Transcriptional responses to water stress and recovery in a drought-tolerant fescue wild grass (Festuca ovina; Poaceae).

Water stress associated with drought-like conditions is a major factor limiting plant growth and impacts productivity of natural plant communities and agricultural crops. Molecular responses of plants to water stress have been studied most extensively in model species and crops, few of which have evolved natural drought tolerance. In the current study, we examined physiological and transcriptomic responses at multiple timepoints during increasing water stress and following initial recovery from stress in a drought-tolerant C3 species, Festuca ovina. Results demonstrated non-linear transcriptomic changes during increasing stress, but largely linear declines in physiological measurements during this same period. Transcription factors represented approximately 12.7% of all differentially expressed genes. In total, 117 F. ovina homologs of previously identified and molecularly characterized drought-responsive plant genes were identified. This information will be valuable for further investigations of the molecular mechanisms involved in drought tolerance in C3 plants.

Phylogenetic trends and environmental correlates of nuclear genome size variation in Helianthus sunflowers.

Flowering plants serve as a powerful model for studying the evolution of nuclear genome size (GS) given the tremendous GS variation that exists both within and across angiosperm lineages. Helianthus sunflowers consist of c. 50 species native to North America that occupy diverse habitats and vary in ploidy level. In the current study, we generated a comprehensive GS database for 49 Helianthus species using flow cytometric approaches. We examined variability across the genus and present a comparative phylogenetic analysis of GS evolution in diploid Helianthus species. Results demonstrated that different clades of diploid Helianthus species showed evolutionary patterns of GS contraction, expansion and relative stasis, with annual diploid species evolving smaller GS with the highest rate of evolution. Phylogenetic comparative analyses of diploids revealed significant negative associations of GS with temperature seasonality and cell production rate, indicating that the evolution of larger GS in Helianthus diploids may be more permissible in habitats with longer growing seasons where selection for more rapid growth may be relaxed. The Helianthus GS database presented here and corresponding analyses of environmental and phenotypic correlates will facilitate ongoing and future research on the ultimate drivers of GS evolution in this well-studied North American plant genus.

Genomic abundance and transcriptional activity of diverse gypsy and copia long terminal repeat retrotransposons in three wild sunflower species.

BACKGROUND: Long terminal repeat (LTR) retrotransposons are highly abundant in plant genomes and require transcriptional activity for their proliferative mode of replication. These sequences exist in plant genomes as diverse sublineages within the main element superfamilies (i.e., gypsy and copia). While transcriptional activity of these elements is increasingly recognized as a regular attribute of plant transcriptomes, it is currently unknown the extent to which different sublineages of these elements are transcriptionally active both within and across species. In the current report, we utilize next generation sequencing methods to examine genomic copy number abundance of diverse LTR retrotransposon sublineages and their corresponding levels of transcriptional activity in three diploid wild sunflower species, Helianthus agrestis, H. carnosus and H. porteri. RESULTS: The diploid sunflower species under investigation differ in genome size 2.75-fold, with 2C values of 22.93 for H. agrestis, 12.31 for H. carnosus and 8.33 for H. porteri. The same diverse gypsy and copia sublineages of LTR retrotransposons were identified across species, but with gypsy sequences consistently more abundant than copia and with global gypsy sequence abundance positively correlated with nuclear genome size. Transcriptional activity was detected for multiple copia and gypsy sequences, with significantly higher activity levels detected for copia versus gypsy. Interestingly, of 11 elements identified as transcriptionally active, 5 exhibited detectable expression in all three species and 3 exhibited detectable expression in two species. CONCLUSIONS: Combined analyses of LTR retrotransposon genomic abundance and transcriptional activity across three sunflower species provides novel insights into genome size evolution and transposable element dynamics in this group. Despite considerable variation in nuclear genome size among species, relatively conserved patterns of LTR retrotransposon transcriptional activity were observed, with a highly overlapping set of copia and gypsy sequences observed to be transcriptionally active across species. A higher proportion of copia versus gypsy elements were found to be transcriptionally active and these sequences also were expressed at higher levels.

Investigation of mitochondrial-derived plastome sequences in the Paspalum lineage (Panicoideae; Poaceae).

