Development and application of molecular tools to elucidate the influence of mycorrhizal interactions on the spatial distribution of terrestrial orchids.

Orchids have long been a source a scientific curiosity, even Darwin himself found them to be a source of intrigue, when he noted that “[seeds] are produced by orchids in vast profusion.... What checks the unlimited multiplication of the Orchideae throughout the world is not known.” (Darwin, 1877). We now understand that this preponderance of seed production and correspondingly rare event of germination and seed development are a consequence of the Orchidaceae’s dependence upon symbiosis with compatible mycorrhizal fungi. However, many of the specific details of these dependent associations between orchid and fungi remained poorly understood at the onset of this research. Who are the participating fungi? Where are they found? How exclusive are these relationships? What impact do orchid mycorrhizal associations have on orchid germination, recruitment, and therefore their distribution?

The overarching objecting of this thesis was to apply modern DNA-based methods, primarily high-throughput “next-generation” DNA sequencing, to test the hypothesis that  mycorrhizal associations have a profound influence on the large scale spatial distribution and local abundance of terrestrial orchids. More specifically, the following research questions were addressed:

In chapter two, we evaluated the performance of different combinations of existing PCR primers to characterize orchid mycorrhizal communities using 454 amplicon pyrosequencing by analysis of both an artificially assembled community of orchid mycorrhizal fungi and root samples from three different orchid species, Anacamptis morio, Ophrys tenthredinifera and Serapias lingua. Our results indicated 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 (OTUs) 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.

In chapter three, 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. 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 indicated that availability of fungal associates is not necessarily the determining factor driving the distribution of mycorrhizal plant species.

In chapter four, we investigated spatial variations in the community of potential orchid mycorrhizal fungi within the roots of three co-habitating orchid species, Anacamptis morio, Gymnadenia conopsea, and Orchis mascula, and within the surrounding soil in an orchid-rich calcareous grassland in Southern Belgium using 454 amplicon pyrosequencing. We found that putative OrM fungi were broadly distributed in the soil, although variations in community composition were strongly related to the proximal host plant. The diversity and frequency of sequences corresponding to OrM fungi in the soil declined with increasing distance from orchid plants, suggesting that the clustered distribution of orchid species may to some extent be explained by the localised distribution of species-specific mycorrhizal associates.

In chapter five, we investigated the abundance and distribution of orchid mycorrhizal fungi in the soil and how they relate to spatial patterns of adults and seedling recruitment in two related orchid species, Orchis mascula and Orchis purpurea, by combining 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. Our qPCR analyses showed that fungal abundance declined rapidly with distance from adult host plants. Spatial point pattern analyses showed that successful recruitment was in both species significantly clustered 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.

In the final chapter, we discuss the most important findings of this thesis, describe ongoing related experiments and their implications, and conclude with some general shortcomings and suggestions for future research in the fields of orchid mycorrhizae and the use of high-throughput sequencing techniques in their investigation.