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Sibling recognition in the prairie vole, Microtus ochrogaster
Anita. Behav., 1984, 32, 362-366
SIBLING RECOGNITION IN THE PRAIRIE VOLE, MICROTUS OCHROGASTER BY LEAH GAVISH*, JOYCE E. H O F M A N N & LOWELL L. GETZ Department of Ecology, Ethology, and Evolution, University of Illinois, Urbana-Champaign, IL 61801 U.S.A. Abstract. Since mating is seldom observed between sibling prairie voles, Microtus ochrogaster, this, behavioural discrimination was used to investigate sibling recognition in this speciesl Cross-fostering of 1-3-day-old pups demonstrated that unrelated pups reared together did not breed, whereas siblings reared apart bred readily when paired at weaning. When unrelated voles were paired at 14 days of age, prior to sexual maturity, significantly fewer pairs bred than when strangers were paired at 21 days of age. Separation of 21-day-old siblings for 8 days before pairing overcame incest avoidance; a 15-day separation was required for breeding by siblings that had remained together until they were 50 days old. These results indicate that sibling recognition and, consequently, incest avoidance depend on association prior to weaning. The prairie vole, Microtus oehrogaster, has been shown to exhibit incest avoidance. McGuire & Getz (1981) found that when brothers and sisters were weaned artd paired at 21 days of age only 6 % of the pairs produced a litter, whereas 78 % of non-siblings paired at the same age bred. Suppression of breeding among prairie vole littermates has also been reported by Hasler & Nalbandov (1974) and Batzli et al. (1977). The consistency of results obtained under different experimental conditions suggested that this form of behavioural discrimination could be used to investigate the mechanism of sibling recognition in the prairie vole. The present study was designed to determine whether recognition involves a genetic mechanism by which siblings could recognize each other even without prior association, or whether it is dependent upon the association of siblings early irt life. Recent studies indicate that kin recognition cart occur independently of previous association. Species which have shown this ability to recognize unfamiliar relatives include Cascades frog tadpoles, Rana easeadae (Blaustein & O'Hara 1981; O'Hara & Blaustein 1981); pigtail macaques, Maeaea nemestrina (Wu et al. 1980); Richardson's ground squirrels, Spermophilus riehardsonii (Davis 1982); arctic and Belding's ground squirrels, S. parryii and S. beldingi (Holmes & Sherman 1982); white-footed mice, Peromyseus leueopus (Grau 1982; although Hoffman 1981 obtained contradictory results); and sweat bees, Lasioglossum zephyrum (Greerlberg 1979).
Unfamiliar relatives might recognize each other because of the action of shared recognition alleles which would confer (1) a phenotypic trait, (2) the ability to recognize this trait in other individuals, and (3) a tendency to behave differentially towards individuals which shared the trait (Hamilton 1964; the 'green beard' effect of Dawkirts 1976). It has been suggested that such a genetic system is improbable (e.g. Hamilton 1964; Dawkins 1976; Sherman 1980). However, it has been shown that laboratory mice (Mus musculus) discriminated, in mate preference tests, between individuals that differed only in their major histocompatibility complex genotypes (Yamazaki et al. 1976). Alternatively, recognition could be the result of phenotype matching (Holmes & Sherman 1982; Lacy & Sherman 1983; called 'signature matching' by Beecher 1982). By this mechanism an individual would compare the phenotype of a newlyencountered stranger with its own phenotype or that of familiar kin. Thus, recognition would involve phenotypic traits shared as a consequence of genetic relatedness and the learning of reference phenotypes. On the other hand, recognition might require association among individuals. Art animal could learn the characteristics of the individuals with which it interacts in the social context of early life. Whenever close proximity during early life is highly correlated with kinship, this mechanism would normally result irt effective kin recognition. This may be the most common proximate mechanism of kin recognitiort (Sherman 1980; Bekoff 1981; Holmes & Sherman 1982). Familiarity due to association
*Present address: Department of Zoology, The Hebrew University, Jerusalem, Israel. 362
GAVISH ET AL.: SIBLING RECOGNITION IN PRAIRIE VOLES appears to be responsible for sibling recognition in spiny mice, Acomys cahirinus (Porter et al. 1978; Porter & Wyrick 1979); prairie deer mice, Peromyscus maniculatus (Hill 1974); cactus mice, P. eremicus (Dewsbury 1982); and, perhaps, white-footed mice, P. leucopus (Hoffman 1981). Association is also partially involved in kin recognition in Spermophilus parryii and S. beldingi (Holmes & Sherman 1982). The proximate mechanisms of kin recognition have been discussed in depth by Holmes & Sherman (1982, 1983). In this study prairie voles from a laboratory colony were used to manipulate the relative degree of genetic relatedness and social familiarity between individuals. We performed a crossfostering experiment to investigate whether incest avoidance among siblings depends upon innate recognition of genetic relatedness or upon association prior to weaning. We also studied the effect of separation for different lengths of time and at different ages upon sibling recognition and incest avoidance.
