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New Phytol. (1998), 140, 599–624 Tansley Review No. 102 Plant hybridization B L O R E N H. R I E S E B E R G S H A N N A E. C A R N E Y Dept of Biology, Indiana University, Bloomington, IN 47405, USA (Received 19 March 1998 ; accepted 18 July 1998) I. II. III. IV. V. Summary Introduction Concepts and terminology Historical background Studies of experimental hybrids 1. Isolating mechanisms 2. Prezygotic barriers (a) Gametic barriers to hybridization 3. Postzygotic barriers (a) Chromosomal rearrangements (b) Genic sterility or inviability 4. Hybrid vigour 5. Introgression 6. Hybrid speciation Experimental manipulations of natural hybrid populations 1. Hybrid-zone formation 2. Pollinator-mediated selection 599 599 600 600 601 601 602 602 603 604 604 605 606 607 609 610 610 3. Habitat selection VI. The biology of different classes of hybrids 1. Character expression (a) Morphological characters (b) Chemical characters (c) Molecular characters 2. The fitness of different classes of hybrids (a) The importance of variance (b) Estimating hybrid fitness 3. Interactions with parasites and herbivores 4. Patterns of mating (a) Outcrossing rate (b) Hybridization frequency (c) Mate choice VII. Conclusions and future research Acknowledgements References 612 612 613 613 613 613 614 614 615 616 617 617 618 618 619 620 620 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. Key words : Hybridization, introgression, reproductive isolation, speciation. . In the conclusions from his 1979 benchmark volume on hybridization, Levin predicted that most major advances in understanding hybridization would ‘ come from experimental analyses (of hybridization E-mail : lriesebe!indiana.edu phenomena), and manipulations of natural hybrid swarms or hybrid zones, and a very thorough analysis of the biology of first-generation, advanced generation, and backcross hybrids relative to the parental taxa under a range of environments ’. He also argued that major advances were unlikely to ‘ come from further documentation of hybridization in the fashion of our time ’. Printed from the C JO service for personal use only by... 600 L. H. Rieseberg and S. E. Carney Although many studies continue to document hybridization phenomena, the past two decades have seen a large increase in the kinds of experimental, microevolutionary studies that Levin predicted would lead to advances in our knowledge of the process and implications of hybridization in natural populations. Here, we discuss the three kinds of studies Levin thought would be important : studies of experimental hybrids ; studies that experimentally manipulate natural hybrid populations ; and studies that describe the biology of different generational classes of hybrids. As with Levin’s volume, the focus of the review is restricted to homoploid hybrids. These recent studies have, as predicted, led to major new discoveries and advances. . Hybridization can have several different meanings for evolutionary biologists. The term ‘ hybrid ’ can be restricted to organisms formed by cross-fertilization between individuals of different species. Alternatively, hybrids can be defined more broadly as the offspring between individuals from populations ‘ which are distinguishable on the basis of one or more heritable characters ’ (Harrison, 1990). Similarly, introgression can be defined narrowly as the movement of genes between species mediated by backcrossing or more broadly defined as the transfer of genes between genetically distinguishable populations. We prefer the broader definitions of hybridization and introgression, as they provide greater flexibility in usage. Nonetheless, here our focus will be on hybridization and introgression between species. A focus on interspecific hybrids requires consideration of species concepts. Unfortunately, the term species has a wide variety of definitions that range from concepts based on the ability to interbreed to those based on common descent. Perhaps the most widely accepted of these is Mayr’s (1963) biological species concept : ‘ species are groups of interbreeding natural populations which are reproductively isolated from all other such groups ’. This concept is useful for studies of hybridization and speciation, but it would deny species status of hybridizing taxa if applied stringently. Thus, here we will refer to biological species as groups of interbreeding populations that are ‘ genetically isolated ’ rather than ‘ reproductively isolated ’ from other such groups. This may seem to be a trivial distinction, but it is clear that most hybrid zones serve as an effective barrier to interspecific genetic exchange, even if local introgression is extensive. . Hybridization has been important to humans since the Neolithic era when the domestication and breeding of plants and animals began (Zirkle, 1935). However, at least in plants, hybridization appears to have been inadvertent rather than intentional, because of a lack of understanding of plant sexuality and pollen function. Hand pollination of dioecious date palms in the Old World and of monoecious maize in the New World are exceptions to this general rule, but this knowledge does not appear to have been transferred to other species. As a result, knowledge and study of plant hybridization lagged behind that of animals until the 16th century. The modern history of plant hybridization was initiated by Camerarius, who in 1694 speculated that it might be possible to fertilize a female plant of one species with pollen from a male plant of another species (Zirkle, 1935). The first written reference to spontaneous plant hybridization, found in a letter written by Cotton Mather in 1716, describes naturally occurring crosses between Indian and yellow corn and between gourds and squash that were planted together (Zirkle, 1935). However, it appears that the first intentional artificial hybrid was generated by Thomas Fairchild in a cross between carnation (Dianthus caryophyllus) and sweet William (Dianthus barbatus). Other authors have ascribed the first artificial hybrid to Linnaeus’ experiments on Tragopogon in 1759, but it is clear that Fairchild’s hybrid was generated almost 50 yr earlier (Zirkle, 1935). Rigorous scientific study of plant hybridization began with the publication of Ko$ lreuter’s hybridization experiments in 1761 (Roberts, 1929). Ko$ lreuter made several discoveries about hybridization that have endured the test of time. First, he demonstrated that hybrids from interspecific crosses are sometimes sterile or ‘ botanical mules ’. As a result, Ko$ lreuter concluded that hybrid plants are produced only with difficulty and are unlikely to occur in nature without human intervention or disturbance of the habitat. This is the first explicit reference to the importance of ecological factors, particularly disturbance, in mediating hybridization. Second, Ko$ lreuter showed that hybrids are usually intermediate morphologically relative to their parents. Third, he discovered that if later-generation hybrids are produced, they tend to revert back to the parental forms. This discovery refuted an earlier suggestion by Linnaeus that hybrids were constant or true-breeding and represented new species (Linne! , 1760). In the latter part of the 18th century and the early 19th century hybridization techniques were widely applied to plant breeding, a focus that continues today. Early botanical hybridizers were also interested in the validity of hybrid sterility as a species criterion (Roberts, 1929). This work was accompanied by increasing numbers of reports of spontaneous hybrids between wild plant species. The implications of these early hybridization studies were summarized by Focke (1881). He noted Printed from the C JO service for personal use only by... Plant hybridization that some taxonomic groups hybridize readily, whereas others do not. These taxonomic correlates of hybridization have been quantified in a recent survey of five biosystematic flora (Ellstrand, Whitkus & Rieseberg, 1996). Focke also suggested that plants with zygomorphic flowers were more likely to hybridize than plants with actinomorphic flowers – a tendency that is still recognized (Stebbins, 1957). Finally, Focke hypothesized that natural hybridization is most likely when flowers from one of the parental species are in a quantitative minority. Biased ratios of parental species flowers could be caused by the rarity of one of the parental species or by variation between species in flowering phenology. Recent studies of Louisiana iris hybrids by Arnold and co-workers (e.g. Arnold, Hamrick & Bennett, 1993 ; Carney, Cruzan & Arnold, 1994) confirm Focke’s hypothesis and suggest that interspecific pollen competition is the mechanism that largely prevents hybrid production when flowers of both parental species are abundant. Although there was little discussion of an evolutionary role for hybridization during this period, there was speculation concerning the possible origin of new species via hybridization. For example, Naudin (1863) suggested that hybrid characters may become fixed in later generations and that this might facilitate species formation. This idea was expanded by Kerner (1894–1895), who discussed the possible role habitat might play in mediating the establishment of hybrid species. Kerner realized that although hybrids were formed frequently in nature, their successful establishment required suitable open habitat that was free of the parental species. This was a significant contribution because ecological factors play an important role in current models of hybrid speciation (e.g. Templeton, 1981). In the early 20th century, three key discoveries laid the foundation of modern evolutionary studies of hybridization. The first discovery was by Winge$ (1917), who showed that new and constant hybrid species could be derived instantaneously by the duplication of a hybrid’s chromosome complement (i.e. allopolyploidy). This hypothesis was quickly confirmed experimentally in a variety of plant species, and allopolyploidy is now recognized to be a prominent mechanism of speciation in flowering plants and ferns (Soltis & Soltis, 1993 ; Leitch & Bennett, 1997). A second important discovery resulted from the work of Mu$ ntzing (1930) on homoploid hybrid or ‘ recombinational ’ speciation. He postulated that the sorting of chromosomal rearrangements in latergeneration hybrids could, by chance, lead to the formation of new population systems that were homozygous for a unique combination of chromosomal sterility factors. The new hybrid population would be fertile, stable, and at the same ploidal level as its parents, yet partially reproductively isolated 601 from both parental species because of a chromosomal sterility barrier. Although early authors focused on chromosomal rearrangements (e.g. Mu$ ntzing, 1930 ; Grant, 1958), it is clear that the sorting of genic sterility factors should generate similar results. Thus, current models incorporate both genic and chromosomal sterility factors. Modern contributions to the study of this process, termed recombinational speciation by Grant (1958), include rigorous experimental and theoretical tests of the model, as well as the gradual accumulation of well-documented case studies from nature (Rieseberg, 1997). A third key advance resulted from studies of natural hybrid populations by Anderson and coworkers (Anderson, 1936 ; Anderson & Hubricht, 1938). Anderson suggested that products of interspecific hybridization, particularly those resulting from backcrossing or introgression, might be favoured by selection and thus contribute to adaptive evolution within populations. Over the past two decades, molecular markers have been used to document introgression in many groups of plants, but its adaptive significance remains poorly understood (Rieseberg & Wendel, 1993 ; Arnold, 1997). Although these advances provided the conceptual foundations for most recent studies of hybridization, scientific interest has expanded considerably to include theoretical modelling of the structure and maintenance of hybrid zones, the detection and consequences of hybridization in phylogenetic reconstruction, the interactions of hybrids with pathogens and herbivores, the potential extinction of rare species through hybridization, and the role of hybridization in mediating the escape of genetically engineered genes. The experimental, microevolutionary studies advocated by Levin (1979) have informed each of these areas. . Natural hybridization phenomena can often be replicated in the glasshouse, allowing the process of hybridization and introgression to occur under controlled conditions. Artificial hybridization experiments provide a means for investigating genetic isolating mechanisms and for testing models of hybrid speciation and introgression. 1. Isolating mechanisms Reproductive isolating mechanisms are generally divided into two categories based on whether they act before or after fertilization. Mechanisms that act to prevent mating or fertilization are referred to as prezygotic, whereas those that act to reduce the viability or fertility of the hybrid zygote or latergeneration hybrid offspring are referred to as postzygotic. Hybridization experiments have been used to study both kinds of mechanisms, although Printed from the C JO service for personal use only by... 602 L. H. Rieseberg and S. E. Carney the emphasis has been on mechanisms that act after mating, because these are more easily studied under greenhouse conditions. 