Unfamiliar Kin Research Articles

Kin recognition and inbreeding avoidance in zebrafish, Danio rerio, is based on phenotype matching

Differentiating kin from nonkin enables organisms of many species to allocate resources or altruistic behaviour towards related conspecifics and to avoid mating with close relatives. Kin recognition mechanisms can vary among species and may reflect the social environment. Learned familiarity with nest or shoalmates may serve as a good indication that individuals are related, as long as the social system is sufficiently stable to avoid intermingling of unrelated individuals with siblings. Phenotype matching allows for recognition of even unfamiliar kin because individuals establish an olfactory, visual or acoustic template for their kin during early development and compare this template to cues from unfamiliar individuals later in life. We tested which kin recognition mechanism is used by zebrafish and we found that the preference for kin changes with sexual maturity. The olfactory preference of laboratory-bred juveniles and reproductively active adults were tested in an odour choice flume. Juveniles of mixed-sex groups spent more time on the side of unfamiliar kin than unfamiliar nonkin, indicating that kin recognition and preference are based on a phenotype matching mechanism. They also preferred familiar kin to unfamiliar kin, showing that familiarity with individuals enhances kin preference. Preference changed with sexual maturity. Adult females preferred the odour of unfamiliar, unrelated males to unfamiliar brothers, indicating inbreeding avoidance. Adult male zebrafish showed no preference for the odour of related or unrelated females. To our knowledge, this is the first study testing different kin recognition mechanisms and switching of kin preference in the same species of fish.

Kin and population recognition in sympatric Lake Constance perch (Perca fluviatilis L.): can assortative shoaling drive population divergence?

Prior studies have shown that perch (Perca fluviatilis L.) of Lake Constance belong to two genetically different but sympatric populations and that local aggregations of juveniles and adults contain closely related kin. In this study, we analysed the genetic structure of pelagic perch larvae to investigate if kin-structured shoals already exist during early ontogenetic development or might be the result of homing to natal sites. Analysis of the gene frequencies at five microsatellite loci revealed that three out of five pelagic aggregations of larvae showed significant accumulation of kin. To investigate possible mechanisms of shoal formation, we tested if perch use olfactory cues to recognize their kin. Choice tests in a fluviarium showed preference for odours of unfamiliar kin vs unfamiliar non-kin. Additionally, we showed that perch could differentiate between the odours of the two sympatric populations and significantly preferred unfamiliar and unrelated conspecifics of their own over the foreign population. Our results present a behavioural mechanism that can lead to the observed formation of kin-structured shoals in perch. We further discuss if the ability to discriminate between their own and a foreign population can result in assortative mating within populations and thus form the basis of “socially mediated speciation” in perch.

Kin Recognition in Juvenile Zebrafish (Danio rerio) Based on Olfactory Cues

Genetic analyses of numerous fish species have shown that shoals formed by larvae often consist of closely related kin (1). Aggregating with kin may be an altruistic trait that evolved through kin selection (2). Individuals would increase their inclusive fitness by sharing the benefits of shoaling among related individuals (3). Laboratory experiments on recognition of kin vs. non-kin groups of Atlantic Salmon (Salmo salar) (4) demonstrated possible advantages: kin groups had fewer aggressive interactions, used a greater proportion of “threat” behavior as opposed to fighting, and subordinates especially had improved growth. The mechanisms by which these kin groups develop and stay separate from each other are not known. The genes of the major histocompatibility complex (MHC) are a source of individual odors released into the water via urine (5). Such pheromones might be involved in olfactory kin recognition. Here, we tested the hypothesis that zebrafish can recognize kin based on olfactory cues. Zebrafish (Danio rerio) live in freshwater streams and rice paddies in the Ganges River of East India, Bangladesh, and Burma. Although this species is widely used as a model in genetic and developmental research, little is known about its natural behavior. Zebrafish spawn up to several hundred eggs at a time, and these develop in the substrate without any parental care. Larvae (G.G. pers. obs.), and sometimes adults, can be observed in shoals (6, 7), but their genetic relatedness is unknown. We observed wild-type juvenile zebrafish, aged 6–8 weeks; the fish were kept in 2.5-1 aquaria under a day/night cycle of 14/10 h and fed on a standard diet of brine shrimp nauplii and dry fish food. Twenty-four hours before an experiment started, 2 separate kin groups consisting of 12 full siblings each were placed into two 9-1 aquaria with standing water. From each kin group, 3 fish were tested in the flume, one at a time, for a total of 6 fish. This procedure was repeated 3 times for a total of 6 kin groups and 18 test animals. Water from each of the two aquaria was used as the two stimuli in an olfactory preference test. Single individuals of either group were used as test fish and were placed into a choice flume (20 cm long 4 cm wide, water level 2.5 cm) that maintained two separate water columns (Fig. 1) (8). Uniform and unidirectional water flow was maintained at a constant rate of 40 ml/min ( 3.5 mm/s). Periodic dye tests showed that the two water columns remained well separated. Prior to each trial, formulated fresh water was run through both channels of the flume for 5 min to allow the subject to acclimate. Each trial consisted of four 3-min periods, during which water from kin and non-kin aquaria was presented on alternate sides of the flume to correct for the possibility of unrelated side bias. Every 10 s, we recorded which side of the flume (A or B, Fig. 1) the fish was swimming on. The number of times each animal was recorded on the side with kin stimulus was expressed as a percentage of the total number of recorded observations (i.e., kin plus non-kin). A score greater than 50% (random distribution) indicated a preference for the kin stimulus; and when the percentages of all the fish were compared using a Wilcoxon matched-pairs signed-ranks (WSR) test, the preference for kin was significant (WSR 45.0, P 0.050) (Fig. 1). Our study is the first to show that juvenile zebrafish can recognize and prefer their siblings to unrelated conspecifics based on olfactory cues. There are two general categories of kin recognition mechanisms, both based on learning processes as Tang-Martinez (9) emphasized. The first (“indirect”) mechanism is based on familiarity, where individuals behave nepotistically to conspecifics with whom they grow up. The second (“direct”) kin recognition mechanism allows individuals to identify even unfamiliar kin. Direct recognition is thought to be based on ‘phenotype matching’, in which an individual must learn cues, either from the phenotypes of close relatives (familial imprinting) (10), or from itself, to form a template for comparison with the phenotype of other individuals. Our results cannot distinguish between these recognition mechanisms.

