The analysis area include c. 2 km dos of varieties-rich dry calcareous grassland within the west Estonia (°N, °E). The latest grasslands setup historically on account of a lot of time-name extensive management, somewhat grazing by the residential animals, get together existe and you will reducing shrubs and you will trees to own electricity. , 1999 ). The fresh grasslands traditionally got a maximum juniper (Juniperus communis) shelter away from 29%, but they are now even more overgrown which have Scots pine (Pinus sylvestris; up to 80% cover).
Testing try used during the during the websites in which constantly managed (mown) discover grassland stands (GR) had been surrounded by stands out-of different degrees of overgrowth: transformation grasslands (TR) and you can younger oak forests (FO). The 3 successional values (GR, TR, FO) setting a-temporal colombian cupid reddit succession out-of regeneration sequence layer c. sixty yr, and generally are characterised of the a steady over growing out-of discover grasslands by the shrubs and woods, that is typical for given up calcareous grasslands in the investigation part (Neuenkamp mais aussi al., 2016 ; Support Guidance Fig. S1). Into the transitional grasslands government stopped c. 30–thirty-five year prior to sampling and you may grasslands was partially overgrown of the pine and you can juniper; younger oak forest was basically previous grasslands where government ceased c. 60 yr before testing and you will that have been totally overgrown by oak and you can juniper. A webpage is actually identified as a location where most of the about three successional degree was indeed located in this fifty m of any almost every other (GR–TR–FO). Altogether, twenty five internet sites was in fact tested. An arbitrarily discovered step one ? step 1 m spot is actually created in for every successional stage (GR, TR, FO) of each webpages, leading to a total of 75 plots (25 internet ? about three successional degree). Next characteristics was discussed for every single spot: the field covering plant society, Am fungal communities (in plant root, and you can rhizosphere ground) and you may ecological variables.
For every step 1 ? step 1 m spot, the vascular bush kinds in the soil-covering area had been submitted in addition to their abundance was estimated since fee safeguards (Peet & Roberts, 2013 ). At exactly the same time, commission safety rates regarding shrubbery and you can woods contained in this a beneficial 10 ? ten m city as much as for each and every spot had been registered. Hereafter, a floor-covering plant neighborhood is called ‘bush community’ as well as analyses is presented with this specific society unless of course shown or even.
Good ten ? ten ? 10 cm floor center are accumulated about center of each area and you can plant roots were separated regarding the floor, dehydrated having twenty-four h in the fifty°C and you can kept dry for additional molecular studies out of supply-colonising In the morning fungus. Have always been yeast diversity in-plant origins ended up being analysed out-of blended sources examples. Roots for the for each and every test was indeed soil playing with drinking water nitrogen and you can carefully combined just like the explained by Garcia de Leon ainsi que al. ( 2016 ). Good subsample from 70 milligrams of every mixed means try was useful molecular analyses.
Plant groups was indeed explained within a bigger spatial size weighed against Am fungal teams, since this size should approximately echo this new plant resources society seized from the a good 10 ? ten ? 10 cm surface key. Small-scale bush range for the grasslands is actually large below ground than just a lot more than ground (Hiiesalu et al., 2014 ), partly while the roomy options system and you can much time rhizomes of several grassland plants link this new roots present in a localised ground center with significantly more than-ground organs expanding several ).
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).