Animal Evolution During Domestication: the Domesticated Fox As a Model

Home , Lyudmila Trut, Silver fox (animal), Wild ancestor

D OI 10.1002/bies.200800070 My favorite animal

A nimal evolution during domestication: the domesticated fox as a model Lyudmila Trut,* Irina O skina, and Anastasiya Kharlamova Institute of Cytology and G enetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia

We review the evolution of domestic animals, emphasiz- fertilization or embryonic development has profound effects on ing the effect of the earliest steps of domestication on its the characteristics of the offspring. Darwin emphasized that the course. Using the first domesticated species, the dog constitution of the organism will largely determine the kind of (C anis familiaris), for illustration, we describe the evolutionary peculiarities during the historical domestication, changes induced by the environment, and acknowledged the such as the high level and wide range of diversity. We occurrence of peculiarities as being due to unknown laws acting suggest that the process of earliest domestication via on individual constitution. unconscious and later conscious selection of human- Besides environmental influences on variation, Darwin defined behavioral traits may accelerate phenotypic var- noted the effects of crosses and inbreeding known in his time iations. The review is based on the results of a long-term experiment designed to reproduce early mammalian from the experience of animal breeders. Relying on their domestication in the silver fox (Vulpes vulpes) selected observations, Darwin presented vivid examples of the results for tameability or amenability to domestication. We of breeding selection apparent in a comparatively short time. describe changes in behavior, morphology and physiol- However, he also assigned a great role to unconscious ogy that appeared in the fox during its selection for selection acting on animals for thousands of years. Because tameability, which were similar to those observed in all our domestic animals have been first exposed to the domestic dog. B ased on the data of the fox experiment and survey of relevant data, we discuss the devel- domestication at very remote periods, it is unknown when opmental, genetic and possible molecular genetic animals started to change and at what rates. Darwin mechanisms underlying these changes. We ascribe the maintained that animals continued to be variable for long causative role in evolutionary transformation of domes- periods after their early domestication, suggesting that the tic animals to the selection for behavior and to the early domesticates were even more variable than the now neurospecific regulatory genes it affects. existing ones, and proposed that the capacity to become more variable under domestication is common to all species. K eywords: animal domestication; behavior; developmental Darwin also raised the problem of similarity between the rates; evolution; morphological diversity; regulatory system; changes observed in different domestic animals. He regarded selection certain features shared by many domestic species as the result of their domestication. Darwin’s observations and conclusions on variation under domestication hold true today. The evolutionary peculiarities Domestication in evolutionary of domestication, such as the wide phenotypic diversity of terms – from Darwin to the present day domesticates, remain puzzling. Indeed, the variation range of certain traits within a domestic species occasionally exceeds It is well known that Darwin has focused much attention on that within whole families or even orders.(2–5) Morphological domestication as the process during which striking variation variation in dogs, our earliest domesticates, is most illustrative arises. Although believing that the range of changes in any in this respect (Fig. 1). direction may be different, he admitted that the ‘‘tendency to According to conventional genetic theory, rare (10 5–10 6 general variability is unlimited’’(1). He repeatedly raised the per gene per generation) random mutations are the most question why domestic animals are so variable. In his analysis common mechanisms of phenotypic changes.(6) The diver- of the causes of variation under domestication, Darwin thought gence of the dog from the wolf might have happened some the changes to be exclusively due to environmental influences. 12,000–15,000 years ago,(3,7,8) which is a short span of time He maintained that the state of the parent organism during on the evolutionary scale. Nevertheless, variability has accumulated at immense rates which are incommensurate with random mutations. Therefore, the nature and sources of *C orrespondence to: L. Trut, Institute of Cytology and G enetics, Siberian the variation under domestication are intriguing. Branch of Russian Academy of Sciences, Lavrentieva ave, 10, 630090, Novosibirsk, Russia. Another hallmark feature of variation under domestication E-mail: trut@ bionet.nsc.ru is its similar pattern in different domestic mammalian

