Inbreeding in Plants
Inbreeding is defined as the production of offspring via the mating between relatives. As the majority of plant species are hermaphroditic, they possess the capability of producing offspring from the union of gametes within one individual, i.e. selfing. Plants therefore seemingly possess the ultimate adaptation for reproductive assurance, not to mention the ability of many species to reproduce vegetatively. By selfing, plants would be putatively freed from the capricious whims of mate availability, environmental stochasticity and countless other impediments inherent to sexual reproduction. Indeed some species have given up outbreeding altogether, and they produce indistinct flowers which do not even open to allow for cross-pollination. However, the sheer variety and exuberance of flowers in the plant kingdom clearly indicates that plants as a whole have not abandoned outbreeding. Indeed, some species display mating strategies which meticulously prevent selfing and/or inbreeding. The impetus that leads to the maintenance of outbreeding is the negative consequences of inbreeding for offspring fitness, known as inbreeding depression (Charlesworth and Charlesworth 1987).
Inbreeding depression in plants has provided ample material to fascinate many scientists from Darwin (1876) to modern-day molecular geneticists (Slate and Pemberton 2002). The interest has lead to extensive genetic theory to explain the influence of inbreeding on offspring fitness and its subsequent impact at the population level. In many cases, the quantitative theory has synergistically arisen from experimental studies of plants in both natural and laboratory settings. Inbreeding depression effects have become especially important in the context of rare and threatened species (Frankham et al. 2003). Through habitat destruction and other human-induced changes, many species of plants now occur in small, isolated populations. Plants in small populations are then in an ‘extinction vortex’, whereby they are vulnerable to the combined influences of demographic and environmental stochasiticity, as well as genetic factors (Gilpin and Soulé 1986). The primary genetic factor in the extinction vortex is increased inbreeding in small populations; this can lead to reduced offspring survival via inbreeding depression, exacerbating small population size (Barrett and Kohn 1991, Ellstrand and Elam 1993). However, the importance of genetic vs. environmental effects in population extinction is the subject of many scholarly debates (Caro and Laurenson 1994).
The purpose of this review will be to synthesize the theoretical and empirical studies of inbreeding in plants. Throughout the review emphasis will be placed on the effects of inbreeding in natural populations of plants, rather than domesticated species or laboratory settings. Included in this review will be a discussion of the evolution of selfing and breeding systems in plants, the quantitative genetic theory behind inbreeding depression and the methods used to investigate inbreeding depression. The empirical studies of the effects of inbreeding on offspring traits will also be summarized, beginning with Darwin’s (1876) investigations. The last part of the review will focus on the effects of inbreeding in natural populations of rare or threatened plant species.
Inbreeding is defined as the mating between individuals related by a shared ancestry (Falconer and Mackay 1996). However, inbreeding can occur in plants via selfing, or the union of haploid gametes produced by the same genetic individual, also known as autogamy (Silvertown and Charlesworth 2001). As most flowers are hermaphroditic, selfing is a reproductive strategy present in the majority of plants (Bawa and Beach 1981). Furthermore, many plants have multiple flowers or inflorescences per individual, allowing for another type of self-fertilization, termed geitonogamy (Jain 1976). Darwin (1876) showed in early studies that this type of selfing differs from selfing within flowers in terms of the quality of offspring produced. Because plants can reproduce vegetatively and apomictically (seeds having the same genotype as their mother) discrete individuals can actually be genetic clones (Bawa and Beach 1981, Silvertown and Charlesworth 2001). Therefore, another class of selfing will be mating between genetically identical individuals (Silvertown and Charlesworth 2001). Inbreeding of course can also occur in plants through the mating between close relatives, which is termed biparental inbreeding to differentiate it from selfing (Griffin and Eckert 2003). Where dispersal of propagules is limited, genetically related individuals tend to occur in patches or clumps across a landscape, increasing biparental inbreeding (Loveless and Hamrick 1984, Griffin and Eckert 2003). Similarly, when small, less vagile insects achieve pollen dispersal, pollination between relatives within a patch will prevail (Loveless and Hamrick 1984).
