Environmental DNA metabarcoding reflects spatiotemporal fish community shifts in the Scheldt estuary.

Estuarine ecosystems face increasing anthropogenic pressures, necessitating effective monitoring methods to mitigate their impacts on the biodiversity they harbour. The use of environmental DNA (eDNA) based detection methods is increasingly recognized as a promising tool to complement other, potentially invasive monitoring techniques. Integrating such eDNA analyses into monitoring frameworks for large ecosystems is still challenging and requires a deeper understanding of the scale and resolution at which eDNA patterns may offer insights in species presence and community composition space and time. The Scheldt estuary, characterized by its diverse habitats and complex currents, is one of the largest Western European tidal river systems. Until now, it remains challenging to obtain accurate information on fish communities living in and migrating through this ecosystem, consequently confining our knowledge to specific locations. To explore the potential of eDNA based monitoring, we simultaneously combine stow net fishing with eDNA metabarcoding, to assess spatiotemporal shifts in the Scheldt estuary's fish communities. In total, we detected 71 fish species in the estuary using eDNA metabarcoding, partly overlapping with historic fish community data gathered at the different study locations and in contrast to only 42 species using stow net fishing during the same survey period. Community compositions found by both detection methods varied among sampling locations, driven by a clear correlation to the salinity gradient. Limited effects of sampling depth and tide were observed on the eDNA metabarcoding data, allowing a significant reduction of the eDNA sampling effort for future eDNA fish monitoring campaigns in this study system. Our results further demonstrate that seasonal shifts in fish species occurrence can be detected using eDNA metabarcoding. Combining eDNA metabarcoding and stow net fishing further enhances our understanding of this vital waterway's diverse fish populations, allowing a higher resolution and more efficient monitoring strategy.

Using environmental DNA metabarcoding to monitor fish communities in small rivers and large brooks: Insights on the spatial scale of information.

Monitoring fish communities is central to the evaluation of ecological health of rivers. Both presence/absence of fish species and their relative quantity in local fish assemblages are crucial parameters to measure. Fish communities in lotic systems are traditionally monitored via electrofishing, characterized by a known limited efficiency and high survey costs. Analysis of environmental DNA could serve as a non-destructive alternative for detection and quantification of lotic fish communities, but this approach still requires further insights in practical sampling schemes incorporating transport and dilution of the eDNA particles; optimization of predictive power and quality assurance of the molecular detection method. Via a controlled cage experiment, we aim to extend the knowledge on streamreach of eDNA in small rivers and large brooks, as laid out in the European Water Framework Directive's water typology. Using a high and low source biomass in two river transects of a species-poor river characterized by contrasting river discharge rates, we found strong and significant correlations between the eDNA relative species abundances and the relative biomass per species in the cage community. Despite a decreasing correlation over distance, the underlying community composition remained stable from 25 to 300 m, or up to 1 km downstream of the eDNA source, depending on the river discharge rate. Such decrease in similarity between relative source biomass and the corresponding eDNA-based community profile with increasing distance downstream from the source, might be attributed to variation in species-specific eDNA persistence. Our findings offer crucial insights on eDNA behaviour and characterization of riverine fish communities. We conclude that water sampled from a relatively small river offers an adequate eDNA snapshot of the total fish community in the 300-1000 m upstream transect. The potential application for other river systems is further discussed.

Environment and shipping drive environmental DNA beta-diversity among commercial ports.