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Figure 1. Variability range in dogs for A: body size (Chinese crested, pekingese and bullmastiff). B: body shape (greyhound and English bulldog). C: coat types (hairless terrier and komondor). species.(9,10) When subjected to domestication, animals During evolution the same phenotypic changes can be whose evolutionary pathways did not cross, started to evolve generated through different developmental pathways under- in the same direction. They all lost the species-specific wild- lain by different genes. However, there may be developmental type behavioral response to humans. The activity of their processes underlain by key genes with many regulatory reproductive system became enhanced and relatively functions which under certain recurring selective conditions uncoupled from the environmental photoperiod, and they may most likely be targeted by selection. According to all, unlike their wild ancestors, acquired the capacity to breed Belyaev, the crucial selective factor during early domestication in any season and more often than once a year.(10,11) In was the new social environment, i.e., the first encounter of a contrast, the activity of the hypothalamic–pituitary–adrenal wild species with humans. This extremely stressful setting (HPA) axis, the key hormonal regulator of adaptation to stress, rendered the behavior – tolerance, docility toward humans became attenuated in the very few domesticates studied in and the correlated stress resistance – the main target of this respect.(12–14) The same morphological changes, first in selection. In his opinion, the genes that control behavioral terms of overall body size and its proportions and also color, variation play a key regulatory role during development. length, and texture of the coat, appeared in many domes- Belyaev therefore suggested that behavioral variation was the ticates.(2,3–5,7,8,11) Some of these attributes (white spotting, causative variation during domestication. According to his line floppy ears, curly tails) have been aptly called the morpho- of thought, the relationship between behavioral variation and logical markers of domestication (Fig. 2). It seems unlikely transformation of domestic animals would become more that these similar trends of morphological and physiological intelligible when domestication would be traced from the transformation of different domestic animals depend on beginning, i.e., when this process would be reproduced homology-independent mutations of structural homologous experimentally. Such a sophisticated model was set up using genes. The Russian evolutionary biologist Belyaev has silver foxes (Vulpes vulpes) at the Institute of Cytology and suggested more than 50 years ago that domestication might G enetics, Novosibirsk, Russia, about 50 years ago.(16–19) involve other mechanisms that contribute to phenotypic Belyaev was the initiator of this experiment. There were two variation, mainly changes in the activity of genes regulating reasons why silver foxes were given preference for experi- development.(2,15,16) mental domestication. One was the close taxonomic relation-

Figure 2. Representatives of different families and orders show the most specific morphological markers of domestication, white spotting on the head (top row) and floppy ears (bottom row): A: Horse (Equus caballus): Order Perissodactyla, family Equidae, breed Russian heavy draught horse. B: Cow (Bos taurus): Order Artiodactyla, family Bovidae, breed Aberdeen-Angus (top), banteng (bottom). C: Pig (Sus scrofa domestica): Order Artiodactyla, family Suidae, breed Vietnamese hybrid (top), Landrace (bottom). D: Sheep (Ovis): Order Artiodactyla, family Bovidae, breed Romanov (top), balbas (bottom). E: Dog (Canis familiaris): Order Carnivora, family Canidae, breed Boston terrier (top), pug (bottom). F: Rabbit (Oryctolagus cunticulus): Order Lagomorpha, family Leporidae, breed Holland white-black (top), G erman ram (bottom).

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ship between the fox and the dog; the other was that cage The rationale of the experiment was that subjection of an breeding of foxes had already been started at the beginning experimental fox population to selection for a single of the 20th century. Thus, by the onset of the experiment, behavioral trait, tameability, is sufﬁcient for domestication to captive foxes had already been subjected to rigorous occur, and the experimental design followed this ratio- selection for adaptation to the new social environment. This nale.(20,21) The researcher tested the tameability during the considerably facilitated the long life experiment and reduced time-dosed contacts with pups on the basis of their responses its duration. to hand feeding and to attempts to touch or pet them. All the pups underwent time-dosed contacts for 2.0–2.5 months. Those pups that continued to show aggressive-avoidance Development of the experimental responses despite the contacts were discarded from the model of domestication experimental population. The pressure of selection was very rigorous: less than 10% of the most tame individuals of every Although silver foxes had been farm-bred for about 50 years generation were used as parents of the next generation.(17–21) before our experiment, they retained the standard morpho- As a result of such rigorous selection, the offspring exhibiting logical phenotype, seasonal pattern of breeding speciﬁc to the the aggressive and fear avoidance responses were eliminated foxes of natural populations, and the wild-type aggressive from the experimental population in just two to three (growing and biting) or avoidance response to humans.(20) generations of selection. Figures 3A–C shows the polymorphism of the expression of In the fourth selected generation, there appeared pups that these behavioral responses in foxes of large farm-bred responded to humans by dog-like tail wagging. The offspring populations. Altogether several thousands foxes were tested of the next selected generations displayed more and more at fur farms. In the behavioral test, the experimenter dog-like behavior. In the sixth generation, there appeared approached the home cage, tried to open it and monitored pups that eagerly sought contacts with humans, not only the expression of the response. This observational test was wagging, but also whining, whimpering, and licking in a dog- highly reproducible. About 10% of farm foxes displayed the like manner. Such foxes were assigned to the ‘‘elite of wild-type responses very weakly or not at all (Fig. 3C). Of domestication’’ (Fig. 3 D E F). Elite pups constituted only 1.8% these foxes, 100 females and 30 males were chosen from (4 of the total 213 foxes) in the sixth generation of selection, different farm-bred populations as the initial parental 17.9% (66 of 379) in the tenth and 35% (503 of 1438) in the generation for selection for tolerance towards humans or twentieth generation. In the thirtieth generation, the propor- docility, then for tameability. The fox population was out- tion of elite pups was as high as 49%, (804 of 1641).(19) By breeding.(20) 2005/2006 almost all the foxes of the domesticated population

