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, ß-myrcene and E-ß-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.

Taxon-specific, phased siRNAs underlie a speciation locus in monkeyflowers.

Taxon-specific small RNA loci are widespread in eukaryotic genomes, yet their role in lineage-specific adaptation, phenotypic diversification, and speciation is poorly understood. Here, we report that a speciation locus in monkeyflowers (Mimulus), YELLOW UPPER (YUP), contains an inverted repeat region that produces small interfering RNAs (siRNAs) in a phased pattern. Although the inverted repeat is derived from a partial duplication of a protein-coding gene that is not involved in flower pigmentation, one of the siRNAs targets and represses a master regulator of floral carotenoid pigmentation. YUP emerged with two protein-coding genes that control other aspects of flower coloration as a "superlocus" in a subclade of Mimulus and has contributed to subsequent phenotypic diversification and pollinator-mediated speciation in the descendant species.

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.

Two MYB Proteins in a Self-Organizing Activator-Inhibitor System Produce Spotted Pigmentation Patterns.

Many organisms exhibit visually striking spotted or striped pigmentation patterns. Developmental models predict that such spatial patterns can form when a local autocatalytic feedback loop and a long-range inhibitory feedback loop interact. At its simplest, this self-organizing network only requires one self-activating activator that also activates a repressor, which inhibits the activator and diffuses to neighboring cells. However, the molecular activators and inhibitors fully fitting this versatile model remain elusive in pigmentation systems. Here, we characterize an R2R3-MYB activator and an R3-MYB repressor in monkeyflowers (Mimulus). Through experimental perturbation and mathematical modeling, we demonstrate that the properties of these two proteins correspond to an activator-inhibitor pair in a two-component, reaction-diffusion system, explaining the formation of dispersed anthocyanin spots in monkeyflower petals. Notably, disrupting this pattern impacts pollinator visitation. Thus, subtle changes in simple activator-inhibitor systems are likely essential contributors to the evolution of the remarkable diversity of pigmentation patterns in flowers.

Morphospace exploration reveals divergent fitness optima between plants and pollinators.

The obligate mutualism and exquisite specificity of many plant-pollinator interactions lead to the expectation that flower phenotypes (e.g., corolla tube length) and corresponding pollinator traits (e.g., hawkmoth proboscis length) are congruent as a result of coevolution by natural selection. However, the effect of variation in flower morphology on the fitness of plants and their pollinators has not been quantified systematically. In this study, we employed the theoretical morphospace paradigm using a combination of 3D printing, electronic sensing, and machine vision technologies to determine the influence of two flower morphological features (corolla curvature and nectary diameter) on the fitness of both parties: the artificial flower and its hawkmoth pollinator. Contrary to the expectation that the same flower morphology maximizes the fitness of both plant and pollinator, we found that the two parties have divergent optima for corolla curvature, with non-overlapping fitness peaks in flower morphospace. The divergent fitness optima between plants and pollinators could lead to evolutionary diversification in both groups.

Using experimental evolution to investigate geographic range limits in monkeyflowers.

Every species occupies a restricted geographic distribution, but it is unclear why natural selection at the range margin fails to increase tolerance to limiting environmental variables and thereby allow continual range expansion. Models indicate that the interplay of demographic asymmetries, dispersal, divergent natural selection, and adaptive trade-offs across spatially varying environments can give rise to stable range limits. Here we examine sister species of the monkeyflowers Mimulus cardinalis and M. lewisii to identify traits that might contribute to the evolution of the species' ranges and to ask whether adaptive trade-offs between environments can limit their geographic distribution. In the Sierra Nevada Mountains of California, M. cardinalis is found from low to mid elevation and M. lewisii is found from mid to high elevation. We transplanted segregating populations of interspecific hybrids to low and high elevation and cross-pollinated those that survived to flowering to create selected populations that evolved at low or high elevation. When grown in a common environment, the progeny of hybrids selected at high elevation flowered earlier compared to a greenhouse control population, whereas hybrids selected at low elevation displayed increased warm-temperature photosynthetic capacity. If adaptation to one environment entails a cost to adaptation in other environments, then selected hybrid populations should display reduced fitness, relative to an unselected control population, when grown in an environment in which they were not selected. Two such trade-offs were observed in this study, where hybrids selected at high elevation displayed reduced biomass when grown in temperatures characteristic of low elevation and hybrids selected at low elevation showed reduced resistance to freezing. These results identify traits under selection for range expansion and suggest that adaptive trade-offs can contribute to limiting the geographic distribution of species.

Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells.

A cDNA containing the entire coding region of the human thymidine kinase gene has been molecularly cloned. The cDNA is under the control of a simian virus 40 promoter and is expressible in mammalian cells. The complete nucleotide sequence of the human thymidine kinase cDNA has been determined. The cDNA is 1,421 base pairs in length and has a large open reading frame of 702 base pairs capable of specifying a protein with a molecular weight of 25,504. Genomic Southern blotting experiments show that sequences homologous to the human thymidine kinase cDNA are conserved among many vertebrates, including prosimians (lemur), tree shrews, rats, mice, and chickens. Direct comparison of the nucleotide sequences of the human thymidine kinase cDNA and the chicken thymidine kinase gene reveals ca. 70% overall homology. This homology is extended further at the amino acid sequence level, with greater than 74% amino acid residues matched between the human and chicken thymidine kinase proteins.

Molecular genetics of growth and development in populus. IV. Mapping QTLs with large effects on growth, form, and phenology traits in a forest tree.

We have mapped quantitative trait loci (QTLs) for commercially important traits (stem growth and form) and an adaptive trait (spring leaf flush) in a Populus F2 generation derived from a cross between interspecific F1 hybrids (P. trichocarpa x P. deltoides). Phenotypic data were collected over a 2-year period from a replicated clonal trial containing ramets of the parental, F1, and F2 trees. Contrary to the assumptions of simple polygenic models of quantitative trait inheritance, 1-5 QTLs of large effect are responsible for a large portion of the genetic variance in each of the traits measured. For example, 44.7% of the genetic variance in stem volume after 2 years of growth is controlled by just two QTLs. QTLs governing stem basal area were found clustered with QTLs for sylleptic branch leaf area, sharing similar chromosomal position and mode of action and suggesting a pleiotropic effect of QTLs ultimately responsible for stem diameter growth.

Molecular genetics of rust resistance in poplars (Melampsora larici-populina Kleb/Populus sp.) by bulked segregant analysis in a 2 x 2 factorial mating design.

With random amplified polymorphic DNA (RAPD) markers, we have tagged a genomic region in Populus sp. involved in qualitative resistance to Melampsora larici-populina. Our approach was based on three steps: use of RAPD markers that can be quickly and efficiently researched: application of "bulked segregant analysis" technique on individuals of one interspecific family P. trichocarpa x P. deltoides to search for RAPD markers linked to resistance; and validation of these markers in two other families linked with the first one in a 2 x 2 factorial mating design. Of five detected markers, only one marker M03/04_480 was polymorphic in the three segregating families, involving 89 individuals and four different parents. We have estimated the recombination value of 1 cM with 1 cM sampling error.

Quantitative trait loci affecting differences in floral morphology between two species of monkeyflower (Mimulus).

Conspicuous differences in floral morphology are partly responsible for reproductive isolation between two sympatric species of monkeyflower because of their effect on visitation of the flowers by different pollinators. Mimulus lewisii flowers are visited primarily by bumblebees, whereas M. cardinalis flowers are visited mostly by hummingbirds. The genetic control of 12 morphological differences between the flowers of M. lewisii and M. cardinalis was explored in a large linkage mapping population of F2 plants n = 465 to provide an accurate estimate of the number and magnitude of effect of quantitative trait loci (QTLs) governing each character. Between one and six QTLs were identified for each trait. Most (9/12) traits appear to be controlled in part by at least one major QTL explaining >/=25% of the total phenotypic variance. This implies that either single genes of individually large effect or linked clusters of genes with a large cumulative effect can play a role in the evolution of reproductive isolation and speciation.

