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Commentary Blackwell Publishing, Ltd.
New functions for electrical signals in plants Electrical signals in plants have been known for over 100 years, but scientists are still looking for a function. The bulk of the work has focused on heat-wound evoked responses in aboveground parts, but evidence exists for their role in many processes, including transcription, translation and respiration. An additional function elegantly demonstrated in this issue of New Phytologist (Koziolek et al. pp. 713–720) is a transient reduction in photosynthesis. Koziolek et al. measured membrane potential, chlorophyll fluorescence and PSII quantum yield, and showed that the electrical signal preceded the changes in photosynthetic parameters – transport of chemicals in the phloem was far too slow to account for these changes.
‘One of the beauties of this study is that the authors recognize the need to take great care to avoid generating a wound during the act of measurement, and so use techniques imposing little or no stress on the plant’
Untouchable research to accepted phenomenon For many years, the idea of electrical signaling in plants hovered in the research background, a minor interest topic even though Burdon-Sanderson (1873) and Charles Darwin (1875) had demonstrated the existence of electrical signals in insectivorous (‘motorized’) plants. However, by the early 1970s, with the publication of ‘The secret life of plants ’ by Tompkins & Bird (1973) the credibility of the area had become severely strained in the research community making it essentially untouchable in the eyes and minds of funding agencies (Box 1). Just over 20 yr ago we presented some research showing that wounding one location on a plant caused very rapid changes in gene expression (translation/polyribosomes) in distant regions (Davies & Schuster, 1981). Joe Varner, a very eminent plant biochemist and molecular biologist, discussed this research with me and suggested that I might be looking at electrical signals. I remember my response: ‘But plants don’t have electrical signals, do they?’. Joe assured me that they did, and arranged for me to work in Barbara Pickard’s lab at Washington University in St. Louis. I went there, did my first experiment by taping a plant in place on a styrofoam block, attaching electrodes, hooking them up to recorders, and then cutting the plant. The recorders, in sequence, starting with the one nearest the cut, exhibited a clear transient 70 mV change in membrane potential. I was now a ‘believer’ that electrical signals existed in normal plants, but was unsure whether they actually evoked any important responses.
Box 1 Electrical signaling in plants, 1791–1973 Over 200 years ago Galvani (1791) showed that animals had electrical activity, while about 130 years ago, both Burden-Sanderson (1873) and Charles Darwin (1875) demonstrated the existence of electrical signals in insectivorous (‘motorized’) plants. Shortly after that, Darwin (1881) working primarily on circumnutation provided evidence for chemical signals in plants. For some reason this evidence was so compelling that others soon forgot about plant electrical activity and focused on chemical signals. So the belief now arose that animals had electrical signals and plants had chemical ones – a rather nice classification – especially since one could see the need for rapid (electrical) signals in motile animals, but less need for such signals in sessile plants. Then, around the turn of the century (early 19.00 s) more and more evidence accumulated to suggest that animals also had chemical signals – thus arose the field of endocrinology. This then gave rise to the concept that animals have both electrical and chemical signals, while plants have only chemical signals (again, elevating animals to a more ‘lively’ status). Based on research by Bose (1924), and more recently by Barbara Pickard (1973) substantial evidence was presented for the existence of electrical signals (action potentials) in a wide array of plants, not just insectivorous or other ‘motorized’ plants. Hope arose that the existence of both electrical and chemical signals in both animals and plants might become widely accepted. This hope was, however, very short-lived. In the same year as Pickard’s major review (1973), there appeared a book ‘ The secret life of plants’ by Tompkins & Bird (1973). This book may be one of the best examples of pseudoscience to catch the public’s attention and has been castigated by Galston & Slayman (1979) among others. The most fascinating topics discussed in this book ( Tompkins & Bird, 1973) were the ‘metaphysical’, ‘paranormal’ or ‘extrasensory perception’ processes attributed to plants by a criminologist who specialized in lie detectors (Backster, 1968). This book accomplished at least two things. First, it held the imagination of many of the public who seem far more interested in pseudoscience (magic) than in science. Second, it effectively extinguished any prestige the topic of electrical signals in plants might have had in the research community, making it essentially untouchable in the eyes and minds of funding agencies.
