Genetic diversity, compatibility patterns and seed quality in isolated populations of Cypripedium calceolus (Orchidaceae)

Cypripedium calceolus has suffered an alarming decline, and today mainly occurs in small and isolated populations. In Denmark there are only two populations, close to each other and situated far from other European stands. One population is stagnant or in slow decline, whereas the other is in rapid increase. We examined the levels of genetic diversity and compatibility and seed quality following experimental crosses. No genetic variation could be detected in plastid and nuclear markers within or between the two populations—in contrast to results previously reported from other European populations of C. calceolus. This may indicate a founder effect in both populations, but it could also be the outcome of prolonged inbreeding or reflect a genetic bottleneck after the populations were established. According to fruit dimensions and frequency of fully developed seeds there was full self-compatibility in the stagnant population, and partial late-acting self-incompatibility in the proliferating population. In combination with previous reports from other countries, this suggests that several self-incompatibility systems may occur in C. calceolus. Seeds from the older and stagnant population performed more poorly in germination tests in vitro than seeds from the thriving population. The difference needs not be genetically based, but could be due to environmental differences during seed maturation, producing different seed quality or dormancy characteristics. However, low level of genetic diversity within the populations may affect their ability to adapt and the possibility of inbreeding depression should be investigated.     

Dynamics of island populations of Cypripedium calceolus in the Biebrza river valley (north-east Poland)

Three Cypripedium calceolus populations were observed in the Biebrza National Park (north-east Poland) for 11 years (1989–99). The populations differed in the number of genets and ramets and in their dynamics. Differences were in the proportion of flowering ramets, number of flowers, fruiting and recruitment. Fruiting and recruitment are low in populations of Cypripedium calceolus . Fruiting is probably pollinators limited, and recruitment is limited by environmental conditions. Changes in the size of populations of this species are caused by the following processes: changes in the number of genets (their appearance and death), changes in the size of clones (growth of individuals and their disintegration, death of individual ramets), dormancy of genets, and animal pressure. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 67–77.     

The reintroduction project of Cypripedium calceolus in Switzerland

In 2013, a project to reintroduce Cypripedium calceolus in the Swiss Jura was organised between the Swiss Orchid Foundation at the Herbarium of Jany Renz, Basel, Switzerland and Anthura B.V., Bleiswijk, Holland with the approval of the Nature Conservation authorities of nine cantons in the Swiss Jura. Seed was legally collected from sites in the nine cantons and was successfully grown to flowering size by the commercial nursery in the Netherlands. Three thousand flowering sized plants were successfully reintroduced to 43 sites in the summer of 2018. Monitoring in successive years has shown a success rate of over 50% and much higher in some sites. The successful collaboration of conservation authorities, a charitable foundation (Swiss Orchid Foundation) and a commercial company (Anthura B.V.) in the conservation of an endangered plant might serve as a model of how other projects might be run. A version of this paper, in German may be found in Bauhinia.     

Allozyme differentiation and genetic structure of the Lady's slipper (Cypripedium calceolus) island populations in north-east Poland

The population genetic structure in the rare and endangered perennial plant, Cypripedium calceolus , from north-east Poland was investigated. A total of 11 loci were analysed, of which five were polymorphic for the species and in every population. The 11 loci resolved gave a total of 19 alleles with the mean number of 1.73 alleles per locus. The proportion of polymorphic loci (P) was the same for the species and in every population, 45.5%. The average observed and expected heterozygosities for the total Lady's slipper data set were 0.156 and 0.184, respectively. The average expected heterozygosity for the populations under study ranged from 0.176 to 0.188 and did not differ significantly among populations. Genetic diversity within C. calceolus populations from the Biebrza Valley is relatively high compared with rare taxa and taxa with similar life histories. The genetic diversity among populations of C. calceolus studied ( F _ST = 0.016) is much smaller than the genetic diversity within populations. In the species studied, there was no significant correlation between genetic and geographical distances ( r = −0.73, Mantel test). This may indicate the relatively recent origin of the populations studied, or a high level of gene flow among populations. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 138 , 433–440.     

