The use of genetic information expressed in isozymic form has enhanced our understanding of heritable variation within and among plant populations. For over three decades now, protein electrophoresis coupled with histochemical staining has provided data on the concordance of observable features in plants (e.g., morphology, cytology, ecological adaptation) with isozyme phenotypes. Yet inadequate dissemination of procedural details and varied investigative approaches have limited the comparative value of electrophoretic data and have made it difficult for novices to apply the technique to other experimental systems. Information gathered from over 25 laboratories utilizing enzyme electrophoresis is treated comparatively, providing access to protocols that often are either unpublished or widely dispersed in the literature. Analyzed in the review are methods and guidelines for enzyme extraction, effective storage ofplant samples and buffers, efficient screening of taxa for enzymatic activity, and interpretation of diploid and polyploid banding patterns.

Inferences regarding hybridization rely on genetic markers to differentiate parental taxa from one another. Intersimple sequence repeat (ISSR) markers are based on single-primer PCR reactions where the primer sequence is derived from di- and trinucleotide repeats. These markers have successfully been used to assay genetic variability among cultivated plants, but have not yet been tested in natural populations. We used genetic markers generated from eight ISSR primers to examine patterns of hybridization and purported examples of hybrid speciation in Penstemon (Scrophulariaceae) in a hybrid complex involving P. centranthifolius, P. grinnellii, P. spectabilis and P. clevelandii. This hybrid complex has previously been studied using three molecular data sets (allozymes, and restriction-site variation of nuclear rDNA and chloroplast DNA). These studies revealed patterns of introgression involving P. centranthifolius, but were unsuccessful in determining whether gene flow occurs among the other species, and support for hypotheses of diploid hybrid speciation was also lacking. In this study, we were able to fingerprint each DNA accession sampled with one to three ISSR primers and most accessions could be identified with a single primer. We found population- and species-specific markers for each taxon surveyed. Our results: (i) do not support the hybrid origin of P. spectabilis; (ii) do support the hypothesis that P. clevelandii is a diploid hybrid species derived from P. centranthifolius and P. spectabilis; and (iii) demonstrate that pollen-mediated gene flow via hummingbird vectors is prevalent in the hybrid complex.

SummaryNursery pollinators, and the plants they use as hosts for offspring development, function as exemplary models of coevolutionary mutualism. The two pre-eminent examples -fig wasps and yucca moths -show little variation in the interaction: the primary pollinator is an obligate mutualist. By contrast, nursery pollination of certain Caryophyllaceae, including Silene spp., by two nocturnal moth genera, Hadena and Perizoma , ranges from antagonistic to potentially mutualistic, offering an opportunity to test hypotheses about the factors that promote or discourage the evolution of mutualism. Here, we review nursery pollination and host-plant interactions in over 30 caryophyllaceous plants, based on published studies and a survey of researchers investigating pollination, seed predation, and moth morphology and behavior. We detected little direct evidence of mutualism in these moth-plant interactions, but found traits and patterns in both that are nonetheless consistent with the evolution of mutualism and merit further attention.New Phytologist (2006) 169 : 667-680

Hybrid speciation has played a significant role in the evolution of angiosperms at the polyploid level. However, relatively little is known about the importance of hybrid speciation at the diploid level. Two species of Penstemon have been proposed as diploid hybrid derivatives based on morphological data, artificial crossing studies, and pollinator behavior observations: Penstemon spectabilis (derived from hybridization between Penstemon centranthifolius and Penstemon grinnellii) and Penstemon clevelandii (derived from hybridization between P. centranthifolius and P. spectabilis). Previous studies were inconclusive regarding the purported hybrid nature of these species because of a lack of molecular markers sufficient to differentiate the parental taxa in the hybrid complex. We developed hypervariable nuclear markers using inter-simple sequence repeat banding patterns to test these classic hypotheses of diploid hybrid speciation in Penstemon. Each species in the hybrid complex was genetically distinct, separated by 10-42 species-specific inter-simple sequence repeat markers. Our data do not support the hybrid origin of P. spectabilis but clearly support the diploid hybrid origin of P. clevelandii. Our results further suggest that the primary reason diploid hybrid speciation is so difficult to detect is the lack of molecular markers able to differentiate parental taxa from one another, particularly with recently diverged species.Hybridization has long been considered a potential mechanism for plant evolution (1-21). The primary effect of hybridization is an increase in genetic variation, both locally and beyond an obvious hybrid zone (3,7,8,11). Hybridization often results in the formation of ecological races, a critical step in speciation (12). Its importance in polyploid speciation is widely recognized (5, 16, 17), but its evolutionary role has been variously questioned or corroborated over the past 50 years, particularly with respect to diploid hybrid speciation (1-21).The reticulate nature of angiosperm evolution has been acknowledged from numerous studies supporting organelle capture (22). However, of the estimated 300,000 species of angiosperms, there are Ͻ10 clearly documented diploid or homoploid hybrid species based on molecular data (3-5, 6, 9, 18-19, 21). To understand the process of evolution, one first needs the tools to detect the patterns. In the case of reticulate evolution, the necessary tools include morphological, cytological, and molecular characters. Many hypotheses of diploid hybrid speciation are based on morphological and cytological characters (8), but support for this mechanism of evolution has been lacking due to the absence of molecular markers that can clearly distinguish closely related parental species.The PCR was instrumental in the development of new molecular markers for population studies (23). New methods for generating molecular markers based on PCR include random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), amplified fragment length polymorphism, a...

