| The ectomycorrhizal (ECM) interaction between fungi and plant roots is a common, worldwide symbiotic interaction that contributed to land colonization. ECM fungi (EMF) are essential for the growth and survival of many important and endangered forest tree species. For they can improve the survival and growth of trees by enhancing uptake of nutrients and water, lengthening the root life span, protecting the root system against soil-borne pathogens and increasing tolerance to adverse condition. Fundamental of this kind of symbioses is the switch of nutrients between the plant and fungi linked by mycelial networks. Pseudotsuga, as an important tree species, is distributed separately in Asia and North America. North American lineage migrated across the Bering land bridge, as an important high-latitude link between eastern Asia and North America for floras and faunas, and gave rise to P. japonica, which then gave rise to P. sinensis and P. wilsoniana. And P. sinensis, a kind of Pseudotsuga species in China, which locate in Hunan, Shaanxi, Sichuan, Yunnan, Guizhou and other provinces, belongs to national protected and endangered species. Asian Pseudotsuga species, P. japonica, P. sinensis and P. wilsoniana as a synonym of P. sinensis, are distributed as small isolated populations and the existence of specific lineage provide a research opportunity in host effects on ECM fungi structuring.Here, molecular techniques were used to study ECM fungal communities in relict forests dominated by Chinese Douglas-fir (Pseudotsuga sinensis), which is designated as Vulnerable in the IUCN Red List. We collected 73 soil samples from three forest sites. Fungal and host species in individual ECM tips were identified by sequences of the internal transcribed spacer regions of rDNA and plastid DNA, respectively. Of 86 ECM fungal species identified,66 were confirmed on Chinese Douglas-fir. An average of 2.8 ECM fungi species were detected from each soil sample with the maximum of 7 species. We found one Rhizopogon sp. that belongs to the Pseudotsugα-speciffic lineage Villosuli, but it was quite rare on ECM tips (0.4% in relative abundance). At the family level, the relative abundances of Russulaceae and Sebacinaceae were 33% and 32%. Russulaceae (35 spp.) were the most species-rich ECM fungal lineages, followed by Sebacinaceae (12 spp.), Thelephoraceae (12 spp.), Clavulinaceae (7 spp.). Sebacina sp.1, Sebacina sp.7, and Cenococcum geophilum were dominant in P. sinensis associate ECM fungal species, of which the important value reached to 19.2%,8.8% and 7.9%, respectively.Host identity strongly influence ECM fungal community composition, and responsible for forming the differences in the ECM fungal community structure. But how it happens remains unclear. Except comparing EMF with coexisting host tree species to confirm host effects in ECM fungal communities in our study system, we also compared the EMF on P. sinensis with those on P. japonica in Asia to examine the possibility of co-evolution with the host. The Sorensen similarity index value for the ECM fungal community on P. sinensis comparing with other hosts showed that it was highest between P. sinensis and Pinus spp. (0.27), followed by P. sinensis paring with Quercus spp (0.21). According Nonmetric Multidimensional Scaling (NMS) and Permutational Multivariate Analysis of Variance (perMANOVA), most of frequent fungal species were shared between coexisting hosts (Pinus and Quercus), between which no significant difference was found in ECM fungal communities (F2,s= 0.838, p= 0.843). In contrast, the communities were significantly different between three sites in China (F2,s= 1.417, p= 0.021), and more clearly between Chinese and Japanese Douglas-fir forests (F1.18= 3.851, p= 0.001), but still no difference between hosts (F2.17= 1.027,p= 0.359). These results indicated that host monophyly is not a major determinant of ECM fungal communities in this spatial and geological time scale. The strong spatial structure of ECM fungal communities (even in similar forests between China and Japan) over host monophyly also suggested that biogeography of most ECM fungi could be fundamentally independent of host biogeography in this time scale. We suggested that ECM community was shaped by locally evolved EMF and have evolved with the effects of host in each region separately.Spores are another important factor for root colonization by EMF besides external mycelia. In the present study, glasshouse experiments were used to study the effects of host on ECM fungal