Fitness costs associated with evolved herbicide resistance alleles in plants

Authors

  • Martin M. Vila-Aiub,

    1. Western Australian Herbicide Resistance Initiative (WAHRI), School of Plant Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia
    2. Present address: Av. San Martin 4453, University of Buenos Aires (UBA), Faculty of Agronomy, IFEVA-CONICET, Buenos Aires (1417), Argentina
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  • Paul Neve,

    1. Western Australian Herbicide Resistance Initiative (WAHRI), School of Plant Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia
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  • Stephen B. Powles

    1. Western Australian Herbicide Resistance Initiative (WAHRI), School of Plant Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia
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Author for correspondence:
Martin M. Vila-Aiub
Tel: +54 11 45248070
Email: vila@ifeva.edu.ar

Abstract

Contents

 Summary751
I.Introduction752
II.Methods for the detection and estimation of herbicide resistance fitness costs753
III.Fitness costs associated with AHAS target site resistance756
IV.Fitness costs associated with ACCase target site resistance757
V.Fitness costs associated with resistance to phenoxy herbicides758
VI. Arabidopsis thaliana as a model plant to understand pleiotropic effects of herbicide target-site based resistance alleles759
VII.Fitness costs associated with herbicide resistance endowed by enhanced rates of herbicide metabolism catalysed by P450 enzymes760
VIII.Fitness costs associated with glyphosate resistance760
IX.Discussion761
X.Conclusion762
 Acknowledgements763
 References763

Summary

Predictions based on evolutionary theory suggest that the adaptive value of evolved herbicide resistance alleles may be compromised by the existence of fitness costs. There have been many studies quantifying the fitness costs associated with novel herbicide resistance alleles, reflecting the importance of fitness costs in determining the evolutionary dynamics of resistance. However, many of these studies have incorrectly defined resistance or used inappropriate plant material and methods to measure fitness. This review has two major objectives. First, to propose a methodological framework that establishes experimental criteria to unequivocally evaluate fitness costs. Second, to present a comprehensive analysis of the literature on fitness costs associated with herbicide resistance alleles. This analysis reveals unquestionable evidence that some herbicide resistance alleles are associated with pleiotropic effects that result in plant fitness costs. Observed costs are evident from herbicide resistance-endowing amino acid substitutions in proteins involved in amino acid, fatty acid, auxin and cellulose biosynthesis, as well as enzymes involved in herbicide metabolism. However, these resistance fitness costs are not universal and their expression depends on particular plant alleles and mutations. The findings of this review are discussed within the context of the plant defence trade-off theory and herbicide resistance evolution.

I. Introduction

Weeds are a major constraint to crop production and herbicides are key elements of weed control in most global crop production systems. Herbicides applied to large populations of genetically diverse weedy plants exert a very strong selection pressure for the evolution of resistance, and evolved resistance in weed populations has been reported worldwide (Powles & Holtum, 1994; Powles & Shaner, 2001; Powles & Yu, in press).

It is a basic tenet of evolutionary genetics that adaptation to a new environment will often have negative pleiotropic effects on fitness in the original environment, the so-called ‘cost of adaptation’ (Purrington, 2000; Strauss et al., 2002). These fitness costs can prevent the fixation of novel adaptive alleles (Tian et al., 2003) and contribute to the maintenance of genetic polymorphisms within populations (Antonovics & Thrall, 1994). However, as reviewed here, it has been shown that some herbicide resistance alleles have no observable impact on plant fitness. An understanding of the fitness consequences of herbicide resistance alleles in the presence and absence of a herbicide is important for predicting the evolutionary dynamics of herbicide resistance (Neve et al., 2003) and, therefore, in conceiving strategies by which fitness costs can be manipulated to result in selection against resistance alleles (resistance management) (Jordan et al., 1999; Friesen et al., 2000; Beckie, 2006; Walsh & Powles, 2007).

When a herbicide resistance allele confers a fitness cost, there are at least three explanations for the origin of the fitness cost. First, fitness costs may result when novel, resistance-conferring mutations in herbicide target enzymes (target site resistance) also compromise or interfere at some level with normal plant function or metabolism (Cohan et al., 1994; Groeters et al., 1994; Chevillon et al., 1995). For example, a single amino acid substitution may cause a structural modification in the target enzyme that limits herbicide binding, but also compromises the efficiency of enzyme function and kinetics (Tranel & Wright, 2002; Délye, 2005; Powles & Preston, 2006; Powles & Yu, in press).

Second, resource-based allocation theory predicts a trade-off between plant reproduction, growth and defence functions (Coley et al., 1985; Chapin et al., 1993; Herms & Mattson, 1994). Herbicide resistance is an evolved plant defence mechanism that could potentially divert resources away from growth and reproduction. For example, herbicide resistance endowed by enhanced metabolism may rely on the novel or increased production of cytochrome P450 enzymes (Werck-Reichhart et al., 2000). According to the resource-based allocation model, when this novel enzyme production is constitutive, the additional energy and resource investments to synthesize these enzymes will divert resources away from growth and reproduction and may impose a resistance fitness cost in the absence of the herbicide.

Third, fitness costs may arise as a consequence of altered ecological interactions (Purrington, 2000; Strauss et al., 2002). If a resistance allele has pleiotropic effects such that the resistant phenotype becomes, for instance, less attractive to pollinators or more susceptible to diseases, fitness costs may occur independently or in addition to any energetic drain or alteration of normal metabolism (Salzmann et al., 2008). It is emphasized that, where there is a fitness cost associated with recently evolved herbicide resistance alleles, co-adaptation and integration of the resistance allele into the genome can reduce the fitness cost over generations (Fisher, 1928).

Beyond their importance for informing herbicide resistance evolution and management, costs of evolved resistance to herbicides provide a good model for answering wider fundamental questions about the origin of the cost of plant adaptation. In addition, understanding ecologically and evolutionarily based changes in plant resource acquisition and allocation patterns in response to environmental disturbance, stress and heterogeneity provides useful insights into the theory of weed community assembly (Weiher & Keddy, 1999), biodiversity and energy flow in agroecosystems (Swift & Anderson, 1994), as well as the potential for weed invasions into natural communities (Sax et al., 2007).

