A MOLECULAR REEXAMINATION OF INTROGRESSION BETWEEN HELIANTHUS ANNUUS AND H. BOLANDERI (COMPOSITAE)

Heiser (1949) hypothesized that a weedy race of Helianthus bolanderi had originated by the introgression of genes from H. annum into a serpentine race of H. bolanderi. Although Heiser's investigation of these species is frequently cited as one of the best examples of introgression in plants, definitive evidence of gene exchange is lacking (Heiser, 1973). To determine whether the weedy race of H. bolanderi actually originated via introgression, we analyzed allozyme, chloroplast-DNA (cpDNA), and nuclear-ribosomal-DNA (rDNA) variation. Evidence from enzyme electrophoresis did not support the proposed introgressive origin of weedy H. bolanderi. We detected a total of 37 low-frequency alleles distinguishing the serpentine race of H. bolanderi from H. annuus. Weedy H. bolanderi possessed only four of the 37 marker alleles. Further analysis demonstrated that serpentine H. bolanderi combined seven of the 35 alleles distinguishing H. annuus from weedy H. bolanderi, indicating that serpentine H. bolanderi shares three more marker alleles with H. annuus than does weedy H. bolanderi. These results are similar to expectations for race divergence from a single common ancestor and suggest that, if introgression occurred, the majority of marker alleles were rapidly lost following the initial hybridization event. Even more compelling evidence opposing Heiser's (1949) hypothesis, however, was from restriction-fragment analysis of cpDNA and nuclear rDNA. We detected a total of 17 cpDNA and five rDNA restriction-site mutations among the 19 populations examined. No parallel or back mutations were observed in phylogenetic trees constructed using either cpDNA or rDNA mutations, and both phylogenies were completely congruent regarding the alignment of all three taxa. In addition, the weedy race of H. bolanderi possessed a unique cpDNA, which was outside the range of variation observed among populations of either of the presumed parental species. Mean sequence divergences between the cpDNAs of weedy H. bolanderi and those of serpentine H. bolanderi and H. annuus were 0.30% and 0.35%, respectively. These estimates are comparable to sequence-divergence values observed between closely related species in other plant groups. Given the lack of parallel or convergent mutations in the cpDNA and rDNA phylogenetic trees, the complete congruence of these trees with flavonoid- and allozyme-variation patterns, and the presence of a unique and divergent chloroplast genome in the weedy race of H. bolanderi, we suggest that the weedy race of H. bolanderi was not derived recently through introgression, as hypothesized, but is relatively ancient in origin.     

Leaf expansion as related to plant water availability in a wild and a cultivated sunflower

This study aimed to determine if two species of sunflower, Helianthus annus L. cv. Hysun 31 (cultivated, single-stemmed genotype) and Helianthus petiolaris Nuttall ssp. fallax (wild, many-hranched genotype) differed in the response of leaf growth to water deficits. Earlier published studies, concerned only with H. annuus, failed to reveal differences in the response of sunflowers to water stress. Plants of the two species were paired in large containers of soil and grown under high radiation in a glasshouse. One batch of plants was irrigated and the other allowed to dry so that predawn leaf water potentials declined at an average of 0.072 MPa day^?1. The dry batch was rewatered when predawn leaf water potentials reached ?0.85 MPa. The stress imposed was sufficient to curtail leaf growth so that plants in the dry treatment had only 60% of the leaf area of irrigated plants at the onset of rewatering. Both species were affected by stress to the same relative extent, though their leaf areas at this stage differed 7-fold. Both genotypes also recovered to the same degree in the long term, finally having leaf areas and gross dry matter distribution patterns which were indistinguishable from plants which were irrigated throughout. However, water stress resulted in different distribution patterns of leaf area: H. annuus produced larger leaves at the top of its single stem which compensated for the reduced area in lower leaves, whereas H. petiolaris compensated in the leaves on its branches. Leaves which emerged after the time of stress were most able to compensate in area subsequently. For example, those leaves of H. annuus which emerged one week after stress-relief were more than three times larger than comparable leaves on plants irrigated continuously. Leaf expansion rates were affected earlier in the stress cycle than leaf conductance in H. annuus, but not in H. petiolaris. But as with other plant responses to water stress, the differences between the two species were small.     

