Evidence for phenotypic plasticity in brain size and the size of different brain parts is widespread, but experimental investigations into this effect remain scarce and are usually conducted using individuals from a single population. As the costs and benefits of plasticity may differ among populations, the extent of brain plasticity may also differ from one population to another. In a common garden experiment conducted with three‐spined sticklebacks (Gasterosteus aculeatus) originating from four different populations, we investigated whether environmental enrichment (aquaria provided with structural complexity) caused an increase in the brain size or size of different brain parts compared to controls (bare aquaria). We found no evidence for a positive effect of environmental enrichment on brain size or size of different brain parts in either of the sexes in any of the populations. However, in all populations, males had larger brains than females, and the degree of sexual size dimorphism (SSD) in relative brain size ranged from 5.1 to 11.6% across the populations. Evidence was also found for genetically based differences in relative brain size among populations, as well as for plasticity in the size of different brain parts, as evidenced by consistent size differences among replicate blocks that differed in their temperature.

In heterogametic species, biological differences between the two sexes are ubiquitous, and hence, errors in sex identification can be a significant source of noise and bias in studies where sex-related sources of variation are of interest or need to be controlled for. We developed and validated a universal multimarker assay for reliable sex identification of three-spined sticklebacks (Gasterosteus aculeatus). The assay makes use of genotype scores from three sex-linked loci and utilizes Bayesian probabilistic inference to identify sex of the genotyped individuals. The results, validated with 286 phenotypically sexed individuals from six populations of sticklebacks representing all major genetic lineages (cf. Pacific, Atlantic and Japan Sea), indicate that in contrast to commonly used single-marker-based sex identification assays, the developed multimarker assay should be 100% accurate. As the markers in the assay can be scored from agarose gels, it provides a quick and cost-efficient tool for universal sex identification of three-spined sticklebacks. The general principle of combining information from multiple markers to improve the reliability of sex identification is transferable and can be utilized to develop and validate similar assays for other species.

Snapshot analyses have demonstrated dramatic intraspecific variation in the degree of brain sexual size dimorphism (SSD). Although brain SSD is believed to be generated by the sex-specific cognitive demands of reproduction, the relative roles of developmental and populationspecific contributions to variation in brain SSD remain little studied. Using a common garden experiment, we tested for sex-specific changes in brain anatomy over the breeding cycle in three-spined stickleback (Gasterosteus aculeatus) sampled from four locations in northern Europe. We found that the male brain increased in size (ca. 24%) significantly more than the female brain towards breeding, and that the resulting brain SSD was similar (ca. 20%) for all populations over the breeding cycle. Our findings support the notion that the stickleback brain is highly plastic and changes over the breeding cycle, especially in males, likely as an adaptive response to the cognitive demands of reproduction (e.g. nest construction and parental care). The results also provide evidence to suggest that breeding-related changes in brain size may be the reason for the widely varying estimates of brain SSD across studies of this species, cautioning against interpreting brain size measurements from a single time point as fixed/static.

Freshwater communities and especially pond‐breeding amphibians are extremely vulnerable to land‐use change, alien species introductions and the use of pesticides or other toxic chemicals, as reflected in their worldwide decline. Effective conservation and management of ponds requires a better understanding of the biotic and abiotic factors that shape diversity patterns and species distribution, especially in cases of habitat loss The present study aimed to reveal and classify which pond and landscape characteristics are the most important determinants for the occurrence patterns of amphibian species as well as for the overall amphibian species richness in an urban pond network. To achieve this aim, 17 biotic and abiotic variables were measured for 61 ponds and the dataset was analysed by means of a machine‐learning approach, suitability indices and co‐occurrence modelling The highest probability of Triturus macedonicus occurrence was found at fishless artificial and semi‐natural ponds. The persistence of Lissotriton graecus in ponds was predicted by high pond connectivity and the absence of fish reflecting the species dispersive potential. Pelophylax kurtmuelleri showed a higher probability of occurrence with increased pond connectivity and macrophyte cover. Amphibian species richness was higher in fishless ponds with well‐oxygenated waters located in sites with a low to intermediate road density network. Ponds categorized as ‘average’ in terms of newt suitability were more frequently inhabited by fish than Triturus macedonicus. Alien fish species showed negative associations with both newt species, while both newt species showed a positive association between them. The results support the view that conserving a greater number of water bodies with increased landscape connectivity and without any fish presence could provide amphibian species with alternative habitat choices, especially in sites with human pressure where pond stability is at stake owing to continuous landscape changes.