Gregory E. Maes

Application of SNPs for population genetics of nonmodel organisms: new opportunities and challenges

Recent improvements in the speed, cost and accuracy of next generation sequencing are revolutionizing the discovery of single nucleotide polymorphisms (SNPs). SNPs are increasingly being used as an addition to the molecular ecology toolkit in nonmodel organisms, but their efficient use remains challenging. Here, we discuss common issues when employing SNP markers, including the high numbers of markers typically employed, the effects of ascertainment bias and the inclusion of nonneutral loci in a marker panel. We provide a critique of considerations specifically associated with the application and population genetic analysis of SNPs in nonmodel taxa, focusing specifically on some of the most commonly applied methods.

The european eel (Anguilla anguilla, Linnaeus), its lifecycle, evolution and reproduction: a literature review

The European eel (Anguilla anguilla Linnaeus 1758) is a species typical for waters of Western Europe. Thanks to early expeditions on the Atlantic Ocean by the Danish biologist Johannes Schmidt who found small (<10mm) leptocephali larvae in the Sargasso Sea about 100 years ago, we have now a strong indication where the spawning site for this species is located. The American eel (Anguilla rostrata, LeSueur) also spawns in the Sargasso Sea. The spawning time and location of both species have been supported and refined in recent analyses of the available historical data. Subsequent ichthyoplankton surveys conducted by McCleave (USA) and Tesch (Germany) in the 1980s indicated an increase in the number of leptocephali <10 mm , confirming and refining the Sargasso Sea theory of Johannes Schmidt. Distinctions between the European and American eel are based on morphological characteristics (number of vertebrae) as well as molecular markers (allozymes, mitochondrial DNA and anonymous genomic-DNA. Although recognised as two distinct species, it remains unclear which mechanisms play a role in species separation during larval drift, and what orientation mechanism eels use during migration in the open sea. The current status of knowledge on these issues will be presented. The hypothesis that all European eel migrate to the Sargasso Sea for reproduction and comprise a single randomly mating population, the so called panmixia theory, was until recently broadly accepted. However, based on field observations, morphological parameters and molecular studies there are some indications that Schmidt’s claim of complete homogeneity of the European eel population and a unique spawning location may be an overstatement. Recent molecular work on European eel indicated a genetic mosaic consisting of several isolated groups, leading to a rejection of the panmixia theory. Nevertheless, the latest extensive genetic survey indicated that the geographical component of genetic structure lacked temporal stability, emphasising the need for temporal replication in the study of highly vagile marine species. Induced spawning of hormone treated eels in the aquarium was collective and simultaneous. In this work for the first time group spawning behaviour has ever been observed and recorded in eels. Studies in swim-tunnels indicate that eels can swim four to six times more efficiently than non-anguilliform fish such as trout. After a laboratory swim trial of eels over 5,500 km, the body composition did not change and fat, protein and carbohydrate were used in the same proportion. This study demonstrated for the first time that European eel are physiologically able of reaching the Sargasso Sea without feeding. Based on catches of newly hatched larvae, temperature preference tests and telemetry tracking of mature hormone treated animals, it can be hypothesised that spawning in the Sargasso Sea is collective and simultaneous, while presumably taking place in the upper 200 m of the ocean. Successful satellite tracking of longfin female eels in New Zealand has been performed to monitor migration pathways. Implementation of this new technology is possible in this species because it is three times larger than the European eel. In the future, miniaturisation of tagging technology may allow European eels to be tracked in time by satellite. The most interesting potential contribution of telemetry tracking of silver eels is additional knowledge about migration routes, rates, and depths. In combination with catches of larvae in the Sargasso Sea, it may elucidate the precise spawning locations of different eel species or groups. Only then, we will be able to define sustainable management issues by integrating this novel knowledge into spawners escapement and juvenile fishing quota.

Panmixia in the European eel: a matter of time...

The European eel (Anguilla anguilla L.) has been a prime example of the panmixia paradigm because of its extraordinary adaptation to the North Atlantic gyral system, semelparous spawning in the Sargasso Sea and long trans-oceanic migration. Recently, this view was challenged by the suggestion of a genetic structure characterized by an isolation-by-distance (IBD) pattern. This is only likely if spawning subpopulations are spatially and/or temporally separated, followed by non-random larval dispersal. A limitation of previous genetic work on eels is the lack of replication over time to test for temporal stability of genetic structure. Here, we hypothesize that temporal genetic variation plays a significant role in explaining the spatial structure reported earlier for this species. We tested this by increasing the texture of geographical sampling and by including temporal replicates. Overall genetic differentiation among samples was low, highly significant and comparable with earlier studies (FST=0.0014; p<0.01). On the other hand, and in sharp contrast with current understandings, hierarchical analyses revealed no significant inter-location genetic heterogeneity and hence no IBD. Instead, genetic variation among temporal samples within sites clearly exceeded the geographical component. Our results provide support for the panmixia hypothesis and emphasize the importance of temporal replication when assessing population structure of marine fish species.

