Differentiation in phenotypic plasticity among populations of Arabis serrata Fr. & Sav. (Brassicaceae)

Studies on divergence of phenotypic plasticity in closely related species have suggested that character means and plasticity of these characters may evolve independently. Similar patterns of divergence between populations within a species have been reported although few plant species have been studied. Thus, in this paper, the patterns of differentiation between character means and phenotypic plasticity among eight populations of Arabis serrata are documented. Mean response and magnitude and pattern of phenotypic plasticity were measured and compared in plants growing under an environmental gradient of nutrients. Differences in means and coefficients of variation (CV as indicators of plasticity) among populations were compared using the Canberra metric and generating unrooted Wagner trees. Populations showed significant differences in character means in nine morphological traits. Magnitude and patterns of phenotypic plasticity showed a complex pattern of differentiation for each trait and population. Biomass traits were more plastic, in general, than characters associated with linear size. Comparisons between pairs of populations for nine morphological traits showed that in 28.6% of 252 possible cases, populations differed in means, magnitude and patterns of phenotypic plasticity. In almost 90% of the cases, populations differed in magnitude and/or pattern of plasticity. Considering all characters together, populations from similar habitats and with common life history features tended to respond in similar ways. The patterns of divergence, however, suggest that character means and character plasticities among populations are able to evolve independently.     

Individual differences in behavioural plasticities

Interest in individual differences in animal behavioural plasticities has surged in recent years, but research in this area has been hampered by semantic confusion as different investigators use the same terms (e.g. plasticity, flexibility, responsiveness) to refer to different phenomena. The first goal of this review is to suggest a framework for categorizing the many different types of behavioural plasticities, describe examples of each, and indicate why using reversibility as a criterion for categorizing behavioural plasticities is problematic. This framework is then used to address a number of timely questions about individual differences in behavioural plasticities. One set of questions concerns the experimental designs that can be used to study individual differences in various types of behavioural plasticities. Although within‐individual designs are the default option for empirical studies of many types of behavioural plasticities, in some situations (e.g. when experience at an early age affects the behaviour expressed at subsequent ages), ‘replicate individual’ designs can provide useful insights into individual differences in behavioural plasticities. To date, researchers using within‐individual and replicate individual designs have documented individual differences in all of the major categories of behavioural plasticities described herein. Another important question is whether and how different types of behavioural plasticities are related to one another. Currently there is empirical evidence that many behavioural plasticities [e.g. contextual plasticity, learning rates, IIV (intra‐individual variability), endogenous plasticities, ontogenetic plasticities) can themselves vary as a function of experiences earlier in life, that is, many types of behavioural plasticity are themselves developmentally plastic. These findings support the assumption that differences among individuals in prior experiences may contribute to individual differences in behavioural plasticities observed at a given age. Several authors have predicted correlations across individuals between different types of behavioural plasticities, i.e. that some individuals will be generally more plastic than others. However, empirical support for most of these predictions, including indirect evidence from studies of relationships between personality traits and plasticities, is currently sparse and equivocal. The final section of this review suggests how an appreciation of the similarities and differences between different types of behavioural plasticities may help theoreticians formulate testable models to explain the evolution of individual differences in behavioural plasticities and the evolutionary and ecological consequences of individual differences in behavioural plasticities.     

Comparative studies of evolutionary responses to light environments in Arabidopsis

In this article, we compare the reaction norms to foliage shade (changes in light quality, spatially fine-grained environmental variation) and photoperiod (day length, spatially coarse-grained environmental variation) in several haplotypes of Arabidopsis thaliana from Scandinavia. We found that both across-environment means and phenotypic plasticities evolved continuously and very rapidly within this group. Both character means and trait plasticities were highly integrated, in part as predicted by the adaptive plasticity hypothesis for response to foliage shade (the so-called shade-avoidance syndrome). We found that a significant but small fraction of the variance in across-treatment trait means and plasticities in response to one environmental factor is explained by variation of the same traits in response to the other factor. Genetic relatedness based on chloroplast DNA sequence variation among haplotypes was not associated with variation in across-treatment character means or their plasticities, suggesting that evolution of these characters has occurred on a local geographic scale via reticulation (outcrossing) among maternal lines rather than by the differential survival of selfing lineages.     

