Disturbance did not influence OTU composition
This study was driven by the general assumption that there is a trade-off between competitive ability and disturbance tolerance and that disturbance increases the abundance of OTUs that are rare under undisturbed conditions. Disturbance has previously caused successional shifts in AM fungal communities in agricultural systems (Jansa et al. 2002, 2003; Rosendahl & Matzen 2008) as well as in natural plant communities (Husband et al. 2002; Sykorova et al. 2007; Fitzsimons, Miller & Jastrow 2008), but had no significant effect here. The fact that the fungal community in the DB treatment did not differ from the one observed in the undisturbed, surrounding vegetation suggests that abundant OTUs in this grassland possess a similar – and high – degree of disturbance tolerance. This was unexpected given the apparent lack of any severe disturbance in this grassland. It is possible that less-obvious forces, such as frost heave, wind and trampling from humans and animals are sufficient to select for disturbance tolerance, although these disturbances – if present – are considerably less intense than what was employed in this study. Furthermore, a tolerance to other stresses, such as recurrent droughts (Hawkes et al. 2011), changing environmental conditions, and a temporal and spatial flux in host carbon may select for resilient traits, which makes it less likely to observe treatment effects. Regardless, we find the lack of treatment effects remarkable given that only 4.5 months had passed after the severe disturbance was applied. This high disturbance tolerance could arise from the ability to colonize seedlings from pieces of external mycelia and colonized roots rather than spores, because the AM colonization in the DB treatment was high (Table 2) even though spore numbers are consistently low (Y. Lekberg & S. Rosendahl, pers. observ.). Because fungal communities did not differ between the two disturbance treatments, there does not appear to be a great difference among AM fungi in their ability to resist and recover from disturbance. Based on the overall lack of treatment effects, we reject our first hypothesis, which predicted an inverse relationship between OTU abundances and disturbance in this grassland. Further experiments are required to verify the commonality of these findings in other systems. Our second hypothesis regarding phylogenetic trait conservatism is difficult to address because of the dominance of Glomus group A fungi, but it is noteworthy that the one Scutellospora with reasonable abundance was not reduced in the DB treatment (Table 1), which would be expected based on previous studies (Klironomos & Hart 2002). This suggests that caution may be warranted before extrapolating traits observed in cultured fungi to their uncultured relatives.
We cannot exclude the possibility that the lack of treatment effects was an artefact of our experimental protocol, but for reasons outlined below, we find this to be unlikely. For example, if hyphae were able to colonize DB plants from the bottom of the barrier (something we find improbable for reasons stated earlier), differences between the D and DB treatments would be reduced. Even if that were the case, it would not influence our overall finding that this AM fungal community is highly resilient. One can also argue that the immediate planting of P. lanceolata into the disturbed soil may have facilitated a rapid recovery by AM fungi by providing an immediate host plant. However, significant differences were observed among AM fungi in their ability to colonize plants from spores, hyphal and root fragments using a similar approach in a previous study (Klironomos & Hart 2002). Furthermore, opportunistic, early colonizing fungi may have been missed because we chose not to analyse samples from the first harvest. However, roots in all treatments were poorly colonized (<10%), which suggests that even if this group of fungi was present, they constituted a relatively small proportion of the community that was rapidly replaced. Including a third sampling of older roots is also unlikely to have affected the results, because AM colonization and OTU composition did not differ among our treatments and undisturbed surrounding vegetation 4.5 months after a severe disturbance event, which indicates that the fungal community had reached some sort of equilibrium.
Similar to other community studies, we found some OTUs to be common and others to be rare, but disturbance did not influence this relationship because abundance numbers were positively correlated across treatments. That is, OTUs that were common in the control samples were also common after severe disturbances. What factors may cause this cannot be deduced from this study, but clearly some abiotic and/or biotic conditions favour certain OTUs. It is interesting to note that many of the OTUs found here are also abundant in a similar coastal grassland in Sweden (Schnoor et al. 2011), indicating that significant landscape level or vegetation type structuring of AM fungal communities occurs. It is also noteworthy that large-scale perturbation did influence the AM fungal community composition in the Swedish grassland (Schnoor et al. 2011), suggesting that treatment effects may be scale-dependent, especially if there are concomitant changes in the plant community composition.