Methods and Results Subjects The prairie voles used in all experiments were laboratory-reared. The colon), had been established with approximately 25 pairs of wildcaught voles from several sites in the vicinity of Urbana, Illinois and has been maintained for over 20 generations. Additional wild-caught voles have been introduced into the colony from time to time. The breeding colony and the experimental animals were maintained in two rooms under a 14L : 10D photoperiod. The voles were housed in 25 • 45-cm plastic breeding cages with hardwood-chip bedding; Purina rabbit chow and water were provided ad libitum. There was no olfactory isolation of the animals in either room. The chemical signal (pheromone) which initiates reproductive activation in female prairie voles is not air-transmitted; dir.ect naso-genital contact is necessary (Carter et al. 1980). Experiment 1: Pups Cross-fostered Shortly After Birth Methods. In the first part of this experiment pairs were formed from non-siblings that had been reared together (NSRT). Litters of 1-2-dayold pups were removed from their parents' cages. A few minutes later each lactating female was given a 'litter' consisting of one of her own pups and pups from other like-aged litters. Each
363
female received the same number of pups (three to five) that she had had originally. Pups were weaned and paired at 22-23 days of age; each pair consisted of male and female non-siblings that had been reared by the same female. In the second part of the experiment pairs were formed from full siblings that had been reared apart (SRA). Newborn pups ( < 24 h old) were toe-clipped for identification and returned to their pare,Its. One or two days later, pups from these litters were exchanged so that each lactating female received one or two of her own pups and at least two foster pups. Most females were given five or six pups to rear. Pups were weaned at 22-23 days of age and male and female siblings that had been reared by different females were paired. Cages were checked frequently for newborn pups. After 60 days any pair that had not produced a litter was separated and each individual was paired with a stranger. These animals were maintained for an additional 60 days to determine if they were capable of breeding. Only those pairs in which both animals were determined to be fertile were included in the statistical analysis of results. The incidence of successful mating in the two groups of cross-fostered animals was compared with that of a group of non-siblings paired at 21-22 days of age (in experiment 2) and a group of siblings paired at the same age (in experiment 3). These groups were maintained under the same conditions as the cross-fostered groups. Results. Thirteen pairs o f non-siblings that had been reared together (NSRT) were established, but animals in three pairs were apparently infertile. Only one of the 10 fertile NSRT pairs produced a litter within 60 days (Table I). When the individuals in the other nine pairs were placed with strangers, pups were borI1 to each newly-established pair 22-25 days later. Twelve pairs of unfamiliar siblings (SRA) were established. Of these, 11 pairs produced a litter within 60 days (Table I). The earliest litter was born 36 days after pairing, the latest 46 days (median interval, 41 days). The male and female of the remaining pair both bred when placed with new mates, producing litters within 4 weeks. The number of SRA pairs that bred was significantly greater than the number of NSRT pairs that produced litters (x 2 = 11.8, 1 df, P < 0.001). The response of the NSRT group did not differ significantly from that of familiar siblings paired at weaning (Table I; Fisher exact test, P ---- 1.0). There was also no difference in the
364
ANIMAL
BEHAVIOUR,
Table L The Incidence of Successful Breeding in Prairie Voles Paired at 21-23 Days of Age Group
No. of pairs*
No. of pairs that bred
Non-siblings reared together (NSRT) (experiment 1)
10
1
Siblings reared apart (SRA) (experiment 1)
12
11~
Non-siblings reared apart (experiment 2)
10
lOt
Siblings reared together (experiment 3)
13
1w
*Includes only pairs in which both animals were fertile (see text). tSignificantly different from NSRT (xe = 11.8, 1 df, P < 0.001). ,Not significantly different from SRA (Fisher exact test, P = 1.0). w significantlydifferent from NSRT (Fisher exact test, P = 1.0). results for the SRA group and non-siblings paired at weaning (Table I; Fisher exact test, P = 1.0). Thus, males and females that had been reared together as juveniles rarely mated when given the opportunity, regardless of their genetic relatedness. Experiment 2: Non-siblings Paired Prior to Reproductive Maturity Methods. Males and females from different litters were weaned and paired at 14 days of age. This is as young as M. ochrogaster cart be safely weaned; most female prairie voles probably are not capable of achieving oestrus until 20 days of age (Carter et al. 1980). For comparison, males and females from different litters were weaned and paired at 21-22 days of age. All pairs were maintained until they produced a litter or until they were 60 days old. Individuals fi'om pairs that had not produced a litter were then switched so that each was paired with a stranger. Only pairs in which both individuals were fertile were included in the analysis. Results. Of the 14 pairs of fertile animals paired before reproductive maturity, only two produced a litter by 60 days of age; a third female was pregnant at the end of the experiment. The earliest litter was born when the parents were 53 days old. In contrast all 10 pairs formed from non-siblings at 21-22 days of age produced
32,
2
a litter before they were 60 days of age. The difference between these two groups is highly significant (Xz = 11.61, 1 df, P < 0.0001). These results suggest that social familiarity between non-sibling males and females before reproductive maturity lowers the likelihood that they will mate. Experiment 3: Effect of Separation on Incest Avoidance Methods. The effect of separation on recognition and incest avoidance was examined at two ages: newly-weaned 21-day-old siblings and 50-day-old siblings that had been housed together since weaning at 21 days of age. Three treatment groups were established with a n i m a l s of each age. In the first group male and female siblings were removed from their family groups and immediately paired (sib 21-0, sib 50-0). The second group consisted of siblings that had been separated f r o m their littermates and housed individually for 8 days before pairing (sib 21-8, sib 50-8). I n the third group siblings were separated for 15 days prior to pairing (sib 21-15, sib 50-15). The cages of all pairs were checked daily for newborn pups. Females that had not produced a litter within 60 days of pairing were sacrificed and autopsied. Females with embryos were combined with those that had given birth to arrive at the total number of pregnancies in each group. Results. With no separation, the incidence of pregnancy was < 10% (Fig. 1); there was no significant difference between the sib 21-0 and I00"
t~50 q~ O-
0~..
