Reconstructing the origin of Helianthus deserticola: survival and selection on the desert floor

The diploid hybrid species Helianthus deserticola inhabits the desert floor, an extreme environment relative to its parental species Helianthus annuus and Helianthus petiolaris. Adaptation to the desert floor may have occurred via selection acting on transgressive, or extreme, traits in early hybrids between the parental species. We explored this possibility through a field experiment in the hybrid species' native habitat using H. deserticola, H. annuus, H. petiolaris, and two populations of early-generation (BC(2)) hybrids between the parental species, which served as proxies for the ancestral genotype of the ancient hybrid species. Character expression was evaluated for each genotypic class. Helianthus deserticola was negatively transgressive for stem diameter, leaf area, and flowering date, and the latter two traits are likely to be advantageous in a desert environment. The BC(2) hybrids contained a range of variation that overlapped these transgressive trait means, and an analysis of phenotypic selection revealed that some of the selective pressures on leaf size and flowering date, but not stem diameter, would move the BC(2) population toward the H. deserticola phenotype. Thus, H. deserticola may have originated from habitat-mediated directional selection acting on hybrids between H. annuus and H. petiolaris in a desert environment.     

Genetic architecture of leaf ecophysiological traits in Helianthus

We investigated quantitative trait loci (QTLs) for several leaf chemistry traits in early-generation hybrids between Helianthus annuus and Helianthus petiolaris, the parental species of the ancient diploid hybrid sunflower species Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus. We grew individuals of a second-generation backcross (BC(2)) toward H. petiolaris under optimum conditions in a glass house experiment. Trait values were measured once for each individual. In addition, genotypic data previously determined for each individual were employed for composite interval mapping of QTLs. We detected QTLs for leaf carbon concentration, leaf nitrogen concentration, leaf nitrogen per unit area, and photosynthetic nitrogen use efficiency. Leaf carbon isotope discrimination (delta(13)C) and leaf nitrogen isotopic composition (delta(15)N) were analyzed, but no significant QTLs were found for these traits. Interestingly, two neighboring loci explained a relatively large percentage of the variation in leaf nitrogen per unit area. This was notable because leaf nitrogen has been shown to strongly affect the fitness of early-generation sunflower hybrids in the H. anomalus habitat, and QTLs of large effect are expected to respond relatively quickly to selection. We speculate that the genetic architecture underlying leaf nitrogen may have facilitated the colonization of active desert sand dunes by H. anomalus.     

Phenotypic selection on leaf water use efficiency and related ecophysiological traits for natural populations of desert sunflowers

Plant water-use efficiency (WUE) is expected to affect plant fitness and thus be under natural selection in arid habitats. Although many natural population studies have assessed plant WUE, only a few related WUE to fitness. The further determination of whether selection on WUE is direct or indirect through functionally related traits has yielded no consistent results. For natural populations of two desert annual sunflowers, Helianthus anomalus and H. deserticola, we used phenotypic selection analysis with vegetative biomass as the proxy for fitness to test (1) whether there was direct and indirect selection on WUE (carbon isotope ratio) and related traits (leaf N, area, succulence) and (2) whether direct selection was consistent with hypothesized drought/dehydration escape and avoidance strategies. There was direct selection for lower WUE in mesic and dry H. anomalus populations, consistent with dehydration escape, even though it is the longer lived of the two species. For mesic H. anomalus, direct selection favored lower WUE and higher N, suggesting that plants may be “wasting water” to increase N delivery via the transpiration stream. For the shorter lived H. deserticola in the direr habitat, there was indirect selection for lower WUE, inconsistent with drought escape. There was also direct selection for higher leaf N, succulence and leaf size. There was no direct selection for higher WUE consistent with dehydration avoidance in either species. Thus, in these natural populations of two desert dune species higher fitness was associated with some combination direct and indirect selection for lower WUE, higher leaf N and larger leaf size. Our understanding of the adaptive value of plant ecophysiological traits will benefit from further consideration of related traits such as leaf nitrogen and more tests in natural populations.     

