Distinct root-associated bacterial communities on three wild plant species growing in a common field

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 sampled endophytic and epiphytic bacteria in root tissues 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) to determine if plant species hosted unique root microbiota. Our results show 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 gamma Proteobacteria and Acidobacteria occupied a larger portion of the community. 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 explored.

Plant roots function as distinct habitats within the soil and bacterial communities in root systems have repeatedly been shown to differ from those of the surrounding bulk soil (Smalla et al. 2001;Haichar et al. 2008;Gottel et al. 2011;Lundberg et al. 2012). Even though root associated bacterial communities (both rhizospheric -in the soil surrounding the roots, epiphytic -living at the surface of roots and endophytic -living inside root tissues) have been under investigation for many years, there is still little consensus in how these communities are formed and what determines their composition (Berg & Smalla 2009;Aleklett & Hart 2013;Bulgarelli et al. 2013).
Traditionally, the composition of bacterial communities living in association with plants has been attributed to environmental factors. For example, soil type has been suggested as the strongest determinant of community structure in root associated microbial communities (De Ridder-Duine et al. 2005;Singh et al. 2007;Lundberg et al. 2012;Bulgarelli et al. 2013). At the same time, it has also been argued that the host plant may play an equally large role in determining the composition of its root microbiota (Marschner et al. 2005;Costa et al. 2006;Hartmann et al. 2008;Doornbos et al. 2011), especially endophytic bacterial communities (Haichar et al. 2008).
Recent work has demonstrated that hosts can alter their root microbiota by regulating soil conditions in the vicinity of the root system through root exudation of sugars, phenolics and amino acids that could also function as signaling molecules with the microbes in the surrounding soil (Chaparro et al. 2013). Since root exudation patterns and composition can be associated with plant gene expression, variation in host genetics has the potential to create large differences in the chemical profile of plants and consequently the composition of microbes able to inhabit the root system. Several studies have found that different plant species or genotypes of the same species host distinct microbial communities (Bailey et al. 2005;Marschner et al. 2005;van Overbeek & van Elsas 2008;Schweitzer et al. 2008;Micallef et al. 2009a;Manter et al. 2010;Becklin et al. 2012;Peiffer et al. 2013). Even studies where soil type was considered to have the strongest effect on structuring the root microbiota, differences in bacterial community composition between genotypes was still detected (Bulgarelli et al. 2012;Lundberg et al. 2012).
The root environment varies greatly among plant species; these differences may lead to the selection of distinct bacterial communities. Plants can differ in terms of both root lifespan (Roumet et al. 2006), root architecture (Hodge et al. 2009), root surface structure and components and patterns of root exudation (Bais et al. 2006). Root exudates are known to provide a food source for the microbes (Farrar et al. 2003), instigators of symbiotic associations (such as mycorrhizal infection or nodule formation) (Bais et al. 2004), and defend the plant against pathogens (Doornbos et al. 2011). All these plant characteristics could contribute to shaping root systems of different plants into local habitats and potentially distinct niches for microbial colonizers.
The role of intra-species variation among root associated microbial communities has been overlooked, but might represent a significant proportion of variation in natural systems (Bell et al. 2013). Since we know that natural populations exhibit variation in root exudation patterns and root morphology, one would expect there to be variation among individual plants in their root microbiota as well (Micallef et al. 2009). But variation among plants might also be driven by environmental heterogeneity because we know that small-scale environmental heterogeneity exists in soil systems. Is this variation static across plant taxa, or do different taxa exhibit more variation than others? If plant genetics are determining bacterial community composition, then certainly, populations with low genetic diversity (i.e. asexually reproducing, or metapopulations), would be expected to have less variation than sexually reproducing populations with high levels of gene flow. Because we sampled a plant species known to reproduce clonally through stolons and apomixis (P. aurantiaca), we also examined whether individuals within that species had less dispersion in their microbial community composition than individuals within the other two plant species.
The majority of studies characterizing bacterial communities in the root microbiota have been conducted with model plants in artificial greenhouse settings or agricultural contexts (Marschner & Yang 2001;Garbeva et al. 2004;Micallef et al. 2009;Manter et al. 2010;Doornbos et al. 2011;Lundberg et al. 2012) where the study of genetically modified plants have been especially informing when it comes to understanding slight differences between plant genotypes (van Overbeek & van Elsas 2008;Weinert et al. 2009;İnceo lu et al. 2010).
g While these studies are crucial for understanding the mechanistic basis of plant:microbe interactions, they do not reflect how natural environmental conditions contribute to variation in bacterial community composition across individual plants, particularly in complex environments where a wide diversity of plants and biota are interacting.
In this study, we explored variation in bacterial community composition between individual root systems of neighboring plants in a common field in order to determine how much variation exists within and between plant taxa. We sampled the root microbiota of three plant species growing within 10 meters from each other in a field and asked -are bacterial root communities distinct among plant species growing in a common location? And -do certain plant species contain more intra-species variance in bacterial communities than others?

