Aim: We investigated the phylogeographical history of a clonal-sexual orchid, to test the hypothesis that current patterns of genetic diversity and differentiation retain the traces of climatic fluctuations and of the species reproductive system. Location: Europe, Siberia and Russian Far East.Taxon: Cypripedium calceolus L. (Orchidaceae).Methods: Samples (>900, from 56 locations) were genotyped at 11 nuclear microsatellite loci and plastid sequences were obtained for a subset of them. Analysis of genetic structure and approximate Bayesian computations were performed.Species distribution modelling was used to explore the effects of past climatic fluctuations on the species range. Results: Analysis of genetic diversity reveals high heterozygosity and allele diversity, with no geographical trend. Three genetic clusters are identified with extant gene pools derived from ancestral demes in glacial refugia. Siberian populations exhibit different plastid haplotypes, supporting an early divergence for the Asian gene pool. Demographic results based on genetic data are compatible with an admixture event explaining differentiation in Estonia and Romania and they are consistent with past climatic dynamics inferred through species distribution modelling. Current population differentiation does not follow isolation by distance model and is compatible with a model of isolation by colonization. Main conclusions: The genetic differentiation observed today in C. calceolus preserves the signature of climatic fluctuations in the historical distribution range of the species. Our findings support the central role of clonal reproduction in the reducing loss of diversity through genetic drift. The dynamics of the clonal-sexual reproduction are responsible for the persistence of ancestral variation and stability during glacial periods and post-glacial expansion.

Mixotrophic species use both organic and mineral carbon sources. Some mixotrophic plants combine photosynthesis and a nutrition called mycoheterotrophy, where carbon is obtained from fungi forming mycorrhizal symbiosis with their roots. These species can lose photosynthetic abilities and evolve full mycoheterotrophy. Besides morphological changes, the latter transition is associated with a deep alteration of the plastid genome. Photosynthesis-related genes are lost first, followed by housekeeping genes, eventually resulting in a highly reduced genome. Whether relaxation of selective constraints already occurs for the plastid genome of mixotrophic species, which remain photosynthetic, is unclear. This is partly due to the difficulty of comparing plastid genomes of autotrophic, mixotrophic, and mycoheterotrophic species in a narrow phylogenetic framework. We address this question in the orchid tribe Neottieae, where this large assortment of nutrition types occurs. We sequenced 13 new plastid genomes, including 9 mixotrophic species and covering all 6 Neottieae genera. We investigated selective pressure on plastid genes in each nutrition type and conducted a phylogenetic inference of the group. Surprisingly, photosynthesis-related genes did not experience selection relaxation in mixotrophic species compared with autotrophic relatives. Conversely, we observed evidence for selection intensification for some plastid genes. Photosynthesis is thus still under purifying selection, maybe because of its role in fruit formation and thus reproductive success. Phylogenetic analysis resolved most relationships, but short branches at the base of the tree suggest an evolutionary radiation at the beginning of Neottieae history, which, we hypothesize, may be linked to mixotrophy emergence.

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This study of populations of Cypripedium calceolus L., Cypripedium macranthon Sw. and their hybrid Cypripedium ventricosum Sw. in several regions of Siberia was carried out in 2009-2014. Isolated and mixed populations of the two species and their hybrid were surveyed. In sympatric populations, C. macranthon is most common in the central part and those of C. calceolus prevail in the western part of the range. Mature vegetative and/or reproductive shoots were present but vegetative reproduction was clearly most important in most of the populations investigated. Five colour variations were recorded for C. ventricosum. All species benefit from the increase in the intensity of light reaching ground level in forests after ground fires in spring destroy young trees and undergrowth. Vegetative reproduction and the size of the plants increases in burnt areas. Important factors determining the size and the ontogenetic structure of Cypripedium populations are: the duration of the growing season, the ecological conditions, the activities of pollinators and the relationships among the three species in sympatric populations.

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 (FST = 0.106 for nuclear microsatellites and FST = 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 (Rr = 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.