- ~
~
zl-o
50-0
i
i
zl-8
=
50-8
m-t5
50-15
GROUPS
Fig. 1. Percentage of pregnant females among pairs of siblings separated for different lengths of time prior to pairing. See text (experiment 3) for an explanation of the groups. The number of pairs in each group is within the bar. Groups 21-0 and 21-8 (*) are significantly different from each other (X2 = 6.9, ldf, P < 0.01), and groups 50-8 and 50-15 ('~) also differ significantly (Fisher exact text, P = 0.013).
GAVISH ET AL.: SIBLINGRECOGNITION IN PRAIRIE VOLES sib 50-0 groups. The number of pregnancies (60~, Fig. 1) among 21-day-old females was significantly greater when the siblings were separated for 8 days (X2 =- 6.9, 1 df, P < 0.01). A 15-day separation resulted in an even higher pregnancy rate (90 ~, Fig. 1), but the difference between the sib 21-8 and sib 21-15 groups was not statistically significant. The incidence of pregnancy among 50-day-old females paired following an 8-day separation (15~, Fig. 1) did not differ significantly from that for siblings that had not been separated. For 50-day-old sibling pairs that had been separated for 15 days the number of pregnancies (64 ~, Fig. 1) was significantly greater than that of the sib 50-8 group (Fisher exact test, P = 0.013). Thus, incest avoidance in siblings that had spent a relatively short time together (the 21 days from birth to weaning) was overcome by a separation of 8 days. However, for siblings that had remained together well past weaning (i.e. until 50 days old), a separation of 8 days was not sufficiently long to induce breeding. Discussion The results of this study indicate that prairie voles did not innately recognize siblings with whom they had no prior contact. Full siblings separated shortly after birth and reared apart reacted like strangers (i.e. they bred readily) when paired at weaning. On the other hand, nonsibling males and females reared together by the same pair of adults since shortly after birth did not breed when paired at weaning; these animals reacted to each other as do siblings. Juveniles did not breed with their nestmates, regardless of genetic relatedness. We did not investigate how the pups recognize each other. On the basis of other studies of rodents (e.g. Bowers & Alexander 1967; Halpin 1976; Porter et al. 1978; Huck & Banks 1979; Yamaguchi et al. 1981), we believe olfactory cues may be of major importance. Behavionral interactions between familiar individuals differ from those in which unfamiliar animals engage. There is very little naso-genital grooming among sibling prairie voles in undisturbed social groups (Getz & Carter 1980); thus, females normally would not acquire the male urinary pheromone required for reproductive activation (Carter et al. 1980) from their brothers in this way. In this study incest avoidance was overcome when 21-day-old siblings were separated for 8
365
days and then paired, although this length of separation had no effect on siblings that had been together until 50 days of age. The effect of separation argues against an innate mechanism involving recognition alleles (Hamilton 1964) or phenotype matching (Holmes & Sherman 1982) in which an individual's own phenotype serves as the basis of comparison; such mechanisms should not require reinforcement through continued association. Separation has also been shown to decrease sibling recognition in Acomys cahirinus (Porter & Wyrick 1979), Peromyscus erernicus (Dewsbury 1982) and P. maniculatus (Hill 1974), and recognition of familiar individuals in the collared lemming, Dicrostonyx groenlandicus (Huck & Banks 1979). Holmes & Sherman (1982) proposed that association during a critical period just prior to the time when non-kin are first encountered, rather than the total length of association (Bekoff 1981), is important for kin recognition. Our study was not specifically designed to determine a critical period between birth and weaning when siblings learn to recognize each other. However, few of the unrelated males and females that had been paired at 14 days of age bred upon reaching reproductive maturity; in this regard they behaved as if they were siblings. Thus the critical period for sibling recognition is not restricted to the first few days of life. Similarly, sibling recognition in Spermophilus beIdingi and S. parryii develops around the time of weaning (Sherman 1980; Holmes & Sherman 1982). Non-sibling Peromyscus maniculatus, paired at weaning but prior to sexual maturity, exhibited a reproductive delay similar to that of paired siblings (Hill 1974) 9That sibling recognition is based on proximity within the social context of early life is consistent with the life history and proposed social organization of the prairie vole (Getz & Carter 1980). Since the young are altricial and are reared in underground nests within spatially-discrete burrows, there would be little chance of unrelated pups encountering each other for two or three weeks after birth. In fact, during the course of live-trapping with multiple-catch traps at prairie vole burrows and on 10-m demographic grids, pups were rarely caught independently of their parents if they weighed less than 8-10 g (Hofmann & Getz, unpublished data); this corresponds to the size of 10-14-day-old pups in our laboratory colony. Thus there is time for siblings to learn to recognize each other before pups begin wandering above ground and first