100 Expected IH × IF (a) Gametic barriers to hybridization. Probably the most detailed studies of gametic barriers to hybridization have been in the Louisiana irises. Studies in this group of naturally hybridizing species have focused on pollen competition or conspecific pollen advantage, including measurements of pollen-tube lengths in conspecific and heterospecific styles and examination of hybrid seed production following different types of controlled pollinations. Measurements of Iris fulva and I. hexagona pollen-tube lengths 3n5 h after pure conspecific and heterospecific pollinations revealed that heterospecific pollen tubes grew at least as well as conspecific pollen tubes in the initial stages of growth (Carney et al., 1994). This result suggests that relative pollen-tube growth rates are unlikely to act as a barrier to hybridization in these species. However, only pure parental progeny were obtained from controlled crosses of I. fulva and I. hexagona with 1 : 1 mixtures of pollen from the two species (Arnold et al., 1993). These experimental crosses were later repeated as part of a larger crossing study, in which 1 : 9, 1 : 3, 1 : 1, 3 : 1 and 9 : 1 ratios of pollen from the two species were used in addition to pure conspecific and heterospecific crosses (Carney et al., 1994). Although some hybrid seeds were formed from the crosses using mixed pollen loads, there were significantly fewer than would be expected if fertilization was random (Fig. 1). Similar results were obtained from crosses involving I. fulva Hybrid seeds (%) Prezygotic isolating mechanisms in plants include habitat, temporal and ethological barriers, as well as gametic competition or incompatibility. The role of the first three isolating mechanisms in mediating hybridization are most easily studied under natural conditions or in experimentally manipulated hybrid populations (e.g. Campbell, Waser & MelendezAckerman, 1997 ; Emms & Arnold, 1997 ; Nagy, 1997 a, b). Thus, the few artificial hybridization experiments that investigate habitat and temporal isolation have largely focused on the genetic basis of these traits (e.g. Macnair & Christie, 1983 ; Macnair, Smith & Cumbes, 1993 ; Schat, Vooijs & Kuiper, 1996 ; Kuittinen, Sillanpa$ a$ & Savolainen, 1997). Glasshouse experiments offer little for studying how hybridization affects pollinator preferences, although substantial information has been gathered on the genetic basis of floral traits that affect these preferences (e.g. Macnair & Cumbes, 1989 ; Bradshaw et al., 1995 ; Kuittinen et al., 1997). By contrast, artificial hybridization studies have yielded extensive data on how gametic competition and incompatibility serve as isolating mechanisms. IF × IH 75 2. Prezygotic barriers IB × IF IF × IB 50 25 0 0 25 50 75 100 Interspecific pollen (%) Figure 1. Percentage of hybrid seeds formed from pollinations of Iris hexagona (IH), I. fulva (IF) and I. brevicaulis (IB) with mixtures of conspecific and heterospecific pollen. For each cross listed, the first species is the maternal species and the second is the competing pollen type. The ‘ expected ’ line shows the percentage of hybrid seeds that would be formed assuming equal fertilization by pollen grains present on the stigma. For 0 % and 100 % heterospecific pollen, the observed percentage of hybrid seeds did not differ significantly from that expected, but significant differences were seen for all other values (IBiIF, P 0n05 ; all other crosses, P 0n005). Adapted from Carney et al., 1994 and Emms et al., 1996. and a third species, I. brevicaulis (Emms, Hodges & Arnold, 1996). Iris fulva and I. brevicaulis pollen tubes did not differ significantly in length in I. brevicaulis flowers, but after 3 h of growth in I. fulva, conspecific pollen tubes were significantly longer than heterospecific tubes, suggesting that pollen competition is a stronger barrier in I. fulva flowers than in I. brevicaulis flowers. When 1 : 1 mixtures of I. fulva and I. brevicaulis pollen were used to pollinate flowers of each of the parental species, a significantly larger fraction of seeds was conspecific than heterospecific (Fig. 1). Although these studies suggest that faster growth of conspecific pollen tubes might be acting as a barrier to hybridization, its importance relative to postzygotic factors could not be determined. Sequential pollination studies were used to investigate further the role of relative pollen-tube growth rates in isolating the pairs of iris species described (Carney, Hodges & Arnold, 1996 ; Carney & Arnold, 1997). If pollen-tube growth factors isolate species, the fraction of hybrid seed produced should vary with the length of time between stylar applications of heterospecific and conspecific pollen. No differences would be expected if postzygotic mechanisms, such as hybrid seed abortion, are responsible for reduced hybrid seed set. For both Printed from the C JO service for personal use only by... Plant hybridization Iris sp. pairs, increasing frequencies of hybrid seed were produced with increasing ‘ head starts ’ afforded to heterospecific pollen, confirming that conspecific pollen-tube growth advantage and\or heterospecific attrition (or prefertilization pollen-tube growth failure) play an important role in isolating the Louisiana irises. Similar studies have been performed in other naturally hybridizing plant species. Rieseberg, Desrochers & Youn (1995 a) pollinated Helianthus annuus and H. petiolaris with mixed pollen loads and obtained significantly fewer hybrid progeny than expected. In addition, no hybrids were formed when H. annuus pollen was given a 15- or 30-min head start over H. petiolaris pollen in H. petiolaris styles. However, conspecific and heterospecific pollen-tube lengths did not differ significantly in flowers of either species. Although pollen-tube measurements did not accurately predict relative fertilization success, these experiments suggest that gametic barriers to hybridization also exist between these species. Smith (1968, 1970) was able to correlate the relatedness of Haplopappus species with the strength of their reproductive barriers using sequential pollinations. Specifically, he investigated the temporal advantage necessary to equalize the competitive ability of pollen for each species pair (i.e. to produce 50 % hybrid seed). Chen & Gibson (1972) studied pollen germination, pollen-tube growth and fertilization in conspecific and heterospecific crosses of Trifolium repens with T. nigrescens, T. occidentale, T. hybridum, T. ambiguum and T. uniflorum. Pollen germination frequency was lower in heterospecific crosses than in conspecific crosses. Heterospecific pollen tubes often grew abnormally, with swelling, coiling and bursting in the styles. Pollen-tube growth rate and fertilization frequency were found to be correlated with relatedness of the pollen donor species to T. repens. An interesting discovery of many studies of prezygotic barriers to hybridization has been that reproductive isolation is often asymmetrical. For all Haplopappus sp. pairs, the direction of the cross has been shown to affect the pollination interval needed to produce 50 % hybrid seeds (Smith, 1970). Similarly, in the Louisiana irises, I. fulva is the father of hybrids far more frequently than it is the mother (Carney et al., 1994, 1996 ; Emms et al., 1996 ; Hodges, Burke & Arnold, 1996 ; Fig. 1), and H. petiolaris sired many more hybrid seeds than did H. annuus following pollinations with mixed pollen loads (Rieseberg et al., 1995 a). Other studies have demonstrated a correlation between self- and hetero-incompatibility (Lewis & Crowe, 1958). In general, heterospecific incompatibility between a self-incompatible and selfcompatible species is unidirectional ; only the selfcompatible parent is able to produce hybrid offspring. 603 Although pollen competition has been studied as a barrier to hybridization in several naturally hybridizing plants, studies of gametic barriers in commercially important plant species have been more prevalent. These studies have generally been undertaken to learn what barriers exist so that they can be overcome and the desired crosses can be performed (e.g. Sanyal, 1958 ; Gore et al., 1990 ; Beharav & Cohen, 1995 ; Marcella! n & Camadro, 1996). Other investigations have sought to estimate the risk of escape of transgenes into wild populations via cropiweed hybridization (e.g. Lefol, Fluery & Darmency, 1996). 3. Postzygotic barriers Much of the biosystematic work in the early 1900s focused on postzygotic isolating barriers. Part of this work was motivated by a desire to understand speciation. An even greater motivation was the prevailing belief that the strength of postmating barriers was proportional to the degree of relatedness among species. The use of crossing studies to investigate evolutionary relationships was extended to the analysis of chromosome pairing relationships. This approach assumes that the degree of chromosome pairing is proportional to the level of overall genomic divergence – an assumption now largely invalidated by the discovery of genes that control chromosome pairing (Jackson, 1985). Nevertheless, numerous crossing studies have been performed, and substantial evidence has been gathered on the kinds and strength of postmating reproductive barriers in both closely and distantly related plants species (Heiser, 1949 ; Stebbins, 1957 ; Levin, 1979 ; Grant, 1981 ; Jackson, 1985). Common postmating barriers include hybrid weakness or inviability, hybrid sterility and hybrid breakdown in which first generation (F ) hybrids are robust and fertile, but " later generation hybrids are weak or inviable. An important early observation was the high degree of variability in viability and fertility observed in both first- and later-generation hybrids (Stebbins, 1959). This is perhaps best exemplified by F hybrids " of Primula elatioriP. vulgaris, which vary dramatically in vigour, ranging from significant positive to significant negative heterosis (Valentine, 1947).The variation is even more extreme between different interspecific combinations, with some combinations generating inviable or sterile hybrids, and others producing hybrids that are robust and fertile and show no evidence of breakdown in later hybrid generations (Gillett, 1972). All gradations in fertility and viability have been observed between these extremes. Extensive variability in viability and fertility is also observed within and between hybrid generations from the same interspecific cross. Variability levels Printed from the C JO service for personal use only by... 604 L. H. Rieseberg and S. E. Carney tend to be greatest in F and first back-cross (BC ) # " generations. Successive hybrid generations are characterized by an increase in fertility and viability and a reduction in variation as a result of natural selection (e.g. Grant, 1966 a). The large variability observed in the viability and fertility of natural hybrids and the rapid response of these characteristics to selection were interpreted correctly by Anderson (1949), Stebbins (1957), Grant (1981) and others to indicate that hybridization need not be a ‘ blind alley ’ of evolution. (a) Chromosomal rearrangements. Over the past few decades, considerable progress has been made toward understanding the genetic basis of hybrid inviability, sterility and breakdown. In crosses between chromosomally divergent species, sterility is typically attributed to the effects of chromosomal rearrangements on meiotic pairing. However, this assumption has been questioned recently because individuals heterozygous for chromosomal rearrangements often show little meiotic impairment or loss of fertility (Sites & Moritz, 1987 ; Coyne et al., 1993). These authors have suggested that genic factors may explain much of the loss of fertility typically attributed to chromosomal rearrangements. Unfortunately, it has been difficult until recently to distinguish unambiguously between chromosomal and genic effects. Two approaches have been employed successfully to separate the effects of chromosomal rearrangements and genic factors on sterility in interspecific crosses. One approach involves genetic mapping of quantitative trait loci (QTLs) for sterility. An early example involved the analysis of hybrids between two lentil species, Lens culinaris and L. ervoides (Tadmor, Zamir & Ladizinsky, 1987). The two species appear to differ by a single translocation, and it was thought that the translocation was responsible for reduced fertility in F hybrids. To test this " hypothesis, Tadmore et al. (1987) used a segregating F population between the two species to generate a # map based on 18 isozyme markers. Correlations between four of the isozyme markers and quadrivalent formation in meiosis allowed precise identification of the translocation end-points. All plants with pollen viability 65 % were heterozygous for the translocation, whereas plants with pollen viability 85 % were homozygous for the translocation. Thus, the chromosomal translocation does appear to represent the primary postmating reproductive barrier between these two species. A similar study was conducted recently in Helianthus. Most species in the genus appear to differ by one or more chromosomal translocations, and these chromosomal rearrangements are generally well correlated with the pollen viability of F hybrids " (Chandler, Jan & Beard, 1986). To provide a quantitative estimate of the influence of chrom- osomal rearrangements on pollen viability, Quillet et al. (1995) analysed the segregation of 48 genetic markers in BC progeny of an interspecific hybrid " between H. argophyllus and the common sunflower, H. annuus. Helianthus argophyllus is the sister species of H. annuus (Rieseberg, 1991), and cytogenetic analyses indicate that the two species differ by two reciprocal translocations (Chandler et al., 1986). As predicted by cytogenetic studies, a wide range of variability in pollen viability was observed in the mapping family (27–93 %). Over 80 % of this variation was explained by three genetic intervals located on linkage groups 1, 2 and 3, respectively. Analyses of meiosis in the backcross hybrids revealed that meiotic abnormalities were also tightly correlated with the markers, indicating that chromosomal rearrangements are largely responsible for reducing fertility in hybrids between these species. A second approach that has been employed successfully to distinguish between chromosomal and genic effects involves analysis of introgression patterns across the sterility barrier. If the chromosomal rearrangements contribute to reduced hybrid fitness, then linkage blocks carrying these rearrangements will be selected against in introgressed progeny. An example of this approach comes from the analysis of introgression lines between two sunflower species, H. annuus and H. petiolaris (Rieseberg, Linder & Seiler, 1995 b, Rieseberg et al., 1996 a). Comparative genetic mapping studies have allowed the identification of 10 chromosomes that differ in gene order between the two species. The remaining seven chromosomes appear to be collinear. Analysis of the distribution of interspecific genetic material in the introgression lines revealed that introgression is significantly reduced in the rearranged chromosomes as compared with the collinear chromosomes. These data support the view that chromosomal