Ontogenetic phenotypic plasticity during the reproductive phase in Arabidopsis thaliana (Brassicaceae)

While phenotypic plasticity has been the focus of much research and debate in the recent ecological and evolutionary literature, the developmental nature of the phenomenon has been mostly overlooked. A developmental perspective must ultimately be an integral part of our understanding of how organisms cope with heterogeneous environments. In this paper I use the rapid cycling Arabidopsis thaliana to address the following questions concerning developmental plasticity. (1) Are there genetic and/or environmental differences in parameters describing ontogenetic trajectories? (2) Is ontogenetic variation produced by differences in genotypes and/or environments for two crucial traits of the reproductive phase of the life cycle, stem elongation and flower production? (3) Is there ontogenetic variability for the correlation between the two characters? I found genetic variation, plasticity, and variation for plasticity affecting at least some of the growth parameters, indicating potential for evolution via heterochronic shifts in ontogenetic trajectories. Within-population differences among families are determined before the onset of the reproductive phase, while among-population variation is the result of divergence during the reproductive phase of the ontogeny. Finally, the ontogenetic profiles of character correlations are very distinct between the ecologically meaningful categories of early- and late-flowering “ecotypes” in this species, and show susceptibility to environmental change.     

PHENOTYPIC PLASTICITY IN PHLOX. I. WILD AND CULTIVATED POPULATIONS OF P. DRUMMONDII

We investigated the changes in amounts and patterns of phenotypic plasticity which have arisen in the Texas annual Phlox drummondii during domestication. Character means and plasticities were compared for five populations: a wild population, three cultivated varieties (a Tall cultivar and two Dwarf cultivars), and a population of an escaped Tall cultivar naturalized in Texas. To measure plasticity, we scored the responses of 10 characters to six treatments and analyzed both the amount and direction of plastic response. Wild plants are phenotypically distinct from the Tall and Escaped cultivar and from the two Dwarf cultivars. Despite its substantial phenotypic divergence from the Wild population, the Tall cultivar's plasticity has changed little during domestication. Traits most strongly correlated with fitness show the least change in their plasticities. The two Dwarf varieties have very similar plasticities, despite strong phenotypic divergence from the Tall population and despite the fact that they were derived from different Tall lines. This suggests that indirect selection on phenotypic plasticity related to selection for the Dwarf habit has resulted in the characteristic plasticity of the Dwarf lines. The Escaped cultivar has substantially different plastic responses from those of the Wild or cultivated populations.     

Density dependence and population differentiation of genetic architecture in Impatiens capensis in natural environments

We identified environment-dependent constraints on the evolution of plasticity to density under natural conditions in two natural populations of Impatiens capensis. We also examined the expression of population divergence in genetic variance-covariance matrices in these natural environments. Inbred lines, originally collected from a sunny site with high seedling densities and a woodland site with low seedling densities, were planted in both original sites at natural high densities and at low density. Morphological and life-history characters were measured. More genetic variation for plastic responses to density was expressed in the sun site than in the woodland site, so the evolutionary potential of plasticity was greater in the sun site. Strong genetic correlations between the same character expressed at different densities and correlations among different characters could constrain the evolution of plasticity in both sites. Genetically based trade-offs in meristem allocation to vegetative growth and reproduction were apparent only in the high-resource environment with no overhead canopy and no intraspecific competition. Therefore, genetic constraints on the evolution of plasticity depended on the site and density in which plants were grown, and correlated responses to selection on plastic characters are also expected to differ between sites and densities. Population differentiation in genetic variance-covariance matrices was detected, but matrix structural differences, as opposed to proportional differences, were detected between populations only in the sun site at natural high density. Thus, population divergence in genetic architecture can occur rapidly and on a fine spatial scale, but the expression of such divergence may depend on the environment.     

Interspecific aggression and character displacement in the damselfly Calopteryx splendens

Problems in species recognition are thought to affect the evolution of secondary sexual characters mainly through avoidance of maladaptive hybridization. Another, but much less studied avenue for the evolution of sexual characters due to species recognition problems is through interspecific aggression. In the damselfly, Calopteryx splendens, males have pigmented wing spots as a sexual character. Large-spotted males resemble males of another species, Calopteryx virgo, causing potential problems in species recognition. In this study, we investigate whether there is character displacement in wing spot size and whether interspecific aggression could cause this pattern. We found first that wing spot size of C. splendens in populations decreased with increasing relative abundance of C. virgo. Secondly, C. virgo males were more aggressive towards large- than small-spotted C. splendens males. Thirdly, in interspecific contests C. virgo males had better territory holding ability than C. splendens males. These results suggest that interspecific aggression may have caused character displacement in wing spot size of C. splendens, because the intensity of aggression towards large-spotted males is likely to increase with relative abundance of C. virgo males. Thus, interspecific aggression may be an evolutionarily significant force that is able to cause divergence in secondary sexual characters.     

