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Background: The diversity and phylogeny of whitefish of the genus Coregonus is complex, and includes many endemic species of high conservation concern. However, because of commercial importance of whitefish fisheries, stockings and translocations have occurred repeatedly, which challenges the identification of local populations as conservation units. This study analyses the phylogenetic relationships of 15 contemporary and two historical populations of lake-resident and anadromous whitefish (Coregonus spp.) from the southern Baltic and North Sea basins. We elucidated the complex history of Lake Schaal (northern Germany) whitefish, for which a local tale suggests that the devil threw whitefish from the Central European Lake Constance into this lake. Studies from the early twentieth century indeed suggested numerous stocking events for Lake Schaal from Lake Constance, from Estonian/Russian Lake Peipsi and from the anadromous whitefish of the Baltic Sea., Results: Analyses of 13 microsatellite markers showed that Lake Constance whitefish are unrelated to any northern Germany whitefish population, including the contemporary whitefish population from Lake Schaal. Comparison with four historical specimens further showed that the native Lake Schaal whitefish (C. holsatus) vanished from the lake, but has survived as a non-native population in the north German Lake Drewitz. The whitefish currently occurring in Lake Schaal and three adjacent lakes are identified as C. maraenoides, introduced from Lake Peipsi. The contemporary anadromous whitefish populations from the Baltic (German and Finnish coast) and the German River Treene (North Sea basin, stocked from Danish River Vida) grouped together, but showed significant genetic differentiation. The 14 historical specimens of C. oxyrinchus from Rivers Rhine and Schelde were assigned to several contemporary whitefish populations, but among them only one specimen was assigned to the contemporary River Treene population. Therefore, we do not support the view that the whitefish from River Vida/Treene are identical with the historical C. oxyrinchus., Conclusions: Our study demonstrates that lake and anadromous whitefish in the Baltic and North Sea basins reflect a complex phylogeography, which is further blurred by the effects of repeated stocking and translocations. To identify conservation units, the genetic identity of each population has to be scrutinized.

Background: It is well established that symbionts have considerable impact on their host, yet the investigation of the possible role of the holobiont in the host's speciation process is still in its infancy. In this study, we compared the intestinal microbiota among five sympatric pairs of dwarf (limnetic) and normal (benthic) lake whitefish Coregonus clupeaformis representing a continuum in the early stage of ecological speciation. We sequenced the 16s rRNA gene V3-V4 regions of the intestinal microbiota present in a total of 108 wild sympatric dwarf and normal whitefish as well as the water bacterial community from five lakes to (i) test for differences between the whitefish intestinal microbiota and the water bacterial community and (ii) test for parallelism in the intestinal microbiota of dwarf and normal whitefish., Results: The water bacterial community was distinct from the intestinal microbiota, indicating that intestinal microbiota did not reflect the environment, but rather the intrinsic properties of the host microbiota. Our results revealed a strong influence of the host (dwarf or normal) on the intestinal microbiota with pronounced conservation of the core intestinal microbiota (mean ~ 44% of shared genera). However, no clear evidence for parallelism was observed, whereby non-parallel differences between dwarf and normal whitefish were observed in three of the lakes while similar taxonomic composition was observed for the two other species pairs., Conclusions: This absence of parallelism across dwarf vs. normal whitefish microbiota highlighted the complexity of the holobiont and suggests that the direction of selection could be different between the host and its microbiota.

Background: The detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Therefore, accurate diagnostic procedures are a must. Real-time PCR has been a mainstay in diagnostics over the years due to its speed, specificity, sensitivity, reproducibility and throughput; as such, real-time PCR is a target for improvement. Nevertheless, to validate a novel diagnostic tool, correct setup of the assay, including proper endogenous controls to evaluate the quantity and quality of the samples and to detect possible sample degradation, is compulsory. This work aims to design a unique RT-qPCR assay for pathogen detection in the three salmonid species reared in Chile. The assay uses elongation factor 1 alpha as the single endogenous control, thus avoiding the need for multiple endogenous controls, as well as multiple validations and non-comparable quality control parameters., Results: The in vivo and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch showed that when primers were accurately selected to target conserved regions of the elongation factor 1 alpha (ELF1α) gene, a single novel RT-qPCR assay yielding similar and reproducible Ct values between the three species could be designed. The opposite occurred when an assay originally designed for Salmo salar was tested in samples from the two species of the genus Oncorhynchus., Conclusions: Here, we report the design and evaluation of an accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha (ELF1α) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile.

