Searching for the bull's eye: agents and targets of selection vary among geographically disparate cyanogenesis clines in white clover (Trifolium repens L.)

The recurrent evolution of adaptive clines within a species can be used to elucidate the selective factors and genetic responses that underlie adaptation. White clover is polymorphic for cyanogenesis (HCN release with tissue damage), and climate-associated cyanogenesis clines have evolved throughout the native and introduced species range. This polymorphism arises through two independently segregating Mendelian polymorphisms for the presence/absence of two required components: cyanogenic glucosides and their hydrolyzing enzyme linamarase. Cyanogenesis is commonly thought to function in herbivore defense; however, the individual cyanogenic components may also serve other physiological functions. To test whether cyanogenesis clines have evolved in response to the same selective pressures acting on the same genetic targets, we examined cyanogenesis cline shape and its environmental correlates in three world regions: southern New Zealand, the central United States and the US Pacific Northwest. For some regional comparisons, cline shapes are remarkably similar despite large differences in the spatial scales over which clines occur (40–1600 km). However, we also find evidence for major differences in both the agents and targets of selection among the sampled clines. Variation in cyanogenesis frequency is best predicted using a combination of minimum winter temperature and aridity variables. Together, our results provide evidence that recurrent adaptive clines do not necessarily reflect shared adaptive processes.     

Classic clover cline clues

Adaptive clines are striking examples of natural selection in action, yet few have been studied in depth. In this issue of Molecular Ecology, Kooyers & Olsen (2012) introduce modern analyses and thinking towards studies of a classical example of the rapid and repeated evolution of latitudinal and altitudinal clines in cyanogenesis in white clover, Trifolium repens L. Recognizing that adaptive clines represent trade-offs in the selective benefits of traits at different ends of a geographical transect, these researchers focus on whether evidence for selection can be found at regional (coarse) and local (fine) scales. After adjusting for population genetic patterns generated by demographic processes, Kooyers and Olsen provide evidence that the cyanogenesis cline is adaptive across a transect from Louisiana to Wisconsin, USA. Within local populations, divergent selection on coupling dominant and recessive alleles that underlie cyanogenesis is predicted to drive populations to gametic phase disequilibrium (LD), a pattern that has been found in several other studies reviewed by Kooyers and Olsen. The absence of LD within any sampled populations in this study leads the authors to suggest that selective patterns within these clines may be more complex than previously proposed, perhaps even following theoretical predictions of a geographic mosaic.     

Aridity shapes cyanogenesis cline evolution in white clover (Trifolium repens L.)

Adaptive differentiation between populations is often proposed to be the product of multiple interacting selective pressures, although empirical support for this is scarce. In white clover, populations show adaptive differentiation in frequencies of cyanogenesis, the ability to produce hydrogen cyanide after tissue damage. This polymorphism arises through independently segregating polymorphisms for the presence/absence of two required cyanogenic components, cyanogenic glucosides and their hydrolysing enzyme. White clover populations worldwide have evolved a series of recurrent, climate‐associated clines, with higher frequencies of cyanogenic plants in warmer locations. These clines have traditionally been hypothesized to reflect a fitness trade‐off between chemical defence in herbivore‐rich areas (warmer climates) and energetic costs of producing cyanogenic components in areas of low herbivore pressure (cooler climates). Recent observational studies suggest that cyanogenic components may also be beneficial in water‐stressed environments. We investigated fitness trade‐offs associated with temperature‐induced water stress in the cyanogenesis system using manipulative experiments in growth chambers and population surveys across a longitudinal precipitation gradient in the central United States. We find that plants producing cyanogenic glucosides have higher relative fitness in treatments simulating a moderate, persistent drought stress. In water‐neutral treatments, there are energetic costs to producing cyanogenic components, but only in treatments with nutrient stress. These fitness trade‐offs are consistent with cyanogenesis frequencies in natural populations, where we find clinal variation in the proportion of plants producing cyanogenic glucosides along the precipitation gradient. These results suggest that multiple selective pressures interact to maintain this adaptive polymorphism and that modelling adaptation will require knowledge of environment‐specific fitness effects.     

