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.

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.

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.

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.

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.

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 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.

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.

Nonrandom seedling establishment corresponds with distance-dependent decline in mycorrhizal abundance in two terrestrial orchids.

In plant species that critically rely on mycorrhizal symbionts for germination and seedling establishment, distance-dependent decline of mycorrhizal fungi in the soil can be hypothesized to lead to significant spatial clustering as a result of nonrandom spatial patterns of seedling establishment. To test this hypothesis, we investigated the abundance and distribution of mycorrhizal fungi in the soil and how they relate to spatial patterns of adults and seedling recruitment in two related orchid species. We combined assessments of spatial variation in fungal abundance using quantitative PCR (qPCR) with spatial point pattern analyses based on long-term demographic data and cluster point process models. qPCR analyses showed that fungal abundance declined rapidly with distance from the adult host plants. Spatial point pattern analyses showed that successful recruitment in both species was clustered significantly around adult plants and that the decline in the neighborhood density of recruits around adults coincided with the decline of fungal abundance around adult plants. Overall, these results indicate that the distribution and abundance of fungal associates in the soil may have a strong impact on the aboveground distribution of its partner.

Differences in mycorrhizal communities between Epipactis palustris, E. helleborine and its presumed sister species E. neerlandica.

BACKGROUND AND AIMS: In orchid species that have populations occurring in strongly contrasting habitats, mycorrhizal divergence and other habitat-specific adaptations may lead to the formation of reproductively isolated taxa and ultimately to species formation. However, little is known about the mycorrhizal communities associated with recently diverged sister taxa that occupy different habitats. METHODS: In this study, 454 amplicon pyrosequencing was used to investigate mycorrhizal communities associating with Epipactis helleborine in its typical forest habitat and with its presumed sister species E. neerlandica that almost exclusively occurs in coastal dune habitats. Samples of the phylogenetically more distant E. palustris, which co-occurred with E. neerlandica, were also included to investigate the role of habitat-specific conditions on mycorrhizal communities. RESULTS: A total of 105 operational taxonomic units (OTUs) of putative orchid mycorrhizal fungi were observed in the three studied species. The majority of these fungi were endophytic fungi of Helotiales and ectomycorrhizal fungi belonging to Thelephoraceae, Sebacinaceae and Inocybaceae. In addition, a large number of other ectomycorrhizal taxa were detected, including Cortinarius, Cenococcum, Tuber, Geopora, Wilcoxina, Meliniomyces, Hebeloma, Tricholoma, Russula and Peziza Mycorrhizal communities differed significantly between the three species, but differences were most pronounced between the forest species (E. helleborine) and the two dune slack species (E. neerlandica and E. palustris). CONCLUSION: The results clearly showed that recently diverged orchid species that occupy different habitats were characterized by significantly different mycorrhizal communities and call for more detailed experiments that aim at elucidating the contribution of habitat-specific adaptations in general and mycorrhizal divergence in particular to the process of speciation in orchids.

The impact of hybridization on long-term persistence of polyploid Dactylorhiza species.

PREMISE OF THE STUDY: Hybridization may pose severe threats to the long-term survival of the parental taxa through introgression and the formation of hybrid swarms. However, when the resulting hybrids show strong male and female sterility, backcrossing and introgression are unlikely to occur, but the parental species may suffer from reduced male and female fitness. METHODS: We assessed the impact of hybridization on the long-term persistence of two food-deceptive orchids in the genus Dactylorhiza (the common Dactylorhiza maculata and the rare D. sphagnicola). The extent of hybridization was investigated using both molecular markers and morphometric measurements. To determine the strength of postmating reproductive isolation, hand pollinations were conducted between pure and hybrid individuals. Finally, fruit set and seed viability of open-pollinated plants were determined in sympatric and allopatric populations to investigate the impact of hybridization on the reproductive output of the pure parental species. KEY RESULTS: Our results showed that postmating reproductive isolation was weak and that hybridization occurred frequently within the studied sympatric population. Although hybrids were characterized by very low female fitness, mainly because of strongly reduced seed viability, backcrossing appeared to occur and was asymmetric toward the rare D. sphagnicola. Fruit set and seed viability of open-pollinated plants were also significantly lower in the sympatric population than in the allopatric populations, indicating that hybridization and ongoing introgression incurred fitness costs in the pure parental species. CONCLUSIONS: Overall, our results suggest that extensive hybridization can affect the long-term viability of the parental species through the combined effect of introgression following interspecific hybrid fertilization and reduced fitness of the parental species.

Mycorrhizal networks and coexistence in species-rich orchid communities.