Background: Sympatric species pairs are particularly common in freshwater fishes associated with postglacial lakes in northern temperate environments. The nature of divergences between co-occurring sympatric species, factors contributing to reproductive isolation and modes of genome evolution is a much debated topic in evolutionary biology addressed by various experimental tools. To the best of our knowledge, nobody approached this field using molecular cytogenetics. We examined chromosomes and genomes of one postglacial species pair, sympatric European winter-spawning Coregonus albula and the local endemic dwarf-sized spring-spawning C. fontanae, both originating in Lake Stechlin. We have employed molecular cytogenetic tools to identify the genomic differences between the two species of the sympatric pair on the sub-chromosomal level of resolution., Results: Fluorescence in situ hybridization (FISH) experiments consistently revealed a distinct variation in the copy number of loci of the major ribosomal DNA (the 45S unit) between C. albula and C. fontanae genomes. In C. fontanae, up to 40 chromosomes were identified to bear a part of the major ribosomal DNA, while in C. albula only 8-10 chromosomes possessed these genes. To determine mechanisms how such extensive genome alternation might have arisen, a PCR screening for retrotransposons from genomic DNA of both species was performed. The amplified retrotransposon Rex1 was used as a probe for FISH mapping onto chromosomes of both species. These experiments showed a clear co-localization of the ribosomal DNA and the retrotransposon Rex1 in a pericentromeric region of one or two acrocentric chromosomes in both species., Conclusion: We demonstrated genomic consequences of a rapid ecological speciation on the level undetectable by neither sequence nor karyotype analysis. We provide indirect evidence that ribosomal DNA probably utilized the spreading mechanism of retrotransposons subsequently affecting recombination rates in both genomes, thus, leading to a rapid genome divergence. We attribute these extensive genome re-arrangements associated with speciation event to stress-induced retrotransposons (re)activation. Such causal interplay between genome differentiation, retrotransposons (re)activation and environmental conditions may become a topic to be explored in a broader genomic context in future evolutionary studies.

Background: Most fishes possess two paralogs for myostatin, a muscle growth inhibitor, while salmonids are presumed to have four: mstn1a, mstn1b, mstn2a and mstn2b, a pseudogene. The mechanisms responsible for preserving these duplicates as well as the depth of mstn2b nonfunctionalization within the family remain unknown. We therefore characterized several genomic clones in order to better define species and gene phylogenies., Results: Gene organization and sequence conservation was particularly evident among paralog groupings and within salmonid subfamilies. All mstn2b sequences included in-frame stop codons, confirming its nonfunctionalization across taxa, although the indels and polymorphisms responsible often differed. For example, the specific indels within the Onchorhynchus tshawytscha and O. nerka genes were remarkably similar and differed equally from other mstn2b orthologs. A phylogenetic analysis weakly established a mstn2b clade including only these species, which coupled with a shared 51 base pair deletion might suggest a history involving hybridization or a shared phylogenetic history. Furthermore, mstn2 introns all lacked conserved splice site motifs, suggesting that the tissue-specific processing of mstn2a transcripts, but not those of mstn2b, is due to alternative cis regulation and is likely a common feature in salmonids. It also suggests that limited transcript processing may have contributed to mstn2b nonfunctionalization., Conclusions: Previous studies revealed divergence within gene promoters while the current studies provide evidence for relaxed or positive selection in some coding sequence lineages. These results together suggest that the salmonid myostatin gene family is a novel resource for investigating mechanisms that regulate duplicate gene fate as paralog specific differences in gene expression, transcript processing and protein structure are all suggestive of active divergence.

Background: The structure and diversity of grayling (Thymallus thymallus) populations have been well studied in most of its native habitat; however the southernmost populations of the Balkan Peninsula remain largely unexplored. The purpose of this study was to assess the genetic diversity of Serbian grayling populations, detect the impact of stocking and provide guidelines for conservation and management., Methods: Eighty grayling individuals were collected from four rivers (Ibar, Lim, Drina and Rzav). The mitochondrial DNA control region (CR; 595 bp of the 3'end and 74 bp of flanking tRNA) and the ATP6 gene (630 bp fragment) were sequenced for 20 individuals (five from each locality). In addition, all individuals were genotyped with 12 microsatellite loci. The diversity and structure of the populations as well as the recent and ancient population declines were studied using specialized software., Results: We detected three new haplotypes in the mtDNA CR and four haplotypes in the ATP6 gene of which three had not been described before. Previously, one CR haplotype and two ATP6 gene haplotypes had been identified as allochthonous, originating from Slovenia. Reconstruction of phylogenetic relations placed the remaining two CR haplotypes from the River Danube drainage of Serbia into a new clade, which is related to the previously described sister Slovenian clade. These two clades form a new Balkan clade. Microsatellite marker analysis showed that all four populations are genetically distinct from each other without any sign of intra-population structure, although stocking of the most diverse population (Drina River) was confirmed by mtDNA analysis. Recent and historical population declines of Serbian grayling do not differ from those of other European populations., Conclusions: Our study shows that (1) the Ibar, Lim and Drina Rivers grayling populations are genetically distinct from populations outside of Serbia and thus should be managed as native populations in spite of some introgression in the Drina River population and (2) the Rzav River population is not appropriate for further stocking activities since it originates from stocked Slovenian grayling. However, the Rzav River population does not represent an immediate danger to other populations because it is physically isolated from these.

