Identification of novel microRNA genes in freshwater and marine ecotypes of the three‐spined stickleback (Gasterosteus aculeatus)

The three‐spined stickleback (Gasterosteus aculeatus L.) is an important model organism for studying the molecular mechanisms of speciation and adaptation to salinity. Despite increased interest to microRNA discovery and recent publication on microRNA prediction in the three‐spined stickleback using bioinformatics approaches, there is still a lack of experimental support for these data. In this paper, high‐throughput sequencing technology was applied to identify microRNA genes in gills of the three‐spined stickleback. In total, 595 miRNA genes were discovered; half of them were predicted in previous computational studies and were confirmed here as microRNAs expressed in gill tissue. Moreover, 298 novel microRNA genes were identified. The presence of miRNA genes in selected ‘divergence islands’ was analysed and 10 miRNA genes were identified as not randomly located in ‘divergence islands’. Regulatory regions of miRNA genes were found enriched with selective SNPs that may play a role in freshwater adaptation.     

Computational identification of miRNAs,their targets and functions in three‐spined stickleback (Gasterosteus aculeatus)

An intriguing question in biology is how the evolution of gene regulation is shaped by natural selection in natural populations. Among the many known regulatory mechanisms, regulation of gene expression by microRNAs (miRNAs) is of critical importance. However, our understanding of their evolution in natural populations is limited. Studying the role of miRNAs in three‐spined stickleback, an important natural model for speciation research, may provide new insights into adaptive polymorphisms. However, lack of annotation of miRNA genes in its genome is a bottleneck. To fill this research gap, we used the genome of three‐spined stickleback to predict miRNAs and their targets. We predicted 1486 mature miRNAs using the homology‐based miRNA prediction approach. We then performed functional annotation and enrichment analysis of these targets, which identified over‐represented motifs. Further, a database resource (GAmiRdb) has been developed for dynamically searching miRNAs and their targets exclusively in three‐spined stickleback. Finally, the database was used in two case studies focusing on freshwater adaptation in natural populations. In the first study, we found 44 genomic regions overlapping with predicted miRNA targets. In the second study, we identified two SNPs altering the MRE seed site of sperm‐specific glyceraldehyde‐3‐phosphate gene. These findings highlight the importance of the GAmiRdb knowledge base in understanding adaptive evolution.     

The role of calcium and predation on plate morph evolution in the three‐spined stickleback (Gasterosteus aculeatus)

While the genetic basis to plate morph evolution of the three‐spined stickleback (Gasterosteus aculeatus) is well described, the environmental variables that select for different plate and spine morphs are incompletely understood. Using replicate populations of three‐spined sticklebacks on North Uist, Scotland, we previously investigated the role of predation pressure and calcium limitation on the adaptive evolution of stickleback morphology and behavior. While dissolved calcium proved a significant predictor of plate and spine morph, predator abundance did not. Ecol. Evol., xxx, 2014 and xxx performed a comparable analysis to our own to address the same question. They failed to detect a significant effect of dissolved calcium on morphological evolution, but did establish a significant effect of predation; albeit in the opposite direction to their prediction.     

A test for within‐lake niche differentiation in the nine‐spined sticklebacks (Pungitius pungitius)

Specialization for the use of different resources can lead to ecological speciation. Accordingly, there are numerous examples of ecologically specialized pairs of fish “species” in postglacial lakes. Using a polymorphic panel of single nucleotide variants, we tested for genetic footprints of within‐lake population stratification in nine‐spined sticklebacks (Pungitius pungitius) collected from three habitats (viz. littoral, benthic, and pelagic) within a northern Swedish lake. Analyses of admixture, population structure, and relatedness all supported the conclusion that the fish from this lake form a single interbreeding unit.     

Local adaptation through genetic differentiation in highly fragmented Tilia cordata populations

We assessed the level of geographic differentiation of Tilia cordata in Denmark based on tests of 91 trees selected from 12 isolated populations. We used quantitative analysis of spring phenology and population genetic analysis based on SSR markers to infer the likely historical genetic processes within and among populations. High genetic variation within and among populations was observed in spring phenology, which correlated with spring temperatures at the origin of the tested T. cordata trees. The population genetic analysis revealed significant differentiation among the populations, but with no clear sign of isolation by distance. We infer the findings as indications of ongoing fine scale selection in favor of local growth conditions made possible by limited gene flow among the small and fragmented populations. This hypothesis fits well with reports of limited fruiting in the investigated Danish T. cordata populations, while the species is known for its ability to propagate vegetatively by root suckers. Our results suggest that both divergent selection and genetic drift may have played important roles in forming the genetic patterns of T. cordata at its northern distribution limit. However, we also speculate that epigenetic mechanism arising from the original population environment could have created similar patterns in regulating the spring phenology.     