The spread of nonindigenous species by shipping is a large and growing global problem that harms coastal ecosystems and economies and may blur coastal biogeographical patterns. This study coupled eukaryotic environmental DNA (eDNA) metabarcoding with dissimilarity regression to test the hypothesis that ship-borne species spread homogenizes port communities. We first collected and metabarcoded water samples from ports in Europe, Asia, Australia and the Americas. We then calculated community dissimilarities between port pairs and tested for effects of environmental dissimilarity, biogeographical region and four alternative measures of ship-borne species transport risk. We predicted that higher shipping between ports would decrease community dissimilarity, that the effect of shipping would be small compared to that of environment dissimilarity and shared biogeography, and that more complex shipping risk metrics (which account for ballast water and stepping-stone spread) would perform better. Consistent with our hypotheses, community dissimilarities increased significantly with environmental dissimilarity and, to a lesser extent, decreased with ship-borne species transport risks, particularly if the ports had similar environments and stepping-stone risks were considered. Unexpectedly, we found no clear effect of shared biogeography, and that risk metrics incorporating estimates of ballast discharge did not offer more explanatory power than simpler traffic-based risks. Overall, we found that shipping homogenizes eukaryotic communities between ports in predictable ways, which could inform improvements in invasive species policy and management. We demonstrated the usefulness of eDNA metabarcoding and dissimilarity regression for disentangling the drivers of large-scale biodiversity patterns. We conclude by outlining logistical considerations and recommendations for future studies using this approach.

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.

Accurate detection and quantification of seasonal abundance of American bullfrog (Lithobates catesbeianus) using ddPCR eDNA assays.

The invasive American bullfrog (Lithobates catesbeianus) imperils freshwater biodiversity worldwide. Effective management hinges on early detection of incipient invasions and subsequent rapid response, as established populations are extremely difficult to eradicate. Although environmental DNA (eDNA) detection methods provide a highly sensitive alternative to conventional surveillance techniques, extensive testing is imperative to generate reliable output. Here, we tested and compared the performance of two primer/probe assays to detect and quantify the abundance of bullfrogs in Western Europe in silico and in situ using digital droplet PCR (ddPCR). Although both assays proved to be equally target-specific and sensitive, one outperformed the other in ddPCR detection resolution (i.e., distinguishing groups of target-positive and target-negative droplets), and hence was selected for further analyses. Mesocosm experiments revealed that tadpole abundance and biomass explained 99% of the variation in eDNA concentration. Because per individual eDNA emission rates did not differ significantly among tadpoles and juveniles, and adults mostly reside out of the water, eDNA concentration can be used as an approximation of local bullfrog abundance in natural populations. Seasonal eDNA patterns in three colonized ponds showed parallel fluctuations in bullfrog eDNA concentration. An increase in eDNA concentration was detected in spring, followed by a strong peak coinciding with the breeding season (August, September or October), and continuously low eDNA concentrations during winter. With this study, we report the validation process required for appropriately implementing eDNA barcoding analyses in lentic systems. We demonstrate that this technique can serve as a solid and reliable tool to detect the early stages of bullfrog invasions and to quantify temporal changes in abundance that will be useful in coordinating large-scale bullfrog eradication programs and evaluating their efficiency.

Mycorrhizal Communities and Isotope Signatures in Two Partially Mycoheterotrophic Orchids.

Partial mycoheterotrophy, the ability of plants to obtain carbon from fungi throughout their life cycle in combination with photosynthesis, appears to be more common within the Plant Kingdom than previously anticipated. Recent studies using stable isotope analyses have indicated that isotope signatures in partially mycoheterotrophic plants vary widely among species, but the relative contributions of family- or species-specific characteristics and the identity of the fungal symbionts to the observed differences remain unclear. Here, we investigated in detail mycorrhizal communities and isotopic signatures in four co-occurring terrestrial orchids (Platanthera chlorantha, Epipactis helleborine, E. neglecta and the mycoheterotrophic Neottia nidus-avis). All investigated species were mycorrhizal generalists (i.e., associated with a large number of fungi simultaneously), but mycorrhizal communities differed significantly between species. Mycorrhizal communities associating with the two Epipactis species consisted of a wide range of fungi belonging to different families, whereas P. chlorantha and N. nidus-avis associated mainly with Ceratobasidiaceae and Sebacinaceae species, respectively. Isotopic signatures differed significantly between both Epipactis species, with E. helleborine showing near autotrophic behavior and E. neglecta showing significant enrichment in both carbon and nitrogen. No significant differences in photosynthesis and stomatal conductance were observed between the two partially mycoheterotrophic orchids, despite significant differences in isotopic signatures. Our results demonstrate that partially mycoheterotrophic orchids of the genus Epipactis formed mycorrhizas with a wide diversity of fungi from different fungal families, but variation in mycorrhizal community composition was not related to isotope signatures and thus transfer of C and N to the plant. We conclude that the observed differences in isotope signatures between E. helleborine and E. neglecta cannot solely be explained by differences in mycorrhizal communities, but most likely reflect a combination of inherent physiological differences and differences in mycorrhizal communities.