Photosynthesis,dry matter accumulation and distribution in the wild sunflower Helianthus petiolaris and the cultivated sunflower Helianthus annuus as influenced by water deficits

Summary This study reports on the effects of water deficits on photosynthesis, plant growth and carbon allocation in the wild sunflower Helianthus petiolaris and in the cultivated sunflower Helianthus annuus grown under controlled conditions in the glasshouse. Water deficits reduced the rate of net photosynthesis and the dry weight of leaves, stems, roots and reproductive parts in both species. The root-to-shoot ratio of about 0.05 in H. petiolaris was lower than the root-to-shoot ratio of about 0.15 in H. annuus. Water stress did not affect the root-to-shoot ratio, but increased the percentage of roots at depth in H. annuus. The decrease in growth induced by water deficits was a consequence of a reduction in both leaf area production and net photosynthesis. Flowering occurred earlier in H. petiolaris than in H. annuus with a consequent earlier allocation of carbon to reproductive parts in the wild compared to the cultivated sunflower. The time to budding and flowering of either species was not altered by mild water stress, but was delayed by severe water deficits. During mild water stress carbon allocation to stems decreased, but that to reproductive parts increased. When plants were severely stressed and then rewatered the proportion af carbon allocated to leaves increased and the proportion allocated to stems decreased when compared to unstressed plants. The adaptative role of these features is discussed.     

Interspecific pollen competition as a reproductive barrier between sympatric species of Helianthus (Asteraceae)

Artificial crosses between Helianthus annuus and H. petiolaris using 1:9, 1:1, and 9:1 mixtures of intraspecific: interspecific pollen were conducted to determine the role of interspecific pollen competition as a reproductive barrier in Helianthus. Of 1,245 achenes analyzed from the pollen competition experiments, only 49 were hybrids. The number of hybrids observed was significantly less than expectations for all three pollen mixtures, regardless of the identity of maternal parent (P < 0.01). Stigma age and pollen ratio had no significant impact on hybrid frequency. However, hybrids were significantly more frequent with H. annuus than with H. petiolaris as the maternal parent (P < 0.01). Analysis of pollen tube growth rates revealed no differences in the rate of growth of intraspecific vs. interspecific pollen. Likewise, pollinations with either intraspecific or interspecific pollen or with different pollen ratios did not affect the percentage of filled achenes. Thus, the mechanism responsible for selective fertilization by intraspecific pollen in mixed pollen loads remains unclear. Nonetheless, these findings suggest that interspecific pollen competition plays an important role in controlling the formation of hybrids between H. annuus and H. petiolaris and may partially account for patterns or differential cytoplasmic vs. nuclear introgression in Helianthus.     

Molecular traits to elucidate the ancestry of Helianthus x multiflorus

The triploid perennial Helianthus x multiflorus L. is a beloved garden ornamental, but its phylogenetic origin has long been a source of discussion. Sequence comparison of genes involved in the biosynthesis of the sesquiterpene lactones was used to identify the common sunflower Helianthus annuus L. and the diploid taxon Helianthus decapetalus L. as the most likely parental combination in the origin of the triploid hybrid species. A pair of deletions (22 and 26 bp in length) in an intron part of the germacrene A synthase 1 gene differentiated the diploid from the tetraploid karyotype of H. decapetalus. The same deletion was found in one of the two alleles (299 bp in length) in H. x multiflorus, whereas a second allele (350 bp in length) showed a very high similarity with the sequence found in H. annuus. Quantification of the two amplification products in H. x multiflorus showed a higher concentration of the larger amplicon thus suggesting that H. annuus has contributed an unreduced genome to the hybrid. Comparison of variable regions of the cpDNA showed highest identity of H. x multiflorus with cpDNA sequences of the diploid H. decapetalus. This suggests that H. decapetalus served as the maternal parent in a cross with H. annuus to produce H. x multiflorus.     