Spatial genetic structure and clonal diversity of island populations of lady's slipper (Cypripedium calceolus) from the Biebrza National Park (northeast Poland)

Three populations of the rare and endangered plant species Cypripedium calceolus were included in a study of genetic diversity and spatial genetic structure in the Biebrza National Park, northeast Poland. Analysis of 11 allozyme loci indicate that the populations of this species contained high genetic variability (P = 45.5%, A= 1.73). On the other hand, the genetic differentiation (FST = 0.014, P < 0.05) among C. calceolus populations was very low when compared to other species with similar life history characteristics. The observed high rate of gene flow (Nm = 18) may suggest that the populations studied derived from each other in the recent past. Five polymorphic allozyme markers identified 109 multilocus genotypes in three populations and the majority of them (67%) were population-specific. One of the populations studied, characterized by particularly extensive vegetative reproduction, showed the lowest clonal diversity (G/N = 0.15) and heterozygosity (HO = 0.111) values and the highest FIS(0.380), when compared to other two populations (G/N = 0.26-0.27, HO= 0.166-178, FIS = 0.024-0.055). This may indicate that clonal reproduction has an important influence on the genetic structure of C. calceolus populations. The longevity of genets, the out-crossing breeding system and the presence of recruitment from seeds are factors maintaining genetic diversity in C. calceolus.     

Genetic diversity in Cypripedium calceolus (Orchidaceae) with a focus on north-western Europe, as revealed by plastid DNA length polymorphisms

Background and Aims

Cypripedium calceolus, although widespread in Eurasia, is rare in many countries in which it occurs. Population genetics studies with nuclear DNA markers on this species have been hampered by its large nuclear genome size. Plastid DNA markers are used here to gain an understanding of variation within and between populations and of biogeographical patterns.

Methods

Thirteen length-variable regions (microsatellites and insertions/deletions) were identified in non-coding plastid DNA. These and a previously identified complex microsatellite in the trnL-trnF intergenic spacer were used to identify plastid DNA haplotypes for European samples, with sampling focused on England, Denmark and Sweden.

Key Results

The 13 additional length-variable regions identified were two homopolymer (polyA) repeats in the rps16 intron and a homopolymer (polyA) repeat and ten indels in the accD-psa1 intergenic spacer. In accD-psa1, most of these were in an extremely AT-rich region, and it was not possible to design primers in the flanking regions; therefore, the whole intergenic spacer was sequenced. Together, these new regions and the trnL-trnF complex microsatellite allowed 23 haplotypes to be characterized. Many were found in only one or a few samples (probably due to low sampling density), but some commoner haplotypes were widespread. Most of the genetic variation was found within rather than between populations (83 vs. 18%, respectively). Two haplotypes occurred from the Spanish Pyrenees to Sweden.

Conclusions

Plastid DNA data can be used to gain an understanding of patterns of genetic variation and seed-mediated gene flow in orchids. Although these data are less information-rich than those for nuclear DNA, they present a useful option for studying species with large genomes. Here they support the hypothesis of long-distance seed dispersal often proposed for orchids.Key words: Biogeography, Cypripedium calceolus, genome size, plastid microsatellites, population genetics, seed dispersal     

Fruit-set and recruitment in populations of Cypripedium calceolus L. in Estonia

The overall mean percentage of fruiting for over 3500 flowers observed in eight Estonian populations of a self-compatible clonal orchid Cypripedium calceolus over 11 years was 10.5%. The larger clones set relatively fewer fruits, otherwise the general pattern of fruiting was close to random, despite several significant local deviations from random pollination. There is no cost associated with the fruit-set at the clonal level. The fruit-set which appears pollinator limited was not correlated with the frequency of seedlings in a population. Data presented provide information on the relationship between fruit and seedling production in orchids. The sites suitable for seedling establishment are characterized as having relatively more extensive moss cover, less vascular plant cover, more moisture and better light conditions. Recruitment is concluded to be microsite limited, and the fruit-set to be without significant influence on the fitness of populations.     

