.3 \textbar{} Bioassay using the \emph{M. humile} seeds}

We performed a bioassay using 20 fresh untreated or solvent-treated
seeds of \emph{M. humile} . For the treated seeds, 20 fresh seeds
of\emph{M. humile} were soaked in ethanol (99.5\%, Wako Pure Chemical
Industries, Ltd.) for 30 min. Four replicate colonies resulted in a
total of 80 seeds for each treatment. Untreated and treated seeds were
assayed separately to avoid odor transfer. The colonies were starved
(fed with water only) for 72 h before performing the assays. After the
seeds were set at the center of the arena, the number of seeds carried
to the nest by the ants was recorded every 15 min for 90 min.

\textbf{2.4 \textbar{} Bioassay using the dummy seeds of filter paper}

We performed a bioassay using dummy seeds made of square bits filter
paper of 2 mm x 2 mm. The dummy seeds were soaked with the volatile
compounds of the \emph{M. humile} seeds (Table 1). These compounds were
purchased from Wako Pure Chemical Industries (Osaka, Japan) or Tokyo
Kasei (Tokyo, Japan) with \textgreater{}98.0\% purities.

In this dummy-seed assay, 20 pieces of filter paper numerically numbered
with a carbon pencil to track the outcome of the dummy seeds were used.
The odor treatment in this assay consisted of seven conditions, that is,
each of the six odors identified (six conditions) and a mixture of
equivalent amounts of the six odors (one condition). Each odor component
was diluted to a final concentration of 100 ng / \selectlanguage{greek}µ\selectlanguage{english}L using the 99.5\%
ethanol as a solvent. Twenty pieces of filter paper were soaked with 20
\selectlanguage{greek}µ\selectlanguage{english}l odor solution (1 \selectlanguage{greek}µ\selectlanguage{english}l for each filter paper). The dummy seeds soaked
only with the solvent were used as the control. Each of the odor
treatments was replicated six times using the six different colonies,
and each colony experienced each treatment only once. The colonies were
starved (fed with water only) for 72 h before performing the assays.

The order of the odor treatments that were served to each colony was
randomized using the 'sample' function of the base package of R 3.5.2
for Mac OSX (R core team, 2018). Dummy seeds were placed at the center
of the arena and the number of dummy seeds carried to the nest by the
ants was recorded every 15 min for 90 min. After 72 h from setting the
dummy seeds, the number of dummy seeds inside the nest and discarded
outside the nest was recorded. For discarded dummy seeds, we also
recorded their destination and the numerical number on the dummy seeds.

\textbf{2.5 \textbar{} Statistical analysis}

All analyses were executed with R 3.5.2 for Mac OSX. The package
'tidyverse' was used for data shaping and arrangement. The factors
affecting the number of seeds (or dummy seeds) carried by ants were
analyzed using a generalized linear mixed model (GLMM) and likelihood
ratio test. We used the zero inflated Poisson distribution with the
function glmmTMB from the package 'glmmTMB'. In this analysis, colony
identity was set as the random effect and the odor conditions were the
explanatory variables. Chi-square tests were performed to examine
whether the destination of the dummy seeds was biased.

\textbf{RESULTS}

\textbf{3.1 \textbar{} Volatile chemical composition of \emph{M.
humile}seeds}

Chemical analyses of seed volatiles revealed the presence of several
substances. By comparing the results with blank analyses, we found eight
compounds that were derived from the \emph{M. humile seeds} (Figure 2).
Among eight peaks, 6 peaks were identified as isobutyl alcohol, isoamyl
alcohol, isoamyl hexanoate, linalool, isobutyric acid, and \selectlanguage{greek}α-\selectlanguage{english}terpineol
(Table 1). Peaks 2 and 8 could not be identified from the obtained mass
spectra.

\textbf{3.2 \textbar{} Bioassay using \emph{M. humile} seeds}

The ants carried away the \emph{M. humile} seeds in 90 min. Table 2
shows the number of \emph{M. humile} seeds carried to the nest within 90
min. Nine of the 80 untreated seeds were carried to the nest although
none of the treated seeds were carried to the nest (Figure 3, Table S1).
GLMM analysis revealed that untreated seeds were carried away
significantly more than treated seeds (Table S2).

\textbf{3.3 \textbar{} Bioassay using filter paper as dummy seed}

Ants did not carry the dummy seeds to the nest in 90 min but did in 72 h
(Table S3). A total of 30 out of 840 odor-treated dummy seeds were
carried to the nest. Comparing the number of removed dummy seeds for
each odor treatment, the six-odor-mixture treatment were carried most to
the nest according to the GLMM analysis (Table 2, Figure 4).

Of the 30 dummy seeds carried, 17 were discarded (Figure 5). The rest of
the seeds carried was left inside the nests. The chi-square test was
performed on the six categories and included the four compartments, the
empty area, and the nest, and there was a significant bias (P = 0.0002).
When we performed the chi-square test on four compartments of the dummy
seeds, glass beads, water-moistened glass beads, cotton wool, and
water-moistened cotton wool, the distribution was also significantly
biased (P = 0.0032) and most of seeds were discarded on moistened glass
beads (Figure 5).