366
ANIMAL
BEHAVIOUR,
e n c o u n t e r n o n - k i n as well as kin. A s l o n g as y o u n g voles r e m a i n t o g e t h e r at their natal b u r r o w , familiarity a m o n g t h e m w o u l d result in the a v o i d a n c e o f incestuous breeding. However, if voles disperse f r o m the n a t a l b u r r o w shortly after weaning, it seems t h a t there w o u l d b e no i m p e d i m e n t to siblings p a i r i n g a n d breeding should they meet after several d a y s ' separation. O u r experimental results are consistent with Bekoff's (1981) c o n t e n t i o n t h a t the p r o x i m a t e meclqanism of sibling recognition in m a n y m a m mals, p a r t i c u l a r l y those with multi-individual litters a n d altrieial young, d e p e n d s on associa t i o n d u r i n g early social experience. Aeknowledgments This study was f u n d e d b y N S F g r a n t D E B 78-25864 a n d N I H g r a n t H D 09328 to L. L. Getz. D r C. Sue C a r t e r p r o v i d e d s u p p o r t for a p r e l i m i n a r y study to e x p e r i m e n t 3. Brian K l a t t a n d B a r b a r a F r a s e offered helpful c o m m e n t s on the manuscript. REFERENCES
Batzli, G. O., Getz, L. L. & Hurley, S. S. 1977. Suppression of growth and reproduction of microtine rodents by social factors. J. Mammal., 58, 583-591. Beecher, M. D. 1982. Signature systems and kin recognition. Am. Zool., 22, 477-490. Bekoff, M. 1981. Mammalian sibling interactions: genes, facilitative environments, and the coefficient of familiarity. In: Parental Care in Mammals (Ed. by D. J. Gubernick & P. H. Klopfer), pp. 307-346. New York: Plenum Press. Blaustein, A. R. & O'Hara, R. K. 1981. Genetic control for sibling recognition? Nature, Lond., 290, 246-248. Bowers, J. M. & Alexander, B. K. 1967. Mice: individual recognition by olfactory cues. Science, N.Y., 158, 1208-1210. Carter, C. S., Getz, L. L., Gavish, L., McDermott, J. L. & Arnold, P. 1980. Male-related pheromones and the activation of female reproduction in the prairie vole (Microtus ochrogaster). Biol. Reprod., 23, 1038-1045. Davis, L. S. 1982. Sibling recognition in Richardson's ground squirrels (Spermophilus richardsonii). Behav. Ecol. Sociobiol., 11, 65-70. Dawkins, R. 1976. The Selfish Gene. Oxford: Oxford University Press. Dewsbury, D. A. 1982. Avoidance of incestuous breeding between siblings in two species of Peromyscus mice. Biol. Behav., 7, 157-169. Getz, L. L. & Carter, C. S. 1980. Social organization in Microtus ochrogaster populations. Biologist, 62, 56-69. .~
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Grau, H. J. 1982. Kin recognition in white-footed deermice (Peromyscus Ieucopus). Anim. Behav., 30, 497-505. Greenberg, L. 1979. Genetic component of bee odor in kin recognition. Science, N.Y., 266, 1095-1097. Halpin, Z. T. 1976. The role of individual recognition by odors in the social interactions of the Mongolian gerbil (Meriones unguiculatus). Behaviour, 58, 117-130. Hamilton, W. D. 1964. The genetical evolution of social behaviour. II. J. theoret. Biol., 7, 17-52. Hasler, M. J. & Nalbandov, A. V. 1974. The effect of weanling and adult males on sexual maturation in female voles (Microtus ochrogaster). Gen. comp. Endocrinol., 23, 237-238. Hill, J. L. 1974. Peromyscus: effect of early pairing on reproduction. Science, N.Y., 186, 1042-1044. Hoffman, M. D. 1981. Kin recognition and preference in Peromyscus leucopus: innate recognition or social familiarity. M.S. thesis, University of Missouri-St Louis. Holmes, W. G. & Sherman, P. W. 1982. The ontogeny of kin recognition in two species of ground squirrels. Am. Zool., 22, 491-517. Hohnes, W. G. & Sherman, P. W. 1983. Kin recognition in animals. Am. Scient., 71, 46-55. Huck, U. W. & Banks, E. M. 1979. Behavioral components of individual recognition in the collared lemming ( Dicrostonyx groenlandicus). Behav. EcoL SociobioL, 6, 85-90. Lacy, R. C. & Sherman, P. W. 1983. Kin recognition by phenotype matching. Am. Nat., 121, 489-512. McGuire, M. R. & Getz, L. L. I981. Incest taboo between sibling Microtus ochrogaster. J. Mammal., 62, 213-215. O'Hara, R. K. & Blaustein, A. R. 1981. An investigation of sibling recognition in Rana cascadae tadpoles. Anim. Behav., 29, 1121-1126. Porter, R. H. & Wyrick, M. 1979. Sibling recognition in spiny mice (Acomys cahirinus): influence o f age and isolation. Anim. Behav., 27, 761-766. Porter, R. H., Wyrick, M. & Pankey, J. 1978. Sibling recognition in spiny mice (Acomys cahirinus). Behav. EcoL SociobioL, 3, 61-68. Sherman, P. W. 1980. The limits of ground squirrel nepotism. In: Sociobiology: Beyond Nature] Nurture? (Ed. by G. W. Barlow & J. Silverberg), pp. 505-544. Boulder, Colorado: Westview Press. Wu, H. M. M., I-Iollaaes,W. G., Medina, S. R. & Sackett, G. P. 1980. Kin preference in infant Macaca nemestrina. Nature, Lond., 285, 225-227. Yamaguchi, M., Yamazaki, K., Beauchamp, G. K., Bard, J., Thomas, L. & Boyse, E: A. 1981. Distinctive urinary odors governed by the major histocompatibility locus of the mouse. Proc. Nat. Acad. Sci., 78, 5817-5820. Yamazaki, K., Boyse, E. A., Mikr, V., Thaler, H. T., Mathieson, B. J., Abbott, J., Boyse, J., Zayas, Z. A. & Thomas, L. 1976. Control of mating preferences in mice by genes in the major histocompatibility complex. J. exp. Med., 144, 13241335. (Received 18 April 1983 ; revised 26 May 1983 ; MS. number: A2876)