Adaptive cyanogenesis clines evolve recurrently through geographical sorting of existing gene deletions

Identifying the genetic basis of parallel phenotypic evolution provides insight into the process of adaptation and evolutionary constraint. White clover (Trifolium repens) has evolved climate‐associated adaptive clines in cyanogenesis (the ability to produce hydrogen cyanide upon tissue damage) in several world regions where it has been introduced. Gene‐deletion polymorphisms at the CYP79D15 and Li loci underlie the presence/absence of the cyanogenic phenotype. Both loci have undergone multiple independent gene‐deletion events, which are identifiable through molecular signatures in flanking regions. To investigate whether cyanogenesis clines in introduced populations have evolved through the sorting of standing genetic variation or de novo gene deletions, we examined cyanogenesis gene‐flanking regions in three world regions. In comparison with native Eurasian populations, we find no evidence for novel gene deletion events in any introduced region, which suggests that these adaptive clines have evolved through the geographical sorting of pre‐existing genetic variation.     

How much can history constrain adaptive evolution? A real‐time evolutionary approach of inversion polymorphisms in Drosophila subobscura

Chromosomal inversions are present in a wide range of animals and plants, having an important role in adaptation and speciation. Although empirical evidence of their adaptive value is abundant, the role of different processes underlying evolution of chromosomal polymorphisms is not fully understood. History and selection are likely to shape inversion polymorphism variation to an extent yet largely unknown. Here, we perform a real‐time evolution study addressing the role of historical constraints and selection in the evolution of these polymorphisms. We founded laboratory populations of Drosophila subobscura derived from three locations along the European cline and followed the evolutionary dynamics of inversion polymorphisms throughout the first 40 generations. At the beginning, populations were highly differentiated and remained so throughout generations. We report evidence of positive selection for some inversions, variable between foundations. Signs of negative selection were more frequent, in particular for most cold‐climate standard inversions across the three foundations. We found that previously observed convergence at the phenotypic level in these populations was not associated with convergence in inversion frequencies. In conclusion, our study shows that selection has shaped the evolutionary dynamics of inversion frequencies, but doing so within the constraints imposed by previous history. Both history and selection are therefore fundamental to predict the evolutionary potential of different populations to respond to global environmental changes.     

Frequency of cyanogenesis in tropical rainforests of far north Queensland, Australia

BACKGROUND AND AIMS: Plant cyanogenesis is the release of toxic cyanide from endogenous cyanide-containing compounds, typically cyanogenic glycosides. Despite a large body of phytochemical, taxonomic and ecological work on cyanogenic species, little is known of their frequency in natural plant communities. This study aimed to investigate the frequency of cyanogenesis in Australian tropical rainforests. Secondary aims were to quantify the cyanogenic glycoside content of tissues, to investigate intra-plant and intra-population variation in cyanogenic glycoside concentration and to appraise the potential chemotaxonomic significance of any findings in relation to the distribution of cyanogenesis in related taxa. METHODS: All species in six 200 m(2) plots at each of five sites across lowland, upland and highland tropical rainforest were screened for cyanogenesis using Feigl-Anger indicator papers. The concentrations of cyanogenic glycosides were accurately determined for all cyanogenic individuals. KEY RESULTS: Over 400 species from 87 plant families were screened. Overall, 18 species (4.5 %) were cyanogenic, accounting for 7.3 % of total stem basal area. Cyanogenesis has not previously been reported for 17 of the 18 species, 13 of which are endemic to Australia. Several species belong to plant families or orders in which cyanogenesis has been little reported, if at all (e.g. Elaeocarpaceae, Myrsinaceae, Araliaceae and Lamiaceae). A number of species contained concentrations of cyanogenic glycosides among the highest ever reported for mature leaves-up to 5.2 mg CN g(-1) d. wt, for example, in leaves of Elaeocarpus sericopetalus. There was significant variation in cyanogenic glycoside concentration within individuals; young leaves and reproductive tissues typically had higher cyanogen content. In addition, there was substantial variation in cyanogenic glycoside content within populations of single species. CONCLUSIONS: This study expands the limited knowledge of the frequency of cyanogenesis in natural plant communities, includes novel reports of cyanogenesis among a range of taxa and characterizes patterns in intra-plant and intra-population variation of cyanogensis.     