Field site description
Samples were collected in August, 2011, from a subalpine meadow near Chute Lake, British Columbia, Canada (49.698859N,-119.533133W). The sampling area has not been used for agriculture or forestry but is in proximity to a forestry road as well as a camp site. Since it also contains a high number of invasive plant species it could therefore be considered disturbed site.
The dominant soil at the site is a sandy loam and the site is classified under the biogeoclimatic

Target plant
Our target plant was P. aurantiaca (formerly known as Hieracium aurantiacum), which is native to Europe and invasive in North America. Genetic diversity within P. aurantiaca has previously been examined across 48 locations in North America, and results showed that there were only three genotypes, of which two were found only in isolated locations (one in Alaska and one in Oregon) (Loomis & Fishman 2009). By choosing to work with a plant expressing this low diversity in wild populations, we hoped to minimize genetic variance within the population that we sampled.
To clarify the role of host identity and intra-species variance in bacterial root microbiota, we additionally sampled two of the co-occurring plant species, L. vulgare and T. hybridum that were in the same developmental stage (flowering) as P. aurantiaca.

Experimental design
Root systems of P. aurantiaca were collected one meter apart along two 10 m transects (n=20).
Additional samples of T. hybridum (n=10) and L. vulgare (n=10) were collected where present along the transects, several of which were growing within centimeters of P. aurantiaca samples.
Each root system was rinsed from surrounding rhizospheric soil in de-ionized water in order to separate it from roots of neighboring plants. Root systems were then cut up in pieces and a subsample of root tissue, representative of the whole root system, including young fresh roots as well as older root tissues (with no exclusion of nodules in T.hybridum), was collected and further used for classification of bacterial community composition. Since no further treatment was performed in order to remove rhizoplane microbes, we assume that the communities extracted could be of either endophytic or epiphytic origin.  Processing raw sequence data All sequences were de-multiplexed and further analyzed using the Quantitative Insights Into

Bacterial community analysis
Microbial Ecology (QIIME) toolkit (Caporaso et al. 2010 et al. 1990). In order to correct for differences in the number of sequences analyzed per sample, a randomly selected subset of 400 sequences per root sample was used to compare relative differences in taxonomic diversity. Only samples from which we obtained a minimum of 400 bacterial sequences per sample or more were considered in the study, eliminating 3 samples from the study (one P. aurantiaca and two L. vulgare). Though 400 sequences cannot fully capture the rare biosphere, it allowed us to compare samples while still maintaining as many samples as possible. It has previously been shown that studies of bacterial communities show similar results even at a lower rarefaction (Hamady & Knight 2009;Kuczynski et al. 2010). In fact, re-analyzing our data set with a higher rarefaction limit showed the same general trends but drastically lowered our number of samples available to analyze.

