Intraspecific divergence in the lateral line system in the nine-spined stickleback (Pungitius pungitius)

The mechanosensory lateral line system of fishes is an important organ system conveying information crucial to individual fitness. Yet, our knowledge of lateral line diversity is almost exclusively based on interspecific studies, whereas intraspecific variability and possible population divergence have remained largely unexplored. We investigated lateral line system variability in four marine and five pond populations of nine-spined stickleback (Pungitius pungitius). We found significant differences in neuromast number between pond and marine fish. In particular, three of seventeen lateral line regions (viz. caudal peduncle superficial neuromasts; canal neuromasts from the anterior trunk and caudal peduncle) showed strong divergence between habitats. Similar results were obtained with laboratory-reared individuals from a subset of populations, suggesting that the patterns found in nature likely have a genetic basis. Interestingly, we also found habitat-dependent population divergence in neuromast variability, with pond populations showing greater heterogeneity than marine populations, although only in wild-caught fish. A comparison of neutral genetic (F(ST)) and phenotypic (P(ST)) differentiation suggested that natural selection is likely associated with habitat-dependent divergence in neuromast counts. Hence, the results align with the conclusion that the mechanosensory lateral line system divergence among marine and pond nine-spined sticklebacks is adaptive.     

Parasites of the nine-spined stickleback Pungitius pungitius (L)

Eleven species of parasite are reported from the nine-spined stickleback Pungitius pungitius (L). Nine of these, namely, Trichodina domerguei, T. tenuidens, Vorticella sp., Epistylis?, Diplostomum spathaceum, Cryptocotyle lingua, Proteocephalus filicollis, Schistocephalus solidus and glochidia of Anodonta cygnea, are new host records for Great Britain. Gyrodactylus spp. and Thersitina gasterostei were also found.     

Predation mediated population divergence in complex behaviour of nine‐spined stickleback (Pungitius pungitius)

The proximate and ultimate explanations for behavioural syndromes (correlated behaviours – a population trait) are poorly understood, and the evolution of behavioural types (configuration of behaviours – an individual trait) has been rarely studied. We investigated population divergence in behavioural syndromes and types using individually reared, completely predator‐ or conspecific‐naïve adult nine‐spined sticklebacks (Pungitius pungitius) from two marine and two predatory fish free, isolated pond populations. We found little evidence for the existence of behavioural syndromes, but population divergence in behavioural types was profound: individuals from ponds were quicker in feeding, bolder and more aggressive than individuals from marine environments. Our data reject the hypothesis that behavioural syndromes exist as a result of genetic correlations between behavioural traits, and support the contention that different behavioural types can be predominant in populations differing in predation pressure, most probably as a result of repeated independent evolution of separate behavioural traits.     

Quantitative genetics of body size and timing of maturation in two nine-spined stickleback (Pungitius pungitius) populations

Due to its influence on body size, timing of maturation is an important life-history trait in ectotherms with indeterminate growth. Comparison of patterns of growth and maturation within and between two populations (giant vs. normal sized) of nine-spined sticklebacks (Pungitius pungitius) in a breeding experiment revealed that the difference in mean adult body size between the populations is caused by differences in timing of maturation, and not by differential growth rates. The fish in small-sized population matured earlier than those from large-sized population, and maturation was accompanied by a reduction in growth rate in the small-sized population. Males matured earlier and at smaller size than females, and the fish that were immature at the end of the experiment were larger than those that had already matured. Throughout the experimental period, body size in both populations was heritable (h² = 0.10–0.64), as was the timing of maturation in the small-sized population (h² = 0.13–0.16). There was a significant positive genetic correlation between body size and timing of maturation at 140 DAH, but not earlier (at 80 or 110 DAH). Comparison of observed body size divergence between the populations revealed that Q _ST exceeded F _ST at older ages, indicating adaptive basis for the observed divergence. Hence, the results suggest that the body size differences within and between populations reflect heritable genetic differences in the timing of maturation, and that the observed body size divergence is adaptive.     

Heteromorphic sex chromosomes in the ninespine stickleback Pungitius pungitius

Specimens from two freshwater populations of the ninespine stickleback Pungitius pungitius in Poland showed morphologically differentiated sex chromosomes. A heteromorphic pair of chromosomes appeared only in male diploid cells. The Y chromosome was the largest chromosome in the P. pungitius karyotype.     

