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.

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.

Variation in ectomycorrhizal fungal communities associated with Silver linden (Tilia tomentosa) within and across urban areas.

Trees in urban areas face harsh environmental conditions. Ectomycorrhizal fungi (EcM) form a symbiosis with many tree species and provide a range of benefits to their host through their extraradical hyphal network. Although our understanding of the environmental drivers and large scale geographical variation of EcM communities in natural ecosystems is growing, our knowledge of EcM communities within and across urban areas is still limited. Here, we characterized EcM communities using Illumina miseq sequencing on 175 root samples of the urban tree Tilia tomentosa from three European cities, namely Leuven (Belgium), Strasbourg (France) and Porto (Portugal). We found strong differences in EcM richness and community composition between cities. Soil acidity, organic matter and moisture content were significantly associated with EcM community composition. In agreement, the explained variability in EcM communities was mostly attributed to general soil characteristics, whereas very little variation was explained by city and heavy metal pollution. Overall, our results suggest that EcM communities in urban areas are significantly associated with soil characteristics, while heavy metal pollution and biogeography had little or no impact. These findings deliver new insights into EcM distribution patterns in urban areas and contribute to specific inoculation strategies to improve urban tree vitality.

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.

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.

Assessing the composition of microbial communities in textile wastewater treatment plants in comparison with municipal wastewater treatment plants.

It is assumed that microbial communities involved in the biological treatment of different wastewaters having a different chemical composition harbor different microbial populations which are specifically adapted to the environmental stresses encountered in these systems. Yet, little is known about the composition of these microbial communities. Therefore, the aim of this study was to assess the microbial community composition over two seasons (winter and summer) in activated sludge from well-operating textile wastewater treatment plants (WWTPs) in comparison with municipal WWTPs, and to explain observed differences by environmental variables. 454-pyrosequencing generated 160 archaeal and 1645 bacterial species-level Operational Taxonomic Units (OTUs), with lower observed richness in activated sludge from textile WWTPs compared to municipal WWTPs. The bacterial phyla Planctomycetes, Chloroflexi, Chlorobi, and Acidobacteria were more abundant in activated sludge samples from textile WWTPs, together with archaeal members of Thaumarchaeota. Nonmetric multidimensional scaling analysis of the microbial communities showed that microbial communities from textile and municipal WWTPs were significantly different, with a seasonal effect on archaea. Nitrifying and denitrifying bacteria as well as phosphate-accumulation bacteria were more abundant in municipal WWTPs, while sulfate-reducing bacteria were almost only detected in textile WWTPs. Additionally, microbial communities from textile WWTPs were more dissimilar than those of municipal WWTPs, possibly due to a wider diversity in environmental stresses to which microbial communities in textile WWTPs are subjected to. High salinity, high organic loads, and a higher water temperature were important potential variables driving the microbial community composition in textile WWTPs. This study provides a general view on the composition of microbial communities in activated sludge of textile WWTPs, and may provide novel insights for identifying key players performing important functions in the purification of textile wastewaters.

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.

Mycorrhizal Fungal Diversity and Community Composition in Two Closely Related Platanthera (Orchidaceae) Species.

While it is generally acknowledged that orchid species rely on mycorrhizal fungi for completion of their life cycle, little is yet known about how mycorrhizal fungal diversity and community composition vary within and between closely related orchid taxa. In this study, we used 454 amplicon pyrosequencing to investigate variation in mycorrhizal communities between pure (allopatric) and mixed (sympatric) populations of two closely related Platanthera species (Platanthera bifolia and P. chlorantha) and putative hybrids. Consistent with previous research, the two species primarily associated primarily with members of the Ceratobasidiaceae and, to a lesser extent, with members of the Sebacinales and Tulasnellaceae. In addition, a large number of ectomycorrhizal fungi belonging to various families were observed. Although a considerable number of mycorrhizal fungi were common to both species, the fungal communities were significantly different between the two species. Individuals with intermediate morphology showed communities similar to P. bifolia, confirming previous results based on the genetic architecture and fragrance composition that putative hybrids essentially belonged to one of the parental species (P. bifolia). Differences in mycorrhizal communities between species were smaller in mixed populations than between pure populations, suggesting that variation in mycorrhizal communities was largely controlled by local environmental conditions. The small differences in mycorrhizal communities in mixed populations suggests that mycorrhizal fungi are most likely not directly involved in maintaining species boundaries between the two Platanthera species. However, seed germination experiments are needed to unambiguously assess the contribution of mycorrhizal divergence to reproductive isolation.

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.

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.

Food availability affects the strength of mutualistic host-microbiota interactions in Daphnia magna.

The symbiotic gut microbial community is generally known to have a strong impact on the fitness of its host. Nevertheless, it is less clear how the impact of symbiotic interactions on the hosts' fitness varies according to environmental circumstances such as changes in the diet. This study aims to get a better understanding of host-microbiota interactions under different levels of food availability. We conducted experiments with the invertebrate, experimental model organism Daphnia magna and compared growth, survival and reproduction of conventionalized symbiotic Daphnia with germ-free individuals given varying quantities of food. Our experiments revealed that the relative importance of the microbiota for the hosts' fitness varied according to dietary conditions. The presence of the microbiota had strong positive effects on Daphnia when food was sufficient or abundant, but had weaker effects under food limitation. Our results indicate that the microbiota can be a potentially important factor in determining host responses to changes in dietary conditions. Characterization of the host-associated microbiota further showed that Aeromonas sp. was the most prevalent taxon in the digestive tract of Daphnia.

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.

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.

Impact of primer choice on characterization of orchid mycorrhizal communities using 454 pyrosequencing.

Although the number of studies investigating mycorrhizal associations in orchids has increased in recent years, the fungal communities associating with orchids and how they differ between species and sites remain unclear. Recent research has indicated that individual orchid plants may associate with several fungi concurrently, implying that to study mycorrhizal associations in orchids the fungal community should be assessed, rather than the presence of individual dominant fungal species or strains. High-throughput sequencing methods, such as 454 pyrosequencing, are increasingly used as the primary tool for such analyses. However, many studies combine universal primers from previous phylogenetic or ecological studies to generate amplicons suitable for 454 pyrosequencing without first critically evaluating their performance, potentially resulting in biased fungal community descriptions. Here, following in silico primer analysis we evaluated the performance of different combinations of existing PCR primers to characterize orchid mycorrhizal communities using 454 pyrosequencing by analysis of both an artificially assembled community of mycorrhizal fungi isolated from diverse orchid species and root samples from three different orchid species (Anacamptis morio, Ophrys tenthredinifera and Serapias lingua). Our results indicate that primer pairs ITS3/ITS4OF and ITS86F/ITS4, targeting the internal transcribed spacer-2 (ITS-2) region, outperformed other tested primer pairs in terms of number of reads, number of operational taxonomic units recovered from the artificial community and number of different orchid mycorrhizal associating families detected in the orchid samples. Additionally, we show the complementary specificity of both primer pairs, making them highly suitable for tandem use when studying the diversity of orchid mycorrhizal communities.

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.