With some exceptions, inbreeding reduces offspring fitness in essentially all naturally outcrossing plants and to a lesser extent in selfing species. The negative effects of mating between relatives have been noticed for many centuries. The careful breeding studies of Darwin (1876) first empirically demonstrated inbreeding depression in a wide variety of taxa. The negative effects of inbreeding have since been observed in both outcrossing and selfing species for a variety of traits with consequences for offspring fitness (Charlesworth and Charlesworth 1987, Keller and Waller 2002). Examples of traits shown to be subjects to inbreeding depression include pollen quantity, number of ovules, amount of seed, germination rate, growth rate and competitive ability (Keller and Waller 2002, Frankham et al. 2003). Genetic models have been developed to functionally explain reduction in fitness-related traits caused by inbreeding.
The primary genetic consequence of inbreeding is increased homozygosity (Falconer and MacKay 1996). Two hypotheses for the genetic basis of inbreeding depression are put forth, both of which depend on the idea that homozygosity wll increase with inbreeding. Either the overdominance or partial dominance hypotheses are invoked to model the negative consequences of inbreeding (Charlesworth and Charlesworth 1987; Lynch 1991; Karkkainen et al. 1999). In the overdominance hypothesis, inbreeding depression is attributable to higher fitness of heterozygotes versus homozygotes for the loci in question (Frankham et al. 2003). For the partial dominance hypothesis, negative fitness consequences for inbred lines are due to the fixation of recessive or partially recessive deleterious alleles (Frankham et al. 2003). Current thought favors the latter hypothesis, where inbreeding depression is attributable to many genes of small effect (Keller and Waller 2002, Frankham et al. 2003). However, distinguishing between the two genetic mechanisms is complicated by linked sets of deleterious recessives that imitate overdominance effects (Keller and Waller 2002).
Because self-fertilization will produce the most profound inbreeding (Falconer and MacKay 1996), discussion of the evolution of inbreeding in plants primarily focuses on the evolution of selfing. Altogether, plants provide an excellent system in which to study the evolution of inbreeding. Members of the same genus that are otherwise closely related often display a variety of breeding systems (Stebbins 1957, Johnston and Schoen 1996). The ability to self-fertilize is almost universally a derived characteristic, with self-fertilizing species arising from outcrossing species (Stebbins 1957; Bawa and Beach 1981; Schemske and Lande 1985). By comparing predominantly outcrossing versus selfing species, the selective pressure surrounding the evolution of self-fertilization versus outcrossing can be estimated.
The evolution of inbreeding in plants is the product of the interplay between several selective forces, with inbreeding depression being the primary cost associated with self-fertilization (Darwin 1876; Stebbins 1957; Jain 1976; Lande and Schemske 1985). Outcrossing has inherent costs for the parent plant that will influence fitness (Solbrig 1976). For example, the direct costs of copious pollen production for effective fertilization take away from the potential for seed production (Jain 1976, Solbrig 1976). The so-called ‘meiotic cost’ for sexual reproduction occurs because only transmitting half of the genome to the next generation lowers parental fitness (Solbrig 1976). Outcrossing will also have fitness costs for offspring if it breaks up co-adapted gene complexes that confer adaptation to a local environmental conditions, i.e. outbreeding depression (Lynch 1991).
Various other ecological phenomena also foster the evolution of selfing in plants. For example, stochastic fluctuations in pollinator populations could lead to reduced seed set for insect cross-pollinated plants in years with low pollinator numbers (Stebbins 1957, Jain 1976). Populations that are subject to environmental stochasticity benefit by selfing when years are either too dry or too wet for pollen to be spread between plants (Stebbins 1957; Jain 1976; Lande and Schemske 1985). Also, founding of new populations by single individual, as is evidenced in many ruderal (weedy) species, favors self-fertilizing species (Stebbins 1957;Jain 1976; Loveless and Hamrick 1984; Lande and Schemske 1985). In the absence of inbreeding depression via partial dominance, selection against outcrossing would lead to the predominance of selfing (Jain 1976, Lande and Schemske 1985). However, outcrossing is necessary to maintain genetic variability to ensure the long-term evolutionary potential of a species (Solbrig 1976, Frankham et al. 2003).
Quantitative genetic models have been developed for the evolution of inbreeding and self-fertilization in plants. Fisher (1941) first demonstrated that a gene leading to self-fertilization would rapidly spread in an outcrossing population until the entire population was selfing. However, it is unlikely that selfing is conferred by a single gene of major effect, but rather a shift to selfing is likely due to a suite of genes of small effect (Lande and Schemske 1985). As demonstrated by Lande and Schemske (1985) predominant outcrossing and predominant selfing can exist as alternative stable states of a mating system. Their quantitative genetic models also confirm that highly self-fertilizing species can evolve more readily from primarily outcrossing species than vice versa (Lande and Schemske 1985). However, primarily selfing species are not necessarily an evolutionary dead end as sufficient quantitative variation can be maintained between inbred lines of a species (Lande and Schemske 1985, Schemske and Lande 1985).
