Paleoclimate reconstruction methods brGDGTs (a bacterial lipid biomarker proxy), pollen and chironomid assemblages will be used as paleothermometers (pollen also as paleo-precipitation proxy) for reconstructing  the  mean annual air temperature (MAAT), the mean temperature above freezing (MAF) and the mean temperature of the warmest month (MTW). BrGDGTs have been used during the last decades as paleothermometers providing continental temperature reconstructions in marine and lacustrine sediment cores [49], [50]. These compounds are a family of bacterial membrane-spanning lipids consisting of two alkyl chains ether-bound to glycerol molecules. The structural diversity of the lipids used in paleoclimate proxies is caused by the changing numbers of methyl branches (4-6) and cyclopentane moieties (0-2) incorporated in the two alkyl chains. Furthermore, the outer methyl branch(es) can be located on C5 (5-methyl) or C6 (6-methyl) position of the alkyl chain. Variation in the methylation of 5-methyl brGDGTs is summarized as the MBT’5ME ratio. In a set of globally distributed lakes, the MBT’5ME ratio reveals a strong temperature dependency, which is at the basis of the temperature calibration to be used in this project [53], [54]. This research method will be applied in collaboration with Cindy de Jonge and Maria Ramos-Roman. Fossil pollen have been used in paleoecological and paleoclimatic reconstructions for many decades. The quantitative reconstruction from fossil pollen assemblages is based on a comparison with modern pollen assemblages and their associated modern climatic parameters using different transfer functions and training sets. In this project we will use the weighted average partial least squares (WAPLS) and modern analogue techniques (MAT) to derive annual and seasonal mean temperatures and annual rainfall estimates. Chironomid based July mean temperature reconstructions have been performed by our group in recent years on South-Carpathian alpine lakes. In these studies the Norwegian-Swiss training set was used in combination with the WAPLS transfer function. In scope of this project, the focus will be on the 6-2 kyr cal BC interval from 3 alpine lakes in Romania, Bulgaria and Montenegro using the techniques described in Tóth et al. In high altitude lakes with prolonged winter ice cover, temporary decreases in diatom inferred pH were previously connected with extended winter ice cover. Since CO2 formed by cellular respiration is trapped under the ice in a cold early spring period, this turns oligotrophic alpine and arctic lakes more acidic, which relationship can be used to track RCC events. As demonstrated above, the intensification of the Siberian High effects the SE European Region by the intrusion of extreme cold air during the late winter/early spring season, therefore episodic diatom inferred pH decreases can potentially be used to reconstruct Holocene RCC events. Although the technique works better in the Late Glacial, we will use this technique in combination with pollen, chironomid and brGDGT reconstruction in two alpine lakes to reconstruct climate change for several climatic variables. Diatom analyses will follow the method described in Buczkó et al. (2018). d18O analysis of diatom valves can be used to reconstruct temperature changes during the main diatom bloom periods in alpine lakes characterised with low water retention times. This method will be applied in collaboration with Thorsten Vennemann (Uni Lousanne) and Attila Demény on two alpine lakes in combination with other climate proxies and the monitoring of the modern diatom bloom and associated d18O values from both diatom valves and lake water.

Archaeobotany & charcoal analyses will be applied on 5 LN archeological excavation material for which site details are given in the Study site section.

Environmental DNA (edNA) analysis of lowland lake sediment samples will be used to trace the first appearance and intensity of animal husbandry around the lakes. The lakes are situated in the proximity of LN archaeological sites. Steps will include extraction, amplification with tagged primers and high throughput next-generation sequencing.