The ontogeny of kin recognition in tadpoles of the toad Bufo melanostictus (Anura; bufonidae).

The ontogeny of kin recognition and influence of social environment on the development of kin recognition behaviour was experimentally investigated in tadpoles of Bufo melanostictus that lived in aggregations and showed low larval dispersion. Embryos and tadpoles of the toad were reared as (i) kin only, (ii) with kin and non-kin (separated by a mesh screen), and (iii) in isolation. They were tested for the ability to discriminate between (i) familiar siblings and unfamiliar non-siblings, (ii) familiar siblings and familiar non-siblings and, (iii) unfamiliar siblings and unfamiliar non-siblings. All tadpoles were fed on boiled spinach before conducting trials. Preference of test tadpoles to associate near the end compartments whether empty or containing members of specific stimulus groups was assessed using a rectangular choice tank. When tested in tanks with empty end compartments, the test tadpoles showed random distribution and thus no bias for the apparatus or the procedure. In the presence of kin/non-kin in the end compartments a significantly greater number of test tadpoles spent the majority of the time near familiar or unfamiliar kin rather than near familiar or unfamiliar non-kin. Kin discrimination ability persisted throughout larval development. Familiarity with siblings is not required for discriminating kin from non-kin, and kin discrimination ability is not modified following exposure to non-kin. Also, involvement of dietary cues is unlikely to be the prime mechanism of kin recognition in B. melanostictus unlike in some other anurans.

Schooling decisions in guppies ( Poecilia reticulata ) are based on familiarity rather than kin recognition by phenotype matching

Evidence from a number of freshwater species indicates that fish prefer to school with familiar individuals. Do they also choose to associate with kin? Our experiment tested this idea using the Trinidadian guppy, Poecilia reticulata, a species whose reproductive biology favours the association of kin groups. Juveniles reared together were able to recognise one another on the basis of either visual or chemical cues, but showed no preference for schooling with unfamiliar kin. We therefore conclude that any naturally occurring kin groups in this species will occur as a result of familiarity rather than as a consequence of kin recognition based on phenotype matching.

Kin discrimination in salmonids

The data presented here suggest that significant selection pressures towards kin discrimination behaviour patterns result from kin-biased territorial defence behaviour patterns. Salmonids employ a phenotype matching recognition mechanism allowing individuals to discriminate unfamiliar kin. Kin discrimination abilities allow individuals to reduce the levels of aggression associated with territorial defence towards related conspecifics and to defend smaller territories near kin versus non-kin. This kin-biased territorial defence behaviour is observed in at least one species under a wide range of territorial quality conditions. Within kin groups, subordinate individuals obtain a greater number of foraging attempts, resulting in kin-biased foraging within the social group. As a result of this kin-bias, individuals within kin groups show significantly higher mean weight increases (increased direct fitness benefits) and reduced variance in these increases (increased indirect benefits). Since all individuals within the kin groups obtained higher, less variable weight increases, we can argue that individuals are increasing their inclusive fitness as a result of these kin-biased behaviour patterns.

Kin recognition by tadpoles and froglets of the wood frog Rana sylvatica.