Female preference and larval performance of sunflower moth,Homoeosoma electellum,on sunflower pre‐breeding lines

The preference–performance hypothesis for insect herbivores predicts that adult females should preferentially choose hosts on which their offspring perform better. We tested this hypothesis for the sunflower moth, Homoeosoma electellum (Hulst) (Lepidoptera: Pyralidae), using 16 sunflower (pre‐breeding) lines, derived from a number of wild species of Helianthus, including Helianthus annuus L., Helianthus deserticola Heiser, Helianthus paradoxus Heiser, Helianthus praecox Engelm. & Gray ssp. hirtus (Heiser) Heiser, Helianthus praecox Engelm. & Gray ssp. runyonii (Heiser) Heiser, Helianthus petiolaris Nutt., Helianthus resinosus Small, and Helianthus tuberosus L. (Asteraceae), that are suitable for introducing wild sunflower germplasm into commercial cultivars. Female moths showed a range of ovipositional preference measures to the various lines. Combined data for three Helianthus species represented by multiple lines showed significant differences in female preference with respect to the parental species. Larval performance, determined by proportion of infested neonate larvae reaching the pupal stage, or mean pupal weight, varied across the lines and, as for the female preference data, also showed significant differences among the three parental Helianthus species represented by multiple lines. These data suggest that the characteristics in the pre‐breeding lines influencing female sunflower moth preference and larval performance likely originate from the parental species and may be consistently transferred to the derived pre‐breeding lines. Of particular note with regard to potential plant resistance mechanisms, lines derived from H. tuberosus showed consistent low preference–performance measures. Female preference and larval performance (for both measures) were strongly correlated, indicating that females preferred plants and lines on which larvae performed better, in support of the preference–performance hypothesis.     

Microsporogenesis Abortion in Cytoplasmic Male Sterile Plants from H. petiolaris or H. petiolaris fallax Crossed by Sunflower (Helianthus annuus)

The microsporogenesis of two sunflower lines carrying two differentcytoplasmic male sterilities (CMS) from H. petiolaris (PL) andfrom H. petiolaris fallax (PF) has been studied and comparedto the normal process in male fertile lines. The first signsof abortion are, respectively, the vacuolation of the endoplasmicreticulum in PL during the first division of meiosis and theabnormal deposition of the endexine layer in PF during the vacuolationof the microspore. The genotype of the male line seems to modulate the stage ofpollen abortion, Furthermore, two ways of abortion have beenobserved for a single male sterile plant. Consequently, we suggestthat the ultrastructural observations reflect only the mainconsequences of a primary unknown event which takes place earlier. Cytoplasmic male-sterility, meiosis, Helianthus annuus L, H. petiolaris Nutt, microsporogenesis, electron microscopy     

A comparison of the water relations characteristics of Helianthus annuus and Helianthus petiolaris when subjected to water deficits

The effect of water deficits on the water relations and stomatal responses of Helianthus annuus and Helianthus petiolaris were compared in plants growing in the glasshouse under controlled conditions. Unirrigated plants of both genotypes were subjected to two different stress rates in which predawn leaf water potentials declined steadily at either 0.15 MPa day^?1 or 0.50 MPa day^?1. In both genotypes water stress induced a gradual and similar decrease in leaf conductance from 1.6 to 0.3 cm s^?1 as water potential decreased from-0.5 to-2.0 MPa. The relationship between leaf conductance and leaf water potential was not affected by the rate of stress development. Development of predawn leaf water potentials of-1.3 MPa had no significant effect on the relative water content at zero turgor, the apoplastic water content or the volumetric elastic modulus of whole leaves in either species, but decreased the osmotic potential at full turgor and zero turgor by 0.22 MPa and decreased the turgid weight: dry weight ratio from 10.6 to 8.4 in H. annuus, but not in H. petiolaris. In H. annuus leaves expanded during stress development, changes in the osmotic potential at full turgor induced by water deficits did not disappear on rewatering.     

Characterization of a <Emphasis Type="Italic">Plasmopara</Emphasis> isolate from <Emphasis Type="Italic">Helianthus × laetiflorus</Emphasis> based on cross infection,morphological, fatty acids and molecular phylogenetic data

A collection of Plasmopara halstedii s. l. from Helianthus × laetiflorus is recorded, described and characterised by laboratory cross infection studies, light microscopical studies, fatty acid pattern and LSU rDNA data. Its ability to infect Helianthus annuus was demonstrated in laboratory cross infection studies, however, it could not be unequivocally ascribed to a known pathotype. Microscopically, it shows no differences from isolates originating from Helianthus annuus, its fatty acid pattern is indistinguishable, and also its LSU rDNA sequence is identical. Due to this identity in a broad range of characters, the investigated downy mildew isolates from H. × laetiflorus and from H. annuus are regarded as conspecific.     