Reliable eDNA detection and quantification of the European weather loach (Misgurnus fossilis).

The European weather loach (Misgurnus fossilis) is a cryptic and poorly known fish species of high conservation concern. The species is experiencing dramatic population collapses across its native range to the point of regional extinction. Although environmental DNA (eDNA)-based approaches offer clear advantages over conventional field methods for monitoring rare and endangered species, accurate detection and quantification remain difficult and quality assessment is often poorly incorporated. In this study, we developed and validated a novel digital droplet PCR (ddPCR) eDNA-based method for reliable detection and quantification, which allows accurate monitoring of M. fossilis across a number of habitat types. A dilution experiment under laboratory conditions allowed the definition of the limit of detection (LOD) and the limit of quantification (LOQ), which were set at concentrations of 0.07 and 0.14 copies µl-1 , respectively. A series of aquarium experiments revealed a significant and positive relationship between the number of individuals and the eDNA concentration measured. During a 3 year survey (2017-2019), we assessed 96 locations for the presence of M. fossilis in Flanders (Belgium). eDNA analyses on these samples highlighted 45% positive detections of the species. On the basis of the eDNA concentration per litre of water, only 12 sites appeared to harbour relatively dense populations. The other 31 sites gave a relatively weak positive signal that was typically situated below the LOQ. Combining sample-specific estimates of effective DNA quantity (Qe ) and conventional field sampling, we concluded that each of these weak positive sites still likely harboured the species and therefore they do not represent false positives. Further, only seven of the classified negative samples warrant additional sampling as our analyses identified a substantial risk of false-negative detections (i.e., type II errors) at these locations. Finally, we illustrated that ddPCR outcompetes conventional qPCR analyses, especially when target DNA concentrations are critically low, which could be attributed to a reduced sensitivity of ddPCR to inhibition effects, higher sample concentrations being accommodated and higher sensitivity obtained.

Monitoring of spatiotemporal occupancy patterns of fish and amphibian species in a lentic aquatic system using environmental DNA.

To effectively monitor, manage and protect aquatic species and understand their interactions, knowledge of their spatiotemporal distribution is needed. In this study, we used a fine-scale spatiotemporal water sampling design, followed by environmental DNA (eDNA) 12S metabarcoding, to investigate occupancy patterns of a natural community of fish and amphibian species in a lentic system. In the same system, we experimentally estimated the spatial and temporal dispersion of eDNA by placing a community of different fish and amphibian species in cages at one side of the pond, creating a controlled point of eDNA emission. Analyses of this cage community revealed a sharp spatial decline in detection rates and relative eDNA quantities at a distance of 5-10 m from the source, depending on the species and its abundance. In addition, none of the caged species could be detected 1 week after removal from the system. This indicates high eDNA decay rates and limited spatial eDNA dispersal, facilitating high local resolution for monitoring spatial occupancy patterns of aquatic species. Remarkably, for seven of the nine cage species, the presence of a single individual could be detected by pooling water of subsamples taken across the whole water body, illustrating the high sensitivity of the eDNA sampling and detection method applied. Finally, our work demonstrated that a fine-scale sampling design in combination with eDNA metabarcoding can cover total biodiversity very precisely and allows the construction of consistent spatiotemporal patterns of relative abundance and local distribution of free-living fish and amphibian species in a lentic ecosystem.

