Landscape Connectivity and Genetic Structure in a Mainstem and a Tributary Stonefly (Plecoptera) Species Using a Novel Reference Genome.

Understanding how environmental variation influences population genetic structure can help predict how environmental change influences population connectivity, genetic diversity, and evolutionary potential. We used riverscape genomics modeling to investigate how climatic and habitat variables relate to patterns of genetic variation in 2 stonefly species, one from mainstem river habitats (Sweltsa coloradensis) and one from tributaries (Sweltsa fidelis) in 40 sites in northwest Montana, USA. We produced a draft genome assembly for S. coloradensis (N50 = 0.251 Mbp, BUSCO > 95% using "insecta_ob9" reference genes). We genotyped 1930 SNPs in 372 individuals for S. coloradensis and 520 SNPs in 153 individuals for S. fidelis. We found higher genetic diversity for S. coloradensis compared to S. fidelis, but nearly identical genetic differentiation among sites within each species (both had global loci median FST = 0.000), despite differences in stream network location. For landscape genomics and testing for selection, we produced a less stringently filtered data set (3454 and 1070 SNPs for S. coloradensis and S. fidelis, respectively). Environmental variables (mean summer precipitation, slope, aspect, mean June stream temperature, land cover type) were correlated with 19 putative adaptive loci for S. coloradensis, but there was only one putative adaptive locus for S. fidelis (correlated with aspect). Interestingly, we also detected potential hybridization between multiple Sweltsa species which has never been previously detected. Studies like ours, that test for adaptive variation in multiple related species are needed to help assess landscape connectivity and the vulnerability of populations and communities to environmental change.

Evidence for adaptive introgression of exons across a hybrid swarm in deer.

BACKGROUND: Secondary contact between closely related lineages can result in a variety of outcomes, including hybridization, depending upon the strength of reproductive barriers. By examining the extent to which different parts of the genome introgress, it is possible to infer the strength of selection and gain insight into the evolutionary trajectory of lineages. Following secondary contact approximately 8000 years ago in the Pacific Northwest, mule deer (Odocoileus hemionus hemionus) and black-tailed deer (O. h. columbianus) formed a hybrid swarm along the Cascade mountain range despite substantial differences in body size (up to two times) and habitat preference. In this study, we examined genetic population structure, extent of introgression, and selection pressures in freely interbreeding populations of mule deer and black-tailed deer using mitochondrial DNA sequences, 9 microsatellite loci, and 95 SNPs from protein-coding genes. RESULTS: We observed bi-directional hybridization and classified approximately one third of the 172 individuals as hybrids, almost all of which were beyond the F1 generation. High genetic differentiation between black-tailed deer and mule deer at protein-coding genes suggests that there is positive divergent selection, though selection on these loci is relatively weak. Contrary to predictions, there was not greater selection on protein-coding genes thought to be associated with immune function and mate choice. Geographic cline analyses were consistent across genetic markers, suggesting long-term stability (over hundreds of generations), and indicated that the center of the hybrid swarm is 20-30 km to the east of the Cascades ridgeline, where there is a steep ecological transition from wet, forested habitat to dry, scrub habitat. CONCLUSIONS: Our data are consistent with a genetic boundary between mule deer and black-tailed deer that is porous but maintained by many loci under weak selection having a substantial cumulative effect. The absence of clear reproductive barriers and the consistent centering of geographic clines at a sharp ecotone suggests that ecology is a driver of hybrid swarm dynamics. Adaptive introgression in this study (and others) promotes gene flow and provides valuable insight into selection strength on specific genes and the evolutionary trajectory of hybridizing taxa.

Assessing thermal adaptation using family-based association and FST outlier tests in a threatened trout species.

Discovering genetic markers associated with phenotypic or ecological characteristics can improve our understanding of adaptation and guide conservation of key evolutionary traits. The Lahontan cutthroat trout (Oncorhynchus clarkii henshawi) of the northern Great Basin Desert, USA, demonstrated exceptional tolerance to high temperatures in the desert lakes where it resided historically. This trait is central to a conservation hatchery effort to protect the genetic legacy of the nearly extinct lake ecotype. We genotyped full-sibling families from this conservation broodstock and samples from the only two remaining, thermally distinct, native lake populations at 4,644 new single nucleotide polymorphisms (SNPs). Family-based genome-wide association testing of the broodstock identified nine and 26 SNPs associated with thermal tolerance (p < 0.05 and p < 0.1), measured in a previous thermal challenge experiment. Genes near the associated SNPs had complex functions related to immunity, growth, metabolism and ion homeostasis. Principal component analysis using the thermotolerance-related SNPs showed unexpected divergence between the conservation broodstock and the native lake populations at these loci. FST outlier tests on the native lake populations identified 18 loci shared between two or more of the tests, with two SNPs identified by all three tests (p < 0.01); none overlapped with loci identified by association testing in the broodstock. A recent history of isolation and the complex genetic and demographic backgrounds of Lahontan cutthroat trout probably limited our ability to find shared thermal tolerance loci. Our study extends the still relatively rare application of genomic tools testing for markers associated with important phenotypic or environmental characteristics in species of conservation concern.

