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- Materials and Methods
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Arbuscular mycorrhizal (AM) fungi (Ph. Glomeromycota) are ubiquitous plant root symbionts that can be considered as ‘keystone mutualists’ in terrestrial ecosystems, forming a link between biotic and abiotic ecosystem components via carbon and nutrient fluxes that pass between plants and fungi in the soil (O’Neill et al., 1991). AM fungal diversity affects plant community diversity and productivity (van der Heijden et al., 1998). There can be large differences in functional complementarity between coexisting plants and AM fungi (Helgason et al., 2002; Moora et al., 2004a,b). Therefore it is essential to understand the fine-scale structure and dynamics of AM fungal communities in natural and managed ecosystems. However, possible approaches to link the taxon diversity of Glomeromycota communities with functional significance are still under debate because of the practical difficulties of working with such obligate symbiotic organisms (van der Heijden & Scheublin, 2007).
The diversity and composition of intraradical AM fungal communities vary among habitat types around the globe (Öpik et al., 2006a). When comparing studies using the small subunit ribosomal RNA gene (SSU rDNA) region to identify AM fungi, the highest taxon richness in a single site has been reported from tropical rain forest in Panama – 30 taxa in the roots of three host species (Husband et al., 2002a,b; 29 according to the taxon synonymy used by Öpik et al., 2006a). Taxon-rich fungal communities (over 20 taxa) are also known from temperate grassland and forest locations (Vandenkoornhuyse et al., 2002; Saito et al., 2004; Helgason et al., 2007). Tropical forest may exhibit significantly higher mean fungal richness than other ecosystems (expressed as number of fungal taxa per plant species: 18; Öpik et al., 2006a). By contrast, human-impacted habitats such as arable fields (Helgason et al., 1998; Daniell et al., 2001) or sites polluted with heavy metals (Whitfield et al., 2004) may exhibit low AM fungal taxon diversity. However, recent evidence of higher richness in such habitats (Hijri et al., 2006; Vallino et al., 2006) suggests that the relationship with management is complex.
AM fungal communities in deciduous forests of the temperate zone have been described from the UK by Helgason and colleagues (Helgason et al., 1998, 1999, 2002, 2007). They have recorded > 20 AM fungal taxa from roots of six host plant species in a single forest location. Nine taxa, most of them unique, were detected in 25 pooled root samples from a warm-temperate broadleaved forest in Japan (Yamato & Iwase, 2005). However, the presence of AM fungi in coniferous forests has been largely ignored. Only two AM fungal taxa were detected in the roots of Taxus baccata in a Norway spruce (Picea abies) forest in Germany (Wubet et al., 2003) and 10 taxa in the roots of two Pulsatilla species in a Scots pine (Pinus sylvestris) forest in Estonia (Öpik et al., 2003). To our knowledge, no further information (including that from spore surveys) is available relating to AM fungal richness in boreal forest ecosystems. The land area covered by boreal forest constitutes one-third of the world's forest and provides important ecological functions as well as 20–50% of the world's pulp, newsprint, sawn wood, paper and paper board (Reich et al., 2001). There is a considerable wealth of information regarding the diversity of ectomycorrhizal fungi – the symbionts of the dominant overstorey plants in temperate/boreal coniferous forests (Horton & Bruns, 2001; Johnson et al., 2005; Tedersoo et al., 2006). However, in herb-rich temperate/boreal coniferous forests, where there are plentiful AM plant species, Glomeromycota should not be overlooked as an ecosystem component. In order to obtain a good overview of biodiversity in coniferous forests, more information about AM fungi is required.
Different management practices may have significant impacts on the diversity and composition of boreal forest plant communities (Reich et al., 2001; Ramovs & Roberts, 2003). The impact of logging can directly influence vegetation via the disturbance of soil or forest floor, altered habitat structure, removal of nutrients, or altered microclimate (Roberts & Gilliam, 1995; Bergeron & Harvey, 1997). Clearcut logging may have a significant impact on ectomycorrhizal fungal communities (Jones et al., 2003). There is, however, no information about the impact of boreal forest management on AM fungi.
