Author Correction: Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antibiotic Resistance Plasmids Cointegrated into a Megaplasmid Harboring the blaOXA-427 Carbapenemase Gene.

OXA-427 is a new class D carbapenemase encountered in different species of Enterobacteriaceae in a Belgian hospital. To study the dispersal of this gene, we performed a comparative analysis of two plasmids containing the blaOXA-427 gene, isolated from a Klebsiella pneumoniae strain and an Enterobacter cloacae complex strain. The two IncA/C2 plasmids containing blaOXA-427 share the same backbone; in the K. pneumoniae strain, however, this plasmid is cointegrated into an IncFIb plasmid, forming a 321-kb megaplasmid with multiple multiresistance regions.

GBSX: a toolkit for experimental design and demultiplexing genotyping by sequencing experiments.

BACKGROUND: Massive parallel sequencing is a powerful tool for variant discovery and genotyping. To reduce costs, sequencing of restriction enzyme based reduced representation libraries can be utilized. This technology is generally referred to as Genotyping By Sequencing (GBS). To deal with GBS experimental design and initial processing specific bioinformatic tools are needed. RESULTS: GBSX is a package that assists in selecting the appropriate enzyme and the design of compatible in-line barcodes. Post sequencing, it performs optimized demultiplexing using these barcodes to create fastq files per barcode which can easily be plugged into existing variant analysis pipelines. Here we demonstrate the usability of the GBSX toolkit and demonstrate improved in-line barcode demultiplexing and trimming performance compared to existing tools. CONCLUSIONS: GBSX provides an easy to use suite of tools for designing and demultiplexing of GBS experiments.

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.

Comparative phylogeography of three trematomid fishes reveals contrasting genetic structure patterns in benthic and pelagic species.

Population genetics patterns of marine fish in general and of Southern Ocean fish in particular range from virtual panmixia over ocean-wide scale to deeply fragmented populations. However the causes underlying these different patterns are not properly understood. In this paper, we tested the hypotheses that population connectivity is positively related to a combination of life history traits, namely duration of pelagic larval period and the tendency towards pelagic life style in the adulthood. To do so, we analysed the variability of six microsatellite and one mitochondrial marker (cytochrome b) in three Southern Ocean fish species (Trematomus newnesi, Trematomus hansoni and Trematomus bernacchii). They share a recent common ancestor but notably differ in their duration of pelagic larval period as well as pelagic versus benthic lifestyle. We sampled over a range of more than 5000 km for all three species and used a number of population genetics tools to investigate past and contemporary levels of connectivity. All species experienced population fluctuations, but coalescent simulations suggested that contemporary populations are in migration-drift equilibrium. Although global F(ST) values were rather low, a significant population structure separated the High-Antarctic from the Peninsular regions in all species. The level of genetic differentiation was much lower in the pelagic versus benthic species. Present data suggest that past and present genetic structuring in the Southern Ocean are indeed related with the ecological traits of Antarctic fish, however the relative importance of individual factors remains unclear.

Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

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.

Heterozygous missense mutations in SMARCA2 cause Nicolaides-Baraitser syndrome.

Nicolaides-Baraitser syndrome (NBS) is characterized by sparse hair, distinctive facial morphology, distal-limb anomalies and intellectual disability. We sequenced the exomes of ten individuals with NBS and identified heterozygous variants in SMARCA2 in eight of them. Extended molecular screening identified nonsynonymous SMARCA2 mutations in 36 of 44 individuals with NBS; these mutations were confirmed to be de novo when parental samples were available. SMARCA2 encodes the core catalytic unit of the SWI/SNF ATP-dependent chromatin remodeling complex that is involved in the regulation of gene transcription. The mutations cluster within sequences that encode ultra-conserved motifs in the catalytic ATPase region of the protein. These alterations likely do not impair SWI/SNF complex assembly but may be associated with disrupted ATPase activity. The identification of SMARCA2 mutations in humans provides insight into the function of the Snf2 helicase family.

Differential modes of selection on the rhodopsin gene in coastal Baltic and North Sea populations of the sand goby, Pomatoschistus minutus.

An excellent model to elucidate the mechanisms and importance of evolution in the marine environment is the spectral tuning mechanism of the visual pigment in vertebrates. In the sand goby Pomatoschistus minutus (Teleostei; Gobiidae), a distribution-wide study showed that spatial variation at the rhodopsin gene (RH1) matches the characteristics of specific light environments. This match suggests that populations are locally adapted to selective light regimes targeting the RH1 gene. If so, then the direction of selection should depend on the regional spatial and temporal stability of the light conditions. We tested this prediction by comparing goby populations from two regions: the Baltic Sea, characterized by divergent, but temporally stable light conditions, and the North Sea, characterized by locally heterogeneous and temporally variable light conditions. RH1 sequences of 491 Pomatoschistus minutus individuals from 15 locations were analysed. We found that variation at the RH1 gene in the Baltic populations showed signatures of diversifying selection, whereas the RH1 gene in the North Sea showed signatures of stabilizing selection. These different modes of selection are consistent with the regional light conditions and hence support our predictions, but may also be influenced by migration between the open sea and more turbid estuarine environments. An interesting observation is that within one gene, synonymous and non-synonymous SNPs show a totally different pattern between populations. Population differentiation based on non-synonymous SNPs of the RH1 gene correlated with spectral variation of the local environment of the sand goby populations. In contrast, the differentiation based on synonymous SNPs of RH1 reflects more the neutral historical pattern of the species.