SIBLING RECOGNITION IN THE PRAIRIE VOLE, MICROTUS OCHROGASTER BY LEAH GAVISH*, JOYCE E. H O F M A N N & LOWELL L. GETZ Department of Ecology, Ethology, and Evolution, University of Illinois, Urbana-Champaign, IL 61801 U.S.A. Abstract. Since mating is seldom observed between sibling prairie voles, Microtus ochrogaster, this, behavioural discrimination was used to investigate sibling recognition in this speciesl Cross-fostering of 1-3-day-old pups demonstrated that unrelated pups reared together did not breed, whereas siblings reared apart bred readily when paired at weaning. When unrelated voles were paired at 14 days of age, prior to sexual maturity, significantly fewer pairs bred than when strangers were paired at 21 days of age. Separation of 21-day-old siblings for 8 days before pairing overcame incest avoidance; a 15-day separation was required for breeding by siblings that had remained together until they were 50 days old. These results indicate that sibling recognition and, consequently, incest avoidance depend on association prior to weaning. The prairie vole, Microtus oehrogaster, has been shown to exhibit incest avoidance. McGuire & Getz (1981) found that when brothers and sisters were weaned artd paired at 21 days of age only 6 % of the pairs produced a litter, whereas 78 % of non-siblings paired at the same age bred. Suppression of breeding among prairie vole littermates has also been reported by Hasler & Nalbandov (1974) and Batzli et al. (1977). The consistency of results obtained under different experimental conditions suggested that this form of behavioural discrimination could be used to investigate the mechanism of sibling recognition in the prairie vole. The present study was designed to determine whether recognition involves a genetic mechanism by which siblings could recognize each other even without prior association, or whether it is dependent upon the association of siblings early irt life. Recent studies indicate that kin recognition cart occur independently of previous association. Species which have shown this ability to recognize unfamiliar relatives include Cascades frog tadpoles, Rana easeadae (Blaustein & O'Hara 1981; O'Hara & Blaustein 1981); pigtail macaques, Maeaea nemestrina (Wu et al. 1980); Richardson's ground squirrels, Spermophilus riehardsonii (Davis 1982); arctic and Belding's ground squirrels, S. parryii and S. beldingi (Holmes & Sherman 1982); white-footed mice, Peromyseus leueopus (Grau 1982; although Hoffman 1981 obtained contradictory results); and sweat bees, Lasioglossum zephyrum (Greerlberg 1979).
Unfamiliar relatives might recognize each other because of the action of shared recognition alleles which would confer (1) a phenotypic trait, (2) the ability to recognize this trait in other individuals, and (3) a tendency to behave differentially towards individuals which shared the trait (Hamilton 1964; the 'green beard' effect of Dawkirts 1976). It has been suggested that such a genetic system is improbable (e.g. Hamilton 1964; Dawkins 1976; Sherman 1980). However, it has been shown that laboratory mice (Mus musculus) discriminated, in mate preference tests, between individuals that differed only in their major histocompatibility complex genotypes (Yamazaki et al. 1976). Alternatively, recognition could be the result of phenotype matching (Holmes & Sherman 1982; Lacy & Sherman 1983; called 'signature matching' by Beecher 1982). By this mechanism an individual would compare the phenotype of a newlyencountered stranger with its own phenotype or that of familiar kin. Thus, recognition would involve phenotypic traits shared as a consequence of genetic relatedness and the learning of reference phenotypes. On the other hand, recognition might require association among individuals. Art animal could learn the characteristics of the individuals with which it interacts in the social context of early life. Whenever close proximity during early life is highly correlated with kinship, this mechanism would normally result irt effective kin recognition. This may be the most common proximate mechanism of kin recognitiort (Sherman 1980; Bekoff 1981; Holmes & Sherman 1982). Familiarity due to association
*Present address: Department of Zoology, The Hebrew University, Jerusalem, Israel. 362
GAVISH ET AL.: SIBLING RECOGNITION IN PRAIRIE VOLES appears to be responsible for sibling recognition in spiny mice, Acomys cahirinus (Porter et al. 1978; Porter & Wyrick 1979); prairie deer mice, Peromyscus maniculatus (Hill 1974); cactus mice, P. eremicus (Dewsbury 1982); and, perhaps, white-footed mice, P. leucopus (Hoffman 1981). Association is also partially involved in kin recognition in Spermophilus parryii and S. beldingi (Holmes & Sherman 1982). The proximate mechanisms of kin recognition have been discussed in depth by Holmes & Sherman (1982, 1983). In this study prairie voles from a laboratory colony were used to manipulate the relative degree of genetic relatedness and social familiarity between individuals. We performed a crossfostering experiment to investigate whether incest avoidance among siblings depends upon innate recognition of genetic relatedness or upon association prior to weaning. We also studied the effect of separation for different lengths of time and at different ages upon sibling recognition and incest avoidance.
