The effects of background coloration and dark spots on the risk of predation in poison frog models

Protective coloration is a well-known predator avoidance strategy in prey species. Aposematic species often display a contrasting color pattern consisting of dark spots of different shapes and sizes on a bright background coloration. Both elements, background color and spots are expected to serve different purposes. While the ecological function of the bright coloration has been addressed in many studies, the question of whether the interaction with differently sized spots influences predator behavior has received less attention by researchers. In a lowland rain forest in Costa Rica we used 2700 clay models that imitated the polytypic strawberry poison frog (Oophaga pumilio) as a proxy for an aposematic prey species. We manipulated the dorsal color pattern by using a local and a non-local aposematic and a non-local cryptic background color and combined them with black spots increasing in size (none, small, medium, large). The major objective was to test if spot size alters the survival rate of differently colored models. Background coloration and spot size were significant predictors of being attacked. However, the interaction between both effects was not. During five trials predators avoided the non-local aposematic color morph and did not discriminate between local aposematic and non-local cryptic models. Spot size and attack rate were negatively linear correlated which suggests that predator selection promotes the evolution of dark spots. We further conclude that spot size matters in a contrasting color pattern and plays an important role in predator avoidance.     

Investigating signal modalities of aposematism in a poison frog

Aposematic species combine a conspicuous signal with a secondary defence, the majority of which are studied in the context of a visual signal. While multimodality of the aposematic signal appears to be common in invertebrate species, we know very little about the presence or absence of multimodality in vertebrates. Here, we examine the possibility of multimodality of aposematism in the green-and-black poison frog, Dendrobates auratus. Using a non-visual predator (the cat-eyed snake, Leptodeira annulata) and extractions of chemicals in frog skins, we test whether there is sufficient non-visual information for predators to avoid this aposematic species without using visual cues. We found that experienced predators avoid chemicals in this poison frog's skin by olfactory cues alone in trials with live frogs and extracts from captive poison frogs, whereas extracts from wild poison frogs did not lead to avoidance behaviours in predators. Further, in our limited sampling, naïve predators demonstrate no avoidance. This not only indicates that predators can make informed decisions from the frog's odour, but also indicates that avoidance based on olfactory cue is a learned response.     

An Analysis of Predator Selection to Affect Aposematic Coloration in a Poison Frog Species

Natural selection is widely noted to drive divergence of phenotypic traits. Predation pressure can facilitate morphological divergence, for example the evolution of both cryptic and conspicuous coloration in animals. In this context Dendrobatid frogs have been used to study evolutionary forces inducing diversity in protective coloration. The polytypic strawberry poison frog (Oophaga pumilio) shows strong divergence in aposematic coloration among populations. To investigate whether predation pressure is important for color divergence among populations of O. pumilio we selected four mainland populations and two island populations from Costa Rica and Panama. Spectrometric measurements of body coloration were used to calculate color and brightness contrasts of frogs as an indicator of conspicuousness for the visual systems of several potential predators (avian, crab and snake) and a conspecific observer. Additionally, we conducted experiments using clay model frogs of different coloration to investigate whether the local coloration of frogs is better protected than non-local color morphs, and if predator communities vary among populations. Overall predation risk differed strongly among populations and interestingly was higher on the two island populations. Imprints on clay models indicated that birds are the main predators while attacks of other predators were rare. Furthermore, clay models of local coloration were equally likely to be attacked as those of non-local coloration. Overall conspicuousness (and brightness contrast) of local frogs was positively correlated with attack rates by birds across populations. Together with results from earlier studies we conclude that conspicuousness honestly indicates toxicity to avian predators. The different coloration patterns among populations of strawberry poison frogs in combination with behavior and toxicity might integrate into equally efficient anti-predator strategies depending on local predation and other ecological factors.     