rearrangements do provide significant barriers to gene exchange, particularly within the rearranged linkage groups. These results also suggest that species genomes are often differentially permeable to introgression, where certain portions of the genome are open to the incorporation of alien alleles, but introgression is restricted in other parts of the genome. (b) Genic sterility or inviability. Substantial efforts have also been made in understanding the mechanistic basis of genic sterility or inviability in hybrids. The most widely accepted model was first proposed by Dobzhansky (1937). In this ‘ standard model ’, a gene from one species interacts negatively with a gene from another species, causing some degree of inviability or sterility. Wu & Palopoli (1994) argue that the most plausible interpretation of this model is that the hybrid sterility\inviability gene acts like a mutation whose deleterious effects are suppressed by another gene in the source species ; Printed from the C JO service for personal use only by... Plant hybridization however, when placed in the genetic background of another species, the deleterious effects of the sterility\inviability gene are expressed. A somewhat different model for the evolution of hybrid inviability\sterility is that a much larger number of diverging loci interact negatively in a hybrid genetic background, and that these weak interactions act cumulatively to cause inviability or sterility (Wu & Palopoli, 1994). Others have suggested that meiotic drive plays a key role in the evolution of postmating reproductive isolation (e.g. Frank, 1991). Data that support the standard model come from the many studies of plant hybrids in which one or two genes appear to have major effects on hybrid sterility or inviability. Examples include barley (Wiebe, 1934), cowpea (Saunders, 1952), Crepis (Hollingshead, 1930), cotton (Gerstel, 1954), Melilotus (Sano & Kita, 1978), Mimulus (Macnair & Christie, 1983 ; Christie & Macnair, 1984), rice (Oka, 1974 ; Wan et al., 1996 ; Li et al., 1997) and wheat (Hermson, 1963). However, these observations do not rule out the possibility that many additional genes may affect inviability and sterility in these species as well. Indeed, detailed studies of the genetic basis of hybrid sterility and hybrid breakdown between subspecies of rice suggest that several mechanisms are involved (Li et al., 1997). These mechanisms include a cytoplasmic gene that causes both male and female sterility ; and interactions between a pair of complementary genes that lead to greatly reduced fertility. Both of these mechanisms fit the standard model. In addition, Li et al. (1997) found that recombination between differentiated supergenes represents a major source of sterility. Map comparisons suggest that these regions may contain inversion polymorphisms, and that sterility may be caused by crossing over between cryptic structural rearrangements (cytologically undetectable chromosomal aberrations) (Stebbins, 1958). Thus, chromosomal mutations might also play an important role in the evolution of hybrid sterility in rice. Li et al. (1997) also provide evidence that hybrid breakdown in rice largely fits the polygenic model and results from the uncoupling of coadapted subspecific gene complexes by recombination. In later-generation hybrids, semisterility appears to be caused largely by incompatibility interactions between many loci, and hybrid weakness appears to result from the break-up of coadapted gene complexes that affect fitness traits such as heading age and floret number per panicle. The presence of these coadapted gene complexes in rice has long been suspected as a result of observations that intersubspecific hybrids tend to revert quickly back to one of the parental types in successive hybrid generations. The complex genetic basis of postmating reproductive isolation in rice accords well 605 with studies of Drosophila (Wu & Palopoli, 1994) that indicate that sterility and breakdown in fly hybrids involve many genes and higher-order epistatic interactions. A complex genetic basis for postmating reproductive isolation in many species of plants is also suggested by introgression mapping studies. For instance, the presence of genomic intervals where introgression is reduced or absent is often reported in map-based studies of introgression between crop plants and their wild relatives (Jena, Khush & Kochert, 1992 ; Williams et al., 1993 ; Garcia, Stalker & Kochert, 1995 ; Wang, Dong & Paterson, 1995 ; McGrath, Wielgus & Helgeson, 1996 ; Fulton, Nelson & Tanksley, 1997). Presumably, many of these genomic regions harbour genes that contribute to reproductive isolation. Likewise, strong segregation distortion is often observed in interspecific crosses, suggesting that many genes are negatively selected in hybrids. For example, Zamir & Tadmor (1986) report segregation distortion at 54 % of loci from interspecific crosses of lentil, pepper and tomato, compared with only 13 % in intraspecific crosses. These results suggest that both the standard model and the polygenic model are necessary to account for the behaviour of first- and later-generation hybrids. Meiotic drive might also play an important role, but there are few data that directly test the meiotic-drive hypothesis. 4. Hybrid vigour Interspecific hybrids are highly variable in fertility and vigour. However, one general rule is that F " hybrids, particularly between geographic races or closely related species, tend to exceed their parents in vegetative vigour or robustness (Grant, 1975). This phenomenon – hybrid vigour (heterosis) – is often used to maximize yields in crop plants. Heterosis has major implications for evolutionary biology and at least partly explains the success of allopolyploid species and many clonal hybrid lineages (e.g. Huskins, 1931 ; Grootjans, Allersma & Kik, 1987). It may also contribute to the successful establishment of introgressive hybrid races or hybrid species, but this argument is less convincing because hybrid vigour is more difficult to maintain in segregating hybrid generations. Although heterosis is a likely contributor to the evolutionary success of hybrids, its genetic basis is still poorly understood. Possible models for the evolution of heterosis are listed in Mitchell-Olds (1995) and include : dominance (the masking of deleterious recessives) ; overdominance (single locus heterosis) ; and epistasis (enhanced performance of traits derived from different lineages). It is difficult to distinguish between these models using classical quantitative genetic approaches because the effects Printed from the C JO service for personal use only by... 606 L. H. Rieseberg and S. E. Carney of individual loci cannot be distinguished. The models have been tested in a few marker-based quantitative genetic studies, but the data are too few to permit generalizations. The first study of this type was conducted in maize by Stuber et al. (1992). Of nine QTLs affecting yield, eight showed significant overdominance. A reanalysis of the data by Cockerham & Zeng (1996) suggests that the apparent overdominance observed may represent an example of pseudo-overdominance caused by the presence of several linked QTLs. Cockerham & Zeng argue that the data are most consistent with the model of dominance of favourable genes, but admit that epistasis could also play an important role and that overdominance could not be ruled out. There is convincing evidence for the overdominance model in Arabidopsis, in which Mitchell-Olds (1995) identified a QTL that resulted in a 50 % increase in viability in heterozygotes relative to homozygotes. Mitchell-Olds argues that overdominance will be most important in partially inbred species because major deleterious recessives are likely to be rare and recessives with minor effects are likely to be purged from the population by inbreeding. 5. Introgression Most experimental studies of introgression have focused on the best methods of moving important agronomic traits from a wild species to a cultivated relative. To move a gene across a reproductive barrier, the allele must recombine into a new genetic background before it is eliminated by selection against the alleles with which it is initially associated (Barton & Hewitt, 1985). As a result, successful introgression, whether in the glasshouse or in the wild, will depend in part on the genetic architecture of the reproductive barriers. If many genes contribute to hybrid unfitness, then much of the genome may be resistant to introgression because of linkage (Whittemore & Schaal, 1991 ; Rieseberg & Wendel, 1993), particularly if recombination rates are low. This problem may be exacerbated if the genes interact epistatically or are ‘ co-adapted ’ (Harlan, 1936 ; Carson, 1975). However, if reproductive barriers are under simple genetic control, then most of the genome should be permeable to introgression. Only those traits tightly linked to sterility or inviability genes will be difficult to introgress. These predictions have largely been confirmed by experimental crossing programmes. In interspecific backcrosses involving divergent parental species, the donor parent genome is often eliminated much more rapidly than would be predicted under neutral conditions. For example, Stephens (1949) noted that in backcrosses between Gossypium spp., the donor parent genotype is selectively eliminated, regardless of the direction of the backcrosses. Similar obser- vations have been made for species hybrids in Antirrhinum (Mather, 1947), Hordeum (Koba, Handa & Shimada, 1991), Helianthus (Rieseberg et al., 1995 a), Melilotus (Baenziger & Greenshields, 1958), Lycopersicon (Rick, 1963), Zea (Mangelsdorf, 1958), Nicotiana (Neelam & Narayah, 1994), Oryza (Mao et al., 1995 ; Harushima et al., 1996) and Phaseolus (Wall, 1968). Skewed segregation ratios in hybrids now appear to be the rule rather than the exception. For example, Zamir & Tadmor (1986) report segregation distortion in 54 % of loci from interspecific crosses of Lenz, Capsicum and Lycopersicon, compared with only 13 % in intraspecific crosses. In Helianthus, segregation distortion has been observed at 7–13 % of loci in intraspecific mapping populations (Rieseberg et al., 1993 ; Berry et al., 1995 ; Gentzbittel et al., 1995) compared with 23–90 % of loci in interspecific crosses (Quillet et al. 1995 ; Rieseberg et al., 1995 b, 1996 a). Not only are distorted ratios prevalent, but they can also be extreme. For example, segregation ratios that were skewed 12 : 1 in favour of ‘ wild ’ alleles have been reported in crosses between cultivated pearl millet (Pennisetum glaucum) and one of its wild relatives (P. violaceum) (Liu et al., 1996). An overall result of these skewed segregation ratios is that hybrid progeny receive more alleles from one parent than would be expected under Mendelian rules of segregation and thus resemble that parent more closely than Mendelian rules would predict. Although most deviating ratios observed in species backcrosses have favoured the genes of the recurrent parent, there have been several exceptions to this general rule. For example, the white lint gene of the donor parent was favoured over brown lint alleles of the recipient parent in backcrosses from G. barbadense into G. hirsutum (Stephens, 1949). Likewise, 5 % of H. petiolaris markers introgressed at significantly higher than predicted rates into an H. annuus genetic background (Rieseberg et al., 1995 b). This is a relatively small fraction, however, when compared with the 85 % that introgressed at significantly lower than expected rates (Rieseberg et al., 1995 b). Finally, Wang et al. (1995) noted that the same G. hirsutum chromosome fragments were maintained in independently generated G. barbadense introgression lines. It is not clear whether these loci or chromosomal fragments are selectively favoured in the recurrent parent or whether they represent examples of ‘ self genes ’ – genes that enhance the success of gametes they inhabit even if they pose a significant fitness cost during the diploid phase of the life cycle (Haldane, 1932). Although this discussion focuses on factors that directly contribute to reproductive isolation, genome-wide introgression can also be reduced by lower recombination rates, because the alien chromosomal blocks must recombine into a new genetic background for introgression to occur. Not surprisingly, Printed from the C JO service for personal use only by... Plant hybridization recombination rates appear to be reduced in crosses between genetically divergent taxa such as maize and teosinte (Doebley & Stec, 1993), rice spp. (Causse et al., 1994), sunflower spp. (Quillet et al., 1995) and tomato spp. (Paterson et al., 1988 ; Miller & Tanksley, 1990). Recombination is often completely eliminated in somatic hybrids involving species that are too divergent to allow successful sexual crosses (e.g. Parokenney et al., 1994). Experimental studies have also been used to assess the efficiency of different mating designs for moving alleles across species barriers. From a theoretical standpoint, it appears clear that mating designs that enhance recombination rates will be most effective in facilitating gene transfer (Hanson, 1959 a, b ; Stephens, 1961 ; Wall, 1970). Unfortunately, recurrent backcrossing, the mating design typically employed for breeding purposes and invoked for natural introgression scenarios, has been demonstrated theoretically to be an extremely inefficient mechanism for the break-up of parental linkage blocks (Hanson, 1959 a, b). This problem is exacerbated for chromosomes with short map lengths, where the disruption of parental linkage blocks will be even slower because of lower recombination rates (Hanson, 1959 a). Hanson (1959 b) also noted that chromosomal structural differences greatly reduce effective recombination rates and map lengths. Thus, he recommended that mating designs that enhance recombination between the parental genomes be employed in these types of situations to ensure the disruption of parental linkage groups. Selfing and sib-mating represent two such systems, because these mating systems result in twice the recombination found in the simple backcross method (Wall, 1970 ; Liu et al., 1996). A second requirement for the successful introgression of alleles is the maintenance of reasonable levels of fertility and viability in hybrid or backcross populations. Unfortunately, populations resulting from