Quantitative trait loci and candidate gene mapping of bud set and bud flush in populus.

The genetic control of bud phenology in hybrid poplar was studied by mapping quantitative trait loci (QTL) affecting the timing of autumn bud set and spring bud flush. The founders of the mapping pedigree were collected from widely separated latitudes to maximize segregating variation for dormancy-related traits in the F(2) generation-the female Populus trichocarpa parent is from Washington State (48 degrees N) and the male P. deltoides parent is from Texas (31 degrees N). Bud set and bud flush timing were measured on the F(2) generation in a replicated clonal field trial. Using a linkage map constructed of AFLP and microsatellite markers, three QTL controlling bud set and six QTL controlling bud flush were detected. Additionally, five candidate genes believed to be involved in perception of photoperiod (PHYB1, PHYB2) or transduction of abscisic acid response signals (ABI1B, ABI1D, and ABI3) were placed on the QTL map. PHYB2 and ABI1B were found to be coincident with QTL affecting bud set and bud flush.

Shape matters: corolla curvature improves nectar discovery in the hawkmoth Manduca sexta.

1. We measured the effects of variation in corolla curvature and nectary aperture radius on pollinator foraging ability using the hawkmoth Manduca sexta and 3D-printed artificial flowers whose shapes were mathematically specified. 2. In dimorphic arrays containing trumpet-shaped flowers and flat-disk flowers, hawkmoths were able to empty the nectaries of significantly more trumpet-shaped flowers regardless of nectary aperture size. Interestingly, trumpet-shaped flowers needed to deviate only slightly from the flat-disk morphotype in order to significantly increase hawkmoth foraging ability. 3. Whole-flower three-dimensional shape, particularly corolla curvature, has the potential to act as a mechanical guide for Manduca sexta, further implicating direct flower-proboscis contact as an important contributor to foraging success during flower handling in hawkmoths.

Sequence, structure and promoter characterization of the human thymidine kinase gene.

The 12.9-kb human thymidine kinase gene (tk) has been sequenced in its entirety along with flanking regions. Consistent with the previously sequenced chicken tk sequence, the human tk is composed of seven exons. The intron sizes differ substantially, and are responsible for the four-fold greater size of the human relative to the chicken gene. Within the introns are found 13 Alu family repeated sequences and a polypyrimidine stretch. A functional promoter region has been located by fusing sequences from the 5' end of the tk gene to the chloramphenicl acetyl transferase (CAT) gene and assaying CAT activity following transfection into mouse L cells. Several putative transcription signals have been identified in the 5' end including 'TATAA' and 'CCAAT' sequences and 'G-C' elements, two of which are arranged in a 27-bp inverted repeat. There is also a 12-bp repeat, containing an inverted 'CCAAT' element. This repeat shows strong homology to a repeat in the chicken tk promoter as well as the 5' regions of other cell-cycle regulated genes, suggesting that it may be part of the promoter or a regulatory signal. The 5' flanking sequence is G + C-rich and has a high concentration of CpG dinucleotides.

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

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: ecogeographic 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 of M. 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.

DNA fragmentation in S49 lymphoma cells killed with glucocorticoids and other agents.

The effects of glucocorticoids on DNA integrity in the mouse S49 lymphoma cell line were assessed. DNA cleavage at the internucleosomal regions was observed, and this response was correlated to the dose of hormone used and the time of treatment. Also, an apparent steroid specificity was observed: internucleosomal cleavage was associated only with treatment of the cells with glucocorticoids. Cells treated with dibutyryl cyclic AMP (which also causes lymphocytolysis) also exhibited DNA cleavage. However, when cells were killed with various DNA synthesis inhibitors and other lethal agents, the same DNA cleavage pattern was observed. Furthermore, new protein synthesis did not seem to be required, since cells killed with puromycin and cycloheximide also exhibited internucleosomal DNA fragmentation. Although DNA fragmentation may not be a specific early effect of glucocorticoid-mediated lymphocytolysis, it may be the final, irrevocable step in this complex process.