Signatures of polygenic adaptation associated with climate across the range of a threatened fish species with high genetic connectivity.

Adaptive differences across species' ranges can have important implications for population persistence and conservation management decisions. Despite advances in genomic technologies, detecting adaptive variation in natural populations remains challenging. Key challenges in gene-environment association studies involve distinguishing the effects of drift from those of selection and identifying subtle signatures of polygenic adaptation. We used paired-end restriction site-associated DNA sequencing data (6,605 biallelic single nucleotide polymorphisms; SNPs) to examine population structure and test for signatures of adaptation across the geographic range of an iconic Australian endemic freshwater fish species, the Murray cod Maccullochella peelii. Two univariate gene-association methods identified 61 genomic regions associated with climate variation. We also tested for subtle signatures of polygenic adaptation using a multivariate method (redundancy analysis; RDA). The RDA analysis suggested that climate (temperature- and precipitation-related variables) and geography had similar magnitudes of effect in shaping the distribution of SNP genotypes across the sampled range of Murray cod. Although there was poor agreement among the candidate SNPs identified by the univariate methods, the top 5% of SNPs contributing to significant RDA axes included 67% of the SNPs identified by univariate methods. We discuss the potential implications of our findings for the management of Murray cod and other species generally, particularly in relation to informing conservation actions such as translocations to improve evolutionary resilience of natural populations. Our results highlight the value of using a combination of different approaches, including polygenic methods, when testing for signatures of adaptation in landscape genomic studies.

Legacy introductions and climatic variation explain spatiotemporal patterns of invasive hybridization in a native trout.

Hybridization between invasive and native species, a significant threat to worldwide biodiversity, is predicted to increase due to climate-induced expansions of invasive species. Long-term research and monitoring are crucial for understanding the ecological and evolutionary processes that modulate the effects of invasive species. Using a large, multidecade genetics dataset (N = 582 sites, 12,878 individuals) with high-resolution climate predictions and extensive stocking records, we evaluate the spatiotemporal dynamics of hybridization between native cutthroat trout and invasive rainbow trout, the world's most widely introduced invasive fish, across the Northern Rocky Mountains of the United States. Historical effects of stocking and contemporary patterns of climatic variation were strongly related to the spread of hybridization across space and time. The probability of occurrence, extent of, and temporal changes in hybridization increased at sites in close proximity to historical stocking locations with greater rainbow trout propagule pressure, warmer water temperatures, and lower spring precipitation. Although locations with warmer water temperatures were more prone to hybridization, cold sites were not protected from invasion; 58% of hybridized sites had cold mean summer water temperatures (<11°C). Despite cessation of stocking over 40 years ago, hybridization increased over time at half (50%) of the locations with long-term data, the vast majority of which (74%) were initially nonhybridized, emphasizing the chronic, negative impacts of human-mediated hybridization. These results show that effects of climate change on biodiversity must be analyzed in the context of historical human impacts that set ecological and evolutionary trajectories.

Vive la résistance: genome-wide selection against introduced alleles in invasive hybrid zones.

Evolutionary and ecological consequences of hybridization between native and invasive species are notoriously complicated because patterns of selection acting on non-native alleles can vary throughout the genome and across environments. Rapid advances in genomics now make it feasible to assess locus-specific and genome-wide patterns of natural selection acting on invasive introgression within and among natural populations occupying diverse environments. We quantified genome-wide patterns of admixture across multiple independent hybrid zones of native westslope cutthroat trout and invasive rainbow trout, the world's most widely introduced fish, by genotyping 339 individuals from 21 populations using 9380 species-diagnostic loci. A significantly greater proportion of the genome appeared to be under selection favouring native cutthroat trout (rather than rainbow trout), and this pattern was pervasive across the genome (detected on most chromosomes). Furthermore, selection against invasive alleles was consistent across populations and environments, even in those where rainbow trout were predicted to have a selective advantage (warm environments). These data corroborate field studies showing that hybrids between these species have lower fitness than the native taxa, and show that these fitness differences are due to selection favouring many native genes distributed widely throughout the genome.

Genomic patterns of introgression in rainbow and westslope cutthroat trout illuminated by overlapping paired-end RAD sequencing.