either selfing or sib-mating will generally exhibit lower levels of fertility than those resulting from recurrent backcrossing (Wall, 1970). In particular, selfing tends to result in high proportions of ‘ subvital ’ plants because of hybrid breakdown (Stebbins, 1950 ; Stephens, 1950). Given these considerations, Wall (1970) suggested that there is an optimal level of recombination between genetically or chromosomally divergent populations. If recombination is too low, no introgression occurs, whereas if it is too high it may severely reduce overall viability and fertility in the resulting population. Thus, Wall (1970) argued that mating designs employing one or more generations of sib-mating interspersed with backcrossing will be more effective than backcrossing alone for moving alleles across linkage groups where effective recombination rates are low, such as in chromosomally divergent linkages. Alternatively, Haghighi & Ascher (1988) have 607 proposed the use of congruency backcrossing, in which backcrosses in the direction of one parent are alternated with backcrosses in the direction of the other parent. This approach quickly leads to the formation of fertile hybrids that can be used as a bridge for gene flow between widely divergent species. Experimental studies have largely confirmed the utility of these approaches. Substantial increases in heterospecific recombination have been observed in Arabidopsis (Liu et al., 1996), maize (Horner, 1968) and Phaseolus (Wall, 1968, 1970) using sib-mating and selfing breeding designs. Rieseberg et al. (1996 a) compared the effectiveness of three different mating designs on the movement of genes across a species barrier in Helianthus. Mating designs that employed sib-mating early in the hybridization process resulted in a close to twofold increase in the total length of introgressed fragments per individual. Haghighi & Ascher (1988) were able to generate fertile intermediate hybrids between Phaseolus vulgaris and P. acutifolius by congruity backcrossing, suggesting that this approach may be particularly useful in divergent species crosses. Two conclusions can be drawn from these studies. First, the genetic architecture of the reproductive isolation plays a critical role in controlling patterns of introgression. Thus, the successful movement of an allele across a species barrier will depend both on overall genome architecture and on the genomic location and linkage relationships of the allele in question. Second, recurrent backcrossing is unlikely to be the mating design of choice for breeding programmes, and successful introgression in breeding populations and in natural hybrid zones probably requires a more diverse history of matings. 6. Hybrid speciation This section discusses several studies that both exemplify the utility of hybrid speciation experiments and suggest the most fruitful directions for future study ; see Rieseberg (1997) for a detailed review of experimental studies of hybrid speciation. The most widely accepted model for homoploid hybrid speciation is the recombinational model already described. Briefly, the sorting of parental chromosomal and genic sterility factors in hybrid populations can, under appropriate conditions, lead to the formation of a hybrid neospecies that is homozygous for some combination of parental sterility factors. The new hybrid lineage would be fertile, but at least partially intersterile with both parental species. Factors that appear to play a critical role in recombinational speciation include : strong natural selection for the most fertile or viable hybrid segregants (Templeton, 1981 ; McCarthy, Asmussen & Anderson, 1995) ; rapid chromosomal evolution Printed from the C JO service for personal use only by... 608 L. H. Rieseberg and S. E. Carney (Shaw, Wilkinson & Coates, 1983 ; Rieseberg, Van Fossen & Desrochers, 1995 c) ; and the availability of habitat suitable for the establishment of hybrid neospecies (Templeton, 1981 ; Arnold, 1997). This discussion has assumed that the establishment of the hybrid neospecies will occur in sympatry with both parents. Although hybrid speciation must be initiated in sympatry, Charlesworth (1995) argues that this mode is most likely when ‘ a group of hybrid plants colonize a new locality and are by chance spatially or ecologically isolated from the parental species ’. Thus, hybrid founder events might be viewed as foci of speciation. The possibility that a hybrid derivative might be stabilized in parapatry or allopatry should not be seen as minimizing the importance of the development of reproductive barriers. As the hybrid derivative becomes established and expands its geographical distribution, it probably will come back in contact with its parents. Presumably, the existence of reproductive barriers will allow it to survive the challenge of sympatry. Hybrid speciation experiments are useful because they can test the feasibility of the recombinational model, as well as provide insights regarding the evolutionary conditions under which speciation is most likely. The first rigorous experimental study of hybrid speciation was conducted by Stebbins (1957), who crossed microspecies of two grass genera, Elymus and Sitantion. Most F individuals were " sterile and could not produce seed, but four plants produced a small number of seeds. In three cases, the F seeds were not useful because the offspring " had either recovered the morphology of their maternal parent, undergone polyploidization, or were sterile. However, a single seed from the fourth F appeared to result from a backcross toward E. " glaucus. This plant had a seed fertility of 30 % and was selfed for two generations. The resulting progeny were vigorous and had normal seed fertility (88–100 %). Moreover, crosses with the original E. glaucus parent indicated almost complete reproductive isolation ; pollen fertility in the progeny of these crosses was 0–3 %. These experiments not only verified the feasibility of the recombinational speciation model, but also indicated that the origin of homoploid hybrid species is likely to involve backcrosses when the F hybrids are highly sterile. " Backcross progeny are typically more easily generated and more fertile than self- or sib-crosses in early hybrid generations. A series of elegant studies involving hybrids of Gilia malioriG. modocensis also provide experimental validation of the recombinational model (Grant, 1966 a, b). The two species are selfing annual tetraploids with a relatively high chromosome number (2n l 36). The F hybrids are semi-sterile with " pollen and seed fertility of 2 and 0n007 %, respectively. Abnormal meiotic pairing suggests that this reduction in fertility is caused by chromosomal structural differences between the parental genomes. To generate fertile and meiotically normal hybrid lines, the most fertile and viable plants were artificially selected from each generation, thus augmenting natural selection on the same traits. Although early-generation plants were weak and partially sterile, vigour and fertility improved rapidly. By the F or F generation, full vigour, normal ) * chromosomal pairing and full fertility had been recovered in three hybrid lineages or branches. Branch I and branch III each possessed a new combination of morphological and cytogenetic features (Grant, 1966 a), whereas branch II reverted largely to the G. modocenis parent both morphologically and in terms of crossability (Grant, 1966 b). As in the case of Elymus, the two recombinant Gilia lineages were isolated strongly from their parents (4–18 % pollen fertility). This is concordant with theoretical expectations that the strength of genetic isolation between hybrid derivatives and their parents should be correlated strongly with barrier strength between the parents themselves (Grant, 1954). In the Elymus and Gilia studies, the experimentally synthesized hybrid lineages could not be compared with natural hybrid species from the same parental combinations, making it difficult to evaluate how closely the glasshouse experiments replicated the natural speciation process. However, Rieseberg et al. (1996 b) recently performed a similar experiment using the wild sunflower species H. annuus, H. anomalus and H. petiolaris, except that the experimental sunflower hybrid lineages were directly compared to H. anomalus, a wild species that also originated from H. annuusiH. petiolaris. To facilitate these comparisons, mapped molecular markers were used to assess precisely the genomic composition of the experimental hybrid lineages and to compare their genomes to H. anomalus. As was observed for Elymus and Gilia, the three experimental sunflower hybrid lineages recovered fertility in a small number of generations (Fig. 2). Comparison of the genomic composition of the natural (H. anomalus) and synthetic hybrid lineages revealed that all three synthetic hybrid lineages had converged to nearly identical gene combinations, and that this set of gene combinations was statistically concordant with that of H. anomalus (Fig. 3). Concordance in genomic composition between the synthetic and natural hybrid lineages suggests that deterministic forces such as selection, rather than stochastic forces, largely govern the formation of ‘ recombinational ’ species. Because the synthetic hybrid lineages were generated in the greenhouse, fertility selection probably played a greater role than ecological selection in shaping hybrid genomic composition. Congruence in genomic composition also implies that the genomic structure and composition of hybrid species may essentially be fixed Printed from the C JO service for personal use only by... Plant hybridization 609 100 90 Pollen fertility (%) 80 70 60 50 40 30 Lineage I 20 Lineage II 10 Lineage III 0 1 0 3 2 Generation 4 5 Figure 2. Mean pollen fertility in three synthetic hybrid lineages between Helianthus annuus and H. petiolaris : lineage I, P-F -BC -BC -F -F ; lineage II, P-F -F -BC " " # # $ " # " BC -F ; and lineage III, P-F -F -F -BC -BC . For each # $ " # $ " # line, 100 pollen grains from each of 20 plants per generation were tested by viability staining. Standard error bars are shown. 135 225 296 A4 261 tr1 215 226 254 R T rp1 D4 487 Q 199 313 B6 443 227 nf 73 239 103 Synthesized H. anomalus hybrids S 295 A15 H. petiolaris markers or linkage blocks H. annuus markers or linkage blocks 471 40 cM Figure 3. Genomic composition of ancient and experimental hybrid sunflower lineages for selected linkage groups. Letters at the left of each linkage group designate linkage blocks in the ancient hybrid, Helianthus anomalus, and indicate their relationship to homologous linkages in the parental species, H. annuus and H. petiolaris. The distribution of parental markers within the H. anomalus genome is indicated by gray or black bars within linkage groups, whereas bars at the left of each linkage group indicate the distribution of parental genomic regions in the synthesized hybrids. Adapted from Rieseberg et al., 1996 b. after a small number of generations of hybridization and remain relatively static thereafter. Although congruence between the genomes of the synthetic hybrid lineages and H. anomalus is quite high (rs l 0n68 ; P 0n0001), substantial differences remain. Because the synthetic hybrid lineages were exposed to fertility selection alone, it is possible that the observed differences in genomic composition are a result of chromosomal segments that affect morphological or ecological traits rather than fertility. Maps are currently being generated for H. deserticola and H. paradoxus, two other species that appear to have originated in the wild following hybridization between H. annuus and H. petiolaris (Rieseberg, 1991). Interspecific gene combinations shared by the three natural hybrid species and the synthetic hybrid lineages should be attributable to fertility selection, whereas those exclusive to the natural hybrid species will provide evidence for habitat selection. Although substantial progress has been made in studying homoploid hybrid speciation, much remains to be understood. A major gap in our knowledge relates to the origin of hybrid species that are isolated from their parents by premating barriers. Empirical data indicate that species have arisen in this manner (e.g. Arnold, Hamrick & Bennett, 1990, Wang et al., 1990 ; Arnold et al., 1993 ; Wang & Szmidt, 1994 ; Sang, Crawford & Stuessy, 1995 ; Wolfe, Xiang & Kephart, 1997), but experimental and theoretical studies have focused on the strict recombinational model, which involves the sorting of genic and chromosomal sterility factors. Because hybrid speciation is both reticulate and rapid, it is particularly amenable to experimental manipulation and replication. Thus, it should be feasible experimentally to synthesize new homoploid hybrid species that are isolated by premating barriers only. The design of these experimental studies could be improved by theoretical studies that identify parameters critical to this mode. . By manipulating naturally hybridizing species in the field, scientists have been able to study a number of habitat-dependent phenomena of crucial importance to our understanding of hybridization and its ramifications. These studies have provided insights into the requirements for the formation and establishment of hybrids and the effects of habitat and pollinator-mediated selection on hybrid-zone structure and dynamics. There have been relatively few studies in which hybrid zones are experimentally manipulated. It is possible that this is because scientists are wary about interfering with the natural processes occurring within hybrid zones. However, these experiments provide crucial data involving the formation, main- Printed from the C JO service for personal use only by... 610 L. H. Rieseberg and S. E. Carney tenance and evolutionary outcome of hybrid zones that cannot be obtained in any other way. For example, reciprocal transplant experiments are the only truly accurate method for obtaining environment-dependent fitness data of parental and hybrid individuals. More experimental manipulations of hybrid zones are needed. Perhaps scientists should focus their efforts on groups where a number of independent hybrid zones exist. Thus, disturbance of individual hybrid zones for scientific study would not eliminate the existence of ‘ pristine ’ hybrid zones. Additionally, in some instances, experiments can be engineered to minimize interference with natural processes in the hybrid zone. For example, in the case of reciprocal transplants, anthers of transplanted individuals can be removed to prevent the introduction of foreign pollen, and plants can be harvested before their seeds are dispersed into the natural population. 