Chromosomal Fusions Facilitate Adaptation to Divergent Environments in Threespine Stickleback.

Chromosomal fusions are hypothesized to facilitate adaptation to divergent environments, both by bringing together previously unlinked adaptive alleles and by creating regions of low recombination that facilitate the linkage of adaptive alleles; but, there is little empirical evidence to support this hypothesis. Here, we address this knowledge gap by studying threespine stickleback (Gasterosteus aculeatus), in which ancestral marine fish have repeatedly adapted to freshwater across the northern hemisphere. By comparing the threespine and ninespine stickleback (Pungitius pungitius) genomes to a de novo assembly of the fourspine stickleback (Apeltes quadracus) and an outgroup species, we find two chromosomal fusion events involving the same chromosomes have occurred independently in the threespine and ninespine stickleback lineages. On the fused chromosomes in threespine stickleback, we find an enrichment of quantitative trait loci underlying traits that contribute to marine versus freshwater adaptation. By comparing whole-genome sequences of freshwater and marine threespine stickleback populations, we also find an enrichment of regions under divergent selection on these two fused chromosomes. There is elevated genetic diversity within regions under selection in the freshwater population, consistent with a simulation study showing that gene flow can increase diversity in genomic regions associated with local adaptation and our demographic models showing gene flow between the marine and freshwater populations. Integrating our results with previous studies, we propose that these fusions created regions of low recombination that enabled the formation of adaptative clusters, thereby facilitating freshwater adaptation in the face of recurrent gene flow between marine and freshwater threespine sticklebacks.

Population transcriptomics reveals weak parallel genetic basis in repeated marine and freshwater divergence in nine-spined sticklebacks.

The degree to which adaptation to similar selection pressures is underlain by parallel versus non-parallel genetic changes is a topic of broad interest in contemporary evolutionary biology. Sticklebacks provide opportunities to characterize and compare the genetic underpinnings of repeated marine-freshwater divergences at both intra- and interspecific levels. While the degree of genetic parallelism in repeated marine-freshwater divergences has been frequently studied in the three-spined stickleback (Gasterosteus aculeatus), much less is known about this in other stickleback species. Using a population transcriptomic approach, we identified both genetic and gene expression variations associated with marine-freshwater divergence in the nine-spined stickleback (Pungitius pungitius). Specifically, we used a genome-wide association study approach, and found that ~1% of the total 173,491 identified SNPs showed marine-freshwater ecotypic differentiation. A total of 861 genes were identified to have SNPs associated with marine-freshwater divergence, but only 12 of these genes have also been reported as candidates associated with marine-freshwater divergence in the three-spined stickleback. Hence, our results indicate a low degree of interspecific genetic parallelism in marine-freshwater divergence. Moreover, 1,578 genes in the brain and 1,050 genes in the liver were differentially expressed between marine and freshwater nine-spined sticklebacks, ~5% of which have also been identified as candidates associated with marine-freshwater divergence in the three-spined stickleback. However, only few of these (e.g., CLDND1) appear to have been involved in repeated marine-freshwater divergence in nine-spined sticklebacks. Taken together, the results indicate a low degree of genetic parallelism in repeated marine-freshwater divergence both at intra- and interspecific levels.

A phylogenomic perspective on diversity, hybridization and evolutionary affinities in the stickleback genus Pungitius.

Hybridization and convergent evolution are phenomena of broad interest in evolutionary biology, but their occurrence poses challenges for reconstructing evolutionary affinities among affected taxa. Sticklebacks in the genus Pungitius are a case in point: evolutionary relationships and taxonomic validity of different species and populations in this circumpolarly distributed species complex remain contentious due to convergent evolution of traits regarded as diagnostic in their taxonomy, and possibly also due to frequent hybridization among taxa. To clarify the evolutionary relationships among different Pungitius species and populations globally, as well as to study the prevalence and extent of introgression among recognized species, genomic data sets of both reference genome-anchored single nucleotide polymorphisms and de novo assembled RAD-tag loci were constructed with RAD-seq data. Both data sets yielded topologically identical and well-supported species trees. Incongruence between nuclear and mitochondrial DNA-based trees was found and suggested possibly frequent hybridization and mitogenome capture during the evolution of Pungitius sticklebacks. Further analyses revealed evidence for frequent nuclear genetic introgression among Pungitius species, although the estimated proportions of autosomal introgression were low. Apart from providing evidence for frequent hybridization, the results challenge earlier mitochondrial and morphology-based hypotheses regarding the number of species and their affinities in this genus: at least seven extant species can be recognized on the basis of genetic data. The results also shed new light on the biogeographical history of the Pungitius-complex, including suggestion of several trans-Arctic invasions of Europe from the Northern Pacific. The well-resolved phylogeny should facilitate the utility of this genus as a model system for future comparative evolutionary studies.