In selfing plant populations, the ratio of the fitness of selfed versus outcrossed progeny is used as the measure of inbreeding depression. In Darwin’s (1876) classic studies of the effects of inbreeding, carefully controlled crosses were used to determine the effects of inbreeding. Controlled crosses remain to this day the primary method for measuring inbreeding depression (Keller and Waller 2003). Generally, crossing individuals of a known or inferred relatedness produces outcrossed offspring in these types of studies. Where the fine-scale genetic structure or pedigree of a natural population is unknown, distance between parental plants is used as a proxy measure of relatedness (Waser and Price 1983). The inbred offspring can be similarly produced by crossing individuals within a short distance of one another, or by enclosing hermaphroditic flowers to allow them to self while preventing outcrossing (Waser and Price 1983). Differences in seed set between outcrossed and inbred flowers can indicate inbreeding depression if fewer seeds are produced through inbreeding (Waser and Price 1983). In plants without gametophytic self-incompatibility loci, reduced seed set in inbred flowers in is often attributable to lethal recessives that prevent embryo formation or other essential processes for seed set (Keller and Waller 2002).
The breeding studies described above are amenable to greenhouse experiments, where manipulation of pollination can be easily achieved and controlled. However, a study by Dudash (1990) indicates that offspring fitness measured in greenhouse or garden environments may significantly underestimate inbreeding depression. More indirect methods are needed to study the effects of inbreeding depression in natural populations. Inbreeding depression has been investigated at the population level in several recent studies by examining how fitness-related traits vary between small and large populations (Menges 1990; Oostermeijer et al. 1994; Heschel and Paige 1995; Fischer and Matthies 1998). Reduced fitness in populations may not always be attributable to inbreeding depression due to confounding environmental variation. Experimental studies have also demonstrated density-dependent seed set known as the Allee effect to occur in small populations of plants (Widen 1993).
Studies have made use of advances in molecular genetics for detecting inbreeding in plant populations. The advantage of this approach is that pedigree information and complex breeding experiments are not always requisite, which are often difficult to apply to natural populations. The similarity in state between two alleles at a locus determined by molecular markers can be used to estimate F, the inbreeding coefficient (Ritland 1990, Falconer and MacKay 1996). Changes in F across two generations of a selfing plant can be used to estimate selection against inbred individuals, i.e. inbreeding depression (Ritland 1990). Heterozygosity at marker loci is assumed to be indicative of general levels of heterozygosity in the genome, which is expected to decrease with inbreeding (Britten 1996). Positive correlations between fitness-related traits and heterozygosity are typically taken as evidence for inbreeding depression (Britten 1996, Slate and Pemberton 2002). The application of quantitative trait loci (QTL) methods to the issue of inbreeding depression is in its early stages of development, but has provided significant clues to its underlying genetic basis (Karkainnen et al. 1999, Remington and O’Malley 2000). QTLs allow for the association of observable inbreeding depression effects with discrete regions of the genome (Karkainnen et al. 1999, Remington and O’Malley 2000). For example in Pinus taeda, genes of major effect detected by marker linkage were primarily responsible for inbreeding depression (Remington and O’Malley 2000); this result supports the partial dominance hypothesis described above.
Inbreeding Effects in Wild Populations
The effects of inbreeding in natural population are of interest to ecologists seeking to determine the underlying causes for observed demographic phenomena. Population level investigations of inbreeding have recently documented these effects in a number of species. Menges (1990) compared populations of an endemic prairie species Silene regia (royal catchfly) and found that seed set increased with population size. In a related study, Oostermeijer and colleagues found that offspring fitness of Gentiana pneumonanthe (marsh gentian) increased with population size. In a related rare species, Gentianella germanica (chiltern gentian), steeper population declines via reduced seed set were found in smaller populations; this reduced plant fitness occurred independently of environmental variation, indicating the inbreeding effects were the primary cause (Fischer and Matthies 1998). As indicated in a study of Ipomopsis agregata (scarlet gilia), the effects of inbreeding depression may make populations more susceptible to environmental stress (Heschel and Paige 1995).