Predator experience on cryptic prey affects the survival of conspicuous aposematic prey

Initially, aposematism, which is an unprofitable trait, e.g. noxiousness conspicuously advertised to predators, appears to be a paradox since conspicuousness should increase predation by naive predators. However, reluctance of predators for eating novel prey (e.g. neophobia) might balance the initial predation caused by inexperienced predators. We tested the novelty effects on initial predation and avoidance learning in two separate conspicuousness levels of aposematic prey by using a 'novel world' method. Half of the wild great tits (Parus major) were trained to eat cryptic prey prior to the introduction of an aposematic prey, which potentially creates a bias against the aposematic morph. Both prey types were equally novel for control birds and they should not have shown any biased reluctance for eating an aposematic prey. Knowledge of cryptic prey reduced the expected initial mortality of the conspicuous morph to a random level whereas control birds initially ate the conspicuous morph according to the visibility risk. Birds learned to avoid conspicuous prey in both treatments but knowledge of cryptic prey did not increase the rate of avoidance learning. Predators' knowledge of cryptic prey did not reduce the predation of the less conspicuous aposematic prey and additionally predators did not learn to avoid the less conspicuous prey. These results indicate that predator psychology, which was shown as reluctance for attacking novel conspicuous prey, might have been important in the evolution of aposematism.     

Not all colors are equal: predation and color polytypism in the aposematic poison frog Oophaga pumilio

Aposematic organisms are not predicted to show high levels of warning signal diversity because they are expected to be under stabilizing selection to decrease costs of ‘educating’ predators about their unpalatability. However, systematic changes in warning signals (polytypism) can be expected if they represent adaptations to local predators. The aposematic strawberry poison frog (Oophaga pumilio) is red throughout its mainland distribution in Costa Rica and Panamá, but displays high levels of warning signal diversity in the Bocas del Toro Archipelago of Panamá. Both coloration and spot pattern vary in a polytypic sense. Sexual selection contributes to maintaining the polytypism, but little work has investigated the potential influence of predation. We used unspotted models of O. pumilio to determine if predation might help explain the color polytypism on Isla Colón in the Bocas del Toro Archipelago of Panamá. We tested whether attack rates differed among the red mainland morph, green/yellow Isla Colón morph, and the brown control. We found that frog color significantly predicted being attacked. The local green Isla Colón models were attacked more than foreign red or brown models. No difference in attack rate existed between red and brown control models. Our results suggest that the red mainland morph possesses a more effective warning signal, even when it is not the local morph. Honest signaling of unpalatability, neophobia, and the use of search images by local predators are potential explanations. Similarity of the brown model to other local poison frogs might explain the lower attack rate compared to previous work. The attack rate was lower on Isla Colón compared to mainland Costa Rica, which supports the hypothesis that less overall predation in the Bocas del Toro Archipelago may contribute to the overall warning signal diversity in O. pumilio there by relaxing selection for aposematic traits.     

Behavioural elements reflect phenotypic colour divergence in a poison frog

The coexistence of both aposematic and cryptic morphs as different anti-predator strategies within a species seems to be an unusual phenomenon in nature. The strawberry poison frog, Oophaga pumilio, shows an astonishing colour diversity among populations in western Panama. In this study we selected a red and a green colour morph from two Panamanian islands (Isla Solarte and Isla Colón) for behavioural observations and measurements of conspicuousness. We found that red frogs were more visible to both conspecific frogs and potential predators than green frogs. Interestingly the difference in conspicuousness was most pronounced at the substrate that males used as principal calling places. Red males were more active and spent more time foraging than green males, which spent more time hidden. The association between conspicuousness of colouration and behaviour results in a more aposematic and a more cryptic anti-predator strategy. This is the first study which links differences in conspicuousness between animals on their natural backgrounds to differences in foraging as well as anti-predator behaviour and discusses the results in light of previous findings of toxicity analyses and potential costs and benefits of aposematism. To this end, our study adds a novel perspective for explaining extreme colour diversity between populations within an initially aposematic species.     

Different colour morphs of the poison frog Andinobates bombetes (Dendrobatidae) are similarly effective visual predator deterrents

Aposematism is the use of warning signals to advertise unpleasant or dangerous defences to potential predators. As the effectiveness of this strategy depends on predator learning, little variation is expected in aposematic warning signals, as similar signals facilitate predator learning. However, warning signals are frequently variable in aposematic species. Such variability could arise as a result of geographic variation in the interpretation that local predators give warning signals. We tested this divergent learning hypothesis in the polytypic poison frog Andinobates bombetes (Anura: Dendrobatidae), focusing on visual predators. Our study was conducted in two populations of this species located in the Western Andes of Colombia, where individuals at some localities exhibit red dorsolateral stripes, while those in others exhibit yellow dorsolateral stripes. We deployed paraffin models imitating both forms of A. bombetes in size and colouration, as well as dull‐coloured controls, at sites inhabited by either red‐striped or yellow‐striped frogs. Red and yellow models were attacked at similar rates at both sites, and brown models were attacked more frequently at one of the sites. These results suggest that red and yellow colourations function as similarly effective aposematic signals for primarily visual predators, regardless of the form previously experienced by these predators. Therefore, our results do not support the hypothesis of divergent predator learning as a driver of the polytypism present in this species. Finally, we discuss other mechanisms that may be involved in the evolution and maintenance of this polytypism.     

