Summary 599

I. Introduction 599

II. Concepts and terminology 600

III. Historical background 600

IV. Studies of experimental hybrids 601

1. Isolating mechanisms 601

2. Prezygotic barriers 602

(a) Gametic barriers to hybridization 602

3. Postzygotic barriers 603

(a) Chromosomal rearrangements 604

(b) Genic sterility or inviability 604

4. Hybrid vigour 605

5. Introgression 606

6. Hybrid speciation 607

V. Experimental manipulations of natural hybrid populations 609

1. Hybrid-zone formation 610

2. Pollinator-mediated selection 610

3. Habitat selection 612

VI. The biology of different classes of hybrids 612

1. Character expression 613

(a) Morphological characters 613

(b) Chemical characters 613

(c) Molecular characters 613

2. The fitness of different classes of hybrids 614

(a) The importance of variance 614

(b) Estimating hybrid fitness 615

3. Interactions with parasites and herbivores 616

4. Patterns of mating 617

(a) Outcrossing rate 617

(b) Hybridization frequency 618

(c) Mate choice 618

VII. Conclusions and future research 619

Acknowledgements 620

References 620

Most studies of plant hybridization are concerned with documenting its occurrence in different plant groups. Although these descriptive, historical studies are important, the majority of recent advances in our understanding of the process of hybridization are derived from a growing body of experimental microevolutionary studies. Analyses of artificially synthesized hybrids in the laboratory or glasshouse have demonstrated the importance of gametic selection as a prezygotic isolating barrier; the complex genetic basis of hybrid sterility, inviability and breakdown; and the critical role of fertility selection in hybrid speciation. Experimental manipulations of natural hybrid zones have provided critical information that cannot be obtained in the glasshouse, such as the evolutionary conditions under which hybrid zones are formed and the effects of habitat and pollinator-mediated selection on hybrid-zone structure and dynamics. Experimental studies also have contributed to a better understanding of the biology of different classes of hybrids. Analyses of morphological character expression, for example, have revealed transgressive segregation in the majority of later-generation hybrids. Other studies have documented a high degree of variability in fitness among different hybrid genotypes and the rapid response of such fitness to selection – evidence that hybridization need not be an evolutionary dead end. However, a full accounting of the role of hybridization in adaptive evolution and speciation will probably require the integration of experimental and historical approaches.

Following a precultivation with pedospheric nitrogen nutrition, Ricinus plants were supplied with nitrogen solely by spraying nitrate or ammonium solution onto the leaves during the experimental period. The chemical composition of tissues, xylem and phloem exudates was determined and on the basis of the previously determined nitrogen flows (Peuke et al., New Phytologist (1998), 138, 657–687) the flows of potassium, sodium, magnesium, calcium, chloride and ABA were modelled. These data, which permit quantification of net-uptake, transport in xylem and phloem, and utilization in shoot and root, were compared with results obtained in plants with pedospherically-supplied nitrate or ammonium and data in the literature. Although the overall effects on the chemical composition of supplying ammonium to the leaves were not as pronounced as in pedospherically supplied plants, there were some typical responses of plants fed with ammonium (ammonium syndrome). In particular, in ammonium-sprayed plants uptake and transport of magnesium decreased and chloride uptake was increased compared with nitrate-sprayed plants. Furthermore, acropetal ABA transport in the xylem in ammonium-sprayed Ricinus was threefold higher than in nitrate-sprayed plants. Additionally, concentrations of anions were more or less increased in tissues, particularly in the roots, and transport fluids. The overall signal from ammonium-sprayed leaves without a direct effect of ammonium ions on uptake and transport systems in the root is discussed.

Circumstantial evidence suggests that plants that have evolved metal tolerance are at a disadvantage on normal soil, i.e. there is a cost of tolerance. One hypothesis for the cause of this cost is that individuals have a greater requirement for copper, and so suffer micronutrient deficiency on normal soils, as a result of a reduced uptake, distribution and/or utilization of copper. We provided highly and less copper-tolerant plants of Mimulus guttatus Fischer ex DC. (the common monkey flower) with sub-optimal copper, and demonstrated the importance of copper as an essential micronutrient during the reproductive phase, both in the production of viable pollen and in seed set. We also looked at the effect of sub-optimal copper supply on the growth of the microgametophyte, and the efficiency with which seed was set. No evidence was found that highly tolerant plants have an increased copper requirement during the reproductive phase. This is in agreement with earlier work on Mimulus guttatus, which investigated the copper requirement of highly tolerant plants during vegetative growth and found that any differences in copper requirement were small. The ‘metal requirement hypothesis’ is, therefore, not the sole explanation for the cost of copper tolerance in M. guttatus.