Lack of strong selection pressures maintains wide variation in floral traits in a food-deceptive orchid.

BACKGROUND AND AIMS: Angiosperms vary remarkably in traits such as colour, size and shape of flowers, yet such variation generally tends to be low within species. In deceptive orchids, however, large variation in floral traits has been described, not only between but also within populations. Nonetheless, the factors driving variation in floral traits in deceptive orchids remain largely unclear. METHODS: To identify determinants of variation in floral traits, we investigated patterns of fruit set and selection gradients in the food-deceptive orchid Orchis purpurea, which typically presents large within-population variation in the colour and size of the flowers. Using long-term data, fruit set was quantified in two populations over 16 consecutive years (2004-2019). Artificial hand pollination was performed to test the hypothesis that fruit set was pollinator-limited and that selfing led to decreased seed set and viability. Annual variation (2016-2019) in selection gradients was calculated for three colour traits (brightness, contrast and the number of spots on the labellum), flower size (spur length, labellum length and width) and plant size (number of flowers, plant height). KEY RESULTS: Fruit set was, on average, low (~12 %) and severely pollinator-limited. Opportunities for selection varied strongly across years, but we found only weak evidence for selection on floral traits. In contrast, there was strong and consistent positive selection on floral display. Selfing led to reduced production of viable seeds and hence severe inbreeding depression (δ = 0.38). CONCLUSION: Overall, these results demonstrate that the large variation in flower colour and size that is regularly observed in natural O. purpurea populations is maintained by the consistent lack of strong selection pressures on these traits through time.

Combining ddPCR and environmental DNA to improve detection capabilities of a critically endangered freshwater invertebrate.

Isogenus nubecula is a critically endangered Plecoptera species. Considered extinct in the UK, I. nubecula was recently rediscovered (in one location of the River Dee, Wales), after 22 years of absence. In a similar way to many other species of Perlodidae, I. nubecula could be utilised as a bio-indicator, for assessing water quality and health status of a given freshwater system. However, conventional monitoring of invertebrates via kick-sampling, is invasive and expensive (time consuming). Further, such methods require a high level of taxonomic expertise. Here, we compared the traditional kick-sampling method with the use of eDNA detection using qPCR and ddPCR-analyses. In spring 2018, we sampled eDNA from twelve locations on the River Dee. I. nubecula was detected using kick-sampling in five of these locations, three locations using both eDNA detection and kick-sampling and one location using eDNA detection alone - resulting in a total of six known and distinct populations of this critically endangered species. Interestingly, despite the eDNA assay being validated in vitro and in silico, and results indicating high sensitivity, qPCR analysis of the eDNA samples proved to be ineffective. In contrast, ddPCR analyses resulted in a clear detection of I. nubecula at four locations suggesting that inhibition most likely explains the large discrepancy between the obtained qPCR and ddPCR results. It is therefore important to explore inhibition effects on any new eDNA assay. We also highlight that ddPCR may well be the best option for the detection of aquatic organisms which are either rare or likely to shed low levels of eDNA into their environment.

Influence of accuracy, repeatability and detection probability in the reliability of species-specific eDNA based approaches.

Environmental DNA (eDNA) barcoding has a high potential to increase the cost-efficiency of species detection and monitoring in aquatic habitats. However, despite vast developments in the field, many published assays often lack detailed validation and there is little to no commonly (agreed upon) standardization of protocols. In this study, we evaluated the reliability of eDNA detection and quantification using published primers and assays targeting the Freshwater Pearl Mussel as a model organism. We first assessed limits of detection for two different target genes (COI and 16S) following the MIQE guidelines, and then tested the reliability of quantification in a double-blind mesocosm experiment. Our results reveal that different methodological indicators, namely accuracy, repeatability and detection probability affected the reliability of eDNA measurement at the different levels tested. The selection of the optimal analytical method was mainly determined by detection probability. Both the COI and 16S assays were highly specific for the targeted organism and showed similar accuracy and repeatability, whilst the limit of detection was clearly lower for the COI based approach. In contrast, the reliability of eDNA quantification hinged on repeatability, reflected by the scattering (r2 = 0.87) around the relationship between eDNA and mussel density in mesocosms. A bootstrapping approach, which allowed for the assignment of measures associated with repeatability of samples, revealed that variability between natural replicates (i.e. accuracy) strongly influenced the number of replicates required for a reliable species detection and quantification in the field.