Evolution of coadaptation in a two-locus epistatic system

Although recent advances in genome biology have dramatically increased our understanding of the contribution of gene interactions to the development of complex phenotypes, we still lack general agreement on the process and mechanisms responsible for the evolution of epistatic systems. Even if genes in a species are indeed integrated into coadapted complexes of interacting components, simple additive evolution may eventually result in epistatic differentiation of populations. Consequently, the prevalence of epistatic gene action does not tell us anything about the role of epistatic selection in the history of population divergence. To elucidate the contribution of epistatic selection in the evolution of coadaptation, we investigate the fixation process of two mutations that interact synergistically to enhance fitness. We show by diffusion analysis and simulations that epistatic selection on cosegregating variants does not by itself promote the evolution of epistatic systems; rather, accumulation of neutral mutations may play a crucial role, creating an appropriate genetic milieu for adaptive evolution in the future generations.     

Natural selection drives altitudinal divergence at the albumin locus in deer mice, Peromyscus maniculatus

In populations that are distributed across steep environmental gradients, the potential for local adaptation is largely determined by the spatial scale of fitness variation relative to dispersal distance. Since altitudinal gradients are generally characterized by dramatic ecological transitions over relatively short linear distances, adaptive divergence across such gradients will typically require especially strong selection to counterbalance the homogenizing effect of gene flow. Here we report the results of a study that was designed to test for evidence of adaptive divergence across an altitudinal gradient in a natural population of deer mice, Peromyscus maniculatus. We conducted a multilocus survey of allozyme variation across a steep altitudinal gradient in the southern Rocky Mountains that spanned several distinct biomes, from prairie grassland to alpine tundra. As a control for the effects of altitude, we also surveyed the same loci in mice sampled along a latitudinal transect through the prairie grassland that ran perpendicular to the east-west altitudinal transect. We used a coalescent-based simulation model to identify loci that deviated from neutral expectations, and we then assessed whether locus-specific patterns of variation were nonrandom with respect to altitude. Results indicated that the albumin locus (Alb) reflects a history of diversifying selection across the altitudinal gradient. This conclusion is supported by two main lines of evidence: (1) Alb was characterized by levels of divergence across the altitudinal transect that exceeded neutral expectations in two consecutive years of sampling (in contrast to the spatial pattern of variation across the latitudinal transect), and (2) levels of divergence at the Alb locus exhibited a positive association with altitudinal distance in both years (in contrast to the pattern observed at unlinked loci). We conclude that clinal variation at the Alb locus reflects a balance between gene flow and diversifying selection that results from elevational changes in fitness rankings among alternative genotypes.     

The genomics of rapid climatic adaptation and parallel evolution in North American house mice

Parallel changes in genotype and phenotype in response to similar selection pressures in different populations provide compelling evidence of adaptation. House mice (Mus musculus domesticus) have recently colonized North America and are found in a wide range of environments. Here we measure phenotypic and genotypic differentiation among house mice from five populations sampled across 21° of latitude in western North America, and we compare our results to a parallel latitudinal cline in eastern North America. First, we show that mice are genetically differentiated between transects, indicating that they have independently colonized similar environments in eastern and western North America. Next, we find genetically-based differences in body weight and nest building behavior between mice from the ends of the western transect which mirror differences seen in the eastern transect, demonstrating parallel phenotypic change. We then conduct genome-wide scans for selection and a genome-wide association study to identify targets of selection and candidate genes for body weight. We find some genomic signatures that are unique to each transect, indicating population-specific responses to selection. However, there is significant overlap between genes under selection in eastern and western house mouse transects, providing evidence of parallel genetic evolution in response to similar selection pressures across North America.     