Molecular Evidence and the Origin and Development of the Domesticated Sunflower (Helianthus annum, Asteraceae)

The domesticated sunflower,Helianthus annuus, is an important economic crop, yet molecular data regarding its evolution are limited. Here we review morphological, geographical, archaeological, and molecular evidence pertaining to its origin and development. New isozyme and chloroplast DNA (cpDNA) evidence is also presented. Morphological, geographical, and archaeological evidence has led to the hypothesis that the domesticated sunflower was derived from a wild/weedy form ofH. annuus possibly in the Midwest. Molecular evidence was concordant with this hypothesis. A high degree of enzymatic and cpDNA sequence similarity was observed between wild and domesticatedH. annuus, and domesticatedH. annuus contained a subset of the alleles and cpDNAs found in wildH. annuus. The extensive polymorphism in the wild plants and the virtual monomorphism in cultivated lines for both isozyme and cpDNA phenotypes further suggest a single origin of the domesticated sunflower from a very limited gene pool. In addition, Native American varieties of the domesticated sunflower were genetically more variable than other cultivated lines, possibly indicating that they gave rise to the other cultivated stocks. Molecular evidence did not, however, allow conclusions as to the exact geographic origin of the domesticated sunflower.     

Genome scan of hybridizing sunflowers from Texas (Helianthus annuus and H. debilis) reveals asymmetric patterns of introgression and small islands of genomic differentiation

Although the sexual transfer of genetic material between species (i.e. introgression) has been documented in many groups of plants and animals, genome‐wide patterns of introgression are poorly understood. Is most of the genome permeable to interspecific gene flow, or is introgression typically restricted to a handful of genomic regions? Here, we assess the genomic extent and direction of introgression between three sunflowers from the south‐central USA: the common sunflower, Helianthus annuus ssp. annuus; a near‐endemic to Texas, Helianthus debilis ssp. cucumerifolius; and their putative hybrid derivative, thought to have recently colonized Texas, H. annuus ssp. texanus. Analyses of variation at 88 genetically mapped microsatellite loci revealed that long‐term migration rates were high, genome‐wide and asymmetric, with higher migration rates from H. annuus texanus into the two parental taxa than vice versa. These results imply a longer history of intermittent contact between H. debilis and H. annuus than previously believed, and that H. annuus texanus may serve as a bridge for the transfer of alleles between its parental taxa. They also contradict recent theory suggesting that introgression should predominantly be in the direction of the colonizing species. As in previous studies of hybridizing sunflower species, regions of genetic differentiation appear small, whether estimated in terms of F_ST or unidirectional migration rates. Estimates of recent immigration and admixture were inconsistent, depending on the type of analysis. At the individual locus level, one marker showed striking asymmetry in migration rates, a pattern consistent with tight linkage to a Bateson–Dobzhansky–Muller incompatibility.     

Oil concentration and fatty acid composition of achenes ofHelianthus species (Asteraceae) from Canada

The cultivated sunflower (Helianthus annuus) is one of the four most important annual crops in the world grown for its edible oil. Species of the genusHelianthus served as the ancestral stock for the present cultivated sunflower. The wild relatives are a valuable genetic reservoir for improving the crop species. This study examined oil concentration and fatty acid composition in oil of two annualHelianthus species (31 populations) and four perennial species (32 populations) from the prairie provinces of Canada. The highest average oil concentration was observed in annualH. petiolaris with 31.3%. Among the perennials,H. maximiliani Schrad. had the highest average oil concentration with 31.1%.Helianthus tuberosus L. had the highest average palmitic (16:0) acid concentration with 8.0%, while the lowest value was observed inH. petiolaris with 4.0%. The highest average stearic (18:0) acid concentration was found inH. annuus with 2.9%, whileH. petiolaris had the lowest with 2.3%. The highest average oleic acid (18:1) was observed in H. petiolaris (18.4%), and the highest linoleic (18:2) was observed inH. maximiliani (77.4%). The variability within the wild species appears to be sufficient for altering oil and fatty acid characteristics in cultivated sunflower.     