BACKGROUND: The grass family (Poaceae), ca. 12,075 species, is a focal point of many recent studies that aim to use complete plastomes to reveal and strengthen relationships within the family. The use of Next Generation Sequencing technology has revealed intricate details in many Poaceae plastomes; specifically the trnI - trnL intergenic spacer region. This study investigates this region and the putative mitochondrial inserts within it in complete plastomes of Paspalum and other Poaceae. RESULTS: Nine newly sequenced plastomes, seven of which contain an insert within the trnI - trnL intergenic spacer, were combined into plastome phylogenomic and divergence date analyses with 52 other species. A robust Paspalum topology was recovered, originating at 10.6 Ma, with the insert arising at 8.7 Ma. The alignment of the insert across Paspalum reveals 21 subregions with pairwise homology in 19. In an analysis of emergent self-organizing maps of tetranucleotide frequencies, the Paspalum insert grouped with mitochondrial DNA. CONCLUSIONS: A hypothetical ancestral insert, 17,685 bp in size, was found in the trnI - trnL intergenic spacer for the Paspalum lineage. A different insert, 2808 bp, was found in the same region for Paraneurachne muelleri. Seven different intrastrand deletion events were found within the Paspalum lineage, suggesting selective pressures to remove large portions of noncoding DNA. Finally, a tetranucleotide frequency analysis was used to determine that the origin of the insert in the Paspalum lineage is mitochondrial DNA.

Freeze-induced cyanide toxicity does not maintain the cyanogenesis polymorphism in white clover (Trifolium repens).

PREMISE OF THE STUDY: The maintenance of adaptive polymorphisms within species requires fitness trade-offs reflecting selection for each morph. Cyanogenesis, the ability to produce hydrogen cyanide (HCN) after tissue damage, occurs in >3000 plant species and exists as a discrete polymorphism in white clover. This polymorphism is spatially distributed in recurrent clines, with higher frequencies of cyanogenic plants in warmer climates. The HCN autotoxicity hypothesis proposes that cyanogenic plants are selected against where frosts are common, as freezing liberates HCN and could impair cellular respiration. METHODS: We tested the HCN autotoxicity hypothesis using a freezing chamber to examine survival, tissue damage, and physiological recovery as assessed via chlorophyll fluorescence following mild and severe freezing treatments. We utilized 65 genotypes from a single polymorphic population to eliminate effects of population structure. KEY RESULTS: Cyanogenic plants did not differ from acyanogenic plants in survival, tissue damage, or recovery following freezing. However, plants producing either of the two required cyanogenic precursors had lower survival and tissue damage after freezing than plants lacking both precursors. CONCLUSIONS: These results suggest that freezing-induced HCN toxicity is unlikely to be responsible for the maintenance of the cyanogenesis polymorphism in white clover. However, energetic trade-offs associated with costs of producing the cyanogenic precursors may confer a fitness benefit to acyanogenic plants under stressful climatic conditions. The lack of evidence for HCN toxicity suggests that cyanogenic clover uses physiological mechanisms mediated by ß-cyanoalanine synthase and alternative oxidase to maintain cellular function in the presence of HCN.

The value of long-term ecological research for evolutionary insights.

Scientists must have an integrative understanding of ecology and evolution across spatial and temporal scales to predict how species will respond to global change. Although comprehensively investigating these processes in nature is challenging, the infrastructure and data from long-term ecological research networks can support cross-disciplinary investigations. We propose using these networks to advance our understanding of fundamental evolutionary processes and responses to global change. For ecologists, we outline how long-term ecological experiments can be expanded for evolutionary inquiry, and for evolutionary biologists, we illustrate how observed long-term ecological patterns may motivate new evolutionary questions. We advocate for collaborative, multi-site investigations and discuss barriers to conducting evolutionary work at network sites. Ultimately, these networks offer valuable information and opportunities to improve predictions of species' responses to global change.

The sunflower (Helianthus annuus L.) genome reflects a recent history of biased accumulation of transposable elements.

