Effective population size in a partially clonal plant is not predicted by the number of genetic individuals.

Estimating effective population size (N e) is important for theoretical and practical applications in evolutionary biology and conservation. Nevertheless, estimates of N e in organisms with complex life-history traits remain scarce because of the challenges associated with estimation methods. Partially clonal plants capable of both vegetative (clonal) growth and sexual reproduction are a common group of organisms for which the discrepancy between the apparent number of individuals (ramets) and the number of genetic individuals (genets) can be striking, and it is unclear how this discrepancy relates to N e. In this study, we analysed two populations of the orchid Cypripedium calceolus to understand how the rate of clonal versus sexual reproduction affected N e. We genotyped >1000 ramets at microsatellite and SNP loci, and estimated contemporary N e with the linkage disequilibrium method, starting from the theoretical expectation that variance in reproductive success among individuals caused by clonal reproduction and by constraints on sexual reproduction would lower N e. We considered factors potentially affecting our estimates, including different marker types and sampling strategies, and the influence of pseudoreplication in genomic data sets on N e confidence intervals. The magnitude of N e/N ramets and N e/N genets ratios we provide may be used as reference points for other species with similar life-history traits. Our findings demonstrate that N e in partially clonal plants cannot be predicted based on the number of genets generated by sexual reproduction, because demographic changes over time can strongly influence N e. This is especially relevant in species of conservation concern in which population declines may not be detected by only ascertaining the number of genets.

From UAV to PlanetScope: Upscaling fractional cover of an invasive species Rosa rugosa.

Invasive plant species pose a direct threat to biodiversity and ecosystem services. Among these, Rosa rugosa has had a severe impact on Baltic coastal ecosystems in recent decades. Accurate mapping and monitoring tools are essential to quantify the location and spatial extent of invasive plant species to support eradication programs. In this paper we combined RGB images obtained using an Unoccupied Aerial Vehicle, with multispectral PlanetScope images to map the extent of R. rugosa at seven locations along the Estonian coastline. We used RGB-based vegetation indices and 3D canopy metrics in combination with a random forest algorithm to map R. rugosa thickets, obtaining high mapping accuracies (Sensitivity = 0.92, specificity = 0.96). We then used the R. rugosa presence/absence maps as a training dataset to predict the fractional cover based on multispectral vegetation indices derived from the PlanetScope constellation and an Extreme Gradient Boosting algorithm (XGBoost). The XGBoost algorithm yielded high fractional cover prediction accuracies (RMSE = 0.11, R2 = 0.70). An in-depth accuracy assessment based on site-specific validations revealed notable differences in accuracy between study sites (highest R2 = 0.74, lowest R2 = 0.03). We attribute these differences to the various stages of R. rugosa invasion and the density of thickets. In conclusion, the combination of RGB UAV images and multispectral PlanetScope images is a cost-effective method to map R. rugosa in highly heterogeneous coastal ecosystems. We propose this approach as a valuable tool to extend the highly local geographical scope of UAV assessments into wider areas and regional evaluations.

Historical biogeography and local adaptation explain population genetic structure in a widespread terrestrial orchid.

BACKGROUND AND AIMS: Historical changes in environmental conditions and colonization-extinction dynamics have a direct impact on the genetic structure of plant populations. However, understanding how past environmental conditions influenced the evolution of species with high gene flow is challenging when signals for genetic isolation and adaptation are swamped by gene flow. We investigated the spatial distribution and genetic structure of the widespread terrestrial orchid Epipactis helleborine to identify glacial refugia, characterize postglacial population dynamics and assess its adaptive potential. METHODS: Ecological niche modelling was used to locate possible glacial refugia and postglacial recolonization opportunities of E. helleborine. A large single-nucleotide polymorphism (SNP) dataset obtained through genotyping by sequencing was used to define population genetic diversity and structure and to identify sources of postglacial gene flow. Outlier analyses were used to elucidate how adaptation to the local environment contributed to population divergence. KEY RESULTS: The distribution of climatically suitable areas was restricted during the Last Glacial Maximum to the Mediterranean, south-western Europe and small areas in the Alps and Carpathians. Within-population genetic diversity was high in E. helleborine (mean expected heterozygosity, 0.373 ± 0.006; observed heterozygosity, 0.571 ± 0.012; allelic richness, 1.387 ± 0.007). Italy and central Europe are likely to have acted as important genetic sources during postglacial recolonization. Adaptive SNPs were associated with temperature, elevation and precipitation. CONCLUSIONS: Forests in the Mediterranean and Carpathians are likely to have acted as glacial refugia for Epipactis helleborine. Postglacial migration northwards and to higher elevations resulted in the dispersal and diversification of E. helleborine in central Europe and Italy, and to geographical isolation and divergent adaptation in Greek and Italian populations. Distinguishing adaptive from neutral genetic diversity allowed us to conclude that E. helleborine has a high adaptive potential to climate change and demonstrates that signals of adaptation and historical isolation can be identified even in species with high gene flow.