There have been a wealth of studies unequivocally documenting a fitness cost associated with target site resistance to triazine herbicides (Holt, 1990; Warwick, 1991; Gronwald, 1994; Holt & Thill, 1994; Bergelson & Purrington, 1996), and therefore this will not be reviewed further here. However, a few unique features of the effects of triazine resistance on fitness costs that have partly motivated the present review are worth mentioning. In the great majority of cases, evolved triazine resistance is endowed by a chloroplastic psbA gene mutation that encodes for a serine-264 to glycine (Ser-264–Gly) amino acid substitution in the photosystem II (PSII) D1 protein. This mutation endows resistance because it reduces the affinity of the binding site for triazine herbicides (Trebst, 1996), but also leads to a reduced photosynthetic capacity as a result of an inefficiency of electron transfer within the PSII complex (Jansen & Pfister, 1990). As a result, many triazine-resistant weeds possessing the Ser-264–Gly mutation show significantly reduced photosynthetic potential, growth rates, resource competitive ability and sexual reproduction (reviewed by Holt & Thill, 1994). It is understandable that the light reactions of photosynthesis have been optimized over evolutionary time and that the triazine resistance-endowing mutation reduces the efficiency of the light reactions of photosynthesis. In addition, an interesting aspect of the Ser-264–Gly mutation is that the expression and magnitude of its associated fitness cost have been shown to be modulated by abiotic and biotic factors. Contrasting combinations of light and temperature may amplify, neutralize or even reverse its negative effect on photosynthesis and plant growth (Ducruet & Ort, 1988; Hart & Stemler, 1990; Dekker & Sharkey, 1992; Plowman & Richards, 1997; Arntz et al., 1998, 2000; Jordan et al., 1999; Plowman et al., 1999). Similarly, triazine-resistant plants are more susceptible to fungal infections and insect herbivory, further contributing to the fitness cost of the resistance-endowing Ser-264–Gly mutation (Gassmann, 2005; Gassmann & Futuyma, 2005; Salzmann et al., 2008). Triazine-resistant plants have been shown to possess higher concentrations of nitrogen in leaf tissues. It is speculated that the increase in leaf-level nitrogen is potentially a photosynthetic compensatory trait which ultimately translates into an ecologically based cost of triazine resistance resulting from a greater susceptibility to herbivores (Gassmann, 2005).

The magnitude of the costs associated with triazine resistance has meant that these costs have been easily detected, despite the often flawed methodologies that have been employed to measure fitness (Bergelson & Purrington, 1996; Jasieniuk et al., 1996). A major motivation for this review is to highlight that the triazine resistance example of the near-universality of a single mutation with a strong fitness cost is the exception rather than the rule. As fully elucidated in the sections that follow, there is mounting evidence for the existence of a plethora of herbicide resistance mechanisms and mutations, some of which may lead to pleiotropic effects with negative, positive or neutral consequences on plant fitness.

The purpose of this article is twofold: to consider the methodological and experimental requirements to unequivocally measure fitness and fitness costs in plant populations; and to review evidence from published studies for costs of resistance associated with nontriazine herbicides.

To achieve these objectives, this review critically examines the molecular, physiological and ecological factors influencing the expression and magnitude of fitness costs associated with evolved resistance to nontriazine herbicides in weedy plant species. A total of 56 published articles has been identified in citation databases (Scopus® (Elsevier, Amsterdam, the Netherlands) and Web of Science® (Thomson Reuters, Philadelphia, PA, USA)) (Supporting Information Notes S1 and S2). We provide a critical analysis of the experimental methodologies that have been used to assess the expression and magnitude of fitness costs and their correlation with the physiological/molecular bases of herbicide resistance.

II. Methods for the detection and estimation of herbicide resistance fitness costs

We propose five experimental factors that should be considered in the design of studies to estimate and interpret fitness costs associated with resistance to herbicides. In this section, we present the rationale for considering each of these factors before reviewing the extent to which each of them has been considered in published fitness studies.

1. Control of genetic background

In response to the varying abiotic and biotic selective forces, ecotypic differentiation is a common process occurring at different spatial scales among weed species (Snaydon, 1971; Ransom et al., 1998; Keller & Kollmann, 1999). Thus, herbicide-resistant and herbicide-susceptible individuals from different plant populations will probably exhibit genetic variability at a number of fitness-related loci (Bergelson & Purrington, 1996; Jasieniuk et al., 1996). Therefore, to unequivocally attribute costs to the herbicide resistance-endowing allele, relative fitness should be measured in resistant and susceptible individuals that share a similar genotype, except for the alleles endowing herbicide resistance.

More than a decade ago, it was concluded that numerous studies assessing herbicide resistance fitness costs (most of them on triazine resistance) were flawed as they did not minimize differences in the genetic background between resistant and susceptible populations or individuals (Bergelson & Purrington, 1996). Our literature review reveals that only 25% of studies assessing fitness costs explicitly met the criteria of the control of genetic background. Among the studies that did control genetic background, the experimental protocols to create suitable herbicide-resistant and herbicide-susceptible lines for comparison included genetic transformation, the production of segregating F2 lines and the identification of cosegregating herbicide-resistant and herbicide-susceptible individuals from within single populations (Purrington & Bergelson, 1997; Roux et al., 2004; Vila-Aiub et al., 2005a; Menchari et al., 2008).

An alternative approach may be to reduce the effect of differences in genetic background by comparing multiple resistant and susceptible populations (Cousens et al., 1997; Strauss et al., 2002). When multiple comparisons between resistant and susceptible genotypes are also considered, the proportion of the studies satisfying the control of genetic background criterion rises to about one-half of the published literature.

2. Knowledge of the biochemical basis of herbicide resistance

Where possible, it is desirable to characterize the biochemical and molecular bases of resistance before conducting a fitness study. This knowledge is essential to ascribe identified pleiotropic effects to particular genes and mutations, and to comprehend their biochemical and physiological origins and causes.

The importance of this proposed experimental requirement is evident as, too often, more than one resistance-endowing mechanism may be present at the population and individual level and different resistance mutations may be involved. For example, it is now well established that either or both enhanced detoxification and an insensitive target enzyme may endow resistance to acetyl-CoA carboxylase (ACCase)- or acetohydroxyacid synthase (AHAS)-inhibiting herbicides in Alopecurus myosuroides and Lolium rigidum populations (Tardif & Powles, 1994; Preston, 2004; Powles & Yu, in press). Moreover, for both target site ACCase and AHAS herbicide resistance, there are several different mutations (Tranel & Wright, 2002; Délye, 2005; Yu et al., 2007a), some associated with a fitness cost (Roux et al., 2004; Menchari et al., 2008) and others not (Vila-Aiub et al., 2005a). Given this reality, the almost 50% of published studies that evaluated fitness costs without a knowledge of the biochemical/molecular basis of resistance have evident limitations.

Although it is best that fitness studies be conducted with plants in which the biochemical basis of resistance is known, we emphasize that, where this knowledge cannot be obtained, good fitness studies can still be conducted. Results from these experiments may be useful for the design of weed management strategies to exploit those traits that result in reduced ecological performance. This is the case for triallate and difenzoquat herbicide-resistant Avena fatua populations that exhibit significantly higher germination rates when compared with susceptible genotypes (O’Donovan et al., 1999). Significant differences (or a lack of them) (Murphy et al., 1986) in life history weed traits should provide knowledge for the adoption of agronomic tools to manage herbicide-resistant populations.

