Local adaptation in the Linum marginale-Melampsora lini host-pathogen interaction

The potential for local adaptation between pathogens and their hosts has generated strong theoretical and empirical interest with evidence both for and against local adaptation reported for a range of systems. We use the Linum marginale-Melampsora lini plant-pathogen system and a hierarchical spatial structure to investigate patterns of local adaptation within a metapopulation characterised by epidemic dynamics and frequent extinction of pathogen populations. Based on large sample sizes and comprehensive cross-inoculation trials, our analyses demonstrate strong local adaptation by Melampsora to its host populations, with this effect being greatest at regional scales, as predicted from the broader spatial scales at which M. lini disperses relative to L. marginale. However, there was no consistent trend for more distant pathogen populations to perform more poorly. Our results further show how the coevolutionary interaction between hosts and pathogens can be influenced by local structure such that resistant hosts select for generally virulent pathogens, while susceptible hosts select for more avirulent pathogens. Empirically, local adaptation has generally been tested in two contrasting ways: (1) pathogen performance on sympatric versus allopatric hosts; and (2) sympatric versus allopatric pathogens on a given host population. In situations where no host population is more resistant or susceptible than others when averaged across pathogen populations (and likewise, no pathogen population is more virulent or avirulent than others), results from these tests should generally be congruent. We argue that this is unlikely to be the case in the metapopulation situations that predominate in natural host-pathogen interactions, thus requiring tests that control simultaneously for variation in plant and pathogen populations.     

Spatial scale of local adaptation in a plant-pathogen metapopulation

The rate and scale of gene flow can strongly affect patterns of local adaptation in host-parasite interactions. I used data on regional pathogen occurrence to infer the scale of pathogen dispersal and to identify pathogen metapopulations in the interaction between Plantago lanceolata and its specialist phytopathogen, Podosphaera plantaginis. Frequent extinctions and colonizations were recorded in the metapopulations, suggesting substantial gene flow at this spatial scale. The level of pathogen local adaptation was assessed in a laboratory inoculation experiment at three different scales: in sympatric host populations, in sympatric host metapopulations and in allopatric host metapopulations. I found evidence for adaptation to sympatric host populations, as well as evidence indicating that local adaptation may extend to the scale of the sympatric host metapopulation. There was also variation among the metapopulations in the degree of pathogen local adaptation. This may be explained by regional differences in the rate of migration.     

Does selection by resistant hosts trigger local adaptation in plant–pathogen systems?

Understanding the consequences of selection by host resistance on pathogen population structure provides useful insights into the dynamics of host-parasite co-evolution processes and is crucial for effective disease management through resistant cultivars. We tested general vs. local population adaptation to host cultivars, by characterizing a French collection of Phytophthora infestans (the causal organism of potato late blight) sampled during two consecutive years on cultivars exhibiting various levels of resistance. Local populations were structured by the host for virulence (qualitative pathogenicity) but also for aggressiveness (quantitative pathogenicity). All populations had a low genotypic diversity for amplified fragment length polymorphisms (AFLPs), and presumably consisted of a few closely related clonal lineages. No correlation was detected between pathogenicity traits and AFLP genotypes. The data support the hypothesis of general adaptation for aggressiveness, to which directional selection for virulence is superimposed when race-specific resistance is introduced.     

Detecting local adaptation in a natural plant-pathogen metapopulation: a laboratory vs. field transplant approach

Antagonistic coevolution between hosts and parasites in spatially structured populations can result in local adaptation of parasites. Traditionally parasite local adaptation has been investigated in field transplant experiments or in the laboratory under a constant environment. Despite the conceptual importance of local adaptation in studies of (co)evolution, to date no study has provided a comparative analysis of these two methods. Here, using information on pathogen population dynamics, I tested local adaptation of the specialist phytopathogen, Podosphaera plantaginis, to its host, Plantago lanceolata at three different spatial scales: sympatric host population, sympatric host metapopulation and allopatric host metapopulations. The experiment was carried out as a field transplant experiment with greenhouse-reared host plants from these three different origins introduced into four pathogen populations. In contrast to results of an earlier study performed with these same host and parasite populations under laboratory conditions, I did not find any evidence for parasite local adaptation. For interactions governed by strain-specific resistance, field studies may not be sensitive enough to detect mean parasite population virulence. Given that parasite transmission potential may be mediated by the abiotic environment and genotype-by-environment interactions, I suggest that relevant environmental variation should be incorporated into laboratory studies of parasite local adaptation.     