A universal and reliable assay for molecular sex identification of three‐spined sticklebacks (Gasterosteus aculeatus)

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.     

High genetic diversity of the endangered Iberian three‐spined stickleback (Gasterosteus aculeatus) at the Mediterranean edge of its range

1. The three‐spined stickleback (Gasterosteus aculeatus) on the Iberian Peninsula is only distributed in freshwater habitats and has completely disappeared from most of its range, mainly as a consequence of habitat degradation and invasive fish introductions. Genetic investigations have shown that Mediterranean‐Adriatic sticklebacks constitute an evolutionarily significant unit. Here, we present the first genetic data for Iberian populations living on the southern edge of the stickleback’s range. We used microsatellite markers to study gene diversity, population structure and genetic demography of stickleback populations. 2. High genetic differentiation among collections yielded a model of four genetically homogeneous units related to geography. The observed pattern of isolation by distance resulted mainly from the hydrographical pattern and limited gene flow among rivers. Moreover, low levels of gene diversity, high isolation and recent bottleneck events, which have led to small or even critical effective population sizes in several locations, could be explained by additional recent anthropogenic fragmentation. 3. We defined at least four evolutionarily significant units threatened by habitat fragmentation in north‐eastern Iberian sticklebacks. Because they retain long evolutionary histories, these populations should be considered of high conservation priority, and urgent management measures should be implemented.     

StAMPP: an R package for calculation of genetic differentiation and structure of mixed‐ploidy level populations

Statistical Analysis of Mixed‐Ploidy Populations (StAMPP) is a freely available R package for calculation of population structure and differentiation based on single nucleotide polymorphism (SNP) genotype data from populations of any ploidy level, and/or mixed‐ploidy levels. StAMPP provides an advance on previous similar software packages, due to an ability to calculate pairwise F_ST values along with confidence intervals, Nei's genetic distance and genomic relationship matrixes from data sets of mixed‐ploidy level. The software code is designed to efficiently handle analysis of large genotypic data sets that are typically generated by high‐throughput genotyping platforms. Population differentiation studies using StAMPP are broadly applicable to studies of molecular ecology and conservation genetics, as well as animal and plant breeding.     

Global patterns of population genetic differentiation in seed plants

Evaluating the factors that drive patterns of population differentiation in plants is critical for understanding several biological processes such as local adaptation and incipient speciation. Previous studies have given conflicting results regarding the significance of pollination mode, seed dispersal mode, mating system, growth form and latitudinal region in shaping patterns of genetic structure, as estimated by F_ST values, and no study to date has tested their relative importance together across a broad scale. Here, we assembled a 337‐species data set for seed plants from publications with data on F_ST from nuclear markers and species traits, including variables pertaining to the sampling scheme of each study. We used species traits, while accounting for sampling variables, to perform phylogenetic multiple regressions. Results demonstrated that F_ST values were higher for tropical, mixed‐mating, non‐woody species pollinated by small insects, indicating greater population differentiation, and lower for temperate, outcrossing trees pollinated by wind. Among the factors we tested, latitudinal region explained the largest portion of variance, followed by pollination mode, mating system and growth form, while seed dispersal mode did not significantly relate to F_ST. Our analyses provide the most robust and comprehensive evaluation to date of the main ecological factors predicted to drive population differentiation in seed plants, with important implications for understanding the basis of their genetic divergence. Our study supports previous findings showing greater population differentiation in tropical regions and is the first that we are aware of to robustly demonstrate greater population differentiation in species pollinated by small insects.     

Inbreeding in three‐spined sticklebacks (Gasterosteus aculeatus L.): effects on testis and sperm traits

Mating between relatives often results in inbreeding depression, and is assumed to have a strong effect on fitness traits such as fertility and gonad/gamete quality. However, data concerning this topic are contradictory and particularly scarce in fishes. Three‐spined sticklebacks (Gasterosteus aculeatus L.) show inbreeding depression in fertilization and hatching success, survival rates, body symmetry and behavioural traits. To date, any knowledge of the impact of inbreeding on males' gonads and gametes is lacking in this species. In the present study, testis and sperm traits were quantified in outbred and inbred males. Overall, these traits were not generally impaired by inbreeding, and this result was not changed by a second/third generation of brother–sister matings. However, testes brightness, a potential measure of oxidative stress, was negatively correlated with sperm number. Additionally, inbred males with higher body condition had significantly brighter testes, whereas their sperm number was significantly negatively correlated with sperm quality (as estimated by head volume). Such a trade‐off did not appear in outbred males. The comparatively small impact of inbreeding on testis and sperm traits might be explained by the low number of inbred individuals that reached the reproductive phase. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 510–520.     