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In order to ascertain whether there were any likely functions of electrical signals in plants, I spent a lot of time reviewing the literature until I was convinced that they really did exist, were worth working on, and might, in fact, explain several then inexplicable phenomena (Davies, 1987). Then came the breakthrough – the paper by Wildon et al. (1992) showing that electrical signals are involved in turning on protease inhibitor (PIN ) genes in tomato. They showed that a local flame wound evoked rapid changes in membrane potential in distant tissue, while transport of chemical signals in the phloem took an hour or more. The electrical signals arrived in responding tissue much sooner than transcript accumulation began, while transcript accumulation preceded arrival of the chemical signal. The authors (Wildon et al., 1992) claimed, however, that the signal they were monitoring was a ‘genuine’ electrical signal, or action potential (AP). However, we had already worked on tomatoes and knew that a flame wound invariably evoked another type of ‘electrical’ signal known as a variation potential (VP). The AP is described as a ‘genuine’ electrical signal. It can be evoked electrically, it has an all-or-nothing character (below a threshold stimulus there is no response, while above this threshold, there is a maximal response), and it travels with constant velocity and magnitude (Zawadzki et al., 1991). The AP is a self-perpetuating signal based on the activity of voltage-gated channels which respond to (and cause) changes in membrane potential (Davies, 1993). By contrast, the VP cannot be evoked electrically, it varies with the degree of stimulus, and appears to ‘travel’ with decreasing magnitude and velocity away from the site of stimulus. The VP is a nonself-perpetuating signal based on mechanosensitive (stretch-activated) and/or ligand-activated channels (Davies, 1993; Stankovic et al., 1997). Thus the VP is a local change (in living cells with ion channels) to either a hydraulic surge or chemicals transmitted in the dead xylem. Nevertheless, despite the misunderstandings concerning the different types of electrical signals in plants, this report (Wildon et al., 1992) helped make the study of electrical signals more respectable and closer to mainstream biology.
a plant is attacked by insects (especially if they harbor a virus), the plant needs to put up systemic defenses as quickly as possible to prevent spread of the pathogen/herbivore. The second is ‘ubiquity’, insofar as all cells near the xylem will be affected by the VP, and those near the phloem by the AP. Similarly, ubiquity is important in sofar as the entire plant needs to be defended against potentially hostile threats, there may be little point in leaving some regions susceptible while defending others. This underlies the systemic nature of electrical signals as well as the local responses to them. The third property is ‘information’. This is important because the plant may or may not actually ‘recognize’ the exact insult. This is possible for the flame wound, which is the ‘insult’ used in this paper and the one preferred by us to evoke predictable, large, rapid, electrical signals and responses at the level of gene expression. It is conceivable that the plant does, indeed, ‘recognize’ the flame wound for what it is – fire – a very damaging threat which is pervasive throughout many ecosystems. However, it is equally conceivable that the plant does not recognize specifically that the insult is a local flame and so mounts a general stress response. It should be noted here that there is a huge difference between electrical signaling in the animal nervous system compared with that in a plant. In the animal nervous system, the whole point of an AP is to send information from one specific location to another, without affecting the intervening tissue – rather like a telephone system (Davies, 1993). By contrast, both the AP and VP in a plant are designed to inform as much of the plant as quickly as possible so that all the intervening tissue is informed – rather like a megaphone message warning everyone within hearing distance (Davies, 1993). The final property is ‘transience’. Not only is the signal (change in membrane potential) short-lived, but so is the response. After the wounded leaf has informed the rest of the plant, the recipient regions seem to ‘deal with the emergency and then get back to normal business’. Thus transience is important if the signal is merely to warn distant regions of impending disaster, and after receipt of such a warning, the (forewarned) distant tissue regains homeostasis.
Special precautions needed for wound research Rapidity, ubiquity, information, transience Why do plants have electrical signals? Not only can this involve ‘What plant functions are governed by electrical signals?’, it also concerns ‘Why is there a need for these signals?’ The latter question can also be posed as ‘What properties do electrical signals have that chemical signals do not have?’ The first is ‘rapidity’. The VP in living cells is an almost direct aftermath of a virtually instantaneous loss of tension in the xylem, while the AP is a very rapidly transmitted signal predominantly in the phloem. Why might rapidity be important? In those instances where plants have a movement response, such as in the capture of insects, then speed is essential otherwise the prey would escape. Similarly, when
When studying wounding, great care needs to be taken to avoid generating a wound during the act of measurement, and so noninvasive techniques have to be used. One of the beauties of the study in this issue by Koziolek et al. is that the authors recognize this need, and so used techniques imposing little or no stress on the plant. They used standard techniques including microelectrodes (which impose a very slight puncturing stress) to measure electrical signals, a porometer to measure gas exchange, and autoradiography to estimate time of arrival of an chemical signal. The major feature, however, was their use of a recently developed method involving fluorescence imaging to measure the quantum yield efficiency of photosystem II.