1. Hybrid-zone formation Although many hybrid zones have been described in plants, little is known about how these zones form. Experimental manipulation is an excellent tool for investigating the early stages of hybrid-zone formation, as has been illustrated by studies of Louisiana irises. Genetic marker surveys in several Louisiana iris hybrid zones failed to identify F hybrids (Arnold " et al., 1990 ; Arnold, Buckner & Robinson, 1991 ; Arnold, 1993 b), leading (Arnold, 1993 b) to the suggestion that F hybrid formation was rare. To " investigate the rate of hybridization in nature, the initial stages of hybrid-zone formation were simulated by introducing a block of I. hexagona rhizomes into a natural population of I. fulva (Arnold, Hamrick & Bennett 1993). For each species, a subset of fruits derived from flowers that were open on days when both species were blooming was collected, and a total of 710 seeds were genotyped. Only seven seeds, 1 %, were hybrids (Arnold et al., 1993), and all of them had I. hexagona mothers. This bias could be because of differences in the strength of reproductive barriers in the two species (Emms, Hodges & Arnold, 1996) or because approximately three times as many I. fulva flowers were produced as I. hexagona flowers. This would lead to pollen loads consisting mostly of heterospecific pollen being deposited on I. hexagona stigmas. Arnold and Hamrick continued to monitor the population for 3 yr, and 5000 seeds were genotyped with the percentage of hybrid seeds remaining below 1 % (Hamrick & Arnold, unpublished). The results of this study support the hypothesis that F hybrids " rarely form in the Louisiana irises. A hypothesis that F formation is rate might seem " unlikely because of the large number of advancedgeneration hybrid irises found in nature. However, this conflict is resolved if later-generation hybrids are formed more easily than F hybrids. Hodges, " Burke and Arnold (1996) investigated how a hybrid zone might be formed once an initial hybridization event has occurred by introducing F hybrids " produced in the glasshouse into the population already described. Seeds from F , I. fulva and I. " hexagona plants were genotyped using two diagnostic alozyme loci. A maximum likelihood programme was used to estimate the frequency of seeds from hybrid mothers that were F , Bf and Bh hybrids (Bf # and Bh, backcrossed to I. fulva or to I. hexagona, respectively). Five of the 68 fruits collected from parental plants contained hybrids. The three fruits from I. hexagona had seed genotypes consistent with Bh hybrids, and the two from I. fulva had a mixture of I. fulva and Bf seeds. F plants located near the " I. hexagona plot produced 95 % Bh seeds and 5 % Bf seeds. Those near I. fulva plants produced 90 % F seeds and 10 % Bh seeds, as estimated by the # maximum likelihood model. In I. hexagona, backcross formation was 10 times more likely than the formation of F hybrids and, in I. fulva, backcrosses " were formed 60 times more frequently than F " hybrids. The introduced F hybrids also contributed " to the formation of backcrossed and F hybrids. # Thus, once an F hybrid is formed and established in " nature, it can lead to the formation of advancedgeneration hybrids. However, the initial formation of F hybrids may act as a bottleneck in the formation " of hybrid zones in this group. 2. Pollinator-mediated selection Observations and experiments involving pollinator preferences and visitation in hybrid zones supply information on interspecific gene flow and the strength and mode of selection on reproductive traits. Patterns of pollinator visitation in animalpollinated plants may limit gene flow among those plants (e.g. through positive assortative mating). Interspecific pollinator foraging, and thus interspecific pollen transfer, may occur only rarely. However, in hybrid zones, hybrids can act as a bridge for gene flow between the parental species if they share pollinators. Alternatively, hybrids could potentially have pollination syndromes that differ from either parent, isolating them from the parental species. Pollinators are a major source of selection on floral traits in angiosperms, since appropriate pollinator visitation greatly affects reproductive success (e.g. Waser & Price, 1983 ; Stanton, Snow & Handel, 1986 ; Nilsson, 1988 ; Campbell et al., 1991, Campbell, Waser & Price, 1996). Pollinator choice is determined by the presence of rewards (e.g. nectar, pollen, or scents), and a variety of floral traits can be used as cues to the value of the reward in a given plant. Flower colour, size, or shape characters may Printed from the C JO service for personal use only by... Plant hybridization be associated with larger volumes or higher quality rewards. Additionally, the ease of obtaining the reward in question will affect a pollinator’s choice of flowers. For example, hummingbirds are known to prefer flowers with wider corolla tubes, which enable them to obtain nectar more easily, than flowers with narrow corolla tubes, which restrict their entry (Waser, 1983 ; Campbell et al., 1991, 1996). When pollinators associate specific floral traits with increased rewards, they are more likely to forage on plants with those traits, increasing the reproductive success and thus fitness of individuals that possess the traits in question. The most common pollinator of a given plant taxon will impose the strongest selection on its floral characters. Campbell et al. (1997) investigated pollinatormediated selection in an Ipomopsis aggregatai I. tenuituba hybrid zone. The most frequent pollinators of these species and their hybrids are broadtailed and rufus hummingbirds and, more rarely, hawkmoths. Ipomopsis aggregata has red, trumpetshaped flowers characteristic of hummingbird-pollinated species, while I. tenuituba has longer, narrower corollas that range from white to pink or pale violet. The latter is more characteristic of hawkmoth pollination. The species are separated altitudinally, with hybrid zones forming in intermediate altitudes. To observe pollinator visitation, Campbell et al. (1997) used artificial arrays of pure parental and hybrid plants as well as a mixed array at a lowaltitude site within the range of I. aggregata ; mixed arrays within the hybrid zone ; and natural parental and hybrid populations. Corolla length, corolla width and flower colour were chosen for study because they influence pollinator visitation and vary across the hybrid zone. Selection differentials and gradients were calculated for the three focal traits using pollinator visitation rates as a fitness component. As expected, hummingbirds were found to prefer the red, wide corollas of I. aggregata plants to hybrids and I. tenuituba. These pollinators appear to impose directional selection for I. aggregata-like flowers in the hybrid zone. Hawkmoths were only present at the study site in statistically relevant numbers during 1 yr of the 3-yr study (and a total of 3 yr out of the last 20). However, when present, they preferred plants with narrow corolla tubes, resulting in disruptive selection on floral traits during those years. A recent study of pollinator-mediated selection has focused on the Louisiana irises (Emms & Arnold, unpublished). Mixed experimental arrays of Iris fulva, I. hexagona and their F hybrids were observed " in a population of pure I. hexagona and at the I. fulva site into which I. hexagona and F hybrids were " introduced. Pollinators of these species include rubythroated hummingbirds and queen and worker bumble bees. Pollinator preference was measured as approach frequency, the percentage of approaches 611 that led to a legitimate visit (acceptance rate), and the total number of legitimate visits to each flower type (overall performance). Hummingbirds were the most common pollinator at the I. fulva site, and they preferred I. fulva flowers. At the I. hexagona site, queen bumble bees were the most common pollinator, and they preferred I. hexagona and hybrids over I. fulva. Worker bees, the least common pollinator at both sites, preferred F flowers. Ap" proach and acceptance rates were used to infer the types of cues used by pollinators in selecting flowers for visitation. Preferences of queen bees appeared to be a result of differences in long-distance attractiveness of flowers, because they made more approaches to I. hexagona and hybrid flowers than to I. fulva, but their acceptance rates did not differ. Worker bees approached the three flower types with equal frequency, but accepted few I. hexagona flowers, suggesting that they were rejecting them based on close-range cues. Hummingbirds seemed to use a mixture of short- and long-range cues, because they exhibited variation in approach and acceptance rates at both sites. The following were measured and compared among the species and their hybrids : petal, anther, nectary and stigma length ; anther exsertion ; nectar volume ; and nectar concentration. For most characters, I. fulva is smaller than I. hexagona with F " hybrids intermediate. Nectary length, nectar volume and nectar concentration did not differ significantly among the three flower types, except that F hybrids " had significantly more-concentrated nectar. Therefore, the authors suggest that differences in floral preferences are most likely related to foraging efficiency on flowers of different sizes. Colour, the most obvious long-distance cue, is probably used to indicate differences in efficiency. Close-range cues may be based on an assessment of rewards prior to acceptance or on scent marks left by previous visitors. To infer the effect of pollinator preferences and movement patterns on hybridization frequencies, Emms and Arnold (unpublished) analysed intertaxon patterns of hummingbird and queen bumble bee movements. They compared expected movement frequencies based on overall pollinator preferences with observed movements. Combining data from both pollinator types revealed a pattern of overrepresentation of parental–F movements and an " under-representation of heterospecific and F –F " " movements. These data agree with the results of the study by Hodges et al. (1996), which showed that once an F is established, the formation of back" crossed individuals is much more frequent than the initial hybridization event. These manipulative studies have provided insights into the selection pressures exerted by pollinators in hybrid zones. Because pollinator-mediated selection acts on floral characters, it has implications for the Printed from the C JO service for personal use only by... 612 L. H. Rieseberg and S. E. Carney strength of reproductive barriers and speciation. Additionally, gene flow is determined by pollinator movement patterns in animal-pollinated plants. Thus, observing pollinator movements provides information about the fitness of different phenotypes in hybrid zones. 3. Habitat selection Because plants are so intimately associated with their environment, the importance of ecological factors in hybrid establishment and mating success is paramount. As discussed earlier, Ko$ lreuter was the first to recognize the role of ecology, noting the importance of habitat disturbance in creating opportunities for spontaneous hybridization. This observation formed the basis for Anderson’s (1948) classic paper on hybridization of the habitat. Anderson’s argument was simple. Habitat disturbance creates an array of different habitats that are best exploited by the extraordinary diversity of genotypes created by hybridization. Disturbance also leads to the breakdown of premating reproductive barriers, thus increasing hybridization frequency. Anderson’s conclusions appear to be corroborated by empirical evidence. Hybridization is strongly associated with disturbance. Disturbance often provides corridors for the movement of species and leads to sympatry and hybridization between otherwise allopatric species (Levin et al., 1996). When disturbance is reduced, the number of hybrids at the site declines (Heiser, 1979). Other evidence for the importance of habitat includes the genotype–habitat associations often reported for hybrid swarms (Cruzan & Arnold, 1993, 1994) and the mosaic nature of most plant hybrid zones (Rieseberg & Ellstrand, 1993). However, these correlative studies are problematic because each taxon need not be found in the habitat in which it is most fit. For example, one taxon could be outcompeted from its optimal habitat and forced to live in a less ideal habitat. Another possibility is that correlations between genotypes and environments could be explained by history (Barton & Hewitt, 1985). Reciprocal transplant experiments are extremely useful for identifying the ecological preferences of plants, and they have been used to assess the effects of varied environments on fitness for nearly sixty years (e.g., Clausen, Keck & Hiesey, 1940, 1948 ; Bradshaw, 1960 ; Briggs, 1962 ; Barton, 1980 ; Antonovics & Primack, 1982 ; Potts, 1985 ; Helenurm, 1998). This experimental approach allows the comparison of fitness of taxa or adaptation of genotypes to a number of habitats, but it has been used infrequently in the study of hybrid zones. Wang et al. (1997) used a reciprocal transplant study to determine whether endogenous or exogenous (i.e. habitat-independent or -dependent) selection stabilizes a big sagebrush hybrid zone (Artemesia tridentata ssp. tridentataiA. tridentata ssp. vaseyana). After planting seeds and 1-yr-old seedlings of each subspecies and their hybrids into the native habit of each taxon, Wang et al., 1997 assessed survivorship, size and reproduction. Each taxon did significantly better than the others in its native habitat, demonstrating that selection on big sagebrush is habitat-dependent, and that each taxon occurs in the habitat in which it is most fit. Similarly, Levin & Schmidt (1985) transplanted seeds of Phlox drummondii ssp. drummondii, P. drummondii ssp. mcallisteri and their hybrids into the habitats of each and monitored germination, survivorship, fecundity and finite rate of increase. In contrast to the previous study, they found no significant differences between plant types in the different habitats. Therefore, it is unlikely that extrinsic selection maintains the Phlox hybrid zone ; it is probably maintained by restricted gene flow. Emms & Arnold (1997) used rhizomes rather than seeds of Iris fulva, I. hexagona and their F and F " # hybrids in their reciprocal transplant study, because previous experiments involving transplantation of seeds into natural populations were largely unsuccessful (M. Arnold, pers. comm.). The rhizomes were transplanted into pure parental and hybrid sites, and survival, growth and clonal and sexual reproduction were studied. Hybrid leaf production exceeded that of the parental species at all sites, and hybrid rhizome production was greater than that of both parents in all but one hybrid site. Hybrids were intermediate in the percentage of flowering plants. Fitness measures in this study seem to be affected largely by habitat-independent hybrid vigour. In addition, there are problems with estimating fitness of long-lived perennial plants from data obtained in one or a few years. These reciprocal transplant experiments have increased our understanding of the role of habitat selection in the genetic structure and maintenance of plant hybrid zones. However, more studies of this type are needed before generalizations are possible. . Much of what we know about the biology of different classes of hybrids comes from the study of experimental hybrids and experimental manipulations of hybrid zones already described. However, substantial knowledge of hybrid behaviour has also come from studies in which natural hybrid zones themselves served as the experimental system. This approach has been particularly useful for studying the response or resistance of different kinds of hybrids to pathogens or herbivores, as well as for studying the mating behaviour of different hybrid Printed from the C JO service for personal use only by... Plant hybridization classes. In these studies, hybrid genotypes have been classified using morphological or molecular markers or a combination of both. Our discussion of the biology of different classes of hybrids attempts to incorporate information from all three kinds of studies. 