Facilitating population genomics of non-model organisms through optimized experimental design for reduced representation sequencing.

BACKGROUND: Genome-wide data are invaluable to characterize differentiation and adaptation of natural populations. Reduced representation sequencing (RRS) subsamples a genome repeatedly across many individuals. However, RRS requires careful optimization and fine-tuning to deliver high marker density while being cost-efficient. The number of genomic fragments created through restriction enzyme digestion and the sequencing library setup must match to achieve sufficient sequencing coverage per locus. Here, we present a workflow based on published information and computational and experimental procedures to investigate and streamline the applicability of RRS. RESULTS: In an iterative process genome size estimates, restriction enzymes and size selection windows were tested and scaled in six classes of Antarctic animals (Ostracoda, Malacostraca, Bivalvia, Asteroidea, Actinopterygii, Aves). Achieving high marker density would be expensive in amphipods, the malacostracan target taxon, due to the large genome size. We propose alternative approaches such as mitogenome or target capture sequencing for this group. Pilot libraries were sequenced for all other target taxa. Ostracods, bivalves, sea stars, and fish showed overall good coverage and marker numbers for downstream population genomic analyses. In contrast, the bird test library produced low coverage and few polymorphic loci, likely due to degraded DNA. CONCLUSIONS: Prior testing and optimization are important to identify which groups are amenable for RRS and where alternative methods may currently offer better cost-benefit ratios. The steps outlined here are easy to follow for other non-model taxa with little genomic resources, thus stimulating efficient resource use for the many pressing research questions in molecular ecology.

Isolation and characterisation of 14 novel microsatellite markers through Next Generation Sequencing for the commercial Atlantic seabob shrimp Xiphopenaeus kroyeri.

Assessing population genetic structure is a crucial step to support fisheries and conservation management. DNA microsatellite molecular markers are a widely used tool in population genotyping. In the present study, we characterised and developed 14 novel polymorphic microsatellite markers for a decapod crustacean, the Atlantic seabob shrimp Xiphopenaeus kroyeri (Heller, 1862), through rapid and cost-effective Illumina shotgun sequencing and a Galaxy-based bioinformatic pipeline. We genotyped 60 individuals from 2 populations with the newly developed microsatellites, resulting in the detection of 3 to 29 alleles per locus. Four loci deviated from Hardy-Weinberg equilibrium. Cross-amplification in a cryptic congeneric species was successful for eight loci (57%). The microsatellite loci developed in this study will be highly relevant for genetic and evolutionary studies of X. kroyeri, and for the stock management of this commercially exploited species.

A gene with major phenotypic effects as a target for selection vs. homogenizing gene flow.

Genes with major phenotypic effects facilitate quantifying the contribution of genetic vs. plastic effects to adaptive divergence. A classical example is Ectodysplasin (Eda), the major gene controlling lateral plate phenotype in three-spined stickleback. Completely plated marine stickleback populations evolved repeatedly towards low-plated freshwater populations, representing a prime example of parallel evolution by natural selection. However, many populations remain polymorphic for lateral plate number. Possible explanations for this polymorphism include relaxation of selection, disruptive selection or a balance between divergent selection and gene flow. We investigated 15 polymorphic stickleback populations from brackish and freshwater habitats in coastal North-western Europe. At each site, we tracked changes in allele frequency at the Eda gene between subadults in fall, adults in spring and juveniles in summer. Eda genotypes were also compared for body size and reproductive investment. We observed a fitness advantage for the Eda allele for the low morph in freshwater and for the allele for the complete morph in brackish water. Despite these results, the differentiation at the Eda gene was poorly correlated with habitat characteristics. Neutral population structure was the best predictor of spatial variation in lateral plate number, suggestive of a substantial effect of gene flow. A meta-analysis revealed that the signature of selection at Eda was weak compared to similar studies in stickleback. We conclude that a balance between divergent selection and gene flow can maintain stickleback populations polymorphic for lateral plate number and that ecologically relevant genes may not always contribute much to local adaptation, even when targeted by selection.