Methods and Results Subjects The prairie voles used in all experiments were laboratory-reared. The colon), had been established with approximately 25 pairs of wildcaught voles from several sites in the vicinity of Urbana, Illinois and has been maintained for over 20 generations. Additional wild-caught voles have been introduced into the colony from time to time. The breeding colony and the experimental animals were maintained in two rooms under a 14L : 10D photoperiod. The voles were housed in 25 • 45-cm plastic breeding cages with hardwood-chip bedding; Purina rabbit chow and water were provided ad libitum. There was no olfactory isolation of the animals in either room. The chemical signal (pheromone) which initiates reproductive activation in female prairie voles is not air-transmitted; dir.ect naso-genital contact is necessary (Carter et al. 1980). Experiment 1: Pups Cross-fostered Shortly After Birth Methods. In the first part of this experiment pairs were formed from non-siblings that had been reared together (NSRT). Litters of 1-2-dayold pups were removed from their parents' cages. A few minutes later each lactating female was given a 'litter' consisting of one of her own pups and pups from other like-aged litters. Each
363
female received the same number of pups (three to five) that she had had originally. Pups were weaned and paired at 22-23 days of age; each pair consisted of male and female non-siblings that had been reared by the same female. In the second part of the experiment pairs were formed from full siblings that had been reared apart (SRA). Newborn pups ( < 24 h old) were toe-clipped for identification and returned to their pare,Its. One or two days later, pups from these litters were exchanged so that each lactating female received one or two of her own pups and at least two foster pups. Most females were given five or six pups to rear. Pups were weaned at 22-23 days of age and male and female siblings that had been reared by different females were paired. Cages were checked frequently for newborn pups. After 60 days any pair that had not produced a litter was separated and each individual was paired with a stranger. These animals were maintained for an additional 60 days to determine if they were capable of breeding. Only those pairs in which both animals were determined to be fertile were included in the statistical analysis of results. The incidence of successful mating in the two groups of cross-fostered animals was compared with that of a group of non-siblings paired at 21-22 days of age (in experiment 2) and a group of siblings paired at the same age (in experiment 3). These groups were maintained under the same conditions as the cross-fostered groups. Results. Thirteen pairs o f non-siblings that had been reared together (NSRT) were established, but animals in three pairs were apparently infertile. Only one of the 10 fertile NSRT pairs produced a litter within 60 days (Table I). When the individuals in the other nine pairs were placed with strangers, pups were borI1 to each newly-established pair 22-25 days later. Twelve pairs of unfamiliar siblings (SRA) were established. Of these, 11 pairs produced a litter within 60 days (Table I). The earliest litter was born 36 days after pairing, the latest 46 days (median interval, 41 days). The male and female of the remaining pair both bred when placed with new mates, producing litters within 4 weeks. The number of SRA pairs that bred was significantly greater than the number of NSRT pairs that produced litters (x 2 = 11.8, 1 df, P < 0.001). The response of the NSRT group did not differ significantly from that of familiar siblings paired at weaning (Table I; Fisher exact test, P ---- 1.0). There was also no difference in the
364
ANIMAL
BEHAVIOUR,
Table L The Incidence of Successful Breeding in Prairie Voles Paired at 21-23 Days of Age Group
No. of pairs*
No. of pairs that bred
Non-siblings reared together (NSRT) (experiment 1)
10
1
Siblings reared apart (SRA) (experiment 1)
12
11~
Non-siblings reared apart (experiment 2)
10
lOt
Siblings reared together (experiment 3)
13
1w
*Includes only pairs in which both animals were fertile (see text). tSignificantly different from NSRT (xe = 11.8, 1 df, P < 0.001). ,Not significantly different from SRA (Fisher exact test, P = 1.0). w significantlydifferent from NSRT (Fisher exact test, P = 1.0). results for the SRA group and non-siblings paired at weaning (Table I; Fisher exact test, P = 1.0). Thus, males and females that had been reared together as juveniles rarely mated when given the opportunity, regardless of their genetic relatedness. Experiment 2: Non-siblings Paired Prior to Reproductive Maturity Methods. Males and females from different litters were weaned and paired at 14 days of age. This is as young as M. ochrogaster cart be safely weaned; most female prairie voles probably are not capable of achieving oestrus until 20 days of age (Carter et al. 1980). For comparison, males and females from different litters were weaned and paired at 21-22 days of age. All pairs were maintained until they produced a litter or until they were 60 days old. Individuals fi'om pairs that had not produced a litter were then switched so that each was paired with a stranger. Only pairs in which both individuals were fertile were included in the analysis. Results. Of the 14 pairs of fertile animals paired before reproductive maturity, only two produced a litter by 60 days of age; a third female was pregnant at the end of the experiment. The earliest litter was born when the parents were 53 days old. In contrast all 10 pairs formed from non-siblings at 21-22 days of age produced
32,
2
a litter before they were 60 days of age. The difference between these two groups is highly significant (Xz = 11.61, 1 df, P < 0.0001). These results suggest that social familiarity between non-sibling males and females before reproductive maturity lowers the likelihood that they will mate. Experiment 3: Effect of Separation on Incest Avoidance Methods. The effect of separation on recognition and incest avoidance was examined at two ages: newly-weaned 21-day-old siblings and 50-day-old siblings that had been housed together since weaning at 21 days of age. Three treatment groups were established with a n i m a l s of each age. In the first group male and female siblings were removed from their family groups and immediately paired (sib 21-0, sib 50-0). The second group consisted of siblings that had been separated f r o m their littermates and housed individually for 8 days before pairing (sib 21-8, sib 50-8). I n the third group siblings were separated for 15 days prior to pairing (sib 21-15, sib 50-15). The cages of all pairs were checked daily for newborn pups. Females that had not produced a litter within 60 days of pairing were sacrificed and autopsied. Females with embryos were combined with those that had given birth to arrive at the total number of pregnancies in each group. Results. With no separation, the incidence of pregnancy was < 10% (Fig. 1); there was no significant difference between the sib 21-0 and I00"
t~50 q~ O-
0~..
