Several reduced-representation bisulfite sequencing methods have been developed in recent years to determine cytosine methylation de novo in nonmodel species. Here, we present epiGBS2, a laboratory protocol based on epiGBS with a revised and userfriendly bioinformatics pipeline for a wide range of species with or without a reference genome. epiGBS2 is cost-and time-efficient and the computational workflow is designed in a user-friendly and reproducible manner. The library protocol allows a flexible choice of restriction enzymes and a double digest. The bioinformatics pipeline was integrated in the Snakemake workflow management system, which makes the pipeline easy to execute and modular, and parameter settings for important computational steps flexible. We implemented biSmark for alignment and methylation analysis and we preprocessed alignment files by double masking to enable single nucleotide polymorphism calling with FreebayeS (epiFreebayeS). The performance of several critical steps in epiGBS2 was evaluated against baseline data sets from Arabidopsis thaliana and great tit (Parus major), which confirmed its overall good performance. We provide a detailed description of the laboratory protocol and an extensive manual of the

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Plant interactions are as important belowground as aboveground. Belowground plant interactions are however inherently difficult to quantify, as roots of different species are difficult to disentangle. Although for a couple of decades molecular techniques have been successfully applied to quantify root abundance, root identification and quantification in multispecies plant communities remains particularly challenging. Here we present a novel methodology, multispecies genotyping by sequencing (msGBS), as a next step to tackle this challenge. First, a multispecies meta‐reference database containing thousands of gDNA clusters per species is created from GBS derived High Throughput Sequencing (HTS) reads. Second, GBS derived HTS reads from multispecies root samples are mapped to this meta‐reference which, after a filter procedure to increase the taxonomic resolution, allows the parallel quantification of multiple species. The msGBS signal of 111 mock‐mixture root samples, with up to 8 plant species per sample, was used to calculate the within‐species abundance. Optional subsequent calibration yielded the across‐species abundance. The within‐ and across‐species abundances highly correlated (R2 range 0.72–0.94 and 0.85–0.98, respectively) to the biomass‐based species abundance. Compared to a qPCR based method which was previously used to analyse the same set of samples, msGBS provided similar results. Additional data on 11 congener species groups within 105 natural field root samples showed high taxonomic resolution of the method. msGBS is highly scalable in terms of sensitivity and species numbers within samples, which is a major advantage compared to the qPCR method and advances our tools to reveal hidden belowground interactions.

Organisms must cope with both short-and long-term environmental changes to persist.In this study we investigated whether life histories trade-off between their robustness to short-term environmental perturbations and their ability to evolve directional trait changes. We could confirm the tradeoff by modeling the eco-evolutionary dynamics of lifehistories along the fast-slow pace-of-life continuum. Offspring dormancy and high adult survival rates allowed for large population sizes to be maintained in face of interannual environmental fluctuations but limited the speed of trait evolution with ongoing environmental change. In contrast, precocious offspring maturation and short-living adults promoted evolvability while lowering demographic robustness. This tradeoff had immediate consequences on extinction dynamics in variable environments. High evolvability allowed short-lived species to cope with long-lasting gradual environmental change, but came at the expense of more pronounced population declines and extinction rates from environmental variability. Higher robustness of slow life-histories helped them persist better on short timescales.