Persistence of sunflower crop traits and fitness in Helianthus petiolaris populations

Transgenic plants have increased interest in the study of crop gene introgression in wild populations. Genes (or transgenes) conferring adaptive advantages persist in introgressed populations, enhancing competitiveness of wild or weedy plants. This represents an ecological risk that could increase problems of weed control. Introgression of cultivar alleles into wild plant populations via crop–wild hybridisations is primarily governed by their fitness effect. To evaluate this, we studied the second generation of seven wild–crop interspecific hybrids between weedy Helianthus petiolaris and cultivated sunflower, H. annuus var. macrocarpus. The second generation comprised open‐pollinated progeny and backcrosses to the wild parent, mimicking crosses that occur in natural situations. We compared a number of morphological, life history and fitness traits. Multivariate analysis showed that the parental species H. annuus and H. petiolaris differed in a number of morphological traits, while the second hybrid generation between them was intermediate. Sunflower crop introgression lowered fitness of interspecific hybrids, but fitness parameters tended to recover in the following generation. Relative frequency of wild/weedy and introgressed plants was estimated through four generations, based on male and female parent fitness. In spite of several negative selection coefficients observed in the second generation, introgressed plants could be detected in stands of <100 weedy H. petiolaris populations. The rapid recovery of fecundity parameters leads to prediction that any trait conferring an ecological advantage will diffuse into the wild or weedy population, even if F1 hybrids have low fitness.     

Allozyme variation in domesticated annual sunflower and its wild relatives

 The annual sunflower (Helianthus annuus L.) is a morphologically and genetically variable species composed of wild, weedy, and domesticated forms that are used for ornament, oilseed, and edible seeds. In this study, we evaluated genetic variation in 146 germplasm accessions of wild and domesticated sunflowers using allozyme analysis. Results from this survey showed that wild sunflower exhibits geographically structured genetic variation, as samples from the Great Plains region of the central United States were genetically divergent from accessions from California and the southwestern United States. Sunflower populations from the Great Plains harbored greater allelic diversity than did wild sunflower from the western United States. Comparison of genetic variability in wild and domesticated sunflower by principal coordinate analysis showed these groups to be genetically divergent, in large part due to differences in the frequency of common alleles. Neighbor-Joining analyses of domesticated H. annuus, wild H. annuus and two closely related wild species (H. argophyllus T. & G. and H. petiolaris Nutt.) showed that domesticated sunflowers form a genetically coherent group and that wild sunflowers from the Great Plains may include the most likely progenitor of domesticated sunflowers. Received: 2 December 1996/Accepted: 4 April 1997     

Physical map and gene localization on sunflower (Helianthus annuus) chloroplast DNA: evidence for an inversion of a 23.5-kbp segment in the large single copy region

As a first step in the study of chloroplast genome variability in the genus Helianthus, a physical restriction map of sunflower (Helianthus annuus) chloroplast DNA (cpDNA) has been constructed using restriction endonucleases BamH I, Hind III, Pst I, Pvu II and Sac. I. Sunflower circular DNA contains an inverted repeat structure with the two copies (23 kbp each) separated by a large (86 kbp) and a small (20 kbp) single copy region. Its total length is therefore about 152 kbp. Sunflower cpDNA is essentially colinear with that of tobacco with the exception of an inversion of a 23.5-kbp segment in the large single copy region. Gene localization on the sunflower cpDNA and comparison of the gene map with that from tobacco chloroplasts have revealed that the endpoints of the inversion are located between the trnT and trnE genes on the one hand, and between the trnG and trnS genes on the other hand.Analysis of BamH I restriction fragment patterns of H. annuus, H. occidentalis ssp. plantagineus, H. grossesseratus, H. decapetalus, H. giganteus, H. maximiliani and H. tuberosus cpDNAs suggests that structural variations are present in the genus Helianthus.     

