Analysis site
The analysis city include c. dos kilometres 2 from kinds-rich inactive calcareous grassland from inside the west Estonia (°Letter, °E). The new grasslands put up over the years because of long-term thorough government, significantly grazing from the residential pets, collecting hay and you will cutting shrubs and trees for energy. , 1999 ). The brand new grasslands traditionally got a maximum juniper (Juniperus communis) safeguards off 29%, but they are now all the more overgrown having Scots pine (Pinus sylvestris; up to 80% cover).
Sampling build
Testing was held when you look at the at the websites where continuously managed (mown) discover grassland stands (GR) was indeed enclosed by really stands off different quantities of over growing: transformation grasslands (TR) and more youthful oak forest (FO). The 3 successional values (GR, TR, FO) mode a temporal series away from regeneration series coating c. 60 year, and tend to be characterised because of the a gradual over growing out of unlock grasslands of the bushes and you will woods, that's normal having abandoned calcareous grasslands about study part (Neuenkamp et al., 2016 ; Help Suggestions Fig. S1). Within the transitional grasslands management ceased c. 30–thirty five yr in advance of testing and you can grasslands was partly overgrown because of the oak and you will juniper; younger oak forests was in fact previous grasslands in which government stopped c. 60 yr before testing and which have been youngsterally overgrown from the pine and juniper. A web page was recognized as a place in which most of the around three successional values were located contained in this fifty yards of every most other (GR–TR–FO). Completely, twenty-five internet sites was in fact tested. A randomly discover step one ? step 1 m patch was created in for each 
and every successional stage (GR, TR, FO) of each webpages, resulting in a total of 75 plots (twenty five sites ? around three successional amounts). The second services was indeed described for each patch: the field level bush neighborhood, Was fungal communities (in-plant sources, and you may rhizosphere soil) and you may environmental details.
Analysis range
Each step one ? step one m patch, the vascular plant types about crushed-layer society was in fact submitted in addition to their variety try projected since the percentage safety (Peet & Roberts, 2013 ). Likewise, payment defense rates out of shrubs and you may trees inside an effective 10 ? ten meters town around each plot had been recorded. Hereafter, the ground-covering plant people is called ‘plant community' as well as analyses try held using this community unless of course conveyed if not.
An excellent ten ? 10 ? 10 cm surface core are gathered regarding the center of each spot and plant sources was in fact broke up throughout the ground, dried to have 24 h at the 50°C and held dead for further molecular analysis of options-colonising Am fungus. In the morning fungal assortment in-plant sources ended up being analysed out of mixed root products. Sources within the each test was basically crushed having fun with h2o nitrogen and you can carefully mixed as explained by the Garcia de- Leon ainsi que al. ( 2016 ). A beneficial subsample off 70 mg each and every combined supply test was utilized for molecular analyses.
Plant groups had been explained during the more substantial spatial scale weighed against Have always been yeast communities, that size is to up to reflect the latest plant supply community grabbed by a beneficial 10 ? ten ? ten cm ground center. Small-level plant assortment in the grasslands try high underground than just over ground (Hiiesalu ainsi que al., 2014 ), partially since roomy root system and you can much time rhizomes of many grassland plants hook up the latest root contained in a localised surface core that have over-crushed organs expanding numerous ).
To characterise AM fungal diversity in the soil, 10 g of soil was collected from each soil core, dried with silica gel and stored airtight at room temperature. A second subsample of 10 g of soil was collected from each soil core for measuring the concentration of an AM fungal neutral lipid fatty acid fungal abundance in the soil. This second soil sample was freeze-dried for 15 h at ?80°C and stored airtight at room temperature. The remainder of each soil core was air-dried for analysis of soil chemical properties (pHKCl, Ntot, P, K, Mg, Ca, Corg) following International Organization for Standardization (ISO) protocols (see Methods S1).