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Characterizing genetic diversity and structure of populations is essential for the effective conservation of threatened species.Orchis patenssensu lato is a narrowly distributed tetraploid species with a disjunct distribution (i.e., Northern Italy, North Africa and the Canary Islands), which is facing a severe decline. In this study, we evaluated levels of genetic diversity and population structuring using 12 new nuclear microsatellite markers. Our analyses of genetic differentiation based on multiple approaches (Structure analysis, PCA analysis, andF-statistics using the ploidy-independentRho-index) showed that gene flow is low across the range ofO. patens s.l., particularly in the Canary Islands. Clear differences in allele frequencies between Italy, Algeria and the Canary Islands underlie the genetic differentiation retrieved. Our study provides support for the recognition ofO. canariensisas a sister species toO. patensand the separation of the Italian populations as a new subspecies ofO. patens. Despite the high heterozygosity values found in all populations (ranging from 0.4 to 0.7), compatible with the tetraploid status of the species, small population sizes and reduced gene flow will be likely detrimental for the different populations in the long term, and we recommend immediate conservation actions to counteract further fragmentation and population decline.

The loss of orchid habitats has increased the investment on orchid conservation efforts to reduce the risk of extinction of rare species. In northwestern Italy, orchids are among the most threatene...

As orchids rely on their mycorrhizal fungi for nutrient supply, their spatial range is dependent on the distribution of orchid mycorrhizal (OM) fungi. We addressed possible correlations between mycorrhizal specificity and the geographic distribution of orchids and OM fungi in three populations of the rare sister species Orchis patens and O. canariensis. Metabarcoding of the fungal ITS2 region indicated that, although adult plants of either species were colonized by several ceratobasidioid, tulasnelloid, sebacinoid and serendipitoid fungi, the mycobiont spectra were dominated by Tulasnella helicospora (which occurred in 100% of examined plants with high read numbers), which is a globally distributed fungus. In vitro assays with a T. helicospora isolate obtained from O. patens indicated the effectiveness of this OM fungus at germinating seeds of its native host. At a local scale, higher read numbers for T. helicospora were found in soil samples collected underneath O. patens roots than at locations unoccupied by the orchid. Although these findings suggest that the geographical pattern of the main fungal symbiont does not limit the distribution of O. patens and O. canariensis at this scale, the actual causal link between orchid and OM fungal occurrence/abundance still needs to be better understood.