Coevolution at multiple spatial scales: Linum marginale–Melampsora lini – from the individual to the species

Coevolutionary processes are intrinsically spatial as well as temporal, and occur at many different scales. These range from single populations dominated by demographic and genetic stochasticity, to metapopulations in which colonisation/extinction dynamics have a large influence, and larger geographic regions where phylogenetic patterns and historical events become important. We present data for the genetically and demographically well-characterised plant host–pathogen interaction, the Linum marginale–Melampsora lini system, and use this to demonstrate the varying nature of resistance and virulence structure across these spatial scales. At the within population level, our results indicate considerable variability in resistance and virulence, but little evidence of coordinated changes in host and pathogen. Studies involving comparisons among multiple demes within a single metapopulation show that adjacent populations often have asynchronous disease dynamics and large differences in diversity and frequency of resistance and virulence phenotypes. Nevertheless, at this scale, there is also evidence of spatial structure in that more closely adjacent host populations are significantly more likely to have similar resistance phenotypes and mean levels of resistance. At larger scales, comparisons among adjacent metapopulations indicate that quantitative differences in host mating system and other life history features can have further major consequences for how host and pathogen variation is packaged. Finally, comparisons at continental and among host-species levels show variation consistent with specialisation and speciation in the pathogen. This revised version was published online in July 2006 with corrections to the Cover Date.     

Host ecotype generates evolutionary and epidemiological divergence across a pathogen metapopulation

The extent and speed at which pathogens adapt to host resistance varies considerably. This presents a challenge for predicting when—and where—pathogen evolution may occur. While gene flow and spatially heterogeneous environments are recognized to be critical for the evolutionary potential of pathogen populations, we lack an understanding of how the two jointly shape coevolutionary trajectories between hosts and pathogens. The rust pathogen Melampsora lini infects two ecotypes of its host plant Linum marginale that occur in close proximity yet in distinct populations and habitats. In this study, we found that within-population epidemics were different between the two habitats. We then tested for pathogen local adaptation at host population and ecotype level in a reciprocal inoculation study. Even after controlling for the effect of spatial structure on infection outcome, we found strong evidence of pathogen adaptation at the host ecotype level. Moreover, sequence analysis of two pathogen infectivity loci revealed strong genetic differentiation by host ecotype but not by distance. Hence, environmental variation can be a key determinant of pathogen population genetic structure and coevolutionary dynamics and can generate strong asymmetry in infection risks through space.     

Host resistance and pathogen infectivity in host populations with varying connectivity

Theory predicts that hosts and pathogens will evolve higher resistance and aggressiveness in systems where populations are spatially connected than in situations in which populations are isolated and dispersal is more local. In a large cross‐inoculation experiment we surveyed patterns of host resistance and pathogen infectivity in anther‐smut diseased Viscaria alpina populations from three contrasting areas where populations range from continuous, through patchy but spatially connected to highly isolated demes. In agreement with theory, isolated populations of V. alpina were more susceptible on average than either patchily distributed or continuous populations. While increased dispersal in connected systems increases disease spread, it may also increase host gene flow and the potential for greater host resistance to evolve. In the Viscaria–Microbotryum system, pathogen infectivity mirrored patterns of host resistance with strains from the isolated populations being the least infective and strains from the more resistant continuous populations being the most infective on average, suggesting that high resistance selects for high infectivity. To our knowledge this study is the first to characterize the impacts of varying spatial connectivity on patterns of host resistance and pathogen infectivity in a natural system.     

Local adaptation in a plant herbivore interaction depends on the spatial scale

Local adaptation has central importance in the understanding of co-evolution, maintenance of sexual reproduction, and speciation. We investigated local adaptation in the alkaloid-bearing legume Crotalaria pallida and its seed predator, the arctiid moth Utetheisa ornatrix , at different spatial scales. When we studied three populations from south-east Brazil (150 km apart), we did not find evidence of local adaptation, although we did find interpopulational differences in herbivore performance, and a significant interaction between herbivore sex and plant population. These results indicate that both moth and plant populations are differentiated at the regional scale. In a comparison of populations from Brazil and Florida, the herbivore showed local adaptation to its host plant; for both moth populations, the pupae were heavier when the larvae ate the sympatric than the allopatric host population. We discuss the scale dependence of our results and the possible causes for the lack of local adaptation at the regional scale, even in the presence of plant and moth differentiation. The results obtained demonstrate the importance of studying co-evolution and local adaptation at different geographical scales.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 494–502.     