Figure 3. Behavior of foxes of the farm-bred populations (top row) and of the population selected for tameability (bottom row): A: A fox showing the aggressive response to humans; B: A fox showing the avoidance response to humans; C: A fox weakly expressing the wild-type aggressive- avoidance response to humans; D–F: Domesticated foxes referred to the behavioral elite. F: from left to right: Prof. K.G. Lark (University of Utah, Salt Lake City), Prof. L.N. Trut (Institute of Cytology and G enetics, Novosibirsk), Prof. G.M. Acland (Cornell University, Ithaca) with domestic foxes.

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were assigned to the behavioral elite. Quantitative differences characteristic of domestic animals, might have occurred as a between the behavior of elite foxes are now estimated using correlated consequence of selection for tameability. video records of behavioral tests. From these records, fifty The consequences of selection for behavior are thought- most informative behavioral parameters (for example fre- provoking (Fig. 4). In the foxes of the domesticated quency of wagging, specific vocalizations, posture of body, population, several morphological traits that mirror dog and its communicative parts such as tail, ears, and others) morphological features began to appear at different frequen- were analyzed by the principal component analysis cies (Table 2). The changes in standard coat color pattern (P C A).(22,23) This statistical method identifies the quantitative appeared earlier than the other changes, namely in the 8th– phenotypic variation in tameness as linear combinations of 10th selected generation. Specific piebald Star spotting (Figs. correlated parameters or principal components (P Cs).(24) 4F and 5A,B) and brown mottling on the background of These new integrated independent traits are genetically standard silver-black color (Fig. 5C) are the most typical analyzable phenotypes.(25,26) patterns. The Star spotting is determined by the incompletely Throughout the experiment, altogether 10,500 foxes were dominant autosomal Star mutation.(29) The gene control of used as parents. All in all, about 50,000 offspring were brown mottling in foxes is still unclear. In dogs, it is controlled obtained and tested for their amenability to domestication.(19) by one of the mutations at the Agouti locus. According to The result of this directional selection is impressive: a unique cultural-historical evidence, similar changes in coat color domestic fox behaving very similar to another species, the occurred in early dogs.(8) domestic dog, has been developed through methodically In the domesticated foxes, morphological aberrations such applied selection. as floppy ears and curly tails occurred in addition to changes in standard coat color. These morphological traits are also characteristic of many domesticates, particularly dogs Multifaceted results (Figs. 4F–J). At the more advanced steps of selection, changes in the parameters of the skeletal system began to The rigorous selection of the silver fox solely for tameability arise. They included shortened legs, tail, snout, upper jaw, brought about correlated changes in certain features of and widened skull (Fig. 4). behavior, physiology, and morphology. Some of the phenotypic changes appeared not only in the Thus, the rise of the dog-like ability of foxes to ‘‘read’’ domesticated foxes, but also in those foxes of the farm-bred human social cues, for example gestures and glances, was a populations that were not subjected to selection for tame- noteworthy change in their behavior. Domestic foxes became ability (Table 2). The above observations suggest a relation more skillful than farm-bred foxes in using human cues for between selection for tameability and the appearance of a coping with the man-made environment not only during brief subset of phenotypic changes characteristic of domestic experimental tests and selection procedures, but also during animals. The appearance of some phenotypic changes in the daily routine care.(27) foxes of the nonselected populations is not at variance with The most important physiological consequence of fox this suggestion. These populations have been bred in domestication is the transformation of the seasonal repro- captivity for about a hundred years, during which they have ductive pattern. The mating season of foxes in nature as well been inevitably subjected to selection for adaptation to as at fur farms occurs only once a year during increasing of captivity or amenability to domestication. The analysis of this daylight (January to March). In some genealogical groups of subset in terms of traditional quantitative genetics of foxes that were more advanced with regard to tameness, correlated variability is difficult.(30) In fact, strong selection sexual activity and mating were observed outside the limits of pressure on any quantitative trait, especially on those having the breeding season (Table 1),(20,28) which is normally adaptive significance, breaks down the genetic system kept in strongly restricted by natural selection.(11) Moreover, a few balance by preceding evolution and, as a consequence, vixens have even mated twice a year. The total number of inevitably leads to the appearance of deviants from the animals exhibiting changes in their reproductive pattern was phenotypic means whose reproductive capacity is reduced. small throughout the years of the experiment. However, their C ertainly, the correlated responses of any population to any emergence among the tame foxes is of crucial importance. selection depend on the structure of its genetic pool, and Reproduction seasonality is under strong selective con- therefore they are, in a sense, unique, and irreproducible. straint.(11) Variations in the mating dates of animals within the However, domestication of different populations of one mating season are mainly due to environmental factors. As a species, different species or even orders is, as well as consequence, direct selection for mating timing during the selection of foxes for tameability, consistently associated with season is ineffective and does not change the limits of the the same morphological, and physiological changes. This breading period. However, the data in Table 1 suggest that the remarkable parallelism can be hardly regarded as usual capacity to breed at any time throughout the year, which is correlated responses to selection for any quantitative trait. In