1. Character expression The expression of morphological, chemical, and molecular characters in different classes of hybrids has been summarized by Rieseberg & Ellstrand (1993). This discussion highlights the most interesting results from this summary and discusses both the implications of these data for identifying hybrids and the possible creative role of hybridization in the origin of evolutionary novelties. (a) Morphological characters. Rieseberg & Ellstrand (1993) compiled a list of 46 studies that report morphological character expression in hybrids. The list included 32 examples of character expression in F hybrids, nine examples from later-generation " hybrids, and four examples of homoploid hybrid speciation. For each hybrid, the number of intermediate, parental and extreme or transgressive characters was determined. The studies analysed were not parallel in terms of their treatment of hybrids. For example, several of the data sets were taken from cladistic studies, where quantitative variation may be partitioned into discrete classes. Thus, partially intermediate character states might have been scored as parental because of the lack of an intermediate state for that character (e.g. McDade, 1990). In other instances, means and\or ranges of values were given for hybrids rather than absolute values. Quantitative traits were emphasized by some studies, particularly those interested in hybrid identification or morphological genetics, whereas other studies, particularly phylogenetic ones, tended to emphasize qualitative characters. Finally, some studies employed floral characters only, others emphasized vegetative characters, and still others reported on both floral and vegetative characters. Given these caveats, Rieseberg & Ellstrand (1993) recommended that the results from this compilation be interpreted with caution. Nonetheless, analysis of these studies revealed several surprising tendencies. First, F hybrids were " shown to be a mosaic of both parental (45n2 %) and intermediate (44n7 %) morphological characters rather than just intermediate ones. A possible explanation for the high proportion of parental characters expressed in hybrids is that many morphological traits that differentiate closely related species display dominant inheritance patterns (Hilu, 1983 ; Gottlieb, 1984). Thus, the expression of parental or intermediate character states in hybrids will depend on the nature of the genetic control of a 613 particular character, as well as interactions with the environment. A second important finding from this survey was the high frequency of transgressive or novel characters observed in hybrids. Over 10 % of morphological characters in F hybrids were transgressive, " and over 30 % were transgressive in later-generation hybrids. The expression of transgressive characters was not restricted to a few interspecific combinations, but rather seems to be a predictable feature of most first- (64 %) and later-generation (89 %) hybrids. Several explanations have been offered to account for the expression of novel or transgressive characters in hybrids, including : an increased mutation rate in hybrids ; the complementary action of new combinations of normal alleles ; the placement of unexpressed (or expressed) alleles in a new genetic background (epistasis), as has been suggested to explain novel floral pigmentation in Clarkia gracilis (Gottlieb & Ford, 1988) ; the fixation of recessive alleles present in the heterozygous form in the parents (dominance) ; reduced developmental stability (Wagner, 1962 ; Levin, 1970 ; Grant, 1975) ; and simple heterosis (overdominance). However, we are aware of only one study that provides a definitive genetic basis for transgressive segregation. DeVicente & Tanksley (1993) performed a QTL analysis of eight transgressive characters in an interspecific cross. The major cause of transgression was the complementary action of genes from the two parental species. No evidence was observed for epistasis, but this might be because of the low power of the data analysis method used. However, overdominance was implicated as a secondary cause of transgression. Surprisingly, the more similar the phenotype of the parents, the more likely transgressive segregation was to be observed at that trait. The high frequency of transgressive segregation supports the view of hybridization as a source of variability upon which selection can act. From a systematic perspective, however, the unpredictability of hybrid character expression diminishes the utility of morphological characters for hybrid identification and suggests that some traits may be more predictive of hybridity than others. (b) Chemical characters. Tabulation of results from 24 studies of secondary compound expression in hybrids from 22 plant genera revealed that the majority of compounds were expressed additively in both F hybrids (67n7 %) and hybrid species (53n6 %). " The lack of complete complementation for chemical compounds in some taxa seems to result from differences in genetic control, whereas in other cases it appears to result from polymorphism of the parental loci that control the biosynthesis of secondary compounds (Harborne & Turner, 1984). Thus, a parent heterozygous for the controlling Printed from the C JO service for personal use only by... 614 L. H. Rieseberg and S. E. Carney locus in question may yield progeny with or without that particular compound. Alternatively, the interaction of two disparate genomes in the hybrid may disrupt biosynthesis, resulting in the loss of compounds. Other deviations from a strictly complementary pattern of character expression involve the production of novel compounds in many plant hybrids. Indeed, novel compounds were observed in F " hybrids of 11 of the 24 genera listed (46 %). However, these compounds were relatively infrequent compared with the total number of compounds assayed in the hybrids and their parents (about 4 %). As might be predicted, the frequency of novel compounds increased in later-generation hybrids (7n8 %) and hybrid species (17n9 %). In almost all cases, novel compounds produced in hybrids can be explained relatively easily from a genetic standpoint. One explanation is that the enzymes necessary for the formation of the compounds already exist in one of the parents, but are not expressed until the parental genomes are combined in the hybrids. Alternatively, the new compounds require the additive effects of both parental sets of enzymes on the same basic chemical skeleton (Stace, 1975). Initial interests in chemical character expression focused on their utility as markers for identifying hybrids. However, with the advent of more powerful and efficient macromolecular tools, interest in secondary compounds in hybrids has shifted to their role in herbivore and pathogen defence. For example, the loss of parental compounds may partly explain the observations that many hybrid zones act as pest sinks. It is also possible that the production of novel secondary chemicals in hybrids might stimulate the evolution of pathogen or herbivore genotypes that are tolerant of the new compounds. (c) Molecular characters. The expression of molecular characters in hybrids is inherently less interesting than that of morphological or chemical characters because in most instances genotypes (i.e. DNA polymorphisms) are directly assayed and few epigenetic effects have to be considered. Molecular markers generally follow Mendelian laws of inheritance, although biases are often observed because of selection. There are some exceptions to these general rules. Null alleles are observed infrequently in isozyme assays and in PCR-mediated assays of DNA markers such as microsatellites (Gottlieb, 1981 ; Pemberton et al., 1995). Hybridization has also been reported to generate novel isozyme alleles (Woodruff, 1989), but this result may simply be an artifact of sampling error. Restriction fragment patterns are known to change because of different patterns of methylation in hybrids (Jablonka & Lamb, 1995 ; Song et al., 1995), and genes introduced through sexual hybrid- ization or genetic engineering are often inactivated (Wallace & Landbridge, 1971 ; Martin-Tanguy et al., 1996). Perhaps the most serious problems arise from analyses of repetitive sequences in which concerted evolution can lead to the replacement or loss of alleles from one of the parental species (Arnold, Contreras & Shaw, 1988). Other problems relate to spurious band formation or artifactual variation in marker systems that employ arbitrary primers (Riedy, Hamilton & Aquadro, 1992), but these should be no more common in hybrids than in intraspecific progenies. 2. The fitness of different classes of hybrids Any discussion of the role of hybridization in evolution must address the issue of hybrid fitness (Arnold & Hodges, 1995). If hybrids were uniformly less fit than the parental species, the role of hybridization in adaptive evolution would be minimal : species would be unlikely to merge as a result of hybridization ; speciation by reinforcement would increase in likelihood ; tension (Barton & Hewitt, 1985) and mosaic (Harrison, 1986) hybrid-zone models would be validated ; and outbreeding depression would be a greater threat to hybridizing rare species than genetic assimilation. However, if hybrids were uniformly more fit than either parental species, regardless of ecology, stable hybrid zones would not exist, sympatric or parapatric speciation would not occur and species merging would be the primary outcome of hybridization events. The true situation is much more complex. Hybrid genotypes are highly heterogeneous with regard to fitness, both within and between generations. As discussed earlier, ecological factors, particularly habitat may influence fitness relationships. Moreover, estimates of lifetime fitness, which are crucial to discussions of this topic, are difficult to obtain, particularly for long-lived organisms. Thus, sweeping conclusions about hybrid fitness are difficult to make, but some generalizations are possible. (a) The importance of variance. In discussing hybrid fitness, it is important to distinguish between average fitness of a genealogical class of hybrids and the fitnesses of particular genotypes. The average viability and fertility of early hybrid generations (e.g. F and F hybrids) is predicted to be lower than that # $ of the parental species because of the break-up of adaptive gene combinations (Dobzhansky, 1937). This is generally what is found, particularly for species with strong postmating reproductive barriers. Well-characterized examples include Gilia (Grant, 1966 a), Helianthus (Heiser, 1947), Layia (Clausen, 1951), Oryza (Li et al., 1997) and Zauschneria (Clausen, 1951). This makes sense, because hybridizing species would merge if the Printed from the C JO service for personal use only by... Plant hybridization average fitness of the early-generation hybrids was greater than that of the parents. The fact that most plant hybrid zones are limited in extent also implies that early-generation hybrids are on average less fit than their parents (Coyne, 1996), at least in parental habitats. Although several recent studies have described the replacement of populations of rare taxa by hybrid swarms (Brochmann, 1984 ; Rieseberg & Gerber, 1995 ; Levin et al., 1996), this appears to be because of genetic swamping by a numerically larger congener rather than by higher average hybrid fitness. Low average fitness of a particular class or classes of hybrids does not rule out the possibility that certain hybrid genotypes may be as fit or more fit than either parental species, particularly latergeneration hybrid segregates. Substantial empirical evidence supports this hypothesis. First, heterosis is commonly observed in hybrids and has often been shown to result in increased vigour and fecundity. Although the effects of heterosis are often partly masked by disharmonious interspecific genomic interactions in early-generation hybrids, strong fertility and viability selection will favour the elimination of negative gene combinations and the maintenance of heterosis. Thus, after just a few generations of selection, fertile hybrid genotypes can be generated that sometimes outperform both parental species. Second, studies that describe fertility, viability, or