Field studies also can provide evidence to support theoretical results about the evolutionary consequences of inbreeding. The relationship between natural rates of self-fertilization and inbreeding depression can answer questions about their relative role in the evolution of breeding systems (Johnston and Schoen 1996). Holtsford and Ellstrand (1990) examined how inbreeding effects vary with natural outcrossing rates in populations of Clarkia tembloriensis. The theory that inbreeding effects will be reduced in populations with high natural rates of inbreeding was not supported by their results; rather, all populations showed significant inbreeding depression for fecundity traits (Holtsford and Ellstrand 1990). In a similar study of Collinsia heterophylla, inbreeding depression effects also did not decline with as the level of natural inbreeding increased (Mayer et al. 1996). However, these studies provide conflicting evidence for the role of purging of deleterious recessives from populations with high natural inbreeding rates (Holtsford and Ellstrand 1990; Johnston and Schoen 1996; Mayer et al. 1996).
Mechanisms to Avoid Inbreeding Depression
Plants display a number of variations in morphology and breeding systems that reduce the likelihood of inbreeding through selfing or mating with close relatives (Darwin 1876). However, avoidance of inbreeding is unlikely the only selective force leading to the evolution of these features (Charlesworth and Charlesworth 1987). Many of the adaptations provide overt benefits to plant fitness beyond reducing selfing (Charlesworth and Charlesworth 1987).
Varying the spatial distribution of male and female reproductive organs has evolved in many taxa of plants, and this breeding system can take on several different forms (Bawa and Beach 1981, Charlesworth and Charlesworth 1987). Though most species have hermaphroditic flowers, some species have separate flowers with male and female functions (Bawa and Beach 198, Charleworth and Charlesworth 1987). To reduce selfing, these flowers can be spatially distinct on the same individual plant (monoecy) or on separate individuals (dioecy) (Bawa and Beach 1981, Charlesworth and Charlesworth 1987). Many variations of these breeding systems exist, where plants may display combination of hermaphroditic and functionally male or female flowers (Bawa and Beach 1981). Plants also vary the spatial distribution of male and female organs within hermaphroditic flowers, which is known as heterostyly (Charlesworth and Charlesworth 1987). This mechanism of self-pollination avoidance is present in 22 angiosperm families likely of independent evolutionary origin (Charlesworth and Charlesworth 1987). In distylous species (e.g. primrose Primula vulgaris), two morphs of flowers are present: the ‘pin’ flowers with long styles and short stamens and the ‘thrum’ flowers with short styles and long stamens (Bawa and Beach 1981, Charlesworth and Charlesworth 1987). The function of distyly in preventing inbreeding depends on interactions with the pollinators; pollen transfer between morphs rather than within morphs is facilitated by pollen deposition on different parts of the pollinator (Bawa and Beach 1981). Similarly, tristyly occurs when three flower morphs are present, e.g. the long-, mid- and short-styled morphs of purple loosestrife, Lythrum salicaria (Charlesworth and Charlesworth 1987). Tristyly has most been studied in the context of its breakdown, where the advantage of self-fertilization outweighs the consequences of inbreeding depression (Charlesworth and Charlesworth 1987, Silvertown and Charlesworth 2001).
Plants may also vary the distribution of male and female function in time, known collectively as dichogamy (Bawa and Beach 1981). Where pollen is dispersed from the anthers before the stigmas are receptive, the flowers are considered to be protandrous (Bawa and Beach 1981). Similarly, when the stigmas are receptive before pollen is shed from the anthers, the flowers are protogynous (Bawa and Beach 1981). Protandry is present in many more taxa than protogyny, indicating that dichogamy is mainly a means to control the amount of pollen exiting a flower or inflorescence (Bawa and Beach 1981).
Another means of inbreeding avoidance is via genetically mediated self-incompatibility, in which pollen with the same genotype as the maternal plant is unable to bring about fertilization (Charlesworth and Charlesworth 1987, Silvertown and Charlesworth 2001). An example of this type of self-incompatibility is many apple cultivars that require other varieties to fertilize flowers for fruit to be produced (Silvertown and Charlesworth 2001). Rejection of pollen is controlled by a self-incompatibility locus known as the S-locus, where self-pollen and pollen from other plants with the same allele is rejected (Charlesworth and Charlesworth 1987). Typically there are many different alleles at this locus in a population (Silvertown and Charlesworth 2001). There are two types of self-incompatibility in plants. Gametophytic incompatibility occurs where rejection is controlled by haploid genotype of the pollen itself (Silvertown and Charlesworth 2001). Sporophytic incompatibility occurs where rejection is controlled by the diploid genotype of the plant that produces the pollen (Silvertown and Charlesworth 2001).
