The analysis urban area contains c. dos kilometres dos regarding kinds-rich dead calcareous grassland when you look at the western Estonia (°N, °E). This new grasslands arranged typically on account of a lot of time-name detailed administration, notably grazing because of the home-based dogs, meeting hay and cutting shrubbery and you may woods for strength. , 1999 ). Brand new grasslands generally got an optimum juniper (Juniperus communis) defense off 29%, however they are now even more overgrown that have Scots oak (Pinus sylvestris; around 80% cover).
Sampling are conducted from inside the on web sites where continuously managed (mown) discover grassland stands (GR) was in fact enclosed by really stands away from different levels of overgrowth: transitional grasslands (TR) and you may more youthful pine forest (FO). The 3 successional grade (GR, TR, FO) means a temporal sequence out-of regeneration sequence layer c. sixty yr, and they are characterised by the a slow over growing out of unlock grasslands by the shrubs and you may woods, which is typical getting quit calcareous grasslands in the studies area (Neuenkamp et al., 2016 ; Supporting Advice Fig. S1). Into the transformation grasslands administration ceased c. 30–35 yr prior to sampling and you may grasslands was basically partially overgrown by pine and you may juniper; young pine forest was indeed former grasslands where management stopped c. 60 yr just before testing and you may that have been entirely overgrown of the pine and you can juniper. A site are recognized as a location where all about three successional amounts were discovered within this 50 meters of every most other (GR–TR–FO). Altogether, twenty-five internet sites were tested. A randomly receive step one ? 1 meters plot are created in for every single successional stage (GR, TR, FO) of every website, resulting in a total of 75 plots of land (twenty five sites ? three successional stages). The second attributes was demonstrated for every single plot: the field layer bush area, Are fungal organizations (in-plant roots, and you can rhizosphere ground) and environment variables.
Per 1 ? step one m plot, the vascular plant species on the crushed-covering people was submitted as well as their wealth was projected just like the percentage defense (Peet & Roberts, 2013 ). Concurrently, fee defense rates out of shrubbery and you can woods in this a 10 ? ten meters city around per patch was indeed filed. Hereafter, the floor-level bush community is called ‘bush community’ and all analyses are used using this type of community unless conveyed otherwise.
A ten ? ten ? 10 cm floor center are amassed on the hub of any spot and you can bush root were separated about crushed, dehydrated having twenty four h within fifty°C and stored dry for additional unit investigation out of resources-colonising Am fungus. Was yeast diversity in-plant roots was then analysed away from mixed resources products. Root within the each decide to try was crushed having fun with water nitrogen and very carefully combined because discussed by the Garcia de Leon mais aussi al. ( 2016 ). An effective subsample out-of 70 mg of any combined options attempt was utilized for unit analyses.
Bush organizations was in fact explained during the a more impressive spatial level compared with Am yeast teams, as this size is always to approximately reflect the fresh bush root people captured of the a 10 ? 10 ? ten cm ground center. Small-level plant assortment in the grasslands was highest underground than just over floor (Hiiesalu et al., 2014 ), partially due to the fact roomy supply system and long rhizomes of numerous grassland flowers connect new roots found in a localized surface center that have significantly more than-crushed areas 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 established men desktop (ISO) protocols (see Methods S1).
