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.
We showed the absence of pre-zygotic barriers for autogamy in E. aphyllum Self-pollination and self-fertilization are possible; however, natural self-pollination is unlikely or rare due to the position of the pollinia. Incidental parthenogenesis in E. aphyllum is very likely, given the biology of ovule development of this mycoheterotrophic orchid. This species therefore has the potential to produce seeds via both sexual and asexual means, although the contribution of apomixis to this process appears largely negligible.
Lady's slipper orchid (Cypripedium calceolus) serves as a flagship species for plant conservation in many European countries. Its populations are threatened by overcollecting 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.Keywords Genetic diversity Á Genetic structure Á Microsatellites Á Orchidaceae Lady's slipper orchid (Cypripedium calceolus L.) is one of the largest and the most spectacular elements of European flora. This Euro-Asiatic species has suffered a marked decrease of localities and area occupied (Terschuren 1999). It is legally protected in all European countries, and listed in various national conventions and directives (Terschuren 1999). Studies of genetic diversity and fine-scale spatial genetic structure in relation to habitat management will improve management strategies of this vulnerable species.Two, out of four recently published microsatellite loci for C. calceolus, may pose scoring problems (Pedersen et al. 2012). Therefore a higher number of variable loci is needed to estimate genetic structure and distinguish close relatives. Here we report the isolation and characterization of polymorphic loci that will be useful in future population genetic studies.Seeds of C. calceolus were collected from population Bukówki (Poland) and asymbiotically germinated in in vitro culture. Total genomic DNA was isolated from three seedlings using procedure described by Bekesiova et al. (1999). Extracted DNA was used for a library preparation with a NEBNext Ò DNA Library Prep Master Mix Set for Illumina. The sequencing was performed on the MiSeq Benchtop Sequencer (Illumina) using the 2 9 250 bp read mode. The obtained data (10,19 Mega reads) was assembled using CLCGenomicWorkbench (CLCBio) into 513,225 contigs and the microsatellites were then detected using QDD 2.1 Beta (Meglecz et al. 2010). A total of 22,162 contigs contained at last one microsatellite of which 53 loci were selected for initial screening. We screened 32 plants from two Polish populations (Bukówki and Prokowo) for polymorphisms at these loci. All forward primers were tagged with M13(-21) (5 0 -TGTAAAACGACGGCCAGT-3 0 ) at the 5 0 end. The 10 lL PCR volume contained: 4.5 lL MyTaq TM HS Mix (Bioline), 0.4 lM of both forward and reverse primers, 0.2 lM dye labelled primer, 0.3 % DMSO, 1.2 lL water and 1 lL DNA template (*50 ng). The following PCR conditions were used: 2 min initial denaturation at 96°C, followed by 35 cycles of 95°C for 30 s/primer specific annealing temperature for 30 s/72°C for 45 s.
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.
In order to estimate the best germination conditions of Encyclia aff. oncidioides seeds, five different media (Fast, Knudson C modified by Vajrabhaya, Murashige and Skoog, PB2 and modified Vacin and Went) with different concentrations of plant growth regulators such as benzyladenine (BA), naphthaleneacetic acid (NAA) and gibberellic acid (GA3) were tested. No beneficial effect was observed when BA and NAA were applied to the germination medium and GA3 inhibited germination. The effect of light, activated charcoal, coconut water and casein hydrolysate on seed germination was also studied. The growth rate of seedlings on three different media supplemented with activated charcoal and plant growth regulators was checked. The applied plant growth regulators had no beneficial effect on the further growth of seedlings. Fast and PB2 media with 0.2% activated charcoal proved to be the best for E. aff. oncidioides seed germination, seedling development and plantlet propagation
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