Bayesian estimation of genomic clines

We developed a Bayesian genomic cline model to study the genetic architecture of adaptive divergence and reproductive isolation between hybridizing lineages. This model quantifies locus‐specific patterns of introgression with two cline parameters that describe the probability of locus‐specific ancestry as a function of genome‐wide admixture. ‘Outlier’ loci with extreme patterns of introgression relative to most of the genome can be identified. These loci are potentially associated with adaptive divergence or reproductive isolation. We simulated genetic data for admixed populations that included neutral introgression, as well as loci that were subject to directional, epistatic or underdominant selection, and analysed these data using the Bayesian genomic cline model. Under many demographic conditions, underdominance or directional selection had detectable and predictable effects on cline parameters, and ‘outlier’ loci were greatly enriched for genetic regions affected by selection. We also analysed previously published genetic data from two transects through a hybrid zone between Mus domesticus and M. musculus. We found considerable variation in rates of introgression across the genome and particularly low rates of introgression for two X‐linked markers. There were similarities and differences in patterns of introgression between the two transects, which likely reflects a combination of stochastic variability because of genetic drift and geographic variation in the genetic architecture of reproductive isolation. By providing a robust framework to quantify and compare patterns of introgression among genetic regions and populations, the Bayesian genomic cline model will advance our understanding of the genetics of reproductive isolation and the speciation process.     

Evidence on the molecular basis of the Ac/ac adaptive cyanogenesis polymorphism in white clover (Trifolium repens L)

White clover is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in nature. This chemical defense polymorphism is one of the longest-studied and best-documented examples of an adaptive polymorphism in plants. It is controlled by two independently segregating genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolyzing enzyme, linamarase. Whereas Li is well characterized at the molecular level, Ac has remained unidentified. Here we report evidence that Ac corresponds to a gene encoding a cytochrome P450 of the CYP79D protein subfamily (CYP79D15), and we describe the apparent molecular basis of the Ac/ac polymorphism. CYP79D orthologs catalyze the first step in cyanogenic glucoside biosynthesis in other cyanogenic plant species. In white clover, Southern hybridizations indicate that CYP79D15 occurs as a single-copy gene in cyanogenic plants but is absent from the genomes of ac plants. Gene-expression analyses by RT-PCR corroborate this finding. This apparent molecular basis of the Ac/ac polymorphism parallels our previous findings for the Li/li polymorphism, which also arises through the presence/absence of a single-copy gene. The nature of these polymorphisms may reflect white clover's evolutionary origin as an allotetraploid derived from cyanogenic and acyanogenic diploid progenitors.     

Variation of cyanogenesis in some plant species of the midwestern United States

The frequency of cyanogenesis of 48 species of vascular plants was examined by testing 30 individuals from five populations of each species for release of cyanide. The rate at which cyanide was released and the amount of cyanide released varied widely among individuals of a population and among populations of a species. For many taxa, the frequency of cyanogenesis was highly variable among populations. Of the species examined, 20 have not been reported previously as being cyanogenic.     

Clinal resistance structure and pathogen local adaptation in a serpentine flax-flax rust interaction