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These fluorescence images are very persuasive. They show that PSII efficiency is transiently inhibited in the distant leaf beginning within 90 s of the heat pulse, traveling from the base to apex of the leaf ( pinna) before returning to normal after about 450 s. The images are magnified to show that the vein region is inhibited initially, implying that the vein is the pathway of transmission, but that the intervein region is affected more massively, implying greater photosynthetic activity in that region. They show that generation and transmission of the electrical signal (variation potential) precedes the changes in stomatal conductance and gas exchange in fixed leaflets, which in turn slightly precedes inhibition of PSII quantum yield. Furthermore, the fact that wound responses tend to be transient adds another facet to the experimental approach. If the techniques require tissue extraction or fixation (as ours mostly do), then very detailed time courses need to be made – which adds to the time and expense of obtaining statistically reliable data. This problem can be circumvented by using ‘real-time’ imaging techniques, where the sample can be assayed repeatedly or even continuously. The authors achieve this, not only with their use of microelectrodes and porometers, both of which give continuous recordings, but especially with chlorophyll fluorescence measurements, which gives beautiful (false)-color images. In this context, it is worth noting that a new, non-invasive technique might have become available to monitor electrical signals. Recently, Hanstein & Felle (2004) described a ‘nanoinfusion’ technique where microelectodes are immersed in liquid infused into the substomatal cavity to measure membrane potential – in a totally non-invasive way – thus circumventing the (minor) damage elicited by micoelectrodes inserted into cells.
Processes governed by flame- (or heat pulse-) induced electrical signals The bulk of research in the area has focused on responses in above-ground parts and evidence exists for their role in many processes, including transcription (Wildon et al., 1992), translation (Stankovic & Davies, 1997) and respiration (Filek & Koscielniak, 1997), and in all cases the responses are transient. This paper (Koziolek et al.) adds inhibition of photosynthesis to this list, and also shows that PSII efficiency, as well as changes in gas exchange and stomatal aperture, are transient. It is still not known whether the transience of the response is a direct result of the transience of the signal itself (the change in membrane potential and concomitant fluxes of ions) or whether it signifies that the plant has successfully ‘coped with the problem’, or whether both of these are true. Nevertheless, there do seem to be some underlying similarities between the results from several labs working on many different responses to flame- (or heat-pulse-) induced variation potentials. The values shown here for lag times, time of maximum response, and time of return to baseline were
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Fig. 1 Three-wk old tomato plants were flame-wounded on leaf 3 and leaf 4, harvested at 15 s intervals, frozen immediately in liquid Nitrogen, and finely pulverized. One aliquot of the frozen powder was extracted to assay IP3 using the release of labeled IP3 from rat brain IP3 receptors. The other aliquot was extracted, electrophoresed, blotted and probed with a Rubisco small subunit probe according to published methods (Vian et al., 1999). Data are from three independent experiments, where the standard deviation was < 12% at all time points (Salinas-Mondragon, Atkinson and Davies, unpublished).
