Plant species that are capable of propagating clonally are expected to be less vulnerable to habitat fragmentation due to their long life span. Cypripedium calceolus L. is a rare, clonal, long-lived orchid species. It has suffered marked decline because of habitat loss and fragmentation and over-collection, yet an IUCN report on this species does not regard fragmentation as a major threat to the species. We applied 13 nuclear microsatellites and cpDNA sequences to identify the patterns of population structure, genetic diversity and connectivity of six remnant local populations of C. calceolus in highly fragmented Gdańsk Pomerania region (N Poland). Despite severe (80%) loss of localities in the studied area we found that the local populations retain high levels of clonal (R 0.86–1) and genetic diversity (H_e = 0.572). However, their differentiation is relatively high (F_ST = 0.132 for nuclear SSR and F_ST = 0.363 for cpDNA) despite close geographic proximity (0.6–57 km). Bayesian clustering classified populations according to their geographic origin with little admixture. Low genetic connectivity between the remnant populations shows that the current gene flow is too low to serve as a cohesive force in a fragmented habitat, which may impede a quick response to environmental change. The species’ ability to retain ancestral variation may help withstand fragmentation, but in the light of observed extirpation rate it should be rather considered as a factor that only delays local populations’ extinction. This leads to the conclusion that habitat loss and fragmentation should be regarded as a real threat to stability of C. calcelolus populations.

Summary: While orchids germinate thanks to carbon from their symbiotic fungi, variable carbon exchanges exist between adult orchids and their mycorrhizal fungi. Although some truly autotrophic orchids reward their fungi with carbon at adulthood, some species remain achlorophyllous and fully dependent on fungal carbon (mycoheterotrophy). Others are photosynthetic but also import fungal carbon: The so‐called mixotrophic (MX) orchids rely on fungi of diverse taxonomy and ecology. Here, we classify MX nutrition of orchids into three types. Type I mixotrophy associates with diverse Asco‐ and Basidiomycota that are either saprotrophic or ectomycorrhizal, entailing enrichment of the orchids in ² H, ¹³ C, and ¹⁵ N. The two other types associate with rhizoctonias, a polyphyletic assemblage of Basidiomycotas that is ancestrally mycorrhizal in orchids. Type II mixotrophy associates with rhizoctonias that secondarily evolved into saprotrophic or ectomycorrhizal ecology, and thus enrich the orchid in ² H, ¹³ C, and ¹⁵ N. Type III mixotrophy, which remains debated, associates with rhizoctonias that have retained their ancestral lifestyle, that is saprotrophic and/or endophytic in nonorchids, and only entail orchid enrichment in ² H and ¹⁵ N. Based on a case study of achlorophyllous variants in Mediterranean Ophrys and on published data, we discuss the distinct nature and research perspectives of type III mixotrophy.

Background: Mycorrhiza is a ubiquitous form of symbiosis based on the mutual, beneficial exchange of resources between roots of autotrophic (AT) plants and heterotrophic soil fungi throughout a complex network of fungal mycelium. Mycoheterotrophic (MH) and mixotrophic (MX) plants can parasitise this system, gaining all or some (respectively) required nutrients without known reciprocity to the fungus. We applied, for the first time, an ecological stoichiometry framework to test whether trophic mode of plants influences their elemental carbon (C), nitrogen (N), and phosphorus (P) composition and may provide clues about their biology and evolution within the framework of mycorrhizal network functioning. Results: We analysed C:N:P stoichiometry of 24 temperate orchid species and P concentration of 135 species from 45 plant families sampled throughout temperate and intertropical zones representing the three trophic modes (AT, MX and MH). Welch’s one-way ANOVA and PERMANOVA were used to compare mean nutrient values and their proportions among trophic modes, phylogeny, and climate zones. Nutrient concentration and stoichiometry significantly differentiate trophic modes in orchids. Mean foliar C:N:P stoichiometry showed a gradual increase of N and P concentration and a decrease of C: nutrients ratio along the trophic gradient AT < MX < MH, with surprisingly high P requirements of MH orchids. Although P concentration in orchids showed the trophy-dependent pattern regardless of climatic zone, P concentration was not a universal indicator of trophic modes, as shown by ericaceous MH and MX plants. Conclusion: The results imply that there are different evolutionary pathways of adaptation to mycoheterotrophic nutrient acquisition, and that the high nutrient requirements of MH orchids compared to MH plants from other families may represent a higher cost to the fungal partner and consequently lead to the high fungal specificity observed in MH orchids.