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Table 1. The time course of mating in the tame foxes showing extraseasonal sexual activity. The circles next to the order number of males indicate their matings in the years when they mated outside the breeding season. White circles, sterile mating; black circles, fertile mating. 10 10 15 20 25 30 10 15 25 30 10 20 30 10 20 28 10 30 5 30 5 10 15 Male O ct Nov Nov Nov Nov Nov Dec Dec Dec Dec Jan Jan Jan F eb F eb F eb Mar Mar A pr A pr May May May 1 * 2 * 3 * 4 * 5 * 6 7 8 * 9 *** *** * *** 10 * 11 ** * *** * 12 * * *** * 13 * * * *** 14 ** * 15 ** ** 16 **** * * 17 * ** 18 * 19 ** * * 20 21 * 22 * 23 ** 24 * 25 * 26 ** * 27 * 28 29 Mating season addition, the reproductive performance of all animals under independent gene, since the offspring of parents with one or domestication improved, in contrast to what happened in the another morphological alteration showed, contrary to case of the correlated responses in terms of traditional expectation, quite different morphological changes. Only quantitative genetics.(30) the Star depigmentation phenotype showed an independent The inbreeding coefficients of the population were genetic basis. The results of genetic analysis of the other estimated on the basis of its reproductive performance(19,20) phenotypic changes demonstrated that a common genetic and of a set of randomly chosen polymorphic microsatellite basis may underlie the set of different morphological markers(31) at different stages of selection; the coefficient aberrations. All this strongly suggests that the phenotypic values were not smaller than 0.02 and never greater then 0.07 variation within the domesticated fox population may result in both the domesticated and farm populations. These from changes in the regulation of development by the key estimates indicate that the probability of appearance of genes caused by selection for tameability.(18–20) changes due to stochastic fixation of the alleles pre-existing in the initial population and controlling each and every observed phenotypic change was very low. A ssociation between phenotypic changes Finally, it is unlikely that the changes in the domesticated and developmental rate foxes were a result of randomly arisen new mutations. Thus, in the same litter of phenotypically standard parents and The association between the newly generated traits and even in the same offspring of parents assigned to the tame changes in the developmental rates of domestic foxes is elite, several different changes in the standard phenotype noteworthy. C ertain behavioral and morphological changes appeared (Fig. 5A). This is incompatible with the mutational are correlates of delayed development of normal traits. The nature of those changes. The results of the genetic analysis emotional expression of positive responses to human, of morphological changes are also incompatible with the widened skulls, shortened snouts, floppy ears, curly tails view that each phenotypic alteration is due to a single are all juvenile traits that certain domesticated individuals