History vs. habitat type: explaining the genetic structure of European nine‐spined stickleback (Pungitius pungitius) populations

The genetic structure of contemporary populations can be shaped by both their history and current ecological conditions. We assessed the relative importance of postglacial colonization history and habitat type in the patterns and degree of genetic diversity and differentiation in northern European nine‐spined sticklebacks (Pungitius pungitius), using mitochondrial DNA (mtDNA) sequences and 12 nuclear microsatellite and insertion/deletion loci. The mtDNA analyses identified – and microsatellite analyses supported – the existence of two historically distinct lineages (eastern and western). The analyses of nuclear loci among 51 European sites revealed clear historically influenced and to minor degree habitat dependent, patterns of genetic diversity and differentiation. While the effect of habitat type on the levels of genetic variation (coastal > freshwater) and differentiation (freshwater > coastal) was clear, the levels of genetic variability and differentiation in the freshwater sites were independent of habitat type (viz. river, lake and pond). However, levels of genetic variability, together with estimates of historical effective population sizes, decreased dramatically and linearly with increasing latitude. These geographical patterns of genetic variability and differentiation suggest that the contemporary genetic structure of freshwater nine‐spined sticklebacks has been strongly impacted by the founder events associated with postglacial colonization and less by current ecological conditions (cf. habitat type). In general, the results highlight the strong and persistent effects of postglacial colonization history on genetic structuring of northern European fauna and provide an unparalleled example of latitudinal trends in levels of genetic diversity.     

Comparative study of the olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius)

Summary The olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius) has been studied with a conventional histochemical and a novel immunological staining technique. In both species, the sensory epithelium is arranged in folds separated by non-sensory epithelial tissue. In the nine-spined stickleback, intrinsic folds consisting of non-sensory cells are found in the apical part of the sensory epithelium where they divide the surface of the sensory epithelium into small islets. These non-sensory cells are non-ciliated, flattened and piled on top of each other; they contain numerous electron-translucent vesicles. The intrinsic folds are absent from the sensory epithelium of the three-spined stickleback. In both species, axons of receptor cells form a layer of fibers in the sensory epithelium immediately above the basal cells. In the three-spined stickleback, thick branches of the olfactory nerve are frequently found in this layer. These branches are only occasionally observed in the sensory epithelium of the nine-spined stickleback. Thus, the three-spined stickleback and the nine-spined stickleback show considerable differences in the organization of the sensory regions of the olfactory epithelium.     

Habitat-dependent and -independent plastic responses to social environment in the nine-spined stickleback (Pungitius pungitius) brain

The influence of environmental complexity on brain development has been demonstrated in a number of taxa, but the potential influence of social environment on neural architecture remains largely unexplored. We investigated experimentally the influence of social environment on the development of different brain parts in geographically and genetically isolated and ecologically divergent populations of nine-spined sticklebacks (Pungitius pungitius). Fish from two marine and two pond populations were reared in the laboratory from eggs to adulthood either individually or in groups. Group-reared pond fish developed relatively smaller brains than those reared individually, but no such difference was found in marine fish. Group-reared fish from both pond and marine populations developed larger tecta optica and smaller bulbi olfactorii than individually reared fish. The fact that the social environment effect on brain size differed between marine and pond origin fish is in agreement with the previous research, showing that pond fish pay a high developmental cost from grouping while marine fish do not. Our results demonstrate that social environment has strong effects on the development of the stickleback brain, and on the brain''s sensory neural centres in particular. The potential adaptive significance of the observed brain-size plasticity is discussed.     

Baiting improves CPUE in nine‐spined stickleback (Pungitius pungitius) minnow trap fishery

Whether or not baiting influences stickleback catch per unit effort (CPUE) remains a matter of debate among stickleback researchers: While the opinions about the impact of baiting on CPUE differ, supporting quantitative data are scarce. The effect of baiting and trap type on nine‐spined stickleback (Pungitius pungitius) CPUE was studied in a field experiment conducted over four consecutive days in a small pond in northeastern Finland. The results show that baited traps yielded better (mean CPUE = 1.24 fish/trap/d) catches than unbaited traps (mean CPUE = 0.66); however, there were also differences in CPUE depending on the type of collapsible trap that was used. The trap type effect on CPUE seemed to differ among age classes – the finer meshed trap caught more young‐of‐the‐year fish than the coarse‐meshed one, whereas the opposite was true for the older and larger individuals. The results agree with those of an earlier more restricted study conducted in the same locality: Together, these results provide strong evidence for the positive impact of baiting on nine‐spined stickleback CPUE.     

