Evolution is more repeatable in the introduction than range expansion phase of colonization.

How repeatable is evolution at genomic and phenotypic scales? We studied the repeatability of evolution during 8 generations of colonization using replicated microcosm experiments with the red flour beetle, Tribolium castaneum. Based on the patterns of shared allele frequency changes that occurred in populations from the same generation or experimental location, we found adaptive evolution to be more repeatable in the introduction and establishment phases of colonization than in the spread phase, when populations expand their range. Lastly, by studying changes in allele frequencies at conserved loci, we found evidence for the theoretical prediction that range expansion reduces the efficiency of selection to purge deleterious alleles. Overall, our results increase our understanding of adaptive evolution during colonization, demonstrating that evolution can be highly repeatable while also showing that stochasticity still plays an important role.

Gene expression and alternative splicing contribute to adaptive divergence of ecotypes.

Regulation of gene expression is a critical link between genotype and phenotype explaining substantial heritable variation within species. However, we are only beginning to understand the ways that specific gene regulatory mechanisms contribute to adaptive divergence of populations. In plants, the post-transcriptional regulatory mechanism of alternative splicing (AS) plays an important role in both development and abiotic stress response, making it a compelling potential target of natural selection. AS allows organisms to generate multiple different transcripts/proteins from a single gene and thus may provide a source of evolutionary novelty. Here, we examine whether variation in alternative splicing and gene expression levels might contribute to adaptation and incipient speciation of dune-adapted prairie sunflowers in Great Sand Dunes National Park, Colorado, USA. We conducted a common garden experiment to assess transcriptomic variation among ecotypes and analyzed differential expression, differential splicing, and gene coexpression. We show that individual genes are strongly differentiated for both transcript level and alternative isoform proportions, even when grown in a common environment, and that gene coexpression networks are disrupted between ecotypes. Furthermore, we examined how genome-wide patterns of sequence divergence correspond to divergence in transcript levels and isoform proportions and find evidence for both cis and trans-regulation. Together, our results emphasize that alternative splicing has been an underappreciated mechanism providing source material for natural selection at short evolutionary time scales.

Heritable differences in abundance of bacterial rhizosphere taxa are correlated with fungal necrotrophic pathogen resistance.

Host-microbe interactions are increasingly recognized as important drivers of organismal health, growth, longevity and community-scale ecological processes. However, less is known about how genetic variation affects hosts' associated microbiomes and downstream phenotypes. We demonstrate that sunflower (Helianthus annuus) harbours substantial, heritable variation in microbial communities under field conditions. We show that microbial communities co-vary with heritable variation in resistance to root infection caused by the necrotrophic pathogen Sclerotinia sclerotiorum and that plants grown in autoclaved soil showed almost complete elimination of pathogen resistance. Association mapping suggests at least 59 genetic locations with effects on both microbial relative abundance and Sclerotinia resistance. Although the genetic architecture appears quantitative, we have elucidated previously unexplained genetic variation for resistance to this pathogen. We identify new targets for plant breeding and demonstrate the potential for heritable microbial associations to play important roles in defence in natural and human-altered environments.

Estimating phylogenies from genomes: A beginners review of commonly used genomic data in vertebrate phylogenomics.

Despite the increasing feasibility of sequencing whole genomes from diverse taxa, a persistent problem in phylogenomics is the selection of appropriate genetic markers or loci for a given taxonomic group or research question. In this review, we aim to streamline the decision-making process when selecting specific markers to use in phylogenomic studies by introducing commonly used types of genomic markers, their evolutionary characteristics, and their associated uses in phylogenomics. Specifically, we review the utilities of ultraconserved elements (including flanking regions), anchored hybrid enrichment loci, conserved nonexonic elements, untranslated regions, introns, exons, mitochondrial DNA, single nucleotide polymorphisms, and anonymous regions (nonspecific regions that are evenly or randomly distributed across the genome). These various genomic elements and regions differ in their substitution rates, likelihood of neutrality or of being strongly linked to loci under selection, and mode of inheritance, each of which are important considerations in phylogenomic reconstruction. These features may give each type of marker important advantages and disadvantages depending on the biological question, number of taxa sampled, evolutionary timescale, cost effectiveness, and analytical methods used. We provide a concise outline as a resource to efficiently consider key aspects of each type of genetic marker. There are many factors to consider when designing phylogenomic studies, and this review may serve as a primer when weighing options between multiple potential phylogenomic markers.

Adaptation to distinct habitats is maintained by contrasting selection at different life stages in sunflower ecotypes.

