Navigating the labyrinth: a guide to sequence-based, community ecology of arbuscular mycorrhizal fungi.

Data generated from next generation sequencing (NGS) will soon comprise the majority of information about arbuscular mycorrhizal fungal (AMF) communities. Although these approaches give deeper insight, analysing NGS data involves decisions that can significantly affect results and conclusions. This is particularly true for AMF community studies, because much remains to be known about their basic biology and genetics. During a workshop in 2013, representatives from seven research groups using NGS for AMF community ecology gathered to discuss common challenges and directions for future research. Our goal was to improve the quality and accessibility of NGS data for the AMF research community. Discussions spanned sampling design, sample preservation, sequencing, bioinformatics and data archiving. With concrete examples we demonstrated how different approaches can significantly alter analysis outcomes. Failure to consider the consequences of these decisions may compound bias introduced at each step along the workflow. The products of these discussions have been summarized in this paper in order to serve as a guide for any researcher undertaking NGS sequencing of AMF communities.

Community Assembly and Stability in the Root Microbiota During Early Plant Development.

Little is known about how community composition in the plant microbiome is affected by events in the life of a plant. For example, when the plant is exposed to soil, microbial communities may be an important factor in root community assembly. We conducted two experiments asking whether the composition of the root microbiota in mature plants could be determined by either the timing of root exposure to microbial communities or priority effects by early colonizing microbes. Timing of microbial exposure was manipulated through an inoculation experiment, where plants of different ages were exposed to a common soil inoculum. Priority effects were manipulated by challenging roots with established microbiota with an exogenous microbial community. Results show that even plants with existing microbial root communities were able to acquire new microbial associates, but that timing of soil exposure affected root microbiota composition for both bacterial and fungal communities in mature plants. Plants already colonized were only receptive to colonizers at 1 week post-germination. Our study shows that the timing of soil exposure in the early life stages of a plant is important for the development of the root microbiota in mature plants.

Fungal behaviour: a new frontier in behavioural ecology.

As human beings, behaviours make up our everyday lives. What we do from the moment we wake up to the moment we go back to sleep at night can all be classified and studied through the concepts of behavioural ecology. The same applies to all vertebrates and, to some extent, invertebrates. Fungi are, in most people's eyes perhaps, the eukaryotic multicellular organisms with which we humans share the least commonalities. However, they still express behaviours, and we argue that we could obtain a better understanding of their lives - although they are very different from ours - through the lens of behavioural ecology. Moreover, insights from fungal behaviour may drive a better understanding of behavioural ecology in general.

Fungal foraging behaviour and hyphal space exploration in micro-structured Soil Chips.

How do fungi navigate through the complex microscopic maze-like structures found in the soil? Fungal behaviour, especially at the hyphal scale, is largely unknown and challenging to study in natural habitats such as the opaque soil matrix. We monitored hyphal growth behaviour and strategies of seven Basidiomycete litter decomposing species in a micro-fabricated "Soil Chip" system that simulates principal aspects of the soil pore space and its micro-spatial heterogeneity. The hyphae were faced with micrometre constrictions, sharp turns and protruding obstacles, and the species examined were found to have profoundly different responses in terms of foraging range and persistence, spatial exploration and ability to pass obstacles. Hyphal behaviour was not predictable solely based on ecological assumptions, and our results obtained a level of trait information at the hyphal scale that cannot be fully explained using classical concepts of space exploration and exploitation such as the phalanx/guerrilla strategies. Instead, we propose a multivariate trait analysis, acknowledging the complex trade-offs and microscale strategies that fungal mycelia exhibit. Our results provide novel insights about hyphal behaviour, as well as an additional understanding of fungal habitat colonisation, their foraging strategies and niche partitioning in the soil environment.

Microfluidic chips provide visual access to in situ soil ecology.

Microbes govern most soil functions, but investigation of these processes at the scale of their cells has been difficult to accomplish. Here we incubate microfabricated, transparent 'soil chips' with soil, or bury them directly in the field. Both soil microbes and minerals enter the chips, which enables us to investigate diverse community interdependences, such as inter-kingdom and food-web interactions, and feedbacks between microbes and the pore space microstructures. The presence of hyphae ('fungal highways') strongly and frequently increases the dispersal range and abundance of water-dwelling organisms such as bacteria and protists across air pockets. Physical forces such as water movements, but also organisms and especially fungi form new microhabitats by altering the pore space architecture and distribution of soil minerals in the chip. We show that soil chips hold a large potential for studying in-situ microbial interactions and soil functions, and to interconnect field microbial ecology with laboratory experiments.

Build your own soil: exploring microfluidics to create microbial habitat structures.

Soil is likely the most complex ecosystem on earth. Despite the global importance and extraordinary diversity of soils, they have been notoriously challenging to study. We show how pioneering microfluidic techniques provide new ways of studying soil microbial ecology by allowing simulation and manipulation of chemical conditions and physical structures at the microscale in soil model habitats.

Wild plant species growing closely connected in a subalpine meadow host distinct root-associated bacterial communities.

Plant roots are known to harbor large and diverse communities of bacteria. It has been suggested that plant identity can structure these root-associated communities, but few studies have specifically assessed how the composition of root microbiota varies within and between plant species growing under natural conditions. We assessed the community composition of endophytic and epiphytic bacteria through high throughput sequencing using 16S rDNA derived from root tissues collected from a population of a wild, clonal plant (Orange hawkweed-Pilosella aurantiaca) as well as two neighboring plant species (Oxeye daisy-Leucanthemum vulgare and Alsike clover-Trifolium hybridum). Our first goal was to determine if plant species growing in close proximity, under similar environmental conditions, still hosted unique root microbiota. Our results showed that plants of different species host distinct bacterial communities in their roots. In terms of community composition, Betaproteobacteria (especially the family Oxalobacteraceae) were found to dominate in the root microbiota of L. vulgare and T. hybridum samples, whereas the root microbiota of P. aurantiaca had a more heterogeneous distribution of bacterial abundances where Gammaproteobacteria and Acidobacteria occupied a larger portion of the community. We also explored the extent of individual variance within each plant species investigated, and found that in the plant species thought to have the least genetic variance among individuals (P. aurantiaca) still hosted just as diverse microbial communities. Whether all plant species host their own distinct root microbiota and plants more closely related to each other share more similar bacterial communities still remains to be fully explored, but among the plants examined in this experiment there was no trend that the two species belonging to the same family shared more similarities in terms of bacterial community composition.