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Figure 4. A schematic representation of the results of the fox domestication experiment. A: Transformation of fox behavior resulting from selection for tameability. Left: a fox of the farm-bred population not selected for behavior. Foxes of this population show the typical aggressive response to humans. Right: a fox of the experimental domesticated population. The dog-like behavior of foxes of this population is the result of many years of selection for tameability. B–E: Activity of the HPA axis in farm-bred and domesticated foxes. Farm-bred foxes are shown in grey, the domesticated in red. B: Hypothalamic C R F (C R FmRNA/18SmRNA) and pituitary POMC (POMCmRNA/18SmRNA) gene expression, as well as AC TH (pg/ml) and cortisol (mg/dl) levels in farm-bred and domesticated foxes. C: Age-related changes in plasma cortisols level in farm-bred and domesticated foxes: 1) farm-bred foxes exhibiting aggressive responses to humans, 2, 3) foxes of the domesticated population with low (2) and high (3) domestication scores. D: Total time of locomotion, an indicator of exploratory behavior, and plasma cortisol levels in farm-bred and domesticated foxes at the age of 1–2 months: locomotion is plotted on the graph; plasma cortisol levels are represented as bars. E: Plasma cortisol in silver foxes during pregnancy and lactation. F–J: Dog-like morphological changes arisen in foxes of the domesticated population: F: Similarity of coat depigmentation between dogs and foxes: Left, border collie; Right, tame fox. G: Tail carriage, curly tail: Left, Islandsk Farehund; Right, tame fox. H: E ars are floppy and face skull is widened in some pups of tame foxes: Left, pug; Right, tame fox pup. I: Long jaw (elongation of the lower jaw), characteristic of the English bulldog, occurs among tame foxes: Left, English bulldog; Right, tame fox. J: Elongation of face skull in certain dog breeds and tame foxes: Left, Pharaoh hound, Right, tame fox. retain to adulthood.(3,32,33) Delay in developmental rates was levels of sexual hormones in the plasma and increased observed as early as during embryonic morphogenesis. As gonadal weight during the prepubertal period are indicators of already noted, the specifically located Star depigmented spots earlier sexual maturation of tame foxes. Judging by these are the specific markers of domestication (Figs. 2 and 5). It was shown that retardation of the development (proliferation and Table 2. Frequencies of phenotypic changes newly arisen in fox migration from the neural crest) of the embryonic precursors of populations. melanocytes, or primary melanoblasts, is the mechanism underlying depigmentation.(34) This retardation leads to the Frequency in populations absence of melanocytes from specific areas of the coat and, Traits Domesticated F arm-bred hence, to its depigmentation. Star 1.24 10 1 0.71 10 2 The shifts in the timing of development brought about by Brown mottling 0.45 10 2 0.9 10 3 selection of foxes for tameability have a neotenic-like Floppy ears 0.23 10 2 0.17 10 3 2 3 tendency; the development of individual somatic traits is Shortened tails 0.14 10 0.2 10 Curly tails 0.94 10 1 0.8 10 2 decelerated, while sexual maturation is accelerated. Higher

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Figure 5. Specific markers of domestication. A: Two different phenotypic changes in an Ss heterozygote: the Star depigmentation and a floppy right ear. B: Homozygote for the Star mutation (SS). C: Phenotypic similarity between brown mottling in fox and dog.

parameters, tame foxes reach sexual maturity, on average, glucocorticoid (G C) status in animals. This assumption is one month earlier than their farm-bred counterparts.(35) Many based on the close association between social behavior and researchers regard the neotenic tendency of development as the formation of the G C status; the reduction in exploratory a mechanism of evolutionary transformation of domestic activity, which, as noted above, sets an end to the sensitive animals. However, the causes of the emergence of this socialization period, was found to be correlated with a rise in tendency during domestication remain unclear.(8,32,33,36) Our plasma cortisol levels during early postnatal development.(40) fox experiment demonstrated that neotenic shifts in devel- In the tame pups, whose socialization period was longer, this opmental rate may arise as a correlated consequence of rise occurred later (Fig. 4D). Not only the rate of social selection for tameability, i.e., social adaptation to humans. development, but also of the embryonic precursors of The point is that there is a sensitive socialization period in melanocytes were found to be associated with the G C mammals when they explore their environment and get used status.(41,42) These observations suggested that G Cs may to particular social factors.(37,38) This period starts early with function as coordinators of the parameters characterizing the the functioning of the sense organs and the formation of timing of development. Evidence exists that during embry- locomotion, and enables the animal to learn about its ogenesis G Cs may have an inhibitory effect on cell surroundings and to form attachments. The maturation of proliferation and thereby promote cell transition to differentia- the neurophysiological substrate of the fear response tion, which is the next step of early development.(43–46) hampers socialization. Increasingly fearful pups start to avoid Only few studies were concerned with the function of the impact of social factors, thus limiting their further glucocorticoids under domestication. It has been reported that socialization. It was found that selection of foxes for domestication of grey rats is associated with a decrease in the tameability slowed down the development of the fear weight of adrenals, changes in their morphology and altera- response and shifted it to later dates. In foxes that were tions in the glucocorticoid synthesis under basal conditions and not selected for tameability, the exploratory activity (estimated stress or under the effect of exogenous adrenocorticotropic on the basis of locomotion in an unknown setting) became hormone (AC TH).(12,13) Comparison of domestic guinea pigs sharply reduced by the age of 45 days or so when fear was with their wild ancestors revealed a decrease in the stress first manifested.(39) This reduction was lacking in the tame response of the HPA axis in domesticates.(14) It is of foxes until the age of four months.(19) Quite plausibly, importance that the function of the HPA axis decreased in retardation of the development of the neurophysiological foxes with advancing selection for tameness. We started the substrate of fear is just one of the numerous manifestations of comparative analysis of the HPA axis from generation 10 of the regulatory effects of genes that affect developmental selection for tame behavior. In this generation, the levels of rates and are targeted by selection for domestication. It plasma glucocorticoids in the tame foxes were significantly cannot be excluded that these genes perform a broader lower in all seasons of the year compared with the farm-bred function by regulating the developmental rates of other traits. foxes.(47) The hormone levels decreased with advancing selection. Basal cortisol levels in the blood of the domesticated foxes in generation 20 were almost two-fold lower than of the Neuroendocrine changes under non-domesticated foxes, and stress-induced levels were about domestication 30% lower. In generation 45, basal and stress-induced blood cortisol levels in foxes of the tame population were already Which putative genes affecting the developmental rate may three- and five-fold lower than in the farm-bred foxes.(48) These possibly be involved in selection for domestication? We data suggest that genes controlling plasma glucocorticoids assumed that some of these genes might perhaps control the were possibly the targets during selection for tameability.

