New resources for genetic studies in Populus nigra: genome‐wide SNP discovery and development of a 12k Infinium array

Whole genome resequencing of 51 Populus nigra (L.) individuals from across Western Europe was performed using Illumina platforms. A total number of 1 878 727 SNPs distributed along the P. nigra reference sequence were identified. The SNP calling accuracy was validated with Sanger sequencing. SNPs were selected within 14 previously identified QTL regions, 2916 expressional candidate genes related to rust resistance, wood properties, water‐use efficiency and bud phenology and 1732 genes randomly spread across the genome. Over 10 000 SNPs were selected for the construction of a 12k Infinium Bead‐Chip array dedicated to association mapping. The SNP genotyping assay was performed with 888 P. nigra individuals. The genotyping success rate was 91%. Our high success rate was due to the discovery panel design and the stringent parameters applied for SNP calling and selection. In the same set of P. nigra genotypes, linkage disequilibrium throughout the genome decayed on average within 5–7 kb to half of its maximum value. As an application test, ADMIXTURE analysis was performed with a selection of 600 SNPs spread throughout the genome and 706 individuals collected along 12 river basins. The admixture pattern was consistent with genetic diversity revealed by neutral markers and the geographical distribution of the populations. These newly developed SNP resources and genotyping array provide a valuable tool for population genetic studies and identification of QTLs through natural‐population based genetic association studies in P. nigra.     

Novel Y‐chromosome short tandem repeats in Sus scrofa and their variation in European wild boar and domestic pig populations

Y‐chromosome markers are important tools for studying male‐specific gene flow within and between populations, hybridization patterns and kinship. However, their use in non‐human mammals is often hampered by the lack of Y‐specific polymorphic markers. We identified new male‐specific short tandem repeats (STRs) in Sus scrofa using the available genome sequence. We selected four polymorphic loci (5–10 alleles per locus), falling in one duplicated and two single‐copy regions. A total of 32 haplotypes were found by screening 211 individuals from eight wild boar populations across Europe and five domestic pig populations. European wild boar were characterized by significantly higher levels of haplotype diversity compared to European domestic pigs (H_D = 0.904 ± 0.011 and H_D = 0.491 ± 0.077 respectively). Relationships among STR haplotypes were investigated by combining them with single nucleotide polymorphisms at two linked genes (AMELY and UTY) in a network analysis. A differentiation between wild and domestic populations was observed (F_ST = 0.229), with commercial breeds sharing no Y haplotype with the sampled wild boar. Similarly, a certain degree of geographic differentiation was observed across Europe, with a number of local private haplotypes and high diversity in northern populations. The described Y‐chromosome markers can be useful to track male inheritance and gene flow in wild and domestic populations, promising to provide insights into evolutionary and population genetics in Sus scrofa.     

Evolutionary history inferred from the de novo assembly of a nonmodel organism,the blue‐eyed black lemur

Lemurs, the living primates most distantly related to humans, demonstrate incredible diversity in behaviour, life history patterns and adaptive traits. Although many lemur species are endangered within their native Madagascar, there is no high‐quality genome assembly from this taxon, limiting population and conservation genetic studies. One critically endangered lemur is the blue‐eyed black lemur Eulemur flavifrons. This species is fixed for blue irises, a convergent trait that evolved at least four times in primates and was subject to positive selection in humans, where 5′ regulatory variation of OCA2 explains most of the brown/blue eye colour differences. We built a de novo genome assembly for E. flavifrons, providing the most complete lemur genome to date, and a high confidence consensus sequence for close sister species E. macaco, the (brown‐eyed) black lemur. From diversity and divergence patterns across the genomes, we estimated a recent split time of the two species (160 Kya) and temporal fluctuations in effective population sizes that accord with known environmental changes. By looking for regions of unusually low diversity, we identified potential signals of directional selection in E. flavifrons at MITF, a melanocyte development gene that regulates OCA2 and has previously been associated with variation in human iris colour, as well as at several other genes involved in melanin biosynthesis in mammals. Our study thus illustrates how whole‐genome sequencing of a few individuals can illuminate the demographic and selection history of nonmodel species.     

SNP discovery and genotyping using restriction‐site‐associated DNA sequencing in chickens

Single nucleotide polymorphisms (SNPs) are essential to the understanding of population genetic variation and diversity. Here, we performed restriction‐site‐associated DNA sequencing (RAD‐seq) on 72 individuals from 13 Chinese indigenous and three introduced chicken breeds. A total of 620 million reads were obtained using an Illumina Hiseq2000 sequencer. An average of 75 587 SNPs were identified from each individual. Further filtering strictly validated 28 895 SNPs candidates for all populations. When compared with the NCBI dbSNP (chicken_9031), 15 404 SNPs were new discoveries. In this study, RAD‐seq was performed for the first time on chickens, implicating the remarkable effectiveness and potential applications on genetic analysis and breeding technique for whole‐genome selection in chicken and other agricultural animals.     