Herbicide resistance mechanisms can be broadly divided into target site and nontarget site mechanisms. The majority of fitness cost studies in the literature are on target site herbicide resistance. However, although there are many cases of nontarget site herbicide resistance (Preston, 2004; Preston & Wakelin, 2008), there are thus far only few studies of fitness costs of nontarget site resistance mechanisms (see Sections VII and VIII).

In total, only 28% of fitness studies combine a knowledge of the molecular basis of resistance with adequate control of genetic background (Supporting Information Notes S1 and S2). These 28% of studies are valuable in fully understanding herbicide resistance fitness costs in plants.

3. Life history traits

Plant fitness may be defined as the relative number of offspring contributing to future generations by one form compared with another (Harper, 1977; Primack & Hyesoon, 1989). This focus on plant reproduction as a measure of fitness has led to many fitness studies that have compared seed production between herbicide-resistant and herbicide-susceptible individuals. However, the assumption that a genotype that produces many seeds is fitter than a genotype producing fewer seeds is only true if seed dispersal, germination and colonization rates, seed longevity, seedling vigour and resistance to pathogens, diseases or herbivores are identical (Primack & Hyesoon, 1989; Hanley, 1998). In addition, the calculation of the number of seeds produced by an individual only estimates the female reproductive fitness, when, in fact, the number of copies of a resistance allele that are transmitted to the next generation is the sum of the male and female reproductive fitness. For example, in an outcrossing species, estimates of the seed production of resistant plants may considerably overestimate the fitness of the resistance allele if female gametes are preferentially pollinated by neighbouring susceptible plants. Most fitness studies which measure seed production do not reveal whether they account for male reproductive fitness. A number of potential ways to account for male reproductive fitness are possible, such as the evaluation of the production, viability, growth and competition between resistant and susceptible pollen (Delph et al., 1998; Song et al., 2002), and pollen discounting in species with both selfing and outcrossing reproductive systems (Chang & Rausher, 1998). Similarly, ovule size and number may be estimated to evaluate female reproductive fitness (Burd et al., 2009). Some methodological protocols, such as the prevention of cross-pollination between resistant and susceptible plants (Vila-Aiub et al., 2009) and the genotypic determination of seeds produced in mixed resistant and susceptible populations (Roux et al., 2005a), are designed to minimize the confounding potential differences in male and female reproductive fitness when assessing costs between herbicide-resistant and herbicide-susceptible genotypes.

The reality is that there are trade-offs between different life history stages, and changes in one component of plant fitness may involve compromises in other traits (Harper, 1977). Seed production is a crucial determinant of fitness which will integrate and be influenced by other life history variations between resistant and susceptible plants. However, it remains important to understand how resistance impacts on other life history processes, as this knowledge may be applied to the design of appropriate weed management to maximize fitness costs. The majority of studies infer costs associated with resistance alleles after exploring phenological, morphological, physiological and growth plant traits at the vegetative and/or reproductive stages. The exploration of the expression of fitness costs and the life history variations associated with traits and processes that occur early in the plant life cycle are important, as any fitness costs evident in young plants will have a significant adverse impact in a competitive world. Of a total of 114 experiments (replicated experiments were counted as one) identified in the reviewed literature, only 7% and 10% evaluated the occurrence of fitness costs during seed germination and plant establishment, respectively.

We believe that the best indication of the fitness of individuals carrying a resistance allele is evident when the frequency of the given allele or genotype is followed over several generations (Roux et al., 2005a, 2006; Wang et al., 2009). Observed variations in the resistance allele frequency are compared with an expected allele frequency given no fitness costs and no genetic drift. A similar methodological approach involves the assessment of the resistant phenotypic frequencies after a certain time of discontinued herbicide use (Bourdôt et al., 1996; Andrews & Morrison, 1997). The ‘allele frequency approach’ enables the evaluation of the evolutionary trajectories of resistance genes without identifying the particular plant traits involved, accounts for male and female reproductive fitness and, by default, integrates fitness effects across all life history stages. This experimental protocol has been proven to be a robust tool to assess fitness costs associated with insecticide (Boivin et al., 2003; Hardstone et al., 2009) and antibiotic (Gustafsson et al., 2003) resistance, and can be extended to the study of fitness costs associated with polygenic traits and traits of unknown biochemical resistance mechanisms. In this regard, studies which are conducted in the field in appropriate natural environments can best simulate the conditions in which fitness costs are expressed in agroecosystems.

4. Resource competition

Ecological fitness costs are differentially expressed depending on interactions with other organisms (Strauss et al., 2002). Ecological costs may become evident as a consequence of biotic interactions, such as predation (Gassmann, 2005), disease (Brown, 2003) and/or competition (Van Dam & Baldwin, 2001). If a herbicide resistance allele results in impaired ability to capture resources or a less efficient use of captured resources, ecological fitness costs should be more evident under intense resource competitive conditions. For this reason, it is easier to measure herbicide resistance fitness costs under competitive conditions (Harper, 1977; Weiner, 1990; Reboud & Till-Bottraud, 1991; van Dam & Baldwin, 1998; Glawe et al., 2003).

Around one-half of the 56 reviewed studies have measured fitness costs under competitive conditions. Unfortunately, the results of many of these studies are inconclusive and difficult to interpret, as there was no control of genetic background. Interestingly, in studies whose experimental design involved both plant resource competitive conditions and control of genetic background between compared resistant and susceptible ecotypes, the expression of fitness costs was almost always reported (Roux et al., 2004; Tardif et al., 2006; Pedersen et al., 2007; Menchari et al., 2008; Vila-Aiub et al., 2009).

5. Environmental gradient

The expression and magnitude of the fitness costs associated with resistance alleles may be environmentally specific (Heidel et al., 2004; Martin & Lenormand, 2006; Jessup & Bohannan, 2008). There is a general premise that fitness costs may be more evident when plants are growing under more extreme, stressful environmental conditions (Coley et al., 1985). Although this prediction is not always true (Bergelson & Purrington, 1996; Marak et al., 2003), it may be more relevant when a mechanism endowing herbicide resistance depends on limited environmental resources to operate. For instance, if resistance depends on herbicide sequestration or detoxification, or cellular processes that require the synthesis of constitutively expressed nitrogen-rich proteins, plant growth may be compromised in nitrogen-poor environments (Lerdau & Gershenzon, 1997; Baldwin et al., 1998). This knowledge may help managers to ‘design environments that potentially exploit functional weaknesses of herbicide-resistant weeds’ (Jordan et al., 1999).