We investigated kin recognition by the wood frog Rana sylvatica in blind laboratory experiments using spatial proximity as a recognition assay. Tadpoles were tested for the ability to discriminate between: 1) familiar full-sibs and unfamiliar non-kin, 2) unfamiliar paternal half-sibs and unfamiliar non-kin, and 3) familiar and unfamiliar full-sibs. Tadpoles discriminated full- and paternal half-sibs from unrelated conspecifics, but did not discriminate between familiar and unfamiliar full-sibs. Froglets from the same laboratory population were tested for the ability to discriminate between 1) familiar full-sibs and unfamiliar non-kin, and 2) unfamiliar paternal half-sibs and unfamiliar non-kin. Froglets preferentially associated with full- and half-sibs over unrelated conspecifics. Our results show that familiarity, i.e., prior association, is not necessary for kin recognition in tadpoles and froglets. The ability of tadpoles and froglets to recognize unfamiliar paternal half-sibs demonstrates that a common maternal factor is not necessary for kin recognition, and indicates that the recognition cue has a genetic component. Our results add to the increasing evidence that a variety of vertebrate and invertebrate animals have the ability to recognize unfamiliar kin by using genetically specified recognition cues.

The kin recognition system of carpenter ants (Camponotus spp.)

Previous investigations of small colonies (<10 workers each) of Camponotus spp. indicated that queen-derived recognition cues, transferred to and learned by workers, are of major importance in between-colony discrimination (Carlin and Holldobler 1983, 1986). However, queens may have contributed disproportionately to a communal label or “gestalt” in small colonies. Here we report experiments on groups of approximately 190 sister workers of C. floridanus, adopted to alien conspecific queens. In 1200 neutral arena encounters, adoptees were introduced to their own unfamiliar sisters, reared in their colony of origin; unfamiliar non-sisters were introduced as controls. Following tests of worker behavior, the queens were dissected. Aggression was equally strong in encounters between unfamiliar kin and encounters between nonkin, if the adoptive queen had fully-developed ovaries, was completely inseminated, and produced abundant brood. However, workers were significantly less aggressive in encounters between kin if their queens had less active ovaries and were incompletely inseminated. Thus the presence of a fully functional queen continues to prevent recognition of unfamiliar kin in larger colonies, but worker cues can become more important when the queen is ineffective. In addition, adoptees of productive queens were significantly less aggressive toward pure-colony sisters than the latter were to them, suggesting the involvement of a foreignlabel rejection mechanism (Getz 1982).

Kin recognition in mice: age, sex and parental effects

Mice were tested for their ability to discriminate close but unfamiliar kin in pairwise interactions. Pairs were set up to control for the effects of age, sex, and experience with the maternal parent on kin discrimination measured as differences in social interaction between full sibling, half-sibling and unrelated pairs. Comparisons between single-sex pairs showed that, as in previous studies, males and females differed in their patterns of kin discrimination, males showing more investigative activity towards unrelated individuals. No differences emerged between maternal and paternal half-siblings. In mixed-sex pairings, however, the pattern of discrimination depended on the relative ages of males and females. In adult female-juvenile male pairs there was no evidence of kin discrimination, but in adult male-juvenile female pairs discrimination was evident in a wider range of social interactions than in male pairs. Furthermore, adult male-juvenile female pairs showed significant differences between full and halfsiblings siblings and between maternal and paternal half-siblings. This is in keeping with the findings of other workers that parental effects on social behaviour in mice are most pronounced in inter-sexual interactions. When pairs were analysed for age effects discrimination turned out to be due mainly to differences in the behaviour of adult animals but with some pair-level effects.

Kin recognition by phenotype matching in female Belding's ground squirrels

Female Belding's ground squirrels ( Spermophilus beldingi), that had been reared apart from each other, were observed in paired-encounter tests to determine whether exposure to nestmates' phenotypes during development had influenced their subsequent social discriminations. As a result of cross-fostering, test partners were (1) either unfamiliar sisters (reared apart) or unfamiliar, unrelated females, and were (2) either reared with each other's siblings (indirectly exposed to each other) or were not reared with each other's siblings (not indirectly exposed to each other). Regardless of relatedness, females that were indirectly exposed to each other were significantly less agonistic during tests than females not indirectly exposed to each other. This suggests that females learned something from their nestmates' phenotypes and later recalled what they had learned in order to distinguish between their nestmates' unfamiliar kin and non-kin. Furthermore, sisters that were indirectly exposed to each other were less agonistic than nonsisters that were indirectly exposed to each other. This was true even when the only kin phenotypes females had experienced during rearing were their own, which suggests that females may have compared unfamiliar phenotypes with their own, as well as those of their nestmates. Thus, the phenotypes that females encountered during rearing, both their nestmates' and their own, influenced their later social discriminations, probably by phenotype matching. Under this recognition mechanism, an individual forms a hypothetical ‘kin template’ based on its own or its familiar relatives' phenotypes, and later compares the phenotypes of other conspecifics with the learned template.