Genetic wealth,population health: Major histocompatibility complex variation in captive and wild ring‐tailed lemurs (Lemur catta)

Across species, diversity at the major histocompatibility complex (MHC) is critical to individual disease resistance and, hence, to population health; however, MHC diversity can be reduced in small, fragmented, or isolated populations. Given the need for comparative studies of functional genetic diversity, we investigated whether MHC diversity differs between populations which are open, that is experiencing gene flow, versus populations which are closed, that is isolated from other populations. Using the endangered ring‐tailed lemur (Lemur catta) as a model, we compared two populations under long‐term study: a relatively “open,” wild population (n = 180) derived from Bezà Mahafaly Special Reserve, Madagascar (2003–2013) and a “closed,” captive population (n = 121) derived from the Duke Lemur Center (DLC, 1980–2013) and from the Indianapolis and Cincinnati Zoos (2012). For all animals, we assessed MHC‐DRB diversity and, across populations, we compared the number of unique MHC‐DRB alleles and their distributions. Wild individuals possessed more MHC‐DRB alleles than did captive individuals, and overall, the wild population had more unique MHC‐DRB alleles that were more evenly distributed than did the captive population. Despite management efforts to maintain or increase genetic diversity in the DLC population, MHC diversity remained static from 1980 to 2010. Since 2010, however, captive‐breeding efforts resulted in the MHC diversity of offspring increasing to a level commensurate with that found in wild individuals. Therefore, loss of genetic diversity in lemurs, owing to small founder populations or reduced gene flow, can be mitigated by managed breeding efforts. Quantifying MHC diversity within individuals and between populations is the necessary first step to identifying potential improvements to captive management and conservation plans.     

Chromosome arm‐specific patterns of polymorphism associated with chromosomal inversions in the major African malaria vector,Anopheles funestus

Chromosomal inversions facilitate local adaptation of beneficial mutations and modulate genetic polymorphism, but the extent of their effects within the genome is still insufficiently understood. The genome of Anopheles funestus, a malaria mosquito endemic to sub‐Saharan Africa, contains an impressive number of paracentric polymorphic inversions, which are unevenly distributed among chromosomes and provide an excellent framework for investigating the genomic impacts of chromosomal rearrangements. Here, we present results of a fine‐scale analysis of genetic variation within the genome of two weakly differentiated populations of Anopheles funestus inhabiting contrasting moisture conditions in Cameroon. Using population genomic analyses, we found that genetic divergence between the two populations is centred on regions of the genome corresponding to three inversions, which are characterized by high values of F_ST, absolute sequence divergence and fixed differences. Importantly, in contrast to the 2L chromosome arm, which is collinear, nucleotide diversity is significantly reduced along the entire length of three autosome arms bearing multiple overlapping chromosomal rearrangements. These findings support the idea that interactions between reduced recombination and natural selection within inversions contribute to sculpt nucleotide polymorphism across chromosomes in An. funestus.     

A resource of genome‐wide single‐nucleotide polymorphisms generated by RAD tag sequencing in the critically endangered European eel

Reduced representation genome sequencing such as restriction‐site‐associated DNA (RAD) sequencing is finding increased use to identify and genotype large numbers of single‐nucleotide polymorphisms (SNPs) in model and nonmodel species. We generated a unique resource of novel SNP markers for the European eel using the RAD sequencing approach that was simultaneously identified and scored in a genome‐wide scan of 30 individuals. Whereas genomic resources are increasingly becoming available for this species, including the recent release of a draft genome, no genome‐wide set of SNP markers was available until now. The generated SNPs were widely distributed across the eel genome, aligning to 4779 different contigs and 19 703 different scaffolds. Significant variation was identified, with an average nucleotide diversity of 0.00529 across individuals. Results varied widely across the genome, ranging from 0.00048 to 0.00737 per locus. Based on the average nucleotide diversity across all loci, long‐term effective population size was estimated to range between 132 000 and 1 320 000, which is much higher than previous estimates based on microsatellite loci. The generated SNP resource consisting of 82 425 loci and 376 918 associated SNPs provides a valuable tool for future population genetics and genomics studies and allows for targeting specific genes and particularly interesting regions of the eel genome.     

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 64^oN, we examined the patterns of genome‐wide genetic diversity across locations. We tested for local selection by searching for increased population differentiation using F_ST‐based outlier tests and by testing for significant associations between allele frequencies and environmental variables. The overall low genetic differentiation found (F_ST = 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.     

An evaluation of sequencing coverage and genotyping strategies to assess neutral and adaptive diversity

Whole genome sequences (WGS) greatly increase our ability to precisely infer population genetic parameters, demographic processes, and selection signatures. However, WGS may still be not affordable for a representative number of individuals/populations. In this context, our goal was to assess the efficiency of several SNP genotyping strategies by testing their ability to accurately estimate parameters describing neutral diversity and to detect signatures of selection. We analysed 110 WGS at 12× coverage for four different species, i.e., sheep, goats and their wild counterparts. From these data we generated 946 data sets corresponding to random panels of 1K to 5M variants, commercial SNP chips and exome capture, for sample sizes of five to 48 individuals. We also extracted low‐coverage genome resequencing of 1×, 2× and 5× by randomly subsampling reads from the 12× resequencing data. Globally, 5K to 10K random variants were enough for an accurate estimation of genome diversity. Conversely, commercial panels and exome capture displayed strong ascertainment biases. Besides the characterization of neutral diversity, the detection of the signature of selection and the accurate estimation of linkage disequilibrium (LD) required high‐density panels of at least 1M variants. Finally, genotype likelihoods increased the quality of variant calling from low coverage resequencing but proportions of incorrect genotypes remained substantial, especially for heterozygote sites. Whole genome resequencing coverage of at least 5× appeared to be necessary for accurate assessment of genomic variations. These results have implications for studies seeking to deploy low‐density SNP collections or genome scans across genetically diverse populations/species showing similar genetic characteristics and patterns of LD decay for a wide variety of purposes.     

Bioclimatic regions influence genetic structure of four Jordanian Stipa species

Strong environmental gradients can affect the genetic structure of plant populations, but little is known as to whether closely related species respond similarly or idiosyncratically to ecogeographic variation. We analysed the extent to which gradients in temperature and rainfall shape the genetic structure of four Stipa species in four bioclimatic regions in Jordan. Genetic diversity, differentiation and structure of Stipa species were investigated using amplified fragment length polymorphism (AFLP) molecular markers. For each of the four study species, we sampled 120 individuals from ten populations situated in distinct bioclimatic regions and assessed the degree of genetic diversity and genetic differentiation within and among populations. The widespread ruderals Stipa capensis and S. parviflora had higher genetic diversity than the geographically restricted semi‐desert species S. arabica and S. lagascae. In three of the four species, genetic diversity strongly decreased with precipitation, while genetic diversity increased with temperature in S. capensis. Most genetic diversity resided among populations in the semi‐desert species (Φ_ST = 0.572/0.595 in S. arabica/lagascae) but within populations in the ruderal species (Φ_ST = 0.355/0.387 S. capensis/parviflora). Principal coordinate analysis ( PCoA) and STRUCTURE analysis showed that Stipa populations of all species clustered ecogeographically. A genome scan revealed that divergent selection at particular AFLP loci contributed to genetic differentiation. Irrespective of their different life histories, Stipa species responded similarly to the bioclimatic gradient in Jordan. We conclude that, in addition to predominant random processes, steep climatic gradients might shape the genetic structure of plant populations.     

Genetic diversity in endangered Notopterygium forbesii Boissieu based on intraspecies sequence variation of chloroplast DNA and implications for conservation

A thorough understanding of the levels and partitioning of genetic variation across populations and geographical regions of endangered species is a prerequisite to ensure effective conservation and/or restoration activities. Here, we examined chloroplast DNA (cpDNA) trnH-psbA intergenic spacer sequences variation within Notopterygium forbesii, an endangered and endemic perennial herb in China. Sequence data obtained from 141 individuals in 14 populations revealed twenty-two haplotypes. A high level of haplotype diversity (Hd = 0.81) and low level of nucleotide diversity (Pi = 0.0047) were detected. Low genetic differentiation among populations and also among regions was consistently indicated by both hierarchical analyses of molecular variance (AMOVA) and the structure of a neighbor-joining tree. Low level of population differentiation between populations or between regions in cpDNA sequences may be due to effects of the abundance of ancestral haplotype sharing and the high number of private haplotypes fixed for each population. Based on our results, we proposed some conservation strategies.     

Population structure and local selection yield high genomic variation in Mimulus guttatus

Across western North America, Mimulus guttatus exists as many local populations adapted to site‐specific environmental challenges. Gene flow between locally adapted populations will affect genetic diversity both within demes and across the larger metapopulation. Here, we analyse 34 whole‐genome sequences from the intensively studied Iron Mountain population (IM) in conjunction with sequences from 22 Mimulus individuals sampled from across western North America. Three striking features of these data address hypotheses about migration and selection in a locally adapted population. First, we find very high levels of intrapopulation polymorphism (synonymous π = 0.033). Variation outside of genes is likely even higher but difficult to estimate because excessive divergence reduces the efficiency of read mapping. Second, IM exhibits a significantly positive genomewide average for Tajima's D. This indicates allele frequencies are typically more intermediate than expected from neutrality, opposite the pattern observed in many other species. Third, IM exhibits a distinctive haplotype structure with a genomewide excess of positive associations between rarer alleles at linked loci. This suggests an important effect of gene flow from other Mimulus populations, although a residual effect of population founding might also contribute. The combination of multiple analyses, including a novel tree‐based analytic method, illustrates how the balance of local selection, limited dispersal and metapopulation dynamics manifests across the genome. The overall genomic pattern of sequence diversity suggests successful gene flow of divergent immigrant genotypes into IM. However, many loci show patterns indicative of local adaptation, particularly at SNPs associated with chromosomal inversions.     

Meta‐analysis reveals lower genetic diversity in overfished populations

While population declines can drive the loss of genetic diversity under some circumstances, it has been unclear whether this loss is a general consequence of overharvest in highly abundant marine fishes. We compiled data from 11 049 loci across 140 species and found that allelic richness was lower in overfished populations within 9 of 12 genera and families. A multiple linear regression showed that allelic richness was on average 12% lower (P < 0.0001) in overharvested populations after accounting for the effects of body size, latitude and other factors. Heterozygosity was on average 2% lower (P = 0.030). Simulations confirmed that these patterns are consistent with a recent bottleneck in abundant species and also showed that our analysis likely underestimates the loss of rare alleles by a factor of two or three. This evidence suggests that overharvest drives the decay of genetic diversity across a wide range of marine fishes. Such reductions of genetic diversity in some of the world's most abundant species may lead to a long‐term impact of fishing on their evolutionary potential, particularly if abundance remains low and diversity continues to decay.     

Host‐targeted RAD‐Seq reveals genetic changes in the coral Oculina patagonica associated with range expansion along the Spanish Mediterranean coast

Many organisms are expanding their ranges in response to changing environmental conditions. Understanding the patterns of genetic diversity and adaptation along an expansion front is crucial to assessing a species’ long‐term success. While next‐generation sequencing techniques can reveal these changes in fine detail, ascribing them to a particular species can be difficult for organisms that live in close association with symbionts. Using a novel modified restriction site‐associated DNA sequencing (RAD‐Seq) protocol to target coral DNA, we collected 595 coral‐specific single nucleotide polymorphisms from 189 colonies of the invasive coral Oculina patagonica from the Spanish Mediterranean coast, including established core populations and two expansion fronts. Surprisingly, populations from the recent northern expansion are genetically distinct from the westward expansion and core populations and also harbour greater genetic diversity. We found that temperature may have driven adaptation along the northern expansion, as genome scans for selection found three candidate loci associated with temperature in the north but none in the west. We found no genomic signature of selection associated with artificial substrate, which has been proposed for explaining the rapid spread of O. patagonica. This suggests that this coral is simply an opportunistic colonizer of free space made available by coastal habitat modifications. Our results suggest that unique genetic variation, possibly due to limited dispersal across the Ibiza Channel, an influx of individuals from different depths and/or adaptation to cooler temperatures along the northern expansion front may have facilitated the northward range expansion of O. patagonica in the western Mediterranean.     

SNPs across time and space: population genomic signatures of founder events and epizootics in the House Finch (Haemorhous mexicanus)

Identifying genomic signatures of natural selection can be challenging against a background of demographic changes such as bottlenecks and population expansions. Here, we disentangle the effects of demography from selection in the House Finch (Haemorhous mexicanus) using samples collected before and after a pathogen‐induced selection event. Using ddRADseq, we genotyped over 18,000 SNPs across the genome in native pre‐epizootic western US birds, introduced birds from Hawaii and the eastern United States, post‐epizootic eastern birds, and western birds sampled across a similar time span. We found 14% and 7% reductions in nucleotide diversity, respectively, in Hawaiian and pre‐epizootic eastern birds relative to pre‐epizootic western birds, as well as elevated levels of linkage disequilibrium and other signatures of founder events. Despite finding numerous significant frequency shifts (outlier loci) between pre‐epizootic native and introduced populations, we found no signal of reduced genetic diversity, elevated linkage disequilibrium, or outlier loci as a result of the epizootic. Simulations demonstrate that the proportion of outliers associated with founder events could be explained by genetic drift. This rare view of genetic evolution across time in an invasive species provides direct evidence that demographic shifts like founder events have genetic consequences more widespread across the genome than natural selection.     

Assessment of genetic diversity and mating system of Acacia cyclops restoration and remnant populations

Acacia cyclops, or Ngaamarur, is a common coastal shrub or small tree of the southwest of Western Australia and South Australia used for restoration in these landscapes and elsewhere. Knowledge of genetic diversity and mating systems of restoration populations is often lacking but can help inform likely restoration success. We compared genetic diversity and mating system parameters at three restoration populations of A. cyclops to nearby remnant reference populations. Mean levels of genetic diversity in restoration populations (Na = 1.806, Ne = 1.560, He = 0.270, Ho = 0.359) were not significantly different from those in the reference populations (Na = 1.833, Ne = 1.574, He = 0.269, Ho = 0.352) suggesting diverse seed collections were made for restoration. Allelic genetic divergence among restoration and reference populations was low (D_ST ≤ 0.04) suggesting that seeds were of local provenance. Mating system parameters were similar among restoration (t_m = 0.989, t_m–t_s = 0.010, r_pm = 0.392) and reference populations (t_m = 0.868, t_m–t_s = 0.039, r_pm = 0.410), suggesting functionality of the mating system and restitution of generalist insect pollinator services in restored populations at 7, 10, and 12 years after planting. Lower levels of heterozygosity in progeny compared to adults suggest post‐germination or late‐acting selection against inbred seedlings. Evidence for selection and seedling attrition further emphasizes the benefits of collections from many individuals in well‐connected outcrossed source populations. Overall, results suggest available local genetic diversity was captured and restoration populations have mating systems equivalent to those of reference populations.     

Conservation genomics in the fight to help the recovery of the critically endangered Siamese crocodile Crocodylus siamensis

Endangered species are often characterized by low genetic diversity and it is imperative for conservation efforts to incorporate the knowledge obtained from genetic studies for effective management. However, despite the promise of technological advances in sequencing, application of genome‐wide data to endangered populations remains uncommon. In the present study we pursued a holistic conservation‐genomic approach to inform a field‐based management programme of a Critically Endangered species, the Siamese crocodile Crocodylus siamensis. Using thousands of single nucleotide polymorphisms from throughout the genome, we revealed signals of introgression from two other crocodile species within our sample of both wild and captive‐bred Siamese crocodiles from Cambodia. Our genetic screening of the Siamese crocodiles resulted in the subsequent re‐introduction of 12 individuals into the wild as well as the selection of four individuals for captive breeding programmes. Comparison of intraspecific genetic diversity revealed an alarmingly low contemporary effective population size in the wild (<50) with evidence of a recent bottleneck around Tonle Sap Lake. We also projected a probable future extinction in the wild (within fewer than five generations) in this population in the absence of re‐introduction efforts. However, an increase in the number of potential breeders through re‐introductions, including the one resulting from this project, could counter this trend. Our results have been implemented in ongoing re‐introduction and captive breeding programmes, with major implications for the conservation management of Siamese crocodiles, and provide a blueprint for the rescue effort of other “terminally ill” populations of critically endangered species.     

Evolutionary genomics of gypsy moth populations sampled along a latitudinal gradient

The European gypsy moth (Lymantria dispar L.) was first introduced to Massachusetts in 1869 and within 150 years has spread throughout eastern North America. This large‐scale invasion across a heterogeneous landscape allows examination of the genetic signatures of adaptation potentially associated with rapid geographical spread. We tested the hypothesis that spatially divergent natural selection has driven observed changes in three developmental traits that were measured in a common garden for 165 adult moths sampled from six populations across a latitudinal gradient covering the entirety of the range. We generated genotype data for 91,468 single nucleotide polymorphisms based on double digest restriction‐site associated DNA sequencing and used these data to discover genome‐wide associations for each trait, as well as to test for signatures of selection on the discovered architectures. Genetic structure across the introduced range of gypsy moth was low in magnitude (F_ST = 0.069), with signatures of bottlenecks and spatial expansion apparent in the rare portion of the allele frequency spectrum. Results from applications of Bayesian sparse linear mixed models were consistent with the presumed polygenic architectures of each trait. Further analyses indicated spatially divergent natural selection acting on larval development time and pupal mass, with the linkage disequilibrium component of this test acting as the main driver of observed patterns. The populations most important for these signals were two range‐edge populations established less than 30 generations ago. We discuss the importance of rapid polygenic adaptation to the ability of non‐native species to invade novel environments.