The effects of temperature on the expression of fitness costs have been studied more than any other environmental factor. However, potential temperature-regulated costs have largely focused on seed germination responses. This is the case for AHAS resistance alleles, which have shown a strong correlation of higher germination rates in cool environments (Dyer et al., 1993; Thompson et al., 1994; Park et al., 2004) (see Section III). Overall, most fitness cost studies have not considered environmental variables as they have mostly been performed in controlled environmental conditions, often optimal for growth. A few publications have aimed to assess herbicide resistance-related fitness costs under contrasting light, nutrient and water conditions (Purrington & Bergelson, 1997; Ismail et al., 2002; Vila-Aiub et al., 2005b; Menalled & Smith, 2007; Menchari et al., 2008). When the control of genetic background was carried out, these reports recognized a significant impact of the environment on the expression of fitness costs (see Sections III and IV).

In the following sections, we have distilled the literature on the expression and magnitude of fitness costs associated with herbicide resistance alleles. We focus on studies that satisfy the requirements of the control of genetic background and a knowledge of the specific resistance mechanisms in the plants under study (other reports that corroborate findings in these studies are also discussed). When possible, if not reported, the magnitude of the fitness cost (%) was calculated as (1 − (resistant plant descriptor/susceptible plant descriptor) × 100).

III. Fitness costs associated with AHAS target site resistance

AHAS-inhibiting herbicides inhibit AHAS (also extensively referred to as ALS), a key plastidic enzyme responsible for the synthesis of the amino acids isoleucine (Ile), valine (Val) and leucine (Leu). In response to the widespread use of AHAS-inhibiting herbicides, many weed species have evolved target site-based resistance caused by mutations in the AHAS gene (reviewed by Saari et al., 1994; Tranel & Wright, 2002; Powles & Yu, in press). Resistance-endowing mutations at seven key amino acid residues [alanine-122 (Ala-122), proline-197 (Pro-197), Ala-205, aspartic acid-376 (Asp-376), tryptophan-574 (Trp-574), Ser-653 and Gly-654] in the AHAS gene have been documented (Table 1a,b).

Table 1.    (a) Acetohydroxyacid synthase (AHAS) mutations in evolved field-selected weed species in which fitness studies have been conducted. (b) Known AHAS mutations in evolved field-selected weed species remaining to be investigated for fitness costs
Amino acid residueCross-resistance to AHAS-inhibiting herbicide familiesWeed speciesFitness costReference
Wild-typeResistant
(a)
Pro-197HisSulfonylureaLactuca serriolaYesAlcocer-Ruthling et al. (1992a,b)
Trp-574LeuImidazolinone
Sulfonylurea
Pyrimidinylthiobenzoates
Triazolopyrimidines
Sulfonylaminocarbonyl-triazolinone
Amaranthus powelliiYesTardif et al. (2006)
(b)
Amino acid residueCross-resistance to AHAS-inhibiting herbicide familiesWeed species
Wild-typeResistant
Ala-122ThrImidazolinoneAmaranthus hybridus, A. powellii, A. retroflexus, Solanum ptycanthum, Xanthium strumarium
Pro-197Ala
Arg
Gln
His
Ile
Leu
Lys
Met
Ser
Thr
Trp
SulfonylureaSeveral weed species
Kochia scoparia, Lolium rigidum
K.
 scoparia, Lindernia spp., L. rigidum
Raphanus raphanistrum
Sisymbrium orientale

Many weed species
K. scoparia
K. scoparia
Many weed species
Many weed species
K. scoparia
Ala-205ValImidazolinone
Sulfonylurea
A. retroflexus, Helianthus annus, Solanum ptycanthum, Xanthium strumarium
Asp-376GluImidazolinone
Sulfonylurea
Pyrimidinylthiobenzoates
Triazolopyrimidines
Sulfonylaminocarbonyl-triazolinone
A. hybridus
Trp-574Arg
Leu
SulfonylureaK. scoparia
L. rigidum
Ser-653Thr
Asn
Ile
Imidazolinone
Imidazolinone
A. powellii, A. retroflexus, A. rudis
A. hybridus
, A. rudis, Setaria viridis
Gly-654Glu
Asp
ImidazolinoneOryza sativa
S. viridis

It is evident from studies with several resistant weed species that Pro-197 can be substituted by several amino acids to result in AHAS herbicide resistance (Yu et al., 2007b, 2008; Tranel et al., 2008) (Table 1a,b). Fitness consequences have only been examined for the Pro-197 to histidine (His) substitution. A field study examining changes in the phenotypic frequency of field-evolved, resistant Lactuca serriola individuals possessing the Pro-197–His allele (Guttieri et al., 1992) showed a decline of between 25% and 86% over 3 yr (Alcocer-Ruthling et al., 1992a). This fitness cost was evident as a 15% [estimated as an average over planting mixtures with equal proportion ratios (50 : 50, 100 : 100, 150 : 150)] reduction in vegetative biomass of resistant compared with susceptible L. serriola individuals growing under competitive conditions (Alcocer-Ruthling et al., 1992b).

Strong pleiotropic effects on plant morphology and anatomy, leading to a fitness cost, have been described in field-evolved, AHAS-resistant Amaranthus powellii with the Trp-574–Leu AHAS mutation (Tardif et al., 2006). An examination of several resistant A. powellii populations revealed that this mutation was associated with thinner roots and stems and a severe leaf area reduction, which led to a resistance cost of 67% (above-ground vegetative biomass) as well as a severe reduction in seed production (Tardif et al., 2006). Further evidence for a cost associated with AHAS resistance comes from imidazolinone-resistant rice crops with the Gly-654 to glutamic acid (Glu) AHAS mutation, which show 5–11% lower grain yield when compared with conventional rice cultivars (Sha et al., 2007).

For target site resistance, an amino acid substitution in the herbicide target site enzyme prevents herbicide binding. The mutation may also impair enzyme activity and/or reduce substrate affinity (resulting in insufficient product biosynthesis). Indeed, impaired AHAS activity appears to be the case for the Pro-197–His substitution in L. serriola and L. sativa, and the Ala-205–Val substitution in Solanum ptychanthum. In both cases, the resistant AHAS shows no altered substrate binding but a near 50% reduction in activity when compared with the wild-type (Eberlein et al., 1997, 1999; Ashigh & Tardif, 2007). Similarly, in yeast, Ala-122–Val and Ala-205–Val (Duggleby et al., 2003) and, in tobacco, Trp-574 to phenylalanine (Phe) (Chong et al., 1999) AHAS resistance substitutions result in a reduction in AHAS activity and substrate affinity. However, for other AHAS mutations, there is no evidence of a reduction in AHAS activity, with reports of no change in AHAS activity [Pro-197 to threonine (Thr)] (Preston et al., 2006) or, in other cases, significantly higher AHAS activity (Pro-197–Ser, Trp-574–Leu) (Boutsalis et al., 1999; Purrington & Bergelson, 1999; Yu et al., 2003) (but see Mourad et al., 1995). Obviously, the impact of each specific mutation/amino acid substitution needs to be evaluated on a case-by-case basis, and generalizations should not be made.