In the present paper, we aimed to investigate the taxon composition and community structure of AM fungi in a herb-rich boreal coniferous forest. In particular, we asked: (1) what is the taxon richness and composition of the AM fungal communities of the studied coniferous forest, and (2) what is the impact of local environmental conditions and forest management (clear-felling and cultivating clear-cut areas versus old growth) on the taxon richness and composition of AM fungal communities?
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- Materials and Methods
- Supporting Information
Communities of AM fungi colonizing the roots of five understorey plant species in an Estonian herb-rich boreal coniferous forest were found to be remarkably rich. We recorded 34 AM fungal taxa in total, comparable to the fungal richness described in tropical rain forests in Panama (Husband et al., 2002a,b) and higher than that in temperate grassland and broad-leaved forest locations (Vandenkoornhuyse et al., 2002; Saito et al., 2004; Helgason et al., 2007). To our knowledge the only other boreal or temperate forest systems where AM fungal community dynamics has been studied are a Scots pine forest in Estonia, a Norway spruce forest in Germany, a warm-temperate broadleaved forest in Japan, and a broadleaved forest in the UK (Helgason et al., 2002, 2007; Öpik et al., 2003; Wubet et al., 2003; Yamato & Iwase, 2005).
The number of samples in our study is higher than (90 vs 20–54), and the number of sequenced clones comparable to or lower than (911 vs 558–2001), those in the studies cited above. Forty-one taxa were reported from roots of three host species from tropical forest and pasture locations in Costa Rica, although forest-specific richness cannot be deduced from these data (Aldrich-Wolfe, 2007). The number of root samples processed can significantly affect the number of root-colonizing AM fungi detected (Öpik et al., 2006a). Furthermore, because of the patchy distribution of colonization units, small subsamples of a root system can contain different AM fungi (Öpik et al., 2006b). Here the individual samples were large (20 cm length) but contained on average three AM fungal taxa in an ecosystem supporting at least 34 taxa. This indicates a trade-off between the number of samples and the size of a root sample needed to reasonably describe the diversity of root colonizers in an ecosystem, an issue that needs to be addressed in future field surveys. In conclusion, care is required when comparing AM fungal richness data acquired with different methodologies, including differences in sample number, screened/sequenced clone number, spatiotemporal sampling design, and number of sampled plant species.
In this study we identified eight previously undescribed AM fungal taxa and 23 taxa that are known from molecular analysis of plant roots only. This high proportion of ‘known-as-sequence-only’ taxa reflects the accumulation of molecular diversity data of AM fungi as a result of the increasing number of studies of Glomeromycota in natural ecosystems. More matches with known fungal species would be expected if or when more intensive sequencing, of relevant genome regions, of identified isolates maintained in culture collections occurs.
The taxon richness of AM fungi per root sample was higher for H. nobilis than for F. vesca and O. acetosella. However, there were no differences among forest management types or seasons (sampling times). It has been shown that co-occurring plant species can be colonized by AM fungal communities of different composition (Helgason et al., 2002; Vandenkoornhuyse et al., 2002), but differences in the number of fungal taxa associated with host plant species have not been previously demonstrated. We can hypothesize that these trends are linked to the host preferences of AM fungi, different symbiont ranges of AM plant hosts, or different sizes of fungal colonization units in roots resulting in variable fungal taxon densities. Variable symbiont ranges of plants could be related to plant functional types, for example life forms, plant growth rates, rooting traits and types of clonal growth. However, why fungi or plants should ‘prefer’ one host to another requires further research.