Differential modes of MHC class IIB gene evolution in cichlid fishes.

Cichlid fishes are emblematic models for the study of adaptive radiation, driven by natural and sexual selection. Parasite mediated selection is an important component in these processes, and the evolution of their immune system therefore merits special attention. In this study, light is shed on the phylogeny of the b family of cichlid major histocompatibility complex (MHC) class IIB genes. Full-length coding sequences were used to reconstruct phylogenies using criteria of maximum parsimony, maximum likelihood and Bayesian inference. All analyses suggest monophyly of the b family of cichlid MHC class IIB genes, although sequences of the cichlid sister taxa are currently not available. Two evolutionary lineages of these genes, respectively encompassing the recently defined genomic regions DBB-DEB-DFB and DCB-DDB, show highly contrasting levels of differentiation. To explore putative causes for these differences, exon 2 sequences were screened for variation in recombination rate and strength of selection. The more diversified lineage of cichlid MHC class IIB b genes was found to have higher levels of both recombination and selection. This is consistent with the observation in other taxa that recombination facilitates the horizontal spread of positively selected sites across MHC loci and hence contributes to fast sequence evolution. In contrast, the lineage that showed low diversification might either be under stabilizing selection or is evolutionary constrained by its low recombination rate. We speculate whether this lineage might include MHC genes with non-classical functions.

Heritability of cortisol response to confinement stress in European sea bass dicentrarchus labrax.

BACKGROUND: In fish, the most studied production traits in terms of heritability are body weight or growth, stress or disease resistance, while heritability of cortisol levels, widely used as a measure of response to stress, is less studied. In this study, we have estimated heritabilities of two growth traits (body weight and length) and of cortisol response to confinement stress in the European sea bass. FINDINGS: The F1 progeny analysed (n = 922) belonged to a small effective breeding population with contributions from an unbalanced family structure of just 10 males and 2 females. Heritability values ranged from 0.54 (± 0.21) for body weight to 0.65 (± 0.22) for standard body length and were low for cortisol response i.e. 0.08 (± 0.06). Genetic correlations were positive (0.94) between standard body length and body weight and negative between cortisol and body weight and between cortisol and standard body length (-0.60 and -0.55, respectively). CONCLUSION: This study confirms that in European sea bass, heritability of growth-related traits is high and that selection on such traits has potential. However, heritability of cortisol response to stress is low in European sea bass and since it is known to vary greatly among species, further studies are necessary to understand the reasons for these differences.

Analysis of the microbial community structure in a membrane bioreactor during initial stages of filtration.

Membrane biofouling was investigated during the early stages of filtration in a laboratory-scale membrane bioreactor operated on molasses wastewater. The bacterial diversity and composition of the membrane biofilm and activated sludge were analyzed using terminal restriction fragment length polymorphism coupled with 16S rRNA clone library construction and sequencing. The amount of extracellular polymeric substances produced by bacteria was investigated using spectroscopic methods. The results reveal that the bacterial community of activated sludge differs significantly from that of the membrane biofilm, especially at the initial phase. Phylogenetic analysis based on 16S rRNA gene sequences identified 25 pioneer OTUs responsible for membrane surface colonization. Also, the relationship between the identified bacterial strains and the system specifications was explored.

Contrasting population genetic responses to migration barriers in two native and an invasive freshwater fish.