Conspecific populations living in adjacent but contrasting microenvironments represent excellent systems for studying natural selection. These systems are valuable because gene flow is expected to force genetic homogeneity except at loci experiencing divergent selection. A history of reciprocal transplant and common garden studies in such systems, and a growing number of genomic studies, have contributed to understanding how selection operates in natural populations. While selection can vary across different fitness components and life stages, few studies have investigated how this ultimately affects allele frequencies and the maintenance of divergence between populations. Here, we study two sunflower ecotypes in distinct, adjacent habitats by combining demographic models with genome-wide sequence data to estimate fitness and allele frequency change at multiple life stages. This framework allows us to estimate that only local ecotypes are likely to experience positive population growth (λ > 1) and that the maintenance of divergent adaptation appears to be mediated via habitat- and life stage-specific selection. We identify genetic variation, significantly driven by loci in chromosomal inversions, associated with different life history strategies in neighbouring ecotypes that optimize different fitness components and may contribute to the maintenance of distinct ecotypes.

Mitochondrial genomes do not appear to regulate flowering pattern/reproductive strategy in Cannabis sativa.

Currently, the amount of genetic data for Cannabis is lacking due to the illegal nature of the plant. Our study used 73 Cannabis sativa whole-genome shotgun libraries to reveal eight different mtDNA haplotypes. The most common haplotype contained 60 of the 73 samples studied and was composed of only dioecious individuals. However, other haplotypes contained a mix of both mating strategies (i.e. monoecious and dioecious). From these haplotype groupings we further examined the fully annotated mitochondrial genomes of four hemp individuals with different mt haplotypes and recorded gene content, copy number variation and synteny. Our results revealed highly syntenic mitochondrial genomes that contained ~60 identifiable sequences for protein-coding genes, tRNAs and rRNAs and no obvious rearrangements or chimeric genes. We found no clear evidence that modern reproductive patterns are due to simple cytoplasmic male sterility mutations. It is likely the interaction between nuclear genetic components and the X/Y sex chromosomes that determines reproductive strategy. Additionally, we added 50 % more mitochondrial genomes to the publicly available repository.

Effective strategies for isolating DNA from members of Asteraceae with high concentrations of secondary metabolites.

The Asteraceae are the largest plant family but among the least studied at the genome level. Our work investigated practical methods to reduce the influence of secondary metabolites - specifically, phenolic compounds - on the extraction of DNA from Silphium spp. This genus is in the Heliantheae tribe of Asteraceae that also includes sunflower (Helianthus annuus). Three methods were attempted in Silphium, with varying condition and age of the leaf sample. A modified cetyl trimethylammonium bromide (CTAB) method on young leaves resulted in the best DNA yield, with sufficient sample purity. No perceptible difference was observed between fresh and lyophilized samples for any extraction method or leaf age. These results provide an excellent basis for DNA extraction of difficult plant samples.

Genetic mapping of a pollinator preference trait: Nectar volume in sunflower (Helianthus annuus L.).

Although high pollinator visitation is crucial to ensure the yields of pollinator-dependent crops, the quantitative trait loci (QTL) controlling nectar volume in sunflower (Helianthus annuus L.), a pollinator preference trait, have yet to be identified. To address this, a recombinant inbred line mapping population, derived from lines with contrasting nectar volume, was used to identify loci responsible for the phenotype. As a result, linkage mapping and QTL analysis discovered major loci on chromosomes 2 and 16 that are associated with variation in nectar volume in sunflower. Increased nectar volume is also associated with increased sugars and total energy available per floret. The regions on chromosomes 2 and 16 associated with the nectar phenotype exhibit indications of chromosome structural variation, such that the phenotype is associated with rearrangements affecting regions containing hundreds of genes. Candidate genes underlying QTL on chromosomes 9 and 16 are homologous to genes with nectary function in Arabidopsis. These results have implications for sunflower breeding, to enhance pollination efficiency in sunflower, as well as current and future studies on sunflower evolution.

Genomic Evidence That Governmentally Produced Cannabis sativa Poorly Represents Genetic Variation Available in State Markets.

The National Institute on Drug Abuse (NIDA) is the sole producer of Cannabis for research purposes in the United States, including medical investigation. Previous research established that cannabinoid profiles in the NIDA varieties lacked diversity and potency relative to the Cannabis produced commercially. Additionally, microsatellite marker analyses have established that the NIDA varieties are genetically divergent form varieties produced in the private legal market. Here, we analyzed the genomes of multiple Cannabis varieties from diverse lineages including two produced by NIDA, and we provide further support that NIDA's varieties differ from widely available medical, recreational, or industrial Cannabis. Furthermore, our results suggest that NIDA's varieties lack diversity in the single-copy portion of the genome, the maternally inherited genomes, the cannabinoid genes, and in the repetitive content of the genome. Therefore, results based on NIDA's varieties are not generalizable regarding the effects of Cannabis after consumption. For medical research to be relevant, material that is more widely used would have to be studied. Clearly, having research to date dominated by a single, non-representative source of Cannabis has hindered scientific investigation.