Maternal-environmental effects on subsequent progeny life-history traits were evaluated in squash (Cucurbita pepo L.) in terms of the amount of time available for seed development, and the timing of fruit production. Progeny arising from three kinds of fruit were compared. Plants from which fruits were removed 3 d post-pollination throughout the growing season developed only ‘late’ fruits (during 10–15 d) at the end of the growing season; on control plants both ‘early’ and ‘late’ fruits developed (both types allowed to ripen fully). Seed from each type of fruit was weighed individually and categorized into three size classes, then germinated and raised to maturity, including regular harvesting of all fruits 3 d post-pollination. Maternal effects were evident for both vegetative and reproductive traits and carried over to later stages. In contrast, effects due simply to seed size disappeared by day 30 for leaf variables and day 60 for male flower production. Within a seed-size class, progeny arising from fruit of treated plants produced significantly more leaves, with greater size, and more male flowers than those arising from fruit of control plants, while the reverse was true for fruit number and fruit mass. This result is discussed in terms of possible gibberellic acid involvement. In control plants, progeny arising from seeds in the large, fully mature ‘early’ fruits produced significantly more, and larger leaves by day 30 than did those from late fruits (suggesting differential provisioning in seeds during development). Male flower production had a highly significant positive correlation with vegetative mass and a significant negative correlation with fruit production.

Eragrostis pilosa (Linn.) P Beauv., a C4 grass native to east Africa, was grown at both ambient (350 μmol mol−1 and elevated (700 μmol mol−1) CO2 in either the presence or absence of the obligate, root hemi-parasite Striga hermonthica (Del.) Benth. Biomass of infected grasses was only 50% that of uninfected grasses at both CO2 concentrations, with stems and reproductive tissues of infected plants being most severely affected. By contrast, CO2 concentration had no effect on growth of E. pilosa, although rates of photosynthesis were enhanced by 30–40% at elevated CO2. Infection with S. hermonthica did not affect either rates of photosynthesis or leaf areas of E. pilosa, but did bring about an increase in root[ratio ]shoot ratio, leaf nitrogen and phosphorus concentration and a decline in leaf starch concentration at both ambient and elevated CO2. Striga hermonthica had higher rates of photosynthesis and shoot concentrations of soluble sugars at elevated CO2, but there was no difference in biomass relative to ambient grown plants. Both infection and growth at elevated CO2 resulted in an increase in the Δ13C value of leaf tissue of E. pilosa, with the CO2 effect being greater. The proportion of host-derived carbon in parasite tissue, as determined from δ13C values, was 27% and 39% in ambient and elevated CO2 grown plants, respectively. In conclusion, infection with S. hermonthica limited growth of E. pilosa, and this limitation was not removed or alleviated by growing the association at elevated CO2.

The response of photosynthetic CO2 exchange to tissue water content in five spp. of Sphagnum from contrasting microhabitats (S. fuscum (Schimp.) Klinggr., S. papillosum H. Lindb., S. magellanicum Brid., S. balticum (Russ.) C. Jens. and S. cuspidatum Ehrh. ex Hoffm.) was measured in the laboratory using an infrared gas analyser technique. Experiments were designed to test recovery of net photosynthesis after periodic and long lasting desiccation.

The contact between capitula and basal parts of the mosses seems to be important for survival. Isolated capitula cut off from any contact with the water table were not able to recover after complete desiccation (at 15°C for 2–4 d). When contact with the water table is lost, e.g. during long periods of desiccation, recovery of net photosynthesis can take place but only if the water content of the capitula does not fall too far below c. 10–20% of the water content at compensation point.

There was no relationship between the ability of net photosynthesis to recover from desiccation and the wetness of the natural microhabitat. Sphagna survive dry periods by avoidance of drying out by high capillarity or dense growth form (as in S. fuscum).

A range of marine photosynthetic picoeukaryote phytoplankton species grown in culture were screened for the presence of extracellular carbonic anhydrase (CAext), a key enzyme in inorganic carbon acquisition under carbon- limiting conditions in some larger marine phytoplankton species. Of the species tested, extracellular carbonic anhydrase was detected only in Micromonas pusilla Butcher. The rapid, light-dependent development of CAext when cells were transferred from carbon-replete to carbon-limiting conditions was regulated by the available free- CO2 concentration and not by total dissolved inorganic carbon. Kinetic studies provided support for a CO2- concentrating mechanism in that the K0.5[CO2] (i.e. the CO2 concentration required for the half-maximal rate of photosynthesis) was substantially lower than the Km [CO2] of Rubisco from related taxa, whilst the intracellular carbon pool was at least seven fold greater than the extracellular DIC concentration, for extracellular DIC values [les ]1.0 mm.