Morphological divergence of North‐European nine‐spined sticklebacks (Pungitius pungitius): signatures of parallel evolution

Parallel evolution is characterised by repeated, independent occurrences of similar phenotypes in a given habitat type, in different parts of the species distribution area. We studied body shape and body armour divergence between five marine, four lake, and ten pond populations of nine‐spined sticklebacks [Pungitius pungitius (Linnaeus, 1758)] in Fennoscandia. We hypothesized that marine and lake populations (large water bodies, diverse fish fauna) would be similar, whereas sticklebacks in isolated ponds (small water bodies, simple fish fauna) would be divergent. We found that pond fish had deeper bodies, shorter caudal peduncles, and less body armour (viz. shorter/absent pelvic spines, reduced/absent pelvic girdle, and reduced number of lateral plates) than marine fish. Lake fish were intermediate, but more similar to marine than to pond fish. Results of our common garden experiment concurred with these patterns, suggesting a genetic basis for the observed divergence. We also found large variation among populations within habitat types, indicating that environmental variables other than those related to gross habitat characteristics might also influence nine‐spined stickleback morphology. Apart from suggesting parallel evolution of morphological characteristics of nine‐spined sticklebacks in different habitats, the results also show a number of similarities to the evolution of three‐spined stickleback (Gasterosteus aculeatus Linnaeus, 1758) morphology. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 403–416.     

Ontogenetic and evolutionary effects of predation and competition on nine-spined stickleback (Pungitius pungitius) body size

1.?Individual- and population-level variation in body size and growth often correlates with many fitness traits. Predation and food availability are expected to affect body size and growth as important agents of both natural selection and phenotypic plasticity. How differences in predation and food availability affect body size/growth during ontogeny in populations adapted to different predation and competition regimes is rarely studied. 2.?Nine-spined stickleback (Pungitius pungitius) populations originating from habitats with varying levels of predation and competition are known to be locally adapted to their respective habitats in terms of body size and growth. Here, we studied how different levels of perceived predation risk and competition during ontogeny affect the reaction norms of body size and growth in (i) marine and pond populations adapted to different levels of predation and competition and (ii) different sexes. We reared nine-spined stickleback in a factorial experiment under two levels of perceived predation risk (present/absent) and competition (high/low food supply). 3.?We found divergence in the reaction norms at two levels: (i) predation-adapted marine stickleback had stronger reactions to predatory cues than intraspecific competition-adapted pond stickleback, the latter being more sensitive to available food than the marine fish and (ii) females reacting more strongly to the treatments than males. 4.?The repeated, habitat-dependent nature of the differences suggests that natural selection is the agent behind the observed patterns. Our results suggest that genetic adaptation to certain environmental factors also involves an increase in the range of expressible phenotypic plasticity. We found support for this phenomenon at two levels: (i) across populations driven by habitat type and (ii) within populations driven by sex.     

Lactate dehydrogenase isozymes and allozymes of the nine-spined stickleback Pungitius pungitius (L.) (Osteichthyes: Gasterosteidae)

Zymograms indicate the existence of three loci coding for lactate dehydrogenase in the nine-spined stickleback Pungitius pungitius. The skeletal muscle locus is polymorphic for two codominant alleles: Ldh-A(100). and Ldh-A(140). Differences in allele frequencies among nine samples, obtained from eight geographically isolated populations, proved not significant in 28/36 pairwise comparisons. The frequency of Ldh-A(100) varied from 0.426 to 0.715. Significant differences in apparent Km (pyruvate) values were found among the LDH-A allozymes suggesting a functional basis for the selective maintenance of this polymorphism.     