Background: Teleost fishes of the Coregonidae are good model systems for studying postglacial evolution, adaptive radiation and ecological speciation. Of particular interest is whether the repeated occurrence of sympatric species pairs results from in-situ divergence from a single lineage or from multiple invasions of one or more different lineages. Here, we analysed the genetic structure of Baltic ciscoes (Coregonus albula complex), examining 271 individuals from 8 lakes in northern Germany using 1244 polymorphic AFLP loci. Six lakes had only one population of C. albula while the remaining two lakes had C. albula as well as a sympatric species (C. lucinensis or C. fontanae)., Results: AFLP demonstrated a significant population structure (Bayesian thetaB = 0.22). Lower differentiation between allopatric (thetaB = 0.028) than sympatric (0.063-0.083) populations contradicts the hypothesis of a sympatric origin of taxa, and there was little evidence for stocking or ongoing hybridization. Genome scans found only three loci that appeared to be under selection in both sympatric population pairs, suggesting a low probability of similar mechanisms of ecological segregation. However, removal of all non-neutral loci decreased the genetic distance between sympatric pairs, suggesting recent adaptive divergence at a few loci. Sympatric pairs in the two lakes were genetically distinct from the six other C. albula populations, suggesting introgression from another lineage may have influenced these two lakes. This was supported by an analysis of isolation-by-distance, where the drift-gene flow equilibrium observed among allopatric populations was disrupted when the sympatric pairs were included., Conclusions: While the population genetic data alone can not unambiguously uncover the mode of speciation, our data indicate that multiple lineages may be responsible for the complex patterns typically observed in Coregonus. Relative differences within and among lakes raises the possibility that multiple lineages may be present in northern Germany, thus understanding the postglacial evolution and speciation in the C. albula complex requires a large-scale phylogenetic analysis of several potential founder lineages.

Background: With a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon (Salmo salar) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering approximately 1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library., Results: An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with approximately 30x coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (approximately 0.09x coverage) were incorporated. The addition of paired end sequencing reads (additional approximately 26x coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (approximately 10.5x coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly., Conclusion: These results represent the first use of GS FLX paired end reads for de novo sequence assembly. Our data demonstrated that this improved the GS FLX assemblies; however, with respect to de novo sequencing of complex genomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently, for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sanger technology.

Background: Adaptive radiation within fishes of the Coregonus lavaretus complex has created numerous morphs, posing significant challenges for taxonomy and conservation priorities. The highly endangered North Sea houting (C. oxyrhynchus; abbreviated NSH) has been considered a separate species from European lake whitefish (C. lavaretus; abbreviated ELW) due to morphological divergence and adaptation to oceanic salinities. However, its evolutionary and taxonomic status is controversial. We analysed microsatellite DNA polymorphism in nine populations from the Jutland Peninsula and the Baltic Sea, representing NSH (three populations, two of which are reintroduced) and ELW (six populations). The objectives were to: 1) analyse postglacial recolonization of whitefish in the region; 2) assess the evolutionary distinctiveness of NSH, and 3) apply several approaches for defining conservation units towards setting conservation priorities for NSH., Results: Bayesian cluster analyses of genetic differentiation identified four major groups, corresponding to NSH and three groups of ELW (Western Jutland, Central Jutland, Baltic Sea). Estimates of historical migration rates indicated recolonization in a north-eastern direction, suggesting that all except the Baltic Sea population predominantly represent postglacial recolonization via the ancient Elbe River. Contemporary gene flow has not occurred between NSH and ELW, with a divergence time within the last 4,000 years suggested from coalescence methods. NSH showed interbreeding with ELW when brought into contact by stocking. Thus, reproductive isolation of NSH was not absolute, although possible interbreeding beyond the F1 level could not be resolved., Conclusion: Fishes of the C. lavaretus complex in the Jutland Peninsula originate from the same recolonization event. NSH has evolved recently and its species status may be questioned due to incomplete reproductive isolation from ELW, but it was shown to merit consideration as an independent conservation unit. Yet, application of several approaches for defining conservation units generated mixed outcomes regarding its conservation priority. Within the total species complex, it remains one among many recently evolved unique forms. Its uniqueness and high conservation priority is more evident at a local geographical scale, where conservation efforts will also benefit populations of a number of other endangered species.

Background: Several genome duplications have occurred in the evolutionary history of teleost fish. In returning to a stable diploid state, the polyploid genome reorganized, and large portions are lost, while the fish lines evolved to numerous species. Large scale transposon movement has been postulated to play an important role in the genome reorganization process. We analyzed the DNA sequence of several large loci in Salmo salar and other species for the presence of DNA transposon families., Results: We have identified bursts of activity of 14 families of DNA transposons (12 Tc1-like and 2 piggyBac-like families, including 11 novel ones) in genome sequences of Salmo salar. Several of these families have similar sequences in a number of closely and distantly related fish, lamprey, and frog species as well as in the parasite Schistosoma japonicum. Analysis of sequence similarities between copies within the families of these bursts demonstrates several waves of transposition activities coinciding with salmonid species divergence. Tc1-like families show a master gene-like copying process, illustrated by extensive but short burst of copying activity, while the piggyBac-like families show a more random copying pattern. Recent families may include copies with an open reading frame for an active transposase enzyme., Conclusion: We have identified defined bursts of transposon activity that make use of master-slave and random mechanisms. The bursts occur well after hypothesized polyploidy events and coincide with speciation events. Parasite-mediated lateral transfer of transposons are implicated.