Drosophilid pollination in mycoheterotrophic orchids reveals a brood-site deception-mutualism continuum and...

Although orchid pollination is often highly specialized, fully mycoheterotrophic orchids are generally thought to favor autonomous self-pollination because of carbon limitation, shaded habitats, and patchy population structure. Here, we investigated six nectarless, fully mycoheterotrophic Gastrodia species in Japan using long-term pollinator observations and hand-pollination experiments, together with phylogenomic analyses of all six species, floral scent analyses of three species, and larval rearing experiments in four species. All six species were self-compatible but incapable of autonomous selfing and relied on drosophilid flies for pollination. Pollinator assemblage dissimilarity was significantly correlated with interspecific genetic differentiation, indicating phylogenetically structured pollinator use. Fruit-feeding drosophilids pollinated all species, whereas mycophagous drosophilids contributed substantially to pollination only in G. foetida and G. nipponica. In these two species, larvae frequently developed in decaying floral tissues, consistent with brood-site mutualism. In G. confusa and G. pubilabiata, larval survival was sporadic and humidity-dependent, indicating an intermediate condition between brood-site deception and mutualism. Floral scents of three representative species were dominated by fermentation-related volatiles, but blend composition differed among species. Together, these findings reveal a deception-mutualism continuum within Gastrodia and suggest that evolutionary history, together with floral scent variation, helps shape pollinator interactions in these orchids.