Aberrant RNA splicing due to genetic incompatibilities in sunflower hybrids.

Genome-scale studies have revealed divergent mRNA splicing patterns between closely related species or populations. However, it is unclear whether splicing differentiation is a simple byproduct of population divergence, or whether it also acts as a mechanism for reproductive isolation. We examined mRNA splicing in wild × domesticated sunflower hybrids and observed 45 novel splice forms that were not found in the wild or domesticated parents, in addition to 16 high-expression parental splice forms that were absent in one or more hybrids. We identify loci associated with variation in the levels of these splice forms, finding that many aberrant transcripts were regulated by multiple alleles with nonadditive interactions. We identified particular spliceosome components that were associated with 21 aberrant isoforms, more than half of which were located in or near regulatory QTL. These incompatibilities often resulted in alteration in the protein-coding regions of the novel transcripts in the form of frameshifts and truncations. By associating the splice variation in these genes with size and growth rate measurements, we found that the cumulative expression of all aberrant transcripts was correlated with a significant reduction in growth rate. Our results lead us to propose a model where divergent splicing regulatory loci could act as incompatibility loci that contribute to the evolution of reproductive isolation.

Widely assumed phenotypic associations in Cannabis sativa lack a shared genetic basis.

The flowering plant Cannabis sativa, cultivated for centuries for multiple purposes, displays extensive variation in phenotypic traits in addition to its wide array of secondary metabolite production. Notably, Cannabis produces two well-known secondary-metabolite cannabinoids: cannabidiolic acid (CBDA) and delta-9-tetrahydrocannabinolic acid (THCA), which are the main products sought by consumers in the medical and recreational market. Cannabis has several suggested subspecies which have been shown to differ in chemistry, branching patterns, leaf morphology and other traits. In this study we obtained measurements related to phytochemistry, reproductive traits, growth architecture, and leaf morphology from 297 hybrid individuals from a cross between two diverse lineages. We explored correlations among these characteristics to inform our understanding of which traits may be causally associated. Many of the traits widely assumed to be strongly correlated did not show any relationship in this hybrid population. The current taxonomy and legal regulation within Cannabis is based on phenotypic and chemical characteristics. However, we find these traits are not associated when lineages are inter-crossed, which is a common breeding practice and forms the basis of most modern marijuana and hemp germplasms. Our results suggest naming conventions based on leaf morphology do not correspond to the chemical properties in plants with hybrid ancestry. Therefore, a new system for identifying variation within Cannabis is warranted that will provide reliable identifiers of the properties important for recreational and, especially, medical use.

Evolution of pathogen response genes associated with increased disease susceptibility during adaptation to an extreme drought in a Brassica rapa plant population.

BACKGROUND: Pathogens are key components in natural and agricultural plant systems. There is evidence of evolutionary changes in disease susceptibility as a consequence of climate change, but we know little about the underlying genetic basis of this evolution. To address this, we took advantage of a historical seed collection of a Brassica rapa population, which we previously demonstrated evolved an increase in disease susceptibility to a necrotrophic fungal pathogen following a drought. RESULTS: Previously, we combined a resurrection experiment with genome-wide sequencing of 124 pooled ancestral and descendant plants. Here, using these previously generated sequence data (Franks et al. in Mol Ecol 25(15):3622-3631, 2016), we show that well-characterized necrotrophic fungal pathogen response (NFPR) genes have evolved, as indicated by changes in allele frequency, between ancestors and descendants, with several of them identified as extreme FST outliers. The jasmonic acid (JA) signaling pathway in particular seems to underlie the evolution of disease susceptibility, in addition to its well characterized role in plastic disease response. We identify a list of 260 genes that are both NFPR genes and are differentially expressed in response to drought, based on publicly available data. We present evidence that five of these genes evolved between ancestors and descendants, suggesting that the drought acted as the evolutionary driver, and that the accompanying increase in disease susceptibility may have been a consequence of genetic pleiotropy. CONCLUSIONS: Our study provides evidence that for this population, standing variation in NFPR genes is affected by natural selection related to climate change. Our results reveal potentially important candidates that may underlie trait evolution in both crops and natural systems. Additionally, this trade-off between adaptation to biotic and abiotic stresses is an example of how climate change can have diverse and unexpected consequences.