Standard percent DNA sequence difference for insects does not predict species boundaries

Diagnosis and assessment of species boundaries of economically important insects are often problematic because of limited morphological and/or biological characters. DNA data can help to identify and revise species. Nonoverlapping intra- and interspecific sequence divergences are often used as evidence for species. Thus, the establishment of a standardized percent nucleotide divergence to predict species boundaries would aid in cases where species status is suspect. However, given variation in nucleotide mutation rates and species concepts, association between a standard percent sequence divergence and species is questionable. This review surveys the percent DNA sequence difference found between sister-species of economically important insects, to assess whether a standard divergence associates with all taxa. Sixty-two comparisons of intra- and interspecific pairwise DNA differences were made for mitochondrial and nuclear loci spanning families of Isoptera, Phthiraptera, Hemiptera, Coleoptera, Lepidoptera, Diptera, and Hymenoptera. Intra- and interspecific sequence divergences varied widely among insects, 0.04-26.0 and 1.0-30.7%, respectively. The ranges of intra- and interspecific sequence divergences overlapped in 28 of 62 comparisons. This implies that a standardized percent sequence divergence would fail to correctly diagnose species for 45% of the cases. Common occurrence of nonmonophyly among closely related species probably explains this observation. Nonmonophyly and overlap of intra- and interspecific divergences were significantly associated. The reviewed studies suggest that a standard percent sequence divergence does not predict species boundaries among economically important insects. DNA data can help best to predict species boundaries via its inclusion in nonphenetic phylogenetic analysis and subsequent systematic expert scrutiny.     

Mutational effects on constraints on character evolution and phenotypic plasticity inArabidopsis thaliana

Although the concept of genetic constraints plays an important role in our understanding of the evolution of natural populations, there are still few empirical investigations probing the nature and limits of constraints in plant and animal species, aside from some studies inDrosophila. In the work reported here, we use an induced mutation - artificial selection protocol to analyse constraints on character means and phenotypic plasticity to nutrients inArabidopsis thaliana, an annual crucifer. We induced point mutations in a highly inbred line characterized by an extreme phenotype (very fast life cycle, early flowering, reduced leaf production) and little plasticity. We then selected individuals with increased leaf numbers. The goals were to determine if: (i) it is possible to increase leaf production; (ii) this has an effect on reproductive fitness; (iii) a mutation-selection process simultaneously alters the environmental insensitivity of the plant, thereby allowing phenotypic plasticity; and (iv) changes in the target trait affect other characters or their plasticities. The results demonstrate that: (a) mutations do increase leaf number; (b) this yields a much higher reproductive fitness, owing to the extension of the very short life cycle of the base inbred line; (c) there are no changes in plasticity of leaf number or of any other trait, possibly because few loci are involved in the control of plasticity; (d) changes in leaf number are related to alterations in three other traits comprising a strong set of covarying characters inA. thaliana. Two uncorrelated traits are capable of independent evolution from the constrained set. We therefore suggest that environmentally insensitive ecotypes of A.thaliana can quickly evolve to form ecologically specialized, relatively environmentally invariant genotypes.     

Unravelling phenotypic plasticity -- why should we bother?

The ability of a genotype to change its phenotype was once considered rather a nuisance -- making it difficult to define a genotype. This led to the idea that there was a problem called 'instability'. But quite early it was recognized that stability was under genetic control, and was a character like other attributes of an individual. From this realization came the idea that there were two sides to the character of 'instability', and that the ability to change could be important. This ability was thus given the title of 'plasticity'. Once recognized, it became clear from surveys of different species and populations that plasticity can (i) be a complex character, and (ii) be selected to fit species to the particular demands of different environments. For plants, which cannot meet variations in environment like animals by behavioural responses, phenotypic plasticity can be very important. Plants should therefore be valuable tools for unravelling the mechanisms of plasticity whilst also demonstrating its contribution to fitness experimentally. We ought also to be able to demonstrate that appropriate genetic variability is available through which complex responses can be built up by selection. Genes must exist not only to determine character means, but also to determine character response, which adds interesting complexity to our ideas about evolution.     