Rapid and inexpensive methods for genomewide single nucleotide polymorphism (SNP) discovery and genotyping are urgently needed for population management and conservation. In hybridized populations, genomic techniques that can identify and genotype thousands of species-diagnostic markers would allow precise estimates of population- and individual-level admixture as well as identification of 'super invasive' alleles, which show elevated rates of introgression above the genomewide background (likely due to natural selection). Techniques like restriction-site-associated DNA (RAD) sequencing can discover and genotype large numbers of SNPs, but they have been limited by the length of continuous sequence data they produce with Illumina short-read sequencing. We present a novel approach, overlapping paired-end RAD sequencing, to generate RAD contigs of >300-400 bp. These contigs provide sufficient flanking sequence for design of high-throughput SNP genotyping arrays and strict filtering to identify duplicate paralogous loci. We applied this approach in five populations of native westslope cutthroat trout that previously showed varying (low) levels of admixture from introduced rainbow trout (RBT). We produced 77 141 RAD contigs and used these data to filter and genotype 3180 previously identified species-diagnostic SNP loci. Our population-level and individual-level estimates of admixture were generally consistent with previous microsatellite-based estimates from the same individuals. However, we observed slightly lower admixture estimates from genomewide markers, which might result from natural selection against certain genome regions, different genomic locations for microsatellites vs. RAD-derived SNPs and/or sampling error from the small number of microsatellite loci (n = 7). We also identified candidate adaptive super invasive alleles from RBT that had excessively high admixture proportions in hybridized cutthroat trout populations.

Hybridization alters growth and migratory life-history expression of native trout.

Human-mediated hybridization threatens many native species, but the effects of introgressive hybridization on life-history expression are rarely quantified, especially in vertebrates. We quantified the effects of non-native rainbow trout admixture on important life-history traits including growth and partial migration behavior in three populations of westslope cutthroat trout over five years. Rainbow trout admixture was associated with increased summer growth rates in all populations and decreased spring growth rates in two populations with cooler spring temperatures. These results indicate that non-native admixture may increase growth under warmer conditions, but cutthroat trout have higher growth rates during cooler periods. Non-native admixture consistently increased expression of migratory behavior, suggesting that there is a genomic basis for life-history differences between these species. Our results show that effects of interspecific hybridization on fitness traits can be the product of genotype-by-environment interactions even when there are minor differences in environmental optima between hybridizing species. These results also indicate that while environmentally mediated traits like growth may play a role in population-level consequences of admixture, strong genetic influences on migratory life-history differences between these species likely explains the continued spread of non-native hybridization at the landscape-level, despite selection against hybrids at the population-level.

Mapping of Adaptive Traits Enabled by a High-Density Linkage Map for Lake Trout.

Understanding the genomic basis of adaptative intraspecific phenotypic variation is a central goal in conservation genetics and evolutionary biology. Lake trout (Salvelinus namaycush) are an excellent species for addressing the genetic basis for adaptive variation because they express a striking degree of ecophenotypic variation across their range; however, necessary genomic resources are lacking. Here we utilize recently-developed analytical methods and sequencing technologies to (1) construct a high-density linkage and centromere map for lake trout, (2) identify loci underlying variation in traits that differentiate lake trout ecophenotypes and populations, (3) determine the location of the lake trout sex determination locus, and (4) identify chromosomal homologies between lake trout and other salmonids of varying divergence. The resulting linkage map contains 15,740 single nucleotide polymorphisms (SNPs) mapped to 42 linkage groups, likely representing the 42 lake trout chromosomes. Female and male linkage group lengths ranged from 43.07 to 134.64 centimorgans, and 1.97 to 92.87 centimorgans, respectively. We improved the map by determining coordinates for 41 of 42 centromeres, resulting in a map with 8 metacentric chromosomes and 34 acrocentric or telocentric chromosomes. We use the map to localize the sex determination locus and multiple quantitative trait loci (QTL) associated with intraspecific phenotypic divergence including traits related to growth and body condition, patterns of skin pigmentation, and two composite geomorphometric variables quantifying body shape. Two QTL for the presence of vermiculations and spots mapped with high certainty to an arm of linkage group Sna3, growth related traits mapped to two QTL on linkage groups Sna1 and Sna12, and putative body shape QTL were detected on six separate linkage groups. The sex determination locus was mapped to Sna4 with high confidence. Synteny analysis revealed that lake trout and congener Arctic char (Salvelinus alpinus) are likely differentiated by three or four chromosomal fissions, possibly one chromosomal fusion, and 6 or more large inversions. Combining centromere mapping information with putative inversion coordinates revealed that the majority of detected inversions differentiating lake trout from other salmonids are pericentric and located on acrocentric and telocentric linkage groups. Our results suggest that speciation and adaptive divergence within the genus Salvelinus may have been associated with multiple pericentric inversions occurring primarily on acrocentric and telocentric chromosomes. The linkage map presented here will be a critical resource for advancing conservation oriented genomic research on lake trout and exploring chromosomal evolution within and between salmonid species.