\textbf{4 \textbar{} DISCUSSION}

\textbf{4.1 \textbar{} Dispersal of elaiosome-less seeds by ants}

In this study, we first verified that \emph{M. humile} seeds without
elaiosomes surely attracted ants. We have confirmed that 9 out of 80
untreated seeds were carried to the ant nest, whereas no treated seeds
were carried (Table S1). We also found that by using dummy seeds coated
with the odor components of \emph{M. humile} seeds, those dummy seeds
treated with a mixture of 6 major components were carried to the nest by
the ants, although the dummy seeds treated with the solvent were not
carried away (Table S3). Among the 6 main odor components, linalool and
\selectlanguage{greek}α-\selectlanguage{english}terpineol were also identified as floral scents of \emph{M. humile} ,
but the other 4 components were not identified (Kubo \& Ono, 2014).
These results indicate that \emph{M. humile} seeds attracted ants by
their seed or fruit specific odor mixture. In the pepper family
plant,\emph{Peperomia macrostachya} , the mixture of seed odors
efficiently attracts the ant, \emph{Camponotus femoratus} (Youngstead et
al., 2007). Since the volatile seed odors would have disappeared in 72
h, the dummy seeds were abandoned outside the nest. Similarly, the seeds
are likely to be abandoned after the outer fruity coating components are
consumed. Therefore, even in the absence of elaiosomes, some plants,
including\emph{M. humile} , could disperse their seeds through the ants
using a specific blend of odor components.

Hence, ants are thought to participate in the seed dispersal of \emph{M.
humile,} in addition to herbivorous insects, such as cockroaches and
camel crickets (Suetsugu 2017; Uehara \& Sugiura 2017), and the seed
dispersal by ants could be beneficial for the achlorophyllous species.
With regards to the seed dispersal by ant, the seed-discards or
left-inside-the-nest indicated that dispersal by ants should be
important for plant seeds of such achlorophyllous plants as \emph{M.
humile} . The plant does not depend solely on the myrmecochory, as
Suetsugu 2017 reported many of seeds have been detected from the
herbivorous insects' excretion. Because the number of seeds carried in
our assay is relatively small, myrmecochory of the species could be one
aspect of their zoochory.

\textbf{4.2 \textbar{} Implication by the biased discard of dummy seeds}

Although we could not track where the genuine seeds discarded because of
their small-size, the author M. Y. observed a part of the genuine seeds
carried into the nests are left in a corner of the nest (data not
shown). Thus, the ant nest could be one of the seed directions. The
dummy seeds coated with mixed odors were carried into the nest and
subsequently discarded out of the nest by the ants. Although 13 dummy
seeds were left inside the nest (17 were discarded outside), it appears
that seeds staying in the nest can also be considered as dispersed
since\emph{N. flavipes} nests are located under litter or on rotting
trees (Kallal \& LaPolla, 2012). The direction of discards was biased,
and the dummy seeds were discarded mostly onto the water-moistened glass
beads. In the ant species \emph{Myrmica rubra} , it is known that
workers discard inert items and corpses in different destinations (Diez
et al., 2012). In our study, it was also observed that ant corpses were
dumped into cotton wool compartments, where the dummy seeds were not
dumped. These results suggests that the ants differently use the discard
destinations depending on what they are discarding, and the seeds could
be discarded specific locations by the ants.

Mycorrhizal fungi of \emph{Russula} species as the hosts of \emph{M.
humile} inhabit the top shallow layer of the forest floor (Rachel, 2004;
Courty et al., 2008), and Imamura and Kurogi (2003) reported
that\emph{M. humile} roots inhabit 5-10-cm depth as parasites on
Russulaceae fungi. Thus, where the ant nests are located and where the
seeds are discarded (moist soil near the surface) is considered to be
close to their hosts and to be suitable for germination, growth and
survival of the \emph{M. humile} seeds although the mycorrhizal roots
have not been quantified before.

The dummy seeds were left inside the nest or most frequently discarded
onto the water-moistened glass beads section. This observation can
support two seed-dispersal hypotheses: 1) seeds relocated to the moist
environments including ant nests can escape desiccation and have
enhanced germination and seedling survival; 2) seeds relocated to ant
nests can also escape predators (modified from Hanzawa et al 1988).

Our results suggest that seed dispersal of \emph{M. humile} by ants is
directional. Directed seed dispersal and its ecological significance
have rarely been verified in achlorophyllous herbaceous species
(reviewed by Giladi, 2006). With regards to myco-heterotrophic plants,
large parts of them produce dust-like and anemochorous seeds, which
require host fungus in their germination, and which could disperse over
long distances (Merckx, 2013). Thus, there is a trade-off of seed
dispersal between distance and direction. Since the genus
\emph{Monotropastrum} , producing fleshy fruit, is thought to evolve
from the relative and anemochorous genus \emph{Monotropa} , producing
capsule fruit (Bidartondo \& Bruns, 2001), this speciation is considered
as from anemochory (distance dispersal) to zoochory including
myrmecochory. Therefore, the verification of the role of
myco-heterotrophic herbaceous species in the temperate forests of Asia,
and of the role of seeds without specialized rewards for ants, presents
a new aspect of directed seed dispersal of achlorophyllous and
myco-heterotrophic plants involving ants.