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In the foxes selected for domestication, the activity of the Destabilizing selection as a possible HPA axis is reduced at all levels (Figs. 4B–E). This includes accelerator of evolutionary the total G Cs pool, the adrenal G C production in vitro, the transformation of domestic animals basal AC TH level in plasma, and the adrenal response to stress.(49,50) The expression of the pro-opiomelanocortin Surveying the observations made during the course of (P OMC) gene, whose protein product includes AC TH experimental fox domestication, Belyaev found no satisfac- and b-endorphin in the pituitary, was also reduced, and tory explanation within the traditional genetic framework and the expression of corticotropin-releasing hormone (C RH), the came to the idea of destabilizing selection as a factor of releasing factor for AC TH, tended to decrease in the domestication.(16) As destabilizing selection Belyaev desig- hypothalamus.(51) It is also important that in the tame foxes nated selection causing destabilization of regulatory systems the total G C pool in the blood was considerably decreased controlling development and, hence, destabilization of the during pregnancy and lactation.(52,53) As a consequence, the morphological and physiological organization stabilized by entire embryonic and early postnatal development of the tame previous natural selection.(62) Under domestication, selection offspring proceeded on the background of lower maternal for particular behavioral features accomplishes the destabi- G Cs. Given the multifarious effects of G Cs on develop- lizing function. Belyaev regarded the selection for just these ment,(54) the impact of their changes on developmental features as a major accelerator of the evolutionary transfor- processes is evident. They are directly associated with the mation of domestic animals. It appears likely that destabilizing expression of the G C receptor (G C R) gene, whose promoter selection can affect animals at any organizational level, region has a complex structure. The multiple and tissue- whose populations experience selection pressure on beha- specific promoters provide fine regulation of changes in G C R vioral traits. Illustrative examples of multifaceted conse- gene expression in different early environments.(55,56) Most quences of selection for behavior(63,64) were reported even in importantly, the G C R gene expression in the hippocampus, a Drosophila.(65,66,67) The evolutionary role of selection for brain structure modulating the regulation of behavior and behavior would increase greatly in the case of highly activity of the HPA axis and in the frontal cortex, is higher in the organized mammals, because their phenotypic manifestation domestic animals.(57) Thus, the effects of selection for tame of behavioral traits involves much more regulatory neuro- behavior on the HPA axis are manifest at all levels, ranging transmitters and neurohormonal relationships.(62) from phenotypic parameters to the expression of the C RH, The evolutionary function of destabilizing selection is P OMC, and G C R genes. the reverse to that of stabilizing selection.(68) Stabilizing Domestication also affects the developmental neurotrans- selection acts always in an environment with which a mitter systems. The role of neurotransmitters in the regulation species has coped well; this leads to stabilization of of development has been discussed(58) and recently reval- development to form a phenotype that is optimal under this uated.(59) The serotonin system of the brain deserves special condition. Stabilizing selection acts by suppressing the effects attention with reference to domestication. Its contribution to of mutations that disrupt development and alter the normal the inhibition of aggressiveness in animals, including foxes, phenotype, or even by eliminating them. It maintains has recently been discussed in Bioessays.(60) The studies of variability within the optimum range of the evolutionary the brain’s serotonin system showed that the domesticated established norm, but it does not produce variability foxes differed from the farm-bred by higher levels of serotonin beyond the phenotype of the species. Analysis of the and its main metabolite 5-hydroxyindol acetic acid in a effects of selection for behavior on development and number of brain structures. Differences in the activities of variability in our fox model demonstrated that it sharply monoamine oxidase, the principal enzyme in serotonin destabilized the correlated developmental systems and led to degradation, and of tryptophan hydroxylase, the key an increase in phenotypic variation. What mechanisms can be enzyme in serotonin synthesis, were also demonstrated. It involved in this process? And what is the nature of the was shown that the activity of tryptophan hydroxylase was changes in the specific genes that are induced by destabiliz- higher in the tame than in the non-tame foxes. The higher ing selection? activities of this key enzyme and the higher levels of serotonin It appears plausible that the phenotypic novelties in the in the brains of domesticated foxes agree well with the data experimental fox population could be due to changes in gene concerning the inhibitory influence of serotonin on a number activity, largely in its epigenetic modification. Hormonal of aggressive types of behavior.(49,60) Thus, it is important to changes under domestication may result in either gene emphasize the role of serotonin in development. Previous(58) silencing or gene activation. In this context, the above- and recent(61) publications have shown that serotonin acts as mentioned Star phenotype (Fig. 5), which appears repeatedly a multifarious signal molecule that is important during at high frequencies (10 1–10 2) in the population of tame development and capable of eliciting a cascade of gene foxes and which is determined by the incompletely dominant activations. S gene, is of importance.(29) Segregation analysis of