AHAS activity is feedback regulated by the accumulation of end-product branched-chain amino acids (Miflin & Cave, 1972). Although, for the Pro-197–Ser substitution, contradictory reports may be found (Tourneur et al., 1993; Mourad et al., 1995; Purrington & Bergelson, 1999), it is clear that the Pro-197–His (Eberlein et al., 1997), Pro-197–Thr (Preston et al., 2006) and Ala-205–Val (Ashigh & Tardif, 2007) AHAS mutations lead to a decreased sensitivity to feedback inhibition by Val, Leu and/or Ile.

A possible consequence of reduced feedback sensitivity to inhibition is the accumulation of branched-chain amino acids in plant tissues. An excess and/or imbalance of the amino acid pool can have toxic effects on cell metabolism, and correlates well with diminished plant growth (Höfgen et al., 1995). Higher concentrations of Val, Leu and Ile have been reported in leaves and seeds of plant species carrying AHAS mutations at the Pro-197 residue (Dyer et al., 1993; Eberlein et al., 1999; Purrington & Bergelson, 1999). Interestingly, this higher concentration of free amino acids is correlated with higher seed germination rates at relatively low temperatures (Dyer et al., 1993). Rapid germination at cool temperatures seems to be a characteristic trait associated with AHAS target site resistance (Pro-197–Ser/Arg/Thr), which, depending on the prevailing agroecological conditions, could turn into either a fitness advantage or disadvantage (Thompson et al., 1994; Park et al., 2004). Despite these reports, no published studies have examined and linked this particular germination response with seedling emergence and establishment processes.

Thus, it is clear that the His-197 mutation in L. serriola and the Leu-574 mutation in A. powellii are associated with a fitness cost (Alcocer-Ruthling et al., 1992a,b; Tardif et al., 2006). The origin of this cost may be either decreased AHAS activity or reduced feedback inhibition. These two explanations have compensating effects in terms of the quantity of branched-chain amino acid synthesis, and further research is required to discern the main driving factor for these fitness costs. For the Leu-574 mutation, no AHAS data are available. For other resistance-endowing AHAS mutations listed in Table 1b, their impact on plant fitness remains unknown and these studies are required. In addition, it is important to emphasize that, especially in Lolium, resistant plants frequently have a combination of two different resistance-endowing AHAS alleles (Yu et al., 2008), and fitness studies have not been conducted with such multiple field-selected resistant plants.

IV. Fitness costs associated with ACCase target site resistance

In the majority of monocot (but not dicot) species, the ACCase herbicides are potent inhibitors of the key plastidic enzyme ACCase. The inhibition of ACCase results in a lethal disruption of fatty acid synthesis, and many grass weed species have evolved ACCase herbicide resistance as a result of reduced herbicide sensitivity of ACCase (Devine & Shimabukuro, 1994; Délye, 2005). To date, eight amino acid substitutions have been shown to confer ACCase resistance in field-evolved weeds (Table 2a,b) (reviewed by Délye, 2005; Liu et al., 2007; Yu et al., 2007a; Powles & Yu, in press).

Table 2.    (a) Acetyl-CoA carboxylase (ACCase) mutations in evolved field-selected weed species in which fitness studies have been conducted. (b) Known ACCase mutations in evolved field-selected weed species remaining to be investigated for fitness costs
Amino acid residueCross-resistance to ACCase -inhibiting herbicide familiesWeed speciesFitness costReference
Wild-typeResistant
(a)
Ile-1781LeuAryloxyphenoxypropionates
Cyclohexanediones
Alopecurus myosuroides
Lolium rigidum
No
No
Menchari et al. (2008)
Vila-Aiub et al. (2005a,b)
Ile-2041AsnAryloxyphenoxypropionatesAlopecurus myosuroidesNoMenchari et al. (2008)
Asp-2078GlyAryloxyphenoxypropionates CyclohexanedionesAlopecurus myosuroides
Lolium rigidum
Yes
Yes
Menchari et al. (2008)
M. M. Vila-Aiub & S. B. Powles (unpublished)
Cys-2088ArgAryloxyphenoxypropionates CyclohexanedionesLolium rigidumYesM. M. Vila-Aiub & S. B. Powles (unpublished)
(b)
Amino acid residueCross-resistance to ACCase-inhibiting herbicide familiesWeed species
Wild-typeResistant
Trp-1999CysAryloxyphenoxypropionates (Fenoxaprop only)Avena sterilis
Trp-2027CysAryloxyphenoxypropionatesAlopecurus myosuroides
A. sterilis, A. fatua
Lolium rigidum
Phalaris minor
Ile-2041ValAryloxyphenoxypropionatesL. rigidum
Gly-2096AlaAryloxyphenoxypropionatesA. myosuroides
P. paradoxa

The resistance-endowing Ile-1781–Leu substitution has evolved in several weed species (reviewed by Délye, 2005; Zhang & Powles, 2006; Délye et al., 2007; Yu et al., 2007a; Powles & Yu, in press). This amino acid substitution does not impair ACCase function (Shukla et al., 1997; Délye et al., 2002; Yu et al., 2007a). Studies evaluating several fitness-related traits in L. rigidum and A. myosuroides with the Ile-1781–Leu mutation have shown no physiological or ecological resistance costs and no detectable reduction in plant productivity (Vila-Aiub et al., 2005a,b; Menchari et al., 2008). Interestingly, when introgressed into a Setaria italica population, the Ile-1781–Leu resistance gene has been shown to correlate with an increase in several fitness components, resulting in an increase in frequency of resistant genotypes over time (Wang & Darmency, 1997; Wang et al., 2009).

In one L. rigidum population, the Ile-1781–Leu ACCase mutation has been shown to be associated with a strong environmentally determined dormancy in seed, so that germination and emergence are promoted by light and fluctuating temperatures (Vila-Aiub et al., 2005b). Although this modification of germination dynamics does not represent a fitness cost per se, it may be exploited by agronomic practices to reduce weed infestations in cropping systems. Although this phenotypic response has proved to be a heritable trait, its occurrence across other genetic backgrounds needs to be assessed.

Menchari et al. (2008) evaluated fitness in several A. myosuroides populations with Ile-2041 to asparagine (Asn) and Asp-2078–Gly ACCase resistance-endowing mutations. In a 2-yr field experiment in which plants were grown in competition with wheat, there was clear evidence that Ile-2041–Asn was not associated with any adverse pleiotropic effects on vegetative and reproductive plant traits. However, for individuals with the Asp-2078–Gly mutation, substantially impaired growth was observed when plants were grown with a limited water supply. Individuals possessing the Asp-2078–Gly mutation displayed a reduction in height (6%) and vegetative (42%) and reproductive (36%) biomass when compared with the wild-type. However, this significant resistance cost was only observed in homozygote resistant plants (2078-Gly/2078-Gly), indicating that the fitness cost is recessive and environmentally dependent (Menchari et al., 2008). Similarly, the 2078-Gly resistance mutation in the homozygous state has adverse pleiotropic effects on resistant L. rigidum growth (M. M. Vila-Aiub & S. B. Powles, unpublished).