All roads lead to home: panmixia of European eel in the Sargasso Sea

European eels (Anguilla anguilla) spawn in the remote Sargasso Sea in partial sympatry with American eels (Anguilla rostrata), and juveniles are transported more than 5000 km back to the European and North African coasts. The two species have been regarded as classic textbook examples of panmixia, each comprising a single, randomly mating population. However, several recent studies based on continental samples have found subtle, but significant, genetic differentiation, interpreted as geographical or temporal heterogeneity between samples. Moreover, European and American eels can hybridize, but hybrids have been observed almost exclusively in Iceland, suggesting hybridization in a specific region of the Sargasso Sea and subsequent nonrandom dispersal of larvae. Here, we report the first molecular population genetics study based on analysis of 21 microsatellite loci in larvae of both Atlantic eel species sampled directly in the spawning area, supplemented by analysis of European glass eel samples. Despite a clear East-West gradient in the overlapping distribution of the two species in the Sargasso Sea, we only observed a single putative hybrid, providing evidence against the hypothesis of a wide marine hybrid zone. Analyses of genetic differentiation, isolation by distance, isolation by time and assignment tests provided strong evidence for panmixia in both the Sargasso Sea and across all continental samples of European eel after accounting for the presence of sibs among newly hatched larvae. European eel has declined catastrophically, and our findings call for management of the species as a single unit, necessitating coordinated international conservation efforts.

Prebiotic effects of arabinoxylan oligosaccharides on juvenile Siberian sturgeon (Acipenser baerii) with emphasis on the modulation of the gut microbiota using 454 pyrosequencing

The potential of a novel class of prebiotics, arabinoxylan oligosaccharides (AXOS), was investigated on growth performance and gut microbiota of juvenile Acipenser baerii. Two independent feeding trials of 10 or 12 weeks were performed with basal diets supplemented with 2% or 4% AXOS-32-0.30 (trial 1) and 2% AXOS-32-0.30 or AXOS-3-0.25 (trial 2), respectively. Growth performance was improved by feeding 2% AXOS-32-0.30 in both trials, although not significantly. Microbial community profiles were determined using 454-pyrosequencing with barcoded primers targeting the V3 region of the 16S rRNA gene. AXOS significantly affected the relative abundance of bacteria at the phylum, family, genus and species level. The consumption of 2% AXOS-32-0.30 increased the relative abundance of Eubacteriaceae, Clostridiaceae, Streptococcaceae and Lactobacillaceae, while the abundance of Bacillaceae was greater in response to 4% AXOS-32-0.30 and 2% AXOS-3-0.25. The abundance of Lactobacillus spp. and Lactococcus lactis was greater after 2% AXOS-32-0.30 intake. Redundancy analysis showed a distinct and significant clustering of the gut microbiota of individuals consuming an AXOS diet. In both trials, concentration of acetate, butyrate and total short-chain fatty acids (SCFAs) increased in fish fed 2% AXOS-32-0.30. Our data demonstrate a shift in the hindgut microbiome of fish consuming different preparation of AXOS, with potential application as prebiotics.

Sequencing, de novo annotation and analysis of the first Anguilla anguilla transcriptome: EeelBase opens new perspectives for the study of the critically endangered european eel

BackgroundOnce highly abundant, the European eel (Anguilla anguilla L.; Anguillidae; Teleostei) is considered to be critically endangered and on the verge of extinction, as the stock has declined by 90-99% since the 1980s. Yet, the species is poorly characterized at molecular level with little sequence information available in public databases.ResultsThe first European eel transcriptome was obtained by 454 FLX Titanium sequencing of a normalized cDNA library, produced from a pool of 18 glass eels (juveniles) from the French Atlantic coast and two sites in the Mediterranean coast. Over 310,000 reads were assembled in a total of 19,631 transcribed contigs, with an average length of 531 nucleotides. Overall 36% of the contigs were annotated to known protein/nucleotide sequences and 35 putative miRNA identified.ConclusionsThis study represents the first transcriptome analysis for a critically endangered species. EeelBase, a dedicated database of annotated transcriptome sequences of the European eel is freely available at http://compgen.bio.unipd.it/eeelbase. Considering the multiple factors potentially involved in the decline of the European eel, including anthropogenic factors such as pollution and human-introduced diseases, our results will provide a rich source of data to discover and identify new genes, characterize gene expression, as well as for identification of genetic markers scattered across the genome to be used in various applications.