Inferring Predator Behavior from Attack Rates on Prey-Replicas That Differ in Conspicuousness

Behavioral ecologists and evolutionary biologists have long studied how predators respond to prey items novel in color and pattern. Because a predatory response is influenced by both the predator’s ability to detect the prey and a post-detection behavioral response, variation among prey types in conspicuousness may confound inference about post-prey-detection predator behavior. That is, a relatively high attack rate on a given prey type may result primarily from enhanced conspicuousness and not predators’ direct preference for that prey. Few studies, however, account for such variation in conspicuousness. In a field experiment, we measured predation rates on clay replicas of two aposematic forms of the poison dart frog Dendrobates pumilio, one novel and one familiar, and two cryptic controls. To ask whether predators prefer or avoid a novel aposematic prey form independently of conspicuousness differences among replicas, we first modeled the visual system of a typical avian predator. Then, we used this model to estimate replica contrast against a leaf litter background to test whether variation in contrast alone could explain variation in predator attack rate. We found that absolute predation rates did not differ among color forms. Predation rates relative to conspicuousness did, however, deviate significantly from expectation, suggesting that predators do make post-detection decisions to avoid or attack a given prey type. The direction of this deviation from expectation, though, depended on assumptions we made about how avian predators discriminate objects from the visual background. Our results show that it is important to account for prey conspicuousness when investigating predator behavior and also that existing models of predator visual systems need to be refined.     

Higher survival of an aposematic than of a cryptic form of a distasteful bug

Summary An experiment was performed to assess the relative survival of two forms of 5th instar larvae of Lygaeus equestris (Heteroptera, Lygaeidae) — the normal red form, called aposematic, and a mutant grey form, called cryptic — when given to hand-raised great tits (Parus major).Sixteen birds were presented with aposematic larvae and 16 were presented with cryptic larvae in 10 consecutive trials. One attack per trial was allowed. Both larval forms were presented against a background matching the grey larvae, but since both prey types were presented in a specific place known to the predator, detection rate for both was assumed to be unity.Birds learned to avoid both prey types. However, the survival of the aposematic larvae was higher than that of the cryptic ones due to three aspects of predator behaviour: i) a greater initial reluctance to attack, ii) a more rapid avoidance learning, and iii) a lower frequency of killing in an attack, when the prey was aposematic. Moreover, a greater number of birds learned to avoid prey without killing any individual, when the prey was aposematic. This result is considered to be due to prey coloration alone, since, in a separate test, no difference in prey distastefulness could be detected.This experiment shows that individual prey can benefit from being aposematic and indicates that individual selection can be a sufficient explanation for the evolution of aposematic coloration. It was concluded that, since the survivorship was 6.4 times higher for the aposematic prey, it could have a detection rate that is correspondingly higher than the cryptic in order for the two forms to have equal fitness.     

Decision time and prey gregariousness influence attack probability in naïve and experienced predators

Aposematic coloration often has an element of conspicuousness. One suggested benefit of conspicuousness is that it enables the prey to be detected at a greater distance, allowing a predator more time to make a correct decision about attacking it and thus reducing possible recognition errors made by predators. I conducted an experiment, with chicks, Gallus gallus domesticus, as predators on live aposematic and nonaposematic prey, to investigate the effects of decision time and signal size on predator sampling behaviour. The chicks were subjected to different degrees of competition to influence how quickly decisions had to be made. Chicks in four treatment groups, either in the presence or absence of a competing chick, were presented with either solitary prey or prey in groups. In the presence of a competitor, chicks attacked the prey more often and more quickly and needed more attacks before they started to avoid the prey. With prey in groups, chicks took longer to attack, attacked less often, learnt to avoid prey more quickly and killed fewer aposematic prey. This experiment provides evidence for the importance of time and signal size for predators' attack decisions. More time to view prey prior to attack could produce a stronger image and thus encourage avoidance learning and produce a stronger neophobic avoidance effect. Copyright 2000 The Association for the Study of Animal Behaviour.     