Moderate and strong reductions in ACCase-specific activity have been reported for the Ile-2041–Asn and Asp-2078–Gly mutations, respectively (Délye et al., 2003, 2005; Yu et al., 2007a). The greatly reduced ACCase activity in plants with the Asp-2078–Gly mutation probably explains the fitness cost of this mutation. Recently, a new ACCase resistance mutation [cysteine-2088 to arginine (Cys-2088–Arg)] has been identified in L. rigidum (Yu et al., 2007a). The ACCase activity of plants with this mutation is only one-half of that of wild-type plants (Yu et al., 2007a), and this impaired ACCase activity is probably responsible for the compromised growth observed at the whole-plant level (M. M. Vila-Aiub & S. B. Powles, unpublished).

The results reviewed above clearly show that the expression of fitness costs associated with ACCase resistance alleles is specific to the amino acid substitution conferring resistance, with different resistance substitutions in the same gene impacting very differently on enzyme functionality and associated pleiotropic effects. For several of the resistance-endowing ACCase resistance alleles, fitness studies have yet to be performed (Table 2b).

V. Fitness costs associated with resistance to phenoxy herbicides

Although the precise mode of action of these phenoxy-type herbicides remains unknown, they clearly disrupt endogenous auxin metabolism (reviewed by Coupland, 1994) and, for some field-selected, resistant weed species, there is a reduction in the binding affinity of these herbicides to auxin-binding proteins (Webb & Hall, 1995; Deshpande & Hall, 2000). However, despite decades of use, there have been few cases of evolved resistance to phenoxy herbicides (reviewed by Coupland, 1994). As a result, there are few unequivocal fitness studies in plant species that have evolved resistance to phenoxy herbicides. In a field study conducted in New Zealand, the frequency of 4-chloro-2-methylphenoxy acetic acid (MCPA)-resistant Ranunculus acris individuals declined by c. 5% yr−1 over 5 yr (Bourdôt et al., 1996). This study also reported a concurrent decline of c. 50% in the LD50 (1.41 kg MCPA ha−1) for a population removed from MCPA selection, in comparison with an R. acris population maintained under MCPA selection (LD50 = 2.13 kg MCPA ha−1). The molecular basis of resistance in this field-selected population has not been reported.

Investigating a Sinapsis arvensis population with evolved resistance to various phenoxy herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), dicamba, MCPA and picloram], Hall & Romano (1995) reported numerous pleiotropic effects on plant morphology and physiology. The resistant genotype showed a significant reduction in resource acquisition, leading to short and small plants with reduced leaf area and a less developed root system (Hall & Romano, 1995). Furthermore, higher chlorophyll and cytokinin levels were associated with resistance to auxinic herbicides in this species. The resistance genes endowing auxinic herbicide resistance in S. arvensis have not been elucidated, and potential confounding effects derived from using plant material with different genetic backgrounds are possible. However, the reported pleiotropic effects are similar to those observed in Arabidopsis thaliana carrying the AXR1 resistance gene (see Section VI).

VI.Arabidopsis thaliana as a model plant to understand the pleiotropic effects of herbicide target-site based resistance alleles

The model plant, A. thaliana, presents a number of intriguing opportunities to study the costs associated with herbicide resistance alleles. Known resistance alleles may be transgenically inserted into a standard genetic background, or ethyl methanesulfonate (EMS) mutagenesis may be used to create herbicide-resistant mutant lines. Fitness can be measured by assessing the seed production of transgenic, mutant and wild-type lines, and high-throughput genotyping can be used to confirm the genotype of individual plants. The results from these studies, together with a consideration of the advantages and limitations of these approaches, are reviewed here.

There have been attempts to determine the fitness costs associated with the Pro-197–Ser AHAS mutation in transgenic and EMS-mutagenized A. thaliana plants. Transgenic plants carrying this mutant allele display a reduction (26–34%) in the number of fruits per plant, but not in total plant biomass or seed germinability (Bergelson et al., 1996; Purrington & Bergelson, 1997; respectively). Roux et al. (2004) also found a trade-off (37% reduction) in the allocation of resources to reproduction in segregating A. thaliana mutants possessing the same Pro-197–Ser resistance allele. This reduction in seed production has a drastic effect on plant fitness, as shown by a significant decline in the frequency of the Ser-197 resistance allele over time in the absence of herbicide selection (Roux et al., 2006). This fitness cost has been shown to be greater in magnitude in nutrient-limited environments, probably as a consequence of a higher nitrogen demand driven by the effects of reduced feedback inhibition and promoted higher AHAS activity and amino acid biosynthesis (Bergelson, 1994; Purrington & Bergelson, 1997, 1999). However, the amino acid substitution Ser-653–Asn, which endows resistance to the AHAS-inhibiting herbicide, imazapyr, is not associated with impaired plant growth or seed germination rate in mutant A. thaliana (Roux et al., 2004).

Point mutations endowing resistance to auxin herbicides have been obtained through seed mutagenesis (EMS) in A. thaliana, and have been evaluated for fitness costs. The Gly-459–Asp mutation in the AUX1 gene has no adverse effects on plant fitness, despite changes in root morphology (Maher & Martindale, 1980; Roux et al., 2004, 2005a; Roux & Reboud, 2005). By contrast, the AXR1 (Cys-154–Tyr) and AXR2 (Pro-87–Ser) gene mutations express a severe fitness cost. Both resistance alleles have been shown to have pleiotropic effects on leaf morphology, hypocotyl length and plant height (Lincoln et al., 1990; Timpte et al., 1994, 1995), resulting in impaired plant resource acquisition and extensive resistance costs of 78% and 89% at the reproductive stage (Roux et al., 2004; Roux & Reboud, 2005).

Fitness costs have also been evaluated in three discrete laboratory-originated mutations endowing resistance to isoxaben (cellulose biosynthesis inhibitor) in A. thaliana (Roux et al., 2004). Although the Thr-942–Ile amino acid substitution in the cellulose synthase catalytic isoform CesA3 (Scheible et al., 2001) resulted in a 43% reduction in reproductive biomass, mutations in the CesA3 (Gly-998–Asp) and CesA6 (Arg-1064–Trp) alleles (Desprez et al., 2002) did not incur fitness costs (Roux et al., 2004, 2005a). It is noteworthy that this second mutation of the CesA3 gene is located only 56 amino acid positions upstream of the mutation that incurred a 43% reproductive cost.