Genome-wide single-generation signatures of local selection in the panmictic European eel

Next‐generation sequencing and the collection of genome‐wide data allow identifying adaptive variation and footprints of directional selection. Using a large SNP data set from 259 RAD‐sequenced European eel individuals (glass eels) from eight locations between 34 and 64oN, we examined the patterns of genome‐wide genetic diversity across locations. We tested for local selection by searching for increased population differentiation using FST‐based outlier tests and by testing for significant associations between allele frequencies and environmental variables. The overall low genetic differentiation found (FST = 0.0007) indicates that most of the genome is homogenized by gene flow, providing further evidence for genomic panmixia in the European eel. The lack of genetic substructuring was consistent at both nuclear and mitochondrial SNPs. Using an extensive number of diagnostic SNPs, results showed a low occurrence of hybrids between European and American eel, mainly limited to Iceland (5.9%), although individuals with signatures of introgression several generations back in time were found in mainland Europe. Despite panmixia, a small set of SNPs showed high genetic differentiation consistent with single‐generation signatures of spatially varying selection acting on glass eels. After screening 50 354 SNPs, a total of 754 potentially locally selected SNPs were identified. Candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand–receptor interaction and circadian rhythm. Remarkably, one of the candidate genes identified is PERIOD, possibly related to differences in local photoperiod associated with the >30° difference in latitude between locations. Genes under selection were spread across the genome, and there were no large regions of increased differentiation as expected when selection occurs within just a single generation due to panmixia. This supports the conclusion that most of the genome is homogenized by gene flow that removes any effects of diversifying selection from each new generation.

Evidence for isolation by time in the European eel (Anguilla anguilla L.)

Life history traits of highly vagile marine species, such as adult reproductive success and larval dispersal, are strongly determined by oceanographic and climatic forces. Nevertheless, marine organisms may show restricted dispersal in time and space. Patterns of isolation by distance (IBD) have been repeatedly observed in marine species. If spawning time is a function of geographical location, temporal and spatial isolation, can easily be confounded or misinterpreted. In this study, we aimed at discriminating between various forces shaping the genetic composition of recruiting juveniles of the European eel (Anguilla anguilla L.). By controlling for geographical variation, we assessed temporal variation and tested for possible isolation by time (IBT) between recruitment waves within and between years. Using 12 polymorphic allozyme and six variable microsatellite loci, we show that genetic differentiation was low (FST = 0.01–0.002) and significant between temporal samples. Regression analysis between genetic and temporal distance, was consistent with a subtle interannual pattern of IBT. Our data suggest that the population dynamics of the European eel may be governed by a double pattern of temporal variance in genetic composition: (i) a broad‐scale IBT of spawning cohorts, possibly as a consequence of the large migration loop in anguillids and strong variance in annual adult reproductive contribution; and (ii) a smaller‐scale variance in reproductive success (genetic patchiness) within cohorts among seasonally separated spawning groups, most likely originating from fluctuating oceanic and climatic forces. The consistency of both mechanisms remains to be verified with fine‐scale analyses of both spawning/migrating aged adults and their offspring to confirm the stochastic/deterministic nature of the IBT pattern in eel.

Qualitative assessment of the diet of European eel larvae in the Sargasso Sea resolved by DNA barcoding

European eels (Anguilla anguilla) undertake spawning migrations of more than 5000 km from continental Europe and North Africa to frontal zones in the Sargasso Sea. Subsequently, the larval offspring are advected by large-scale eastward ocean currents towards continental waters. However, the Sargasso Sea is oligotrophic, with generally low plankton biomass, and the feeding biology of eel larvae has so far remained a mystery, hampering understanding of this peculiar life history. DNA barcoding of gut contents of 61 genetically identified A. anguilla larvae caught in the Sargasso Sea showed that even the smallest larvae feed on a striking variety of plankton organisms, and that gelatinous zooplankton is of fundamental dietary importance. Hence, the specific plankton composition seems essential for eel larval feeding and growth, suggesting a linkage between eel survival and regional plankton productivity. These novel insights into the prey of Atlantic eels may furthermore facilitate eel larval rearing in aquaculture, which ultimately may replace the unsustainable use of wild-caught glass eels.

Detecting Holocene divergence in the anadromous–freshwater three-spined stickleback (Gasterosteus aculeatus) system

The anadromous–freshwater three‐spined stickleback (Gasterosteus aculeatus) system allows for inferring the role of adaptation in speciation with a high level of accuracy because the freshwater ecotype has evolved multiple times from a uniform anadromous ancestor. A cause for concern is that independent evolution among drainages is not guaranteed in areas with a poorly resolved glacial history. This is the case for the west European great rivers, whose downstream valleys flanked the southern limit of the late Pleistocene ice sheet. We tested for independent and postglacial colonization of these valleys hypothesizing that the relationships among anadromous and freshwater sticklebacks correspond to a raceme structure. We compared the reduction in plate number accompanying this colonization to the genetic differentiation using 13 allozyme and five microsatellite loci in 350 individuals. Overall microsatellite differentiation (FST = 0.147) was twice as large as allozyme differentiation (FST = 0.066). Although habitat‐specific gene flow may mask the ancestral relationships among both ecotypes, levels of microsatellite differentiation supported the hypothesis of raceme‐like divergence, reflecting independent colonizations rather than the presence of two distinct evolutionary clades. Under an infinite alleles model and in the absence of gene flow, the observed freshwater divergence might be reached after 440 (microsatellites) to 4500 (allozymes) generations. Hence, the anadromous–freshwater stickleback system most likely diverged postglacially. We conclude that the reduction in plate number in two freshwater basins probably occurred independently, and that its considerable variation among populations is not in agreement with the time since divergence.