Aside from polyploidy, transposable elements are the major drivers of genome size increases in plants. Thus, understanding the diversity and evolutionary dynamics of transposable elements in sunflower (Helianthus annuus L.), especially given its large genome size (∼3.5 Gb) and the well-documented cases of amplification of certain transposons within the genus, is of considerable importance for understanding the evolutionary history of this emerging model species. By analyzing approximately 25% of the sunflower genome from random sequence reads and assembled bacterial artificial chromosome (BAC) clones, we show that it is composed of over 81% transposable elements, 77% of which are long terminal repeat (LTR) retrotransposons. Moreover, the LTR retrotransposon fraction in BAC clones harboring genes is disproportionately composed of chromodomain-containing Gypsy LTR retrotransposons ('chromoviruses'), and the majority of the intact chromoviruses contain tandem chromodomain duplications. We show that there is a bias in the efficacy of homologous recombination in removing LTR retrotransposon DNA, thereby providing insight into the mechanisms associated with transposable element (TE) composition in the sunflower genome. We also show that the vast majority of observed LTR retrotransposon insertions have likely occurred since the origin of this species, providing further evidence that biased LTR retrotransposon activity has played a major role in shaping the chromatin and DNA landscape of the sunflower genome. Although our findings on LTR retrotransposon age and structure could be influenced by the selection of the BAC clones analyzed, a global analysis of random sequence reads indicates that the evolutionary patterns described herein apply to the sunflower genome as a whole.

Genetic variation and mating success in managed american plains bison.

The American plains bison (Bison bison) was pushed to the brink of extinction in the late 1800s but has since rebounded. Less than 5% of animals currently exist in conservation herds that are critical for maintaining genetic variability. Here, we use 25 microsatellite loci to assess genetic diversity and patterns of mating success over a 3-year period in a managed conservation herd at Konza Prairie Biological Station, Kansas (total number of individuals genotyped = 587). Heterozygosity was comparable to and allelic diversity higher than that in 11 other wild and managed herds for which similar estimates are available. Parentage analyses revealed that males within the oldest age classes (5-7 years) sired >90% of calves over the study period, consistent with a polygynous breeding system. Asymmetries in siring success also were observed within age classes, with the same males enjoying high siring success over multiple seasons. Empirical results of paternity will facilitate future modeling and empirical efforts to determine how demographic factors, population size, and variation in siring success interact to determine the retention (or loss) of genetic diversity in natural and managed herds, thus allowing informed recommendations for management practices and conservation efforts of this symbolic North American species.

Fitness benefits and costs of cold acclimation in Arabidopsis thaliana.

Abstract When resources are limited, there is a trade-off between growth/reproduction and stress defense in plants. Most temperate plant species, including Arabidopsis thaliana, can enhance freezing tolerance through cold acclimation at low but nonfreezing temperatures. Induction of the cold acclimation pathway should be beneficial in environments where plants frequently encounter freezing stress, but it might represent a cost in environments where freezing events are rare. In A. thaliana, induction of the cold acclimation pathway critically involves a small subfamily of genes known as the CBFs. Here we test for a cost of cold acclimation by utilizing (1) natural accessions of A. thaliana that originate from different regions of the species' native range and that have experienced different patterns of historical selection on their CBF genes and (2) transgenic CBF overexpression and T-DNA insertion (knockdown/knockout) lines. While benefits of cold acclimation in the presence of freezing stress were confirmed, no cost of cold acclimation was detected in the absence of freezing stress. These findings suggest that cold acclimation is unlikely to be selected against in warmer environments and that naturally occurring mutations disrupting CBF function in the southern part of the species range are likely to be selectively neutral. An unanticipated finding was that cold acclimation in the absence of a subsequent freezing stress resulted in increased fruit production, that is, fitness.

Natural selection drives clinal life history patterns in the perennial sunflower species, Helianthus maximiliani.

In plants, ecologically important life history traits often display clinal patterns of population divergence. Such patterns can provide strong evidence for spatially varying selection across environmental gradients but also may result from nonselective processes, such as genetic drift, population bottlenecks and spatially restricted gene flow. Comparison of population differentiation in quantitative traits (measured as Q(ST) ) with neutral molecular markers (measured as F(ST) ) provides a useful tool for understanding the relative importance of adaptive and nonadaptive processes in the formation and maintenance of clinal variation. Here, we demonstrate the existence of geographic variation in key life history traits in the diploid perennial sunflower species Helianthus maximiliani across a broad latitudinal transect in North America. Strong population differentiation was found for days to flowering, growth rate and multiple size-related traits. Differentiation in these traits greatly exceeds neutral predictions, as determined both by partial Mantel tests and by comparisons of global Q(ST) values with theoretical F(ST) distributions. These findings indicate that clinal variation in these life history traits likely results from local adaptation driven by spatially heterogeneous environments.