Mycorrhizal divergence and selection against immigrant seeds in forest and dune populations of the partially mycoheterotrophic Pyrola rotundifolia.

Plant populations occupying different habitats may diverge from each other over time and gradually accumulate genetic and morphological differences, ultimately resulting in ecotype or even species formation. In plant species that critically rely on mycorrhizal fungi, differences in mycorrhizal communities can contribute to ecological isolation by reducing or even inhibiting germination of immigrant seeds. In this study, we investigated whether the mycorrhizal communities available in the soil and associating with the roots of seedlings and adult plants of the partially mycoheterotrophic Pyrola rotundifolia differed between populations growing in sand dunes and forests. In addition, reciprocal germination experiments were performed to test whether native seeds showed higher germination than immigrant seeds. Our results showed that the mycorrhizal communities differed significantly between forest and dune populations, and that within populations seedlings and adults also associated with different mycorrhizal communities. In both forest and dune populations, mycorrhizal communities were dominated by members of the Thelephoraceae, but dune populations showed a higher incidence of members of the Inocybaceae, whereas forest populations showed a high abundance of members of the Russulaceae. Reciprocal germination experiments showed that native seeds showed a higher germination success than immigrant seeds and this effect was most pronounced in dune populations. Overall, these results demonstrate that plants of P. rotundifolia growing in dune and forest habitats associate with different mycorrhizal communities and that reduced germination of non-native seeds may contribute to reproductive isolation. We conclude that selection against immigrants may constitute an important reproductive barrier at early stages of the speciation process.

Variability in DNA Methylation and Generational Plasticity in the Lombardy Poplar, a Single Genotype Worldwide Distributed Since the Eighteenth Century.

In the absence of genetic diversity, plants rely on the capacity of phenotypic plasticity to cope with shifts in environmental conditions. Understanding the mechanisms behind phenotypic plasticity and how local phenotypic adjustments are transferred to clonal offspring, will provide insight into its ecological and evolutionary significance. Epigenetic changes have recently been proposed to play a crucial role in rapid environmental adaptation. While the contribution of epigenetic changes to phenotypic plasticity has been extensively studied in sexual reproducing model organisms, little work has been done on vegetative generations of asexual reproducing plant species. We studied the variability of DNA methylation and bud set phenology of the Lombardy poplar (Populus nigra cv. Italica Duroi), a cultivated tree representing a single genotype worldwide distributed since the eighteenth century. Bud set observations and CpG methyl polymorphisms were studied on vegetative offspring resulting from cuttings grown for one season in a common glasshouse environment. The cuttings were collected from 60 adult Lombardy poplars growing in different environments. The physiological condition of the cuttings was determined by measuring weight and nutrient condition. Methylation sensitive amplified polymorphisms were used to obtain global patterns of DNA methylation. Using logistic regression models, we investigated correlations among epigenotype, bud phenology, and the climate at the home site of the donor trees, while accounting for physiological effects. We found significant epigenetic variation as well as significant variation in bud phenology, in the absence of genetic variation. Remarkably, phenology of bud set observed at the end of the growing season in the common environment was significantly correlated with climate variables at the home site of the mother trees, specifically the average temperature of January and monthly potential evapotranspiration. Although we could not directly detect significant effects of epigenetic variation on phenology, our results suggest that, in the Lombardy poplar, epigenetic marks contribute to the variation of phenotypic response that can be transferred onto asexually reproduced offspring resulting in locally adapted ecotypes. This contributes to the growing evidence that epigenetic-based transgenerational inheritance might be relevant for adaptation and evolution in contrasting or rapidly changing environments.