The measurement of fitness costs in A. thaliana presents a number of opportunities that would not otherwise be available in less easily manipulated weed species. Nevertheless, some caution should be exercised in the interpretation of these results. Laboratory-derived mutants, by their nature, have not been selected in the field, and therefore mutations with severe fitness costs, as seen for AXR1 and AXR2 above, may have little relevance for the understanding of field-evolved resistance as these mutants could not and have not evolved in the field. EMS mutagenesis will create a range of other potentially deleterious mutations that will impact on fitness and, despite crossing programmes to attempt to disrupt linkage between resistance-endowing and other mutations, it is difficult to unequivocally attribute costs to the resistance allele. Transgenic approaches may be influenced by positional effects, so that fitness costs are dependent on where in the genome the transgene is inserted (Purrington & Bergelson, 1999). Finally, in field-evolved populations, there may have been some compensation of the cost of resistance during selection (see Discussion); this cannot occur in laboratory-derived mutants and may result in an overestimation of resistance costs.

VII. Fitness costs associated with herbicide resistance endowed by enhanced rates of herbicide metabolism catalysed by P450 enzymes

Cytochrome P450s are a large class of proteins involved in many biosynthetic functions in plants (reviewed in Schuler & Werck-Reichhart, 2003). In addition to their essential role in metabolism, plant P450 enzymes are paramount in detoxification pathways and can be responsible for herbicide detoxification (Werck-Reichhart et al., 2000). The current understanding of P450-mediated herbicide metabolism suggests that there are multiple P450 isoforms capable of metabolizing a specific herbicide, as well as P450 isoforms that may have broad herbicide specificity (Preston et al., 1996; Werck-Reichhart et al., 2000; Siminszky, 2006). However, the molecular determination of the specifics of P450 involvement in herbicide metabolism remains to be identified.

Many studies have identified L. rigidum populations that have P450-endowed resistance to ACCase, AHAS, PSII and other herbicide chemistries (Christopher et al., 1992; Burnet et al., 1994; McAlister et al., 1995; Preston et al., 1996). We have shown that individual L. rigidum plants with resistance to specific AHAS and ACCase herbicides endowed by cytochrome P450-based enhanced herbicide metabolism exhibit a nearly 20% reduction in vegetative growth (Vila-Aiub et al., 2005a). This reduction in resource acquisition corresponds to a reduced relative plant growth rate resulting from lower net assimilation rates (Vila-Aiub et al., 2005a). P450-based resistant individuals showed a significantly diminished ability to grow under competition with wheat, when compared with herbicide-susceptible plants. This ecological cost was expressed at both the vegetative (30%) and reproductive (23%) stages (Vila-Aiub et al., 2009).

The value of studies examining the cost of P450-based resistance is evident to validate wider ecological assumptions, such as the trade-off between plant growth and defence (Herms & Mattson, 1992; Vila-Aiub et al., 2009). However, no other studies have attempted to assess the fitness costs associated with P450-enhanced herbicide metabolism, and therefore there is limited appreciation of the fitness consequences of metabolism-based herbicide resistance. Given the importance of P450-based resistance and that plants must invest resources to produce P450 enzymes, much more work is required in this area.

VIII. Fitness costs associated with glyphosate resistance

Glyphosate is the world’s most widely used and important herbicide (Duke & Powles, 2008). The widespread adoption of glyphosate-resistant crops in North and South America has resulted in many populations of glyphosate-resistant weeds (reviewed by Powles, 2008). In many weed populations, resistance to glyphosate correlates with reduced rates of glyphosate translocation to active meristematic root and shoot tissues (Powles & Preston, 2006; Preston & Wakelin, 2008; Shaner, 2009). Lolium rigidum individuals with this resistance mechanism exhibited no reduction in vegetative growth under resource competition with wheat when compared with the susceptible genotype (Pedersen et al., 2007). The same study reported that glyphosate-resistant plants produced fewer but larger seeds under very low competition intensity from wheat (Pedersen et al., 2007). The observed reduction in the number of progeny, when competing with wheat, may explain the drastic decline in the resistance phenotypic frequency from 45% to 11% after 3 yr of relaxed glyphosate selection (Preston & Wakelin, 2008).

Ipomoea purpurea has been shown to exhibit high levels of genetic variation associated with glyphosate tolerance (Baucom & Mauricio, 2004, 2008). Interestingly, a negative correlation shows that the most glyphosate-tolerant individuals show the highest cost in fitness (Baucom & Mauricio, 2004).

Glyphosate resistance may also be endowed by a mutation in the target gene [5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase)] (reviewed by Powles & Preston, 2006; Powles & Yu, in press). Thus far, there have been no published studies of the effect of EPSP synthase gene mutations on the fitness performance of resistant plants. Given the importance of glyphosate in world agriculture and the number of weed species displaying glyphosate resistance, it is crucial that studies be conducted on the impact on fitness of resistance-endowing EPSP synthase gene mutations.

IX. Discussion

1. Towards improved protocols for the estimation of herbicide resistance fitness costs

The large body of research aimed at the detection and quantification of herbicide resistance fitness costs indicates the recognition of the importance of this knowledge for the understanding and predicting of the dynamics of resistance evolution and management. Ultimately, resistance may be an inevitable consequence of herbicide use, yet fitness costs can considerably slow the evolution of resistance, and these costs and their impacts on plant life histories can be manipulated to mitigate risks of resistance. Notwithstanding this, the literature contains many studies that have misunderstood, mismeasured or misinterpreted costs of resistance. Bergelson & Purrington (1996) highlighted that very few studies examining the fitness costs of herbicide resistance controlled or minimized the differences in genetic background between the herbicide-resistant and herbicide-susceptible plants under study. Unfortunately, this review, some 14 yr later, has established that researchers conducting fitness studies often continue to ignore the importance of genetic background.

In Section II, we presented five criteria that should inform the design and interpretation of future studies to determine the costs of herbicide resistance alleles. It will not always be possible or practical to fulfil all of these. However, studies which do not control for genetic background and have little knowledge of the mechanism of resistance will be of limited value in ascribing the origin of fitness costs to particular resistance genes and mutations. Beyond these basic requirements, studies should estimate fitness costs in realistic field conditions under which resistance evolved, and consider fitness costs and implications throughout the plant life cycle. Fortunately, in recent years, a relatively small number of unequivocal fitness cost studies have been conducted. We believe that this trend, if it persists, will allow a systematic evaluation of fitness costs which, ultimately, will be of potential significance for the management of resistance and for providing answers to fundamental questions in plant evolutionary ecology.

2. Future advances in the understanding the costs of herbicide resistance

Routine consideration of the five criteria in Section II will considerably advance the studies of herbicide resistance costs. In future, as molecular tools are developed for weed species, further hypotheses relating to the ‘cost of adaptation’ may be addressed, both for their benefit in terms of resistance management and for their ability to answer more fundamental questions in plant adaptation. Some of these future research areas are outlined below.

