Detecting rare terrestrial orchids and associated plant communities from soil samples with eDNA methods

The complex biology and specialized relationships between orchids and both fungi and pollinators can complicate orchid conservation and management. Some terrestrial orchids are able to undergo prolonged periods of vegetative dormancy, likely depending on specific fungi for surviving belowground, which pose an additional challenge in monitoring and targeted conservation. This study tests the feasibility of using environmental DNA to detect orchid tissue from soil samples as a tool in surveillance of terrestrial orchid populations. We developed species-specific probe-based qPCR assays targeting ITS regions of the rare orchids Anacamptis pyramidalis and Cephalanthera rubra and tested these on 183 soil samples from four localities with current or previously recorded occurrence of the species. A subset of samples (22) were additionally analyzed by ITS2 metabarcoding for a small-scale comparison between methods, as well as for characterizing plant communities associated with the orchids. Although the developed assays were highly efficient and specific, orchid DNA was only detected in 21 of the tested soil samples, all within close vicinity to the nearest orchid, and most within 10 cm. No orchid DNA was detected in any sample from the localities with orchid occurrence a few years prior to the study. The metabarcoding analysis only detected orchid DNA in a single sample but proved informative in characterizing the plant communities at the four tested localities. The developed assays would be highly useful to apply in studies aiming at gaining knowledge on dormancy and number of belowground individuals in populations of high conservation concern.