Habitat fragmentation impacts the distribution of genetic diversity and population genetic structure. Therefore, protecting the evolutionary potential of species, especially in the context of the current rate of human-induced environmental change, is an important goal. In riverine ecosystems, migration barriers affect the genetic structure of native species, while also influencing the spread of invasive species. In this study, we compare genetic patterns of two native and one highly invasive riverine fish species in a Belgian river basin, namely the native three-spined stickleback (Gasterosteus aculeatus) and stone loach (Barbatula barbatula), and the non-native and invasive topmouth gudgeon (Pseudorasbora parva). We aimed to characterize both natural and anthropogenic determinants of genetic diversity and population genetic connectivity. Genetic diversity was highest in topmouth gudgeon, followed by stone loach and three-spined stickleback. The correlation between downstream distance and genetic diversity, a pattern often observed in riverine systems, was only marginally significant in stone loach and three-spined stickleback, while genetic diversity strongly declined with increasing number of barriers in topmouth gudgeon. An Isolation-By-Distance pattern characterizes the population genetic structure of each species. Population differentiation was only associated with migration barriers in the invasive topmouth gudgeon, while genetic composition of all species seemed at least partially determined by the presence of migration barriers. Among the six barrier types considered (watermills, sluices, tunnels, weirs, riverbed obstructions, and others), the presence of watermills was the strongest driver of genetic structure and composition. Our results indicate that conservation and restoration actions, focusing on conserving genetic patterns, cannot be generalized across species. Moreover, measures might target either on restoring connectivity, while risking a rapid spread of the invasive topmouth gudgeon, or not restoring connectivity, while risking native species extinction in upstream populations.

Adaptive Divergence under Gene Flow along an Environmental Gradient in Two Coexisting Stickleback Species.

There is a general and solid theoretical framework to explain how the interplay between natural selection and gene flow affects local adaptation. Yet, to what extent coexisting closely related species evolve collectively or show distinctive evolutionary responses remains a fundamental question. To address this, we studied the population genetic structure and morphological differentiation of sympatric three-spined and nine-spined stickleback. We conducted genotyping-by-sequencing and morphological trait characterisation using 24 individuals of each species from four lowland brackish water (LBW), four lowland freshwater (LFW) and three upland freshwater (UFW) sites in Belgium and the Netherlands. This combination of sites allowed us to contrast populations from isolated but environmentally similar locations (LFW vs. UFW), isolated but environmentally heterogeneous locations (LBW vs. UFW), and well-connected but environmentally heterogenous locations (LBW vs. LFW). Overall, both species showed comparable levels of genetic diversity and neutral genetic differentiation. However, for all three spatial scales, signatures of morphological and genomic adaptive divergence were substantially stronger among populations of the three-spined stickleback than among populations of the nine-spined stickleback. Furthermore, most outlier SNPs in the two species were associated with local freshwater sites. The few outlier SNPs that were associated with the split between brackish water and freshwater populations were located on one linkage group in three-spined stickleback and two linkage groups in nine-spined stickleback. We conclude that while both species show congruent evolutionary and genomic patterns of divergent selection, both species differ in the magnitude of their response to selection regardless of the geographical and environmental context.

Association between Chromosome 4 and mercury accumulation in muscle of the three-spined stickleback (Gasterosteus aculeatus).

Anthropogenic stressors, such as pollutants, act as selective factors that can leave measurable changes in allele frequencies in the genome. Metals are of particular concern among pollutants, because of interference with vital biological pathways. We use the three-spined stickleback as a model for adaptation to mercury pollution in natural populations. We collected sticklebacks from 21 locations in Flanders (Belgium), measured the accumulated levels of mercury in the skeletal muscle tissue, and genotyped the fish by sequencing (GBS). The spread of muscle mercury content across locations was considerable, ranging from 21.5 to 327 ng/g dry weight (DW). We then conducted a genome-wide association study (GWAS) between 28,450 single nucleotide polymorphisms (SNPs) and the accumulated levels of mercury, using different approaches. Based on a linear mixed model analysis, the GWAS yielded multiple hits with a single top hit on Chromosome 4, with eight more SNPs suggestive of association. A second approach, a latent factor mixed model analysis, highlighted one single SNP on Chromosome 11. Finally, an outlier test identified one additional SNP on Chromosome 4 that appeared under selection. Out of all ten SNPs we identified as associated with mercury in muscle, three SNPs all located on Chromosome 4 and positioned within a 2.5 kb distance of an annotated gene. Based on these results and the genome coverage of our SNPs, we conclude that the selective effect of mercury pollution in Flanders causes a significant association with at least one locus on Chromosome 4 in three-spined stickleback.

Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods.