The relative importance of host plant identity for OTU composition has been debated, and correlations between plant and fungal communities have been found in some studies (Husband et al. 2002), but not others (Santos, Finlay & Tehler 2006). OTU abundances correlated significantly with the coverage of Carex arenaria and Dianthus deltoides in our study, which is intriguing because these two plant species belong to families (Cyperaceae and Caryophyllaceae) that are either considered to be non-mycorrhizal or poor hosts (which we define here as being poorly colonized and therefore allocating relatively little C to their fungal symbiont) to AM fungi (e.g. Koide & Schreiner 1992; Miller et al. 1999). AM hyphae and vesicles have been observed in both plants at our field site, but arbuscules are rare or absent (Y. Lekberg, pers. observ.). This is in agreement with previous studies of plants in non-mycorrhizal families (Hirrel, Mehravaran & Gerdeman 1978) and may indicate that this symbiosis is non-functional. AM fungi may use poor host plants to store C acquired from better hosts (Lekberg, Hammer & Olsson 2010), but this strategy may not extend to all AM fungal taxa and could be one possible explanation for the correlation between certain OTUs and C. arenaria and D. deltoides observed here. We do not believe that our results indicate host preference, because there was no difference in OTU composition between the P. lanceolata (planted and native) and surrounding plants. This agrees with previous findings from this grassland (Stukenbrock & Rosendahl 2005). Instead, one may speculate that host quality, rather than host identity, could be an important factor for structuring fungal communities.
While the plant community explained a significant portion of the variation in the OTU composition, the great majority remains unexplained, and the OTU distribution among individual samples was largely unpredictable. For example, while Glomus P was absent in the surrounding plants but present – and even highly abundant – in the C, D and DB treatments in plots 1 and 4, the opposite was found in plots 2 and 3 (Table S3). To obtain patterns like these, OTUs must not only harbour a similar disturbance tolerance, but also differ greatly on small spatial scales, because treatments within plots were within 1 m of each other. Small-scale patterns in AM fungal communities have been documented previously within natural plant communities (Wolfe et al. 2007; Mummey & Rillig 2008; Van der Voorde et al. 2010), and a recent survey in this grassland showed that plants less than 1 m apart were sometimes colonized by different AM fungal taxa (I. Galløe & Y. Lekberg, unpubl. data). Clearly, if spatial patterns such as these are common, they need to be considered during the planning, analysis and interpretation of field experiments involving AM fungi.
The observed difference in OTU composition among samples could have been amplified through priority effects, in which the establishment of one OTU influences subsequent colonization by others. Pre-inoculation with certain taxa has been shown to influence subsequent communities for both AM and ectomycorrhizal fungi (Hepper et al. 1988; Kennedy et al. 2009; Mummey, Antunes & Rillig 2009). Dumbrell et al. (2010) proposed that priority effects and subsequent positive feedback could help explain the overdominance observed in AM fungal communities, in which the most abundant taxon constitutes about 40% of the total abundance. Because the dominant OTU differed among sites, Dumbrell et al. (2010) also argued that stochastic processes may play an important – yet underappreciated – role in shaping AM fungal communities. Our results are in agreement with those presented by Dumbrell et al. (2010), because the AM fungal community assembly in the P. lanceolata roots was largely stochastic, and the OTU dominance was almost identical to the one calculated by Dumbrell et al. (2010). This suggests that the mechanisms proposed by Dumbrell et al. (2010) to explain differences among communities also operate on smaller scales. Furthermore, because overdominance and stochasticity were present in the undisturbed surrounding plants in our study, these patterns appear to persist over time. The AM colonization in our experiment ranged between 35% and 60% and may not be considered sufficiently high for competition among OTUs to occur, but AM fungi may not only compete for root space but also for carbohydrates (Pearson, Abbott & Jasper 1994). The occurrence and severity of competition is not only of interest for AM fungal community ecology, but could also have functional consequences as more competitive AM fungi may be poorer mutualists to plants (Bennett & Bever 2009).
