DOI: 10.5061/dryad.dfn2z3516
title: Odor of achlorophyllous plants’ seeds

description: Seed dispersal by ants is an important means of migration for plants. Many
 myrmecochorous plants have specialized appendages in their seeds called
 elaiosome, which provide nutritional rewards for ants, and enable
 effective seed dispersal. However, some non-myrmecochorous seeds without
 elaiosomes are also dispersed by ant species suggesting that additional
 mechanisms other than elaiosomes for seed dispersal by ants. The seeds of
 the achlorophyllous and myco-heterotrophic herbaceous plant Monotropastrum
 humile are very small without elaiosomes, we investigated whether odor of
 the seeds could mediate seed dispersal by ants. 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 the seeds to their nests. 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 toward specific locations, which might be
 conducive to germination. We concluded that, in M. humile seeds, volatile
 odor mixtures were sufficient to induce seed carrying behavior by the ants
 even without elaiosomes.  
description: We performed
 bioassays using seeds of <i>M. humile,</i> 20 fresh untreated
 or solvent-treated seeds (odourless seeds) per turn of the bioassay. For
 the solvent treated seeds, 20 fresh seeds of <i>M. humile</i>
 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 solvent treated seeds were assayed separately to
 avoid odor transfer, in which the treatment order for each colony was
 randomized by the function 'sample' 4.0.3 for Mac OSX (R core
 team, 2018). The colonies were starved (only given water) for 72 h before
 performing the assays. After the seeds were set at the centre of the
 arena, the number of seeds carried to the nest by the ants was recorded
 every 15 min for 90 min. We performed a
 bioassay using dummy seeds made of a square bit of filter paper (2 mm × 2
 mm). Although the size of dummy seeds were larger than the real seeds
 (Figure 1), the size is the minimum to track the outcome of the dummy
 seeds. The dummy seeds were numerically numbered with a carbon pencil,
 enabling to refer the treatment conditions for each assay. The dummy seeds
 were soaked with the volatile compounds of <i>M. humile</i>
 seeds (Table 1). These compounds were purchased from Wako Pure Chemical
 Industries (Osaka, Japan) or Tokyo Kasei (Tokyo, Japan) with &gt;98.0%
 purity.               In this dummy-seed
 assay, 20 pieces of filter paper were set for each assay. The odor
 treatment in this assay consisted of eight conditions: each of the six
 odors identified alone (six conditions), a mixture of equivalent amounts
 of the six odors (one condition), and the control condition (ethanol
 alone). Each odor component was diluted to a final concentration of 100
 ng/µL using 99.5% ethanol as a solvent. Twenty pieces of filter paper were
 soaked in 20 µL odor solution (1 µL for each filter paper). Each of the
 eight odor treatments was replicated six times using six different
 colonies, and each colony experienced each treatment only once. The
 colonies were starved (only given water) for 72 h before performing the
 assays. During each turn of assay (72 h) ant colony were supplied nothing
 other than the dummy seeds. The interval between assay turns for each
 colony was about a week in minimum.