communities structure and the direct of helper bacteria on P. menziesii growth of the ECM fungal spores in soil. We used soils collected beside mature P. sinensis trees and air-dried them to select for resistant ECM fungal spores. We hypothesized that pioneer species, such as Rhizopogon spp. would account for a large proportion of the ECM diversity on P. menziesii seedlings. We also assumed that changes in EMF species composition probably are at least partly caused by inoculation by resistant spores (i.e. soil propagules). Finally, we compare the ECM fungal communities between glasshouse seedlings and mature trees. In total 19 ECM fungal species were identified by this approach. Only 4 species on seedlings in this experiment were previously identified from natural soil samples associated with P. sinensis. And 8 species of the identified species were shared between 3 sites. There were significant differences among three sites soil spore banks that can associate with P. menziesii in glasshouse experiments according to Multiple Response Permutation Procedure (MRPP) analysis (A= 0.373, p< 0.001), but not different (A= 0.038, p= 0.082) between two sites (ZJJ1, ZJJ2) in the same province. Ordination analysis (NMS) showed ECM communities of P. menziesii seedlings in bioassay experiment were distinct from those of mature P. sinensis trees. MRPP also confirmed that they were significantly different (A= 0.48, p= 0.02). Lower Shannon’s and Simpson’s diversity values were observed for P. menziesii seedlings. There was a positive, but not strong, correlation between EMF richness and shoots dry weight (y= 0.05+0.01x, R= 0.36, p= 0.006). Besides, ANOVA showed that height, as well as dry weight of shoots (F5.74= 8.49, p< 0.001; F5.74= 9.26, p< 0.001) differed significantly between the major ECM species, indicating that inoculations of EMF can enhance the growth of seedlings, especially Meliniomyces and Rhizopogon species.Even host and biogeography are important factors in structuring ECM communities, but the micro-ecological condition of soil also can affect ECM community distribution. In this study, we test the soil physical-chemical properties of glasshouse experiments set before in order to find the way soil affect ECM structure. Based on characteristics, soil samples were clustered into 5 types, of which there were significant difference (A= 0.142, p< 0.001) among spore banks that can form ECM. Even there was no correlation was observed between soil chemical properties and ECM richness, the concentrations of OM, N, K and pH value had a significant effect on community structure of ECM as RDA showed, indicating that interaction between the various factors lead to the difference between ECM communities. Two-way cluster analysis of soil and ECM fungi showed that ECM species could be separated into two groups (Jiangxi and Hunan, two provinces). Geography places an important role in structuring the ECM communities.Rhizopogon spp. can form specific symbiotic interaction with Pseudotsuga. In previous study, we found there were 39 ECM species that can only associated with P. sinensis. In this study, we checked whether they belong to P. sinensis specific lineage. Besides, ITS regions of Rhizopogon spp. and their phylogenetic relatedness with maximum likelihood analyses were used to assess the validity of Rhizopogon sp. were identified. Results turned out that except Rhizopogon sp. there were no Pseudotsuga specific ECM fungi in out data set. Sebacinaceae, Russulaceae and Thelephoraceae have broad spectrum in host selection for association. Rhizopogon sp., belongs to R. subg. Villosuli, was a Pseudotsuga-spocific ECM fungi. It is unclear whether Rhizopogon sp. should be classified in either of the two established sections R. sect. Villosuli or R. sect. Vinicolores in R. subg. Villosuli, for it formed an independent branch together with Japanese Pseudotsuga specific Rhizopogon. The ML phylogeny presented here provides strong support for the single-origin theory of the Rhizopogon-Pseudotsuga EM symbiosis of which determined by the genotype of host. However, interspecific relationships between them are not well described and Rhizopogon ECM of other Asian Pseudotsuga spp. are unknown. Additional sampling in the Pseudotsuga forests in Asia as well as more molecular datasets are needed to test whether R. subg. Villosuli, as the same as Pseudotsuga, were also originated in North America and co-evolution with Pseudotsuga and migrated to other regions. |
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