Candidate adaptive genes associated with lineage divergence: identifying SNPs via next-generation targeted resequencing in mule deer (Odocoileus hemionus).

Mule deer (Odocoileus hemionus) are an excellent nonmodel species for empirically testing hypotheses in landscape and population genomics due to their large population sizes (low genetic drift), relatively continuous distribution, diversity of occupied habitats and phenotypic variation. Because few genomic resources are currently available for this species, we used exon data from a cattle (Bos taurus) reference genome to direct targeted resequencing of 5935 genes in mule deer. We sequenced approximately 3.75 Mbp at minimum 20X coverage in each of the seven mule deer, identifying 23 204 single nucleotide polymorphisms (SNPs) within, or adjacent to, 6886 exons in 3559 genes. We found 91 SNP loci (from 69 genes) with putatively fixed allele frequency differences between the two major lineages of mule deer (mule deer and black-tailed deer), and our estimate of mean genetic divergence (genome-wide FST  = 0.123) between these lineages was consistent with previous findings using microsatellite loci. We detected an over-representation of gamete generation and amino acid transport genes among the genes with SNPs exhibiting potentially fixed allele frequency differences between lineages. This targeted resequencing approach using exon capture techniques has identified a suite of loci that can be used in future research to investigate the genomic basis of adaptation and differentiation between black-tailed deer and mule deer. This study also highlights techniques (and an exon capture array) that will facilitate population genomic research in other cervids and nonmodel organisms.

SNP discovery in candidate adaptive genes using exon capture in a free-ranging alpine ungulate.

Identification of genes underlying genomic signatures of natural selection is key to understanding adaptation to local conditions. We used targeted resequencing to identify SNP markers in 5321 candidate adaptive genes associated with known immunological, metabolic and growth functions in ovids and other ungulates. We selectively targeted 8161 exons in protein-coding and nearby 5' and 3' untranslated regions of chosen candidate genes. Targeted sequences were taken from bighorn sheep (Ovis canadensis) exon capture data and directly from the domestic sheep genome (Ovis aries v. 3; oviAri3). The bighorn sheep sequences used in the Dall's sheep (Ovis dalli dalli) exon capture aligned to 2350 genes on the oviAri3 genome with an average of 2 exons each. We developed a microfluidic qPCR-based SNP chip to genotype 476 Dall's sheep from locations across their range and test for patterns of selection. Using multiple corroborating approaches (lositan and bayescan), we detected 28 SNP loci potentially under selection. We additionally identified candidate loci significantly associated with latitude, longitude, precipitation and temperature, suggesting local environmental adaptation. The three methods demonstrated consistent support for natural selection on nine genes with immune and disease-regulating functions (e.g. Ovar-DRA, APC, BATF2, MAGEB18), cell regulation signalling pathways (e.g. KRIT1, PI3K, ORRC3), and respiratory health (CYSLTR1). Characterizing adaptive allele distributions from novel genetic techniques will facilitate investigation of the influence of environmental variation on local adaptation of a northern alpine ungulate throughout its range. This research demonstrated the utility of exon capture for gene-targeted SNP discovery and subsequent SNP chip genotyping using low-quality samples in a nonmodel species.

RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a much improved restriction site-associated DNA (RAD) sequencing protocol and a new method called Rapture ( R: AD c APTURE: ). The new RAD protocol improves versatility by separating RAD tag isolation and sequencing library preparation into two distinct steps. This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses an in-solution capture of chosen RAD tags to target sequencing reads to desired loci. Rapture combines the benefits of both RAD and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. Our results demonstrate that Rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. The methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science.

RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays.

Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.

Next-generation RAD sequencing identifies thousands of SNPs for assessing hybridization between rainbow and westslope cutthroat trout.

The increased numbers of genetic markers produced by genomic techniques have the potential to both identify hybrid individuals and localize chromosomal regions responding to selection and contributing to introgression. We used restriction-site-associated DNA sequencing to identify a dense set of candidate SNP loci with fixed allelic differences between introduced rainbow trout (Oncorhynchus mykiss) and native westslope cutthroat trout (Oncorhynchus clarkii lewisi). We distinguished candidate SNPs from homeologs (paralogs resulting from whole-genome duplication) by detecting excessively high observed heterozygosity and deviations from Hardy-Weinberg proportions. We identified 2923 candidate species-specific SNPs from a single Illumina sequencing lane containing 24 barcode-labelled individuals. Published sequence data and ongoing genome sequencing of rainbow trout will allow physical mapping of SNP loci for genome-wide scans and will also provide flanking sequence for design of qPCR-based TaqMan(®) assays for high-throughput, low-cost hybrid identification using a subset of 50-100 loci. This study demonstrates that it is now feasible to identify thousands of informative SNPs in nonmodel species quickly and at reasonable cost, even if no prior genomic information is available.