- ~
~
zl-o
50-0
i
i
zl-8
=
50-8
m-t5
50-15
GROUPS
Fig. 1. Percentage of pregnant females among pairs of siblings separated for different lengths of time prior to pairing. See text (experiment 3) for an explanation of the groups. The number of pairs in each group is within the bar. Groups 21-0 and 21-8 (*) are significantly different from each other (X2 = 6.9, ldf, P < 0.01), and groups 50-8 and 50-15 ('~) also differ significantly (Fisher exact text, P = 0.013).
GAVISH ET AL.: SIBLINGRECOGNITION IN PRAIRIE VOLES sib 50-0 groups. The number of pregnancies (60~, Fig. 1) among 21-day-old females was significantly greater when the siblings were separated for 8 days (X2 =- 6.9, 1 df, P < 0.01). A 15-day separation resulted in an even higher pregnancy rate (90 ~, Fig. 1), but the difference between the sib 21-8 and sib 21-15 groups was not statistically significant. The incidence of pregnancy among 50-day-old females paired following an 8-day separation (15~, Fig. 1) did not differ significantly from that for siblings that had not been separated. For 50-day-old sibling pairs that had been separated for 15 days the number of pregnancies (64 ~, Fig. 1) was significantly greater than that of the sib 50-8 group (Fisher exact test, P = 0.013). Thus, incest avoidance in siblings that had spent a relatively short time together (the 21 days from birth to weaning) was overcome by a separation of 8 days. However, for siblings that had remained together well past weaning (i.e. until 50 days old), a separation of 8 days was not sufficiently long to induce breeding. Discussion The results of this study indicate that prairie voles did not innately recognize siblings with whom they had no prior contact. Full siblings separated shortly after birth and reared apart reacted like strangers (i.e. they bred readily) when paired at weaning. On the other hand, nonsibling males and females reared together by the same pair of adults since shortly after birth did not breed when paired at weaning; these animals reacted to each other as do siblings. Juveniles did not breed with their nestmates, regardless of genetic relatedness. We did not investigate how the pups recognize each other. On the basis of other studies of rodents (e.g. Bowers & Alexander 1967; Halpin 1976; Porter et al. 1978; Huck & Banks 1979; Yamaguchi et al. 1981), we believe olfactory cues may be of major importance. Behavionral interactions between familiar individuals differ from those in which unfamiliar animals engage. There is very little naso-genital grooming among sibling prairie voles in undisturbed social groups (Getz & Carter 1980); thus, females normally would not acquire the male urinary pheromone required for reproductive activation (Carter et al. 1980) from their brothers in this way. In this study incest avoidance was overcome when 21-day-old siblings were separated for 8
365
days and then paired, although this length of separation had no effect on siblings that had been together until 50 days of age. The effect of separation argues against an innate mechanism involving recognition alleles (Hamilton 1964) or phenotype matching (Holmes & Sherman 1982) in which an individual's own phenotype serves as the basis of comparison; such mechanisms should not require reinforcement through continued association. Separation has also been shown to decrease sibling recognition in Acomys cahirinus (Porter & Wyrick 1979), Peromyscus erernicus (Dewsbury 1982) and P. maniculatus (Hill 1974), and recognition of familiar individuals in the collared lemming, Dicrostonyx groenlandicus (Huck & Banks 1979). Holmes & Sherman (1982) proposed that association during a critical period just prior to the time when non-kin are first encountered, rather than the total length of association (Bekoff 1981), is important for kin recognition. Our study was not specifically designed to determine a critical period between birth and weaning when siblings learn to recognize each other. However, few of the unrelated males and females that had been paired at 14 days of age bred upon reaching reproductive maturity; in this regard they behaved as if they were siblings. Thus the critical period for sibling recognition is not restricted to the first few days of life. Similarly, sibling recognition in Spermophilus beIdingi and S. parryii develops around the time of weaning (Sherman 1980; Holmes & Sherman 1982). Non-sibling Peromyscus maniculatus, paired at weaning but prior to sexual maturity, exhibited a reproductive delay similar to that of paired siblings (Hill 1974) 9That sibling recognition is based on proximity within the social context of early life is consistent with the life history and proposed social organization of the prairie vole (Getz & Carter 1980). Since the young are altricial and are reared in underground nests within spatially-discrete burrows, there would be little chance of unrelated pups encountering each other for two or three weeks after birth. In fact, during the course of live-trapping with multiple-catch traps at prairie vole burrows and on 10-m demographic grids, pups were rarely caught independently of their parents if they weighed less than 8-10 g (Hofmann & Getz, unpublished data); this corresponds to the size of 10-14-day-old pups in our laboratory colony. Thus there is time for siblings to learn to recognize each other before pups begin wandering above ground and first