Proliferation of Ty3/gypsy-like retrotransposons in hybrid sunflower taxa inferred from phylogenetic data.

BACKGROUND: Long terminal repeat (LTR) retrotransposons are a class of mobile genetic element capable of autonomous transposition via an RNA intermediate. Their large size and proliferative ability make them important contributors to genome size evolution, especially in plants, where they can reach exceptionally high copy numbers and contribute substantially to variation in genome size even among closely related taxa. Using a phylogenetic approach, we characterize dynamics of proliferation events of Ty3/gypsy-like LTR retrotransposons that led to massive genomic expansion in three Helianthus (sunflower) species of ancient hybrid origin. The three hybrid species are independently derived from the same two parental species, offering a unique opportunity to explore patterns of retrotransposon proliferation in light of reticulate evolutionary events in this species group. RESULTS: We demonstrate that Ty3/gypsy-like retrotransposons exist as multiple well supported sublineages in both the parental and hybrid derivative species and that the same element sublineage served as the source lineage of proliferation in each hybrid species' genome. This inference is based on patterns of species-specific element numerical abundance within different phylogenetic sublineages as well as through signals of proliferation events present in the distributions of element divergence values. Employing methods to date paralogous sequences within a genome, proliferation events in the hybrid species' genomes are estimated to have occurred approximately 0.5 to 1 million years ago. CONCLUSION: Proliferation of the same retrotransposon major sublineage in each hybrid species indicates that similar dynamics of element derepression and amplification likely occurred in each hybrid taxon during their formation. Temporal estimates of these proliferation events suggest an earlier origin for these hybrid species than previously supposed.

Comparing control options for time-series RNA sequencing experiments in nonmodel organisms: An example from grasses.

RNA sequencing (RNA-seq) is a widely used approach to investigate gene expression and increasingly is used in time-course studies to characterize transcriptomic changes over time. Two primary options are available as controls in time-course experiments: samples collected at the first sampling time are used as controls (temporal control, TC) and samples collected in parallel at each individual sampling time are used as controls (biological control, BC). While both approaches are used in experimental studies, we know of no analyses performed to date that directly compare effects of control type choices on identifying differentially expressed genes (DEGs) and subsequent functional analysis. In the current study, we compare experimental results using these different control types for time-course RNA-seq drought stress experiments in two wild grass species in the genus Paspalum. Our results showed BC assemblies gave a higher number of loci in both species. The number of DEGs increased with increasing stress and then decreased dramatically at the recovery time point using both control types. Expression levels of the same DEGs were highly correlated between control types in both species, ranging from r = .653 to r = .852. We also observed similar rank orders of shared enriched Gene Ontology term lists using the two different control types. Collectively, our findings suggest similar results in differential gene expression and functional annotation between control types. The ultimate choice of control type will rely on the experimental length and organism type, with labour time and sequencing costs as additional factors to be considered.

Relaxed selection on the CBF/DREB1 regulatory genes and reduced freezing tolerance in the southern range of Arabidopsis thaliana.