Conditions for the spread of conspicuous warning signals: a numerical model with novel insights

The initial evolution of conspicuous warning signals presents an evolutionary problem because selection against rare conspicuous signals is presumed to be strong, and new signals are rare when they first arise. Several possible solutions have been offered to solve this apparent evolutionary paradox, but disagreement persists over the plausibility of some of the proposed mechanisms. In this paper, we construct a deterministic numerical simulation model that allows us to derive the strength of selection on novel warning signals in a wide range of biologically relevant situations. We study the effects of predator psychology (learning, rate of mistaken attacks, and neophobia) on selection. We also study the how prey escape, predation intensity, number of predators, and abundance of different prey types affects selection. The model provides several important results. Selection on novel warning signals is number rather than frequency dependent. In most cases, there exists a threshold number of aposematic individuals below which aposematism is selected against and above which aposematism is selected for. Signal conspicuousness (which increases detection rate) and distinctiveness (which allows predator to distinguish defended from nondefended prey) have opposing effects on evolution of warning signals. A more conspicuous warning signal cannot evolve unless it makes the prey more distinctive from palatable prey, reducing mistaken attacks by predators. A novel warning signal that is learned quickly can spread from lower abundance more easily than a signal that is learned more slowly. However, the relative rate at which the resident signal and the novel signal are learned is irrelevant for the spread of the novel signal. Long-lasting neophobia can facilitate the spread of novel warning signals. Individual selection via the ability of defended prey to escape from predator is not likely to facilitate evolution of conspicuous warning signals if both the resident (cryptic) morph and the novel morph have the same escape probability. Predation intensity (defined as the proportion of palatable prey eaten by the predator) has a strong effect on selection. More intense predation results in strong selection against rare signals, but also strong selective advantage to common signals. The threshold number of aposematic individuals is lower when predation is intense. Thus, the evolution of warning signals may be more likely in environments where predation is intense. The effect of numbers of predators depends on whether predation intensity also changes. When predation intensity is constant, increasing numbers of predators raises the threshold number of aposematic individuals, and thus makes evolution of aposematism more difficult. If predation intensity increases in parallel with number of predators, the threshold number of aposematic individuals does not change much, but selection becomes more intense on both sides of the threshold.     

Hybridization promotes color polymorphism in the aposematic harlequin poison frog,Oophaga histrionica

Whether hybridization can be a mechanism that drives phenotypic diversity is a widely debated topic in evolutionary biology. In poison frogs (Dendrobatidae), assortative mating has been invoked to explain how new color morphs persist despite the expected homogenizing effects of natural selection. Here, we tested the complementary hypothesis that new morphs arise through hybridization between different color morphs. Specifically, we (1) reconstructed the phylogenetic relationships among the studied populations of a dart‐poison frog to provide an evolutionary framework, (2) tested whether microsatellite allele frequencies of one putative hybrid population of the polymorphic frog O. histrionica are intermediate between O. histrionica and O. lehmanni, and (3) conducted mate‐choice experiments to test whether putatively intermediate females prefer homotypic males over males from the other two populations. Our findings are compatible with a hybrid origin for the new morph and emphasize the possibility of hybridization as a mechanism generating variation in polymorphic species. Moreover, because coloration in poison frogs is aposematic and should be heavily constrained, our findings suggest that hybridization can produce phenotypic novelty even in systems where phenotypes are subject to strong stabilizing selection.     

Spatial variation in the fitness of divergent aposematic phenotypes of the poison frog, Dendrobates tinctorius

Aposematic species use brightly coloured signals to warn potential predators of their unpalatability. The function of these signals is largely believed to be frequency-dependent. All else being equal, stabilizing selection is expected to constrain the evolution of novel signals. However, despite the expected frequency-dependent function of aposematic signals, interpopulation variation in aposematic signals is ubiquitous in nature. Here, we used clay models of the poison frog Dendrobates tinctorius to test the nature of selection in regions containing varying frequencies of frogs possessing the local aposematic signal. Our findings support a role for stabilizing selection in maintaining the local signal type in a region of high signal frequency; however, we observe a lack of stabilizing selection at one site coincident with a decrease in the density of frogs possessing the local signal. Spatial variation in local aposematic signal frequencies may facilitate the evolution of novel signal types by altering the adaptive landscape for divergent aposematic phenotypes. Our results provide evidence for spatial variation in the selective regime acting on aposematic signals within an established aposematic system and highlight the need for further study of the nature of selection acting across different spatial scales in diverse aposematic systems.     

