Summary1. Laboratory microcosm experiments using protists as model organisms have a long tradition and are widely used to investigate general concepts in population biology, community ecology and evolutionary biology. Many variables of interest are measured in order to study processes and patterns at different spatiotemporal scales and across all levels of biological organization. This includes measurements of body size, mobility or abundance, in order to understand population dynamics, dispersal behaviour and ecosystem processes. Also, a variety of manipulations are employed, such as temperature changes or varying connectivity in spatial microcosm networks. 2. Past studies, however, have used varying methods for maintenance, measurement, and manipulation, which hinders across-study comparisons and meta-analyses, and the added value they bring. Furthermore, application of techniques such as flow cytometry, image and video analyses, and in situ environmental probes provide novel and improved opportunities to quantify variables of interest at unprecedented precision and temporal resolution. 3. Here, we take the first step towards a standardization of well-established and novel methods and techniques within the field of protist microcosm experiments. We provide a comprehensive overview of maintenance, measurement and manipulation methods. An extensive supplement contains detailed protocols of all methods, and these protocols also exist in a community updateable online repository. 4. We envision that such a synthesis and standardization of methods will overcome shortcomings and challenges faced by past studies and also promote activities such as meta-analyses and distributed experiments conducted simultaneously across many different laboratories at a global scale.

The breeding systems of many organisms are cryptic and difficult to investigate with observational data, yet they have profound effects on a species' ecology, evolution, and genome organization. Genomic approaches offer a novel, indirect way to investigate breeding systems, specifically by studying the transmission of genetic information from parents to offspring. Here we exemplify this method through an assessment of self-fertilization vs. automictic parthenogenesis in Daphnia magna. Self-fertilization reduces heterozygosity by 50% compared to the parents, but under automixis, whereby two haploid products from a single meiosis fuse, the expected heterozygosity reduction depends on whether the two meiotic products are separated during meiosis I or II (i.e., central vs. terminal fusion). Reviewing the existing literature and incorporating recombination interference, we derive an interchromosomal and an intrachromosomal prediction of how to distinguish various forms of automixis from self-fertilization using offspring heterozygosity data. We then test these predictions using RAD-sequencing data on presumed automictic diapause offspring of so-called nonmale producing strains and compare them with "self-fertilized" offspring produced by within-clone mating. The results unequivocally show that these offspring were produced by automixis, mostly, but not exclusively, through terminal fusion. However, the results also show that this conclusion was only possible owing to genome-wide heterozygosity data, with phenotypic data as well as data from microsatellite markers yielding inconclusive or even misleading results. Our study thus demonstrates how to use the power of genomic approaches for elucidating breeding systems, and it provides the first demonstration of automictic parthenogenesis in Daphnia.KEYWORDS genome-wide heterozygosity; breeding system; inbreeding; automixis; tychoparthenogenesis; Daphnia magna; nonmale producers W HILE humans and most other mammals reproduce exclusively by sexual reproduction with sexes being determined by the well-known XY sex-chromosome system, the breeding systems of many other organisms, including many pests and parasites, remain unknown (Bell 1982;Normark 2003). The breeding system sensu lato, (including details of meiosis, e.g., recombination patterns and syngamy, e.g., levels of inbreeding, as well as their variants, e.g., modified meiosis in parthenogens) represents a key for understanding the biology of a species and has profound effects on its ecology, evolution, and genomics. Yet investigating breeding systems is often far from straightforward: Many species cannot easily be cultured and bred in the laboratory and observations of breeding behavior in nature are difficult. Even in species than can be bred in the laboratory, parts of the breeding system may be cryptic and not directly observable.The advent of high-throughput genotyping methods opens an alternative possibility that can be used on a much larger array of species: indirect inference of the breeding system usi...

Phenotypic plasticity is increasingly recognized as a key element of eco‐evolutionary dynamics, but it remains challenging to assess because of its multidimensional nature. Indeed, organisms live in complex environments where numerous factors can impact the phenotypic expression of traits (inter‐environment axis), possess multiple traits that can influence each other's expression (inter‐trait axis) and differ in their genetic background (inter‐genotype axis), which can not only impact the traits' values but also their plasticity. We addressed six questions related to phenotypic plasticity: (a) do different environmental gradients show similar effects on a given trait? (b) Are the effects of two environmental gradients on a trait additive? (c) Do different traits show similar plastic response to a given environmental gradient? (d) Do the (co)variances between traits vary across environmental gradients? (e) Do genotypes differ in their plastic response to a given environmental gradient? (f) Are some genotypes more plastic than others across all traits? We designed a microcosm experiment using the protist Tetrahymena thermophila aimed at encompassing all these aspects of phenotypic plasticity. We exposed 15 distinct genotypes to 25 combinations of temperature and nutrient availability and assessed the plasticity of five phenotypic traits. Our results show strong differences in the plastic response depending on the environmental gradient, not only regarding the shape of the reaction norm of the different traits tested, but also in the overall plasticity of the organisms. We did not find any covariance between traits that was consistent across all environments. Overall, our results suggest independent impacts of the environmental dimension considered on the observed plastic response. These results underline potential difficulties in generalizing findings about plasticity to all environments and all traits. A free Plain Language Summary can be found within the Supporting Information of this article.

When salmonid fish that have been raised in hatcheries spawn in the wild, they often produce fewer surviving adult offspring than wild fish. Recent data from steelhead ( Oncorhynchus mykiss ) in the Hood River (Oregon, USA) show that even one or two generations of hatchery culture can result in dramatic declines in fitness. Although intense domestication selection could cause such declines, it is worth considering alternative explanations. One possibility is heritable epigenetic changes induced by the hatchery environment. Here, we show, using methylation-sensitive amplified fragment length polymorphism, that hatchery and wild adult steelhead from the Hood River do not appear to differ substantially in overall levels of genomic methylation. Thus, although altered methylation of specific DNA sites or other epigenetic processes could still be important, the hatchery environment does not appear to cause a global hypo- or hypermethylation of the genome or create a large number of sites that are differentially methylated.