Transgressive character expression in a hybrid sunflower species

Diploid hybrid lineages often are ecologically distinct from their parental species. However, it is unclear whether this niche divergence is typically achieved via hybrid intermediacy, a mixture of parental traits, and/or the evolution of extreme (transgressive) morphological and ecophysiological features. Here we compare an extensively studied hybrid sunflower species, Helianthus anomalus, with its putative parents, H. annuus and H. petiolaris, for 41 morphological and 12 ecophysiological traits. Helianthus anomalus was morphologically intermediate for one trait (2.4%), parental-like for 23 traits (56.1%), and transgressive for 17 traits (41.5%). For ecophysiological traits, H. anomalus was not significantly different from one or both parents for nine traits (75%), and was transgressive for the remaining three (25%). Thus, H. anomalus appears to be a mosaic of parental-like and transgressive phenotypes. Although the fitness effects of the transgressive characters are not yet known, many of these characters are consistent with adaptations reported for other sand dune plants. Genetic studies are currently underway to ascertain whether these extreme characters arose as a direct byproduct of hybridization or whether they evolved via mutational divergence.     

The origin of ecological divergence in Helianthus paradoxus (Asteraceae): selection on transgressive characters in a novel hybrid habitat

Diploid hybrid speciation in plants is often accompanied by rapid ecological divergence between incipient neospecies and their parental taxa. One plausible means by which novel adaptation in hybrid lineages may arise is transgressive segregation, that is, the generation of extreme phenotypes that exceed those of the parental lines. Early generation (BC2) hybrids between two wild, annual sunflowers, Helianthus annuus and Helianthus petiolaris, were used to study directional selection on transgressive characters associated with the origin of Helianthus paradoxus, a diploid hybrid species adapted to extremely saline marshes. The BC2 plants descended from a single F1 hybrid backcrossed toward H. petiolaris. The strength of selection on candidate adaptive traits in the interspecific BC2 was measured in natural H. paradoxus salt marsh habitat. Positive directional selection was detected for leaf succulence and Ca uptake, two traits that are known to be important in salt stress response in plants. Strong negative directional selection operated on uptake of Na and correlated elements. A significant decrease in trait correlations over time was observed in the BC2 population for Na and Ca content, suggesting an adaptive role for increased Ca uptake coupled with increased net exclusion of Na from leaves. Patterns of directional selection in BC2 hybrids were concordant with character expression in the natural hybrid species, H. paradoxus, transplanted into the wild. Moreover, the necessary variation for generating the H. paradoxus phenotype existed only in the BC2 population, but not in samples of the two parental species, H. annuus and H. petiolaris. These results are consistent with the hypothesis that transgressive segregation of elemental uptake and leaf succulence contributed to the origin of salt adaptation in the diploid hybrid species H. paradoxus.     

Re-creating ancient hybrid species' complex phenotypes from early-generation synthetic hybrids: three examples using wild sunflowers

Can the complex phenotypes that characterize naturally occurring hybrid species be re-created in early-generation artificial hybrids? We address this question with three homoploid hybrid species (Helianthus anomalus, Helianthus deserticola, Helianthus paradoxus) and their ancestral parents (Helianthus annuus, Helianthus petiolaris) that are phenotypically distinct and ecologically differentiated. These species, and two synthetic hybrid populations of the ancestral parents, were characterized for morphological, physiological, and life-history traits in greenhouse studies. Among the synthetic hybrids, discriminant analysis identified a few individuals with the multitrait phenotype of the natural hybrid species: 0.7%-1.1% were H. anomalus-like, 0.5%-13% were H. deserticola-like, and only 0.4% were H. paradoxus-like. These relative frequencies mirror previous findings that genetic correlations are favorable for generating the hybrid species' phenotypes, and they correspond well with phylogeographic evidence that demonstrates multiple natural origins of H. deserticola and H. anomalus but a single origin for H. paradoxus. Even though synthetic hybrids with hybrid species phenotypes are rare, their phenotypic correlation matrices share most of the same principal components (eigenvectors), setting the stage for predictable recovery of hybrid species' phenotypes from different hybrid populations. Our results demonstrate past hybridization could have generated hybrid species-like multitrait phenotypes suitable for persistence in their respective environments in just three generations after initial hybridization.     