roughly 90 s, 240 s, 450 s for PSII inhibition, gas exchange and stomatal conductance. These values are quite similar to, although somewhat slower than, the corresponding times for enhancement of respiration (Filek & Koscielniak, 1997) of 20 s, 80 s, 360 (s). We were also impressed by similarities in the kinetics of response of inhibition of photosynthesis shown in the paper with those from our own work on flame-wound induced changes in gene expression in tomatoes. Here we have measured the level of a putative second messenger, inositol phosphate (IP3) as well as accumulation of the transcript encoding the Rubisco small subunit. As shown in Fig. 1, the systemic increase in IP3 levels shows a lag of 75 s, a maximum at 90 s, and a return to the baseline by 120 s, while the Rubisco SS transcript shows a lag of 90 s, a maximum at 120 s and a return to the baseline by 150 s. There are two main differences between these results on transcript accumulation (Fig. 1) and those on respiration (Filek & Koscielniak, 1997) and photosynthesis (Koziolek et al.). First, our system has the disadvantage that we cannot assay the same tissue over time, but must harvest tissue for each time point. Second, we have an advantage insofar as we have identified a putative local signal (IP3) that could have been evoked by the systemic variation potential, and which could, in turn, evoke transcript accumulation. Eric Davies NC State University, Department of Botany, 1231 Gardner Hall, Raleigh, NC 27695 USA (tel +1 919 5131901; fax +1 919 5153436; email
[email protected])
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References Backster C. 1968. Evidence of a primary perception in plant life. International Journal of Parapsychology 10: 329 –348. Bose JC. 1924. The physiology of photosynthesis. London, UK: Longman, Green and Co. Burdon-Sanderson J. 1873. Note on the electrical phenomena which accompany irritation of the leaf of Dionaea muscipula. Proceedings of the Royal Society (London) 21: 495– 496. Darwin C. 1875. Insectivorous plants. London, UK: John Murray. Darwin C. 1881. The power of movement in plants. London, UK: John Murray. Davies E. 1987. Action potentials as multi-functional signals in plants: a unifying hypothesis to explain apparently disparate phenomena. Plant, Cell & Environment 10: 623– 631. Davies E. 1993. Intercellular and intracellular signals in plants and their transduction via the membrane – cytoskeleton interface. Seminars in Cell Biology 4: 139–147. Davies E, Schuster AM. 1981. Intercellular communication in plants: evidence for a rapidly-generated, bidirectionally-transmitted wound signal. Proceedings of the National Academy of Sciences, USA 78: 2422–2426. Filek M, Koscielniak J. 1997. The effect of wounding the roots by high temperature on the respiration rate of the shoot and propagation of electric signal in horse bean seedlings ( Vicia faba L. minor). Plant Science 123: 39 – 46. Galston AW, Slayman CL. 1979. The not-so-secret life of plants. American Scientist 29: 337 – 344. Galvani L. 1791. De Viribus Electricitatis in Motu Musculari Commentarius. Bologna, Italy: Academy of Science. Hanstein SM, Felle HH. 2004. Nanoinfusion – an integrating tool to study elicitor perception and signal transduction in intact leaves. New Phytologist 161: 959 – 606. Pickard. 1973. Action potentials in higher plants. Botanical Reviews 39: 172 – 201. Stankovic B, Davies E. 1997. Wounding evokes rapid changes in tissue deformation, electrical potential, transcription, and translation in tomato. Plant and Cell Physiology 39: 268 – 274. Stankovic B, Zawadzki T, Davies E. 1997. Characterization of the variation potential in sunflower. Plant Physiology 115: 1083 – 1088. Tompkins P, Bird C. 1973. The secret life of plants. New York, USA: Harper & Row. Vian A, Henry-Vian C, Davies E. 1999. Rapid and systemic accumulation of chloroplast mRNA binding protein transcripts after flame stimulus in tomato. Plant Physiology 105: 51 – 58. Wildon DC, Thain JF, Minchin PEH, Gubb IR, Reilly AJ, Skipper YD, Doherty HM, O’Donnell PJ, Bowles DJ. 1992. Electrical signalling and systemic proteinase inhibitor induction in the wounded plant. Nature 360: 62 – 65. Zawadzki T, Davies E, Dziubinska H, Trebacz K. 1991. Characteristics of action potentials in Helianthus annuus L. Physiological Plantarum 83: 601 – 604. Key words: electrical signalling, action potential, insectivorous (‘motorized’) plants, membrane potential, IP3. Commentary Commentary 610 161
One down and thousands to go – dissecting polyploid speciation The literature on speciation is vast, but there are few undisputed examples for any given model of the process. The exception is polyploid speciation. Because of strong postzygotic reproductive isolation of tetraploid derivatives, the most common class of polyploids, from their diploid progenitors – which comes about because triploid hybrids are highly sterile – the observation of more than one established cytotype is prima-facie evidence of polyploid speciation. Given that estimates of polyploid speciation in angiosperms from a minimum estimate of 2%–4% (Otto & Whitton, 2000) to perhaps as much as 20% – 40% (Stebbins, 1938), botanists have thousands of examples of polyploid speciation in flowering plants alone from which to enhance our understanding of this mechanism of diversification. While the literature on polyploidy has exploded in recent years, the emphasis of current polyploidy research largely focuses on genetic and epigenetic consequences of genome duplication. We are now beginning to understand that the impacts of polyploidization on the genome are varied and complex (Osborn et al., 2003), and while such studies inform us about the impacts of genome duplication, they have thus far offered few direct insights into polyploidization as a mechanism of speciation. To understand polyploid speciation, we need to understand how novel cytotypes arise, become established and persist in nature, and this is where the work of Brian Husband on fireweed, on pp. 701–711, is leading the way.