Elucidating the molecular basis of adaptive phenotypic variation represents a central aim in evolutionary biology. Traits exhibiting patterns of clinal variation represent excellent models for studies of molecular adaptation, especially when variation in phenotype can be linked to organismal fitness in different environments. Natural accessions of the model plant species Arabidopsis thaliana exhibit clinal variation in freezing tolerance that follows a gradient of temperature variability across the species' native range (Zhen Y, Ungerer MC. 2008. Clinal variation in freezing tolerance among natural accessions of A. thaliana. New Phytol. 177:419-427). Here, we report that this pattern of variation is attributable, at least in part, to relaxed purifying selection on members of a small family of transcriptional activators (the CBF/DREB1s) in the species' southern range. These regulatory genes play a critical role in the ability of A. thaliana plants to undergo cold acclimation and thereby achieve maximum freezing tolerance. Relative to accessions from northern regions, accessions of A. thaliana from the southern part of their geographic range exhibit levels of nonsynonymous nucleotide polymorphism that are approximately 2.8-fold higher across this small gene subfamily. Relaxed selection on the CBF/DREB1s in southern accessions also has resulted in multiple mutations in regulatory regions resulting in abrogated expression of particular subfamily members in particular accessions. These coding-region and regulatory mutations compromise the ability of these genes to act as efficient transcriptional activators during the cold acclimation process, as determined by reductions in rates of induction and maximum levels of expression in the downstream genes they regulate. This study highlights the potential role of regulatory genes in underlying adaptive phenotypic variation in nature.

Bar-coded pyrosequencing reveals shared bacterial community properties along the temperature gradients of two alkaline hot springs in Yellowstone National Park.

An understanding of how communities are organized is a fundamental goal of ecology but one which has historically been elusive for microbial systems. We used a bar-coded pyrosequencing approach targeting the V3 region of the bacterial small-subunit rRNA gene to address the factors that structure communities along the thermal gradients of two alkaline hot springs in the Lower Geyser Basin of Yellowstone National Park. The filtered data set included a total of nearly 34,000 sequences from 39 environmental samples. Each was assigned to one of 391 operational taxonomic units (OTUs) identified by their unique V3 sequence signatures. Although the two hot springs differed in their OTU compositions, community resemblance and diversity changed with strikingly similar dynamics along the two outflow channels. Two lines of evidence suggest that these community properties are controlled primarily by environmental temperature. First, community resemblance decayed exponentially with increasing differences in temperature between samples but was only weakly correlated with physical distance. Second, diversity decreased with increasing temperature at the same rate along both gradients but was uncorrelated with other measured environmental variables. This study also provides novel insights into the nature of the ecological interactions among important taxa in these communities. A strong negative association was observed between cyanobacteria and the Chloroflexi, which together accounted for approximately 70% of the sequences sampled. This pattern contradicts the longstanding hypothesis that coadapted lineages of these bacteria maintain tightly cooccurring distributions along these gradients as a result of a producer-consumer relationship. We propose that they instead compete for some limiting resource(s).

Distributions of Mutational Effects and the Estimation of Directional Selection in Divergent Lineages of Arabidopsis thaliana.

Mutations are crucial to evolution, providing the ultimate source of variation on which natural selection acts. Due to their key role, the distribution of mutational effects on quantitative traits is a key component to any inference regarding historical selection on phenotypic traits. In this paper, we expand on a previously developed test for selection that could be conducted assuming a Gaussian mutation effect distribution by developing approaches to also incorporate any of a family of heavy-tailed Laplace distributions of mutational effects. We apply the test to detect directional natural selection on five traits along the divergence of Columbia and Landsberg lineages of Arabidopsis thaliana, constituting the first test for natural selection in any organism using quantitative trait locus and mutation accumulation data to quantify the intensity of directional selection on a phenotypic trait. We demonstrate that the results of the test for selection can depend on the mutation effect distribution specified. Using the distributions exhibiting the best fit to mutation accumulation data, we infer that natural directional selection caused divergence in the rosette diameter and trichome density traits of the Columbia and Landsberg lineages.

Long Terminal Repeat Retrotransposon Content in Eight Diploid Sunflower Species Inferred from Next-Generation Sequence Data.

The most abundant transposable elements (TEs) in plant genomes are Class I long terminal repeat (LTR) retrotransposons represented by superfamilies gypsy and copia Amplification of these superfamilies directly impacts genome structure and contributes to differential patterns of genome size evolution among plant lineages. Utilizing short-read Illumina data and sequence information from a panel of Helianthus annuus (sunflower) full-length gypsy and copia elements, we explore the contribution of these sequences to genome size variation among eight diploid Helianthus species and an outgroup taxon, Phoebanthus tenuifolius We also explore transcriptional dynamics of these elements in both leaf and bud tissue via RT-PCR. We demonstrate that most LTR retrotransposon sublineages (i.e., families) display patterns of similar genomic abundance across species. A small number of LTR retrotransposon sublineages exhibit lineage-specific amplification, particularly in the genomes of species with larger estimated nuclear DNA content. RT-PCR assays reveal that some LTR retrotransposon sublineages are transcriptionally active across all species and tissue types, whereas others display species-specific and tissue-specific expression. The species with the largest estimated genome size, H. agrestis, has experienced amplification of LTR retrotransposon sublineages, some of which have proliferated independently in other lineages in the Helianthus phylogeny.