\textbf{5 \textbar{} CONCLUSION AND CAVEATS}

We verified that reward-less seeds of achlorophyllous and
myco-heterotrophic \emph{M. humile} were certainly dispersed by ants. A
part of the seeds was left in the ant nests and the other part of the
seeds could be discarded outside the nest directionally although we
could not evaluate the fate of genuine seeds but evaluated that of dummy
seeds. In future studies, we must track the fate of the genuine seeds
and investigate spatial distribution of the plants and the ant nests to
validate the directed seed dispersal of the achlorophyllous
plant\emph{M. humile.} An expected way of bioassay is to copy various
conditions of the forest floors and to examine the directed dispersal by
ants in detail.

\textbf{ACKNOWLEDGMENTS}

We thank Takafumi Mizuno and Toshiharu Akino for their help with GC-MS
analyses. This work was supported by JSPS KAKENHI Grant no. 18H02512. We
would like to thank Editage (www.editage.com) for English language
editing.

\textbf{Author contributions}

A.I. conceived the study, conducted analyses, and wrote the manuscript.
K.Y. conducted experiments. M.K. Hojo performed a gas chromatograph
analysis and check the statistical analyses.

\textbf{Conflict of interests}

There are no conflicts of interest to declare.

\textbf{Data availability}

All relevant data are within the paper and its Supporting Information
files.

\textbf{REFERENCES}

Bidartondo, M. I., \& Bruns, T. D. (2001). Extreme specificity in
epiparasitic \emph{Monotropoideae} (Ericaceae): widespread phylogenetic
and geographical structure. \emph{Molecular Ecology} 10, 2285--2295.

Brew, C. R., O'Dowd D. J., \& Rae I. D. (1989). Seed dispersal by ants:
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\begin{longtable}[]{@{}lllll@{}}
\toprule
Peak No. & Components & Molecular formula & Molecular weight & Retention
Index\tabularnewline
1 & Isobutyl alcohol 99.0\% & C\textsubscript{4}H\textsubscript{10}O &
74.1 & 1073\tabularnewline
3 & Isoamyl alcohol 99.0\% & C\textsubscript{5}H\textsubscript{12}O &
88.2 & 1208\tabularnewline
4 & Isoamyl hexanoate 98.0\% &
C\textsubscript{11}H\textsubscript{22}O\textsubscript{2} & 186.3 &
1480\tabularnewline
5 & Isobutyric acid 99.0\% &
C\textsubscript{4}H\textsubscript{8}O\textsubscript{2} & 88.1 &
1573\tabularnewline
6 & Linalool 98.0\% & C\textsubscript{10}H\textsubscript{18}O & 154.2 &
1588\tabularnewline
7 & \selectlanguage{greek}α- \selectlanguage{english}terpineol 98.0\% & C\textsubscript{10}H\textsubscript{18}O &
154.2 & 1725\tabularnewline
- & Mixture of six components & & &\tabularnewline
\bottomrule
\end{longtable}

Table 1 Odor components identified by GC/MS analysis. We used samples of
these components in our bioassay.

Peak numbers correspond to those in Figure 2

Table 2 Results of the generalized linear mixed model (GLMM) and
likelihood ratio test on the number of dummy seeds carried by ants with
a zero-inflated Poisson distribution. The colony identity was set as the
random effect. The odor component was the explanatory variable.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Conditional model & Variance & Std. Div. & & &\tabularnewline
(Intercept) & 8.52e-12 & 2.92e-06 & & &\tabularnewline
Components & Estimate & Std. Error & z value &
Pr(\textgreater{}\textbar{}z\textbar{}) &\tabularnewline
Isobutyl alcohol & -22.325 & 3.17e+04 & -0.001 & 0.999 &\tabularnewline
Isoamyl alcohol & -0.866 & 7.78e-01 & -1.113 & 0.266 &\tabularnewline
Isoamyl hexanoate & -0.866 & 7.78e-01 & -1.113 & 0.266 &\tabularnewline
Isobutyric acid & -22.325 & 3.17e+04 & -0.001 & 0.999 &\tabularnewline
Linalool & -0.228 & 5.54e-01 & -0.411 & 0.681 &\tabularnewline
\selectlanguage{greek}α- \selectlanguage{english}terpineol & -0.920 & 7.50e-01 & -1.226 & 0.220 &\tabularnewline
Mixture of six components & 1.370 & 2.31e-01 & 5.927 & \textless{} 0.001
& ***\tabularnewline
Ethanol as control & -22.325 & 3.17e+04 & -0.001 & 0.999
&\tabularnewline
& Estimate & Std. Error & value &
Pr(\textgreater{}\textbar{}z\textbar{}) &\tabularnewline
Estimate of Zero-inflation model & & & & &\tabularnewline
(Intercept) & -1.52 & 1.14 & -1.33 & 0.18 &\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
\FloatBarrier
\end{document}