Weak population spatial genetic structure and low infraspecific specificity for fungal partners in the rare mycoheterotrophic orchid Epipogium aphyllum.

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.

Effective double-digest RAD sequencing and genotyping despite large genome size.

Obtaining informative data is the ambition of any genomic project, but in nonmodel species with very large genomes, pursuing such a goal requires surmounting a series of analytical challenges. Double-digest RAD sequencing is routinely used in nonmodel organisms and offers some control over the volume of data obtained. However, the volume of data recovered is not always an indication of the reliability of data sets, and quality checks are necessary to ensure that true and artefactual information is set apart. In the present study, we aim to fill the gap existing between the known applicability of RAD sequencing methods in plants with large genomes and the use of the retrieved loci for population genetic inference. By analysing two populations of Cypripedium calceolus, a nonmodel orchid species with a large genome size (1C ~ 31.6 Gbp), we provide a complete workflow from library preparation to bioinformatic filtering and inference of genetic diversity and differentiation. We show how filtering strategies to dismiss potentially misleading data need to be explored and adapted to data set-specific features. Moreover, we suggest that the occurrence of organellar sequences in libraries should not be neglected when planning the experiment and analysing the results. Finally, we explain how, in the absence of prior information about the genome of the species, seeking high standards of quality during library preparation and sequencing can provide an insurance against unpredicted technical or biological constraints.

Drivers of vegetative dormancy across herbaceous perennial plant species.

Vegetative dormancy, that is the temporary absence of aboveground growth for ≥ 1 year, is paradoxical, because plants cannot photosynthesise or flower during dormant periods. We test ecological and evolutionary hypotheses for its widespread persistence. We show that dormancy has evolved numerous times. Most species displaying dormancy exhibit life-history costs of sprouting, and of dormancy. Short-lived and mycoheterotrophic species have higher proportions of dormant plants than long-lived species and species with other nutritional modes. Foliage loss is associated with higher future dormancy levels, suggesting that carbon limitation promotes dormancy. Maximum dormancy duration is shorter under higher precipitation and at higher latitudes, the latter suggesting an important role for competition or herbivory. Study length affects estimates of some demographic parameters. Our results identify life historical and environmental drivers of dormancy. We also highlight the evolutionary importance of the little understood costs of sprouting and growth, latitudinal stress gradients and mixed nutritional modes.

Local-scale spatial structure and community composition of orchid mycorrhizal fungi in semi-natural grasslands.

Orchid mycorrhizal (OrM) fungi play a crucial role in the ontogeny of orchids, yet little is known about how the structure of OrM fungal communities varies with space and environmental factors. Previous studies suggest that within orchid patches, the distance to adult orchids may affect the abundance of OrM fungi. Many orchid species grow in species-rich temperate semi-natural grasslands, the persistence of which depends on moderate physical disturbances, such as grazing and mowing. The aim of this study was to test whether the diversity, structure and composition of OrM fungal community are influenced by the orchid patches and management intensity in semi-natural grasslands. We detected putative OrM fungi from 0 to 32 m away from the patches of host orchid species (Orchis militaris and Platanthera chlorantha) in 21 semi-natural calcareous grasslands using pyrosequencing. In addition, we assessed different ecological conditions in semi-natural grasslands but primarily focused on the effect of grazing intensity on OrM fungal communities in soil. We found that investigated orchid species were mostly associated with Ceratobasidiaceae and Tulasnellaceae and, to a lesser extent, with Sebacinales. Of all the examined factors, the intensity of grazing explained the largest proportion of variation in OrM fungal as well as total fungal community composition in soil. Spatial analyses showed limited evidence for spatial clustering of OrM fungi and their dependence on host orchids. Our results indicate that habitat management can shape OrM fungal communities, and the spatial distribution of these fungi appears to be weakly structured outside the orchid patches.

Factors influencing IUCN threat levels to orchids across Europe on the basis of national red lists.