There is evidence that disturbance can decrease AM fungal taxon richness (Helgason et al., 1998; Whitfield et al., 2004). Therefore, we expected to observe smaller numbers of AM fungal taxa in the more intensively managed young forest stands. However, these ecosystems have preserved a high richness and the common management practices have not had an adverse impact on the AM fungal biodiversity. In contrast to the findings of Helgason et al. (1998) and Whitfield et al. (2004), habitats with moderate management intensities may still display rather high numbers of AM fungal taxa (Hijri et al., 2006; Vallino et al., 2006). In the studied ecosystem the soil disturbance associated with clearcut logging and planting of tree saplings is less intense than that associated with recurrent ploughing, which in combination with no soil disturbance during subsequent years may aid maintenance of fungal diversity through management activity. Thus we propose that the severity and recurrence of disturbance events influence the magnitude of the reaction of AM fungal communities following disturbance. Furthermore, it is worth noting the lack of clear management- or environment-related patterns of AM fungal communities in soil despite the obvious differences among these forest ecosystems apparent to the naked eye. The (lack of) variability in diversity of soil micro-organisms in relation to common disturbances and natural environmental gradients warrants further investigation.
The two most common fungi in our study, Glomus sp. MO-3 and MO-13, are related to the G. intraradices group, which has been detected from world-wide locations of both stable and disturbed ecosystems (Öpik et al., 2006a) and many host species (Helgason et al., 2007). Glomus intraradices is sometimes considered to be an aggressive species. It can depress plant growth even if it provides all the P acquired by the plant (Smith et al., 2003). Glomus intraradices isolates from the same or geographically distant locations can affect plant growth differentially (Hart & Reader, 2002; Koch et al., 2006). Such variation may be explained if this group contains several functionally different taxa as proposed by van der Heijden et al. (2004). Thus, it is reasonable to hypothesize that the G. intraradices species group, as identified using the NS31/AM1 primer pair, contains several cryptic taxa with differences in various ecological properties such as disturbance tolerance, mycelial growth, root colonization rate and sporulation traits. Even if it is considered as a group of multiple taxa, this is the most common group in both the studied boreo-nemoral forest and many ecosystems world-wide, and deserves further attention.
The third most common fungus in the studied ecosystem was G. hoi (here Glomus sp. MO-G7), detected in one-third of samples and all plots and host species. A host specificity of G. hoi towards Acer pseudoplatanus has been demonstrated in one ecosystem (Helgason et al., 2002), but otherwise the taxon appears to be widespread with no specialization in regard to habitat type or host species (Öpik et al., 2006a; Helgason et al., 2007).
An old stand-specific taxon, Glomus sp. MO-G5, has been previously reported from forests and grasslands, but not from disturbed habitats (Öpik et al., 2006a), and from 11 host species (as Glo2; Helgason et al., 2007). MO-G5 was dominant in experimental plants inoculated with boreal Scots pine forest soil (Öpik et al., 2003), and in natural grassland habitats (Öpik et al., 2006a). Here, the taxon was detected in c. 10% of samples.
As regards the methodology, it is obvious that the SSU rDNA gene region used in this study does not separate all Glomeromycota groups very well because of limited nucleotide variation among some clades, including the G. intraradices and G. caledonium clades (Fig. 1). Investigations of mitochondrial large subunit (LSU) sequences, including that of G. intraradices, suggest that better marker regions with more interspecific variability might be available (Raab et al., 2005). However, these have not been rigorously tested on a range of related taxa, on isolates of the same taxa and on environmental samples. Apart from these imperfections the SSU region provides comparability of data for ecological studies as a result of the accumulation of database entries and publications based on this gene region (Öpik et al., 2006a).
In conclusion, the observed unexpectedly high richness of Glomeromycota in a temperate coniferous forest indicates the need to obtain comparable descriptive soil fungal community data from a more diverse range of ecosystems. This almost unique richness of Glomeromycota could be speculatively attributed to the relative stability of the ecosystem and/or a high diversity of host species. Comparative data from a range of ecosystems along disturbance and plant richness gradients and from a range of plant hosts would help to test these hypotheses. The described taxon richness patterns and apparent lack of taxon composition patterns deserve further evaluation in order to establish the role of host plant identity, plant species richness, light availability, and soil conditions as determinants of Glomeromycota taxon distribution at a small scale. Furthermore, it is essential to evaluate the observed diversity patterns in functional terms.