Statistics
Differences in community composition between samples were calculated using phylogenetic metric (UniFrac) where weighted UniFrac shows an emphasis on the more abundant taxa in samples and un-weighted UniFrac treats all taxa the same (Lozupone et al. 2007;Hamady et al. 2010). As a comparison, we also included a taxonomic metric (Bray-Curtis distance) to explore whether dissimilarity patterns were the same in terms of presence/absence of taxa. Before calculating Bray-Curtis distances, all relative abundances were log-transformed. 2-D scatterplots

Variation between host species
When comparing the phylogenetic overlap between bacterial root microbiota (UniFrac) across three different species of plant hosts growing in a common field, bacterial communities from samples of the same plant species were significantly more similar to each other than to bacterial communities sampled from plants of the two other species ( Oxalobacteriaceae -especially bacteria of the genus Herbaspirillum (11% of the total bacterial community in T. hybridum and 18% in L. vulgare) (Fig.3).

Host specificity
Our study shows that root bacterial communities vary significantly between plants belonging to three different species, growing in close proximity to each other in natural plant communities.
Although all plant species investigated in this study (P. aurantiaca, T. hybridum and L. vulgare), are perennial, there are significant morphological differences between the species. For example, P. aurantiaca and L. vulgare (both belonging to the family Asteraceae) have creeping root stocks and produce fibrous root systems whereas T. hybridum (family Fabaceae) grows a branching tap root system that is known to form nodules with nitrogen fixing bacteria. This variation in root morphology could contribute to the differences in abundance and composition of bacteria in our results. For example, roots that penetrate deeper soil may encounter different microbes than those in shallow layers (Fierer et al. 2003). Similarly, the thickness and/or texture of the root surface (i.e. woody, fibrous) may be more or less penetrable to colonizing bacteria.
Part of the variance seen in bacterial community composition between the three plant species could also be caused by species-specific root exudation patterns. For example, several members of the Asteraceae family are known to produce allelochemicals that could affect the bacterial community as well as surrounding plants (Alford et al. 2009). However, these differences are difficult to assess in wild plant communities, especially when roots of different plant species grow in close proximity to each other with entangled root systems. In our study, the roots grew so intimately that exudation from one plant species could have influenced root systems of neighboring plants.

Individual variation in root microbiota
We know that genetic differences between plants, even at the genotype level, can affect the Though our data show a significant difference in compositional turnover within different plant species, it rejects the hypothesis that P. aurantiaca had the most similar root communities across individuals. Comparing the average dispersion of bacterial community composition for the three plant species, there was no indication that P. aurantiaca had a smaller dispersion than the two other plant species (Table 2). Instead, it shows that P. aurantiaca had the highest variation within a species comparing dispersion based on taxonomic differences ( Table 2). The fact that we could not detect any differences in dispersion when using phylogenetic metrics suggests that individual root systems differ more in terms of which taxa are present or absent than how related they are, or that there is little phylogenetic conservatism at the individual level.
Overall, this study shows that the extent of individual variation seen in root microbiota varies between species, but that a plant species thought to be more genetically homogenous does not necessarily host more homogeneous root communities. It also indicates that individual variation in bacterial community composition in root systems is determined, not only by plant genetics, but also by the surrounding environment and potentially, events throughout the plant's life that could affect root colonization (Aleklett & Hart 2013).

