A1

State of the art

Paleogenomic tools are increasingly used to infer past changes in microbial dynamics in aquatic systems (Coolen et al. 2013; Capo et al. 2015; Savichtcheva et al. 2015). Using sedimentary DNA, past community composition and diversity (Capo et al. 2015) as well as changes in the genetic structure of individual species (Härnström et al. 2011; Domaizon et al. 2013) have been reconstructed. For specific taxa, DNA may be retrieved from cysts and resting stages (Boere et al. 2009; Härnström et al. 2011), and DNA of many other taxa appears to be rather well preserved in lake sediment as well (Coolen and Overmann 1998). A recent study on planktonic diversity in the sediment layers of Lake Bourget, France (Capo et al. 2015) concluded that the DNA diversity of most protists (including e.g., Chlorophyta, Dinophyceae, Chrysophyceae, Ciliophora) was of similar magnitude as that of planktonic samples. However, DNA of Cryptophyta and Haptophyta was underrepresented in the sediment layers (Capo et al. 2015).

Lake Constance has been exceptionally well studied with regard to the effects of anthropogenic eutrophication and re-oligotrophication on phytoplankton and ciliate long-term dynamics (e.g. Jochimsen et al. 2013). However, long-term records which can be used on a per-species level date back only to 1965 in case of phytoplankton and to 1987 in case of ciliates. Additionally, these data sets might be confounded by methodological and/or observer bias as has been documented in other lakes (e.g. Straile et al. 2015a). The paleo-metagenomic record thus can provide an independent estimate of e.g. changes in diversity that are not affected by changes in methodology.

Preparatory work

16S-rRNA genes from littoral and profundal sediments of Lake Constance have been extracted and a pmoA-based quantitative real-time PCR (Q-PCR) assay was used to study differences in aerobic methanotrophic communities between littoral and profundal sediments (Rahalkar and Schink 2007; Rahalkar et al. 2009).

The experimental resources, expertise and technology required for deep amplicon-sequencing are established at the University of Konstanz and deep amplicon-sequencing has previously been employed to comparatively analyse a variety of fish species (Elmer et al. 2010a; Franchini et al. 2014) as well as microbial communities in upland and water-saturated soils (Pester et al. 2012, 2014; Hausmann et al. 2016; Pelikan et al. 2016). The comparative analysis of the data generated by deep amplicon-sequencing will follow standard bioinformatics procedures as provided by the open source software packages mothr (www.mothur.org) and giime (giime.org) followed by detailed exploratory and statistical analyses as provided by e.g. the R packages phyloseq, edgeR, and Deseq2 (e.g. as outlined in Hausmann et al. 2016).

Proposed project and role within the RTG

In this project, we aim to extend our knowledge of the species succession occurring in the lake over the past century, as well as to increase our understanding of the effects that eutrophication and oligotrophication had on the lake's ecology in order to better predict the effects that future changes may have on the lake ecosystem. In a first step, our focus will be on recovering and sequencing eukaryotic rRNA gene sequences (e.g. 18S rRNA genes) to a) access a record of past change in the species composition of the lake and b) use the long-term historical records available for the lake and the species to correct for systematic biases induced by the sedimentation process and subsequent experimental treatments. The doctoral researcher will use next-generation amplicon sequencing of recovered 18S rRNA and 16S rRNA genes in order to reconstruct past dynamics of eukaryotic and prokaryotic DNA from different sediment layers in both Upper and Lower Lake Constance. 16S rRNA will be used especially to estimate the community composition and dynamics of cyanobacteria. For both basins, the projects will aim at a high temporal resolution (1 cm slices of sediment corresponding to approximately 2-5 years) which will allow the analysis of e.g. structural resilience, regime shifts and reversibility during the overall eutrophication and oligotrophication period. This work will provide a paleolimnological view of protist and cyanobacterial community composition prior to the onset of massive eutrophication in the 1950s. The doctoral researcher will also provide estimates of past diversity changes for different eukaryotic groups and for cyanobacteria. Based on biomass estimates revealed from pigments (cooperation with project A4) she/he will analyse the relationship between diversity and productivity and investigate whether the trophic efficiency of algae is related to their diversity. The project will closely cooperate with projects A2, A3, A4, C1 and C4.