The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were determined using real-time PCR targeting the bacterial 16S rDNA gene. Microbial community fingerprints were assessed by automated ribosomal intergenic spacer analysis. The resulting community profiles were analyzed with canonical correspondence analysis. Our results clearly demonstrate that direct DNA extraction methods can significantly influence the DNA quantity, purity, and observed community patterns of microbiota in activated sludge. Fast and Mobio generated high amounts of good quality DNA compared to Bead and Qiagen. Mobio also resulted in the detection of the highest number of species while Fast scored the best in discriminating between the community patterns of different activated sludge types. With respect to the characterization of community profiles, our analyses demonstrated a strong sludge type dependent variability among methods. Taking into account our results, we recommend Fast as the most suitable DNA extraction method for activated sludge samples used for bacterial community studies.

Community structure, population structure and topographical specialisation of Gyrodactylus (monogenea) ectoparasites living on sympatric stickleback species.

In order to disentangle the contribution of host and parasite biology to host specificity, we compared the structure and population dynamics of the Gyrodactylus (von Nordmann, 1832) flatworm community living on sympatric three-spined Gasterosteus aculeatus L. and nine-spined Pungitius pungitius (L.) stickleback. Between April 2002 and March 2003, a small lowland creek was sampled monthly. Species identity of about 75% of the worms per host was determined with a genetic nuclear marker (ITS1). Each stickleback species hosted a characteristic gill- and fin-parasitic Gyrodactylus: G. arcuatus Bychowsky, 1933 and G. gasterostei Gläser, 1974 respectively infecting the three-spined stickleback, with G. rarus Wegener, 1910 and G. pungitii Malmberg, 1964 infecting the nine-spined stickleback. Host size and seasonal dynamics were strong determinants of parasite abundance. A strong interaction between host and parasite species determined infection levels and affected three levels of parasite organisation: community structure, population structure and topographical specialisation. Community and population structure were shaped by asymmetric cross-infections, resulting in a net transmission of the Gyro-dactylus species typical of the nine-spined stickleback towards the three-spined stickleback. Host density was not a major determinant of parasite exchange. Aggregation and topographical specialisation of the Gyrodactylus species of the three-spined stickleback were more pronounced than that of the nine-spined stickleback.

Adaptive and non-adaptive divergence in a common landscape.

Species in a common landscape often face similar selective environments. The capacity of organisms to adapt to these environments may be largely species specific. Quantifying shared and unique adaptive responses across species within landscapes may thus improve our understanding of landscape-moderated biodiversity patterns. Here we test to what extent populations of two coexisting and phylogenetically related fishes-three-spined and nine-spined stickleback-differ in the strength and nature of neutral and adaptive divergence along a salinity gradient. Phenotypic differentiation, neutral genetic differentiation and genomic signatures of adaptation are stronger in the three-spined stickleback. Yet, both species show substantial phenotypic parallelism. In contrast, genomic signatures of adaptation involve different genomic regions, and are thus non-parallel. The relative contribution of spatial and environmental drivers of population divergence in each species reflects different strategies for persistence in the same landscape. These results provide insight in the mechanisms underlying variation in evolutionary versatility and ecological success among species within landscapes.The three-spined stickleback is a model species for the study of adaptive divergence. Here, Raeymaekers et al. compare how the three-spined stickleback and its relative the nine-spined stickleback vary at the phenotypic and genomic levels in response to the same spatial and environmental drivers.

A microsatellite linkage map of the European sea bass Dicentrarchus labrax L.

A genetic linkage map of the European sea bass (Dicentrarchus labrax) was constructed from 174 microsatellite markers, including 145 new markers reported in this study. The mapping panel was derived from farmed sea bass from the North Adriatic Sea and consisted of a single family including both parents and 50 full-sib progeny (biparental diploids). A total of 162 microsatellites were mapped in 25 linkage groups. Eleven loci represent type I (coding) markers; 2 loci are located within the peptide Y (linkage group 1) and cytochrome P450 aromatase (linkage group 6) genes. The sex-averaged map spans 814.5 cM of the sea bass genome. The female map covers 905.9 cM, whereas the male map covers only 567.4 cM. The constructed map represents the first linkage map of European sea bass, one of the most important aquaculture species in Europe.