Multispecies assemblages often consist of a complex network of interactions. Describing the architecture of these networks is a first step in understanding the stability and persistence of these species-rich communities. Whereas a large body of research has been devoted to the description of above-ground interactions, much less attention has been paid to below-ground interactions, probably because of difficulties to adequately assess the nature and diversity of interactions occurring below the ground. In this study, we used 454 amplicon pyrosequencing to investigate the architecture of the network between mycorrhizal fungi and 20 orchid species co-occurring in a species-rich Mediterranean grasslands. We found 100 different fungal operational taxonomic units (OTUs) known to be mycorrhizal in orchids, most of which were members related to the genera Ceratobasidium and Tulasnella. The network of interactions was significantly compartmentalized (M = 0.589, P = 0.001), but not significantly nested (N = 0.74, NODF = 10.58; P > 0.05). Relative nestedness was negative (N* = -0.014), also suggesting the existence of isolated groups of interacting species. Compartmentalization is a typical feature of ecological systems showing high interaction intimacy, and may reflect strong specialization between orchids and fungi resulting from physiological, physical or spatial constraints.

Mycorrhizal diversity, seed germination and long-term changes in population size across nine populations of the terrestrial orchid Neottia ovata.

In plant species that rely on mycorrhizal symbioses for germination and seedling establishment, seedling recruitment and temporal changes in abundance can be expected to depend on fungal community composition and local environmental conditions. However, disentangling the precise factors that determine recruitment success in species that critically rely on mycorrhizal fungi represents a major challenge. In this study, we used seed germination experiments, 454 amplicon pyrosequencing and assessment of soil conditions to investigate the factors driving changes in local abundance in 28 populations of the orchid Neottia ovata. Comparison of population sizes measured in 2003 and 2013 showed that nearly 60% of the studied populations had declined in size (average growth rate across all populations: -0.01). Investigation of the mycorrhizal fungi in both the roots and soil revealed a total of 68 species of putatively mycorrhizal fungi, 21 of which occurred exclusively in roots, 25 that occurred solely in soil and 22 that were observed in both the soil and roots. Seed germination was limited and significantly and positively related to soil moisture content and soil pH, but not to fungal community composition. Large populations or populations with high population growth rates showed significantly higher germination than small populations or populations declining in size, but no significant relationships were found between population size or growth and mycorrhizal diversity. Overall, these results indicate that temporal changes in abundance were related to the ability of seeds to germinate, but at the same time they provided limited evidence that variation in fungal communities played an important role in determining population dynamics.

Disruption of the distylous syndrome in Primula veris.

BACKGROUND AND AIMS: Distyly is a floral polymorphism characterized by the presence of two discrete morphs with reciprocal positioning of anthers and stigmas in flowers on different plants within the same population. Although reciprocal herkogamy and associated floral traits are generally thought to be discrete and strict polymorphisms, little is known about variation in floral traits related to the distylous syndrome within and among populations of a single species. In this study, variation in floral morphology and reciprocal positioning of the sexual organs in the distylous Primula veris (cowslip) is quantified. METHODS: Data were collected in ten populations occurring in two contrasting habitat types (grasslands and forests), and for each population the average level of reciprocity was assessed, the strength of the self-incompatibility system was determined, and seed production under natural conditions was quantified. RESULTS: In grassland populations, flowers showed clear distyly with low and symmetric reciprocity indices at both the lower and upper level. In forests, P. veris produced larger flowers that showed strong deviations in stigma-anther separation, especially in the L-morph. This deviation was mainly driven by variation in stigma height, resulting in high and asymmetric reciprocity indices and the occurrence of several short-styled homostylous plants. Self-incompatibility was, however, strict in both habitats, and morph ratios did not differ significantly from isoplethy. The observed shift in reciprocity in forest populations was associated with a significant reduction in seed production in the L-morph. CONCLUSIONS: The results indicate that populations of P. veris show habitat-specific variation in flower morphology. Deviations from perfect reciprocal positioning of stigmas and anthers also translate into reduced seed production, suggesting that small changes in sexual organ reciprocity can have far-reaching ecological and evolutionary implications.

Pollen limitation and the contribution of autonomous selfing to fruit and seed set in a rewarding orchid.

UNLABELLED: • PREMISE OF THE STUDY: Although rewarding orchids are believed to have a high pollination efficiency, pollination success is often low, suggesting that rewarding orchids may be prone to pollen limitation. Assuming that selfing contributes significantly to fruit and seed set (i.e., reproductive assurance) and that the quality of selfed seeds is high (i.e., low inbreeding depression), it can be hypothesized that under these circumstances populations evolve to facultative or even complete self-pollination.• METHODS: In this study, we performed emasculation and pollen supplementation experiments in the field to assess the extent of outcross pollen limitation and the contribution of autonomous self-pollination to fruit and seed set in the rewarding orchid Epipactis palustris. Hand-pollinations using cross and self-pollen were performed in the greenhouse to investigate the impact of selfing on seed set and seed quality and to assess the magnitude of inbreeding depression.• KEY RESULTS: Fruit set under natural conditions was high: ca. 70% of all flowers set fruit. Percentage fruit set of emasculated plants was 56%, implying that fruit set in about 14% of all flowers was the result of autonomous self-pollination. Pollen supplementation significantly increased fruit and seed set, indicating strong outcross pollen limitation. Hand-pollination with cross pollen resulted in significantly higher seed set and seed quality compared with manual self-pollination, indicating high levels of early inbreeding depression (δ = 0.46 ± 0.08).• CONCLUSIONS: Overall, these results indicate that, despite the high pollination efficiency, populations of this rewarding orchid species were strongly pollen limited. Costs of early inbreeding depression, on the other hand, were high, which may prevent rapid evolution of complete selfing.