The red list has become a ubiquitous tool in the conservation of species. We analyzed contemporary trends in the threat levels of European orchids, in total 166 species characterized in 27 national red lists, in relation to their reproductive biology and growth form, distribution area, and land cover where they occur. We found that species in central Europe are more threatened than those in the northern, southern, or Atlantic parts of Europe, while species were least threatened in southern Europe. Nectarless and tuberous species are significantly more threatened than nectariferous and rhizomatous taxa. Land cover (ratios of artificial land cover, area of pastures and grasslands, forests and inland wetlands) also significantly impacted the threat level. A bigger share of artificial land cover increases threat, and a bigger share of pasture and grassland lowers it. Unexpectedly, a bigger share of inland wetland area in a country increased threat level, which we believe may be due to the threatened nature of wetlands themselves relative to other natural land cover types. Finally, species occurring in multiple countries are on average less threatened. We believe that large-scale analysis of current IUCN national red lists as based on their specific categories and criteria may particularly inform the development of coordinated regional or larger-scale management strategies. In this case, we advocate for a coordinated EU protection and restoration strategy particularly aimed at central European orchids and those occurring in wetland area.

Trait-based analysis of decline in plant species ranges during the 20th century: a regional comparison between the UK and Estonia.

Although the distribution ranges and abundance of many plant species have declined dramatically in recent decades, detailed analysis of these changes and their cause have only become possible following the publication of second- and third-generation national distribution atlases. Decline can now be compared both between species and in different parts of species' ranges. We extracted data from distribution atlases to compare range persistence of 736 plant species common to both the UK and Estonia between survey periods encompassing almost the same years (1969 and 1999 in the UK and 1970 and 2004 in Estonia). We determined which traits were most closely associated with variation in species persistence, whether these were the same in each country, and the extent to which they explained differences in persistence between the countries. Mean range size declined less in Estonia than in the UK (24.3% vs. 30.3%). One-third of species in Estonia (239) maintained >90% of their distribution range compared with one-fifth (141) in the UK. In Estonia, 99 species lost >50% of their range compared with 127 species in the UK. Persistence was very positively related to original range in both countries. Major differences in species persistence between the studied countries were primarily determined by biogeographic (affiliation to floristic element) and ecoevolutionary (plant strategy) factors. In contrast, within-country persistence was most strongly determined by tolerance of anthropogenic activities. Decline of species in the families Orchidaceae and Potamogetonaceae was significantly greater in the UK than in Estonia. Almost all of the 736 common and native European plant species in our study are currently declining in their range due to pressure from anthropogenic activities. Those species with low tolerance of human activity, with biotic pollination vectors and in the families referred to above are the most vulnerable, especially where human population density is high.

Temporal patterns of orchid mycorrhizal fungi in meadows and forests as revealed by 454 pyrosequencing.

Orchid mycorrhizal (OrM) symbionts play a key role in the growth of orchids, but the temporal variation and habitat partitioning of these fungi in roots and soil remain unclear. Temporal changes in root and rhizosphere fungal communities of Cypripedium calceolus, Neottia ovata and Orchis militaris were studied in meadow and forest habitats over the vegetation period by using 454 pyrosequencing of the full internal transcribed spacer (ITS) region. The community of typical OrM symbionts differed by plant species and habitats. The root fungal community of N. ovata changed significantly in time, but this was not observed in C. calceolus and O. militaris. The rhizosphere community included a low proportion of OrM symbionts that exhibited a slight temporal turnover in meadow habitats but not in forests. Habitat differences in OrM and all fungal associates are largely attributable to the greater proportion of ectomycorrhizal fungi in forests. Temporal changes in OrM fungal communities in roots of certain species indicate selection of suitable fungal species by plants. It remains to be elucidated whether these shifts depend on functional differences inside roots, seasonality, climate or succession.

Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity.

BACKGROUND AND AIMS: Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes. METHODS: Ploidy and proportions of endoreplicated genome were determined using DAPI (4',6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato. KEY RESULTS: Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted <2 % of the individuals sampled, they were found in 35 % of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe. CONCLUSIONS: Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture.

Advantages of volunteer-based biodiversity monitoring in Europe.

Without robust and unbiased systems for monitoring, changes in natural systems will remain enigmatic for policy makers, leaving them without a clear idea of the consequences of any environmental policies they might adopt. Generally, biodiversity-monitoring activities are not integrated or evaluated across any large geographic region. The EuMon project conducted the first large-scale evaluation of monitoring practices in Europe through an on-line questionnaire and is reporting on the results of this survey. In September 2007 the EuMon project had documented 395 monitoring schemes for species, which represents a total annual cost of about 4 million euro, involving more than 46,000 persons devoting over 148,000 person-days/year to biodiversity-monitoring activities. Here we focused on the analysis of variations of monitoring practices across a set of taxonomic groups (birds, amphibians and reptiles, mammals, butterflies, plants, and other insects) and across 5 European countries (France, Germany, Hungary, Lithuania, and Poland). Our results suggest that the overall sampling effort of a scheme is linked with the proportion of volunteers involved in that scheme. Because precision is a function of the number of monitored sites and the number of sites is maximized by volunteer involvement, our results do not support the common belief that volunteer-based schemes are too noisy to be informative. Just the opposite, we believe volunteer-based schemes provide relatively reliable data, with state-of-the-art survey designs or data-analysis methods, and consequently can yield unbiased results. Quality of data collected by volunteers is more likely determined by survey design, analytical methodology, and communication skills within the schemes rather than by volunteer involvement per se.