PHENOTYPIC PLASTICITY OF ANNUAL PHLOX: TESTS OF SOME HYPOTHESES

Phenotypic plasticity is an important means by which individual plants respond to environmental heterogeneity. Relative plasticities were compared for three species of annual Phlox grown in a set of six environmental treatments. Two measures of plasticity were computed for 18 characters per species: the Coefficient of Variation to measure amounts of plasticity and a new measure, the correlation of responses between species, which compares patterns of plasticity. Three hypotheses concerning the species’ amounts and patterns of plasticity were examined. The heterozygosity hypothesis states that levels of heterozygosity and amounts of plasticity should be inversely related. The relatedness hypothesis states that more distantly related species should have less similar patterns and amounts of plasticity. The ecological hypothesis states that species from similar habitats should have more similar amounts and patterns of plasticity than species which evolved under differing conditions. The results suggest that heterozygosity, relatedness, and ecology may all affect patterns and amounts of plasticity, and that no single hypothesis is sufficient to explain the observed results. Some data from previous studies are examined in relation to the proposed hypotheses.     

Potential local adaptation in populations of invasive reed canary grass (Phalaris arundinacea) across an urbanization gradient

Urban stressors represent strong selective gradients that can elicit evolutionary change, especially in non‐native species that may harbor substantial within‐population variability. To test whether urban stressors drive phenotypic differentiation and influence local adaptation, we compared stress responses of populations of a ubiquitous invader, reed canary grass (Phalaris arundinacea). Specifically, we quantified responses to salt, copper, and zinc additions by reed canary grass collected from four populations spanning an urbanization gradient (natural, rural, moderate urban, and intense urban). We measured ten phenotypic traits and trait plasticities, because reed canary grass is known to be highly plastic and because plasticity may enhance invasion success. We tested the following hypotheses: (a) Source populations vary systematically in their stress response, with the intense urban population least sensitive and the natural population most sensitive, and (b) plastic responses are adaptive under stressful conditions. We found clear trait variation among populations, with the greatest divergence in traits and trait plasticities between the natural and intense urban populations. The intense urban population showed stress tolerator characteristics for resource acquisition traits including leaf dry matter content and specific root length. Trait plasticity varied among populations for over half the traits measured, highlighting that plasticity differences were as common as trait differences. Plasticity in root mass ratio and specific root length were adaptive in some contexts, suggesting that natural selection by anthropogenic stressors may have contributed to root trait differences. Reed canary grass populations in highly urbanized wetlands may therefore be evolving enhanced tolerance to urban stressors, suggesting a mechanism by which invasive species may proliferate across urban wetland systems generally.     

Habitat specialization and adaptive phenotypic divergence of anuran populations

We tested for adaptive population structure in the frog Rana temporaria by rearing tadpoles from 23 populations in a common garden experiment, with and without larval dragonfly predators. The goal was to compare tadpole phenotypes with the habitats of their source ponds. The choice of traits and habitat variables was guided by prior information about phenotypic function. There were large differences among populations in life history, behaviour, morphological shape, and the predator-induced plasticities in most of these. Body size and behaviour were correlated with predation risk in the source pond, in agreement with adaptive population divergence. Tadpoles from large sunny ponds were morphologically distinct from those inhabiting small woodland ponds, although here an adaptive explanation was unclear. There was no evidence that plasticity evolves in populations exposed to more variable environments. Much among-population variation in phenotype and plasticity was not associated with habitat, perhaps reflecting rapid changes in wetland habitats.     