other fitness parameters in hybrids almost invariably report the presence of a small fraction of hybrid genotypes that are as fit or fitter than parental individuals, even if the hybrids on average exhibit reduced fitness (Heiser, 1947 ; Valentine, 1947 ; Grant, 1966 a). Third, significant genotype–habitat associations are often reported for hybrid swarms (Stebbins & Daly, 1961 ; Potts & Reid, 1985 ; Cruzan & Arnold, 1993, 1994 ; Arnold, 1997). Presumably, this indicates that a selective advantage accrues for certain hybrid genotypes when found in favourable habitats, although these correlations could also result from historical factors (Barton & Hewitt, 1985). The preceding discussion has assumed that average hybrid fitness in early hybrid generations is less than that of parental species – an assumption that has substantial empirical support. However, there are several examples for which the average fitness of a particular class or classes of hybrids appears to be equivalent or to exceed that of the parents, at least for those fitness parameters measured. For example, Artemisia hybrids were more developmentally stable and had higher seed germination and growth rates than either parent (Freeman et al., 1995 ; Graham, Freeman & McArthur, 1995 ; Wang et al., 1997) ; Iris (Emms & Arnold, 1997) and Oryza (Langevin, Clay & Grace, 1990) hybrids exhibited higher vegetative growth rates than either parental species ; and no differences 615 in fitness were reported between hybrid and parental individuals in a Phlox hybrid zone (Levin & Schmidt, 1985). Unfortunately, none of these studies measured lifetime fitness, so whether they represent valid exceptions to the general rule of reduced average hybrid fitness is unclear. Vegetative growth rates are particularly problematic as a general indicator of hybrid fitness in annuals or short-lived perennials, since hybrid vigour is often observed in even highly sterile plants. In clonal plants such as the Louisiana irises or in long-lived organisms, vegetative growth rates become a much more critical component of hybrid fitness. These results should not be viewed as a challenge to modern speciation theory (Dobzhansky, 1937). First, in the absence of lifetime fitnesses, the fitness measurements reviewed here must be seen as preliminary, albeit exciting. Second, enhanced hybrid fitness should be plausible if postmating isolating barriers are weak and the hybrids occupy a novel habitat (Endler, 1977 ; Moore, 1977), or if environmental conditions change (Anderson, 1948 ; Grant & Grant, 1993 ; Arnold, 1997). These conditions are common in hybridizing plant species, so rank-order fitness estimates that occasionally favour hybrids should be expected (Rieseberg, 1997). (b) Estimating hybrid fitness. An important caveat in all of these discussions relates to the reliability or relevance of most of the fitness estimates provided in the literature to date. One problem is that it is difficult to obtain lifetime fitness estimates for long-lived organisms. Another issue relates to the pooling of data from heterogeneous hybrid genotypes. It is easy to see how pooling data from several hybrid generations could lead to erroneous conclusions (Arnold & Hodges, 1995). However, even pooling individuals from the same hybrid class can lead to faulty conclusions because the variance in fitness is probably more critical than the mean. Another problem is that most experimental studies of hybrid fitness have been restricted to F or F hybrids or backcrosses, " # yet the fitness of stabilized hybrid segregants is of greater importance in terms of predicting the evolutionary consequences of hybridization. Finally, very few measurements of hybrid fitness have been conducted under natural conditions (Levin et al., 1996), even though fitness relationships among plant hybrids appear to be habitat dependent. Conducting experiments that correct these flaws will be difficult or impossible in long-lived plants, but should be feasible in annuals or short-lived perennials. A possible alternative to classic transplant experiments would be the use of molecularmarker-based parentage studies. By tracking all genotypes in a hybrid population over multiple generations with a large number of molecular markers, it should be possible to estimate parental success or true fitness of all genotypes in a popu- Printed from the C JO service for personal use only by... 616 L. H. Rieseberg and S. E. Carney lation. Long-term experiments over several generations will be needed to determine what fraction of observed fitness differences among different genotypic categories is caused by year-to-year environmental variation. These data will be most informative if combined with detailed habitat surveys so that interactions between genotype and habitat can be assessed. Data from experiments such as those just described will probably be required fully to understand hybrid fitness and to make reliable inferences regarding the role of hybridization in adaptive evolution and speciation. 3. Interactions with parasites and herbivores Over the past decade, considerable interest has developed concerning the interactions of hybrids with herbivores and parasites. This area of study was initiated by Sage et al. (1986), who reported higher levels of nematode and cestode parasites in hybrid mice than in parental species individuals. The high parasite loads found in the hybrids were attributed to the disruption of genetically based resistance mechanisms. This was followed by a report of higher aphid herbivore loads in cottonwood hybrids (Whitham, 1989). Whitham suggested that susceptible hybrids might act as a sink for herbivore pests, perhaps even drawing herbivores away from the more resistant parental individuals. However, it soon became apparent that the response of herbivores and pathogens to hybrids could vary considerably and was partly dependent on the hybrid generation or even hybrid genotype being analysed (Boecklen & Spellenberg, 1990 ; Paige, Capman & Jennetten, 1991 ; Aguilar & Boecklen, 1992 ; Fritz, NicholsOrians & Brunsfeld, 1994 ; Whitham, Morrow & Potts, 1994). For example, Boecklen & Spellenberg (1990) found that oak hybrids supported lower densities of herbivores than did parental individuals, possibly suggesting that the hybrids exceed pure parental individuals in resistance. Another study revealed that the response of different herbivores or pathogens to the same hybrids could be strikingly different (e.g. Fritz et al., 1994). This led to the formation of a more explicit theoretical framework for studying the interactions of hybrids and their pests (Aguilar & Boecklen, 1992 ; Frit et al., 1994 ; Strauss, 1994), as well as to a recent review that summarized data from different studies on levels of herbivory and parasitism in hybrid zones (Strauss, 1994). In a survey of 19 studies comprising 17 hybrid zones and a wide variety of herbivores, Strauss (1994) reported the following : 32 cases in which hybrids exhibited greater abundance of herbivores or parasites than either parental species ; 24 cases in which hybrids displayed intermediate levels of pest abundance ; six cases in which pest loads were reduced in the hybrids relative to either parental species ; and 27 cases in which the hybrids did not differ significantly from either parent in pest load. Although these studies were not parallel in terms of methodology or the kinds of hybrid genotypes analysed, the summary does suggest that the response of herbivores to hybrids will be hard to predict across hybrid zones and that increased susceptibility is probably more likely than increased resistance. Studies published after the Strauss (1994) review have largely confirmed these conclusions (e.g. Hanhima$ ki, Senn & Haukioja, 1994 ; Morrow et al., 1994 ; Whitham et al., 1994 ; Christensen, Whitham & Keim, 1995 ; Gange, 1995 ; Fritz et al., 1996 ; Gaylor, Preszler & Boecklin, 1996). Both genetic and ecological hypotheses have been advanced to explain different resistance patterns in hybrids. Fritz et al. (1994) have described a series of explicit hypotheses based on inheritance of resistance in F hybrids : the additive hypothesis, the domi" nance hypothesis, the hybrid-susceptibility hypothesis, the hybrid-resistance hypothesis and the no differences hypothesis. If F hybrids exhibit re" sistance levels intermediate with those of the parental species, this would suggest that hybrid resistance is caused by additive inheritance of the resistance trait from both parents. If hybrid resistance is similar to that of one parent but not of the other, this might imply dominant inheritance of resistance traits. If hybrids are less resistant than either parent, perhaps because of the disruption of resistance mechanisms, the hybrid-susceptibility hypothesis would be supported. Alternatively, heterosis in hybrids could lead to increased resistance – a result that would support the hybrid-resistance hypothesis. Finally, hybrids may be variable in pest resistance and not differ significantly from their parent. Studies of the inheritance and expression of secondary compounds that contribute to resistance support a genetic mechanism for at least some of the differences in pest abundance on hybrids. As noted earlier, a slim majority of secondary compounds are inherited in an additive or complementary manner (Rieseberg & Ellstrand, 1993 ; Orians & Fritz, 1995), which is concordant with the large number of cases in which pest abundance appears to be intermediate between the two parental species. In many other cases, dominant inheritance of defensive chemicals has been reported (e.g. Huesing et al., 1989 ; Levy & Milo, 1991), which may explain the many instances in which hybrid resistance is similar to that of one parent but differs significantly from that of the other. The many examples of hybrid susceptibility are more difficult to explain, because most hybrids do not appear to differ drastically from their parents in the expression of secondary compounds. Although the patterns of pest abundance on hybrid and parental individuals can be attributed to variation in the inheritance of resistance traits, ecological Printed from the C JO service for personal use only by... Plant hybridization factors may play a role as well. For example, Floate, Whitham & Keim (1994) demonstrated that the increase of beetles in a popular hybrid zone resulted in part from the extended time that young leaves were available in hybrid zones relative to their availability in pure populations. In a similar vein, Paige & Capman (1993) showed that both genotype and tree location were required to explain differences in aphid survivorship patterns in the same poplar hybrid zone. Thus, both ecological and genetic factors appear to play an important role in determining pathogen and herbivore loads in natural hybrid zones. Another confounding factor in these studies has been the varied response of herbivores to different hybrid genotypes from the same and different hybrid generations. This has placed a premium on correct hybrid identification, precipitating a debate concerning the most appropriate tools for classifying hybrids. Reliance on morphological characters has been criticized because of their plasticity (Paige & Capman, 1993) and unpredictable expression (Rieseberg & Ellstrand, 1993). Although the expression of molecular markers is largely unaffected by environmental factors, hybrid and parental genotypic classes often differ minimally in terms of expected marker proportions. Thus, extremely large numbers of molecular markers may be required to distinguish between them. A more fundamental difficulty relates to the potential for selection to bias marker proportions in hybrids, thus leading to faulty genealogical assignments (Rieseberg et al., 1995 b, Rieseberg & Linder, 1999). As a result, Rieseberg & Linder (1999) recommended that hybrids be classified according to genetic relatedness or genetic admixture rather than genealogical category. Although understanding of the inheritance and evolution of resistance in hybrid zones is still in its infancy, several possible consequences of these interactions have been suggested. For example, Whitham, Morrow & Potts (1991) and Whitham & Maschinski (1996) have proposed that hybrid zones might serve as important reservoirs of herbivore and pathogen biodiversity and, in some cases, as a bridge for host shifts. The host shift could lead to the formation of a new herbivore species or simply provide an avenue for movement of insect or pathogen pests across a species barrier. Whitham (1989) has also pointed out that if hybrid zones serve as evolutionary sinks for pests, they might be important in limiting the evolution of virulence. From a plant perspective, hybrid resistance may contribute either to hybrid breakdown if resistance is disrupted or to heterosis if resistance is enhanced. Finally, hybrid zones provide an excellent ecological experimental situation for studying the genetic basis of resistance in natural populations and for understanding the ecological and evolutionary aspects of plant–pest interactions (Fritz et al., 1994). 