Effects of genetic background on response to selection in experimental populations of Arabidopsis thaliana.

The extent to which genetic background can influence allelic fitness is poorly understood, despite having important evolutionary consequences. Using experimental populations of Arabidopsis thaliana and map-based population genetic data, we examined a multigeneration response to selection in populations with differentiated genetic backgrounds. Replicated experimental populations of A. thaliana with genetic backgrounds derived from ecotypes Landsberg and Niederzenz were subjected to strong viability and fertility selection by growing individuals from each population at high density for three generations in a growth chamber. Patterns of genome-wide selection were evaluated by examining deviations from expected frequencies of mapped molecular markers. Estimates of selection coefficients for individual genomic regions ranged from near 0 to 0.685. Genomic regions demonstrating the strongest response to selection most often were selected similarly in both genetic backgrounds. The selection response of several weakly selected regions, however, appeared to be sensitive to genetic background, but only one region showed evidence of positive selection in one background and negative selection in another. These results are most consistent with models of adaptive evolution in which allelic fitnesses are not strongly influenced by genetic background and only infrequently change in sign due to variation at other loci.

Genotype-environment interactions at quantitative trait loci affecting inflorescence development in Arabidopsis thaliana.

Phenotypic plasticity and genotype-environment interactions (GEI) play a prominent role in plant morphological diversity and in the potential functional capacities of plant life-history traits. The genetic basis of plasticity and GEI, however, is poorly understood in most organisms. In this report, inflorescence development patterns in Arabidopsis thaliana were examined under different, ecologically relevant photoperiod environments for two recombinant inbred mapping populations (Ler x Col and Cvi x Ler) using a combination of quantitative genetics and quantitative trait locus (QTL) mapping. Plasticity and GEI were regularly observed for the majority of 13 inflorescence traits. These observations can be attributable (at least partly) to variable effects of specific QTL. Pooled across traits, 12/44 (27.3%) and 32/62 (51.6%) of QTL exhibited significant QTL x environment interactions in the Ler x Col and Cvi x Ler lines, respectively. These interactions were attributable to changes in magnitude of effect of QTL more often than to changes in rank order (sign) of effect. Multiple QTL x environment interactions (in Cvi x Ler) clustered in two genomic regions on chromosomes 1 and 5, indicating a disproportionate contribution of these regions to the phenotypic patterns observed. High-resolution mapping will be necessary to distinguish between the alternative explanations of pleiotropy and tight linkage among multiple genes.

Quantitative trait loci for inflorescence development in Arabidopsis thaliana.

Variation in inflorescence development patterns is a central factor in the evolutionary ecology of plants. The genetic architectures of 13 traits associated with inflorescence developmental timing, architecture, rosette morphology, and fitness were investigated in Arabidopsis thaliana, a model plant system. There is substantial naturally occurring genetic variation for inflorescence development traits, with broad sense heritabilities computed from 21 Arabidopsis ecotypes ranging from 0.134 to 0.772. Genetic correlations are significant for most (64/78) pairs of traits, suggesting either pleiotropy or tight linkage among loci. Quantitative trait locus (QTL) mapping indicates 47 and 63 QTL for inflorescence developmental traits in Ler x Col and Cvi x Ler recombinant inbred mapping populations, respectively. Several QTL associated with different developmental traits map to the same Arabidopsis chromosomal regions, in agreement with the strong genetic correlations observed. Epistasis among QTL was observed only in the Cvi x Ler population, and only between regions on chromosomes 1 and 5. Examination of the completed Arabidopsis genome sequence in three QTL regions revealed between 375 and 783 genes per region. Previously identified flowering time, inflorescence architecture, floral meristem identity, and hormone signaling genes represent some of the many candidate genes in these regions.