The influence of herbivory on pre- and postzygotic stages of reproduction following open, self, and outcross pollination.

PREMISE OF THE STUDY: Herbivory affects pollination success and reproductive output in plants. However, the different stages in the process from pollination to seed maturation have hardly been investigated within the context of herbivory. Herbivory might affect these stages via its effect on geitonogamous pollination and thereby the proportion of self pollen delivered to the stigma and/or via its effect on the nutritional capacity of the maternal plant. METHODS: Plants of monocarpic Cynoglossum officinale were experimentally subjected to root herbivory and exposed to natural open pollination in combination with self and outcross hand pollination. We quantified pollen germination, pollen tube competition intensity, pollen tube attrition, fruit set, and seed initiation, abortion, and maturation. KEY RESULTS: Although root herbivory did not affect pollen germination or pollen tube attrition, fruit set and seed initiation and maturation were negatively affected by herbivory, but for seed initiation only in the case of outcross- and open-pollinated flowers. The intensity of pollen tube competition positively affected seed initiation, but only in plants infested with the herbivore. CONCLUSIONS: Our study demonstrates that herbivory did not affect the early stages following pollination, but significantly impacted later postpollination stages such as fruit set and seed maturation and selection based on pollen tube competition intensity on zygote development. Our findings suggest that decreased nutritional capacity of the mother plant in response to root herbivory rather than herbivory effects on pollen quality was responsible for the lower fruit and seed production in infested plants.

Coexisting orchid species have distinct mycorrhizal communities and display strong spatial segregation.

Because orchids are dependent on mycorrhizal fungi for germination and establishment of seedlings, differences in the mycorrhizal communities associating with orchids can be expected to mediate the abundance, spatial distribution and coexistence of terrestrial orchids in natural communities. We assessed the small-scale spatial distribution of seven orchid species co-occurring in 25 × 25 m plots in two Mediterranean grasslands. In order to characterize the mycorrhizal community associating with each orchid species, 454 pyrosequencing was used. The extent of spatial clustering was assessed using techniques of spatial point pattern analysis. The community of mycorrhizal fungi consisted mainly of members of the Tulasnellaceae, Thelephoraceae and Ceratobasidiaceae, although sporadically members of the Sebacinaceae, Russulaceae and Cortinariaceae were observed. Pronounced differences in mycorrhizal communities were observed between species, whereas strong clustering and significant segregation characterized the spatial distribution of orchid species. However, spatial segregation was not significantly related to phylogenetic dissimilarity of fungal communities. Our results indicate that co-occurring orchid species have distinctive mycorrhizal communities and show strong spatial segregation, suggesting that mycorrhizal fungi are important factors driving niche partitioning in terrestrial orchids and may therefore contribute to orchid coexistence.

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.

Differences in dichogamy and herkogamy contribute to higher selfing in contrasting environments in the annual Blackstonia perfoliata (Gentianaceae).

BACKGROUND AND AIMS: The establishment of plant populations in novel environments may generate pronounced shifts in floral traits and plant mating systems, particularly when pollinators are scarce. In this study, floral morphology and mating system functioning are compared between recently established and older populations of the annual plant Blackstonia perfoliata that occur in different pollinator environments. METHODS: Hand-pollination and emasculation experiments were conducted to assess the extent of pollinator-mediated pollen deposition and pollen limitation, and the contribution of autonomous selfing to total seed production. Detailed measurements of key floral traits were performed to compare the flower morphology and mating system functioning between plants from both pollination environments. KEY RESULTS: Pollinator-mediated pollen deposition was about twice as low in the recently colonized and pollinator-poor environment compared with the old and pollinator-rich sites, but total seed set was little affected by any type of pollen limitation. The contribution of autonomous selfing to total seed production was higher in the pollinator-poor sites than in the pollinator-rich sites (index of reproductive assurance = 0·56 and 0·17, respectively), and seed production was only poorly affected by selfing, whereas in the pollinator-rich populations selfing reduced total reproductive output by about 40 % compared with outcross pollination. Plants originating from pollinator-poor environments produced smaller flowers that showed significantly lower levels of dichogamy (i.e. protogyny) and herkogamy. These reductions resulted in a 2-fold higher capacity for autonomous selfing under pollinator-free conditions (index of autonomous selfing = 0·81 and 0·41 in plants originating from the pollinator-poor and pollinator-rich environment, respectively). CONCLUSIONS: The results illustrate that plant populations colonizing novel environments can differ markedly in floral morphology and mating system functioning. Due to a temporal shift in the male phase, the breeding system of B. perfoliata shifted from delayed selfing under pollinator-rich conditions towards competing selfing in recently established populations, providing greater reproductive assurance when pollinators and/or reproductive partners are limited.