617 4. Patterns of mating One of the important factors that affects the outcome of hybridization is the pattern of mating in hybrid populations. Key parameters include outcrossing rate, hybridization frequency and mate choice by different classes of hybrids. Although it is clear why hybridization frequency would be important, outcrossing rate and mate choice are equally critical. Theoretical studies indicate, for example, that hybrid speciation will be facilitated by selfing (McCarthy et al., 1995). Selfing will also lead to lower rates of hybridization. Differences in mate choice can lead to patterns of differential introgression, and either strengthen or weaken reproductive barriers (e.g. Hodges et al., 1996). Surprisingly, there are few studies of mating patterns in natural hybrid zones or experimentally manipulated populations (notable exceptions include Vickery, 1990 ; Hodges et al., 1996 ; Bacilieri et al., 1996 ; Rieseberg, Baird & Desrochers, 1998). Instead, most reports are restricted to static descriptions of population genetic structure. (a) Outcrossing rate. Outcrossing rates in hybrid populations are assumed to be similar to those of pure parental populations in the absence of information to the contrary. However, this line of reasoning creates a paradox when applied to hybrid speciation. Theoretical studies indicate that rates of hybrid speciation should increase with selfing, yet most natural homoploid hybrid species appear to have an outcrossing mating system. A recent review found that close to 90 % of 50 proposed examples of hybrid species were outcrossers (Rieseberg, 1997). Of these, at least ten have been well-documented with molecular markers : Encelia virginensis (Allan, Clark & Rieseberg, 1997) ; H. anomalus, H. deserticola and H. paradoxus (Rieseberg, Carter & Zona, 1990 ; Rieseberg, 1991 ; Rieseberg et al., 1995 c, 1996 b) ; I. nelsonii (Arnold et al., 1990 ; Arnold, 1993 a), Peaonia emodi and Peaonia sp. (Sang et al., 1995) ; Penstemon clevelandii (Wolfe et al., 1997) ; Pinus densata (Wang et al., 1990 ; Wang & Szmidt, 1994) and Stephanomeria diegensis (Gallez & Gottlieb, 1982). All of these are outcrossers. This apparent bias toward outcrossing hybrid species is particularly striking in Helianthus, in which all three putative hybrid species and their parents, H. annuus and H. petiolaris, are obligate outcrossers characterized by a sporophytic self-incompatibility (SI) system. Desrochers & Rieseberg (1998) suggested a possible explanation for this puzzle. Perhaps conditions in hybrid zones favour selfing, even in normally outcrossing species. For example, in many selfincompatible species, selfing can be induced by mixed loads of self and heterospecific pollen (Richards, 1986). This ‘ mentor effect ’ was demon- Printed from the C JO service for personal use only by... L. H. Rieseberg and S. E. Carney 618 Mean outcrossing rate 1 0·95 0·9 0·85 0·8 0·75 10 30 50 Fertility (%) 70 90 Figure 4. The mean outcrossing rate estimate taken over each fertility class for maternal plants from the three hybrid zones between Helianthus annuus and H. petiolaris, shown with standard errors. Adapted from Rieseberg et al., 1998). strated in experimental sunflower hybrids by Desrochers & Rieseberg (1998), and a significant increase in selfing has been reported in three wild sunflower hybrid zones (Rieseberg et al., 1998). However, the increase in selfing was largely restricted to the parental-like fraction in the population. Outcrossing rates in the critical hybrid fraction of the population did not differ significantly from 1n0, suggesting that mentor effects were unlikely to facilitate hybrid speciation in wild sunflowers. Similar results have been reported for self-incompatible oak hybrid populations in which outcrossing rates approach 1n0 (Bacilieri et al., 1996). Rieseberg et al. (1998) suggested that hybrids were less likely to self than parental individuals because of semisterility and the resulting reduction in the probability of gamete union (Fig. 4). Semisterility may inhibit selfing in hybrids of other species as well, suggesting that selfing may be less important in hybrid speciation than has been suggested by theory (Templeton, 1981 ; McCarthy et al., 1995). However, this cannot be the complete answer, since species that are primarily differentiated by premating barriers often produce fully fertile hybrids. Possibly, the advantage of a selfing breeding system for enhancing the rate of establishment of hybrid species is counterbalanced by lower rates of natural hybridization among selfing taxa. It is also possible that the high, observed proportion of outcrossing hybrid species is simply an artifact of small sample size and the preponderance of outcrossing species of flowering plants. (b) Hybridization frequency. Direct estimates of hybridization frequencies in natural or manipulated populations are available from at least seven plant groups : Brassica (Scott & Wilkinson, 1998) ; Helianthus (Rieseberg et al., 1998) ; Iris (Hodges et al., 1996 ; Arnold, 1997) ; Mimulus (Vickery, 1990) ; Quercus (Bacilieri et al., 1996) ; Phlox (D. Levin, pers. comm.) ; and Senecio (Marshall & Abbott, 1980). Hybridization rates in Mimulus (0 %), Iris ( 1 %), Phlox ( 1 %), Senecio ( 1 %) and Brassica (0n4–1n5 %) are extremely low, whereas those in Helianthus (4–7n5 %) and Quercus (31n7 %) are much higher. Arnold (1997) has argued that the major barrier to interspecific gene flow in Iris is the rarity of F formation. Once F hybrids are formed, " " extensive introgression is typically observed. By contrast, sterility and ecological selection, respectively, represent formidable obstacles to introgression after F formation in Helianthus and " Quercus. (c) Mate choice. The pollen competition experiments described earlier indicate that the progeny produced by different genotypes in hybrid populations are unlikely accurately to reflect the genotypic composition of pollen loads. Instead, fertilization success appears to depend largely on pollen–style interactions, with conspecific pollen tending to be favoured relative to interspecific pollen. Analyses of progeny arrays from natural hybrid populations largely confirm the experimental studies. In wild sunflower hybrid zones, for example, parental-like individuals of H. annuus and H. petiolaris are fertilized largely by intraspecific pollen. By contrast, pollen from both parental species successfully fertilizes ovules of semisterile (intermediate) hybrids. However, as hybrids recover fertility by backcrossing, they become more likely to be fertilized by pollen of the backcross parent. In addition, as predicted by pollen competition experiments, hybridization was much more likely with one of the species as the maternal parent than with the other. Strong asymmetric patterns of hybridization similar to those observed in Helianthus have also been reported for Iris and Quercus. For example, in hybrid Quercus populations, interspecific crossing frequencies of 30 % have been reported for maternal plants of Q. robur, whereas negative values (presumably 0 %) were obtained for Q. petraea mothers. Asymmetric patterns of hybridization have also been inferred from analyses of genetic admixture in hybrid zones (e.g. Wheeler & Guries, 1987 ; De Pamphilis & Wyatt, 1990) ; pollen competition experiments (e.g. Emms et al., 1996) ; and estimates of hybrid and parental genotype frequencies (e.g. Keim et al., 1989 ; Paige et al., 1991 ; Nason, Ellstrand & Arnold, 1992). Asymmetry in hybridization frequencies often appears to lead to unidirectional introgression (e.g. Rieseberg, Choi & Ham, 1991 ; Bacilieri et al., 1996 ; Hodges et al., 1996), but this is not always the case. For instance, bidirectional introgression has been reported in some Iris hybrid zones (Arnold, 1997), and introgression was sym- Printed from the C JO service for personal use only by... Plant hybridization metric in three Helianthus hybrid ones analysed by Rieseberg et al. (1998). The direction of introgression will also be affected by the mating patterns of the early-generation hybrids (Hodges et al., 1996) and by the relative proportions of parental and hybrid genotypes in the hybrid swarms (Rieseberg et al., 1991). In Helianthus, F hybrids do not appear to favour the pollen of " one parental species over the other, but in hybrid populations where one species predominates, interspecific gene flow tends to be in the direction of the minority species (Rieseberg et al., 1991 ; Dorado, Rieseberg & Arias, 1992). . This review represents a validation of the predictions of Levin (1979) about the kinds of studies that would produce major advances in our understanding of hybridization. The experimental studies advocated by Levin (1979) have revealed the importance of gametic selection as a reproductive barrier, elucidated the genetic architecture of postmating reproductive barriers, and demonstrated the critical role of gene interactions and fertility selection in hybrid speciation. By experimentally manipulating hybrid zones, students of hybridization have been able to generate reliable estimates of the frequency of spontaneous hybridization and the strength of habitat selection – two parameters that are critical to reliable predictions of the evolutionary or ecological consequences of hybridization. In addition, a great deal of information has been compiled concerning the biology of different classes of hybrids. Morphological character expression and fitness of hybrid genotypes has been found to be surprisingly difficult to predict, as has been the response of pathogens and herbivores to hybrids. By contrast, the mating behaviour of hybrids appears to be largely predictable, as it seems to be governed in large part by strong gametic selection. Although Levin’s predictions are valid, the past two decades have seen other advances in our understanding of hybridization that do not fall easily within Levin’s categories. The most important of these are as follows : (1) Molecular phylogenetic studies have revealed the surprising power of gene trees to detect ancient hybridization events (Smith & Sytsma, 1990 ; Wendel, Stewart & Rettig, 1991 ; Rieseberg, Whitton & Linder, 1996). As a result, we now have strong evidence for reticulate evolution in many plant lineages, some of which are completely unexpected. (2) Theoretical studies by Endler (1977), Moore (1977), Barton & Hewitt (1985), McCarthy et al. (1995) and Baird (1995), among others, have led to a greater understanding of the maintenance of hybrid zones, the genetic architecture of reproductive 619 barriers and the process of hybrid speciation. Theory has also provided a series of explicit predictions regarding the evolutionary dynamics and outcomes of hybridization events and has provided a means for extracting the maximum information content from empirical data sets. (3) Technical developments in molecular biology have made available a virtually unlimited supply of molecular markers. The quality of genotypic resolution afforded by these markers has made it possible to view hybrid zones as natural experiments and to study mating patterns, dispersal and genetic architecture in the absence of manipulative experimentation. (4) Similarly, advances in genetic mapping and marker-based quantitative genetics have made it possible to estimate precisely the genomic composition of natural and experimental hybrids and to determine the genomic location of genes or chromosomal rearrangements involved in reproductive isolation. Both advances facilitate direct comparisons between experimental and historical studies of hybridization. Future advances are more difficult to predict. The experimental studies of artificial and natural hybrids advocated by Levin nearly two decades ago will probably continue to lead to important new discoveries. Also, the use of genetic mapping approaches to analyse the genomes of ancient hybrids and to map important quantitative traits will make valuable contributions. However, the studies that are likely to prove most useful are those that combine experimental ecological and historical genetic approaches. Because hybrid species and introgressive lineages originate rapidly, the majority of genetic processes associated with them can be accurately replicated by experimental studies. Perhaps the best example of this approach to date, already described, involved a comparison of the genomic composition of three experimentally generated hybrid lineages with that of a natural hybrid species that originated from the same two parents (Rieseberg et al., 1996 b). The striking concordance in genomic composition between the ancient and synthetic hybrid lineages attests to the potential utility of this kind of approach. However, these experiments were conducted in the glasshouse, and it is not yet clear how selection under natural conditions might have affected hybrid genomic composition. Clearly, it would be instructive to determine whether a hybrid species could be replicated in the wild simply by allowing natural selection to take its course. Genomic composition of the end product could be compared with that of the natural hybrid species to provide insights into the repeatability of speciation. Knowledge of the genomic location of genes that control important traits such as habitat differentiation would further inform these Printed from the C JO service for personal use only by... 620 L. H. Rieseberg and S. E. Carney kinds of studies. For example, it should be feasible to determine whether important adaptations arose via hybridization (i.e. involved genes from both parental species) or arose via divergent evolution. Similarly, it would be of interest to reconstruct the genome of introgressive races. This could be accomplished in the greenhouse using a combination of fertility and marker-based selection or by natural selection in field experiments. In both cases, genetic mapping data could be used to determine how closely the synthesized introgressive races match those produced historically in nature. Because introgression has often been thought to provide an avenue for the transfer of genetic adaptations, it would be of interest to compare the fitness and ecological amplitude of the synthesized introgressants with the native species and its introgressive races. Moreover, by determining the location of important QTLs that differentiate the hybridizing species, it would be possible to verify the transmission of these QTLs in both the natural and synthetic introgressive races, as well as to test the adaptive significance of individual QTLs. In some senses, the proposed use of historical data to guide experimental studies represents a departure from the classic interplay of theory and experimentation – the deductive approach championed by introductory scientific textbooks. However, this is perhaps inevitable given the historical nature of evolutionary study. Experimental replication of historical events probably represents the best way of estimating the contributions of deterministic and stochastic forces in evolution. This will not be possible with experiments based on optimality theory in model organisms. Of course, the strong historical component of research proposed does not rule out an important role for theory in future studies of hybridization. Clearly, the interpretation of historical phenomena and the choice of the most appropriate conditions for replication will require strong grounding in theory. Moreover, many questions relating to the dynamics and maintenance of hybrid zones largely lack a historical component. Thus, experimental manipulations and theoretical study of hybrid zones will often be highly informative in the absence of historical context. 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