LOCAL MALADAPTATION IN THE ANTHER-SMUT FUNGUS MICROBOTRYUM VIOLACEUM TO ITS HOST PLANT SILENE LATIFOLIA: EVIDENCE FROM A CROSS-INOCULATION EXPERIMENT

Conventional wisdom holds that parasites evolve more rapidly than their hosts and are therefore locally adapted, that is, better at exploiting sympatric than allopatric hosts. We studied local adaptation in the insect-transmitted fungal pathogen Microbotryum violaceum and its host plant Silene latifolia. Infection success was tested in sympatric (local) and allopatric (foreign) combinations of pathogen and host from 14 natural populations from a metapopulation. Seedlings from up to 10 seed families from each population were exposed to sporidial suspensions from each of four fungal strains derived from the same population, from a near-by population (< 10 km distance), and from two populations at an intermediate (< 30 km) and remote (< 170 km) distance, respectively. We obtained significant pathogen X plant interactions in infection success (proportion of diseased plants) at both fungal population and strain level. There was an overall pattern of local maladaptation of this pathogen: average fungal infection success was significantly lower on sympatric hosts (mean proportion of diseased plants = 0.32 ± 0.03 SE) than on allopatric hosts (0.40 ± 0.02). Five of the 14 fungal populations showed no strong reduction in infection success on sympatric hosts, and three even tended to perform better on sympatric hosts. This pattern is consistent with models of time-lagged cycles predicting patterns of local adaptation in host-parasite systems to emerge only on average. Several factors may restrict the evolutionary potential of this pathogen relative to that of its host. First, a predominantly selfing breeding system may limit its ability to generate new virulence types by sexual recombination, whereas the obligately outcrossing host 5. latifolia may profit from rearrangement of resistance alleles by random mating. Second, populations often harbor only a few infected individuals, so virulence variation may be further reduced by drift. Third, migration rates among host plant populations are much higher than among pathogen populations, possibly because pollinators prefer healthy over diseased plants. Migration among partly isolated populations may therefore introduce novel host plant resistance variants more often than novel parasite virulence variants. That migration contributes to the coevolutionary dynamics in this system is supported by the geographic pattern of infectivity. Infection success increased over the first 10–km range of host-pathogen population distances, which is likely the natural range of gene exchange.     

Ecological genetic interactions between a clonal host plant (Spartina pectinata) and associated rust fungi Puccinia seymouriana and Puccinia sparganioides

The spatial scale of genetic diversity among patches of a host plant could affect the likelihood of pathogen adaptation to the host. If host patches are genetically distinct, pathogen adaptation to local host genotypes may occur. To study this issue, we focused on the ecological and genetic interactions between two rust fungi, Puccinia seymouriana and P. sparganioides, and the clonal prairie grass, Spartina pectinata. In a field transplant experiment, disease levels differed among plants from different patches, suggesting variation in resistance. Over a 4.5-km scale, disease levels were not higher on plants transplanted back into their source patch as opposed to other locations, providing no evidence for local adaptation in the pathogen. However, on the spatial scales examined (ranging from 0.2 km to 120 km), there was no relationship between the physical distance separating host patches and their similarity in isozyme banding patterns, implying that plants from more distant patches are not necessarily more genetically distinct than plants from nearby patches. Plants derived from the most distant location had, on average, the lowest mean number of pustules at the end of the summer, suggesting the need for reciprocal transplant studies to be performed on a larger spatial scale.     

