Response of Arbuscular Mycorrhizal Fungi to Simulated Climate Changes by Reciprocal Translocation in Tibetan Plateau

Arbuscular mycorrhiza (AM) fungi are considered as an important factor in predicting plants and ecosystem responses to climate changes on a global scale. The Tibetan Plateau is the highest region on Earth with abundant natural resources and one of the most sensitive region to climate changes. To evaluate the complex response of arbuscular mycorrhizal fungi colonization and spore density to climate changes, a reciprocal translocation experiment was employed in Tibetan Plateau. The reciprocal translocation of quadrats to AM colonization and spore density were dynamic. Mycorrhizal colonization frequency presented contrary changed trend with elevations of quadrat translocation. Colonization frequency reduced or increased in majority quadrats translocated from low to high or from high to low elevation. Responses of colonization intensity to translocation of quadrats were more sensitive than colonization frequency. Arbuscular colonization showed inconsistent trend in increased or decreased quadrat. Vesicle colonization decreased with changed of quadrat from low to high elevations. However, no significant trend was observed. Although spore density was dynamic with signs of decreasing or increasing in translocated quadrats, the majority enhanced and declined respectively in descent and ascent quadrat treatments. It is crucial to understand the interactions between AM fungi and prairie grasses to accurately predict effects of climate change on these diverse and sensitive ecosystems. This study provided an opportunity for understanding the effect of climate changes on AM fungi.


Introduction
Arbuscular mycorrhiza (AM) fungi form symbiosis with ~80% of vascular plants being widely claimed to be an important mechanism for biochemical cycling in natural ecosystems, and more recently is being increasingly studied under current and projected global climate changes (van der Heijden et al., 1998;Hughes et al., 2008;Averill et al. 2014;Mohan et al., 2014).AM fungi are considered as an important factor in predicting plants and ecosystem responses to climate changes on a global scale (Compant et al., 2010;Mohan et al., 2014).
The Tibetan Plateau is known as the 3rd Pole with abundant ice fields and is also the highest region on Earth with abundant natural resources.However, observed current and projected climate changes have produced numerous impacts including increases in temperature (0.25 °C decade -1 ) (Hu et al., 2013), and annual precipitation with the wettest year in 2010 being during 3,500 year by tree-ring record (Yang et al., 2014), which makes this region a susceptive zone.More recently, greater attention has focused on climate changes in the Tibetan plateau vegetation ecology, and how the below-ground microbial communities respond to both variations in the soils environment and above ground vegetation type (Farrington, 2009;Zhang et al., 2013).Moreover, many studies have shown that prairie plant communities are closely connected with their AM fungi community, and this relationship between plant and fungi displays positive reciprocal effects (Hartnett and Wilson, 1999;Eom et al., 2000).There is also evidence that AM fungi community composition are not randomly distributed throughout tall grass prairies landscapes, but show some degree of host specificity (Eom et al., 2000).Consequently, it is crucial to develop a firm understanding of the interactions between AM fungi and prairie grasses to accurately predict effects of climate change on these diverse and sensitive ecosystems (Araújo and Luoto, 2007).However, Jumpponen alpine plants were dormant.The characteristics of each plot including vegetation, soil and climate were showed in Table 1.Then, each plot was divided into sixty-three (7×9) quadrats of 100×100 cm (Fig. 1 B).Among of them, twelve spaced quadrats were selected as the objects of this study (Fig. 1 B).Three quadrats were kept at original elevation, while nine with the depth of 0.3-0.4m were translocated to the three other elevations.Twelve quadrats were semi-randomly distributed at each elevation (Fig. 1 C).

Samples collection
Five soil cores of 0-30 cm depth with 2 cm in diameter were collected in each quadrat according five-spot sampling model in August, 2011.The five soil cores in the same quadrat were mixed as a sample.Therefore, there are three replicas in each treatment (Fig. 1 C).The roots were picked out carefully from soil cores for the measurement of arbuscular mycorrhizal colonization.The soil was reserved for isolating arbuscular mycorrhizal fungal spores.