Patterns of genetic diversity and candidate genes for ecological divergence in a homoploid hybrid sunflower, Helianthus anomalus

Natural hybridization accompanied by a shift in niche preference by hybrid genotypes can lead to hybrid speciation. Natural selection may cause the fixation of advantageous alleles in the ecologically diverged hybrids, and the loci experiencing selection should exhibit a reduction in allelic diversity relative to neutral loci. Here, we analyzed patterns of genetic diversity at 59 microsatellite loci associated with expressed sequence tags (ESTs) in a homoploid hybrid sunflower species, Helianthus anomalus. We used two indices, ln RV and ln RH, to compare variation and heterozygosity (respectively) at each locus between the hybrid species and its two parental species, H. annuus and H. petiolaris. Mean values of ln RV and ln RH were significantly lower than zero, which implies that H. anomalus experienced a population bottleneck during its recent evolutionary history. After correcting for the apparent bottleneck, we found six loci with a significant reduction in variation or with heterozygosity in the hybrid species, compared to one or both of the parental species. These loci should be viewed as a ranked list of candidate loci, pending further sequencing and functional analyses. Sequence data were generated for two of the candidate loci, but population genetics tests failed to detect deviations from neutral evolution at either locus. Nonetheless, a greater than eight-fold excess of nonsynonymous substitutions was found near a putative N-myristoylation motif at the second locus (HT998), and likelihood-based models indicated that the protein has been under selection in H. anomalus in the past and, perhaps, in one or both parental species. Finally, our data suggest that selective sweeps may have united populations of H. anomalus isolated by a mountain range, indicating that even low gene-flow species may be held together by the spread of advantageous alleles.     

Reconstructing the history of selection during homoploid hybrid speciation

This study aims to identify selection pressures during the historical process of homoploid hybrid speciation in three Helianthus (sunflower) hybrid species. If selection against intrinsic genetic incompatibilities (fertility selection) or for important morphological/ecological traits (phenotypic selection) were important in hybrid speciation, we would expect this selection to have influenced the parentage of molecular markers or chromosomal segments in the hybrid species' genomes. To infer past selection, we compared the parentage of molecular markers in high-density maps of the three hybrid species with predicted marker parentage from an analysis of fertility selection in artificial hybrids and from the directions of quantitative trait loci effects with respect to the phenotypes of the hybrid species. Multiple logistic regression models were consistent with both fertility and phenotypic selection in all three species. To further investigate traits under selection, we used a permutation test to determine whether marker parentage predicted from groups of functionally related traits differed from neutral expectations. Our results suggest that trait groups associated with ecological divergence were under selection during hybrid speciation. This study presents a new method to test for selection and supports earlier claims that fertility selection and phenotypic selection on ecologically relevant traits have operated simultaneously during sunflower hybrid speciation.     

Genetic variation of ecophysiological traits in two gynodioecious species of Schiedea (Caryophyllaceae)

Evolution of dimorphic breeding systems may involve changes in ecophysiological traits as well as floral morphology because of greater resource demands on females. Differences between related species suggest that ecophysiological traits should be heritable, and species with higher female frequencies should show greater sexual differentiation. We used modified partial diallel crossing designs to estimate narrow-sense heritabilities and genetic correlations of sex-specific ecophysiological and morphological traits in closely related gynodioecious Schiedea salicaria (13% females) and Schiedea adamantis (39% females). In S. salicaria, hermaphrodites and females differed in photosynthetic rate and specific leaf area (SLA). Narrow-sense heritabilities were significant for stomatal conductance, SLA and inflorescence number in hermaphrodites, and for SLA and inflorescence number in females. Schiedea adamantis had no sexual dimorphism in measured traits; stomatal conductance, stem number and inflorescence number were heritable in females, and stem number was heritable in hermaphrodites. In both species, significant genetic correlations of traits between sexes were rare, indicating that traits can evolve independently in response to sex-differential selection. Significant genetic correlations were detected between certain traits within sexes of both species. Low heritability of some ecophysiological traits may reflect low additive genetic variability or high phenotypic plasticity in these traits.     