Hidden biodiversity in an ancient lake: phylogenetic congruence between Lake Tanganyika tropheine cichlids and their monogenean flatworm parasites.

The stunning diversity of cichlid fishes has greatly enhanced our understanding of speciation and radiation. Little is known about the evolution of cichlid parasites. Parasites are abundant components of biodiversity, whose diversity typically exceeds that of their hosts. In the first comprehensive phylogenetic parasitological analysis of a vertebrate radiation, we study monogenean parasites infecting tropheine cichlids from Lake Tanganyika. Monogeneans are flatworms usually infecting the body surface and gills of fishes. In contrast to many other parasites, they depend only on a single host species to complete their lifecycle. Our spatially comprehensive combined nuclear-mitochondrial DNA dataset of the parasites covering almost all tropheine host species (N = 18), reveals species-rich parasite assemblages and shows consistent host-specificity. Statistical comparisons of host and parasite phylogenies based on distance and topology-based tests demonstrate significant congruence and suggest that host-switching is rare. Molecular rate evaluation indicates that species of Cichlidogyrus probably diverged synchronically with the initial radiation of the tropheines. They further diversified through within-host speciation into an overlooked species radiation. The unique life history and specialisation of certain parasite groups has profound evolutionary consequences. Hence, evolutionary parasitology adds a new dimension to the study of biodiversity hotspots like Lake Tanganyika.

Microsatellite primers for the gynodioecious grassland perennial Saxifraga granulata (Saxifragaceae).

PREMISE OF THE STUDY: Nine polymorphic and 12 monomorphic microsatellite loci (simple sequence repeats [SSRs]) were isolated and characterized for the gynodioecious grassland perennial Saxifraga granulata. • METHODS AND RESULTS: Based on genomic screening of leaf material of four individuals from four populations, a total of 21 microsatellite primer pairs were designed for S. granulata. Nine loci were polymorphic and were optimized into two PCR multiplex reactions and tested on 100 individuals from five riparian populations from central Belgium. The number of alleles of the polymorphic loci ranged from three to 18, and gametic heterozygosity ranged from 0.26 to 0.94. • CONCLUSIONS: The markers that are presented here are the first microsatellite markers reported for S. granulata and will be used to assess how river systems shape the spatial genetic structure and diversity of riparian populations of this species.

Identification and validation of single nucleotide polymorphisms in growth- and maturation-related candidate genes in sole (Solea solea L.).

Genomic methodologies applied in evolutionary and fisheries research have been of great benefit to understand the marine ecosystem and the management of natural resources. Although single nucleotide polymorphisms (SNPs) are attractive for the study of local adaptation, spatial stock management and traceability, and investigating the effects of fisheries-induced selection, they have rarely been exploited in non-model organisms. This is partly due to difficulties in finding and validating SNPs in species with limited or no genomic resources. Complementary to random genome-scan approaches, a targeted candidate gene approach has the potential to unveil pre-selected functional diversity and provides more in depth information on the action of selection at specific genes. For example genes can be under selective pressure due to climate change and sustained periods of heavy fishing pressure. In this study, we applied a candidate gene approach in sole (Solea solea L.), an important member of the demersal ecosystem. As consumption flatfish it is heavy exploited and has experienced associated life-history changes over the last 60years. To discover novel genetic polymorphisms in or around genes linked to important life history traits in sole, we screened a total of 76 candidate genes related to growth and maturation using a targeted resequencing approach. We identified in total 86 putative SNPs in 22 genes and validated 29 SNPs using a multiplex single-base extension genotyping assay. We found 22 informative SNPs, of which two represent non-synonymous mutations, potentially of functional relevance. These novel markers should be rapidly and broadly applicable in analyses of natural sole populations, as a measure of the evolutionary signature of overfishing and for initiatives on marker assisted selection.