Variation in aggressiveness components of Zymoseptoria tritici populations in Iran

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, has emerged and evolved as a pathogen on wheat in the Fertile Crescent. Iran is located in this region, and study on the ancient pathogen populations in the country can improve our understanding on adaptive potential of aggressiveness, the role of local adaptation in shaping population structure and the involvement of selection and genetic drift in favouring aggressiveness adaptation to environments. To this aim, three aggressiveness components including days until first lesion (DUFL), days until first pycnidia (DUFP) and percentage of leaf area covered by pycnidia, in five populations of the pathogen collected from different provinces of Iran were compared in greenhouse conditions. All populations except for Golestan showed high within‐population diversity for the examined traits. No difference in aggressiveness components was found between fungal collections from Khuzestan and Fars; however, significant variation was evident among the populations originated from other provinces. Comparisons of estimated Q_ST values to F_ST indicated that genetic differentiation in pycnidial coverage has been the result of selection imposed by different variables; however, the divergence found for DUFL and DUFP has been achieved by genetic drift. The possible mechanisms involved in aggressiveness diversity of the pathogen populations and the impact of these findings on breeding programs for quantitative resistance are discussed.     

Molecular Genetic Diversity and Variation for Aggressiveness in Populations of Fusarium graminearum and Fusarium culmorum Sampled from Wheat Fields in Different Countries

Fusarium graminearum and Fusarium culmorum are the major pathogenic organisms causing head blight in small-grain cereals. Natural epidemics may result in severe yield losses, reduction in quality, and contamination of the grain by mycotoxins. The genetic diversity of four field populations of F. graminearum from Germany, Hungary, and Canada, and one population of F. culmorum from Russia was investigated by polymerase chain reaction (PCR)-based fingerprinting. Additionally, a world-wide collection and two of the F. graminearum populations were analysed for their aggressiveness on young plants of winter rye in the greenhouse. The number of isolates analysed per population varied from 25 to 70. Significant quantitative variation for aggressiveness was observed within each of the individual field populations amounting to the same range as the world-wide collection. Abundant variation within populations was also revealed by DNA markers. The F. graminearum populations from Hungary and Winnipeg displayed the least genotypic diversity, the two German F. graminearum populations and the Russian F. culmorum population were highly diverse. Population diversity, however, followed no spatial pattern among samples within a German field for aggressiveness or molecular markers. For F. graminearum , sexual recombination is the most likely explanation for the large genetic diversity within field populations. Asexual and/or parasexual recombination, and balancing selection caused by the periodic alternation between the saprophytic and parasitic phase might play an additional role and account for the variation within the F. culmorum population. For improving Fusarium resistance, several resistance genes of different sources should be combined to avoid an unspecific adaptation of the genetically variable pathogen to an increased resistance level.     

Local adaptation and effect of host genotype on the rate of pathogen evolution: an experimental test in a plant pathosystem

Abstract Virulence is thought to be a driving force in host–pathogen coevolution. Theoretical models suggest that virulence is an unavoidable consequence of pathogens evolving towards a high rate of intrahost reproduction. These models predict a positive correlation between the reproductive fitness of a pathogen and its level of virulence. Theoretical models also suggest that the demography and genetic structure of a host population can influence the evolution of virulence. If evolution occurs faster in pathogen populations than in host populations, the predicted result is local adaptation of the pathogen population. In our studies, we used a combination of molecular and physiological markers to test these hypotheses in an agricultural system. We isolated five strains of the fungal pathogen Mycosphaerella graminicola from each of two wheat cultivars that differed in their level of resistance to this pathogen. Each of the 10 fungal strains had distinct genotypes as indicated by different DNA fingerprints. These fungal strains were re‐inoculated onto the same two host cultivars in a field experiment and their genotype frequencies were monitored over several generations of asexual reproduction. We also measured the virulence of these 10 fungal strains and correlated it to the reproductive fitness of each fungal strain. We found that host genotypes had a strong impact on the dynamics of the pathogen populations. The pathogen population collected from the moderately resistant cultivar Madsen showed greater stability, higher genotype diversity, and smaller selection coefficients than the pathogen populations collected from the susceptible cultivar Stephens or a mixture of the two host cultivars. The pathogen collection from the mixed host population was midway between the two pure lines for most parameters measured. Our results also revealed that the measures of reproductive fitness and virulence of a pathogen strain were not always correlated. The pathogen strains varied in their patterns of local adaptation, ranging from locally adapted to locally maladapted.     

