H. D. Bradshaw

Allele substitution at a flower colour locus produces a pollinator shift in monkeyflowers

The role of major mutations in adaptive evolution has been debated for more than a century. The classical view is that adaptive mutations are nearly infinite in number with infinitesimally small phenotypic effect, but recent theory suggests otherwise. To provide empirical estimates of the magnitude of adaptive mutations in wild plants, we conducted field studies to determine the adaptive value of alternative alleles at a single locus, YELLOW UPPER (YUP). YUP controls the presence or absence of yellow carotenoid pigments in the petals of pink-flowered Mimulus lewisii, which is pollinated by bumblebees, and its red-flowered sister species M. cardinalis, which is pollinated by hummingbirds. We bred near-isogenic lines (NILs) in which the YUP allele from each species was substituted into the other. M. cardinalis NILs with the M. lewisii YUP allele had dark pink flowers and received 74-fold more bee visits than the wild type, whereas M. lewisii NILs with the M. cardinalis yup allele had yellow-orange flowers and received 68-fold more hummingbird visits than the wild type. These results indicate that an adaptive shift in pollinator preference may be initiated by a single major mutation.

Components of reproductive isolation between the monkeyflowers Mimulus lewisii and M. cardinalis (Phrymaceae)

Abstract.— Evolutionists have long recognized the role of reproductive isolation in speciation, but the relative contributions of different reproductive barriers are poorly understood. We examined the nature of isolation between Mimulus lewisii and M. cardinalis, sister species of monkeyflowers. Studied reproductive barriers include: ecogeo‐graphic isolation; pollinator isolation (pollinator fidelity in a natural mixed population); pollen competition (seed set and hybrid production from experimental interspecific, intraspecific, and mixed pollinations in the greenhouse); and relative hybrid fitness (germination, survivorship, percent flowering, biomass, pollen viability, and seed mass in the greenhouse). Additionally, the rate of hybridization in nature was estimated from seed collections in a sympatric population. We found substantial reproductive barriers at multiple stages in the life history of M. lewisii and M. cardinalis. Using range maps constructed from herbarium collections, we estimated that the different ecogeographic distributions of the species result in 58.7% reproductive isolation. Mimulus lewisii and M. cardinalis are visited by different pollinators, and in a region of sympatry 97.6% of pollinator foraging bouts were specific to one species or the other. In the greenhouse, interspecific pollinations generated nearly 50% fewer seeds than intraspecific controls. Mixed pollinations ofM. cardinalis flowers yielded > 75% parentals even when only one‐quarter of the pollen treatment consisted of M. cardinalis pollen. In contrast, both species had similar siring success on M. lewisii flowers. The observed 99.915% occurrence of parental M. lewisii and M. cardinalis in seeds collected from a sympatric population is nearly identical to that expected, based upon our field observations of pollinator behavior and our laboratory experiments of pollen competition. F1 hybrids exhibited reduced germination rates, high survivorship and reproduction, and low pollen and ovule fertility. In aggregate, the studied reproductive barriers prevent, on average, 99.87% of gene flow, with most reproductive isolation occurring prior to hybrid formation. Our results suggest that ecological factors resulting from adaptive divergence are the primary isolating barriers in this system. Additional studies of taxa at varying degrees of evolutionary divergence are needed to identify the relative importance of pre‐ and postzygotic isolating mechanisms in speciation.

Emerging model systems in plant biology: poplar (Populus) as a model forest tree.

Forest trees have tremendous economic and ecological value, as well as unique biological properties of basic scientific interest. The inherent difficulties of experimenting on very large long-lived organisms motivates the development of a model system for forest trees. Populus (poplars, cottonwoods, aspens) has several advantages as a model system, including rapid growth, prolific sexual reproduction, ease of cloning, small genome, facile transgenesis, and tight coupling between physiological traits and biomass productivity. A combination of genetics and physiology is being used to understand the detailed mechanisms of forest tree growth and development.