Change in protective coloration in the striated shieldbug <Emphasis Type="Italic">Graphosoma lineatum</Emphasis> (Heteroptera: Pentatomidae): predator avoidance and generalization among different life stages

There are two major forms of protective coloration, camouflage and warning coloration, which often entail different colour pattern characteristics. Some species change strategy between or within life stages and one such example is the striated shieldbug, Graphosoma lineatum. The larvae and the pale brownish-and-black striated pre-diapause adults are more cryptic in the late summer environment than is the red-and black striation that the adults change to after diapause in spring. Here we investigate if the more cryptic pre-diapause adult and larval coloration may affect the aposematic function of the coloration as compared to the red adult form. In a series of trials we presented fifth instar larvae, pale or red adults to shieldbug-naïve domestic chicks, Gallus gallus domesticus, to investigate the birds’ initial wariness, avoidance learning, and generalization between the three prey types. The naïve chicks found the red adults most aversive followed by pale adults, and they found the larvae the least aversive. The birds did not find the larvae unpalatable and did not learn to avoid them, while they learned to avoid the two adult forms and then to a similar degree. Birds generalized asymmetrically between life stages, positively from larvae to adults and negatively from adults to larvae. We conclude that the lower conspicuousness in the pale forms of G. lineatum may entail a reduced aposematic function, namely a reduced initial wariness in inexperienced birds. The maintenance of the colour polymorphism is discussed.     

Experienced chicks show biased avoidance of stronger signals: an experiment with natural colour variation in live aposematic prey

An important factor for understanding the evolution of warning coloration in unprofitable prey is the synergistic effect produced by predator generalisation behaviour. Warning coloration can arise and become stabilised in a population of solitary prey if more conspicuous prey benefit from a predator's previous interaction with less conspicuous prey. This study investigates whether domestic chicks (Gallus gallus domesticus) show a biased generalisation among live aposematic prey by using larvae of three species of seed bugs (Heteroptera: Lygaeidae) that are of similar shape but vary in the amount of red in the coloration. After positive experience of edible brownish prey, chicks in two reciprocal experiments received negative experience of either a slightly red or a more red distasteful larva. Attacking birds were then divided into two treatment groups, – one presented with the same prey again, and one presented with either a less red or a more red larva. Birds with only experience of edible prey showed no difference in attack probability of the two aposematic prey types. Birds with experience of the less red prey biased their avoidance so that prey with a more red coloration was avoided to a higher degree, whereas birds with experience of the more red prey avoided prey with the same, but not less red coloration. Thus, we conclude that bird predators may indeed show a biased generalisation behaviour that could select for and stabilise an aposematic strategy in solitary prey. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sound improves visual discrimination learning in avian predators

Aposematic insects use warning colours to deter predators, but many also produce odours or sounds when attacked by a predator. One possible role for these additional components is that they promote the association between the warning colour and the non-profitability it signals, thus reducing the chance of future attacks from visually hunting predators. This experiment explicitly tests this idea by looking at the effects of sound on a visual discrimination task. Young domestic chicks were trained to look for food rewards under coloured paper cones scattered in an experimental arena. In a subsequent visual discrimination task, they learned to discriminate between rewarded and non-rewarded hats on the basis of colour. Half the chicks performed this task in silence, whilst the other half had a tone played when they attacked non-rewarded hats. The presence of the tone improved the speed of colour discrimination learning. This demonstrates that there could be a selective advantage for aposematic coloured insects to emit sounds when attacked, since avian predators will learn to avoid their coloration more quickly. The role of psychological interactions between signal components in receivers is discussed in relation to the evolution of multimodal displays.     