PATTERNS OF MATING IN WILD SUNFLOWER HYBRID ZONES

Theory predicts that homoploid hybrid speciation will be facilitated by selfing, yet most well-documented hybrid species are outcrossers. One possible explanation for this puzzle is that conditions in hybrid populations may favor selfing, even in otherwise outcrossing species. For example, in self-incompatible plants, mixtures of self and interspecific pollen often induce selfing. Here, we examine patterns of mating in three hybrid zones and four “pure” populations of Helianthus annuus and H. petiolaris, wild, self-incompatible sunflower species that are thought to have parented three homoploid hybrid species. Fourteen to 16 maternal families from each pure population and 44–46 maternal families from each hybrid zone were analyzed for seven polymorphic isozyme loci. Maximum-likelihood (ML) methods were used to estimate multilocus outcrossing rates (T_m) and hybridization frequencies for each maternal family, each phenotypic group within each hybrid zone (annuus-like, hybrid, and petiolaris-like), and each population. As predicted for self-incompatible species, all four parental populations have outcrossing rate ML estimates of 1.0. Within the hybrid zones, outcrossing rates were lowest in the H. annuus–like fraction of the population (0.73, 0.72, and 0.74 in the three hybrid zones, respectively), largely intermediate in the H. petiolaris–like group (0.94, 0.90, and 0.94), and highest in the hybrid group (0.97, 0.93, and 0.97). Although outcrossing rates are lower in hybrid zones than in pure populations, it is unlikely that the observed decrease facilitates hybrid speciation because outcrossing rates in the critical hybrid fraction of the population do not differ significantly from 1.0. Dividing the outcrossed pollen pool into intraspecific and interspecific components revealed that maternal plants are largely fertilized by conspecific pollen, confirming an important role for pollen competition as a reproductive barrier. Highly sterile hybrid plants do not appear to discriminate between parental species pollen, but hybrids with higher fertility tend to be fertilized by pollen from the parental group they resemble genetically. Thus, gametic selection leads to substantial assortative mating in these hybrid zones.     

Intra-individual heterogeneity of rDNA allows the distinction between two closely related species in the Genus Helianthus

The Ribosomal DNAs of Helianthus annuus and H. argophyllus were analysed. Total DNA from single individuals of six cultivated lines, one wild ecotype ofH. annuus, and three ecotypes of H. argophyllus, were digested with various restriction enzymes. Hybridisation of Southern blots with sunflower ribosomal probes containing most of the interspacer regions (R3) or the 25 s coding region (R2) reveals different patterns from those expected: while no difference between H. annuus and H. argophyllus had been observed in previous rDNA RFLP analysis, our study clearly distinguished the two species on the basis of two different patterns when using R3 and BamHI, BstYI, or EcoRI/BamHI. Furthermore, the sum of the fragment weights of the BamHI restriction patterns was much greater than that of the rDNA entire unit-weight space. The co-existence of different rDNA units within single individuals is proposed as a model to explain these results. Four rDNA units were distinguished, which differed in their state of methylation and by the presence of mutations at two BamRI restriction sites. H. annuus individuals displayed two types of rDNA units while H. argophyllus individuals displayed four types.     

DIVERGENCE IS FOCUSED ON FEW GENOMIC REGIONS EARLY IN SPECIATION: INCIPIENT SPECIATION OF SUNFLOWER ECOTYPES

Early in speciation, as populations undergo the transition from local adaptation to incipient species, is when a number of transient, but potentially important, processes appear to be most easily detected. These include signatures of selective sweeps that can point to asymmetry in selection between habitats, divergence hitchhiking, and associations of adaptive genes with environments. In a genomic comparison of ecotypes of the prairie sunflower, Helianthus petiolaris, occurring at Great Sand Dunes National Park and Preserve (Colorado), we found that selective sweeps were mainly restricted to the dune ecotype and that there was variation across the genome in whether proximity to the nondune population constrained or promoted divergence. The major regions of divergence were few and large between ecotypes, in contrast with an interspecific comparison between H. petiolaris and a sympatric congener, Helianthus annuus. In general, the large regions of divergence observed in the ecotypic comparison swamped locus‐specific associations with environmental variables. In both comparisons, regions of high divergence occurred in portions of the genetic map with high marker density, probably reflecting regions of low recombination. The difference in genomic distributions of highly divergent regions between ecotypic and interspecific comparisons highlights the value of studies spanning the spectrum of speciation in related taxa.