High molecular diversity in the rhodopsin gene in closely related goby fishes: A role for visual pigments in adaptive speciation?

The spectral tuning mechanism of visual pigments is an excellent model to elucidate the mechanisms of adaptive evolution and the importance of selection as an evolutionary force. Therefore, we use a phylogenetic approach to determine whether there is evidence for differential adaptive molecular evolution on the rhodopsin (RH1) gene among closely related 'sand goby' species (Teleostei, Gobiidae). Fragments of the RH1 gene (868 bp) were sequenced and analyzed for nine 'sand goby' species that inhabit different photic environments. A high level of interspecific polymorphism at the RH1 gene was observed, including non-synonymous mutations on amino acids known as spectral tuning sites. Clear indications for positive Darwinian selection were provided by three independent methods: (1) by linking functional variation on the RH1 gene to specific light environments of the different fish habitats; (2) by constructing and comparing phylogenies based on RH1 and the 'neutral' 12S and 16S mtDNA fragments; and (3) by performing statistical tests to detect signatures of directional selection on the RH1 gene. This study shows an unusual high variability in the gobiid visual RH1 pigment, and we therefore suggest a possible role for sensory genes in the adaptive radiation of 'sand goby' species.

To see in different seas: spatial variation in the rhodopsin gene of the sand goby (Pomatoschistus minutus).

Aquatic organisms living in a range of photic environments require specific mechanisms to tune their visual pigments. Maximum absorbance (lambda(max)) of retinal rods in populations of the marine demersal sand goby, (Pomatoschistus minutus; Gobiidae, Teleostei) correlates with the local optic environment. It has been shown that this is not regulated through a physiological response by exchanging the rhodopsin chromophore. To test for evolutionary adaptation, the sequence of the rhodopsin (RH1) gene was analysed in 165 Pomatoschistus minutus individuals from seven populations across its distribution range. Analysis showed a high level of intraspecific polymorphism at the RH1 gene, including nonsynonymous mutations on amino acids, known as spectral tuning sites. Population differentiation at these sites was in agreement with the observed differentiation in lambda(max) values. Analyses of d(N)/d(S) substitution rate ratios and likelihood ratio tests under site-specific models detected a significant signal of positive Darwinian selection on the RH1 gene. A strong discrepancy in differentiation was noticed between RH1 gene variation and the presumably neutral microsatellites and mitochondrial data. Samples did not cluster according to geographical or historical proximity with regards to RH1, but according to the general photic conditions of the habitat environment of the sand goby. This study highlights the usefulness of sensory genes, like rhodopsin, for studying the characteristics of local adaptation in marine nonmodel organisms.

Divergent selection as revealed by P(ST) and QTL-based F(ST) in three-spined stickleback (Gasterosteus aculeatus) populations along a coastal-inland gradient.

Three measures of divergence, estimated at nine putatively neutral microsatellite markers, 14 quantitative traits, and seven quantitative trait loci (QTL) were compared in eight populations of the three-spined stickleback (Gasterosteus aculeatus L.) living in the Scheldt river basin (Belgium). Lowland estuarine and polder populations were polymorphic for the number of lateral plates, whereas upland freshwater populations were low-plated. The number of short gill rakers and the length of dorsal and pelvic spines gradually declined along a coastal-inland gradient. Plate number, short gill rakers and spine length showed moderate to strong signals of divergent selection between lowland and upland populations in comparison between P(ST) (a phenotypic alternative for Q(ST)) and neutral F(ST). However, such comparisons rely on the unrealistic assumption that phenotypic variance equals additive genetic variance, and that nonadditive genetic effects and environmental effects can be minimized. In order to verify this assumption and to confirm the phenotypic signals of divergence, we tested for divergent selection at the underlying QTL. For plate number, strong genetic evidence for divergent selection between lowland and upland populations was obtained based on an intron marker of the Eda gene, of which the genotype was highly congruent with plate morph. Genetic evidence for divergent selection on short gill rakers was limited to some population pairs where F(ST) at only one of two QTL was detected as an outlier, although F(ST) at both loci correlated significantly with P(ST). No genetic confirmation was obtained for divergent selection on dorsal spine length, as no outlier F(ST)s were detected at dorsal spine QTL, and no significant correlations with P(ST) were observed.