Avian predators attack aposematic prey more forcefully when they are part of an aggregation

Defended insects often advertise their unprofitability to potential predators using conspicuous aposematic coloration. Many aposematic insects are also gregarious, and it has been suggested that the aggregation of defended prey may have facilitated the evolution of aposematic coloration. Empirical studies have demonstrated that birds are more wary of aggregated aposematic prey, and learn to avoid them more quickly than solitary prey. However, many aposematic insects survive being attacked by birds, and the effect of aggregation on post-attack survival has not previously been investigated. Using domestic chicks as predators and artificially manipulated mealworms as prey, we provide empirical evidence that predators attack aggregated aposematic prey more forcefully than solitary prey, reducing the likelihood of prey surviving an attack. Hence, we suggest that previous works concluding that aggregation was an important pre-requisite for the evolution of aposematism may have overestimated the fitness benefits of aggregation, since aggregated prey may be attacked less but are also less likely to survive an attack.     

NOT EVERYTHING IS BLACK AND WHITE: COLOR AND BEHAVIORAL VARIATION REVEAL A CONTINUUM BETWEEN CRYPTIC AND APOSEMATIC STRATEGIES IN A POLYMORPHIC POISON FROG

Aposematism and crypsis are often viewed as two extremes of a continuum of visual conspicuousness to predators. Theory predicts that behavioral and coloration conspicuousness should vary in tandem along the conspicuousness spectrum for antipredator strategies to be effective. Here we used visual modeling of contrast and behavioral observations to examine the conspicuousness of four populations of the granular poison frog, Oophaga granulifera, which exhibits almost continuous variation in dorsal color. The patterns of geographic variation in color, visual contrast, and behavior support a gradient of overall conspicuousness along the distribution of O. granulifera. Red and green populations, at the extremes of the color distribution, differ in all elements of color, contrast, and behavior, strongly reflecting aposematic and cryptic strategies. However, there is no smooth cline in any elements of behavior or coloration between the two extremes. Instead populations of intermediate colors attain intermediate conspicuousness by displaying different combinations of aposematic and cryptic traits. We argue that coloration divergence among populations may be linked to the evolution of a gradient of strategies to balance the costs of detection by predators and the benefits of learned aversion.     

Behavioural responses of native predators to an invasive toxic prey species

One important impact of invasive species may be to modify the behaviour of native taxa. For example, the invasion of highly toxic cane toads (Bufo marinus) kills many anurophagous native predators, but other predators learn to recognize and avoid the toxic invader. We exposed native fish (northern trout gudgeons, Mogurnda mogurnda) and Dahl's aquatic frogs (Litoria dahlii) to cane toad tadpoles, then monitored the predator's responses during subsequent trials. Both the frogs and fish initially attacked toad tadpoles, but rapidly learned not to do so. Fish and adult frogs retained their aversion for at least a week, whereas recently metamorphosed frogs did not. Clearly, the spread of cane toads through tropical Australia can modify feeding responses of native aquatic predators. For predators capable of rapid avoidance learning, the primary impact of cane toads may be on foraging behaviour rather than mortality.     

Experimental evidence for predator learning and Müllerian mimicry in Peruvian poison frogs (Ranitomeya,Dendrobatidae)

The evolution of mimicry is one of the most powerful examples of evolution driven by natural selection; however it is rare in non-insect taxa and thus is understudied. Ranitomeya imitator underwent a ‘mimetic radiation’ and now mimics three congeneric model species (R. fantastica, R. summersi, and two morphs of R. variabilis), creating geographically distinct populations of the species, including four allopatric mimetic morphs. These complexes are thought to represent a case of Müllerian mimicry, but no prior empirical studies on learned avoidance by predators support this claim. In this study we used young chickens (Gallus domesticus) as naïve predators to determine if a co-mimetic morph of R. imitator and R. variabilis contribute to reciprocal learned avoidance by predators—a key component of Müllerian mimicry. Chickens exposed to either stimulus species demonstrated reciprocal learned avoidance; thus our results indicate that this complex functions as a Müllerian mimicry system. This study provides novel empirical evidence supporting predictions of the Müllerian mimicry hypothesis in anurans. Our study shows no difference between learned avoidance in stimuli frogs and a ‘novel’ morph of R. imitator that differed in both color and pattern, indicating that learned avoidance by predators may be generalized in this system. Generalized learning provides a plausible mechanism for the maintenance of both polytypic mimicry and the maintenance of intrapopulation phenotypic heterogeneity.