Likely multiple origins of a diploid hybrid sunflower species

The recurrent origin of diploid hybrid species is theoretically improbable because of the enormous diversity of hybrid genotypes generated by recombination. Recent greenhouse experiments, however, indicate that the genomic composition of hybrid lineages is shaped in part by deterministic forces, and that recurrent diploid hybrid speciation may be more feasible than previously believed. Here we use patterns of variation from chloroplast DNA (cpDNA), nuclear microsatellite loci, cross-viability and chromosome structure to assess whether a well-characterized diploid hybrid sunflower species, Helianthus anomalus, was derived on multiple occasions from its parental species, H. annuus and H. petiolaris. Chloroplast DNA and crossability data were most consistent with a scenario in which H. anomalus arose three times: three different H. anomalus fertility groups were discovered, each with a unique cpDNA haplotype. In contrast, there was no clear signature of multiple, independent origins from the microsatellite loci. Given the age of H. anomalus (> 100 000 years bp), it may be that microsatellite evidence for recurrent speciation has been eroded by mutation and gene flow through pollen.     

Are hybrid species more fit than ancestral parent species in the current hybrid species habitats?

Hybrid speciation is thought to be facilitated by escape of early generation hybrids into new habitats, subsequent environmental selection and adaptation. Here, we ask whether two homoploid hybrid plant species (Helianthus anomalus, H. deserticola) diverged sufficiently from their ancestral parent species (H. annuus, H. petiolaris) during hybrid speciation so that they are more fit than the parent species in hybrid species habitats. Hybrid and parental species were reciprocally transplanted into hybrid and parental habitats. Helianthus anomalus was more fit than parental species in the H. anomalus actively moving desert dune habitat. The abilities to tolerate burial and excavation and to obtain nutrients appear to be important for success in the H. anomalus habitat. In contrast, H. deserticola failed to outperform the parental species in the H. deserticola stabilized desert dune habitat, and several possible explanations are discussed. The home site advantage of H. anomalus is consistent with environmental selection having been a mechanism for adaptive divergence and hybrid speciation and supports the use of H. anomalus as a valuable system for further assessment of environmental selection and adaptive traits.     

Crossing relationships among ancient and experimental sunflower hybrid lineages

Abstract.— Reproductive barrier formation between newly derived hybrid taxa and their parental species represents a major evolutionary hurdle. Here, I examine the development of a sterility barrier during hybrid speciation by examining the fertility of progeny from all combinations of crosses involving three experimentally synthesized sunflower hybrid lineages, their natural hybrid counterpart, Helianthus anomalus , and their parents, H. annuus and H. petiolaris . Crosses between the parental species and H. anomalus generated almost completely sterile offspring (pollen viability < 5%; seed set < 1%). A fairly strong sterility barrier also has developed between three hybrid lineages and both parental species (pollen viability 11.1–41.6%; seed set 0.84–20.1%). In contrast, the three hybrid lineages are almost fully interfertile (pollen viabilities 83.1–88.6%; seed set 72.1–75.3%), as predicted by molecular mapping studies that indicate they have converged on a similar set of gene combinations and chromosomal rearrangements. A modest decline in compability is observed in crosses between the three hybrid lineages and H. anomalus (pollen viabilities 64.1–70.7%; seed set 37–43%), a result that agrees well with prior data demonstrating significant congruence between the genomes of the natural and experimental hybrid lineages. These observations not only indicate that reproductive isolation can arise as a by-product of fertility selection in hybrid populations, but also testify to the repeatability of this mode of speciation.     

Adaptive introgression of herbivore resistance traits in the weedy sunflower Helianthus annuus

The role of hybridization in adaptive evolution is contentious. While many cases of adaptive trait introgression have been proposed, the relevant traits have rarely been identified, resulting in a lack of clear examples of this process. Here, we examine a purported case of adaptive introgression in which the annual sunflower Helianthus annuus annuus has captured alleles from a congener (Helianthus debilis) to form a stabilized hybrid, Helianthus annuus texanus. We tested the hypotheses that herbivore resistance traits have introgressed from H. debilis to H. annuus and have increased adaptation in the latter. In two common gardens, fitness (estimated by seed production) was on average 55% higher in H. a. texanus than in H. a. annuus. For H. a. texanus, three damage traits (of seven tested) differed significantly from the H. a. annuus parent in one or both sites and were shifted in the direction of the more resistant H. debilis. Natural selection favored H. a. annuusxH. debilis BC(1) hybrids (synthesized to mimic the ancestors of H. a. texanus) with H. debilis-like resistance to seed midges Neolasioptera helianthis and to receptacle/seed feeding Lepidoptera at one or both sites. Assuming similar herbivore pressures in the past, these results suggest that introgression of biotic resistance traits was important in the adaptation of H. annuus to central and southern Texas.     