Molecular genetics of growth and development in Populus. III. A genetic linkage map of a hybrid poplar composed of RFLP, STS, and RAPD markers

We have evaluated three DNA-based marker types for linkage map construction in Populus: RFLPs detected by Southern blot hybridization, STSs detected by a combination of PCR and RFLP analysis, and RAPDs. The mapping pedigree consists of three generations, with the F1 produced by interspecific hybridization between a P. trichocarpa female and a P. deltoides male. The F2 generation was made by inbreeding to the maximum degree permitted by the dioecious mating system of Populus. The applicability of STSs and RAPDs outside the mapping pedigree has been investigated, showing that these PCR-based marker systems are well-suited to breeding designs involving interspecific hybridization. A Populus genome map (343 markers) has been constructed from a combination of all three types. The length of the Populus genome is estimated to be 2400–2800 cM.

Molecular genetics of growth and development in Populus. II. Segregation distortion due to genetic load

Distortion of expected Mendelian segregation ratios, commonly observed in many plant taxa, has been detected in an experimental three-generation inbred pedigree of Populus founded by interspecific hybridization between P. trichocarpa and P. deltoides. An RFLP linkage map was constructed around a single locus showing severe skewing of segregation ratio against F2 trees carrying the P. trichocarpa allele in homozygous form. Several hypotheses for the mechanism of segregation distortion at this locus were tested, including directional chromosome loss, segregation of a pollen lethal allele, conflicts between genetic factors that isolate the parental species, and inbreeding depression as a result of genetic load. Breeding experiments to produce inbred and outcrossed progenies were combined with PCR-based detection of RFLPs to follow the fate of the deficient allele throughout embryo and seedling development. A recessive lethal allele, lth, inherited from the P. trichocarpa parent, was found to be tightly linked to the RFLP marker locus POP1054 and to cause embryo and seedling mortality. Heterozygotes (lth/+) appear to be phenotypically normal as embryos, seedlings, and young trees.

Genetic Dissection of a Major Anthocyanin QTL Contributing to Pollinator-Mediated Reproductive Isolation Between Sister Species of Mimulus

Prezygotic barriers play a major role in the evolution of reproductive isolation, which is a prerequisite for speciation. However, despite considerable progress in identifying genes and mutations responsible for postzygotic isolation, little is known about the genetic and molecular basis underlying prezygotic barriers. The bumblebee-pollinated Mimulus lewisii and the hummingbird-pollinated M. cardinalis represent a classic example of pollinator-mediated prezygotic isolation between two sister species in sympatry. Flower color differences resulting from both carotenoid and anthocyanin pigments contribute to pollinator discrimination between the two species in nature. Through fine-scale genetic mapping, site-directed mutagenesis, and transgenic experiments, we demonstrate that a single-repeat R3 MYB repressor, ROSE INTENSITY1 (ROI1), is the causal gene underlying a major quantitative trait locus (QTL) with the largest effect on anthocyanin concentration and that cis-regulatory change rather than coding DNA mutations cause the allelic difference between M. lewisii and M. cardinalis. Together with the genomic resources and stable transgenic tools developed here, these results suggest that Mimulus is an excellent platform for studying the genetics of pollinator-mediated reproductive isolation and the molecular basis of morphological evolution at the most fundamental level—gene by gene, mutation by mutation.

Two High-Throughput Techniques for Determining Wood Properties as Part of a Molecular Genetics Analysis of Hybrid Poplar and Loblolly Pine

Two new high-through put techniques, computer tomography X-ray densitometry (CT scan) and pyrolysis molecular beam mass spectrometry (pyMBMS), coupled with quantitative trait loci (QTL) analysis, were tested as a means to overcome the time and cost associated with conventional characterization of biomass feedstock components. Applications of these two techniques were evaluated using hybrid poplar for the CT scan and loblolly pine for the pyMBMS. Segregating progeny from hybrid poplar varied in specific gravity, with individual mean estimates ranging from 0.21–0.41. Progeny from loblolly pine varied in lignin, α cellulose, and mannan contents, with individual mean estimates of lignin content ranging from 28.7–33.1%, α cellulose content from 28.8–43.5% and mannan content from 4.2–10.1%. QTL analysis of the loblolly pine data suggested that eleven QTLs were associated with individual feedstock characteristics and that two QTLs for several feedstock components were linked to the same position on the loblolly pine genetic map. Each QTL individually accounted for 7–13% of the total phenotypic variation in associated loblolly pine feedstock components.