Experimental evolution of local parasite maladaptation

Sign and magnitude of local adaptation in host–parasite systems may vary with ecological, epidemiological or genetic parameters. To investigate the role of host genetic background, we established long‐term experimental populations of different genotypes of the protozoan Paramecium caudatum, infected with the bacterial parasite Holospora undulata. We observed the evolution of an overall pattern of parasite local maladaptation for infectivity, indicating a general coevolutionary disadvantage of this parasite. Maladaptation extended to host populations with the same genetic background, similar to extending from the local to a higher regional level in natural populations. Patterns for virulence were qualitatively similar, but with less statistical support. A nonsignificant correlation with levels of (mal)adaptation for infectivity suggests independent evolution of these traits. Our results indicate similar (co)evolutionary trajectories in populations with different genetic backgrounds. Nonetheless, the correlated clines of genetic distance and parasite performance illustrate how genetic background can shape spatial gradients of local adaptation.     

Plant–parasite coevolution: A weak signature of local adaptation between Peruvian Globodera pallida populations and wild potatoes

Plant–parasite coevolution has generated much interest and studies to understand and manage diseases in agriculture. Such a reciprocal evolutionary process could lead to a pattern of local adaptation between plants and parasites. Based on the phylogeography of each partner, the present study tested the hypothesis of local adaptation between the potato cyst nematode Globodera pallida and wild potatoes in Peru. The measured fitness trait was the hatching of cysts which is induced by host root exudates. Using a cross‐hatching assay between 13 populations of G. pallida and root exudates from 12 wild potatoes, our results did not show a strong pattern of local adaptation of the parasite but the sympatric combinations induced better hatching of cysts than allopatric combinations, and there was a negative relationship between the hatching percentage and the geographical distance between nematode populations and wild potatoes. Moreover, a strong effect of the geographic origin of root exudates was found, with root exudates from south of Peru inducing better hatching than root exudates from north of Peru. These results could be useful to develop new biocontrol products or potato cultivars to limit damages caused by G. pallida.     

Host-parasite local adaptation after experimental coevolution of Caenorhabditis elegans and its microparasite Bacillus thuringiensis

Coevolving hosts and parasites can adapt to their local antagonist. In studies on natural populations, the observation of local adaptation patterns is thus often taken as indirect evidence for coevolution. Based on this approach, coevolution was previously inferred from an overall pattern of either parasite or host local adaptation. Many studies, however, failed to detect such a pattern. One explanation is that the studied system was not subject to coevolution. Alternatively, coevolution occurred, but remained undetected because it took different routes in different populations. In some populations, it is the host that is locally adapted, whereas in others it is the parasite, leading to the absence of an overall local adaptation pattern. Here, we test for overall as well as population-specific patterns of local adaptation using experimentally coevolved populations of the nematode Caenorhabditis elegans and its bacterial microparasite Bacillus thuringiensis. Furthermore, we assessed the importance of random interaction effects using control populations that evolved in the absence of the respective antagonist. Our results demonstrate that experimental coevolution produces distinct local adaptation patterns in different replicate populations, including host, parasite or absence of local adaptation. Our study thus provides experimental evidence of the predictions of the geographical mosaic theory of coevolution, i.e. that the interaction between parasite and host varies across populations.     

I. Pathogenic variation in fungi and bacteria and mycorrhizal compatibility: The genetic architecture of aggressiveness in Ustilago maydis

Isolates of Ustilago maydis (D.C.) Cda are known to differ in the degree to which they can parasitise maize and, as no differential reaction occurs between pathogen and host, these differences must be differences in aggressiveness. Inoculation of juvenile maize plants induced morphological changes which provided a means of assessing and distinguishing U. maydis isolates in respect of their aggressiveness. Thus it was possible to carry out a survey of the genetic architecture of aggressiveness in natural populations of the pathogen. The distribution of aggressiveness within a single gall suggested that selection had taken place over a number of generations in favour of genes or gene systems tending to increase aggressiveness. Diallele analyses of isolates from two natural populations revealed that aggressiveness had a low heritability and was determined by genes exhibiting mainly non-allelic interaction. It has been proposed that horizontal resistance in host cultivars would have a more long lasting protective effect against disease. However, the genetic architecture of aggressiveness in U. maydis should enable the pathogen to respond rapidly to changes in host resistance and the skewed distribution found within a single gall tends to support this. Therefore, the use of higher levels of host resistance should rapidly select for higher aggressiveness in the pathogen.     

