Seed dispersal by ants is an important means of migration for plants. Although many 34 myrmecochorous plants have seeds containing elaiosome, a nutritional reward for ants, some 35 non-myrmecochorous seeds without elaiosomes are also dispersed by ant species. However, the 36 mechanism by which seeds without elaiosomes enable efficient dispersal by ants is scarcely 37 investigated. The seeds of the achlorophyllous and myco-heterotrophic herbaceous plant 38 Monotropastrum humile are very small without elaiosomes and require a fungal host for 39 germination and survival. We performed a bioassay using seeds of M. humile and the ant 40 Nylanderia flavipes to demonstrate ant-mediated seed dispersal. We also analyzed the volatile 41 odors emitted from M. humile seeds and conducted bioassays using dummy seeds coated with 42 seed volatiles. Although elaiosomes were absent from the M. humile seeds, the ants carried the 43 seeds to their nests. They also carried the dummy seeds coated with the seed volatile mixture to 44 the nest, and left some dummy seeds inside the nest and discarded the rest of the dummy seeds 45 outside the nest with a bias toward locations with moisture conditions, which might be 46 conducive to germination. We concluded that seeds of M. humile were dispersed by the ants, 47 and that seed odors were sufficient to induce directed dispersal even without elaiosomes. It is 48 probable that the fleshy fruit producing genus Monotropastrum evolved from the related 49 anemochorous genus Monotropa, which produces capsule fruit. This transformation from 50 anemochory to myrmecochory presents a novel evolutionary pathway toward ant-mediated seed 51 dispersal in an achlorophyllous plant.

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.