‘It is a matter of time, and probably not that much time, before we can understand the direct contribution of genome duplication in polyploid speciation’
Studies with fireweed Brian Husband and coworkers’ studies of fireweed (Chamerion angustifolium) provide the most comprehensive set of experiments on the dynamics of cytotype interactions available for any polyploid system. In North America, fireweed includes diploid and tetraploid cytotypes that have generally distinct ranges, with diploids occurring further North and at higher elevations. A broad contact zone exists in the Rocky Mountains, with the cytotypes
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segregating along the elevational gradient (Husband & Schemske, 1998). In this case, the polyploids are presumed autopolyploids that have not been granted taxonomic status (as is typically the case for autopolyploids), though the cytotypes are distinct biological species, with diploids and tetraploids having largely distinct ranges and hybrids between them being almost completely sterile (their fitness relative to diploids was estimated at 0.09; Burton & Husband, 2000). Husband & Sabara (this issue) uses a number of previous studies to develop a comprehensive estimate of the contributions of a suite of six pre- and post– zygotic reproductive isolating barriers in the contact zone. They apply an intuitive method, described by Coyne & Orr (1989, 1997; see also Ramsey et al., 2003), that considers the timing of action of each barrier and uses the isolation achieved at each step to scale the contributions of later– acting barriers. Using this approach, they estimate that the two fireweed cytoypes achieve more than 99.7% reproductive isolation in nature. Perhaps unexpectedly, most of the isolation is achieved before fertilization, via spatial isolation, flowering time divergence and pollinator fidelity. Together these three barriers account for more than 92% of the observed isolation, so while hybrid sterility provides a strong barrier between the cytotypes, its late action decreases its current importance. In all studies of reproductive isolation, a key focus is to understand which isolating barriers were critical in the initial stages of speciation. In the case of polyploid speciation, Levin (1975) and others have pointed out that the future for a newly arisen tetraploid in a sea of diploids is not so bright as it will face minority cytotype disadvantage, the likely fate of being swamped to extinction by maladaptive hybridization with diploid progenitors. Thus it has long been recognized that factors which enhance the probability that a newly arisen tetraploid produces tetraploid offspring are likely critical to the establishment of nascent polyploid species. Such factors include spatial (ecological) isolation, increased self-compatibility, shifts in developmental timing or pollinator preferences. Indeed, each of these traits contributes to total isolation in Chamerion angustifolium. In addition, the well documented tendency for recurrent polyploid formation and/or recurrent formation of unreduced gametes in diploids (and perhaps in triploids as well; Ramsey & Schemske, 1998) may contribute to the gamete pool for successful production of polyploid offspring that could help overcome the initial minority cytotype disadvantage.
Reproductive barriers during the establishment of the polyploid cytotype The difficulty with using established polyploids to study the early stages of establishment is that the barriers that are currently present may have had a variety of origins. First, phenotypic changes that confer reproductive isolation may
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be the direct consequence of genetic or epigenetic changes that accompanied polyploidization. Second, prezygotic isolation may have been enhanced via reinforcement (selection to avoid maladaptive hybridization). Third, some of the additional isolating barriers may have arisen following polyploid establishment, and represent the incidental byproducts of divergence. We must, however, keep in mind that the origin of the polyploid cytotype must have been sympatric or nearly so, and therefore to overcome minority cytotype disadvantage, some barrier other than hybrid sterility likely contributed to initial establishment. This suggests that barriers that have a tendency to arise as an immediate by-product of genome duplication may have been most likely to act in polyploid establishment. Previous work on the phenotypic consequences of genome duplication (Levin, 1983; Ramsey & Schemske, 2002) provide potential insight into which pre–zygotic barriers are most likely to have been in place early in the speciation process. However, as pointed out by Ramsey & Schemske, many, indeed most, reports of phenotypic correlates of polyploidy are difficult to interpret because they confound divergence that may have occurred subsequent to polyploid establishment with effects that are attributable to polyploidy per se. Nonetheless, based on a review of studies of newly synthesized polyploids, Ramsey & Schemske (2002) find evidence that neopolyploids differ in phenology from their progenitors, with flowering typically initiated later. This difference is in the same direction as observed in fireweed. Pollinator fidelity, the tendency for pollinators to fly within rather than between cytotypes, has previously been noted in Heuchera grossularifolia (Segraves & Thompson, 1999), and represents a significant reproductive barrier in fireweed. No studies of neopolyploids indicate pollinator shifts as a direct consequence of genome duplication, though changes in flower size that could potentially be associated with shifts in pollinator fidelity or that could contribute to shifts in selfing rate or success rate of intercytotype pollination have been noted (Ramsey & Schemske, 2002). Spatial isolation resulting from shifts in habitat tolerances are frequently cited as important in polyploid establishment, yet I am unaware of a single study that compares ecological tolerances of newly synthesized polyploids to their diploid progenitors. Thus, although this spatial isolation is important at present in Chamerion, we have no way to evaluate the likelihood that such a shift occurred early in polyploid establishment. To further our understanding of the early stages of polyploid establishment, it would be ideal to use newly synthesized polyploids of Chamerion to evaluate the shifts that accompany genome duplication. In addition, an understanding of the evolutionary history of the contact zone would allow a better understanding as to whether the dynamics observed reflect early stages in polyploid establishment or secondary contact between cytotypes that originated elsewhere and have diverged in allopatry.