Fine-scale genetically based differentiation of life-history traits in the perennial shrub Lupinus arboreus

Across large spatial scales, plants often exhibit genetically based differentiation in traits that allow adaptation to local sites. At smaller spatial scales, sharp boundaries between edaphic conditions also can create strong gradients in selection that counteract gene flow and result in local adaptation. Few studies, however, have examined the degree to which continuous populations of perennial plants exhibit genetically based differentiation in life-history traits over small spatial scales. We quantified the degree of genetically based differentiation in adaptive traits among bush lupine (Lupinus arboreus) from nearby dune and grassland sites (sites separated by < 0.75 km) that formed part of a larger continuous population of L. arboreus. We also investigated the spatial genetic structure of bush lupine by examining how genetic structure differed between seeds and juvenile plants that were less than two years old. We calculated F-statistics from gel electrophoresis of 10 polymorphic loci. We then used these values to infer levels of gene flow. To examine differentiation in adaptive traits, we created full-sibling/half-sibling families of lupine within each area and established reciprocal common gardens at each site. Across two years, we measured canopy volume, flowering time, seed set, and mortality of progeny planted in each garden. Spatial genetic structure among seeds was virtually nonexistent (F(ST) = 0.002), suggesting that gene flow between the three areas could be quite high. However, genetic structure increased 20-fold among juvenile plants (F(ST) = 0.041). We found strong evidence for fine-scale genetically based differentiation and local adaptation in adaptive traits such as plant size, flowering phenology, fecundity, and mortality. Thus, it is likely that strong but differing selection regimes within each area drive spatial differentiation in lupine life-history traits.     

Variation in infectivity and aggressiveness in space and time in wild host-pathogen systems: causes and consequences

Variation in host resistance and in the ability of pathogens to infect and grow (i.e. pathogenicity) is important as it provides the raw material for antagonistic (co)evolution and therefore underlies risks of disease spread, disease evolution and host shifts. Moreover, the distribution of this variation in space and time may inform us about the mode of coevolutionary selection (arms race vs. fluctuating selection dynamics) and the relative roles of G × G interactions, gene flow, selection and genetic drift in shaping coevolutionary processes. Although variation in host resistance has recently been reviewed, little is known about overall patterns in the frequency and scale of variation in pathogenicity, particularly in natural systems. Using 48 studies from 30 distinct host–pathogen systems, this review demonstrates that variation in pathogenicity is ubiquitous across multiple spatial and temporal scales. Quantitative analysis of a subset of extensively studied plant–pathogen systems shows that the magnitude of within‐population variation in pathogenicity is large relative to among‐population variation and that the distribution of pathogenicity partly mirrors the distribution of host resistance. At least part of the variation in pathogenicity found at a given spatial scale is adaptive, as evidenced by studies that have examined local adaptation at scales ranging from single hosts through metapopulations to entire continents and – to a lesser extent – by comparisons of pathogenicity with neutral genetic variation. Together, these results support coevolutionary selection through fluctuating selection dynamics. We end by outlining several promising directions for future research.     

Effect of spatial connectivity on host resistance in a highly fragmented natural pathosystem

Both theory and experimental evolution studies predict migration to influence the outcome of antagonistic coevolution between hosts and their parasites, with higher migration rates leading to increased diversity and evolutionary potential. Migration rates are expected to vary in spatially structured natural pathosystems, yet how spatial structure generates variation in coevolutionary trajectories across populations occupying the same landscape has not been tested. Here, we studied the effect of spatial connectivity on host evolutionary potential in a natural pathosystem characterized by a stable Plantago lanceolata host network and a highly dynamic Podosphaera plantaginis parasite metapopulation. We designed a large inoculation experiment to test resistance of five isolated and five well‐connected host populations against sympatric and allopatric pathogen strains, over 4 years. Contrary to our expectations, we did not find consistently higher resistance against sympatric pathogen strains in the well‐connected populations. Instead, host local adaptation varied considerably among populations and through time with greater fluctuations observed in the well‐connected populations. Jointly, our results suggest that in populations where pathogens have successfully established, they have the upper hand in the coevolutionary arms race, but hosts may be better able to respond to pathogen‐imposed selection in the well‐connected than in the isolated populations. Hence, the ongoing and extensive fragmentation of natural habitats may increase vulnerability to diseases.