It is proposed that when the flux of CO2 into the cell is insufficient to support the photosynthetic rate at an optimum photon irradiance, the development of CAext increases the availability of CO2 at the plasma membrane. This ensures rapid acclimation to environmental change and provides an explanation for the central role of M. pusilla as a carbon sink in oligotrophic environments.

The aim of this work was to assess whether or not oxidative stress had developed in a dwarf shrub bilberry (Vaccinium myrtillus L.) under long-term exposure to enhanced levels of ultraviolet-B (u.v.-B) radiation. The bilberry plants were exposed to increased u.v.-B representing a 15% stratospheric ozone depletion for seven full growing seasons (1991–1997) at Abisko, Swedish Lapland (68°N). The oxidative stress was assessed on leaves and stems by analysing ascorbate and glutathione concentrations, and activities of the closely related enzymes ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The affects of autumnal leaf senescence and stem cold hardening on these variables were also considered. The results showed that the treatment caused scarcely any response in the studied variables, indicating that u.v.-B flux representing a 15% ozone depletion under clear sky conditions is not sufficient to cause oxidative stress in the bilberry. It is suggested that no strain was evoked since adaptation was possible under such u.v.-B increases. The studied variables did, however, respond significantly to leaf senescence and especially to stem cold hardening.

Barley yellow dwarf virus (BYDV) causes significant losses in yield and in overwintering ability of winter cereals. Mechanisms by which the physiology of plants is affected by the virus are not clear. To see how carbohydrates in the crown of winter cereals were affected by BYDV, fructan isomers of degree of polymerization (DP) 3–5, fructan DP>6 and the simple sugars, glucose, fructose and sucrose, were measured before and during cold hardening in three oat (Avena sativa L.) cultivars, ‘Wintok’, ‘Coast Black’ and ‘Fulghum’. On a fresh weight basis fructan DP>6 decreased by 50% in infected ‘Wintok’ and ‘Coast Black’ and by 25% in ‘Fulghum’. Two DP3, one DP4 and one DP5 isomer were significantly higher than non-infected controls. The percentages of simple sugars in infected crowns were significantly higher than controls in all three cultivars in every week except the first week of hardening. Crude enzyme extracts from BYDV infected plants incubated with sucrose suggested higher invertase and lower sucrose-sucrosyl transferase activity. When incubated with 1-kestose and neokestin, no significant difference was found in fructose fructosyl transferase or in hydrolase activity. The activity of unidentified enzymes catalysing the synthesis of larger (DP>5) fructan was altered by BYDV. The decrease of carbohydrates in the crown induced indirectly by BYDV may alter the plant's capacity to regenerate tillers in the spring. The ability of plants to prevent or tolerate carbohydrate fluctuations induced by BYDV infection may be an important genetically regulated characteristic for developing virus-resistant cultivars.

This is the first extensive investigation that quantifies natural mycoparasitic relationships in the phyllosphere. The presence of Ampelomyces spp. was quantified in naturally occurring powdery mildew fungi collected in Hungary and Romania between 1992 and 1995. A total of 570 samples was studied representing 27 species (nine genera) of the Erysiphaceae infecting 41 host plant genera. The incidence of Ampelomyces spp., determined as the proportion of samples in which intracellular pycnidia were present, varied between 4.3 and 68.8% in the host fungal genera studied. The intensity of mycoparasitism, defined as a percentage of the powdery mildew mycelia parasitized by Ampelomyces, ranged from 0.15 to 65%. Both the incidence and the intensity of mycoparasitism showed the lowest values in Blumeria graminis (DC.) Speer collected from wild and cultivated monocotyledons, while the highest values were found in Arthrocladiella mougeotii (Lév.) Vassilkov infecting Lycium halimifolium Mill. plants. The paper reports for the first time the natural occurrence of Ampelomyces in Sawadaea bicornis (Wallr.[ratio ]Fr.) Homma on maple.

Seeds of the orchid species, Spiranthes sinensis (Pers.) Ames, were sterilized and germinated in vitro with the symbiotic fungus Ceratobasidium cornigerum (Bourdot) Rogers. Colonized embryos developed into protocorms and these were examined for changes in microtubule arrays, after initial invasion of fungal hyphae into embryos and during peloton formation and degradation. Methods utilized to detect microtubules included immunofluorescence combined with laser scanning confocal microscopy, conventional transmission electron microscopy combined with morphometric analysis, and immunogold labelling. Microtubules were regularly found in close association with intracellular hyphae and degraded hyphal masses. Cortical microtubules disappear during peloton formation but reappear in cells that show fungal lysis. With conventional transmission electron microscopy and immunogold labelling the microtubules associated with fungal hyphae and degenerated hyphal masses were located close to the perifungal membrane that separates fungal hyphae from protocorm cytoplasm.