Bacterial community composition
Similar patterns of bacterial community composition to what we found in our plants, growing in a subalpine meadow in Canada, have been reported in rhizosphere samples of other studies. For example, roots tissues of the plant species that we sampled were mainly dominated by Betaproteobacteria, (Fig. 2), especially members of the order Burkholderiales and the family Oxalobacteriaceae, which represented as much as 32% of the total bacterial community in L. vulgare (Fig. 3). Seed-and root-colonizing populations of Oxalobacteriaceae have previously shown to be responsive to plant species (Green et al. 2007 in the plant species that we sampled (Fig. 2) and was mainly found in samples of P. aurantiaca that, in general, were less dominated by beta-proteobacteria.
The dominance of sequences belonging to the genus Herbaspirillum was further emphasized when we examined the fourteen most abundant OTUs across all samples (Table 3).
Herbaspirillum spp. are known to colonize apoplastic or intracellular spaces of plant tissues and several species have shown the ablility to fix nitrogen (Schmid et al. 2006). While it is believed that this nitrogen fixing ability could be beneficial to their plant host, it has also been documented that certain Herbaspirillum strains are mild pathogens and a causative agent of "mottled stripe disease" in crops such as sugar-cane (Schmid et al. 2006). Besides Herbaspirillum, we also saw high abundances of sequences belonging to Limnohabitans and Cytophaga (Table 3), two genera more commonly associated with bacterial communities in fresh water (Kirchman 2002;Simek et al. 2010) as well as the species Methylibium petroleiphilum, a recognized methylotroph (Kane et al. 2007) and Janthinobacterium lividum, known to thrive in soils (Shivaji et al. 1991) and produce antibiotics (Johnson et al. 1990). The high presence of these groups in our samples could be due to the inclusion of epiphytic members of the root microbiota, where bacteria associated with water films and soil particles of the root surface would be expected.
In comparison, Bodenhausen and colleagues (2013) found that a Flavobacterium of the phylum Bacteroidetes stood out as the single most abundant OTU in endophytic root samples, making up 10.15% of the total community. Though bacteria of the phylum Bacteroidetes represented a significant part of the community in root samples of the three plant species sampled in our study (Fig.2), they were by no means the most dominant taxonomic group in any of the species (Table 3).
As the genus Trifolium are known to be hosts of nitrogen fixing bacteria that form nodules in their roots, T.hybridum samples were expected to host larger populations of Alphaproteobacteria, specifically belonging to the order Rhizobiales which is a common  -Boivin et al. 2009).This expected pattern was not evident in our results though. The only OTU belonging to the order Rhizobiales detected in notable abudances in our study was a Bradyrhizobium taxa which made up 1% of the collective community of T.
hybridum samples and 1.5% in P. aurantiaca samples (Table 3). Instead, it was evident that the T.
hybridum community was dominated by the family Oxalobacteraceae (23.48%) (Fig.3) and specifically one OTU of the genus Herbaspirillum (10.75%) (Table 3), which is mainly known to colonize roots of non-leguminous plants, and have nitrogen fixing properties (Baldani et al.1997).
What stands out though is that this group of bacteria was even more predominant in L. vulgare samples, where Oxalobacteraceae made up 32.47% of the community and the same Herbaspirillum OTU represented 18.26% of the total community.

Variance in relative abundances of bacterial taxa across plant species
We observed differences in the evenness of bacterial taxa across host plants. While the roots of L.
vulgare and T. hybridum seemed dominated by a few select groups of microbes, samples of P.
aurantiaca supported communities with abundances more evenly distributed among bacterial taxa ( Fig. 2; Table 3). Though few studies have looked specifically at variance in bacterial evenness between plant species, it could be an important source of variation. For example, dominance of single taxon may indicate specialized plant/bacterial associations whereas high evenness in community composition could reflect generalist associations among plants and bacteria. Alternatively, differences in evenness may result from microbial interactions within the plant, not driven by the plant but microbial competition for plant resources.

Conclusions
In this study, we showed that plant identity plays a major role in explaining the variation seen in root microbiota both between and within plant species growing under natural conditions. Further   Table 3 was submitted separately as a PDF due to formatting issues upon submission

Relative abundance of selected phyla
Average relative abundances of Betaproteobacteria families and Oxalobacteraceae genera found in root samples of the three plant species. Values are given as the percentage of sequences belonging to a certain taxa out of the total average bacterial community for each of the three plant species (rarefied at 400 sequences/sample). The heat map is colour coded from blue (low abundance) to red (high abundance).