Dorsal spine evolution in threespine sticklebacks via a splicing change in MSX2A.

BACKGROUND: Dorsal spine reduction in threespine sticklebacks (Gasterosteus aculeatus) is a classic example of recurrent skeletal evolution in nature. Sticklebacks in marine environments typically have long spines that form part of their skeletal armor. Many derived freshwater populations have evolved shorter spines. Changes in spine length are controlled in part by a quantitative trait locus (QTL) previously mapped to chromosome 4, but the causative gene and mutations underlying the repeated evolution of this interesting skeletal trait have not been identified. RESULTS: Refined mapping of the spine length QTL shows that it lies near the MSX2A transcription factor gene. MSX2A is expressed in developing spines. In F1 marine × freshwater fish, the marine allele is preferentially expressed. Differences in expression can be attributed to splicing regulation. Due to the use of an alternative 5 ' splice site within the first exon, the freshwater allele produces greater amounts of a shortened, non-functional transcript and makes less of the full-length transcript. Sequence changes in the MSX2A region are shared by many freshwater fish, suggesting that repeated evolution occurs by reuse of a spine-reduction variant. To demonstrate the effect of full-length MSX2A on spine length, we produced transgenic freshwater fish expressing a copy of marine MSX2A. The spines of the transgenic fish were significantly longer on average than those of their non-transgenic siblings, partially reversing the reduced spine lengths that have evolved in freshwater populations. CONCLUSIONS: MSX2A is a major gene underlying dorsal spine reduction in freshwater sticklebacks. The gene is linked to a separate gene controlling bony plate loss, helping explain the concerted effects of chromosome 4 on multiple armor-reduction traits. The nature of the molecular changes provides an interesting example of morphological evolution occurring not through a simple amino acid change, nor through a change only in gene expression levels, but through a change in the ratio of splice products encoding both normal and truncated proteins.

Tooth microwear formation rate in Gasterosteus aculeatus.

Tooth microwear feature densities were significantly increased in a population of laboratory-reared three-spined stickleback Gasterosteus aculeatus in four days, after they were transferred from a limnetic feeding regime to a benthic feeding regime. These results show that even in aquatic vertebrates with non-occluding teeth, changes in feeding can cause changes in tooth microwear in just a few days, as in mammals.

Strontium isotope analysis of otoliths reveals differences in the habitat salinity among three sympatric stickleback species of the genus Pungitius.

The analysis of otolith Sr isotope ratios (87Sr/86Sr) is a powerful method to study fish migration in freshwater areas. However, few studies have applied this method to study fish movement in brackish-water environments. Furthermore, despite the fact that habitat differentiation has been shown to drive genetic differentiation and reproductive isolation among stickleback fish, no studies have used the otolith 87Sr/86Sr ratios to analyze habitat differentiation between stickleback ecotypes and species. In this study, we analyzed the otolith 87Sr/86Sr ratios of three sympatric stickleback species of the genus Pungitius in the Shiomi River on Hokkaido Island, Japan: P. tymensis, the brackish-water type of the P. pungitius-P. sinensis complex, and the freshwater type of the P. pungitius-P. sinensis complex. First, we created a mixing equation to depict the relationship between habitat salinity and the 87Sr/86Sr ratios of river water. We found that the otolith 87Sr/86Sr ratios differed significantly among the three species, indicating that the three species utilize habitats with different salinities: P. tymensis and the brackish-water type inhabit freshwater and brackish-water environments, respectively, with the freshwater type using intermediate habitats. In addition, we found that some freshwater individuals moved to habitats with higher salinities as they grew. Our study demonstrates that the analysis of otolith 87Sr/86Sr ratios is a useful method for studying the habitat use of fish in brackish-water environments and habitat differentiation among closely related sympatric and parapatric species.

Complete mitochondrial genome of the Greek nine-spined stickleback Pungitius hellenicus (Gasterosteiformes, Gasterosteidae).