Three floral volatiles contribute to differential pollinator attraction in monkeyflowers (Mimulus)

Flowering plants employ a wide variety of signals, including scent, to attract the attention of pollinators. In this study we investigated the role of floral scent in mediating differential attraction between two species of monkeyflowers (Mimulus) reproductively isolated by pollinator preference. The emission rate and chemical identity of floral volatiles differ between the bumblebee-pollinated Mimulus lewisii and the hummingbird-pollinated M. cardinalis. Mimulus lewisii flowers produce an array of volatiles dominated by d-limonene, β-myrcene and E-β-ocimene. Of these three monoterpenes, M. cardinalis flowers produce only d-limonene, released at just 0.9% the rate of M. lewisii flowers. Using the Bombus vosnesenskii bumblebee, an important pollinator of M. lewisii, we conducted simultaneous gas chromatography with extracellular recordings in the bumblebee antennal lobe. Results from these experiments revealed that these three monoterpenes evoke significant neural responses, and that a synthetic mixture of the three volatiles evokes the same responses as the natural scent. Furthermore, the neural population shows enhanced responses to the M. lewisii scent over the scent of M. cardinalis. This neural response is reflected in behavior; in two-choice assays, bumblebees investigate artificial flowers scented with M. lewisii more frequently than ones scented with M. cardinalis, and in synthetic mixtures the three monoterpenes are necessary and sufficient to recapitulate responses to the natural scent of M. lewisii. In this system, floral scent alone is sufficient to elicit differential visitation by bumblebees, implying a strong role of scent in the maintenance of reproductive isolation between M. lewisii and M. cardinalis.

Wound-induced and developmental activation of a poplar tree chitinase gene promoter in transgenic tobacco

Wounding hybrid poplar (Populus trichocarpa × P. deltoides) trees results in the expression of novel wound-inducible (win) mRNAs thought to encode proteins involved in defense against pests and pathogens. Members of thewin6 gene family encode acidic multi-domain chitinases, with combined structure and charge characteristics that differ from previously described chitinases.Win6 expression has been shown to occur in pooled unwounded leaves of a wounded (on multiple leaves) poplar plant. Here we demonstrate that wounding a single leaf induceswin6 expression locally, in the wounded leaf, and remotely, in specific unwounded leaves with strong vascular connections to the wounded leaf. We also demonstrate that awin6 promoter-β-glucuronidase (GUS) gene fusion (win6-GUS) responds to wounding locally and remotely in transgenic tobacco. These data indicate that the poplarwin6 promoter has regulatory elements that are responsive to ‘wound signals’ in the heterologous host. In addition,win6-GUS is developmentally activated in unwounded young leaves and floral tissues of transgenic tobacco. Similar developmental expression patterns are found to occur forwin6 in poplar trees, demonstrating that a herbaceous plant can serve as a host for woody tree transgene analysis and can accurately predict expression patterns in tree tissues (e.g. flowers) that would be difficult to study in free-living trees.

The genetic control of flower-pollinator specificity.

The ca. 275,000 species of flowering plants are the result of a recent adaptive radiation driven largely by the coevolution between plants and their animal pollinators. Identification of genes and mutations responsible for floral trait variation underlying pollinator specificity is crucial to understanding how pollinator shifts occur between closely related species. Petunia, Mimulus, and Antirrhinum have provided a high standard of experimental evidence to establish causal links from genes to floral traits to pollinator responses. In all three systems, MYB transcription factors seem to play a prominent role in the diversification of pollinator-associated floral traits.