The importance of a homogenized genetic background for the measurement of fitness costs has been argued. In selfing species, resistance alleles will arise in a variety of genetic backgrounds and, for outcrossing species, the genetic background in which the resistance allele occurs may vary between individuals within a single population. It is likely that the magnitude and expression of fitness costs will vary between different genetic backgrounds, presenting the possibility that fitness costs will vary between populations and between individuals (Paris et al., 2008). These effects should be investigated. Furthermore, in outcrossing species, there may be selection so that resistance alleles are preferentially expressed in backgrounds in which the cost is lowest. Evolutionary forces will select for genetic backgrounds in which the costs are minimized through: (1) the acquisition of modifiers (i.e. new alleles at other loci) which compensate and moderate pleiotropic costs (Paris et al., 2008); (2) the replacement of alleles, which involves the substitution of the costly allele by a less costly one at the locus involved in resistance; and (3) increased resistance to biotic stresses (parasitism) (Agnew et al., 2004). These amelioration processes will lead to a reduction in the magnitude of fitness costs over plant generations, and represent one form of compensatory evolution such that the costs of resistance evolve over time (resistance co-adaptation). The potential for resistance co-adaptation is poorly understood for herbicide resistance, although it has been demonstrated for insecticide resistance (McKenzie & Clarke, 1988; Bourguet et al., 2004) and antibiotic resistance (Maisnier-Patin & Andersson, 2004; Paulander et al., 2007). These processes create the opportunity for the co-adaptation and integration of the new resistance allele into the genome (Fisher, 1928; Cohan et al., 1994; Guillemaud et al., 1998).

Only a few studies have considered the dominance of the herbicide resistance cost. In the field, new herbicide resistance alleles which arise by spontaneous mutation will occur as heterozygotes, and therefore it is most important to understand the fitness consequences of resistance alleles in the heterozygous state. If the cost of resistance is recessive (i.e. only evident in homozygous individuals), the fitness cost will have little impact on the dynamics of resistance in the early stages of selection where most individuals are heterozygous. Using EMS-mutagenized A. thaliana, Roux et al. (2004) demonstrated recessive, dominant and overdominant herbicide resistance fitness costs.

Herbicide resistance-endowing mutations are likely to occur as independent events, and this may result in the accumulation of diverse herbicide resistance alleles within single individuals under herbicide selection (Hall et al., 1994; Tardif & Powles, 1994; Preston et al., 1996). From a fitness cost viewpoint, an interesting question is to consider the epistatic effects (e.g. multiplicative, additive, synergistic and compensatory) of two or more different resistance alleles on the fitness cost when there are multiple resistance alleles within an individual. A study with the model plant A. thaliana has revealed multiplicative epistatic effects on several fitness traits in double and triple mutants expressing the ALS (Pro-197–Ser), 2,4-D (Cys-154–Tyr) and isoxaben (Thr-942–Ile) resistance alleles (Roux et al., 2005b). This result predicts that the frequency decline of multiple resistance alleles in a herbicide-free environment would be faster than when epistatic interactions among resistance alleles are not evident.

3. The evidence for pleiotropic effects of herbicide resistance alleles

There is unquestionable evidence, as reviewed here, that some herbicide resistance alleles are associated with fitness costs. The observed costs are associated with target site resistance [resistance-endowing amino acid substitutions in proteins involved in amino acid (Section III), fatty acid (Section II), auxin (Section V) and cellulose (Section VI) biosynthesis], as well as nontarget site resistance caused by enhanced rates of herbicide metabolism (Section VII) and transport (Section VIII). However, resistance costs are not universal and thus must be assessed on a case-by-case basis. The fitness costs associated with target site resistance generally result from a compromise in normal enzyme function, whereas the costs associated with metabolic resistance are thought to arise from the allocation of resources to defence rather than growth (Coley et al., 1985; Herms & Mattson, 1994; Strauss et al., 2002; Vila-Aiub et al., 2009).

4. Evolution of herbicide resistance alleles and their fitness costs

Theoretical models predict that resistance alleles with the largest fitness costs are least likely to be fixed (Gillespie, 1975). This prediction appears to be confirmed for some ACCase gene mutations, which show a negative correlation between the magnitude of the fitness costs and their frequency in agroecosystems. An extensive survey in France has revealed that the Asp-2078–Gly ACCase mutation is the least frequent resistance allele in A. myosuroides (Délye et al., 2007). This mutation has been shown to be associated with impaired plant performance at the enzyme and whole-plant level, causing the mutation to be negatively selected in the absence of ACCase herbicide selection pressure. By contrast, the Ile-1781–Leu ACCase mutation in A. myosuroides has no detectable pleiotropic effect on plant fitness (Menchari et al., 2008), and has been found to be the most common resistance allele in field crops (Délye et al., 2007). Similar results have been found for both the Asp-2078–Gly and Ile-1781–Leu resistance alleles in L. rigidum (Vila-Aiub et al., 2005a; Owen et al., 2007; Yu et al., 2007a). The presence of the Ile-1781–Leu ACCase mutation in more weed species than any other ACCase allele, as well as its fixation in naturally resistant species, is a good indication of its lack of fitness cost (Délye, 2005; Délye & Michel, 2005; Liu et al., 2007; Yu et al., 2007a).

The same analogy may be applied to the Pro-197–Ser and Trp-574–Leu AHAS mutations, which have been reported to exhibit moderate and severe pleiotropic effects, respectively, on A. thaliana and A. powellii fitness (Bergelson et al., 1996; Roux et al., 2004, 2005a; Tardif et al., 2006). Although the former has been reported to be the most common resistance allele in several Raphanus raphanistrum populations from Australia, the latter has not evolved within this species to date (L. J. S. Friesen & S. B. Powles, unpublished).

X. Conclusion

The fitness costs associated with herbicide resistance alleles are evident, but not universal, among plant species. Their expression and magnitude are strongly influenced by the abiotic, biotic and genetic environment. Future research efforts should compartmentalize the effects of these ‘environments’ by assessing the pleiotropic effects of resistance alleles on plant fitness with appropriate control of genetic background and, ideally, where the molecular basis of resistance is known. These studies should integrate molecular, physiological and ecological techniques to measure fitness at all life history stages and in a variety of biotic and abiotic environments. Field experiments that record changes in resistance allele frequencies in the absence of herbicide selection should complement these studies. Only then will it be possible to systematically elucidate whether and under which environmental conditions herbicide resistance alleles express fitness costs.

Acknowledgements

The Western Australian Herbicide Resistance Initiative (WAHRI) is funded by the Australian Grains Research and Development Corporation (GRDC). We thank many of our colleagues for useful comments on manuscript drafts, including Drs Roberto Busi, Henry Darmency, Christophe Délye, Jacques Gasquez, Xavier Reboud, Michael Walsh and Qin Yu.

Ancillary