Multiple modes of selection can influence the role of phenotypic plasticity in species' invasions: Evidence from a manipulative field experiment

In exploring the roles of phenotypic plasticity in the establishment and early evolution of invading species, little empirical attention has been given to the importance of correlational selection acting upon suites of functionally related plastic traits in nature. We illustrate how this lack of attention has limited our ability to evaluate plasticity''s role during invasion and also, the costs and benefits of plasticity. We addressed these issues by transplanting clones of European‐derived Plantago lanceolata L. genotypes into two temporally variable habitats in the species'' introduced range in North America. Phenotypic selection analyses were performed for each habitat to estimate linear, quadratic, and correlational selection on phenotypic trait values and plasticities in the reproductive traits: flowering onset and spike and scape lengths. Also, we measured pairwise genetic correlations for our “colonists.” Results showed that (a) correlational selection acted on trait plasticity after transplantation, (b) selection favored certain combinations of genetically correlated and uncorrelated trait values and plasticities, and (c) using signed, instead of absolute, values of plasticity in analyses facilitated the detection of correlational selection on trait value‐plasticity combinations and their adaptive value. Based on our results, we urge future studies on species invasions to (a) measure correlational selection and (b) retain signed values of plasticity in order to better discriminate between adaptive and maladaptive plasticity.     

河北木蓝的叶表型可塑性研究

以中国分布最广、形态变异复杂且分类上存在争议的木蓝属植物河北木蓝(Indigofera bungeana Walp.)为研究对象,运用GIS技术从较大尺度上(17省28县29个居群)进行叶表型可塑性分析,利用表型可塑性指数和变异系数对叶表型可塑性进行评价,并对叶表型性状与环境因子的相关性进行分析。结果显示:河北木蓝叶表型性状在居群间的变异大于居群内;叶长、叶柄长、最少小叶数、最多小叶数、小叶长、小叶宽6个叶表型性状均具有可塑性,其中叶长的可塑性最大,小叶数目的可塑性最小;年均降水量是对叶表型可塑性影响最大的环境因子;6个叶表型性状与海拔均呈负相关,与年均气温呈正相关。研究结果可为河北木蓝的分类、适应性进化和开发利用奠定基础。     

Differences between inter- and intraspecific architectonic adaptations to pharyngeal mollusc crushing in cichlid fishes

Because fish heads are densely packed with muscles, ligaments, skeletal elements and other structures, transformations in one structure may influence surrounding structures. Transformations occur during phylogeny, ontogeny and as environmentally induced alterations, i.e. phenotypic plasticity. We describe differences in intra- and interspecific transformations of the pharyngeal jaw apparatus of haplochromine cichlids. Using multivariate clustering techniques we trace possible correlations in transformations of anatomical characters of the pharyngeal jaws and surrounding structures. The intraspecific transformation analysis is based on two environmentally induced morphs of Astatoreochromis alluaudi : a molluscivorous morph with a hypertrophied pharyngeal jaw apparatus and an insectivorous one with a non-hypertrophied apparatus. For the interspecific analysis five other haplochromine species from Lake Victoria with diets ranging from insects to molluscs were investigated. Although ranges in diet are the same, the anatomical ranges differ between A. alluaudi and the species cline. Besides similarities in anatomical changes of the pharyngeal jaw apparatus in the intra- and interspecific cline, differences were also observed. Apparently there are among haplochromines multiple pathways to achieve similar performance. In A. alluaudi architectonic and intrinsic plasticity constraints limit the adaptability of the pharyngeal jaw apparatus. In the species cline, these constraints have been overcome by genetical adaptation.     

A COMPARISON OF TWO STICKLEBACKS

We present results of an experiment designed to address fundamental issues in the ecology and evolution of plastic trophic morphology: (1) Is observed plasticity adaptive? (2) How much interspecific morphological variation is the result of plasticity? (3) Have different selective regimes resulted in the evolution of different degrees of plasticity? (4) Is genetic variation for phenotypic plasticity present in contemporary populations? We raised fish from two recently diverged species of freshwater threespine sticklebacks on two different diets representative of the natural prey of the two species. Both species exhibited morphological plasticity in an adaptive direction: each species more closely resembled the other when raised on the latter's diet. Dietreversal reduced the natural morphological gap between these two species, -1% to 58%, depending on the trait. One species is known to have a more variable diet in the wild than the other species, and we found that it also exhibited the greater amount of morphological plasticity. Given that the two species have recently diverged, this result is compelling evidence that diet variability is important in the evolution of plastic trophic morphology. Finally, by using a full-sib experimental design, we demonstrated that genetic variation for morphological plasticity exists in contemporary populations, thus confirming that plasticity has evolutionary potential.     