Heterozygosity–behaviour correlations in nine‐spined stickleback (Pungitius pungitius) populations: contrasting effects at random and functional loci

The study of heterozygosity‐fitness correlations (HFCs) has a long history in the fields of ecology and evolutionary biology but remains controversial. Recently, it has been shown that the genetic distance of markers from functional loci can be an important factor to be considered in addition to marker numbers and variability. In this study, we investigated the correlation between individual heterozygosity and behaviour (aggression, boldness and feeding activity) in nine‐spined stickleback (Pungitius pungitius) individuals originating from four populations in two contrasting environments. Offspring of full‐sib families raised in a common garden setting were assessed for behaviour and genotyped using 84 microsatellite markers that were either located within or near behaviourally or physiologically important genes (termed ‘functional’) or were randomly selected. No associations were detected with any behavioural trait in any population or over all populations when genetic variability was measured using all 84 markers combined. However, when the markers were separated into three functional categories (behavioural, physiological and random), several significant associations were observed both with functional markers and random markers in one of the four populations assessed. Interestingly, contrasting correlations with behaviour were observed when using physiological gene (negative) and random (positive) markers. Upon dividing the physiological gene markers into further subcategories based on their specific physiological functions, a strong relationship between the heterozygosity of markers linked to osmoregulation‐related genes, and behaviour was revealed in the brackish water population. Our results indicate that both local (physiological) and general (neutral) effects are important in shaping behaviour and that heterozygosity–behaviour correlations are population dependent.     

Adaptive brain size divergence in nine‐spined sticklebacks (Pungitius pungitius)?

Most studies seeking to provide evolutionary explanations for brain size variability have relied on interspecific comparisons, while intraspecific studies utilizing ecologically divergent populations to this effect are rare. We investigated the brain size and structure of first‐generation laboratory‐bred nine‐spined sticklebacks (Pungitius pungitius) from four geographically and genetically isolated populations originating from markedly different habitats. We found that the relative size of bulbus olfactorius and telencephalon was significantly larger in marine than in pond populations. Significant, but habitat‐independent population differences were also found in relative brain and cerebellum sizes. The consistent, habitat‐specific differences in the relative size of bulbus olfactorius and telencephalon suggest their adaptive reduction in response to reduced (biotic and abiotic) habitat complexity in pond environments. In general, the results suggest that genetically based brain size and structure differences can evolve relatively rapidly and in repeatable fashion with respect to habitat structure.     

Rapid genetic divergence in postglacial populations of threespine stickleback (Gasterosteus aculeatus): the role of habitat type, drainage and geographical proximity

The goal of this study was to evaluate the role of common ancestry, and of geographical or reproductive isolation, in genetic divergence in populations of threespine sticklebacks (Gasterosteus aculeatus). Using seven DNA microsatellite loci we compared the effects of habitat type, drainage system and geographical proximity on genetic distance among 16 populations situated in an area in Schleswig-Holstein (Germany) that became deglaciated approximately 12 000 years ago. Stickleback population structure correlated only weakly with drainage system, whereas the primary divergence was among habitat types. Phylogenetic analysis revealed that lake (n = 7) and river (n = 5) populations formed two distinct clades (Cavalli-Sforza's and Edwards' chord distance, 82-100% bootstrap support) at approximately equal genetic distances to a third clade, comprising putative estuarine (n = 4) ancestors. Allele frequencies in lake and river populations represented different subsets of the genetically more diverse estuarine populations. In nested amovas approximately twice the genetic variance was distributed among lake vs. river vs. estuarine populations as compared with the combined effects of drainage system and geographical distance. Limited gene flow between habitat types must have been established after postglacial colonization, suggesting ecological hybrid inferiority or behavioural mating barriers between ecotypes. Within estuarine and lake populations, population differentiation followed an isolation-by-distance model. Given the high observed heterozygosities within the 16 study populations (HO = 0.65-0.87), the mean divergence between lake and river population pairs (FST = 0.18 +/- 0.007) would be reached after 300-6000 generations in a stepwise mutation model, depending on the size of N(e). This demonstrates both the utility of hypervariable microsatellites for detecting recent population divergences and the danger of operating at temporal or spatial scales which are beyond their resolution.     