Mycorrhizal interactions of orchids colonizing Estonian mine tailings hills.

Northeastern Estonia is home to extensive oil shale mines. Associated with these are desolate and environmentally damaging hills of ash and semicoke tailings. Interestingly, some of the first plants to colonize these hills are rare orchids. Here, we assess the identities of the mycorrhizal fungi associated with these orchids, in particular Epipactis atrorubens, Orchis militaris, and Dactylorhiza baltica, and compare them with mycorrhizal fungi from orchids from pristine habitat. Epipactis atrorubens associated with the widest breadth of fungi, including unnamed members of the basidiomycete family Tulasnellaceae and the potentially ectomycorrhizal ascomycetes Trichophaea woolhopeia and Geopora cooperi. Orchis militaris also associated with unnamed members of the Tulasnellaceae. Dactylorhiza baltica associated with Ceratobasidium albasitensis. In Epipactis and Orchis, the same fungi associated with plants in the pristine habitat as with those on ash hills. The tulasnelloid and ceratobasidioid fungi mycorrhizal with these orchids appear closely related to common orchid mycorrhizal fungi, while one of the ascomycetes mycorrhizal with E. atrorubens is closely related to a mycorrhizal fungus with E. microphylla. Our results suggest that these orchids and their fungi are not limited to pristine habitats and that environmentally polluted sites may present novel habitats that may be exploited for endangered plant conservation.

Conflicts between biodiversity conservation and human activities in the central and eastern European countries.

Conflicts between the conservation of biodiversity and other human activities have been and continue to be of increasing concern in the European Union, often having important political, economic, and environmental repercussions. These conflicts have been addressed in the European Union by using a wide array of top down and bottom up tools, with varying degrees of success. A new challenge is now facing Europe with the integration of 10 new countries in the European Union (EU) and an additional range of biodiversity-rich habitats placed under EU legislation. The rich biodiversity of the central and eastern European countries (CEEC) is likely to be threatened by some aspects of the integration process, and conflicts between the conservation of biodiversity and other human activities are expected. In this paper, we review certain existing conflicts between biodiversity conservation and human activities in the CEEC, expected conflicts associated with integration to the EU, particularly in terms of new policy and legislation implementation, and, finally, explore possible conflict management options.

The evolutionary history of mycorrhizal specificity among lady's slipper orchids.

Although coevolution is acknowledged to occur in nature, coevolutionary patterns in symbioses not involving species-to-species relationships are poorly understood. Mycorrhizal plants are thought to be too generalist to coevolve with their symbiotic fungi; yet some plants, including some orchids, exhibit strikingly narrow mycorrhizal specificity. Here, we assess the evolutionary history of mycorrhizal specificity in the lady's slipper orchid genus, Cypripedium. We sampled 90 populations of 15 taxa across three continents, using DNA methods to identify fungal symbionts and quantify mycorrhizal specificity. We assessed phylogenetic relationships among sampled Cypripedium taxa, onto which we mapped mycorrhizal specificity. Cypripedium taxa associated almost exclusively with fungi within family Tulasnellaceae. Ancestral specificity appears to have been narrow, followed by a broadening after the divergence of C. debile. Specificity then narrowed, resulting in strikingly narrow specificity in most of the taxa in this study, with no taxon rewidening to the same extant as basal members of the genus. Sympatric taxa generally associated with different sets of fungi, and most clades of Cypripedium-mycorrhizal fungi were found throughout much of the northern hemisphere, suggesting that these evolutionary patterns in specificity are not the result of biogeographic lack of opportunity to associate with potential partners. Mycorrhizal specificity in genus Cypripedium appears to be an evolvable trait, and associations with particular fungi are phylogenetically conserved.

High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium.

Lady's slipper orchids (Cypripedium spp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adult Cypripedium mycorrhizae are unclear; however, Cypripedium distribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100 Cypripedium plants, and successfully did so with two Cypripedium calceolus, 10 Cypripedium californicum, six Cypripedium candidum, 16 Cypripedium fasciculatum, two Cypripedium guttatum, 12 Cypripedium montanum, and 11 Cypripedium parviflorum plants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority of Cypripedium mycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus, Phialophora. C. californicum was the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones.