Let's talk about sex: A rigorous statistical framework to assign the sex of individuals from reduced‐representation sequencing data

Molecular markers have been used to identify the sex of sampled individuals for several decades, but the time‐consuming development phase prevented their application in many systems. Recently, a growing number of papers have applied reduced‐representation sequencing (RRS) protocols to the identification of sex‐specific markers without the use of test crosses or prior genomic information. While such an approach has great advantages in terms of versatility and ease of use, the “shotgun sequencing” nature of RRS data sets leads to a high amount of missing data, which results in statistical challenges to the confident assignment of sex to individuals. In this issue of Molecular Ecology Resources, Stovall et al. (Molecular Ecology Resources, 18, 2018) provide a statistical framework to answer two questions: (1) how many individuals of one sex only must possess a genotype for this locus to be considered significantly sex‐specific? and (2) How many sex‐specific loci must an individual of unknown sex possess (in a given data set) to be confidently assigned a sex? The statistical pipeline introduced, and applied to samples of New Zealand fur seal (Arctocephalus forsteri) to identify 90 sex‐specific loci, should be broadly applicable to a large number of species and constitutes a nice addition to the molecular ecology toolkit in the genomics era.     

Divergent and linked selection shape patterns of genomic differentiation between European and North American Atlantic salmon (Salmo salar)

As populations diverge many processes can shape genomic patterns of differentiation. Regions of high differentiation can arise due to divergent selection acting on selected loci, genetic hitchhiking of nearby loci, or through repeated selection against deleterious alleles (linked background selection); this divergence may then be further elevated in regions of reduced recombination. Atlantic salmon (Salmo salar) from Europe and North America diverged >600,000 years ago and despite some evidence of secondary contact, the majority of genetic data indicate substantial divergence between lineages. This deep divergence with potential gene flow provides an opportunity to investigate the role of different mechanisms that shape the genomic landscape during early speciation. Here, using 184,295 single nucleotide polymorphisms (SNPs) and 80 populations, we investigate the genomic landscape of differentiation across the Atlantic Ocean with a focus on highly differentiated regions and the processes shaping them. We found evidence of high (mean F_ST = 0.26) and heterogeneous genomic differentiation between continents. Genomic regions associated with high trans‐Atlantic differentiation ranged in size from single loci (SNPs) within important genes to large regions (1–3 Mbp ) on four chromosomes (Ssa06, Ssa13, Ssa16 and Ssa19). These regions showed signatures consistent with selection, including high linkage disequilibrium, despite no significant reduction in recombination. Genes and functional enrichment of processes associated with differentiated regions may highlight continental differences in ocean navigation and parasite resistance. Our results provide insight into potential mechanisms underlying differences between continents, and evidence of near‐fixed and potentially adaptive trans‐Atlantic differences concurrent with a background of high genome‐wide differentiation supports subspecies designation in Atlantic salmon.     

Do genetic drift and accumulation of deleterious mutations preclude adaptation? Empirical investigation using RADseq in a northern lacustrine fish

Understanding genomic signatures of divergent selection underlying long‐term adaptation in populations located in heterogeneous environments is a key goal in evolutionary biology. In this study, we investigated neutral, adaptive and deleterious genetic variation using 7,192 SNPs in 31 Lake Trout (Salvelinus namaycush) populations (n = 673) from Québec, Canada. Average genetic diversity was low, weakly shared among lakes, and positively correlated with lake size, indicating a major role for genetic drift subsequent to lake isolation. Putatively deleterious mutations were on average at lower frequencies than the other SNPs, and their abundance relative to the entire polymorphism in each population was positively correlated with inbreeding, suggesting that the effectiveness of purifying selection was negatively correlated with inbreeding, as predicted from theory. Despite evidence for pronounced genetic drift and inbreeding, several outlier loci were associated with temperature and found in or close to genes with biologically relevant functions notably related to heat stress and immune responses. Outcomes of gene–temperature associations were influenced by the inclusion of the most inbred populations, in which allele frequencies deviated the most from model predictions. This result illustrates challenge in identifying gene–environment associations in cases of high genetic drift and restricted gene flow and suggests limited adaptation in populations experiencing higher inbreeding. We discuss the relevance of these findings for the conservation and management, notably regarding stocking and genetic rescue, of Lake Trout populations and other species inhabiting highly fragmented habitats.     

Lessons learned from microsatellite development for nonmodel organisms using 454 pyrosequencing

Microsatellites, also known as simple sequence repeats (SSRs), are among the most commonly used marker types in evolutionary and ecological studies. Next Generation Sequencing techniques such as 454 pyrosequencing allow the rapid development of microsatellite markers in nonmodel organisms. 454 pyrosequencing is a straightforward approach to develop a high number of microsatellite markers. Therefore, developing microsatellites using 454 pyrosequencing has become the method of choice for marker development. Here, we describe a user friendly way of microsatellite development from 454 pyrosequencing data and analyse data sets of 17 nonmodel species (plants, fungi, invertebrates, birds and a mammal) for microsatellite repeats and flanking regions suitable for primer development. We then compare the numbers of successfully lab‐tested microsatellite markers for the various species and furthermore describe diverse challenges that might arise in different study species, for example, large genome size or nonpure extraction of genomic DNA. Successful primer identification was feasible for all species. We found that in species for which large repeat numbers are uncommon, such as fungi, polymorphic markers can nevertheless be developed from 454 pyrosequencing reads containing small repeat numbers (five to six repeats). Furthermore, the development of microsatellite markers for species with large genomes was also with Next Generation Sequencing techniques more cost and time‐consuming than for species with smaller genomes. In this study, we showed that depending on the species, a different amount of 454 pyrosequencing data might be required for successful identification of a sufficient number of microsatellite markers for ecological genetic studies.     

A robust sequencing assay of a thousand amplicons for the high-throughput population monitoring of Alpine ibex immunogenetics

Polymorphism for immune functions can explain significant variation in health and reproductive success within species. Drastic loss in genetic diversity at such loci constitutes an extinction risk and should be monitored in species of conservation concern. However, effective implementations of genome-wide immune polymorphism sets into high-throughput genotyping assays are scarce. Here, we report the design and validation of a microfluidics-based amplicon sequencing assay to comprehensively capture genetic variation in Alpine ibex (Capra ibex). This species represents one of the most successful large mammal restorations recovering from a severely depressed census size and a massive loss in diversity at the major histocompatibility complex (MHC). We analysed 65 whole-genome sequencing sets of the Alpine ibex and related species to select the most representative markers and to prevent primer binding failures. In total, we designed ~1,000 amplicons densely covering the MHC, further immunity-related genes as well as randomly selected genome-wide markers for the assessment of neutral population structure. Our analysis of 158 individuals shows that the genome-wide markers perform equally well at resolving population structure as RAD-sequencing or low-coverage genome sequencing data sets. Immunity-related loci show unexpectedly high degrees of genetic differentiation within the species. Such information can now be used to define highly targeted individual translocations. Our design strategy can be realistically implemented into genetic surveys of a large range of species. In conclusion, leveraging whole-genome sequencing data sets to design targeted amplicon assays allows the simultaneous monitoring of multiple genetic risk factors and can be translated into species conservation recommendations.     

Genomics and museum specimens

Nearly 25 years ago, Allan Wilson and colleagues isolated DNA sequences from museum specimens of kangaroo rats (Dipodomys panamintinus) and compared these sequences with those from freshly collected animals (Thomas et al. 1990 ). The museum specimens had been collected up to 78 years earlier, so the two samples provided a direct temporal comparison of patterns of genetic variation. This was not the first time DNA sequences had been isolated from preserved material, but it was the first time it had been carried out with a population sample. Population geneticists often try to make inferences about the influence of historical processes such as selection, drift, mutation and migration on patterns of genetic variation in the present. The work of Wilson and colleagues was important in part because it suggested a way in which population geneticists could actually study genetic change in natural populations through time, much the same way that experimentalists can do with artificial populations in the laboratory. Indeed, the work of Thomas et al. ( 1990 ) spawned dozens of studies in which museum specimens were used to compare historical and present‐day genetic diversity (reviewed in Wandeler et al. 2007 ). All of these studies, however, were limited by the same fundamental problem: old DNA is degraded into short fragments. As a consequence, these studies mostly involved PCR amplification of short templates, usually short stretches of mitochondrial DNA or microsatellites. In this issue, Bi et al. ( 2013 ) report a breakthrough that should open the door to studies of genomic variation in museum specimens. They used target enrichment (exon capture) and next‐generation (Illumina) sequencing to compare patterns of genetic variation in historic and present‐day population samples of alpine chipmunks (Tamias alpinus) (Fig. 1). The historic samples came from specimens collected in 1915, so the temporal span of this comparison is nearly 100 years.     

Standing and flowing: the complex origins of adaptive variation

A population faced with a new selection pressure can only adapt if appropriate genetic variation is available. This genetic variation might come from new mutations or from gene exchange with other populations or species, or it might already segregate in the population as standing genetic variation (which might itself have arisen from either mutation or gene flow). Understanding the relative importance of these sources of adaptive variation is a fundamental issue in evolutionary genetics (Orr & Betancourt 2001 ; Barrett & Schluter 2008 ; Gladyshev et al. 2008 ) and has practical implications for conservation, plant and animal breeding, biological control and infectious disease prevention (e.g. Robertson 1960 ; Soulé & Wilcox 1980 ; Prentis et al. 2008 ; Pennings 2012 ). In this issue of Molecular Ecology, Roesti et al. ( 2014 ) make an important contribution to this longstanding debate.     

Advances in ecological genomics in forest trees and applications to genetic resources conservation and breeding

Forest trees are an unparalleled group of organisms in their combined ecological, economic and societal importance. With widespread distributions, predominantly random mating systems and large population sizes, most tree species harbour extensive genetic variation both within and among populations. At the same time, demographic processes associated with Pleistocene climate oscillations and land‐use change have affected contemporary range‐wide diversity and may impinge on the potential for future adaptation. Understanding how these adaptive and neutral processes have shaped the genomes of trees species is therefore central to their management and conservation. As for many other taxa, the advent of high‐throughput sequencing methods is expected to yield an understanding of the interplay between the genome and environment at a level of detail and depth not possible only a few years ago. An international conference entitled ‘Genomics and Forest Tree Genetics’ was held in May 2016, in Arcachon (France), and brought together forest geneticists with a wide range of research interests to disseminate recent efforts that leverage contemporary genomic tools to probe the population, quantitative and evolutionary genomics of trees. An important goal of the conference was to discuss how such data can be applied to both genome‐enabled breeding and the conservation of forest genetic resources under land use and climate change. Here, we report discoveries presented at the meeting and discuss how the ecological genomic toolkit can be used to address both basic and applied questions in tree biology.     

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

Integration of Random Forest with population‐based outlier analyses provides insight on the genomic basis and evolution of run timing in Chinook salmon (Oncorhynchus tshawytscha)

Anadromous Chinook salmon populations vary in the period of river entry at the initiation of adult freshwater migration, facilitating optimal arrival at natal spawning. Run timing is a polygenic trait that shows evidence of rapid parallel evolution in some lineages, signifying a key role for this phenotype in the ecological divergence between populations. Studying the genetic basis of local adaptation in quantitative traits is often impractical in wild populations. Therefore, we used a novel approach, Random Forest, to detect markers linked to run timing across 14 populations from contrasting environments in the Columbia River and Puget Sound, USA. The approach permits detection of loci of small effect on the phenotype. Divergence between populations at these loci was then examined using both principle component analysis and F_ST outlier analyses, to determine whether shared genetic changes resulted in similar phenotypes across different lineages. Sequencing of 9107 RAD markers in 414 individuals identified 33 predictor loci explaining 79.2% of trait variance. Discriminant analysis of principal components of the predictors revealed both shared and unique evolutionary pathways in the trait across different lineages, characterized by minor allele frequency changes. However, genome mapping of predictor loci also identified positional overlap with two genomic outlier regions, consistent with selection on loci of large effect. Therefore, the results suggest selective sweeps on few loci and minor changes in loci that were detected by this study. Use of a polygenic framework has provided initial insight into how divergence in a trait has occurred in the wild.     

Linking genotypes, phenotypes, and fitness in wild primate populations

In the decade since the first draft of the human genome was announced, genome sequencing projects have been initiated for an additional twenty-some primate species. Within the next several years, genome sequence data will likely become available for all primate genera and for most individuals within some primate populations. At the same time, gene mapping and association studies of humans and other organisms are rapidly advancing our understanding of the genetic bases of behavioral and morphological traits. Primatologists are especially well-placed to take advantage of this coming flood of genetic data. Here we discuss what this new era of primate genomics means for field primatology and highlight some of the unprecedented opportunities it will afford, particularly with regard to examining the genetic basis of primate adaptation and diversity.     

Genotyping‐by‐sequencing for estimating relatedness in nonmodel organisms: Avoiding the trap of precise bias

There has been remarkably little attention to using the high resolution provided by genotyping‐by‐sequencing (i.e., RADseq and similar methods) for assessing relatedness in wildlife populations. A major hurdle is the genotyping error, especially allelic dropout, often found in this type of data that could lead to downward‐biased, yet precise, estimates of relatedness. Here, we assess the applicability of genotyping‐by‐sequencing for relatedness inferences given its relatively high genotyping error rate. Individuals of known relatedness were simulated under genotyping error, allelic dropout and missing data scenarios based on an empirical ddRAD data set, and their true relatedness was compared to that estimated by seven relatedness estimators. We found that an estimator chosen through such analyses can circumvent the influence of genotyping error, with the estimator of Ritland (Genetics Research, 67, 175) shown to be unaffected by allelic dropout and to be the most accurate when there is genotyping error. We also found that the choice of estimator should not rely solely on the strength of correlation between estimated and true relatedness as a strong correlation does not necessarily mean estimates are close to true relatedness. We also demonstrated how even a large SNP data set with genotyping error (allelic dropout or otherwise) or missing data still performs better than a perfectly genotyped microsatellite data set of tens of markers. The simulation‐based approach used here can be easily implemented by others on their own genotyping‐by‐sequencing data sets to confirm the most appropriate and powerful estimator for their data.     

Don't throw the baby out with the bathwater: identifying and mapping paralogs in salmonids

Many eukaryotic genomes contain a large fraction of gene duplicates (or paralogs) as a result of ancient or recent whole‐genome duplications (Ohno 1970 ; Jaillon et al. 2004 ; Kellis et al. 2004 ). Identifying paralogs with NGS data is a pervasive problem in both ancient polyploids and neopolyploids. Likewise, paralogs are often treated as a nuisance that has to be detected and removed (Everett et al. 2012 ). In this issue of Molecular Ecology Resources, Waples et al. ( 2015 ) show that exclusion might not be necessary and how we may miss out on important genomic information in doing so. They present a novel statistical approach to detect paralogs based on the segregation of RAD loci in haploid offspring and test their method by constructing linkage maps with and without these duplicated loci in chum salmon, Oncorhynchus keta (Fig.  1 ). Their linkage map including the resolved paralogs shows that these are mostly located in the distal regions of several linkage groups. Particularly intriguing is their finding that these homoeologous regions appear impoverished in transposable elements (TE). Given the role that TE play in genome remodelling, it is noteworthy that these elements are of low abundance in regions showing residual tetrasomic inheritance. This raises the question whether re‐diploidization is constrained in these regions and whether they might have a role to play in salmonid speciation. This study provides an original approach to identifying duplicated loci in species with a pedigree, as well as providing a dense linkage map for chum salmon, and interesting insights into the retention of gene duplicates in an ancient polyploid.     

The chimerical genome of Isla del Coco feral pigs (Costa Rica), an isolated population since 1793 but with remarkable levels of diversity

The history of domestic species and of their wild ancestors is not a simple one, and feral processes can clarify key aspects of this history, including the adaptive processes triggered by new environments. Here, we provide a comprehensive genomic study of Isla del Coco (Costa Rica) feral pigs, a unique population that was allegedly founded by two individuals and has remained isolated since 1793. Using SNP arrays and genome sequencing, we show that Cocos pigs are hybrids between Asian and European pigs, as are modern international pig breeds. This conclusively shows that, as early as the 18th century, British vessels were loading crossbred pigs in Great Britain and transporting them overseas. We find that the Y chromosome has Asian origin, which has not been reported in any international pig breed. Chinese haplotypes seem to have been transmitted independently between Cocos and other pig breeds, suggesting independent introgression events and a complex pattern of admixing. Although data are compatible with a founder population of N = 2, variability levels are as high in Cocos pigs as in international pig breeds (~1.9 SNPs/kb) and higher than in European wild boars or local breeds (~1.7 SNPs/kb). Nevertheless, we also report a 10‐Mb region with a marked decrease in variability across all samples that contains four genes (CPE, H3F3C, SC4MOL and KHL2) previously identified as highly differentiated between wild and domestic pigs. This work therefore illustrates how feral population genomic studies can help to resolve the history of domestic species and associated admixture events.     

Pike Esox lucius as an emerging model organism for studies in ecology and evolutionary biology: a review

The pike Esox lucius is a large, long‐lived, iteroparous, top‐ predator fish species with a circumpolar distribution that occupies a broad range of aquatic environments. This study reports on a literature search and demonstrates that the publication rate of E. lucius research increases both in absolute terms and relative to total scientific output, and that the focus of investigation has changed over time from being dominated by studies on physiology and disease to being gradually replaced by studies on ecology and evolution. Esox lucius can be exploited as a model in future research for identifying causes and consequences of phenotypic and genetic variation at the levels of individuals, populations and species as well as for investigating community processes.     

Genomic-scale capture and sequencing of endogenous DNA from feces

Genomic-level analyses of DNA from non-invasive sources would facilitate powerful conservation and evolutionary studies in natural populations of endangered and otherwise elusive species. However, the typical low quantity and poor quality of DNA that is extracted from non-invasive samples have generally precluded such work. Here we apply a modified DNA capture protocol that, when used in combination with massively-parallel sequencing technology, facilitates efficient and highly-accurate resequencing of megabases of specified nuclear genomic regions from fecal DNA samples. We validated our approach by comparing genetic variants identified from corresponding fecal and blood DNA samples of six western chimpanzees (Pan troglodytes verus) across more than 1.5 megabases of chromosome 21, chromosome X, and the complete mitochondrial genome. Our results suggest that it is now feasible to conduct genomic studies in natural populations for which constraints on invasive sampling have otherwise long been a barrier. The data we collected also provided an opportunity to examine western chimpanzee genetic diversity at unprecedented scale. Despite high mitochondrial genome diversity (π = 0.585%), western chimpanzees have a low ratio (0.42) of X chromosomal (π = 0.034%) to autosomal (chromosome 21 π = 0.081%) sequence diversity, a pattern that may reflect an unusual demographic history of this subspecies.