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offspring from crosses between Ss heterozygotes for this domestic dogs and their closest wild relatives, wolves gene demonstrated a shortage of SS homozygotes. This (C anis lupus) and coyotes (C anis latrans).(76) In dogs, the shortage cannot be accounted for by selective embryonic, most prominent changes in gene expression occurred in the zygotic, and gametic mortality. Some foxes that were proven hypothalamus, which is a biologically important, evolutionary to be SS homozygotes have the heterozygous phenotype (Ss) conserved brain structure as well as a modulator of behavioral and the inheritance pattern characteristic of heterozygotes. It and neuroendocrine responses to environmental agents. was assumed that one of the two homologous mutant Star Comparisons of the differences in gene expression between alleles became functionally inactive. Also, cases of birth of wolves and coyotes, which have been separated for 2 Myr homozygotes in crosses between a heterozygous and a BP,(77) have demonstrated that the differences are minimal standard parent have been documented (unpublished data). and that the expression profiles of the hypothalamic genes in This suggests the reverse process of activation of a wild-type the wild canids are conserved. This is in striking contrast ‘‘silent’’ allele in rare heterozygotes for the Star gene. A similar with the variable expression of these genes in dogs and phenomenon of heritable gene inactivation/reactivation has with the differences between dogs and wild canids. It could been described for the kinked tail character in the mouse not be ruled out that selection for tame behavior during controlled by the dominant Axin-fused gene, the phenotypic 12,000–15,000 years of dog domestication accelerated expression of which is extremely variable, to the extent that divergence of gene expression in the brain, as postulated some of its carriers have a normal phenotype.(69) Subsequent by the authors.(76) studies showed that the absence of the kinked tail phenotype correlates with the level of DNA methylation in a retro- transposon within AxinFu.(70) Moreover, the epigenetic Molecular genetic implications for modification of the mutant kinked allele of both maternal phenotypic changes under domestication and paternal derivation passes meiosis, i.e., it does not undergo reprogramming during gametogenesis.(70) As yet, little is known about the correlation between the In the mouse, another example of inheritance of epigenetic expression levels of specific genes and phenotypic variability modification in the Agouti locus, which controls wild-type coat in particular traits. However, some data indirectly indicate that color, has been described. The Avy allele bears a transposon this relation does exist. For example, it is known that coding insertion. The number of viable yellow phenotypes was regions abound with presumably noncoding simple sequence related to the methylation pattern of this insertion.(71–74) The repeats (SSRs). It is curious that half of these repeats cluster Agouti locus is implicated to be involved in brown mottling in in regions containing one or several genes playing a key role in the dog. This prompts to study the Agouti locus at the development.(78) This suggests that they are important for the molecular level in the domesticated fox to reveal whether the regulation of expression of these key developmental genes. locus possess structural components that can act as effective Their altered regulation may be implicated as the major carriers of methylation marks. source of changes in many genes under their control. Thus, it Methylation of the DNA promoter regions is implicated as a has been shown that variation in the number of tandem mechanism of the epigenetic alteration in gene expression; repeats in two such genes, Runx-2 and Alx-4, may be related methylation is related to the transcriptional silencing of to the variation in skeletal traits in dogs.(79) Variation rates in regulated genes, while hypomethylation is related to their the number of tandem repeats in genes for neurotransmitters transcriptional activity. As to the Star mutation in foxes, it is and their receptors may be involved in determining variations unknown whether its activity changes at the transcriptional in behavior.(80) The variation in the number of repeats can level or at subsequent steps of gene product conversion. It exceed by far that of point mutations. This suggested that may be speculated that the selection-related genetic changes variation in the length of tandem repeats may be a in the regulatory neurohormonal system of the domesticated predominant source of rapid morphological evolution and foxes could induce changes in the expression of many other that the repeats associated with the developmental genes downstream genes, thereby causing phenotypic destabiliza- increase the pace of evolution.(79) tion. However, only limited changes in gene expression in fox In addition to SSRs, phenotypic variation may be brain have been shown so far.(51,75) As indicated above, these influenced by canid-specific small interspersed nuclear changes involve also genes of the HPA axis.(48,51) This elements (SIN Es).(81) Both seem to be often present at suggests that behavioral as well as morphological and positions influencing gene expression.(82,83) Bsp repeats physiological changes in the domestic foxes may depend similar to canine SIN Es have been detected in the fox on changes in the expression of a very small number of brain genome, in which they may potentially perform regulatory genes with many regulatory downstream effects. functions.(84,85) The presence of SSR, SIN E, or Bsp elements It is of interest that the expression profiles of the brain- in the genes of the hormonal and neurotransmitter systems specific genes showed remarkable differences between may have important consequences. Changes in their