Breeding for sustainable oilseed crop yield and quality in a changing climate.

As the effects of climate change continue to alter crop-growing conditions year-to-year on both prime and marginal agricultural landscapes, we must consider the effects not only on yield but also on quality. This is particularly true for oilseed crops. In this review, we explore the importance of oilseeds in general and the specific uses of major oilseed crops including soybean, sunflower, canola, peanut, and cottonseed. We review the physiology of seed oil production, from the perspective of the plant's adaptation to environmental changes. Of particular importance is the role of temperature and water availability on oil synthesis. We then discuss how this influences genetic variation, phenotype variability due to environment, and the interaction of genetics and environment to affect composition and yield of vegetable oils. The ability to predict these effects using genomics and bioinformatics is an important new frontier for breeders to maximize stability of a desired fatty acid composition for their crop over increasingly extreme agricultural environments.

Genetic loci underlying quantitative resistance to necrotrophic pathogens Sclerotinia and Diaporthe (Phomopsis), and correlated resistance to both pathogens.

KEY MESSAGE: We provide results rooted in quantitative genetics, which combined with knowledge of candidate gene function, helps us to better understand the resistance to two major necrotrophic pathogens of sunflower. Necrotrophic pathogens can avoid or even benefit from plant defenses used against biotrophic pathogens, and thus represent a distinct challenge to plant populations in natural and agricultural systems. Sclerotinia and Phomopsis/Diaporthe are detrimental pathogens for many dicotyledonous plants, including many economically important plants. With no well-established methods to prevent infection in susceptible plants, host-plant resistance is currently the most effective strategy. Despite knowledge of a moderate, positive correlation in resistance to the two diseases in sunflower, detailed analysis of the genetics, in the same populations, has not been conducted. We present results of genome-wide analysis of resistance to both pathogens in a diversity panel of 218 domesticated sunflower genotypes of worldwide origin. We identified 14 Sclerotinia head rot and 7 Phomopsis stem canker unique QTLs, plus 1 co-located QTL for both traits, and observed extensive patterns of linkage disequilibrium between sites for both traits. Most QTLs contained one credible candidate gene, and gene families were common for the two disease resistance traits. These results suggest there has been strong, simultaneous selection for resistance to these two diseases and that a generalized mechanism for defense against these necrotrophic pathogens exists.

Rapid evolutionary changes in gene expression in response to climate fluctuations.

There is now abundant evidence of rapid evolution in natural populations, but the genetic mechanisms of these changes remain unclear. One possible route to rapid evolution is through changes in the expression of genes that influence traits under selection. We examined contemporary evolutionary gene expression changes in plant populations responding to environmental fluctuations. We compared genome-wide gene expression, using RNA-seq, in two populations of Brassica rapa collected over four time points between 1997 and 2014, during which precipitation in southern California fluctuated dramatically and phenotypic and genotypic changes occurred. By combining transcriptome profiling with the resurrection approach, we directly examined evolutionary changes in gene expression over time. For both populations, we found a substantial number of differentially expressed genes between generations, indicating rapid evolution in the expression of many genes. Using existing gene annotations, we found that many changes occurred in genes involved in regulating stress responses and flowering time. These appeared related to the fluctuations in precipitation and were potentially adaptive. However, the evolutionary changes in gene expression differed across generations within and between populations, indicating largely independent evolutionary trajectories across populations and over time. Our study provides strong evidence for rapid evolution in gene expression, and indicates that changes in gene expression can be one mechanism of rapid evolutionary responses to selection episodes. This study also illustrates that combining resurrection studies with transcriptomics is a powerful approach for investigating evolutionary changes at the gene regulatory level, and will provide new insights into the genetic basis of contemporary evolution.

Complete mitochondrial genomes provide current refined phylogenomic hypotheses for relationships among ten Hirundo species.

Hirundo is the most species-rich genus of the passerine swallow family (Hirundinidae) and has a cosmopolitan distribution. Here we report the complete, annotated mitochondrial genomes for 25 individuals from 10 of the 14 extant Hirundo species; these include representatives from four subspecies of the barn swallow, H. rustica. Mitogenomes were conserved in size, ranging from 18,500 to 18,700 base pairs. They all contained 13 protein-coding regions, 22 tRNAs, a control region, and large and small ribosomal subunits. Phylogenetic analysis resolved most of the relationships between the studied species and subspecies which were largely consistent with previously published trees. Several new relationships were observed within the phylogeny that could have only been discovered with the increased amount of genetic material. This study represents the largest Hirundo mitochondrial phylogeny to date, and could serve as a vital tool for other studies focusing on the evolution of the Hirundo genus.