Norway spruce (Picea abies (L.) Karst.) seedlings were inoculated with the ectomycorrhizal fungus Laccaria bicolor ((Marie) Orton), strain S238 N, in axenic conditions. The presence of the fungus slowed tap–root elongation by 26% during the first 15 d after inoculation and then stimulated it by 136%. In addition, it multiplied in vitro lateral root formation by 4.3, the epicotyl growth of the seedlings by 8.4 and the number of needles by 2. These effects were maintained when the fungus was separated from the roots by a cellophane membrane preventing symbiosis establishment, thus suggesting that the fungus acted by non-nutritional effects. We tested the hypothesis that IAA produced by L. bicolor S238 N would be responsible for the stimulation of fungal induced rhizogenesis. We showed in previous work that L. bicolor S238 N can synthesize IAA in pure culture. Exogenous IAA supplies (100 and 500 μm) reproduced the stimulating effect of the fungus on root branching but inhibited root elongation. The presence of 2,3,5-triiodobenzoic acid (TIBA) in the culture medium significantly depressed lateral root formation of inoculated seedlings. As TIBA had no significant effect on IAA released in the medium by L. bicolor S238 N, but counteracted the stimulation of lateral rhizogenesis induced by an exogenous supply of IAA, we suggest that TIBA inhibited the transport of fungal IAA in the root. Furthermore TIBA blocked the colonization of the main root cortex by L. bicolor S238 N and the formation of the Hartig net. These results specified the role of fungal IAA in the stimulation of lateral rhizogenesis and in ectomycorrhizal symbiosis establishment.

The contribution of the extramatrical mycelium to N and P nutrition of mycorrhizal Norway spruce (Picea abies (L.) Karst.) was investigated. Seedlings either inoculated with Paxillus involutus (Batsch) Fr. or non-mycorrhizal were grown in a two compartment sand culture system where hyphae were separated from roots by a 45 μm nylon net. Nutrient solution of the hyphal compartment contained either 1.8 mm NH4+ and 0.18 mm H2PO4− or no N and P. Aluminium added to the hyphal compartment as a tracer of mass flow was not detected in the plant compartment, indicating that measurements of N and P transfer by the mycelium were not biased by solute movement across the nylon net.

The addition of N and P to the hyphal compartment markedly increased dry weight, N and P concentration and N and P content of mycorrhizal plants. Calculating uptake from the difference in input and output of nutrient in solution confirmed a hyphal contribution of 73% and 76% to total N and P uptake, respectively. Hyphal growth was increased at the site of nutrient solution input.

The involvement of the cytoskeleton in symbiotic interactions such as arbuscular mycorrhizas has received little attention. In this paper, we examine the organization of actin in tobacco mycorrhizal roots and compare actin and tubulin patterns within arbuscule-containing cells.

Our results show drastic reorganization of microfilaments and microtubules upon fungal infection and how those new cytoskeletal patterns relate to the host cytoplasm rearrangement and the intracellular fungal structures. Whereas in uninfected cells a network of cortical and perinuclear actin filaments was observed, in infected cells actin filaments closely follow the fungal branches and envelop the whole arbuscule in a dense coating network. Microtubules are less closely connected with the fungus surface. They run across the whole arbuscule mass, linking branches to each other and to the host cell cortex and nucleus.

These major differences between the two cytoskeletal components are used to advance some suggestions concerning their contribution to structural functions in the plant–fungus interactions during the mycorrhizal symbiosis.

Mitochondrial and nuclear genes have different inheritance, thus studies of fungal populations should use both mitochondrial and nuclear markers. Using nuclear markers, the S238N strain of the ectomycorrhizal basidiomycete Laccaria bicolor ((Maire) Orton) has been previously shown to persist for at least 10 yr after outplanting in a plantation of Douglas fir (Pseudotsuga menziesii (Mir.) Franco) inoculated with this strain. In the present study, we have sampled 539 sporophores of Laccaria spp. from this plantation, some of which had the S238N nuclear genotype, to study mitochondrial DNA polymorphism and persistence of the inoculated S238N mitochondrial genome. Length polymorphism in fragments of the large subunit of mitochondrial ribosomal DNA (LrDNA) allowed distinction of the haplotypes present in the plantation at the species level. In addition, heteroduplex analysis and sequencing revealed intraspecific polymorphism of LrDNA among the L. bicolor sporophores and enabled specific identification of S238N LrDNA. This haplotype was only retained in sporophores carrying the S238N nuclear genome, confirming the survival of this introduced strain in a natural population.