Abundant Pathogenic Variation in the New Hybrid Rust Melampsora xcolumbiana on Hybrid Poplar.

ABSTRACT The recently described rust hybrid Melampsora xcolumbiana was discovered as a result of its novel pathogenic variation on Populus trichocarpa x P. deltoides (TxD) hybrid poplar. To characterize this pathogenic variation, 10 commercial TxD clones, all F(1) clones, were chosen as host differentials. Fourteen mononuredinial isolates of Pacific Northwestern field collections of M. xcolumbiana, from 1996 to 1998 inclusive, were determined to be 13 distinct pathotypes. In contrast, four Southeastern isolates of M. medusae could not be distinguished on the same TxD host differentials, although they can be distinguished as pathotypes using P. deltoides differentials. The first three pathotypes of M. xcolumbiana (Mxc1, Mxc2, and Mxc3) and a Mississippi isolate of M. medusae were inoculated onto a three-generation TxD pedigree, formerly used to characterize the Mmd1 gene for resistance to M. medusae. Resistance to the Mxc3 pathotype mapped to the same linkage group (group Q) as the Mmd1 gene. In contrast, linked genes for resistance to Mxc1 and to Mxc2 were located on linkage group O, and were unlike Mmd1 and Mxc3 in that they were inherited from P. deltoides. The latter two genes resembled Mmd1 and Mxc3 in that infection type was correlated with quantitative traits such as uredinial density and latent period. Pathogenic variation in M. xcolumbiana matches resistance genes from both P. trichocarpa and P. deltoides and reveals the vulnerability to hybrid rust of commercial TxD hybrid poplar clones.

Leaf stomatal and epidermal cell development: identification of putative quantitative trait loci in relation to elevated carbon dioxide concentration in poplar.

Genetic variation in stomatal initiation and density, and epidermal cell size and number were examined in a hybrid pedigree of Populus trichocarpa T. & G. and P. deltoides Marsh in both ambient ([aCO2]) and elevated ([eCO2]) concentrations of CO2. We aimed to link anatomical traits with the underlying genetic map of F2 Family 331, composed of 350 markers across 19 linkage groups. Leaf stomatal and epidermal cell traits showed pronounced differences between the original parents. We considered the following traits in the F2 population: stomatal density (SD), stomatal index (SI), epidermal cell area (ECA) and the number of epidermal cells per leaf (ECN). In [eCO2], adaxial SD and SI were reduced in the F2 population, whereas ECA increased and ECN remained unchanged. In [aCO2], four putative quantitative trait loci (QTL) with logarithm of the odds ratio (LOD) scores greater than 2.9 were found for stomatal traits on linkage group B: adaxial SI (LOD scores of 5.4 and 5.2); abaxial SI (LOD score of 3.3); and SD (LOD score of 3.2). These results imply that QTL for SI and SD share linkage group B and are under genetic control. More moderate LOD scores (LOD scores >/= 2.5) suggest QTL for SI on linkage groups A and B and for SD on linkage groups B, D and X with a probable co-locating quantitative trait locus for SI and SD on linkage group D (position 46.3 cM). The QTL in both [aCO2] and [eCO2] for adaxial SD were co-located on linkage group X (LOD scores of 3.5 and 2.6, respectively) indicating a similar response across both treatments. Putative QTL were located on linkage group A (position 89.2 cM) for both leaf size and ECN in [aCO2] and for ECA at almost the same position. The data provide preliminary evidence that leaf stomatal and cell traits are amenable to QTL analysis.

Bulk segregant analysis of an induced floral mutant identifies a MIXTA-like R2R3 MYB controlling nectar guide formation in Mimulus lewisii.

The genetic and developmental basis of many ecologically important floral traits (e.g., carotenoid pigmentation, corolla tube structure, nectar volume, pistil and stamen length) remains poorly understood. Here we analyze a chemically induced floral mutant of Mimulus lewisii through bulk segregant analysis and transgenic experiments and identify a MIXTA-like R2R3 MYB gene that controls nectar guide formation in M. lewisii flowers, which involves epidermal cell development and carotenoid pigmentation.

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.