Bee size and pollen transfer in Cypripedium calceolus (Orchidaceae)

In Denmark Cypripedium calceolus is found in only two small and isolated populations, and its fruit set is suspected to be pollinator-limited. Collection and observations of flower visitors were carried out between 1995 and 1997, and fruit set were estimated. Forty-four visitors were collected. The majority belonged to the genera Halictus, Lasioglossum and Andrena . The latter is known as pollinator of C. calceolus in other parts of Europe, but in this study Andrena spp. were both not regular visitors and too large to function as efficient pollinators. Medium-sized halictine species, especially females of Halictus tumulorum and Lasioglossum calceatum , were the most regular and frequent pollen vectors. Andrena jacobi and Lasioglossum rufitarse are recorded for the first time in Denmark.     

Re-establishment of the lady's slipper orchid (Cypripedium calceolus L.) in Britain

The Sainsbury Orchid Conservation Project (SOCP), based in the Micropropagation Unit of the Royal Botanic Gardens, Kew, has worked for many years with English Nature, and the Species Recovery Programme in particular, in raising plants of the lady's slipper orchid for re-establishment. This is a collaborative venture with the involvement of a large number of organizations and individuals at national and local level.Cypripedium calceolusis one of Britain's rarest plants. Thought at one time to be extinct, its decline is due to overcollection by botanists for herbarium specimens and by gardeners and it is found now on a single, fragile, natural site. It has been wardened since 1970s because of concerns about long-term safety and is protected under Schedule 8 of the Wildlife and Countryside Act 1981. Natural pollination has not been observed and hand pollination is carried out to ensure seed set. Some seed is scattered on site resulting in a few seedlings and the remaining capsules are sent to the Sainsbury Orchid Conservation Project. In addition, some plants of native origin are held in cultivation and molecular techniques are being carried out at Kew to ascertain how these are related. The best pollination strategy to increase genetic diversity is co-ordinated by English Nature and SOCP, as is the optimum collection time post-pollination to achieve maximum germination. Seedlings are propagated in the laboratory using immature green capsules sown on a range of nutrient media. No symbiotic fungus has yet been found for this species. Germination occurs after 6 weeks in the dark and the plants chilled when roots are well developed and shoots beginning to form. Plants potted up at Kew and near the native site have produced leaves and buds and extensive planting trials have now commenced. The main objective is to increase the number of localities whereCypripediumoccurs through re-establishment. As its decline is due to overcollection rather than habitat loss, many old sites have changed very little. Observation of continental populations is important in assessing suitability of potential sites as the extant clone may not be in optimum conditions. An initial planting trial on the wild site in winter 1989/90 resulted in a 75% survival rate of seedlings planted out. However, establishing seedlings is a long process and careful monitoring is required.     

濒危植物扇脉杓兰野生居群遗传多样性的AFLP分析

扇脉杓兰(Cypripedium japonicum)是一种珍稀的地生兰。为了探索并制定针对性的保护措施,采用AFLP分子标记技术对现存于浙江省的6个扇脉杓兰天然居群进行了遗传多样性和遗传结构的研究。筛选出的7对引物共扩增出377条清晰条带,多态性比率为20.95%。居群总Nei基因多样性指数为0.0512,其中居群内基因多样性为0.0247,居群平均Shannon多态性信息指数为0.0417,群体间总遗传分化系数为0.5178,基因流为0.4655,表明扇脉杓兰居群的遗传多样性较低,居群间出现一定程度的遗传分化。UPGMA聚类和Mantel检测分析结果表明,居群间的地理位置与遗传距离之间不存在显著相关性(r=0.16,P=0.242)。基于本研究结果并结合扇脉杓兰的生物学特性,初步提出了相应的保育策略。     

Identity of Cypripedium calceolus (Orchidaceae) in Rebun Island: comparative DNA analysis of related species