Phylogeography and genetic structuring of European nine-spined sticklebacks (Pungitius pungitius)-mitochondrial DNA evidence

As a consequence of colonisation from different glacial refugia, many northern European taxa are split into distinct western and eastern lineages. However, as for the nine-spined stickleback (Pungitius pungitius), the exact location of the contact zone between lineages often remains poorly known. We assessed the genetic differentiation and diversity in the nine-spined stickleback within Europe using 1037 base pairs of cytochrome b sequence for 320 individuals from 57 locations, including pond, lake, river, and coastal habitats. Our main aims were (i) to locate the contact zone between the previously recognized western and eastern lineages, (ii) investigate latitudinal patterns in genetic diversity, (iii) compare genetic diversity among different habitat types, and (iv) date the known split between eastern and western lineages. The data revealed the split between eastern and western to be located across the Danish Straits and roughly following the Norway/Sweden border to the North. Reference sites from Canada form their own clades, and one of the Canadian sites was found to have a haplotype common to the Eastern European lineage, possibly representing an ancestral polymorphism. The split between the two European clades was dated to approximately 1.48 million years ago (Mya), and between Canada and Europe to approximately 1.62 Mya. After controlling for habitat effects, nucleotide (but not haplotype) diversity across populations decreased with increasing latitude. Coastal populations showed significantly higher haplotype diversity (but not nucleotide diversity) than pond populations, but there were no detectable differences in haplotype diversity among different freshwater habitat types (viz. river, lake and pond populations), or between coastal and lake/river populations. Sequences were found to cluster according to their geographic proximity, rather than by habitat type, and all habitat types were found within each major clade, implying that colonisation and adaptation between the coastal and freshwater environments in different regions must have occurred in parallel.     

Hybrid male sterility between the fresh- and brackish-water types of ninespine stickleback Pungitius pungitius (Pisces, Gasterosteidae)

Two ecologically distinct forms, fresh- and brackish-water types, of ninespine stickleback co-exist in several freshwater systems on the coast of eastern Hokkaido. Recent genetic analyses of 13 allozyme loci revealed genetic separation between the two types even though their spawning grounds were in close proximity. On the other hand, there is only a small difference in mitochondrial DNA (mtDNA) sequence between the two types suggesting that they diverged quite recently or that mtDNA introgression occurred between them. To test for postzygotic reproductive isolating mechanisms and hybrid mediated gene flow, we examined the viability and reproductive performance of reciprocal F1 hybrids. The hybrids grew to the adult size normally and both sexes expressed secondary sexual characters in the reciprocal crosses. The female hybrids were reciprocally fertile, while the male hybrids were reciprocally sterile. Histological and flow-cytometric analyses of the hybrid testis revealed that the sterility pattern was classified as 'gametic sterility,' with gonads of normal size but abnormal spermatogenesis. To our knowledge, the present finding is a novel example of one sex hybrid sterility in the stickleback family (Gasterosteidae).     

Quantitative trait loci for growth and body size in the nine‐spined stickleback Pungitius pungitius L.

Body size is an ecologically important trait shown to be genetically variable both within and among different animal populations as revealed by quantitative genetic studies. However, few studies have looked into underlying genetic architecture of body size variability in the wild using genetic mapping methods. With the aid of quantitative trait loci (QTL) analyses based on 226 microsatellite markers, we mapped body size and growth rate traits in the nine‐spined stickleback (Pungitius pungitius) using an F₂‐intercross (n = 283 offspring) between size‐divergent populations. In total, 17 QTL locations were detected. The proportion of phenotypic variation explained by individual body size‐related QTL ranged from 3% to 12% and those related to growth parameters and increments from 3% to 10%. Several of the detected QTL affected either early or late growth. These results provide a solid starting point for more in depth investigations of structure and function of genomic regions involved in determination of body size in this popular model of ecological and evolutionary research.     

Fine scale genetic population structure of the freshwater and Omono types of nine-spined stickleback Pungitius pungitius (L.) within the Omono River system, Japan

The fine scale geographic population structure of two types of nine-spined stickleback Pungitius pungitius (the widely distributed freshwater type and a local endemic, the Omono type) within the Omono River system, Japan, was investigated. A principal components analysis of allele frequencies and neighbour-joining tree for pair-wise F _ST values, based on 10 allozyme loci, revealed that the Omono type was comprised of four regional groups with relatively high genetic divergence. This grouping was also supported by hierarchical analysis of molecular variance (AMOVA) with a higher variance component among the regional groups, and by an exact test with significant genotypic differentiation for all sample pairs among the regional groups. Moreover, in the clustering of individuals using the Bayesian method, most of individuals in each regional group were assigned the corresponding cluster. On the other hand, there were less pronounced regional groups of the freshwater type, although AMOVA, exact test for genotypic differentiation and Bayesian analysis indicated genetic divergence between two sampling sites in lower reach of the Omono River and other sites. The results suggest that the Omono type represented an earlier colonization, with subsequent invasion of the freshwater type.