Plastic responses to parents and predators lead to divergent shoaling behaviour in sticklebacks

Population divergence in antipredator defence and behaviour occurs rapidly and repeatedly. Genetic differences, phenotypic plasticity or parental effects may all contribute to divergence, but the relative importance of each of these mechanisms remains unknown. We exposed juveniles to parents and predators to measure how induced changes contribute to shoaling behaviour differences between two threespine stickleback species (benthics and limnetics: Gasterosteus spp). We found that limnetics increased shoaling in response to predator attacks, whereas benthics did not alter their behaviour. Care by limnetic fathers led to increased shoaling in both limnetic and benthic offspring. Shoaling helps limnetics avoid trout and avian predation; our results suggest that this adaptive behaviour is the result of a combination of paternal effects, predator-induced plasticity and genetic differences between species. These results suggest that plasticity substantially contributes to the rapid divergence in shoaling behaviour across the post-Pleistocene radiation of sticklebacks.     

Evolution of phenotypic plasticity a comparative approach in the phylogenetic neighbourhood of Arabidopsis thaliana

The evolution of phenotypic plasticity has rarely been examined within an explicitly phylogenetic framework, making use of modern comparative techniques. Therefore, the purpose of this study was to determine phylogenetic patterns in the evolution of phenotypic plasticity in response to vegetation shade (the ‘shade avoidance’ syndrome) in the annual plant Arabidopsis thaliana and its close relatives. Specifically, we asked the following questions: (i) Do A. thaliana and related species differ within or among clades in the magnitude and/or pattern of plasticity to shade? (ii) Are the phenotypic variance–covariance matrices (phenotypic integration) of these taxa plastic to the changes in light quality induced by the presence of a canopy? (iii) To what extent does the variation in uni- and multivariate plasticity match the phylogeny of Arabidopsis? In order to address these questions we grew individuals from six taxa of known phylogenetic relationship in a greenhouse under full sun and under a grass canopy. Taxa differed in the magnitude, but not in the pattern, of plasticities for all traits. At the univariate level, the late flowering species, A. pumila and A. griffithiana, as well as the late flowering Moscow ecotype of A. thaliana, showed greater plasticity for allocation to vegetative and reproductive meristems. At the multivariate level, several taxa displayed a very low stability of their variance–covariance structures to environmental change, with only one taxon sharing as many as three principal components across environments. We conclude that both univariate and multivariate plasticities to vegetation shade can evolve rapidly within a genus of flowering plants, with little evidence of historical constraints (phylogenetic inertia).     

How fluctuating competition and phenotypic plasticity mediate species divergence

Causal evidence linking resource competition to species divergence is scarce. In this study, we coupled field observations with experiments to ask if the degree of character displacement reflects the intensity of competition between two closely related spadefoot toads (Spea bombifrons and S. multiplicata). Tadpoles of both species develop into either a small-headed omnivorous morph, which feeds mostly on detritus, or a large-headed carnivorous morph, which specializes on and whose phenotype is induced by fairy shrimp. Previously, we found that S. multiplicata are inferior competitors for fairy shrimp and are less likely to develop into carnivores in sympatry with S. bombifrons. We compared four key trophic characters in S. multiplicata across natural ponds where the frequency of S. bombifrons varied. We found that S. multiplicata became increasingly more omnivore-like as the relative abundance of S. bombifrons increased. Moreover, in controlled laboratory populations, S. multiplicata became increasingly more omnivore-like and S. bombifrons became increasingly more carnivore-like as we increased the relative abundance of the other species. Phenotypic plasticity helped mediate this divergence: S. multiplicata became increasingly less likely to eat shrimp and develop into carnivores in the presence of S. bombifrons, a superior predator on shrimp. However, divergence also reflected differences in canalized traits: When reared under common conditions, S. multiplicata tadpoles became increasingly less likely to produce carnivores as their natal pond decreased in elevation. Presumably, this pattern reflected selection against carnivores in lower-elevation ponds, because S. bombifrons became increasingly more common with decreasing elevation. Local genetic adaptation to the presence of S. bombifrons was remarkably fine grained, with differences in carnivore production detected between populations a few kilometers apart. Our results suggest that the degree of character displacement potentially reflects the intensity of competition between interacting species and that both phenotypic plasticity and fine-scale genetic differentiation can mediate this response. Moreover, these results provide causal evidence linking resource competition to species divergence.