The impact of floral morphology on genetic differentiation in two closely related biennial plant species.

The genetic diversity and structure of plant populations are determined by the interaction of three distinct processes: gene flow, genetic drift and natural selection. These processes are to some extent dependent on the mating system of plants, which in turn is largely determined by floral morphology and the level of herkogamy in particular. In this study, we used molecular markers to investigate the impact of floral morphology on genetic differentiation and structure in two closely related Centaurium species that display large variation in floral morphology across two distinct geographic regions in Europe (mainland Europe and the UK). Our results showed that genetic differences between regions and populations within regions were similar for both species, but that patterns of genetic structure largely depended on the observed variation in floral morphology. Populations of Centaurium erythraea showed higher genetic differentiation and clear isolation by distance (IBD) in mainland Europe, but limited IBD in the UK. Opposite patterns were found in Centaurium littorale, with higher genetic differentiation and significant IBD in populations sampled in the UK and lower genetic differentiation in Continental populations with no pattern of IBD. Overall, these results indicate that variation in floral morphology has a profound impact on structuring of genetic diversity, with populations displaying low levels of herkogamy showing the strongest patterns of genetic structuring and the reverse pattern in populations showing high levels of herkogamy.

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.

Mycorrhizal Associations and Trophic Modes in Coexisting Orchids: An Ecological Continuum between Auto- and Mixotrophy.

Two distinct nutritional syndromes have been described in temperate green orchids. Most orchids form mycorrhizas with rhizoctonia fungi and are considered autotrophic. Some orchids, however, associate with fungi that simultaneously form ectomycorrhizas with surrounding trees and derive their carbon from these fungi. This evolutionarily derived condition has been called mixotrophy or partial mycoheterotrophy and is characterized by 13C enrichment and high N content. Although it has been suggested that the two major nutritional syndromes are clearly distinct and tightly linked to the composition of mycorrhizal communities, recent studies have challenged this assumption. Here, we investigated whether mycorrhizal communities and nutritional syndromes differed between seven green orchid species that co-occur under similar ecological conditions (coastal dune slacks). Our results showed that mycorrhizal communities differed significantly between orchid species. Rhizoctonia fungi dominated in Dactylorhiza sp., Herminium monorchis, and Epipactis palustris, which were autotrophic based on 13C and N content. Conversely, Liparis loeselii and Epipactis neerlandica associated primarily with ectomycorrhizal fungi but surprisingly, 13C and N content supported mixotrophy only in E. neerlandica. This, together with the finding of some ectomycorrhizal fungi in rhizoctonia-associated orchids, suggests that there exists an ecological continuum between the two syndromes. The presence of a large number of indicator species associating with individual orchid species further confirms previous findings that mycorrhizal fungi may be important factors driving niche-partitioning in terrestrial orchids and therefore contribute to orchid coexistence.

Analysis of spatial genetic variation reveals genetic divergence among populations of Primula veris associated to contrasting habitats.

Genetic divergence by environment is a process whereby selection causes the formation of gene flow barriers between populations adapting to contrasting environments and is often considered to be the onset of speciation. Nevertheless, the extent to which genetic differentiation by environment on small spatial scales can be detected by means of neutral markers is still subject to debate. Previous research on the perennial herb Primula veris has shown that plants from grassland and forest habitats showed pronounced differences in phenology and flower morphology, suggesting limited gene flow between habitats. To test this hypothesis, we sampled 33 populations of P. veris consisting of forest and grassland patches and used clustering techniques and network analyses to identify sets of populations that are more connected to each other than to other sets of populations and estimated the timing of divergence. Our results showed that spatial genetic variation had a significantly modular structure and consisted of four well-defined modules that almost perfectly coincided with habitat features. Genetic divergence was estimated to have occurred about 114 generations ago, coinciding with historic major changes in the landscape. Overall, these results illustrate how populations adapting to different environments become structured genetically within landscapes on small spatial scales.