Some plants abandoned photosynthesis and developed full dependency on fungi for nutrition. Most of the so-called mycoheterotrophic plants exhibit high specificity towards their fungal partners. We tested whether natural rarity of mycoheterotrophic plants and usual small and fluctuating population size make their populations more prone to genetic differentiation caused by restricted gene flow and/or genetic drift. We also tested whether these genetic characteristics might in turn shape divergent fungal preferences. We studied the mycoheterotrophic orchid Epipogium aphyllum, addressing the joint issues of genetic structure of its populations over Europe and possible consequences for mycorrhizal specificity within the associated fungal taxa. Out of 27 sampled E. aphyllum populations, nine were included for genetic diversity assessment using nine nuclear microsatellites and plastid DNA. Population genetic structure was inferred based on the total number of populations. Individuals from 17 locations were included into analysis of genetic identity of mycorrhizal fungi of E. aphyllum based on barcoding by nuclear ribosomal DNA. Epipogium aphyllum populations revealed high genetic diversity (uHe = 0.562) and low genetic differentiation over vast distances (F_ST = 0.106 for nuclear microsatellites and F_ST = 0.156 for plastid DNA). Bayesian clustering analyses identified only two genetic clusters, with a high degree of admixture. Epipogium aphyllum genets arise from panmixia and display locally variable, but relatively high production of ramets, as shown by a low value of rarefied genotypic richness (R_r = 0.265). Epipogium aphyllum genotype control over partner selection was negligible as (1) we found ramets from a single genetic individual associated with up to 68% of the known Inocybe spp. associating with the plant species, (2) and partner identity did not show any geographic structure. The absence of mosaicism in the mycorrhizal specificity over Europe may be linked to preferential allogamous habit of E. aphyllum and significant gene flow, which tend to promote host generalism.

Lady’s slipper orchid (Cypripedium calceolus) serves as a flagship species for plant conservation in many European countries. Its populations are threatened by over-collecting and loss of suitable habitat. Information on local and regional genetic structure can help to develop appropriate conservation strategies. A total of fifteen novel microsatellite markers were developed using MiSeq sequencing. All loci found to be polymorphic, with the number of alleles per locus ranging from 2 to 8. Observed heterozygosity ranged from 0.19 to 0.89. The developed microsatellite markers will be useful to analyze genetic diversity and genetic structure of C. calceolus populations.

Results of an analysis of ITS sequences for the subtribe Spiranthinae s.l. (Orchidaceae) are presented, and compared with results of morphological studies. To evaluate the monophyly of the subtribe, nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene were sequenced for 19 taxa of the tribe Spirantheae, with Erythrodes sp., Bonatea speciosa, Cynorkis sp., Pterostylis curta and Chloraea flavescens designated as an outgroup. For 8 taxa, sequences were taken from the GenBank. The results confirmed the current subtribal position of Cyclopogoninae, with members of the subtribe forming a moderately supported clade. The unexpected occurrence of Odontorrhynchos (Spiranthinae) within the Cyclopogoninae is discussed with reference to the results of morphological studies. Although some of the genera representing the subtribe Stenorrhynchidinae Szlach. form a weakly supported clade, the remaining taxa are variously grouped with Spiranthinae representatives, receiving moderately weak bootstrap support. The results do not provide evidence for the strict monophyly of Spiranthinae s.l. and Stenorrhynchidinae.