Identification of pollinators of lesser twayblade Neottia cordata with DNA barcoding reveals strong links with pine forest‐related fauna.

Many European terrestrial orchids are in decline. To curb this negative trend and preserve remaining populations, more ecological knowledge is needed. Surprisingly little is known yet about the identity and efficiency of pollinators of lesser twayblade <italic>Neottia cordata</italic>, a small terrestrial orchid species associated with pine trees through joint mycorrhizae. We identified its small and inconspicuous pollinators with DNA barcoding and assessed its fruit set with the help of observations submitted to various nature platforms. We caught pollinators on Terschelling in the Netherlands during the flowering season of 2013 and 2014. Insects were identified with 28S and COI sequences obtained from both fresh and museum‐preserved material identified by specialists. Several pollinators were detected, belonging to either parasitoid wasps (Braconidae) and spider wasps (Pompilidae), active during sunny periods, or fungus gnats (Mycetophilidae and Sciaridae), active during overcast conditions. Combined pollinator efforts resulted in a continuous average fruit set above 70% in Europe over the past 135 years. The parasitoid wasps were identified as <italic>Bracon pineti</italic> and <italic>Blacus</italic> sp., which strongly depend on pine trees for their prey. The fungus gnats were identified as <italic>Austrosciara hyalipennis, Trichosia lengersdorfi, Allodia lugens</italic> and <italic>Phronia forcipata</italic>. All four species are known to deposit their eggs in the vicinity of fruiting bodies of cone caps <italic>Strobilurus stephanocystis</italic>, mushrooms growing on pine cones, as their larvae feed on the fungi. <italic>Priocnemis pertubator</italic> and <italic>Anoplius viaticus</italic> (Pompilidae) are also important pollinators. Results obtained show that lesser twayblade is even more intricately linked to pine forest ecosystems than previously thought. Management of growth sites of <italic>N. cordata</italic> should be tailored towards preserving pine tree forests with plots that are constantly being rejuvenated to generate young pine trees and prevent succession towards birch–oak forests. Such forests not only provide mycorrhizal fungi and layers of needle litter needed for germination and development of the seeds of this orchid but also pinewood‐decaying fungi that provide egg deposition sites and food for its pollinators. [ABSTRACT FROM AUTHOR]