Genomic differentiation and patterns of gene flow between two long‐tailed tit species (Aegithalos)

Patterns of heterogeneous genomic differentiation have been well documented between closely related species, with some highly differentiated genomic regions (“genomic differentiation islands”) spread throughout the genome. Differential levels of gene flow are proposed to account for this pattern, as genomic differentiation islands are suggested to be resistant to gene flow. Recent studies have also suggested that genomic differentiation islands could be explained by linked selection acting on genomic regions with low recombination rates. Here, we investigate genomic differentiation and gene‐flow patterns for autosomes using RAD‐seq data between two closely related species of long‐tailed tits (Aegithalos bonvaloti and A. fuliginosus) in both allopatric and contact zone populations. The results confirm recent or ongoing gene flow between these two species. However, there is little evidence that the genomic regions that were found to be highly differentiated between the contact zone populations are resistant to gene flow, suggesting that differential levels of gene flow is not the cause of the heterogeneous genomic differentiation. Linked selection may be the cause of genomic differentiation islands between the allopatric populations with no or very limited gene flow, but this could not account for the heterogeneous genomic differentiation between the contact zone populations, which show evidence of recent or ongoing gene flow.     

Predator‐inspection behaviour in female three‐spined sticklebacks Gasterosteus aculeatus is associated with status of gravidity

Differences in predator‐inspection behaviour between gravid and non‐gravid female as well as between male and female three‐spined sticklebacks Gasterosteus aculeatus were investigated. Gravid females confronted with a live rainbow trout Oncorhynchus mykiss showed bolder inspection behaviour than non‐gravid ones. The behaviour of gravid females was comparable with that of males, maybe because both face a high risk of predation. The results indicate that antipredator behaviour in female G. aculeatus is not fixed but adjusted to their reproductive state.     

Population responses to anthropogenic disturbance: lessons from three‐spined sticklebacks Gasterosteus aculeatus in eutrophic habitats

Human‐induced environmental changes differ from most natural changes in which they happen at a faster rate and require quicker responses from populations. The first response of populations is usually phenotypically plastic alterations of morphology, physiology and behaviour. This plasticity can be favourable and move the population closer to an adaptive peak in the altered environment and, hence, maintain a viable population, or be maladaptive and move the population further from the peak and increase the risk of extinction. The radiation of the three‐spined stickleback Gasterosteus aculeatus from the ocean to different freshwater habitats has provided much information on adaptation to new environmental conditions. Currently, human‐induced eutrophication is changing the breeding areas of these fish, which creates a model system for investigation of responses to rapid environmental disturbance. Results show that a primary reaction is plastic alterations of behaviour, with some adjustments being adaptive while others are not. At the same time, the strength of sexual selection on several traits is relaxed, which could increase the relative importance of survival selection. Whether this will restore population viability depends on the amount of standing genetic variation in the right direction. Human disturbances can be dramatic and resolution of the limit of flexibility and the possibility of genetic adaptation should be important targets of future research.     

Non‐parallel divergence across freshwater and marine three‐spined stickleback Gasterosteus aculeatus populations

This work investigated whether multiple freshwater populations of three‐spined stickleback Gasterosteus aculeatus in different freshwater catchments in the Jutland Peninsula, Denmark, derived from the same marine populations show repeated adaptive responses. A total of 327 G. aculeatus collected at 13 sampling locations were screened for genetic variation using a combination of 70 genes putatively under selection and 26 neutral genes along with a marker linked to the ectodysplasin gene (eda), which is strongly correlated with plate armour morphs in the species. A highly significant genetic differentiation was found that was higher among different freshwater samples than between marine–freshwater samples. Tests for selection between marine and freshwater populations showed a very low degree of parallelism and no single nucleotide polymorphism was detected as outlier in all freshwater–marine pairwise comparisons, including the eda. This suggests that G. aculeatus is not necessarily the prime example of parallel local adaptation suggested in much of the literature and that important exceptions exist (i.e. the Jutland Peninsula). While marine populations in the results described here showed a high phenotype–genotype correlation at eda, a low association was found for most of the freshwater populations. The most extreme case was found in the freshwater Lake Hald where all low‐plated phenotypes were either homozygotes for the allele supposed to be associated with completely plated morphs or heterozygotes, but none were homozygotes for the putative low‐plated allele. Re‐examination of data from seven G. aculeatus studies agrees in showing a high but partial association between phenotype–genotype at eda in G. aculeatus freshwater populations and that mismatches occur everywhere in the European regions studied (higher in some areas, i.e. Denmark). This is independent of the eda marker used.     