Cypripedium calceolus was found in 1980 in Rebun Island off the north coast of Hokkaido, Japan, but the origin of this plant has been a controversial issue. In this study, we have made a comparative study by chloroplast DNA sequencing analysis among C. calceolus which occurs in Rebun Island and populations of C. calceolus from western Europe, China and far eastern Russia (Nakhodka), and also as references, C. macranthos in Japan and other Cypripedium species in North America. A Cypripedium cf. "calceolus", found recently in eastern Hokkaido, was also included in this analysis. The C. calceolus samples analyzed were categorized into three groups, i.e., those from Western Europe, from China and far eastern Russia, and from Rebun Island. The C. calceolus in Rebun Island was clearly different from the others in terms of DNA sequence and morphological features. The C. cf. calceolus from eastern Hokkaido and one sample from Nadhodka, Russia, were also classified into the same group as those from Rebun Island, although some differences in their morphological features were observed. It is concluded that the C. calceolus found in Rebun Island is not identical with those growing in Europe and China. In addition, it was found that it may be possible to classify C. macranthos into two groups, namely groups which include or do not include var. rebunense. An unidentified Cypripedium species found in Rebun Island falls into the same group as var. rebunense.     

Pollination of the Lady's slipper orchid (Cypripedium calceolus) in Scandinavia – taxonomic and conservational aspects

The Lady's slipper orchid Cypripedium calceolus L. is considered one of the most beautiful orchids of Europe. Consequently, the species has suffered from over-collecting and is now critically endangered in many countries. Although pollination success is suspected to influence the long-term survival of Cypripedium calceolus , relatively little is known about the identity of its pollinators in mainland Sweden – a region that comprises the largest European populations. In order to identify which species pollinate eight representative populations in mainland Sweden, we observed and sampled visitors to flowers using a standardized protocol. Specimens were identified and any pollen smear found on their body was examined for the presence of Cypripedium pollen. Nine species were recognized as effective pollen vectors ( Andrena cineraria, A. carantonica, A. haemorrohoa, A. helvola, A. nigroaenea, A. praecox, Colletes cunicularius, Lasioglossum fratellum and L. fulvicorne ), four of them for the first time in Scandinavia. This is the first time that a species of Colletes is reported to carry pollen of Cypripedium in this region. All but one specimens were females. Our results suggest a taxonomically heterogeneous pollinator fauna for Cypripedium calceolus and are discussed in light of the management of this species.     

Effect of Lighting Conditions on the Reproductive Success of Cypripedium calceolus L. (Orchidaceae,Liliopsida)

Biology Bulletin - Abstract—The influence of lighting conditions on the reproductive characteristics of Cypripedium calceolus L. was studied on the territory of the Komi Republic, where the...     

Genetic variation and genotypic diversity in Epipactis helleborine populations from NE Poland

Enzyme electrophoreses were used to estimate genetic variation in five populations of Epipactis helleborine from two National Parks (Biebrza and Wigry) in northeast Poland. It has been proved that populations from these two regions differed in genetic structure, with the populations belonging to the Biebrza group having a higher level of genetic variation than those from Wigry. The number of polymorphic loci (P) ranged from 18.2% to 40.9% and the mean number of alleles per polymorphic locus (Ap) from 2.25 to 2.80. Although the observed heterozygosity (H_o) was lower than the expected one (H_e) in each population, the values of H_o and H_e were 2–3 times lower in the populations from Wigry than in those from Biebrza. Excluding the LUK population with the smallest genotypic diversity, the majority of 345 distinct multilocus genotypes occurred only once, sporadically 2–4 times in each population and their frequency ranged from 0.2% to 3.7%. Moreover, factors shaping genetic structure of E. helleborine and their intensity varied in the populations studied. Reproduction from seeds seems to influence greatly the genetic variation of the populations from the Biebrza National Park, while an assortative mating between related individuals or population size appears to be more important in the case of the populations from the Wigry National Park.     