366
ANIMAL
BEHAVIOUR,
e n c o u n t e r n o n - k i n as well as kin. A s l o n g as y o u n g voles r e m a i n t o g e t h e r at their natal b u r r o w , familiarity a m o n g t h e m w o u l d result in the a v o i d a n c e o f incestuous breeding. However, if voles disperse f r o m the n a t a l b u r r o w shortly after weaning, it seems t h a t there w o u l d b e no i m p e d i m e n t to siblings p a i r i n g a n d breeding should they meet after several d a y s ' separation. O u r experimental results are consistent with Bekoff's (1981) c o n t e n t i o n t h a t the p r o x i m a t e meclqanism of sibling recognition in m a n y m a m mals, p a r t i c u l a r l y those with multi-individual litters a n d altrieial young, d e p e n d s on associa t i o n d u r i n g early social experience. Aeknowledgments This study was f u n d e d b y N S F g r a n t D E B 78-25864 a n d N I H g r a n t H D 09328 to L. L. Getz. D r C. Sue C a r t e r p r o v i d e d s u p p o r t for a p r e l i m i n a r y study to e x p e r i m e n t 3. Brian K l a t t a n d B a r b a r a F r a s e offered helpful c o m m e n t s on the manuscript. REFERENCES
Batzli, G. O., Getz, L. L. & Hurley, S. S. 1977. Suppression of growth and reproduction of microtine rodents by social factors. J. Mammal., 58, 583-591. Beecher, M. D. 1982. Signature systems and kin recognition. Am. Zool., 22, 477-490. Bekoff, M. 1981. Mammalian sibling interactions: genes, facilitative environments, and the coefficient of familiarity. In: Parental Care in Mammals (Ed. by D. J. Gubernick & P. H. Klopfer), pp. 307-346. New York: Plenum Press. Blaustein, A. R. & O'Hara, R. K. 1981. Genetic control for sibling recognition? Nature, Lond., 290, 246-248. Bowers, J. M. & Alexander, B. K. 1967. Mice: individual recognition by olfactory cues. Science, N.Y., 158, 1208-1210. Carter, C. S., Getz, L. L., Gavish, L., McDermott, J. L. & Arnold, P. 1980. Male-related pheromones and the activation of female reproduction in the prairie vole (Microtus ochrogaster). Biol. Reprod., 23, 1038-1045. Davis, L. S. 1982. Sibling recognition in Richardson's ground squirrels (Spermophilus richardsonii). Behav. Ecol. Sociobiol., 11, 65-70. Dawkins, R. 1976. The Selfish Gene. Oxford: Oxford University Press. Dewsbury, D. A. 1982. Avoidance of incestuous breeding between siblings in two species of Peromyscus mice. Biol. Behav., 7, 157-169. Getz, L. L. & Carter, C. S. 1980. Social organization in Microtus ochrogaster populations. Biologist, 62, 56-69. .~
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Grau, H. J. 1982. Kin recognition in white-footed deermice (Peromyscus Ieucopus). Anim. Behav., 30, 497-505. Greenberg, L. 1979. Genetic component of bee odor in kin recognition. Science, N.Y., 266, 1095-1097. Halpin, Z. T. 1976. The role of individual recognition by odors in the social interactions of the Mongolian gerbil (Meriones unguiculatus). Behaviour, 58, 117-130. Hamilton, W. D. 1964. The genetical evolution of social behaviour. II. J. theoret. Biol., 7, 17-52. Hasler, M. J. & Nalbandov, A. V. 1974. The effect of weanling and adult males on sexual maturation in female voles (Microtus ochrogaster). Gen. comp. Endocrinol., 23, 237-238. Hill, J. L. 1974. Peromyscus: effect of early pairing on reproduction. Science, N.Y., 186, 1042-1044. Hoffman, M. D. 1981. Kin recognition and preference in Peromyscus leucopus: innate recognition or social familiarity. M.S. thesis, University of Missouri-St Louis. Holmes, W. G. & Sherman, P. W. 1982. The ontogeny of kin recognition in two species of ground squirrels. Am. Zool., 22, 491-517. Hohnes, W. G. & Sherman, P. W. 1983. Kin recognition in animals. Am. Scient., 71, 46-55. Huck, U. W. & Banks, E. M. 1979. Behavioral components of individual recognition in the collared lemming ( Dicrostonyx groenlandicus). Behav. EcoL SociobioL, 6, 85-90. Lacy, R. C. & Sherman, P. W. 1983. Kin recognition by phenotype matching. Am. Nat., 121, 489-512. McGuire, M. R. & Getz, L. L. I981. Incest taboo between sibling Microtus ochrogaster. J. Mammal., 62, 213-215. O'Hara, R. K. & Blaustein, A. R. 1981. An investigation of sibling recognition in Rana cascadae tadpoles. Anim. Behav., 29, 1121-1126. Porter, R. H. & Wyrick, M. 1979. Sibling recognition in spiny mice (Acomys cahirinus): influence o f age and isolation. Anim. Behav., 27, 761-766. Porter, R. H., Wyrick, M. & Pankey, J. 1978. Sibling recognition in spiny mice (Acomys cahirinus). Behav. EcoL SociobioL, 3, 61-68. Sherman, P. W. 1980. The limits of ground squirrel nepotism. In: Sociobiology: Beyond Nature] Nurture? (Ed. by G. W. Barlow & J. Silverberg), pp. 505-544. Boulder, Colorado: Westview Press. Wu, H. M. M., I-Iollaaes,W. G., Medina, S. R. & Sackett, G. P. 1980. Kin preference in infant Macaca nemestrina. Nature, Lond., 285, 225-227. Yamaguchi, M., Yamazaki, K., Beauchamp, G. K., Bard, J., Thomas, L. & Boyse, E: A. 1981. Distinctive urinary odors governed by the major histocompatibility locus of the mouse. Proc. Nat. Acad. Sci., 78, 5817-5820. Yamazaki, K., Boyse, E. A., Mikr, V., Thaler, H. T., Mathieson, B. J., Abbott, J., Boyse, J., Zayas, Z. A. & Thomas, L. 1976. Control of mating preferences in mice by genes in the major histocompatibility complex. J. exp. Med., 144, 13241335. (Received 18 April 1983 ; revised 26 May 1983 ; MS. number: A2876)