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expression may be implicated in changes in the expression of marker of tolerance and successful adaptation to the human many genes under their control. social environment. The experimental model of domestica- The molecular genetic mechanisms underlying the tion, as a kind of forced evolution, was developed by behavioral and morphological traits of the now existing systematically applying selection for tameability on silver dog breeds may not, however, reflect the genetic nature of foxes. the early changes brought about by domestication. The The founders of the selected population were chosen from domestic fox is therefore a unique model of the genetic large farm-bred populations. They represented the genetic events of early domestication. At the IC&G fur farm, foxes variants that had reproductive success and showed the are methodically selected not only for domestication, i.e., weakest wild-type aggressive-avoidance response to elimination of the aggressive genes, but also in the opposite humans. Such foxes had undoubtedly been under natural direction—for retention and enhancement of the expression and artificial selection for tameability through all the years of of aggressive behavior. As a result, a population of cage breeding. However, this selection did not affect the aggressive foxes has also been set up. This population, standard morphological and physiological phenotype of the together with the domesticated one, serves as valuable foxes in all populations we studied. We succeeded in rapidly resource for genetic studies of early domestication. setting up a unique population of domestic foxes following the It should be noted again that an intriguing consequence effect of rigorous selection pressure for tame behavior. The of domestication—historical of the dog and experimental of behavioral patterns of these foxes are dog-like. These foxes the silver fox—were the newly arisen morphological also have a range of morphological and physiological traits variations. This is amazing with respect to the traits of attributable to domestic animals. The fox domestication the skeletal system that define body size and conformation. experiment demonstrated that this selection affected the We are now raising the important evolutionary question of genes controlling the neurohormonal status. Some of the whether behavioral and morphological traits are integrated genes responsible for the association between tameability at the genomic level, i.e., whether there exist loci which co- and hormonal and transmitter levels might have been brought regulate morphological-behavioral traits. We have started together and become fixed by 8–10 selected generations. research in this direction in collaboration with the University Their fixation might have modified the activity of many other of Utah (Prof. Lark) and the Cornell University (Prof. downstream genes, thereby destabilizing and shifting timing Ackland and Dr. Kukekova). The P C A approach was used of development, and uncovering some of the phenotypically for genetic dissection not only of complex behavioral traits hidden potentialities of the genome. This means that the as above but also of skeletal traits.(86,87) This approach interactions between genetic variants altered during selection revealed sets of correlated parameters (P Cs) which are might have produced new patterns of gene expression and structuring variability. Differences between tame, aggres- new phenotypes. sive, and control populations were expressed in terms of the Different molecular mechanisms are implied in the P Cs. The P Cs behaved as individual phenotypes in the phenotypic expression of non-expressing genes and their segregating generations from the Tame Aggressive cross epigenetic modification. They include non-coding sequences and the subsequent backcrosses, as we reported.(22,23,86) and methylation–demethylation of the regulatory regions of Thus, these phenotypes were appropriate for molecular genes. Whatever the molecular mechanisms of changes in genetic analysis. Search for the loci for behavior and gene activity under domestication, the results suggest that morphology, along with their common loci, is of current these changes could be produced by selection for tameability. interest, because co-segregation of some of their P Cs In the light of the results of the fox domestication suggests that they may share certain loci. Construction of experiment, the similar patterns of behavioral as well as the meiotic map of the genome of the fox was followed by morphological and physiological transformation in foxes, dogs genetic mapping of the loci for the P Cs,(88) which will be and other domesticates are suggested to be the result of used to further study the co-regulation of morphology and selection for tameability. This selection is thought to be the behavior. causative and universal mechanism for evolutionary transformation of domestic animals.

C onclusions A cknowledgments: The authors thank Anna Fadeeva for We proceeded on the assumption that regulatory changes in translation of the manuscript from Russian into English and gene activity may generate the remarkable level of diversity Yekaterina Omelchenko for technical assistance. NIH grants # and its similar patterns among domestic animals. These 1R03 TW 008098-01, # R01 MH 077811-01, and the Programs regulatory changes were presumably caused by selection of of Basic Research of the RAS Presidium ‘‘Biodiversity and gene animals for speciﬁc behavior, namely tameability as a pool dynamics’’ and ‘‘Molecular and C ell Biology’’.

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