Because disease resistance is a hallmark signature of pathogen-mediated selection pressure on hosts, studies of resistance structure (the spatial distribution of disease resistance genes among conspecific host populations) can provide valuable insights into the influence of pathogens on host evolution and spatial variation in the magnitude of their effects. To date few studies of wild plant-pathogen interactions have characterized resistance structure by sampling across the host's biogeographic range, and only a handful have paired such investigations with studies of disease levels under natural conditions. I used a greenhouse cross-inoculation experiment to characterize genetic resistance of 16 populations of California dwarf flax (Hesperolinon californicum) to attack by multiple samples of the rust fungus Melampsora lini. I documented a latitudinal cline in resistance structure, manifest across the host's biogeographic range, which mirrored almost identically a cline in infection prevalence documented through field surveys of disease in study populations. These results provide empirical evidence for clinal patterns of antagonistic selection pressure, demonstrate that such patterns can be manifest across broad biogeographic scales, and suggest that rates of disease prevalence in wild plant populations may be tightly linked to the distribution of host resistance genes. Tests for local adaptation of the fungus revealed evidence of the phenomenon (significantly greater infection in sympatric plant-fungal pairings) as well as the potential for substantial bias to be introduced into statistical analyses by spatial patterns of host resistance structure.     

Chromosomal and allelic variation in Drosophila americana: selective maintenance of a chromosomal cline.

Geographically structured genetic variation, as represented by clines and hybrid zones, offers unique opportunities to study adaptation and speciation in natural populations. A hybrid zone has been reported between Drosophila americana americana and Drosophila americana texana, two taxa that are distinguished solely by the arrangement of their X and 4th chromosomes. In this study, samples of D. americana were collected along a latitudinal transect across the inferred hybrid zone, and the frequency of the alternative chromosomal arrangements is reported. These data illustrate that the alternative chromosomal arrangements are distributed along a shallow cline over a broad geographic region, and that the frequency of the arrangements is tightly correlated with latitude. Allelic variants at 13 RFLP loci in three genes on chromosome 4 exhibit no evidence of association with the cline. Presence of a cline for the chromosomal arrangements, as well as a general absence of geographic structure for variation at these genes, is interpreted as evidence that natural selection is responsible for the maintenance of this chromosomal cline. Furthermore, these results demonstrate that taxonomic subdivision of D. americana is unwarranted, because it exists as a cohesive species that is segregating a chromosomal fusion.     

Nonclinality of molecular variation implicates selection in maintaining a morphological cline of Drosophila melanogaster

One general approach for assessing whether phenotypic variation is due to selection is to test its correlation with presumably neutral molecular variation. Neutral variation is determined by population history, the most likely alternative explanation of spatial genetic structure, whereas phenotypic variation may be influenced by the spatial pattern of selection pressure. Several methods for comparing the spatial apportionment of molecular and morphological variation have been used. Here, we present an analysis of variance framework that compares the magnitudes of latitudinal effects for molecular and morphological variation along a body size cline in Australian Drosophila populations. Explicit incorporation of the relevant environmental gradient can result in a simple and powerful test of selection. For the Australian cline, our analysis provides strong internal evidence that the cline is due to selection.     

Rapid evolution of wing size clines in Drosophila subobscura

Parallel latitudinal clines across species and continents provide dramatic evidence of the efficacy of natural selection, however little is known about the dynamics involved in cline formation. For example, several drosophilids and other ectotherms increase in body and wing size at higher latitudes. Here we compare evolution in an ancestral European and a recently introduced (North America) cline in wing size and shape in Drosophila subobscura. We show that clinal variation in wing size, spanning more than 15 degrees of latitude, has evolved in less than two decades. In females from Europe and North America, the clines are statistically indistinguishable however the cline for North American males is significantly shallower than that for European males. We document that while overall patterns of wing size are similar on two continents, the European cline is obtained largely through changing the proximal portion of the wing, whereas the North American cline is largely in the distal portion. We use data from sites collected in 1986/1988 (Pegueroles et al. 1995) and our 1997 collections to compare synchronic (divergence between contemporary populations that share a common ancestor) and allochronic (changes over time within a population) estimates of the rates of evolution. We find that, for these populations, allochronically estimated evolutionary rates within a single population are over 0.02 haldanes (2800 darwins), a value similar in magnitude to the synchronic estimates from the extremes of the cline. This paper represents an expanded analysis of data partially presented in Huey et al. (2000).     

