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utilizing enzyme electrophoresis is treated comparatively, providing access to protocols that often are either unpublished or widely dispersed in the literature.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...