Assessment of arbuscular mycorrhizal colonization
Fresh roots were washed free of soil and cleared in 10% (w/v) KOH at 90°C in a water bath for 20-30 min, the exact and Jones (2014) did not show fungal species richness or diversity changes to abiotic climate changes e.g.elevated CO2, O3, UV and Drought.Shi et al. (2014) indicated AM fungal richness and diversity are positively correlative with temperature in Mount Taibai.Currently, most of the AM fungi studies are laboratory, not field studies and this may be a limitation.
Here, we conduct a in situ study to determine the response of AM fungi colonization and spore density to elevational changes by using a reciprocal translocation experiment at the Haibei Alpine Meadow Research Station (101 o 19'E, 37 o 35'N), located in the northeast of the Tibetan Plateau to study the effects of climate changes on AM fungi colonization and spore density.

Study site and experimental design
This study site located at the Haibei Alpine Meadow Research Station, Chinese Academy of Sciences (101°19′E, 37°35′N) (Fig. 1 A) in the northeast of the Qinghai-Tibetan Plateau of China.Four plots of 7×9 m plots were selected in flat (aspect < 3 o ) and homogenous plots of four altitudinal gradients of 3200 m, 3400 m, 3600 m and 3800 m.Note: Organic matter and total nitrogen of soil are the average in 0 to 30 cm soil.time depending on the degree of lignification of the roots and their pigmentation.The root subsamples were cooled, washed, and cut into 0.5 to 1.0-cm-long segments and stained with 0.5% (w/v) acid fuchsin (Biermann and Linderman, 1981).At least thirty root fragments (ca. 1 cm long) were mounted on slides in polyvinyl alcohol-lactic acid-glycerol (Koske and Tessier, 1983) and examined at ×100-400 magnification using microscope.The colonization parameters were: F% = Colonization frequency in the root system M% = Colonization intensity in the root system m%=Intensity of the mycorrhizal colonization in the root fragments A%=Arbuscule abundance in the root system a%= Arbuscule abundance in mycorrhizal parts of root fragments These specific terms were calculated according to Trouvelot et al. (1986).Moreover, we calculated vesicle abundance in mycorrhizal parts of root fragments v% and V% referring to the formulas of (a%) and (A%) at Trouvelot et al. (1986).
V%=Arbuscule abundance in the root system v%= Arbuscule abundance in mycorrhizal parts of root fragments Extraction and counting of AM fungal spores Spores or sporocarps were extracted from 100 g air-dried sub-samples of each soil sample in triplicate by wet sieving followed by flotation-centrifugation in 50% sucrose (Dalpé, 1993).Spore density was calculated using the spore number in 100 g dried soil.

Statistical analysis
The were subjected to one-way of analysis of variance and means were compared by least significant difference (LSD) at the 5% level.The changes of colonization parameters and spore density were calculated by the following formula: [(translocated quadrats-primary elevation quadrats)/ primary elevation quadrats)]*100%.The statistical tests were applied using SPSS software package version 13.0 developed by SPSS Inc., Chicago, IL 60606, USA.