This study investigates the extent of pollinator sharing and specialization of three co—occurring species of milkweed (Asclepias) in experimental plantings. The potential for competition among plants for pollinators was also assessed from observed levels of pollinator constancy, interspecific pollination, and seed set in experimental populations. All three species (A. incarnata, A. syriaca, and A. verticillata) were visited by a wide variety of Hymenoptera and Lepidoptera. Niche breadth estimates for populations of A. syriaca were comparatively low (.09—.27); Bombus griseocollis and the introduced honeybee, Apis mellifera, were clearly the most common visitors to this species. Niche breadths were greater, implying less specialization, for A. incarnata (x = .30) and A. verticillata (x = .39). Paper wasps (Polistes) and digger wasps (Sphex) were numerous and effective pollinators of A. verticillata, but bumblebees comprised the largest proportion (27%) of the pollinators at some sites. Similarly, utilization of pollinators A. incarnata was highly dependent on the locality of the population studied, with Sphex pennsylvanicus, Apis mellifera, Bombus griseocollis, and Xylocopa virginica constituting the most abundant pollinators. Intraspecific comparisons of the similarity of pollinators among populations of A. syriaca gave values that were higher than corresponding values for different species within a locality, implying possible niche differentiation with respect to this species. However, a hypothesis of pollinator partitioning among species was not accepted for A. incarnata and A. verticillata because estimates of niche overlap between these species were as great as those involving populations of either species alone. Both the timing and amount of flowering, as well as the degree of interplanting of A. verticillata and A. incarnata, affected the abundance and constancy of pollinators on these species in experimental plots. Interspecific flights or pollinators and interspecific insertion of pollinia for A. incarnata were much higher in 1978 when flowering of A. verticillata was earlier and more extensive. During that same year, interspecific pollination of A. verticillata was lower. In addition, the abundance of Bombus griseocollis on this species declined rapidly as A. incarnata came into flower, even though peak flowering of A. verticillata had not been reached. Mixed plantings of these species gave higher levels of interspecific pollination and lower seed set than unispecific plantings at the same density. The lack of specialization in pollination of A. incarnata and A. verticillata is not surprising since these species typically occupy different habitats and have nectar that is readily accessible. However, the current proliferation of disturbed environments where species occur in close proximity provides the potential for exploitative competition between them when pollinators are a scarce or limiting resource. The presence of pollen from other species may also interfere with intraspecific pollination, reducing se...

Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades.

Variable spatial and temporal environments are known to affect the population dynamics of plants, but studies of local scale variability and its relationship to demographic change within a population remain limited. Using mapped plants, we examined the population dynamics of a coastal grassland endemic, Silene douglasii var. oraria, in two habitats over 10 yr. We hypothesized that ecological differences between rocky and grassy habitats might influence demographic parameters, including adult survival, growth, and density. Soil pH, soil moisture. and other abiotic variables differed little between habitats, but microsite differences in light, soil depth. and vegetation height were related to variation in Silene density and plant circumference. We also found significantly higher population densities, lower adult mortality, and more juvenile recruitment in rocky areas. Finite rates of population growth varied across years and habitats (lambda = 0.82-1.12). with different patterns evident in the two habitats. In both, observed population sizes in 1992 were similar to matrix projections using 1982-1985 data. Populations declined in size in some years despite high adult survivorship and variable recruitment. More intensive study of seedlings is needed, including experimental evaluation of the role of light and competition. However, the habitat-specific differences we observed imply that ecological studies and conservation plans developed for rare plants should consider the effect of local scale variability on demography.

Summary• We explored whether mechanical or ethological differences provide pollinatormediated floral isolation capable of reinforcing existing species barriers among sympatric Asclepias with divergent floral morphologies: A. incarnata , A. verticiallata and A. syriaca .• In a common garden, we quantified pollinator visitation and flight patterns, differences in corporal attachment of pollinia to insects, and the potential outcome of putative floral barriers for interspecific pollination and fruit set.• We detected significant variation in the importance, constancy, and behavior of major pollinators on sympatric asclepiads, including Bombus , Xylocopa and large sphecid wasps. Pollinia attach differentially to the arolium on insect legs for A. syriaca , but to the tarsal hairs in other asclepiads. Fruit-set was lower in mixed than unispecific patches of Asclepias .• We detected mechanical isolation between A. syriaca and its congeners and a tendency toward wasp pollination in A. verticillata . All three species appear to show some specialization for long-tongued hymenoptera and lepidopterans. Pre-mating barriers provide a potentially effective means of reducing interspecific pollination, but more study is needed in species visited by generalists.