Association Mapping and the Genomic Consequences of Selection in Sunflower

The combination of large-scale population genomic analyses and trait-based mapping approaches has the potential to provide novel insights into the evolutionary history and genome organization of crop plants. Here, we describe the detailed genotypic and phenotypic analysis of a sunflower (Helianthus annuus L.) association mapping population that captures nearly 90% of the allelic diversity present within the cultivated sunflower germplasm collection. We used these data to characterize overall patterns of genomic diversity and to perform association analyses on plant architecture (i.e., branching) and flowering time, successfully identifying numerous associations underlying these agronomically and evolutionarily important traits. Overall, we found variable levels of linkage disequilibrium (LD) across the genome. In general, islands of elevated LD correspond to genomic regions underlying traits that are known to have been targeted by selection during the evolution of cultivated sunflower. In many cases, these regions also showed significantly elevated levels of differentiation between the two major sunflower breeding groups, consistent with the occurrence of divergence due to strong selection. One of these regions, which harbors a major branching locus, spans a surprisingly long genetic interval (ca. 25 cM), indicating the occurrence of an extended selective sweep in an otherwise recombinogenic interval.     

Quantitative trait locus analysis of the early domestication of sunflower

Genetic analyses of the domestication syndrome have revealed that domestication-related traits typically have a very similar genetic architecture across most crops, being conditioned by a small number of quantitative trait loci (QTL), each with a relatively large effect on the phenotype. To date, the domestication of sunflower (Helianthus annuus L.) stands as the only counterexample to this pattern. In previous work involving a cross between wild sunflower (also H. annuus) and a highly improved oilseed cultivar, we found that domestication-related traits in sunflower are controlled by numerous QTL, typically of small effect. To provide insight into the minimum genetic changes required to transform the weedy common sunflower into a useful crop plant, we mapped QTL underlying domestication-related traits in a cross between a wild sunflower and a primitive Native American landrace that has not been the target of modern breeding programs. Consistent with the results of the previous study, our data indicate that the domestication of sunflower was driven by selection on a large number of loci, most of which had small to moderate phenotypic effects. Unlike the results of the previous study, however, nearly all of the QTL identified herein had phenotypic effects in the expected direction, with the domesticated allele producing a more crop-like phenotype and the wild allele producing a more wild-like phenotype. Taken together, these results are consistent with the hypothesis that selection during the post-domestication era has resulted in the introduction of apparently maladaptive alleles into the modern sunflower gene pool.     

The genomic organization of Ty3/gypsy-like retrotransposons in Helianthus (Asteraceae) homoploid hybrid species

The origin of new diploid, or homoploid, hybrid species is associated with rapid genomic restructuring in the hybrid neospecies. This mode of speciation has been best characterized in wild sunflower species in the genus Helianthus, where three homoploid hybrid species (H. anomalus, H. deserticola, and H. paradoxus) have independently arisen via ancient hybridization events between the same two parental species (H. annuus and H. petiolaris). Most previous work examining genomic restructuring in these sunflower hybrid species has focused on chromosomal rearrangements. However, the origin of all three homoploid hybrid sunflower species also is associated with massive proliferation events of Ty3/gypsy-like retrotransposons in the hybrid species' genomes. We compared the genomic organization of these elements in the parent species and two of the homoploid hybrid species using fluorescence in situ hybridization (FISH). We found a significant expansion of Ty3/gypsy-like retrotransposons confined to the pericentromeric regions of two hybrid sunflower species, H. deserticola and H. paradoxus. In contrast, we detected no significant increase in the frequency or extent of dispersed retrotransposon populations in the hybrid species within the resolution limits of our assay. We discuss the potential role that transposable element proliferation and localization plays in the evolution of homoploid hybrid species.     