The fungal community as revealed by 454-sequencing
Thorough molecular characterization of fungal communities has been limited because of time and cost constraint related to cloning and Sanger sequencing. 454 and other next-generation sequencing techniques hold great promise for thorough community analyses as they eliminate the need for cloning and appear to generate sequences in a quantitative manner (Thomas et al. 2006) as long as care is taken to avoid biological and technical artefacts (Tedersoo et al. 2010). For example, Öpik et al. (2009) compared results from cloning and Sanger sequencing with those from 454-sequencing and found 33% more OTUs with the 454-sequencing using the same PCR protocol and primers. Because we designed new primers for this study, we are unable to compare our richness estimates with those found previously (Rosendahl & Stukenbrock 2004; Stukenbrock & Rosendahl 2005) also targeting the LSU. Nonetheless, abundant OTUs in earlier studies, such as Glomus D and F, were also abundant in this study. The exception was Glomus mosseae, which has been documented previously (Rosendahl & Stukenbrock 2004; Stukenbrock & Rosendahl 2005) but was conspicuously absent here. It is also interesting to note that even though comparisons to other studies are difficult because of differences in OTU delineation and target gene sequences, the 32 OTUs found here are within the range found in other studies (Vandenkoornhuyse et al. 2002; Öpik et al. 2009). It is possible that our reported richness underestimates the actual AM fungal richness, because we excluded some OTUs with no match to know fungal groups. Paraglomeraceae is the most basal group in the Glomeromycota, and there could potentially be more basal groups of non-sporulating – yet unidentified – AM fungi. While these excluded OTUs were rare at our study site, they could be abundant in other vegetation systems. Overall, this surprising fungal diversity may be important, because the long-held assumption that AM fungi are functionally equivalent has been refuted by research showing that fungal species richness influences plant diversity and productivity (van der Heijden et al. 1998) as well as ecosystem properties such as soil aggregation and carbon and nutrient cycling (as discussed in Rillig 2004). Because a majority of functional studies have focused on culturable taxa and been conducted under controlled greenhouse conditions, the function of the majority of AM fungi remains unknown, and field studies assessing mycorrhizal function are sorely needed. For example, two of our three most abundant OTUs have no close match to known taxa in GenBank and may constitute locally adapted taxa with unknown traits. This is also true for many of the fine endophytes whose identity and function are uncertain, and it is unclear to what extent our primers were able to amplify this group of fungi.
As the number of next-generation sequencing studies increases, it becomes imperative that some standards are put in place to allow for comparisons among studies. An initial proposal is put forward by Nilsson et al. (2011), in which guidelines for how data and results should be reported and made available to the scientific community are suggested. Other areas that complicate comparisons include OTU delineation and abundance data, but these potential sources of bias are not necessarily unique to next-generation sequencing techniques. For example, OTUs are often defined based on a 97% sequence similarity in AM fungal community studies (Buée et al. 2009). Such criteria are operational, but may suffer from the problem that the genetic diversity within species may vary considerably, even between related species. The neighbour net analysis (Figs 1 and 2) shows that some OTUs (e.g. Glomus F) belong to well-resolved lineages, whereas others (e.g. Glomus P) show considerable genetic variation and belong to less-resolved lineages. This phenomenon is not unique for AM fungi and is indicative of differences in the speciation process among lineages (Taylor et al. 2000). With the increasing number of sequences available with 454-sequencing, these differences can be more categorically assessed.