Geographic variation in floral traits and the capacity of autonomous selfing across allopatric and sympatric populations of two closely related Centaurium species.

Floral traits and the relative contribution of autonomous selfing to total seed set varies geographically and is often driven by the availability and abundance of suitable pollinators and/or the presence of co-flowering relatives. In the latter case, competition for pollinator services and costs of hybridization can select for floral traits that reduce interspecific gene flow and contribute to prezygotic isolation, potentially leading to geographic variation in floral divergence between allopatric and sympatric populations. In this study, we investigated variation in floral traits and its implications on the capacity of autonomous selfing in both allopatric and sympatric populations of two closely related Centaurium species(Gentianaceae) across two distinct geographic regions(UK and mainland Europe). Although the magnitude and direction of floral differentiation varied between regions, sympatric populations were always significantly more divergent in floral traits and the capacity to self autonomously than allopatric populations. These results indicate that mating systems can vary substantially within a species and that the joint occurrence of plant species can have a major impact on floral morphology and capacity of autonomous selfing, most likely as a way to reduce the probability of interspecific interference.

Mycorrhizal specificity does not limit the distribution of an endangered orchid species.

What factors determine the distribution of a species is a central question in ecology and conservation biology. In general, the distribution of plant species is assumed to be controlled by dispersal or environmentally controlled recruitment. For plant species which are critically dependent on mycorrhizal symbionts for germination and seedling establishment, specificity in mycorrhizal associations and availability of suitable mycorrhizal fungi can be expected to have a major impact on successful colonization and establishment and thus ultimately on a species distribution. We combined seed germination experiments with soil analyses and fungal assessments using 454 amplicon pyrosequencing to test the relative importance of dispersal limitation, mycorrhizal availability and local growth conditions on the distribution of the orchid species Liparis loeselii, which, despite being widely distributed, is rare and endangered in Europe. We compared local soil conditions, seed germination and mycorrhizal availability in the soil between locations in northern Belgium and France where L. loeselii occurs naturally and locations where conditions appear suitable, but where adults of the species are absent. Our results indicated that mycorrhizal communities associating with L. loeselii varied among sites and plant life cycle stages, but the observed variations did not affect seed germination, which occurred regardless of current L. loeselii presence and was significantly affected by soil moisture content. These results indicate that L. loeselii is a mycorrhizal generalist capable of opportunistically associating with a variety of fungal partners to induce seed germination. They also indicate that availability of fungal associates is not necessarily the determining factor driving the distribution of mycorrhizal plant species.

Habitat-driven variation in mycorrhizal communities in the terrestrial orchid genus Dactylorhiza.

Orchid species are critically dependent on mycorrhizal fungi for completion of their life cycle, particularly during the early stages of their development when nutritional resources are scarce. As such, orchid mycorrhizal fungi play an important role in the population dynamics, abundance, and spatial distribution of orchid species. However, less is known about the ecology and distribution of orchid mycorrhizal fungi. In this study, we used 454 amplicon pyrosequencing to investigate ecological and geographic variation in mycorrhizal associations in fourteen species of the orchid genus Dactylorhiza. More specifically, we tested the hypothesis that variation in orchid mycorrhizal communities resulted primarily from differences in habitat conditions where the species were growing. The results showed that all investigated Dactylorhiza species associated with a large number of fungal OTUs, the majority belonging to the Tulasnellaceae, Ceratobasidiaceae and Sebacinales. Mycorrhizal specificity was low, but significant variation in mycorrhizal community composition was observed between species inhabiting different ecological habitats. Although several fungi had a broad geographic distribution, Species Indicator Analysis revealed some fungi that were characteristic for specific habitats. Overall, these results indicate that orchid mycorrhizal fungi may have a broad geographic distribution, but that their occurrence is bounded by specific habitat conditions.