The protection, preservation and restoration of aquatic ecosystems and their functions are of global importance. For European states it became legally binding mainly through the EU-Water Framework Directive (WFD). In order to assess the ecological status of a given water body, aquatic biodiversity data are obtained and compared to a reference water body. The quantified mismatch obtained determines the extent of potential management actions. The current approach to biodiversity assessment is based on morpho-taxonomy. This approach has many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to the varying individual taxonomic expertise of the analysts. Novel genomic tools can overcome many of the aforementioned problems and could complement or even replace traditional bioassessment. Yet, a plethora of approaches are independently developed in different institutions, thereby hampering any concerted routine application. The goal of this Action is to nucleate a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel ecogenomic indices for routine application in biodiversity assessments of European fresh-and marine water bodies. Furthermore, DNAqua-Net will provide a platform for training of the next generation of European researchers preparing them for the new technologies. Jointly with water managers, politicians, and other stakeholders, the group will develop a

BackgroundUnderstanding the molecular basis of craniofacial variation can provide insights into key developmental mechanisms of adaptive changes and their role in trophic divergence and speciation. Arctic charr (Salvelinus alpinus) is a polymorphic fish species, and, in Lake Thingvallavatn in Iceland, four sympatric morphs have evolved distinct craniofacial structures. We conducted a gene expression study on candidates from a conserved gene coexpression network, focusing on the development of craniofacial elements in embryos of two contrasting Arctic charr morphotypes (benthic and limnetic).ResultsFour Arctic charr morphs were studied: one limnetic and two benthic morphs from Lake Thingvallavatn and a limnetic reference aquaculture morph. The presence of morphological differences at developmental stages before the onset of feeding was verified by morphometric analysis. Following up on our previous findings that Mmp2 and Sparc were differentially expressed between morphotypes, we identified a network of genes with conserved coexpression across diverse vertebrate species. A comparative expression study of candidates from this network in developing heads of the four Arctic charr morphs verified the coexpression relationship of these genes and revealed distinct transcriptional dynamics strongly correlated with contrasting craniofacial morphologies (benthic versus limnetic). A literature review and Gene Ontology analysis indicated that a significant proportion of the network genes play a role in extracellular matrix organization and skeletogenesis, and motif enrichment analysis of conserved noncoding regions of network candidates predicted a handful of transcription factors, including Ap1 and Ets2, as potential regulators of the gene network. The expression of Ets2 itself was also found to associate with network gene expression. Genes linked to glucocorticoid signalling were also studied, as both Mmp2 and Sparc are responsive to this pathway. Among those, several transcriptional targets and upstream regulators showed differential expression between the contrasting morphotypes. Interestingly, although selected network genes showed overlapping expression patterns in situ and no morph differences, Timp2 expression patterns differed between morphs.ConclusionOur comparative study of transcriptional dynamics in divergent craniofacial morphologies of Arctic charr revealed a conserved network of coexpressed genes sharing functional roles in structural morphogenesis. We also implicate transcriptional regulators of the network as targets for future functional studies.Electronic supplementary materialThe online version of this article (doi:10.1186/2041-9139-5-40) contains supplementary material, which is available to authorized users.

A major goal of evolutionary science is to understand how biological diversity is generated and altered. Despite considerable advances, we still have limited insight into how phenotypic variation arises and is sorted by natural selection. Here we argue that an integrated view, which merges ecology, evolution and developmental biology (eco evo devo) on an equal footing, is needed to understand the multifaceted role of the environment in simultaneously determining the development of the phenotype and the nature of the selective environment, and how organisms in turn affect the environment through eco evo and eco devo feedbacks. To illustrate the usefulness of an integrated eco evo devo perspective, we connect it with the theory of resource polymorphism (i.e. the phenotypic and genetic diversification that occurs in response to variation in available resources). In so doing, we highlight fishes from recently glaciated freshwater systems as exceptionally well‐suited model systems for testing predictions of an eco evo devo framework in studies of diversification. Studies on these fishes show that intraspecific diversity can evolve rapidly, and that this process is jointly facilitated by (i) the availability of diverse environments promoting divergent natural selection; (ii) dynamic developmental processes sensitive to environmental and genetic signals; and (iii) eco evo and eco devo feedbacks influencing the selective and developmental environments of the phenotype. We highlight empirical examples and present a conceptual model for the generation of resource polymorphism – emphasizing eco evo devo, and identify current gaps in knowledge.

Arctic charr (Salvelinus alpinus) is a highly polymorphic species and in Lake Thingvallavatn, Iceland, four phenotypic morphs have evolved. These differences in morphology, especially in craniofacial structures are already apparent during embryonic development, indicating that genes important in the formation of the craniofacial features are expressed differentially between the morphs. In order to generate tools to examine these expression differences in Arctic charr, the aim of the present study was to identify reference genes for quantitative real-time PCR (qPCR). The specific aim was to select reference genes which are able to detect very small expression differences among different morphs. We selected twelve candidate reference genes from the literature, identified corresponding charr sequences using data derived from transcriptome sequencing (RNA-seq) and examined their expression using qPCR. Many of the candidate reference genes were found to be stably expressed, yet their quality-rank as reference genes varied considerably depending on the type of analysis used. In addition to commonly used software for reference gene validation, we used classical statistics to evaluate expression profiles avoiding a bias for reference genes with similar expression patterns (co-regulation). Based on these analyses we chose three reference genes, ACTB, UB2L3 and IF5A1 for further evaluation. Their consistency was assessed in an expression study of three known craniofacially expressed genes, sparc (or osteonectin), matrix metalloprotease 2 (mmp2) and sox9 (sex-determining region Y box 9 protein) using qPCR in embryo heads derived from four charr groups at three developmental time points. The three reference genes were found to be very suitable for studying expression differences between the morphotypes, enabling robust detection of small relative expression changes during charr development. Further, the results showed that sparc and mmp2 are differentially expressed in embryos of different Arctic charr morphotypes.

Northern freshwater fish may be suitable for the genetic dissection of ecological traits because they invaded new habitats after the last ice age (∼10.000 years ago). Arctic charr (Salvelinus alpinus) colonizing streams and lakes in Iceland gave rise to multiple populations of small benthic morphotypes, often in sympatry with a pelagic morphotype. Earlier studies have revealed significant, but subtle, genetic differentiation between the three most common morphs in Lake Thingvallavatn. We conducted a population genetic screen on four immunological candidate genes Cathelicidin 2 (Cath2), Hepcidin (Hamp), Liver expressed antimicrobial peptide 2a (Leap-2a), and Major Histocompatibility Complex IIα (MHCIIα) and a mitochondrial marker (D-loop) among the three most common Lake Thingvallavatn charr morphs. Significant differences in allele frequencies were found between morphs at the Cath2 and MHCIIα loci. No such signal was detected in the D-loop nor in the other two immunological genes. In Cath2 the small benthic morph deviated from the other two (FST = 0.13), one of the substitutions detected constituting an amino acid replacement polymorphism in the antimicrobial peptide. A more striking difference was found in the MHCIIα. Two haplotypes were very common in the lake, and their frequency differed greatly between the morphotypes (from 22% to 93.5%, FST = 0.67). We then expanded our study by surveying the variation in Cath2 and MHCIIα in 9 Arctic charr populations from around Iceland. The populations varied greatly in terms of allele frequencies at Cath2, but the variation did not correlate with morphotype. At the MHCIIα locus, the variation was nearly identical to the variation in the two benthic morphs of Lake Thingvallavatn. The results are consistent with a scenario where parts of the immune systems have diverged substantially among Arctic charr populations in Iceland, after colonizing the island ∼10.000 years ago.