Evaluation of a non‐invasive tagging system for laboratory studies using three‐spined sticklebacks Gasterosteus aculeatus

A non‐invasive tagging system for individual identification of three‐spined sticklebacks Gasterosteus aculeatus was evaluated. The tags were easily detected via video, and tagged and non‐tagged fish did not differ in terms of growth, activity levels or shoaling behaviour.     

Exploring Jordan's rule in Pacific three‐spined stickleback Gasterosteus aculeatus

Coastal marine Gasterosteus aculeatus were captured from seven locations along the Pacific coast of North America, ranging across 21·8° latitude to test Jordan's rule, i.e. that vertebral number should increase with increasing latitude for related populations of fish. Vertebral number significantly increased with increasing latitude for both total and caudal vertebral number. Increasing length with latitude (sensu Bergmann's rule) was also supported, but the predictions for Jordan's rule held when controlling for standard length. Pleomerism was weakly evidenced. Gasterosteus aculeatus exhibited sexual dimorphism for Jordan's rule, with both sexes having more vertebrae at higher latitudes, but only males showing a positive association between latitude and the ratio of caudal to abdominal vertebrae. The number of dorsal‐ and anal‐fin rays and basals increased with increasing latitude, while pectoral‐fin ray number decreased. This study reinforces the association between phenotypic variation and environmental variation in marine populations of G. aculeatus.     

Food intake rates of inactive fish are positively linked to boldness in three‐spined sticklebacks Gasterosteus aculeatus

To investigate the link between personality and maximum food intake of inactive individuals, food‐deprived three‐spined sticklebacks Gasterosteus aculeatus at rest in their home compartments were provided with ad libitum prey items. Bolder individuals ate considerably more than shyer individuals, even after accounting for body size, while sociability did not have an effect. These findings support pace‐of‐life theory predicting that life‐history strategies are linked to boldness.     

Sexual dimorphism of head morphology in three‐spined stickleback Gasterosteus aculeatus

This study examined sexual dimorphism of head morphology in the ecologically diverse three‐spined stickleback Gasterosteus aculeatus. Male G. aculeatus had longer heads than female G. aculeatus in all 10 anadromous, stream and lake populations examined, and head length growth rates were significantly higher in males in half of the populations sampled, indicating that differences in head size increased with body size in many populations. Despite consistently larger heads in males, there was significant variation in size‐adjusted head length among populations, suggesting that the relationship between head length and body length was flexible. Inter‐population differences in head length were correlated between sexes, thus population‐level factors influenced head length in both sexes despite the sexual dimorphism present. Head shape variation between lake and anadromous populations was greater than that between sexes. The common divergence in head shape between sexes across populations was about twice as important as the sexual dimorphism unique to each population. Finally, much of the sexual dimorphism in head length was due to divergence in the anterior region of the head, where the primary trophic structures were found. It is unclear whether the sexual dimorphism was due to natural selection for niche divergence between sexes or sexual selection. This study improves knowledge of the magnitude, growth rate divergence, inter‐population variation and location of sexual dimorphism in G. aculeatus head morphology.     

Population genetic dynamics of three-spined sticklebacks (Gasterosteus aculeatus) in anthropogenic altered habitats

In industrialized and/or agriculturally used landscapes, inhabiting species are exposed to a variety of anthropogenic changes in their environments. Genetic diversity may be reduced if populations encounter founder events, bottlenecks, or isolation. Conversely, genetic diversity may increase if populations adapt to changes in selective regimes in newly created habitats. With the present study, genetic variability of 918 sticklebacks from 43 samplings (21.3 ± 3.8 per sample) at 36 locations from cultivated landscapes in Northwest Germany was analyzed at nine neutral microsatellite loci. To test if differentiation is influenced by habitat alterations, sticklebacks were collected from ancient running waters and adjacent artificial stagnant waters, from brooks with salt water inflow of anthropogenic and natural origin and adjacent freshwater sites. Overall population structure was dominated by isolation by distance (IBD), which was significant across all populations, and analysis of molecular variance (AMOVA) revealed that 10.6% of the variation was explained by river catchment area. Populations in anthropogenic modified habitats deviated from the general IBD structure and in the AMOVA, grouping by habitat type running/stagnant water explained 4.9% of variation and 1.4% of the variation was explained by salt-/freshwater habitat. Sticklebacks in salt-polluted water systems seem to exhibit elevated migratory activity between fresh- and saltwater habitats, reducing IBD. In other situations, populations showed distinct signs of genetic isolation, which in some locations was attributed to mechanical migration barriers, but in others to potential anthropogenic induced bottleneck or founder effects. The present study shows that anthropogenic habitat alterations may have diverse effects on the population genetic structure of inhabiting species. Depending on the type of habitat change, increased genetic differentiation, diversification, or isolation are possible consequences.