Genetic diversity and structure of the endangered lady's slipper orchid Cypripedium japonicum Thunb. (Orchidaceae) in Japan

Cypripedium japonicum Thunb. (Orchidaceae), once a common perennial herb, is now designated as endangered throughout most of its distribution due to habitat destruction and fragmentation, and the impacts of horticultural collection. We investigated the genetic characteristics of this species for conservation purposes, using microsatellite markers to examine the genetic diversity and structure of 15 native and 5 ex situ populations in Japan. The results imply that although allelic variation is low in Japanese C. japonicum, sexual reproduction by seed, as well as clonal propagation, may occur in some populations. Both native and ex situ populations were found to be genetically differentiated, indicating that some populations may have experienced recent population declines, genetic fragmentation, or bottlenecks. The degree of genetic drift from the putative ancestral population, inferred through STRUCTURE analysis, was more pronounced in northern populations than in southern populations. Some of the ex situ conserved populations exhibited a low degree of differentiation from ancestral native populations. Our results imply that conservation of C. japonicum in Japan is best supported by maintaining individual populations and their unique genetic characteristics.     

Genetic diversity in Dactylorhiza majalis subsp. majalis populations (Orchidaceae) of northern Poland

We analysed 16 populations of Dactylorhiza majalis subsp. majalis from northern Poland, simultaneously utilizing both morphological and molecular data. Genetic differentiation was examined using five microsatellite loci, and morphological variation was assessed for 23 characters. At the species level, our results showed a moderate level of genetic diversity (A = 6.00; A_e = 1.86; H_o = 0.387; F_IS = 0.139) which varied between the studied populations (A = 2.60–4.20; A_e = 1.68–2.39; H_o = 0.270–0.523; F_IS = ?0.064–0.355). A significant excess of homozygotes was detected in five population, while excess of heterozygotes was observed in four populations, but the latter values were statistically insignificant. Moderate, but clear between population genetic differentiation was found (F_ST = 0.101; p < 0.001). Considering pairwise‐F_ST and number of migrants among populations, we recognized three population groups (I, II, III), where the first could be further divided into two subgroups (Ia, Ib). These three groups differed with respect to gene flow values (N_m = 0.39–1.12). The highest number of migrants per generation was noticed among populations of subgroup Ia (8.58), indicative of a central panmictic population with free gene flow surrounded by peripatric local populations (Ib) with more limited gene flow. Geographic isolation, habitat fragmentation and limited seed dispersal are inferred to have caused limitations to gene flow among the three indicated population groups. There was a significant correlation between the morphological and genetic distance matrices. A weak but significant pattern of isolation by distance was also observed (r = 0.351; p < 0.05).     

Contrasting levels of genetic diversity between the historically rare orchid Cypripedium japonicum and the historically common orchid Cypripedium macranthos in South Korea

Many terrestrial orchids are historically rare and occur in small, spatially isolated populations. Theory predicts that such species will harbour low levels of genetic variation within populations and will exhibit a high degree of population genetic divergence, primarily as a result of genetic drift. If the origin of the present‐day populations is relatively recent from the same genetically depauperate source population, a complete lack of genetic differentiation between conspecific populations is expected. If a terrestrial orchid was historically common with moderate or high levels of genetic diversity, but has experienced more recent anthropogenic disturbance as a result of over‐collection, it would still exhibit initial levels of genetic variation within populations and a low degree of genetic divergence between populations. To test these predictions, we examined the genetic diversity in six populations (N = 131) of the historically and currently rare Cypripedium japonicum and in four populations (N = 94) of the historically common but now rare C. macranthos from South Korea. Fourteen putative allozyme loci resolved from eight enzyme systems revealed no variation either within or among populations of C. japonicum, which supports the first prediction. In contrast, populations of C. macranthos harboured high levels of genetic variation (mean percentage of polymorphic loci %P = 46.7; mean expected heterozygosity H_e = 0.185) and exhibited a low degree of population genetic divergence (G_ST = 0.059), supporting the second prediction. The lack of genetic variation both within and among conspecific populations of C. japonicum may suggest that populations originated from the same genetically depauperate ancestral population. The high levels of genetic diversity maintained in populations of C. macranthos suggest that the collection‐mediated decrease in the number of individuals is still too recent for long‐term effects on genetic variation. Based on current demographic and genetic data, in situ and ex situ conservation strategies should be provided to preserve genetic variation and to ensure the long‐term survival of the two species in the Korean Peninsula. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 119–129.