Inbreeding and Conservation of Plants
Inbreeding is of concern to the conservation of rare plants because in many rare or threatened species high levels of inbreeding that occur in small, remnant populations (Barrett and Kohn 1991, Ellstrand and Elam 1993). The level of inbreeding in a population is determined by a number of factors discussed above, but is ultimately determined by population size, as this will generally determine the diversity of available mates (Frankham et al. 2003). Furthermore, human activities like selective logging can directly increase the level of inbreeding by increasing mating between relatives in wild populations of plants (Murawski et al. 2002). The severity of inbreeding depression is overall less in populations that have regularly inbred in the past (Frankham et al 2003); for example, populations of narrowly endemic species with consistent small population sizes throughout their evolutionary history (Loveless and Hamrick 1991). However, many species that are currently endangered or threatened now occur in small populations, which may or may not reflect historical sizes (Ellstrand and Elam 1993). Inbreeding depression will proximately have detrimental consequences for population growth, but long-term population survival will ultimately depend on genetic variability (Barret and Kohn 1991; Ellstrand and Elam 1993; Frankham et al. 2003). Thus, inbreeding depression is an issue in the viability of populations of rare or threatened plants.
Despite the significant negative effects of inbreeding depression demonstrated in studies from Darwin (1876) to today, the relative importance of inbreeding depression as a factor in population extinction is subject to debate (Frankham and Ralls 1998). There is some doubt about the importance of genetic effects in determining the survival of small populations relative to demographic and environmental stochasticity (Lande 1988, Caro and Laurenson 1994). Recent studies have provided evidence for the role of inbreeding in population extinction by demonstrating the importance of genetic diversity (Newman and Pilson 1997, Richards 2000). Genetic ‘rescue’ effects have been demonstrated in a metapopulation of Silene alba (white campion), where gene influx increased the persistence of isolated populations (Richards 2000). Though direct empirical evidence of inbreeding depression contributing to population extinction is limited, other sources of information clearly suggest that the influence of genetic factors cannot be discounted. Demographic models have predicted that inbreeding depression can significantly effect population survival, especially when it impacts the survival of adults (Mills and Smouse 1994, Tanaka 1997). Circumstantial evidence from recent studies also indicates that inbreeding depression plays a role in population survival. Heschel and Paige (1995) demonstrated increased offspring fitness when small, presumedly inbred, populations were outcrossed, but did not observe such increases when large populations were similarly treated. Menges (1991) found that seed germination increased with population size in isolated populations of a threatened prairie species, Silene regia (royal catchfly). Similarly, decreased fecundity and survival in relation to genetic diversity and population size were found in populations of Gentiana pneomonanthe (marsh gentian), a rare plant of European calcareous grasslands (Oostermiejer et al. 1994). However, a similar study of Senecio integrifolius (groundsel) failed to detect a clear relationship between inbreeding effects and population size, indicating that other factors were likely of primary importance for this plant (Widen 1993).
Inbreeding is a force pertinent to both the viability of populations of rare or threatened plants and the evolution of breeding systems in plants. The incredible diversity of floral traits and breeding systems apparent in plants is largely attributable to the opposing influences of avoiding inbreeding depression while assuring reproduction. The interplay between these forces has challenged quantitative geneticists to devise comprehensive models and empiricists beginning with Darwin to demonstrate these phenomena in nature. Empirical studies have demonstrated that levels of inbreeding depression vary across taxa, populations and environments, but that overall, inbreeding depression effects are significant. Though molecular methods have been developed and applied to plants, the detection of inbreeding depression by comparing the fitness of outcrossed versus inbred progeny is as germane today as in Darwin’s time. The effects of inbreeding on individual fitness are well demonstrated in both greenhouse and field settings; however, how these effects alter population dynamics in the wild is still largely unknown. Because demographic and environmental factors interact with genetic issues in small populations, inbreeding depression has received renewed emphasis in the conservation of rare and threatened plants. In light of the pace of human impacts on ecosystems, there will likely be a near inexhaustible supply of plant populations experiencing novel levels of inbreeding. Though unfortunate for the preservation of biodiversity, these may provide opportunities to gather evidence for quantitative genetic models of the effects of inbreeding in plant populations.
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