Cyanogenesis of Wild Lima Bean (Phaseolus lunatus L.) Is an Efficient Direct Defence in Nature

In natural systems plants face a plethora of antagonists and thus have evolved multiple defence strategies. Lima bean (Phaseolus lunatus L.) is a model plant for studies of inducible indirect anti-herbivore defences including the production of volatile organic compounds (VOCs) and extrafloral nectar (EFN). In contrast, studies on direct chemical defence mechanisms as crucial components of lima beans'' defence syndrome under natural conditions are nonexistent. In this study, we focus on the cyanogenic potential (HCNp; concentration of cyanogenic glycosides) as a crucial parameter determining lima beans'' cyanogenesis, i.e. the release of toxic hydrogen cyanide from preformed precursors. Quantitative variability of cyanogenesis in a natural population of wild lima bean in Mexico was significantly correlated with missing leaf area. Since existing correlations do not by necessity mean causal associations, the function of cyanogenesis as efficient plant defence was subsequently analysed in feeding trials. We used natural chrysomelid herbivores and clonal lima beans with known cyanogenic features produced from field-grown mother plants. We show that in addition to extensively investigated indirect defences, cyanogenesis has to be considered as an important direct defensive trait affecting lima beans'' overall defence in nature. Our results indicate the general importance of analysing ‘multiple defence syndromes’ rather than single defence mechanisms in future functional analyses of plant defences.     

Cyanogenesis variation in the Turnera sidoides L. polyploid complex (Turneraceae)

This study constitutes the first investigation of cyanogenesis variation within the Turnera sidoides autopolyploid complex. The qualitative and quantitative analysis carried out in 38 populations showed that the five subspecies of the complex are cyanogenic, although the levels of cyanide released varied both between and within subspecies. Between the subspecies, carnea , holosericea and integrifolia showed higher values of HCN released than subspecies pinnatifida and sidoides . Within subspecies, there is a tendency toward an increment of mean cyanide values with ploidy levels and different hypotheses are considered in order to explain this variation. The study of cyanogenesis in populations from different geographical areas cultivated under uniform greenhouse conditions showed that, in the widespread subspecies pinnatifida , HCN variation may also reflect the action of different selection pressures, since populations having dissimilar cyanide levels grow in different phytogeographical provinces with different climatic regimes. On the other hand, the fact that the variation of HCN released was greater between populations from different geographical areas than that observed between diploid and tetraploid populations of the same area provides additional evidence for the multiple origins of polyploids of the T. sidoides complex.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 141 , 85–94.     

Studies on Variability in White Clover: Growth Habits and Cyanogenic Glucosides

Growth habits and cyanogenesis were studied in a field experimentwith white clover (Trifolium repens L.) cv. Huia. Eighty controlplants propagated by seeds, and 80 clones of an in vivo selectedvariant were examined in mid-late August and late September.The temperature effect on cyanogenic glucoside levels was alsoexamined on in vitro grown plantlets of the variant in growthchambers. Results showed that the upright growth habit dominated in controlplants. The population of the variant plants was mainly composedof prostrate clones and a transition from prostrate to uprightgrowth habit occurred. Considerable variation was observed withregard to all measured morphological characters in both controland variant plants. Great variation was also noted in cyanogenicglucoside content in the leaf laminae of the control plants.Low cyanogenic plants, on the other hand, dominated in the variant.The number of low and high cyanogenic plants increased in thecontrol by the end of the growth season, after the first frosts.A one-way shift of cyanogenic (slight increase) was recordedin the variant. Similar levels of cyanogenic glucoside and linamarasewere determined in the in vitro and field-grown plants. Cyanogenicglucoside content slightly decreased with the age of the invitro plantlets, but variation in temperature did not causeany changes in their level. The response to the environmentalchanges, regarding cyanogenesis, appear to be genetically determined. Trifolium repens, in vitro, somaclonal variation, cyanogenic glucosides