Results and Discussion
Decreasing altitude produced the highest observed colonization frequency in the root system in translocated quadrats from 3800 m to 3400 m (Table 2), which may be explained by the combination of changes in all kinds of factors including climate and soil etc. due to the translocated quadrats among different elevations.The changes of temperature have shown in Table 1 in different elevations.The changes of soil nutrition have also confirmed (Li et al., 2010).Usually, the CO2 concentration also increased with the decrease of elevations.The effect of soil nutrition on mycorrhizal colonization was well known (Smith and Read, 2008).It has also been widely claimed that increased CO2 can improve the AM colonization (Monz et al., 1994;Godbold et al., 1997;Staddon et al., 1999a;Rillig et al., 1999), but increased temperature have variable impacts on colonization (Heinemeyer and Fitter, 2004;Wilson, 2012).Additionally, we tested the effect of stressing temperature on AM fungi colonization, which showed that temperature stress did not significantly affect AM fungi colonization with colonized rate of 56.9% and 60.8 in temperature stress and control treatment, respectively.Further, when the high or low temperature stress was considered, the same findings were obtained with the colonization values of 60.8% in low temperature stress comparing to 66.9% in control.The colonization in high temperature stress and control was 48.9 % and 47.9%, respectively.Interestingly there are some contrary findings, where elevated CO2 decreased or do not significantly change the AM fungi colonization (Monz et al., 1994;Staddon et al., 1999b).The highest of vesicle frequency presented at translocated quadrats from 3600 to 3200 m.Further, the highest colonization intensity in the root system and root fragment, arbuscule abundance in mycorrhizal parts of root fragments, arbuscule and vesicle abundance in the root system and spore density presented the nontranslocated quadrat of 3400 m.The response of AM fungi colonization parameters to climate changes were varied, which is in accordance with by previous reports (Staddon et al., 1998;Mohan et al., 2014).Such changes of host plant community possibly account for the colonization, because host plants regulate tightly internal mycorrhizal colonization, and do not allow the fungal partner to utilize all the soluble carbon in the root potentially .0c Note: F% and M% mean colonization frequency and intensity in the root system, respectively.m% means intensity of the mycorrhizal colonization in the root fragments.A% and a% mean arbuscule abundance in the root system or in mycorrhizal parts of root fragments, respectively.V% and v% mean vesicle abundance in the root system or in mycorrhizal parts of root fragments, respectively.Spore density was calculated using the spore number in 100 g dried soil.Different letters between elevation denote significant differences (Duncan test, P<0.05).
available (Lewis et al., 1994).Indeed, the plant communities have changed with the translocation of quadrats at different elevation (Zhang et al., 2011).Additionally, the sampling season may have affected the responses of AM fungi colonization to climate changes.Zavalloni et al. (2012) showed the responses of AM fungi root colonization to elevated temperature and CO2 varied with the growing season in grassland communities, which was previously confirmed under elevated CO2 by Staddon et al. (1998).
The colonization frequency in the root system has lower changes than colonization intensity in the root system and intensity of the mycorrhizal colonization in the root fragments (Fig. 2), this result supports previous observations in newly established grasslands where AM fungi root colonization intensity, but not colonization frequency increased with elevated CO2 and temperature (Zavalloni et al., 2012).These data show that AM fungi colonization intensity is more sensitive than colonization frequency, which may be attributed to the root characteristics of host plants as AM fungi mainly colonize in absorption roots (Smith and Read, 2008).There were dynamic changes across all AM fungi colonization parameters, with the ascent or descent of translocated quadrats (Figs.2-4), which is in accordance with majority studies in elevated CO2 (Staddon and Fitter, 1998).Moreover, Staddon and Fitter (1998) reported CO2 effects on AM fungi are indirect and are a result of plant growth changes at higher CO2 concentrations.The observation of changes in arbuscule and vesicle are similar with previous findings that these presented different change trends with the elevated CO2 (Sanders, 1996).
The spore density reduced in 4 out of 6 treatments of ascent quadrats (Fig. 5).Contrarily, they increased in 4 out of 6 treatments of descent quadrats.These results showed that climate changes impacted the spore density of AM fungi, which is similar to the findings made by Sun et al. (2013).Gong et al. (2013) confirmed spore density was closely related with climatic and edaphic factors.With the translocation of quadrats among different elevations, the climatic and edaphic factors and plant community and growth inevitably changed.Therefore, the exact response of AM fungi colonization, spore density and even community structure to translocation of quadrats needs to be further studied by the time-course samples owing to the importance of plant developmental stage in analyses of effects of CO2 on mycorrhizal colonization (Staddon et al., 1998).Note: E34-32 means the quadrat that was translocated from elevation 3400 m to 3200 m.The rest can be done in the same manner.The same are as below.F% and M% mean colonization frequency and intensity in the root system, respectively.m% means intensity of the mycorrhizal colonization in the root fragments.The responses of colonization intensity to translocation of quadrats were more sensitive than colonization frequency.The majority spore density decreased or increased in ascent and descent quadrat treatments, respectively.

Fig. 1 .
Fig. 1.The sketch of study site (A) and experimental design (B)

Fig. 2 .
Fig. 2. The changes of total percentage of arbuscular mycorrhizal colonization in translocated quadrats comparing to these in situ quadrats at different elevation in Tibet Fig. 3.The changes of percentage of arbuscule colonization in translocated quadrats comparing to these in situ quadrats at different elevation in TibetNote: A% and a% mean arbuscule abundance in the root system or in mycorrhizal parts of root fragments, respectively.

Table 2 .
Arbuscular mycorrhizal colonization and spores density in translocated quadrats in different elevation in Tibet