The complete mitochondrial genome of the Greek nine-spined stickleback Pungitius hellenicus was obtained using Illumina high-throughput sequencing of genomic DNA. The genome was 16 713 bp long, and contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region. The arrangement of the genes was identical to that of other Gasterosteidae fishes. However, the control region of P. hellenicus contained three copies of imperfect repeated sequences (72-78 bp in single motifs), while P. pungitius and P. tymensis have one or two copies. Nucleotide identity between P. hellenicus and three other Pungitius species across all the 37 genic regions was 93.0% to 95.5%.

Complete mitochondrial genome of the Sakhalin nine-spined stickleback Pungitius tymensis (Gasterosteiformes, Gasterosteidae).

The complete mitochondrial genome of the Sakhalin nine-spined stickleback Pungitius tymensis was determined using Illumina paired-end sequencing of genomic DNA. The genome sequence was 16 481 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a control region. The content and arrangement of the genes were identical to those of other Gasterosteidae species. P. tymensis was phylogenetically positioned with other Pungitius species (P. kaibarae, P. pungitius and P. sinensis) with a clear distinction from them. Nucleotide identity in the 37 genic regions ranged from 94.7% to 94.9% between P. tymensis and the other Pungitius species.

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.

Complete mitochondrial genome of the Ukrainian nine-spined stickleback Pungitius platygaster (Gasterosteiformes, Gasterosteidae).

The complete mitochondrial genome of the Ukrainian nine-spined stickleback Pungitius platygaster was obtained using massive parallel sequencing of genomic DNA. The mitogenome sequence was 16 570 bp long, and the gene order and contents were identical to those of other sequenced Pungitius mitogenomes. In a phylogenetic analysis, the mitogenome of P. platygaster clustered with other Pungitius mitogenomes, yet being clearly distinct from those of P. pungitius, P. sinensis and P. kaibarae.

Complete mitochondrial genomes of the smooth tail nine-spined sticklebacks Pungitius laevis (Gasterosteiformes, Gasterosteidae).

The complete mitochondrial genome sequences of three divergent lineages of the smooth tail nine-spined stickleback (Pungitius laevis) were obtained with massive parallel sequencing of their genomic DNA. The genome sequences were 16 574-16 580 bp long, and the gene order and contents were identical to those of other sequenced Pungitius mitogenomes. Although the mitogenome sequences of all three P. laevis lineages clustered within the genus Pungitius, they were clearly distinct and showed divergence comparable to that seen between some Pungitius species.

Complete mitochondrial genome of the nine-spined stickleback Pungitius pungitius (Gasterosteiformes, Gasterosteidae).

The complete mitochondrial genome of the nine-spined stickleback Pungitius pungitius was obtained with Illumina sequencing of genomic DNA. The assembled mitogenome sequence was 16 582 bp long, and the gene number, order and contents were identical to those of other sequenced Pungitius mitogenomes. The complete mitogenome of P. pungitius from its European range can provide an important template for further phylogenetic and population genetic studies of the Pungitius species complex.

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.

INTEGRATING PHYLOGENY AND EXPERIMENTAL ETHOLOGY: FROM PATTERN TO PROCEs.

Phylogenetic systematics offers ethologists a method for uncovering macroevolutionary patterns of behavioral diversity, which can be used to generate predictions for experimental investigation of microevolutionary processes. I used phylogenetic methods to disentangle the contributions of various selection pressures in the evolution of sexually dimorphic nuptial coloration in gasterosteid fishes in general, and Gasterosteus aculeatus in particular. Based upon the phylogenetic relationships between breeding colors and breeding behaviors in the gasterosteids, three predictions relevant to a discussion of nuptial coloration in G. aculeatus were proposed. The origin and elaboration of nuptial coloration was (1) weakly associated with male/male interactions (intrasexual selection), (2) moderately associated with parental care (natural selection) and (3) strongly associated with male/female interactions (intersexual selection). I tested the hypothesized macroevolutionary relationships between male breeding color and behavior in two sets of experiments studying changes in male nuptial coloration across the breeding cycle and female choice based on intensity of male color. The results of these experiments at the microevolutionary level of analysis corroborated the macroevolutionary predictions and confirmed the original intuition of previous researchers: male body color intensity is an important component of female mate choice in G. aculeatus. This study demonstrates that a phylogenetic hypothesis can generate predictions that are experimentally testable and falsifiable.