QTL mapping of <Emphasis Type="Italic">Sclerotinia</Emphasis> midstalk-rot resistance in sunflower

In many sunflower-growing regions of the world, Sclerotinia sclerotiorum (Lib.) de Bary is the major disease of sunflower (Helianthus annuus L.). In this study, we mapped and characterized quantitative trait loci (QTL) involved in resistance to S. sclerotiorum midstalk rot and two morphological traits. A total of 351 F₃ families developed from a cross between a resistant inbred line from the germplasm pool NDBLOS and the susceptible line CM625 were assayed for their parental F₂ genotype at 117 codominant simple sequence repeat markers. Disease resistance of the F₃ families was screened under artificial infection in field experiments across two sowing times in 1999. For the three resistance traits (leaf lesion, stem lesion, and speed of fungal growth) and the two morphological traits, genotypic variances were highly significant. Heritabilities were moderate to high (h²=0.55–0.89). Genotypic correlations between resistance traits were highly significant (P<0.01) but moderate. QTL were detected for all three resistance traits, but estimated effects at most QTL were small. Simultaneously, they explained between 24.4% and 33.7% of the genotypic variance for resistance against S. sclerotiorum. Five of the 15 genomic regions carrying a QTL for either of the three resistance traits also carried a QTL for one of the two morphological traits. The prospects of marker-assisted selection (MAS) for resistance to S. sclerotiorum are limited due to the complex genetic architecture of the trait. MAS can be superior to classical phenotypic selection only with low marker costs and fast selection cycles.     

Remarkable life history polymorphism may be evolving under divergent selection in the silverleaf sunflower

Substantial intraspecific variation in life history is rare and potentially a signal of incipient ecological speciation, if variation is driven by geographically heterogenous natural selection. We present the first report of extensive life history polymorphism in Helianthus argophyllus, the silverleaf sunflower, and examine evidence for its evolution by divergent selection. In 18 populations sampled from across the species range and grown in a common garden, most quantitative traits covaried such that individuals could be assigned to two distinct life history syndromes: tall and late flowering with small initial flowerheads, or short and early flowering with larger initial flowerheads. Helianthus argophyllus exhibits regional genetic structure, but this population structure does not closely correspond with patterns of phenotypic variation. The early‐flowering syndrome is primarily observed in populations from coastal barrier islands, while populations from the nearby mainland coast, although geographically and genetically close, are primarily late flowering. Additionally, several traits are more differentiated among regions than expected based on neutral genetic divergence (Q_ST > F_ST), including the first principal component score corresponding with life history syndrome. This discordance between patterns of phenotypic and genetic variation suggests that divergent selection is driving genetic differences in life history across the species range. If so, the silverleaf sunflower may be in early stages of ecological speciation.     

Natural selection for salt tolerance quantitative trait loci (QTLs) in wild sunflower hybrids: implications for the origin of Helianthus paradoxus,a diploid hybrid species

For a new diploid or homoploid hybrid species to become established, it must diverge ecologically from parental genotypes. Otherwise the hybrid neospecies will be overcome by gene flow or competition. We initiated a series of experiments designed to understand how the homoploid hybrid species, Helianthus paradoxus, was able to colonize salt marsh habitats, when both of its parental species (H. annuusxH. petiolaris) are salt sensitive. Here, we report on the results of a quantitative trait locus (QTL) analysis of mineral ion uptake traits and survivorship in 172 BC2 hybrids between H. annuus and H. petiolaris that were planted in H. paradoxus salt marsh habitat in New Mexico. A total of 14 QTLs were detected for mineral ion uptake traits and three for survivorship. Several mineral ion QTLs mapped to the same position as the survivorship QTLs, confirming previous studies, which indicated that salt tolerance in Helianthus is achieved through increased Ca uptake, coupled with greater exclusion of Na and related mineral ions. Of greater general significance was the observation that QTLs with effects in opposing directions were found for survivorship and for all mineral ion uptake traits with more than one detected QTL. This genetic architecture provides an ideal substrate for rapid ecological divergence in hybrid neospecies and offers a simple explanation for the colonization of salt marsh habitats by H. paradoxus. Finally, selection coefficients of +0.126, -0.084 and -0.094 for the three survivorship QTLs, respectively, are sufficiently large to account for establishment of new, homoploid hybrid species.