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Linking ecological and genetic studies While the current emphasis of polyploidy research emphasizes genetic and epigenetic shifts, in order to gain a complete understanding of how species arise by polyploidization we must also understand how these entities become established and persist in nature. The chasm between these two fields of endeavor is narrowing: it is now possible to compare gene expression in synthetic and natural polyploids (Adams et al., 2003), and while, thus far, the ecological significance of such expression differences remains elusive, candidate loci for potentially ecologically significant traits are becoming well-characterized. This implies that it is a matter of time, and probably not that much time, before we can understand the direct contribution of genome duplication in polyploid speciation. However in order to fulfil this promise, we will need to have a thorough understanding of the operation of reproductive isolation and the nature of ecological divergence in natural polyploid systems. Jeannette Whitton Department of Botany and Biodiversity Research Centre, The University of British Columbia, Vancouver, BC V6T 1Z4 Canada (tel +1 604 8228863; fax +1 604 8226089; email
[email protected])
References Adams KL, Cronn R, Percifield R, Wendel JF. 2003. Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing. Proceedings of the National Academy of Sciences, USA 100: 4649 – 4654. Burton TL, Husband BC. 2000. Fitness differences among diploids, tetraploids, and their triploid progeny in Chamerion angustifolium: mechanisms of inviability and implications for polyploid evolution. Evolution 54: 1182 – 1191.
Coyne JA, Orr HA. 1989. Patterns of speciation in Drosophila. Evolution 43: 362 – 381. Coyne JA, Orr HA. 1997. Patterns of speciation in Drosophila revisited. Evolution 51: 295 – 303. Husband BC, Sabara HA. 2004. Reproductive isolation between autotetraploids and their diploid progenitors in fireweed, Chamerion angustifolium (Onagraceae). New Phytologist 161: 701 – 711. Husband BC, Schemske DW. 1998. Cytotype distribution at a diploid-tetraploid contact zone in Chamerion (Epilobium) angustifolium (Onagraceae). American Journal of Botany 85: 1688 –1694. Levin DA. 1975. Minority cytotype exclusion in local plant populations. Taxon 24: 35 – 43. Levin DA. 1983. Polyploidy and novelty in flowering plants. American Naturalist 122: 1 – 25. Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee H-S, Comai L, Madlung A, Doerge RW, Colot V, Martienssen RA. 2003. Understanding mechanisms of novel gene expression in polyploids. Trends in Genetics 19: 141 –147. Otto SP, Whitton J. 2000. Polyploid incidence and evolution. Annual Review of Genetics 34: 401 – 437. Ramsey J, Bradshaw Jr HD, Schemske PW. 2003. Components of reproductive isolation between the monkey flowers Mimulus lewisii and M. cardinalis (phrymaceae). Evolution 57: 1520 – 1534. Ramsey J, Schemske DW. 1998. Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annual Review of Ecology and Systematics 29: 467 – 501. Ramsey J, Schemske DW. 2002. Neopolyploidy in flowering plants. Annual Review of Ecology and Systematics 33: 589 – 639. Segraves KA, Thompson JN. 1999. Plant polyploidy and pollination: floral traits and insect visits to diploid and tetraploid Heuchera grossulariifolia. Evolution 53: 1114 –1127. Stebbins Jr GL. 1938. Cytological characteristics associated with the different growth habits in the dicotyledons. American Journal of Botany 25: 189 –198. Key words: polyploidy, reproductive isolation, speciation, fireweed (Chamerion angustifolium), cytotypes.
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